Академический Документы
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Культура Документы
SYLLABUS
2015
University of Texas Medical School at Houston
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Course Description
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Syllabus Page
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COMPLEMENT
Complement
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Timeline of Immunology
Glossary
Cover Description: Principles of Modern Immunobiology. B.H. Park and R.A. Good. 1974.
Lea & Febiger, Henry Kimpton Publishers, Philadelphia. p54.
The purpose of the Immunology course is to provide a basic knowledge of the immune response and
its involvement in health and disease. A series of lectures cover course components; additional
materials are presented through clinical correlations that focus on clinically applied immunological
concepts. An effort has been made to increase clinical relevance and problem-solving skills through
an essay assignment and through a team-learning exercise.
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Session
Date
Time
Instructor
OVERVIEW AND ELEMENTS OF THE IMMUNE SYSTEM
1
1/6/2015 10:00-10:50
Jeffrey Actor
2
1/6/2015 11:00-11:50
Jeffrey Actor
3
1/9/2015 8:00-8:50
Jeffrey Actor
ANTIGENS AND ANTIBODIES
4
1/13/2015 10:00-10:50
5
1/13/2015 11:00-11:50
6
1/16/2015 8:00-8:50
COMPLEMENT
7
1/16/2015
9:00-9:50
Topic
Medical Importance of the Immune System
Cells and Organs of the Immune Sytstem
Innate Immunity/Inflammation
Sudhir Paul
Keri Smith
Keri Smith
Rick Wetsel
Complement
1:00-3:00
1:00-3:00
Midterm Exam
Antibody-Mediated Reactions
1:00-4:00
1:00-4:00
Cell-Mediated Reactions
Immunology:ClinicalScenarios
Disorders of the Immune Response
Immunoprophylaxis (Vaccines)
Cancer Immunology
Team Based Learning
Transplantation
ImmunoTherapy
Final Exam
Essay must be submitted prior to 5:00pm
Course Director:
LECTURERS
OFFICE
TELEPHONE
MSB 2.214
BCM
MSB 2.278
MSB 2.230A
Texas Children's
MSB 2.248
MSE R428
SRB 430A
713-500-5344
713-798-3390
713-500-5338
713-500-5347
832-824-1274
713-500-2250
713-500-5422
713-500-2412
Jeffrey.K.Actor@uth.tmc.edu
skagarwa@bcm.edu
Steven.J.Norris@uth.tmc.edu
Sudhir.Paul@uth.tmc.edu
WTSheare@texaschildrens.org
Keri.C.Smith@uth.tmc.edu
Dat.Q.Tran@uth.tmc.edu
Rick.A.Wetsel@uth.tmc.edu
1) COURSE ORGANIZATION
The purpose of the Immunology course is to provide a basic knowledge of the immune response and its
involvement in health and disease. All lectures will be presented in MSB 2.006. An effort has been
made to increase clinical relevance and problem-solving skills through an essay assignment and facultypresented clinical correlations, and a team based learning exercise.
Any questions on the lecture material should be addressed to Dr. Actor or directly to that lecturer. If you
have general problems or comments regarding the course, your grades, or the faculty, please contact the
course director. If the problem is not resolved, you should make an appointment to see Dr. Robert L.
Hunter (Chairman of Pathology) at MSB 2.136 (500-5301) or, finally, Dr. Patricia Butler (Assoc. Dean
for Educational Programs) or Dr. Margaret McNeese (Assoc. Dean for Student Affairs).
2) COURSE MATERIALS
a)
Lectures. The student is responsible for all material covered in lectures and faculty presented
clinical correlations, as well as for any additional handouts or assignments (whether provided in this
syllabus or at a later time). Immunology is a rapidly advancing area, so the lectures may contain
new information not covered in the textbooks. Therefore you should make every effort to attend
lecture and take complete and accurate notes. Streaming video is available on-line through the UT
Med School student web pages. Additional information may also be available from the Conference
Operations Office (LRC), and can be used to verify your notes.
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b) Reading. Two textbooks are required for the course. Chapter assignments are listed directly in
the syllabus chapter. Required Case Studies are listed separately in this syllabus.
R. Coico and Sunshine, G. Immunology: A Short Course. (6th Ed) John Wiley & Sons, Inc., 2009.
(The 7th edition is set for release in March, 2015.)
R. S. Geha and Notarangelo, L. Case Studies in Immunology: A Clinical Companion. (6th Ed)
Garland Publishing, New York, 2012.
The Coico et al. text was selected because it is well-organized, clearly and concisely written, and
contains chapter summaries, study questions, and case studies. The lecture schedule is loosely organized
to match the Coico et al. book chapters. Knowledge of the assigned reading is required, even if the
material is not covered in the lectures. Modifications of the study questions may be used in the exams.
The Geha and Rosen text provides examples of the role of immunology in health and disease, and is used
extensively in the Clinical Correlations and as Clinical Vignettes in the lectures. Cases from Geha and
Notarangelo presented (in part or in full) during lecture are considered required reading. Please
see the list of required associated cases assigned for each lecture, located under Clinical Correlation
Required Readings.
c) MEDIC IMMUNOLOGY Web Page. You are encouraged to make use of the MEDIC Immunology
web site at: https://med.uth.edu/pathology/courses/immunology/. Materials are also on Blackboard. The
website is actively updated during the course to include links for lecture materials and information that
will assist in understanding of course materials.
Alternative recommended texts available in the bookstore (not required, but potentially helpful):
Actor, J.K. Elseviers Integrated Immunology and Microbiology (2nd Ed.), Mosby/Elsevier,
Philadelphia, 2012.
Actor, J.K. Introductory Immunology: Basic Concepts for Interdisciplinary Applications (1st Ed.),
Academic Press/Elsevier, 2014.
Some other good textbooks (not required, but potentially helpful) may also be available in the bookstore:
Parham, P. The Immune System. 3rd Edition. Garland Publishing, New York, 2009.
o Note: a 4th edition was due for release at the end of 2014.
Abbas, A. K. and Lichtman, A. H. Basic Immunology Functions and Disorders of the Immune System, 4th Edition.
Saunders-Elsevier. Philadelphia, PA. 2012.
Owen, J. Punt, J., and Strandford, S. Kuby Immunology (7th Ed.), W.H. Freeman and Company, New York, 2013.
Murphy, K. Janeways Immunobiology (8th Ed.). Garland Publishing, New York, 2012 (updated 2014).
Each of these texts may be found at the LRC or the HAM Library, or available on-line for purchase
and/or digital download.
d) Essay Assignment. Students must turn in one essay assignment worth 10 points. Students must
attend one of the City-Wide Infectious Disease Rounds and provide a written review of one of the cases.
The assignment and due date are described in detail elsewhere in the syllabus, as well as on Blackboard.
e) Team Based Learning. There will be one team based learning exercise as a portion of the course.
The TBL is detailed later in the syllabus. The TBL is worth a maximum of 10 points.
f) Clinical Scenarios. In addition to the regular lectures, we may have a Clinical Scenario sessions
during the semester. Past experience has shown that immunology (or any other medical topic) is easier
to learn and remember if it is presented as clinical cases involving 'real' patients. In each clinical
correlation, cases relevant to immunology will be discussed by faculty. The correlate scenarios are
related to those presented in the Geha and Notarangelo text, but may vary to accommodate additional
learning materials.
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g) Self Study Questions. Additional study questions are provided at the end of some lecture outlines.
The purpose of these questions is to test your knowledge and extend your learning beyond rote
memorization toward more 'cognitive' learning. The study questions will not be graded, but questions
related to these assignments overlap with examination materials. Answers are typically (but not always)
posted on the Immunology web site or on Blackborad.
h) Streaming video. Lectures will be made available for viewing via streaming video over the internet.
i) Office hours and other assistance. Students are encouraged to approach the lecturers if they need
assistance in understanding the course material. Dr. Actor is also available at his office (MSB 2.214) by
individual appointment or by phone or email.
3) GRADING
a) Examinations. There will be two major exams consisting of multiple choice, matching, and national
board format questions. The midterm exam will contain 60 questions (worth 40% of your grade). A
cumulative final exam will have 80 questions (worth 40% of your grade). Exam answers will be posted
according to accepted policies of the University. Policies for review after each exam are set by the
University (see posted document on University Policy on Exam Grading and Review sessions).*
b) Essay Assignment. The essay assignment is required and will be worth a possible 10 points.
Grading is based on adherence to the format described in this syllabus, thoroughness, and application of
your budding medical knowledge and logic; you are not expected to 'know it all' at this point. The due
date is listed in the Essay instruction page in the syllabus, and posted on Blackboard. Essays may be
turned in early. Assignments turned in late will only receive a maximum of half credit (no exceptions).
c) Team Based Learning. Questions answered for the TBL session are worth a maximum of 10 points.
d) Overall grade. The total possible points and grade assignments are given below. The total value of
points for the course is 100 points.
Midterm
Final
Essay Assignment
Team Based Learning
40 points
40 points
10 points
10 points
(60 questions)
(80 questions)
e) Final grade assignment. The final grade is based on percentage of points earned (max of 100 points)
as related to total possible points (max of 100).
Honors
High pass
Pass
Below Pass
Fail
90-100 %
85.5-89.99 %
69.5-85.49 %
65.5-69.49 %
65.49 % or below
*Immunology Exam Question Review Policy: Review of exams may be done on an individual basis.
Upon written request, students may view questions missed. Any requests to review exams must be
submitted immediately following release of scores; exam questions may only be reviewed within the two
week period following release of scores. The Course Director has the right to limit question viewing.
The Course Director (course policy) limits question viewing to a two week period after release of scores.
vi
immune mechanisms to combat this infectious agent and how they affect the course of
infection (e.g. Macrophages phagocytose and process the antigen and present antigen
fragments in association with MHC Class II proteins to antigen-specific CD4+ helper T cells,
role of complement, cell phenotypes involved, etc.). Be specific and included details! How was
the immune response of this patient different than normal (if this is applicable)? Did the
patient have an underlying condition that contributed to the development of this infection? Did the
patient have cancer, AIDS, hereditary immunodeficiency or some other condition affecting the
immune response? How did the immune response (or lack thereof) affect the outcome of this case?
Did the immune response contribute to the pathogenesis of disease (i.e. is immunopathology
involved)? Describe immunization or other immunologic procedures (such as passive transfer of
antibodies) used in the prevention or treatment of this disease.
d) Cite references used in your analysis of the case. You will need to refer to published journal
articles to obtain specific background information or methods needed to comprehend the case.
Include as many references as needed to support ideas. Points will be subtracted if relevant citations
are absent. You must include at least 2 primary publications (meaning: journal articles)
published within the past 3 years. Web pages are not considered as primary references. You may
also include syllabus chapters as references, but must also include additional references that
demonstrate you have expanded your discussion to materials outside the course lecture presented
materials. Syllabus chapters are NOT primary references. Up to 1 point is subtracted if the
references are missing, incomplete, or inadequate to support your discussion. Recommended: use
PubMed to find related articles for the report.
e) Include a copy of the handout from the Grand Rounds session if one was available. Do
not include copies of the PowerPoint presentation.
f) The length of the essay should not exceed 3 pages (including references). 1 point is
subtracted for going over the set page limit.
ESSAY GRADES: Essays will be returned to students as quickly as grading allows. Inquiries
regarding essay assignment grades received must be submitted within one week after receipt of
returned assignments. Requests for review of essays past the one week period will be denied.
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Policy on Exam Grading (MS1 and MS2 courses) and Exam Review Sessions
Recommended by the Educational Policy Subcommittee: August 12, 2010
Approved by the Curriculum Committee: August 25, 2010
Revised by Educational Programs: August 31, 2010
Approved by the Curriculum Committee: September 15, 2010
Revised and Revisions Approved by the Curriculum Committee: May 18, 2011
The following policy delineates procedures related to exam grading and review/protest sessions
to be followed by all first- and second-year courses.
1. Course directors will score examinations through LXR and post results on MSGradebook as soon as possible.
2. Course directors will use item statistics generated by LXR to identify problematic
questions. Upon review, if a course director determines that a question was written
incorrectly (e.g. had more than one or no correct answer), then the director will give all
students credit for that particular question.
3. Large group post-exam review sessions may be held to provide feedback on difficult
examination topics, in a manner deemed appropriate by the course director. Copies of the
examinations will not be returned to the students during these sessions.
a. Course directors may meet with individual students to review examinations. The
format of these sessions, which may involve reviewing specific examination
questions, will be determined by the course director on a case by case basis.
Immunology Exam Question Review Policy: Review of exams may be done on an individual basis. Upon request, students
may view questions missed. Any requests to review exams must be submitted immediately following release of scores; exam
questions may only be reviewed within the two week period following release of scores. The Course Director has the right to
limit question viewing.
Immunology Essay Grades Review Policy: Discussion of score received for the essay assignment may be done on an
individual basis. Inquiries regarding essay assignment score received must be submitted within one week after receipt of
returned assignments. Requests for review of essays past that one week period will be denied.
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Date
2/12
Time
11:00-11:50 AM
TBL
2/26
8:00-9:50 AM
Case Readings
36. Rheumatoid Arthritis
37. Systemic Lupus Erythematosus
Distributed Reading: Inflammatory Bowel Diseases (Crohns
Disease, Ulcerative Colitis, and Celiac)
39. Crohns Disease
44. Celiac Disease
Required readings complement lectures and presented materials. It is highly encouraged to view these clinical cases.
Case materials may not be covered in full during lectures, however, all required case study readings contain material that
may be tested on exams.
Assigned readings may be discussed in multiple lectures, in addition to the assigned lectures.
February
26th
8:00-9:50 a.m.
Persons missing the session must provide written notice explaining circumstances
for not attending. Written approval must be obtained from the Office of
Educational/Student Affairs prior to consideration for any makeup session or
alternate assignment.
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Students will be assigned to work in small groups of four to six students. These groups
will remain together for all seven of the Integrative Exercises throughout the year.
During the Integrative Exercises, each group will discuss a clinical problem that
integrates material from the current basic science courses and will develop a team answer
to a question regarding that clinical problem. The teams will then prepare a written
justification for their answer for one of these problems. These justifications will be
handed in for grading. Pre-reading and pretests may be posted to Blackboard as
necessary for each exercise. You will receive email notifications regarding any prereading or pretest assignments.
The graded responses from all of the sessions will contribute to the final grade in the
Integrative Exercise course. Each of the group members will receive the same score.
Students who have unexcused absences will receive a score of 0 for all responses for
that Integrative Exercise session.
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immunodeficiency, hypersensitivity
The clinical immunologist is a physician who has specialized in the diagnosis and treatment
of disorders of the immune system. Many other clinical specialties (such as oncology,
hematology, infectious diseases, transplant surgery, etc.) also deal with immunologicallybased diseases in their area of specialization. Much of the work of modern clinical
immunologists revolves around refining diagnostic techniques for greater clinical utility
and evaluating new therapeutic modalities such as recombinant cytokines and cytokine
modulators.
Protection against foreign pathogens
Normal physiologic functions of the immune system include the ability to discern self from
non-self, and recognition of foreign pathogens. This represents recognition of
environmental challenges in an attempt to preserve homeostasis while responding to
pathogenic agents. The goal is to respond with specificity, allowing sufficient intensity and
duration to protect the host without causing damage to self.
The Immune system protects against foreign pathogens, of which four major classes can be
defined. These include (1) Extracellular bacteria, parasites and fungi; (2) Intracellular
bacteria and parasites; (3) Viruses (intracellular); and (4) Parasitic worms (extracellular).
Type I (also called immediate hypersensitivity) is due to aberrant production and activity
of IgE against normally nonpathogenic antigens (commonly called allergens). The IgE
binds to mast cells via high affinity IgE receptors. Subsequent antigen exposure results in
crosslinking of mast cell bound IgE with activation of mast cells that release preformed
mediators (e.g. histamine, leukotrienes, etc.) and synthesize new mediators (i.e.
chemotaxins, cytokines). These mediators are responsible for the signs and symptoms of
allergic diseases. [A = Allergic]
Type II is due to antibody directed against cell membrane-associated antigen that results in
cytolysis. The mechanism may involve complement (cytotoxic antibody) or effector
lymphocytes that bind to target cell-associated antibody and effect cytolysis via a
complement independent pathway (Antibody dependent cellular cytotoxicity, ADCC).
Cytotoxic antibodies mediate many immunologically-based hemolytic anemias while
ADCC may be involved in the pathophysiology of certain virus-induced immunological
diseases. [C = Cytotoxic]
Type III results from soluble antigen-antibody immune complexes that activate
complement. The antigens may be self or foreign (i.e. microbial). Such complexes are
deposited on membrane surfaces of various organs (i.e. kidney, lung, synovium, etc). The
byproducts of complement activation (C3a, C5a) are chemotaxins for acute inflammatory
cells. These result in the inflammatory injury seen in diseases such as rheumatoid arthritis,
systemic lupus erythematosus, postinfectious arthritis, etc). [I = Immune Complexes]
Type IV (also called Delayed Type Hypersensitivity, DTH) involves macrophage-T cellantigen interactions that cause activation, cytokine secretion and potential granuloma
formation. Diseases such as tuberculosis, leprosy and sarcoidosis as well as contact
dermatitis are all clinical examples where the tissue injury is primarily due to the vigorous
immune response rather than the inciting pathogen itself. [D = DTH]
Clinical suspicion for immunodeficiency may be made when patients present with chronic
infection or chronic inflammatory status, poor wound healing, constant fatigue and malaise,
or when unresponsive to vaccine administration. Certain infections with organisms may be
suggestive of deficiency in an immune related component. Alternatively, disruptions in
homeostasis may lead to immunodeficiency, such as those induced inadvertently by a
physician through medical treatment (iatrogenic).
The mechanisms for clinical immunodeficiency are varied, and will be examined (in part)
throughout the remainder of the course.
Therapeutic intervention for immune based diseases
Therapy for these diseases has historically been nonspecific, centering on repair of the
damaged tissues and inhibition of the aberrant immune responses with immunosuppressive
drugs. Recent work using such cutting edge techniques as recombinant DNA technology,
gene therapy, and stem-cell research have opened up an entire new avenue to address these
diseases by providing diagnostic and therapeutic modalities not previously available. For
immunodeficiency states, we have developed the g ability to replace elements through
marrow transplants, recombinant immune molecule administration and, soon, gene therapy.
The chief function of the immune system is to distinguish between self and
non-self.
3.
The antigenic specificity of the adaptive immune system is due to antigenspecific receptors.
4.
The business end of an antibody or TCR is the variable region. This region contains
the antigen-binding site that binds to the epitope (meaning: the conformational shape
recognized).
Variable Region
Coico and Sunshine, 2009. Fig. 1.2.
The variable region is formed during B and T cell development. This process occurs
prior to exposure to a given antigen.
The DNA encoding the variable region is subdivided into V, D, and J gene segments.
There are multiple V, D, and J gene segments in the Ig and TCR genetic loci.
In most cells, these gene segments are spread out, so that all the V segments are
together, all the D segments are together, and all the J segments are together. This is
called the germline configuration, because it is the arrangement seen in sperm and ova.
The V, D, and J gene segments are brought together to form a contiguous exon
encoding the variable region. The V, D, and J segments are selected randomly in each
cell, giving rise to combinatorial diversity. This is similar to the Pick 5 game in
Texas lotto, in which a large number of different number combinations exist.
The light chain gene locus (and some TCR genes) has only V and J regions.
There are several other mechanisms for generating diversity, as will be discussed in a
later lecture.
5.
B and T cells are resting cells that lack functional activity until they undergo
activation, proliferation, and differentiation into plasma cells or activated T
cells. This process takes several days, which explains the lag between being
exposed to an infectious agent and eventually getting better when the immune
response kicks in.
Of the millions of different specificities of B and T cells produced, only a few will
have surface Ig or TCRs that bind the antigen with high affinity. However, we
produce B and T cells that will react with virtually any antigen, including those that
are man-made and are not found in nature (e.g. di-nitrophenol).
In nearly all cases, activation of a B or T cell requires two signals: binding of the
antigen-specific receptor to the antigen, and exposure to proteins called cytokines
expressed by helper T cells.
The blast cells resulting from activation undergo proliferation, resulting in a ~100fold expansion of the number of cells reactive to the antigen.
Some of these cells become effector cells (plasma cells and activated T cells that
express activities that help to eliminate the pathogen. Others become memory cells
that can give rise to secondary responses as described below.
(106-108 clones)
6.
The adaptive immune system has memory, meaning that the response against
an antigen is much greater after the first exposure.
The first response to an antigen is called the primary response, and responses
thereafter are called secondary responses.
The different properties of secondary responses are due to memory cells generated
during the primary responses.
Secondary responses have
o Higher antibody levels
o Increased proportion of IgG and other immunoglobulin isotypes
o Shorter lag period
o Higher affinity for antigen
Vaccination is effective because it primes the immune system to provide secondary
responses when the individual is exposed to an infectious agent.
Each exposure to an antigen tends to increase the secondary response. This is why
booster immunizations are often used in vaccinations.
Figure: Primary and secondary antibody responses. The adaptive immune system
has memory, allowing for maturation of a rapid secondary immune response with
higher specificity and magnitude directed against foreign substances.
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7.
The immune response is designed to interact with the environment to protect the
host against pathogenic invaders.
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1. The chief function of the immune system is to distinguish between self and nonself.
2. The immune system consists of two overlapping compartments: the innate
immune system and the adaptive immune system.
3. The antigenic specificity of the adaptive immune system is due to antigen-specific
receptors.
4. The generation of antigen-binding diversity occurs prior to antigen exposure
through a DNA rearrangement process called VDJ joining.
5. To generate an active immune response against a certain antigen, a small number
of B and T cell clones that bind to the antigen with high affinity undergo
activation, proliferation, and differentiation into plasma cells (for B cells) or
activated T cells. This process is called clonal selection.
6. The adaptive immune system has memory, meaning that the response against an
antigen is much greater after the first exposure.
7. The immune system is tightly regulated.
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13
Figure.
Nomenclature of
Inflammatory
Cells.
Reticuloendothelial System
Cells of the RES provide natural immunity against microorganisms by 1) a coupled process of
phagocytosis and intracellular killing, 2) recruiting other inflammatory cells through the
production of cytokines, and 3) presenting peptide antigens to lymphocytes for the production of
antigen-specific immunity. The RES consists of 1) circulating monocytes; 2) resident
macrophages in the liver, spleen, lymph nodes, thymus, submucosal tissues of the respiratory and
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alimentary tracts, bone marrow, and connective tissues; and 3) macrophage-like cells including
dendritic cells in lymph nodes, Langerhans cells in skin, and glial cells in the central nervous
system.
Leukocytes
Leukocytes provide either innate or specific adaptive immunity. These cells are derived from
myeloid or lymphoid lineage. Myeloid cells include highly phagocytic, motile neutrophils,
monocytes, and macrophages that provide a first line of defense against most pathogens. The other
myeloid cells, including eosinophils, basophils, and their tissue counterparts, mast cells, are
involved in defense against parasites and in the genesis of allergic reactions. Cells from the
lymphoid lineage are responsible for humoral or cell mediated immunity.
Myeloid Cells
Neutrophils: Neutrophils are the most highly adherent, motile, phagocytic leukocytes and are the
first cells recruited to acute inflammatory sites. They ingest, kill, and digest pathogens, with their
functions dependent upon special proteins, such as adherence molecules, or via biochemical
pathways (respiratory burst).
Eosinophils: Eosinophils defend against parasites and participate in hypersensitivity reactions via
cytotoxicity. Their cytotoxicity is mediated by large cytoplasmic granules, which contain
eosinophilic basic and cationic proteins.
Basophils/Mast cells: Basophils, and their tissue counterpart mast cells, produce cytokines that
help defend against parasites, and also cause allergic inflammation. These cells display high
affinity surface membrane receptors for IgE antibodies, and have many cytoplasmic granules
containing heparin and histamine. The cells degranulate when cell-bound IgE antibodies are crosslinked by antigens, and produce low-molecular weight vasoactive mediators (e.g. histamine).
Monocytes/Macrophages: Monocytes and macrophages are involved in phagocytosis and
intracellular killing of microorganisms. Macrophages are differentiated monocytes, which are one
of the principal cells found to reside for long periods in the RES. These monocytes/macrophages
are highly adherent, motile and phagocytic; they marshal and regulate other cells of the immune
system, such as T lymphocytes; they serve as antigen processing-presenting cells.
Dendritic Cells: Dendritic cells provide a link between innate and adaptive immunity by
interacting with T cells in a manner to deliver strong signals for development of memory
responses. Dendritic cells recognize foreign agents and pathogens through a series of pattern
recognition receptors (non-specific), and are able to present antigen to both T helper and T
cytotoxic cells to allow those lymphocytes to mature towards functionality.
Lymphoid Cells
Lymphoid cells provide efficient, specific and long-lasting immunity against microbes/pathogens
and are responsible for acquired immunity. Lymphocytes differentiate into three separate lines: (1)
thymic-dependent cells or T lymphocytes that operate in cellular and humoral immunity; (2) B
lymphocytes that differentiate into plasma cells to secrete antibodies; and (3) natural killer (NK)
15
cells. T and B lymphocytes produce and express specific receptors for antigens while NK cells do
not.
B Lymphocytes: B lymphocytes differentiate into plasma cells to secrete antibodies. The genesis
of mature B cells from pre-B cells is antigen-independent. The activation of B cells into antibody
producing/secreting cells (plasma cells) is antigen-dependent. Mature B cells can have 1-1.5 x 105
receptors for antigen embedded within their plasma membrane. Once specific antigen binds to
surface Ig molecule, the B cells differentiate into plasma cells that produce and secrete antibodies
of the same antigen-binding specificity. If B cells also interact with T helper cells, they proliferate
and switch the isotype (class) of immunoglobulin that is produced, while retaining the same
antigen-binding specificity. T helper cells are thought to be required for switching from IgM to
IgG, IgA, or IgE isotypes. In addition to antibody formation, B cells also process and present
protein antigens.
T Lymphocytes: T lymphocytes are involved in the regulation of the immune response and in cell
mediated immunity, and help B cells to produce antibody. Mature T cells express antigen-specific
T cell receptors (TCR). Every mature T cell also expresses the CD3 molecule, which is associated
with the TCR. In addition mature T cells usually display one of two accessory molecules, CD4 or
CD8, which define whether a T cell will be a helper T lymphocyte, or a cytotoxic T lymphocyte
(CTL). The TCR/CD3 complex recognizes antigens associated with the major histocompatibility
complex (MHC) molecules on target cells (e.g. virus-infected cell).
Development of T lymphocytes
During differentiation in the thymus, immature T cells undergo rearrangement of their TCR and
genes to generate a diverse set of clonotypic TCRs. Immature thymocytes are selected for
further maturation only if their TCRs do not interact with self-peptides presented in the context of
self-major histocompatibility complex (MHC) molecules on antigen presenting cells.
T Helper Cells: T helper cells (Th) are the primary regulators of T cell- and B cell-mediated
responses. They 1) aid antigen-stimulated subsets of B lymphocytes to proliferate and differentiate
toward antibody-producing cells; 2) express the CD4 molecule; 3) recognize foreign antigen
complexed with MHC class II molecules on B cells, macrophages or other antigen-presenting
cells; and 4) aid effector T lymphocytes in cell-mediated immunity.
Currently, it is believed that there are two main functional subsets of Th cells, plus multiple other
helper subsets of importance. T helper 1 (Th1) cells aid in the regulation of cellular immunity,
and T helper 2 (Th2) cells aid B cells to produce certain classes of antibodies (e.g., IgA and IgE).
The functions of these subsets of Th cells depend upon the specific types of cytokines that are
generated, for example interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by Th1 cells; IL4, IL-6 and IL-10 by Th2 cells. Two other classes of T helper cells are thought to be involved in
oral tolerance and serve as regulators for immune function. Th17 cells, characterized by IL-17
secretion, are thought to be involved as effector cells for autoimmune disease progression, and
protect surfaces (skin, gut) from extracellular bacteria. Tfh cells (follicular helper T cells) also
provide help to B cells enabling them to develop into antibody-secreting plasma cells. They
function inside of follicular areas of lymph nodes. Finally, although no longer prevalent in the
literature, a subclass called Th3 cells were historically identified as secreting IL-4 and TGF- to
provide help for IgA production; they were thought to be suppressive for Th1 and Th2 cells.
16
T
Cytotoxic Cells: T cytotoxic cells (CTLs) are cytotoxic against tumor cells and host cells infected
with intracellular pathogens. These cells 1) usually express CD8, and, 2) destroy infected cells in
an antigen-specific manner that is dependent upon the expression of MHC class I molecules on
antigen presenting cells.
T Suppressor/ T Regulatory Cells: T suppressor cells suppress the T and B cell responses and
express CD8 molecules. T regulatory cells (Tregs) also affect T cell response, with many cells
characterized as CD4+CD25+, TGF- secretors. Tregs regulate/suppress other T cell activities,
and help prevent development of autoimmunity.
Natural Killer T Cells: Natural killer T cells (NKT) are a heterogeneous group of T cells that
share properties of both T cells and natural killer (NK) cells. These cells were identified as T cells
that recognize an antigen-presenting molecule (CD1d) able to bind self- and foreign lipids and
glycolipids. They constitute only 0.2% of all peripheral blood T cells. The term NK T cells was
first used in mice to define a subset of T cells that expressed the natural killer (NK) cell-associated
marker NK1.1 (CD161). It is now generally accepted that the term NKT cells primarily refer to
CD1d-restricted T cells co-expressing a heavily biased, semi-invariant T cell receptor (TCR) and
NK cell markers. Natural killer T (NKT) cells should not be confused with natural killer (NK)
cells.
Natural Killer Cells: NK cells are large granular innate lymphocytes that nonspecifically kill
certain types of tumor cells and virus-infected cells. NK cells share many surface molecules with
T lymphocytes. These circulating large granular lymphocytes are able to kill self in the absence
of antigen-specific receptors. NK cells are especially effective against viral infected cells, and
keep the expansion of virus in check until adaptive immunity kicks in. In this regard, they also
secrete interferon-gamma, which is an effective immunoregulator. NK cells can also kill via
antibody-dependent cellular cytotoxic mechanisms (ADCC) via their Fc receptors. NK cells
17
express a large number of receptors that deliver either activating or inhibitory signals, and the
relative balance of these signals controls NK cell
activity.
Antigen Presenting Cells (APCs) are found
primarily in the skin, lymph nodes, spleen and
thymus. They may also be present throughout
the diffuse lymphoid system. Their main role is
to present antigens to antigen-sensitive lymphoid
cells. APCs may be characterized by their ability
to phagocytose antigens, location in body, and
expression of Major Histocompatibility Complex
(MHC) related molecules.
Two main types of APCs are Dendritic Cells and
Macrophages. Of note, B cells are a special class
of APCs; because they have antigen-specific
antibody receptors they are enabled to internalize
and process targeted antigens.
Lymphoid Organs
The lymphatic organs are tissues in which
lymphocytes mature, differentiate and proliferate. Lymphoid organs are comprised of epithelial
and stromal cells arranged either into discretely capsulated organs or accumulations of diffuse
lymphoid tissue. The primary (central) lymphoid organs are the major sites of lymphopoiesis,
where B and T lymphocytes differentiate from stem cells into mature antigen recognizing cells.
The secondary lymphoid organs, therefore, are those tissues in which antigen-driven proliferation
and differentiation take place.
Historically, the primary lymphoid organ was first discovered in birds, in which B cells undergo
maturation in the bursa of Fabricius, an organ situated near the cloaca. Humans do not have a
cloaca, nor do they possess a bursa of Fabricius. In embryonic life, B cells mature and
differentiate from hematopoietic stem cells in the fetal liver. After birth, B cells differentiate in the
bone marrow. Maturation of T cells occurs in a different manner. Progenitor cells from the bone
marrow migrate to the thymus where they differentiate into T lymphocytes. The T lymphocytes
continue to differentiate after leaving the thymus, and are driven to do so by encounter with
specific antigen in the secondary lymphoid organs.
18
Lymph Node: Lymph nodes form part of the network which filters antigen from tissue fluid or
lymph during its passage from the periphery to the thoracic duct. Histologically, the lymph node is
composed of a B cell cortex containing primary and secondary follicles, a T cell paracortex, and a
central medulla which contains cords of lymphoid tissue.
Spleen: The spleen is a filter for blood, and is actively involved in the removal of dying and dead
erythrocytes. There are two main types of tissue; red pulp and white pulp. The white pulp
contains the lymphoid tissue, arranged around a central arteriole as a periarteriolar lymphoid
sheath (PALS). The PALS is composed of T and B cell areas, and contains germinal centers.
Dendritic reticular cells and phagocytic macrophages can be found in germinal centers where they
work to present antigen to lymphocytes.
19
Clinical Vignette - Congenital Asplenia (Case 30 in Geha and Notarangelo): Mr. and Mrs.
Vanderveer had five children. Their 10 month old daughter developed a cold, followed by upper
respiratory infection. The child became feverish, convulsive and died; the causative agent was
Haemophilus influenza which was isolated from the throat and cerebrospinal fluid. At autopsy she was
found to have no spleen.
How does the lack of a spleen affect B cell function, and what implications does this have towards
immune responses to infective agents? In adults? In children?
Review your histology chapters dealing with Hematopoiesis and the Immune System!
Table. Myeloid Leukocytes and Their Properties
Phenotype
Morphology
Circulating Differential Count*
PMN
Neutrophil
granulocyte
2-7.5x109/L
PMN
Eosinophil
granulocyte
0.04-0.44x109/L
PMN
Basophil
granulocyte
0-0.1x109/L
PMN
Mast Cell
granulocyte
Tissue Specific
Monocytes
monocytic
0.2-0.8x109/L
Macrophag
e
monocytic
Tissue Specific
Dendritic
Cell
monocytic
Tissue Specific
* Normal range for 95% of population, +/- 2 standard deviations
Effector Function
Phagocytosis and digestion of microbes
Immediate hypersensitivity (allergic)
reactions; defense against helminths
Immediate hypersensitivity (allergic)
reactions
Immediate hypersensitivity (allergic)
reactions
Circulating macrophage precursor
Phagocytosis and digestion of microbes;
antigen presentation to T cells
Antigen presentation to nave T cells;
initiation of adaptive responses
monocytic
monocytic
Adaptive
Adaptive
Effector Function
Humoral immunity
Terminally differentiated, antibody
secreting B cell
Cell-mediated immunity
monocytic (rare)
monocytic
Adaptive
Innate
20
21
Innate immune mechanisms provide the first line of defense from infectious disease. The innate
immune system is comprised of components which are present prior to the onset of infection and
constitute a set of mechanisms that are not specific for a particular organism. Rather, the innate
components recognize classes of molecules frequently encountered on invading pathogens, so as
to allow defensive measures while the specific immune response is either generated or
upregulated. Innate immune components are present from birth and consist of non-specific
components.
The innate defensive barriers can be divided into four major categories:
1. Anatomic - skin, mucous membranes
2. Physiologic - temperature, low pH, chemical mediators
3. Phagocytic and Endocytic - phagocytose to kill and digest microorganisms
4. Inflammatory - induction of vascular fluid leakage to area of tissue damage
Anatomic Barrier. The skin and mucous membranes provide an effective barrier against
microorganisms. The skin has the thin outer epidermis and the thicker underlying dermis to
impede entry, as well as sebaceous glands to produce sebum. Sebum is made of lactic acid and
fatty acids, which effectively reduce skin pH to between 3 and 5 to inhibit organism growth.
Mucous membranes are covered by cilia which trap organisms in mucous and propel them out of
the body.
Physiologic Barrier. The physiologic barrier includes factors such as temperature, low pH, and
chemical mediators. Many organisms can not survive or multiply in elevated body temperature.
Soluble proteins such as lysozymes, interferons and complement components play a major role in
innate immunity. Lysozmes can interact with bacterial cell walls; interferons alpha and beta are
natural inhibitors of viral growth; complement components use both specific and non-specific
immune components to convert inactive forms to active components that damage membranes of
pathogens. Low pH in the stomach discourages growth.
22
Phagocytic and Endocytic Barriers. Blood monocytes, tissue macrophages and neutrophils
phagocytose and kill microorganisms via multiple complex digestion mechanisms. Bacteria
become attached to cell membranes and are ingested into phagocytic vesicles. Phagosomes fuse
with lysosomes where lysosomal enzymes digest captured organisms.
Inflammatory Barriers. Invading organisms
cause localized tissue damage leading to
complex inflammatory responses. In 1600
BCE, Celsus described the four cardinal signs
of inflammation as rubor (redness), tumor
(swelling), calor (heat), and dolor (pain).
Later, Galen (2nd century) a fifth sign was
added; functio laesa (loss of function).
Inflammatory responses lead to (1)
Vasodilation causing erythema (redness) and
increased temperature; (2) increased capillary
permeability which allows exudates (fluid) to
accumulate leading to tissue swelling
(edema); and (3) influx of cells to site of
tissue damage. Once cells enter area of
damage, they release further chemotactic
factors to call in additional cells to damaged
area, leading to Chemotaxis, Activation,
Margination, Diapedesis (extravasation), and
finally recognition and attachment of these
cells to the damaged site.
23
Hageman factor: Plasma globulin (110 kD), blood clotting factor XII, which is activated
by contact with surfaces to form Factor XIIa, that in turn activates factor XI. Factor XIIa
also generates plasmin from plasminogen and kallikrein from prekallikrein. Both plasmin
and kallikrein activate the complement cascade. Hagemann factor is important both in
clotting and activation of the inflammatory process.
Thrombin: Protease (34 kD) generated in blood clotting that acts on fibrinogen to produce
fibrin. Consists of two chains, A and B, linked by a disulphide bond. Thrombin is
produced from prothrombin by the action either of the extrinsic system (tissue factor +
phospholipid) or, more importantly, the intrinsic system (contact of blood with a foreign
surface or connective tissue). Both extrinsic and intrinsic systems activate plasma factor X
to form factor Xa which then, in conjunction with phospholipid (tissue derived or platelet
factor 3) and factor V, catalyses the conversion.
Kallikrein: Plasma serine proteases normally present as inactive prekallikreins which are
activated by Hageman factor. Act on kininogens to produce kinins, to mediate vascular
reactions and pain.
Plasmin: Trypsin like serine protease that is responsible for digesting fibrin in blood clots.
Generated from plasminogen by the action of another protease, plasminogen activator. The
enzyme catalyses the hydrolysis of peptide bonds at the carbonyl end of lysine or arginine
residues. It also acts on activated Hageman factor and on complement.
24
phospholipases, elastases and collagenases, and lactoferrin. Pus, a yellowish white opaque creamy
matter produced by the process of suppuration consists of innumerable neutrophils (some dead
and dying) and tissue debris.
Figure. Cell membrane adhesion molecules and cytokine activation events associated with
neutrophil transendothelial migration. Left: Weak binding of selectin ligands on the neutrophil to Eselectin on the endothelial cells. Middle: IL-1 and TNF- upregulation of E-selectin, which facilitates
stronger binding. Right: The activation effects of IL-8 on neutrophils cause a conformational change in the
integrins (e.g., LFA-1) to allow them to bind ICAM-1. Coico and Sunshine, 2009. Fig 11.4.
Mononuclear Cells and Macrophages. Mediators such as MIP-1 and MIP-1 attract
monocytes to the site of pathogenic infection. The monocytes express surface ligands which
recognize ligands (VCAM-1) on endothelial cells, leading to
diapedesis. Activated tissue macrophages secrete IL-1, IL-6
and TNF- which further increase expression of adhesion
molecules on endothelial cells to recruit neutrophils and more
monocytes. These molecules also increase release of acutephase proteins from the liver to assist in events leading to
body temperature increase.
Monocytes and macrophages ingest and destroy bacteria.
Multiple factors assist in preparing the particulate for
engulfment and targeting for destruction, including various
opsonins comprised of complement components. Phagocytes
bear several different receptors that recognize microbial
components and induce phagocytosis. Five such receptors on
macrophages are: CD14, Toll-like receptors (such as TLR-4),
the macrophage mannose receptor, the scavenger receptor,
and the glucan receptor. All 5 receptors bind bacterial
carbohydrates. CD14 and CR3 are specific for bacterial
lipopolysaccharide (LPS). In addition, complement receptors
assist in this process.
Figure. Endocytosis and phagocytosis by macrophages.
Figure. Important cytokines secreted by macrophages in response to bacteria and bacterial products
include IL-1, IL-6, CXCL8 (IL-8), IL-12, and TNF-a. TNF-a is an inducer of a local inflammatory response
that helps to contain infections. CXCL8 is also involved in the local inflammatory response, helping to
attract neutrophils to the site of infection. IL-1, IL-6, and TNF-a have a critical role in inducing the acutephase response in the liver and induce fever, which favors effective host defense in several ways. IL-12
may also activate natural killer (NK) cells.
Figure from Immunology (6th ed). 2006. Goldsby, Kindt, Osborn and Kuby. WH Freeman Publisher.
27
NK Cells. NK cells are large granular lymphocytes that nonspecifically kill certain types of tumor
cells and virus-infected cells, and function as both cytolytic effectors and regulators of immune
responses. NK cells express a large number of receptors that deliver either activating or inhibitory
signals; the relative balance of these signals controls NK cell activity. NK cells are activated upon
detection of abnormalities in target cells such as the loss of antigen presentation molecules (MHC
class I expression) or up-regulation of stress-induced ligands. A variety of receptors trigger the
NK cytolytic activity directed toward certain tumor targets, virally infected cells, and even normal
immune system constituents such as immature dendritic cells. NK cells are also important
regulators of the adaptive immune system via their ability to secrete a number of cytokines in
response to immune activation.
Clinical Relevance
Clinical Vignette Case 15. Chediak-Higashi Syndrome: Chediak-Higashi syndrome is a rare inherited disorder
in which a severe immunological deficiency has been linked to deficits in NK cell function and to deficiency in
chemotactic and bactericidal function for neutrophils. Thus, these individuals are more susceptible to bacterial
infections. These individuals have characteristic giant lysosomes within neutrophils. Bone marrow transplantation
is the only effective therapy.
28
Chemokines. Chemokines are specialized cytokines that are chemotactic for leukocytes. They
are small polypeptides that are synthesized by a wide variety of cell types. They act through
receptors that are members of the G-protein coupled signal transducing family. All chemokines
are related in amino acid sequence and their receptors are integral membrane proteins that are
characterized by containing seven membrane-spanning helices.
Chemokines fall mainly into two distinct groups. The CC chemokines have two adjacent cysteine
residues (hence the name "CC"). The CXC chemokines have an amino acid between two cysteine
residues. Each chemokine reacts with one or more receptors, and can affect multiple cell types.
Chemokines and their functions will be covered again in greater depth in the Adaptive
Immunity chapter.
Properties of selected chemokines.
Chemokine
CCL2 (MCP-1)
CCL3 (MIP-1)
CCL5 (Rantes)
CCL11 (Eotaxin)
CXCL8 (IL-8)
29
30
4. Solubilization and clearance of immune complexes. One of the major roles complement plays
is the solubilization and clearance of immune complexes from the circulation. First, C3b and C4b
can covalently bind to the Fc region of insoluble immune complexes, disrupting the lattice, and
making them soluble. C3b and C4b bound to the immune complex are recognized by the CR1
receptor on erythrocytes facilitating their transport to the liver and spleen. In the liver and spleen
the immune complexes are removed and phagocytosed by macrophage-like cells. The RBCs are
returned to the circulation.
5. Enhancement of humoral immune response. Coating of antigens with C3d (a breakdown
product of C3) facilitates their delivery to germinal centers rich in B and follicular dendritic cells.
Clinical Relevance
Clinical Vignette Factor I Deficiency (Case 32, Geha and Notarangelo): The alternative pathway
of complement activation is important in innate immunity. Deficiency in Factor I (as well as deficiency
in Factor H) affects cleavage of C3b, and therefore leads to reduced C3bi. The nonproduction of iC3b
results in defective opsonization, which is critical for removing and destroying invading bacteria.
31
Receptors of the Innate Immune System. Receptors of the innate immune system recognize
broad structural motifs that are highly conserved within microbial species [called PathogenAssociated Molecular Patterns (PAMPs)]. Such receptors are referred to as Pattern-Recognition
Receptors (PRRs). In a similar manner, Damage/Danger-Associated Molecular Pattern molecules
(DAMPs) initiate immune activity as part of the noninfectious inflammatory response. Receptor
engagement leads to triggering of signal pathways that promote inflammation.
Receptor (location)
Target (source)
Effect of Recognition
Receptors of the Innate Immune System. [Table adapted from Immunology (2002) by Goldsby,
Kindt, Osborne and Kuby - W.H.Freeman, et al., NY.] TLR = Toll-like Receptor.
32
Figure 6-4. Link between innate and adaptive (acquired) immunity. Pathogen recognition through
pattern recognition receptors is an important bridge between innate and adaptive immune function.
Recognition leads to activation and maturation of the presenting cell. Here, dendritic cells are
depicted as primary presenting cells, which assist in dictating subsequent responses. Processed
antigen is presented to naive T cells, accompanied by secretion of cytokines to assist development
and maturation of T-cell phenotypic response (e.g., T helper cell-1 maturation via presence of
interleukin-12). Inset box shows important Toll-like receptors and specific ligands involved in
pathogen recognition. At least 15 different Toll-like receptors have been identified. A more
complete list of Toll-like receptors and ligands is provided in the Appendix.
Final word:
33
We will see in later lectures that lymphocyte responses can be divided by specificity and function.
The lymphocytes are considered adaptive historically. Indeed, the classical B lymphocytes and
T lymphocytes we discuss later in the course will be primarily of the adaptive phenotypic groups.
However, there is a population of lymphocytes that respond with innate-like activity. These
include: T-cells, invariant natural killer T-cells, and B-1 cells. All these cell types respond
quickly (1-3 days) to a limited pool of antigens at sites of infection. These cells will be defined as
we discuss them.
34
Hageman factor
Thrombin
Kallikrein
Bradykinin
Leukotrienes and Prostaglandins
Complement
Cytokines and Interferons
Lysozymes
Acute Phase Proteins and Lactoferrin
Inflammatory
Mediators
Complement
Cytokines and Interferons
Lysozymes
Acute Phase Proteins and Lactoferrin
Leukotrienes and Prostaglandins
Cellular
Components
Polymorphonuclear Cells
Neutrophils, Eosinophils
Basophils, Mast Cells
Phagocytic-Endocytic Cells
Monocytes and Macrophages
Dendritic Cells (multiple
subsets)
Other Cells
NK cells
35
- ADCC
ANTIGEN OR IMMUNOGEN?
IMMUNOGEN - Agent capable of binding immune receptors AND inducing an
immune response by B cells and T cells
ANTIGENS - Agent that binds with varying degrees of specificity to immune
receptors (antibodies on B cells; T cell receptor on T cells)
All immunogens are antigens, but not all antigens are immunogens.
IMPORTANCE OF IMMUNOGENICITY
Germfree colostrum-deprived piglets are immunologically "virgin" and extremely
susceptible to microbial infection due to lack of passive maternal immunity. They
are, however, highly immunologically competent as determined by their excellent
immune response to various immunogens. An immunogen is the inducer of
specific antibody formation. The initial step in the primary immune response is
priming of multipotent uncommitted cells ("virgin" X cells) to committed
monopotent cells (Y cells). Y cells proliferate and differentiate into antibodyforming cells (Z cells). Adapted from Y.-B. Kim 1975
Vaccines are the cornerstone of eradicating microbial disease many available
vaccines. See slide.
New vaccines are needed for emerging diseases. See slide.
EPITOPES RECOGNIZED BY T OR B CELLS
Epitopes are the three dimensional arrangements of atoms (sites) on the surface of
an antigen that bind to the paratope of an antibody OR the linear peptides that bind the
MHC molecules/T cell receptor. Epitopes recognized by B cells generally differ from
those recognized by T cells.
36
B cells can mount specific antibody responses without or with help from T cells (Tindependent or T dependent cells).
Antibody binding to antigen does not involve covalent chemical bonds. Instead, several
weaker types of molecular interactions are utilized. Thus, the reactions are reversible.
There are four kinds of forces that stabilize antigen-antibody interactions:
1. Electrostatic interactions. Usually due to the attraction between the charged
amino acid residues in proteins such as lysine, arginine, glutamic acid and aspartic
acid, for instance. The number of such interactions will enhance the affinity of the
interaction dramatically.
2. Hydrogen bonding. Electrostatic binding with covalent character. Example, -H
atom shared by electronegative N and O atoms.
37
3. Van der Waals forces. Attractive and repulsive forces between induced
oscillating dipoles in the electron clouds of two adjacent atoms. The force is
proportional to the 7th power of the distance separating two molecules. This is a
weak force, but is additive, and there are many van der Waals contacts in antibodyantigen complexes.
4. Hydrophobic bonding. Usually involves non-polar amino acids, e.g., leucine,
isoleucine.
38
regions within larger antigens. Thus, the only requirement is that the right
sequence of amino acids is expressed.
40
MULTIVANT ANTIGENS
Macromolecules usually have multiple unique or repeat epitopes. The former type of
immunogens induce heterogenous immune responses (mixtures of antibodies or T cells
directed to individual antigens). The latter type of immunogens are often T-independent.
Both types of antigens can form large complexes with multiple antibodies, a phenomenon
that can cause pathological immune complex deposition, particularly in the kidney.
IMMUNOLOGIC SPECIFICITY AND CROSS-REACTIVITY
The forces mediating antigen-antibody recognition allow for a high degree of specificity.
That is, antibodies specific for one epitope or hapten can easily distinguish that epitope or
hapten from other similar structures. However, this specificity is not absolute because
antibodies specific for one epitope can bind with structurally similar, but non-identical
epitopes although with a lower affinity. Specificity and cross-reactivity can be
distinguished by inspecting the following table reporting antibody reactivity with various
structurally defined carbohydrate epitopes:
41
CROSS-REACTIVITY
Cross reactivity refers to the situation where the cell receptor or antibody can react with
two molecules because a) they share one or more identical epitopes or b) the epitope in
question is similar enough in sequence or in shape to bind to the receptor with a weaker,
yet functional, affinity.
Examples:
1. ToxoidsAntibodies elicited with toxoids react with native toxins
(Clinical Applicationvaccination with tetanus toxoid and with diphtheria toxoid).
2. ABO Blood Group AntigensAntibodies elicited by certain
environmental carbohydrate antigens react with the human A or B blood group antigens.
3. There are 4 strains of the flavivirus that causes Dengue. The virus
infects cells of the monocyte-macrophage lineage. Infection with one strain elicits
antibodies reactive with a common epitope on all 4 strains. Upon infection with a
different strain, the antibody to the common epitope reacts by cross-reaction and
facilitates phagocytosis by macrophages, thus helping the virus gain entry and the second
infection is typically much more severe than the first due to this cross-reactivity.
4. Yet another example of cross-reactivity is the ability of antibodies to
bacterial antigens to attack host tissues, causing autoimmune disease.
42
ADJUVANTS
Definition: An adjuvant is a substance, which when mixed with an immunogen,
enhances the immune response against the immunogen. The adjuvant itself is not
usually immunogenic.
Examples of Immunologic Adjuvants
1. Freunds Complete Adjuvant. This is a mixture of a petroleum
based oil, an emulsifying agent and killed Mycobacteria. A water-inoil emulsion is formed with microdroplets of antigen solution
surrounded by the oil. This works by slowly releasing antigen over a
long period of time while inducing a delayed hypersensitivity reaction.
It is used experimentally, but not in humans.
2. Lipopolysaccharide (LPS). Is experimental
3. Muramyldipeptide. Is experimental
4. Synthetic Polynucleotide (Poly AU). Is experimental
5. Aluminum Hydroxide (alum precipitate). Is used in humans.
Functions to enhance the ingestion and eventual processing of antigen.
6. Cytokines. Are experimental
Currently, adjuvant research is a high priority research area for enhancing the immunogenicity of
the new genetically engineered vaccines. Aluminum hydroxide is currently the only FDA
licensed adjuvant in the US. There are several new adjuvants in phase III clinical trials but the
FDA has not yet licensed any of these. Some other adjuvants are licensed in other countries, but
are not available in the US. Adjuvant MF59 is licensed in Europe, but not in the US. MF59
consists of stable droplets (<250 nm) of the metabolizable oil squalene and two surfactants,
polyoxyethylene sorbitan monooleate and sorbitan trioleate, in an oil-in-water emulsion.
43
SUMMARY
1. Immunogens elicit immune responses; antigens bind to antibodies, lymphocyte
receptors or MHC molecules.
2. The properties of foreignness, size, chemical complexity and degradability all
contribute to the degree of immunogenicity.
3. Haptens are low molecular weight substances that only become immunogenic upon
covalent coupling to an immunogenic molecule. Haptens are models for epitopes.
4. Major classes of antigens include carbohydrates, lipids, nucleic acids and proteins
or glycoproteins.
5. Linear epitopes require only the primary linear structure to be intact but
conformational epitopes require that the 3D integrity of the molecule is intact.
6. Antibody binding to antigen does not utilize covalent interactionsmultiple weak
interactions stabilize the binding such as electrostatic interactions, hydrogen
bonding, hydrophobic interactions and Van der Waals forces.
7. Immunologic specificity is dependent on stereochemical positioning or spatial
arrangement of chemical groupings and on the chemical nature of the group
(mass and charge).
8. Immunologic cross-reactions can be due to antigens sharing identical epitopes or
having epitopes with similar but non-identical chemical groupings.
9. Adjuvants enhance the magnitude of the resulting antibody response when mixed
with an immunogen before injection. Aluminum hydroxide (alum) is the only
licensed adjuvant for human use in the USA. Several others are currently in phase III
clinical trials and some other adjuvants (i.e. MF59) are licensed in foreign countries.
44
IMMUNOLOGY
Antibody Structure and Function
Dr. Keri C. Smith
OBJECTIVES
Develop an understanding of how the structural and molecular features of antibody molecules
mediate both the protective and the pathologic functions of the different classes of antibodies.
KEYWORDS
Immunoglobulin (Ig), isotype, allotype, idiotype, opsonin.
READING
Chapter 4 in the Coico et al textbook, 2009.
Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/antibodystructure-and-function-iii/
INTRODUCTION
The production of circulating antibodies is one of the major functions of the immune system.
Antibodies belong to the general class of glycoproteins called globulins, due to their property of
being insoluble in half-saturated ammonium sulfate solutions. Subsets of antibodies have been
discovered and they are now known collectively as immunoglobulins, abbreviated Ig. Studies of
the molecular structure of the various Ig have clarified many of the properties such as specificity,
cellular reactivity, complement fixation, placental transfer and anaphylactic activity after mast cell
activation.
ISOLATION AND CHARACTERIZATION
Ig are found in large quantities in blood serum. The bulk of Ig migrates in the gamma region when
subjected to electrophoresis at pH 8.2. Tiselius and Kabat proved in 1939 that the gamma region
contained most of the antibodies in an immunized animal:
45
The shape of the gamma peak, being rather broad, suggested very early that the antibody
population was a heterogeneous collection of molecules with slightly different charges. This
heterogeneity was a major early obstacle in attempts to determine the structure of antibody
molecules and relate structure to function. This problem was partially solved by the discovery
of myeloma proteins that are homogeneous Ig produced by cells in a type of cancer called
multiple myeloma, a cancer of plasma cells.
STRUCTURE OF LIGHT AND HEAVY CHAINS
Enzymatic fragmentation and chemical reduction studies showed the basic 4-chain structure
of the predominant Ig, originally called gamma globulin. Pepsin and papain fragments of
Ig were used, along with studies of the reductive products to establish the 4-chain structure
with each molecule consisting of 2 identical heavy chains and 2 identical light chains. Amino
acid sequences then were used to further distinguish constant portions of the chains from
variable portions. The domain concept, hinge region and carbohydrate locations were
determined
46
Light Chains
Studies of Ig from many species showed that nearly all species studied had two types of
Light chains, called and . The difference between the two types of light chains is in the
amino acid sequence of the constant region domain. The overall ratio of the two Light chain
types varies between species (mice have 95% of their Ig with chains whereas human Ig has
60% and 40% ).
Heavy Chains
There are 5 different classes or ISOTYPES of heavy chains. Each class of Heavy chain has a
characteristic amino acid sequence that distinguishes it from the other four classes but all five
classes have significant percentages of amino acid sequence similarities. The five Heavy
chain Ig classes are IgG, IgA, IgM, IgE and IgD. The different Heavy chains corresponding
to their class are given Greek letter designations: , , , and . In many species, there are
two or more subclasses of some heavy chains that differ from one another by only a few
amino acids. Humans have 4 subclasses of the IgG isotype called IgG , IgG , IgG , and
1
Domains
Amino acid sequence studies of Ig shows that there is regularity to the structure in which
there are disulfide-bridged loops of approximately 60 amino acids for each 100-110 amino
acids. This is the case for both Heavy and Light chains. These are called domains.
There are two domains on both and Light chains and either 4 or 5 domains on heavy
chains. The amino acid sequences in the first domain on both Light and Heavy chains are
highly variable from molecule to molecule, and are referred to as the V or V
L
domains, respectively. The other Light chain domain is constant in its amino acid sequence
for the or type of chain and is referred to as the C domain.
L
The constant domains of Heavy chains are numbered from the amino terminal end toward the
carboxy terminal end as C 1, C 2, C 3 and (for IgM and IgE) C 4.
H
The following shows some specific biological functions carried out by Ig domains.
DOMAIN FUNCTIONS OF HUMAN IgG
------------------------------------------------------------------------------------------------------------Domain(s)
Function
V +V
Antigen Binding
H
C 1+C
C 2
Binding C1q
C 3
H
C 1+C 3
H
Bind to Protein A
Interact with Fc Receptors on placental syncitiotrophoblast, neutrophils, and
cytotoxic K-cells
47
Hinge Region
IgG, IgA and IgD genes each have an exon coding for a short span of amino acids that
occupy the space between the C 1 and C 2 domains. This segment is rich in Cys and Pro
H
and permits significant flexibility between the two Fab arms of the antibody and the area is
called the hinge region, accordingly. Since this stretch is open to solvent, it is highly
susceptible to protease cleavage.
Variable Region or Variable Domain
Kabat and Wu developed their variability plot to try to predict which amino acids in the
Variable regions actually contacted antigen. They found that the greatest variability in
sequence between molecules occurred at 3 distinctive regions in Light and Heavy chains.
These were then called hypervariable regions. They were separated by sequences called
framework regions. It has been formally proven that the amino acids comprising the
hypervariable regions are the contact residues for antigen. Since they form the region of
structural complementarity for Ag epitopes, they are termed complementarity-determining
regions (CDRs).
48
In this plot the term VARIABILITY is defined as the ratio of the number of different amino
acids at a given position to the frequency of the most common amino acid at that position.
For example, in the original research at position 7, 63 different light chains had been
sequenced and Serine occurred 41 times so its frequency was 41/63 or 0.65. In all, 4 amino
acids were found at position 7. Thus the Variability value was 4/0.65 or 6.15.
IMMUNOGLOBULIN VARIANTS
Isotypes
The five major classes of Ig (IgG, IgA, IgM, IgE, IgD) are isotypic variants of
immunoglobulins or isotypes. Structural variations in the heavy chains distinguish the
isotypes from one another. The structural elements that define one isotype are the same for
each species. There are subclasses of IgG and IgA.
TABLE 4.2. Important Differences Between Human IgG Subclasses
IgG1
IgG2
IgG3
IgG4
70
20
Half-life
23
23
23
Complement binding
+++
Placental passage
++
++
++
Binding of monocytes
+++
+++
ALLOTYPES
Definition: Ig allotypes are defined as structural variants of the constant regions of L or H
chains of Ig that are coded by germ line genes.
These differences are detected serologically. Specific antibodies are available for analyzing
these differences from one individual to another. The differences are usually due to a single
amino acid difference in the light or heavy chain. Sometimes is it more than one amino acid
difference. The best examples of allotype differences in human Ig are the three allelic
variants of human kappa light chains.
Allotype
Km(1)
Km(1,2)
Km(3)
Mendelian co-dominant autosomal allelic genes code Allotypes. There are 4 subclasses of
human IgG and two subclasses of IgA (described earlier).
Importance: Allotypes are used forensically in cases of disputed parentage or in analysis of
blood. The allotypic variation has no influence on antibody specificity.
49
Clinical Vignette
Results of Pediatric vaccine trials published in 1985 and 1986 show some
correlation between severity of Hemophilus influenzae type b infections and
the IgG2 allotype of the patient (Ambrosino et al, J. Clin. Invest. 75, 19351942, 1985; Granoff et al, J. Infect. Dis. 154, 257-264, 1986). Briefly, they
showed that children with the G2m(23) allotype have higher levels of
immunity than the G2m(23) negative children and Km(1) allotype is more
protective in black children than other L chain allotypes.
IDIOTYPES
Idiotypes are epitopes found in V regions of antibody molecules. They are defined
serologically.
The idiotypic epitopes (Idiotopes) are thought to be located near or even within the
paratope site on the antibody. It is clear that several different kinds of anti-idiotypic
antibodies exist based on the type of idiotope that they recognize. The two most common are
shown in he following diagram:
50
51
Schematic structures for the 5 major isotypes of immunoglobulins are shown below:
may have important structural and/or functional properties. The general function
of carbohydrate on the Igs as well as other glycosylated polypeptides seems to be
that it plays a role in cellular transport and in secretion.
52
th
immunity to the fetus. This begins in the 3 or 4 month of pregnancy in humans. Resistance of
the neonate to most common infections is almost exclusively via this passive IgG. Active transfer
is mediated by the Fc region of IgG molecules.
While this passive immunity is crucial for neonatal protection, some transferred antibodies can be
destructive. Erythroblastosis fetalis, hemolytic disease of the newborn, is mediated by the
passage of anti-Rh (D) antibodies of the IgG class across the placenta in an Rh (D)-negative
mother carrying an Rh (D) positive fetus. She can be sensitized to the Rh antigen by a previous
pregnancy. These antibodies can destroy fetal red blood cells and cause disease that can vary
from mild to fatal, depending on the maternal titer of anti-Rh (D).
Case 46Hemolytic Disease of the NewbornMrs. Waymarsh was 31
and pregnant for the third time. Her blood type was A, Rh-negative. Her
husband was also type A, but Rh-positive. Her first child was born healthy
nd
and she was given RhoGAM following delivery. During her 2 pregnancy,
she developed a 1:16 indirect Coombs titer of 1:16 and a healthy girl was
induced at 36 weeks and she was again given RhoGAM. Five years later in
the third pregnancy, her Coombs titer was 1:16 at 14 weeks and bilirubin
was found in increasing amounts in the amniotic fluid . A low fetal
hematocrit (6.2%, normal is 45%) was detected in fetal blood. 85 ml of type
O, Rh-negative packed RBC were transfused into the umbilical vein. At 30.5
weeks another transfusion of 75 ml was given, and at 33.5 weeks 80ml was
transfused. At 34.5 weeks, labor was induced and a normal female infant
was born.
OPSONIZATION
IgG is a powerful opsonizing antibody (from the Greek opsonin, to prepare for eating). The
antibody reacts with microbial epitopes and its Fc region is then efficiently bound by specific
Fc receptors on macrophages and/or polymorphonuclear cells. The net effect is engulfment of
the bacteria into the phagocyte. The figures on the following page illustrate both the
mechanism of opsonization.
ANTIBODY DEPENDENT CELL MEDIATED CYTOTOXICITY (ADCC)
Certain large granular lymphocytes [also called Natural Killer (NK) cells] have Fc receptors
on their surface and when antibodies bind cellular antigens (for instance on tumor cells) NK
cells can then bind to the antibody via the Fc portion. The NK cell can kill the tumor cells
due to the secretion of substances by the NK cell that are cytotoxic to the tumor cells. Only
53
IgG antibodies are implicated in this type of activity and the importance of ADCC in host
defense or tissue damage is still controversial.
COMPLEMENT ACTIVATION
IgG antibodies are efficient activators of the Complement system. Complement will be
covered in a separate lecture. Briefly, many antigen-antibody interactions trigger a series of
enzymes collectively known as Complement. Some of the by-products of these reactions can
act as opsonins and other components are chemotactic (attract phagocytic cells). IgG
antibody activation of Complement can have profound biological effects, some positive and
some negative. Details will be presented in the lecture on Complement.
BACTERIAL IMMOBILIZATION
Motile bacteria can have their movement arrested by IgG and IgM antibodies by crosslinking their flagella or clumping them via their flagella. The antibody can function in this
regard like handcuffs in stopping the waving of flagella. The result is that the bacteria are less
invasive and less efficient in spreading through tissue.
VIRAL NEUTRALIZATION
Most viruses utilize some form of cellular receptor for initial binding that results in the virus
gaining entry into the cell or moving its DNA or RNA into the cell. IgG and IgM antibodies
specific for those structures on the virus that bind to the cell receptors will inhibit or
eliminate initial binding to the cell, thereby protecting the cell from viral entry. The binding
of IgG also facilitates phagocytosis of the organism.
TOXIN NEUTRALIZATION
Bacterial toxins usually are toxic to cells because the toxin binds to specific cellular receptors
to gain entry to the cell and then toxic effects occur intracellularly. The strategy that the
immune system employs to protect the animal from toxins is to make a variety of antibodies
specific for many different epitopes on the toxin to immobilize it in the form of an antigenantibody aggregate and stop the toxin from reaching the cell receptor. The Ab-Ag aggregates
can be easily phagocytosed and the toxins degraded and rendered non-toxic by acid proteases
in the phagosomes. The problem is in finding a way to use the toxins to trigger an immune
response without killing the host with toxin. This has been done with diphtheria and tetanus
54
toxins very successfully by treating the toxins with formaldehyde. Formaldehyde treatment
eliminates the toxic properties of the molecules without significantly affecting the antigenic
makeup of them (review the section on Cross-reactions in the lecture and chapter on
Immunogens and Antigens). These TOXOIDS then can be used to make vaccines that will
elicit a strong immune response with no toxic effects.
55
COMPLEMENT ACTIVATION
Its pentameric form coupled with the correct amino acid sequence for binding complement
make IgM the most efficient isotype on a mole/mole basis for complement activation. It has
been shown experimentally that the binding of one IgM antibody on the surface of a red
blood cell is sufficient to lyse the cell but that in excess of 100 IgG antibodies are needed to
lyse a red blood cell.
STRUCTURAL FEATURES OF IgA.
IgA appears selectively in the sero-mucous secretions such as saliva, tears, nasal fluids,
sweat, colostrum and secretions of the lung, genitourinary and gastrointestinal tracts where it
has the job of defending the exposed external surfaces of the body against attack by microorganisms from the environment. It is present in these fluids as a dimer stabilized against
proteolysis by combination with another protein, secretory component, which is the cleaved
portion of the polymeric Ig receptor (PIgR) that allows for transport across the epithelial cell
layer (See figure below). sIgA is a single peptide of molecular weight 60,000
Important structural features of the IgA molecule include:
56
57
58
antibodies of a given specificity encounter their antigen while bound to the surface of mast
cells, degranulation of the mast cells ensues with large amounts of histamine and other
vasoactive amines are released that have dramatic physiological consequences. IgE can be
thought of as the allergy or reaginic antibody. IgE is responsible for the symptoms of hay
fever and of extrinsic asthma when patients with atopic allergy come into contact with the
allergen, e.g. grass pollen, ragweed pollen, penicillin etc.
Important structural features of IgE include:
a) the large number of carbohydrate moieties.
b) the 5 distinct domains in the H chain (reminiscent of IgM). The cytotropic regions
appear to be the C 2 and C 3 domains.
H
59
Since inhibitors of methyltransferases and serine esterases inhibit all these events and
mediator release, it is assumed that activation of these enzymes by receptor bridging is the
initial event. Phospholipase c activation generates both IP3 (which mobilizes intracellular
Ca2+), and diacylglycerol which activates protein kinase c. The biochemical cascade
produces membrane-active fusogens such as lysophosphatidic acid that may facilitate
granular membrane fusion and degranulation, and the series of arachidonic acid metabolites
formed by the cyclooxygenase and lipxygenase pathways.
60
CLINICAL VIGNETTES
The first clinical vignette is on reserve in the LRC. It is a story entitled How it feels to
die written by a former editor of Life Magazine. Briefly, he self-prescribed some
penicillin tablets when he woke up one morning. The story describes his experience with
severe anaphylactic shock. It describes his treatment by his family physician who (lucky
for him) lived right next door. She had the right things with her to treat him and he lived.
Case 49Acute systemic AnaphylaxisA life-threatening immediate hypersensitivity
reaction to peanuts. John was a healthy 22 month-old who developed swollen lips while
eating a cookie containing peanut butter. A month later he ate another of the same kind of
cookie. He started to vomit, became hoarse, had difficulty breathing, started to wheeze
and developed a swollen face. He was rushed to the ER but became lethargic and lost
consciousness en route. Upon arrival at the ER his blood pressure was 40/0 (normal
80/60), pulse was 185 (normal 80-90) and respiration was 76 (normal 20). Epinephrine,
saline, anti-histamine, and corticosteroid were administered. Within one hour he was
responsive and after an overnight hospital stay including further epinephrine and antihistamine treatments, he was discharged. The parents were instructed to avoid giving him
any foods containing peanuts or peanut extract. Further tests were scheduled in the
Allergy Clinic.
61
62
ANTIBODY ENGINEERING
Presents a way to make custom made antibodies for therapeutic use. Recapturing in
recombinant form the naturally developed diversity of antibodies
Single chain Fv (ScFv): V domains of expressed antibodies linked with a flexible peptide.
VL and VH domains are cloned into a plasmid vector, then the recombinant protein is
secreted by bacteria. Can also be expressed as Fab fragments containing full length light
chains linked by disulfide bonds to the VH-CH1 fragment of the heavy chain. Half life is
extremely short (hours).
Humanized antibodies: V domains of mouse antibodies against a specific target are cloned
into vectors encoding the constant regions of human antibodies. Resulting antibody may be
specific for antigen, and the potential for antigenicity is decreased.
Immunotoxins: conjugate antibodies with toxins to be able to specifically bind to target cells
and deliver toxin to a specific area (minimizes side effects).
63
CDR affinity
engineering
VL-VH
combinatorial diversity
VL
VH
C
D
R
C
D
R
CL
C
D
R
CH
1
CH2
CH3
CH3
Fc receptor
binding
CH2
Complement
activation
64
C domain
vectors
C
D
R
C
D
R
C
D
R
Fv
domain.
9. Distinguishing features of IgA molecules include 4 domains on each of the two chains, a
larger number of disulfides compared with IgG, three sites of glycosylation, two
subclasses of IgA exist (IgA1 and IgA2) and two allotypic forms of IgA2.
10. Distinguishing features of IgD include 4 domains on each of the two chains with an
extended (64 amino acid) hinge region. Like IgM and IgA, IgD is more highly
glycosylated than IgG. IgD is found in only trace amounts in serum, most is bound to
surfaces of mature lymphocytes.
11. Distinguishing features of IgE include 5 domains on each of the two chains. Like IgM,
IgA, and IgD, IgE is more highly glycosylated than IgG. The C 2 and C 3 domains are
H
cytotropic for mast cells. Only trace amounts are found in serum (except in some
allergy patients) as most is attached to mast cells.
65
12. Allotypes are structural variants of L or H chains. Are germ line encoded Mendelian
autosomal codominant genes. Usually consist of one or a very few amino acid
differences.
13. Idiotypes are epitopes found in the V regions of specific antibodies. The idiotype
defining each antibody specificity is different.
66
67
Antibodies
Table 3-1. Classes of Antibody Isotypes and Functional Properties*
Immunoglobulin Class
IgG
IgE
Isotype
IgM
IgD
Structure
Pentamer
Monomer
Monomer
Monomer
IgA
Monomer,
dimer
Heavy chain
designation
184
146-165
188
160 2
Serum
1.5
concentration(mg/mL)
0.03
0.5-10.0
<0.0001
0.5-3.0
5-10
7-23
2.5
J chain
Yes
No
No
No
Yes
No
Yes, except
IgG4
No
No
Bacterial toxin
neutralization
Yes
No
Yes
No
Yes
Antiviral activity
No
No
Yes
No
Yes
No
No
No
Yes
No
Additional properties
Effective
agglutinator of
particulate
antigens, bacterial
opsonization
Found on surface
of mature B cells,
signaling via
cytoplasmic tail
Antibodydependent
cell
cytotoxicity
Mediation of
allergic
response,
effective
against
parasitic worms
Monomer in
secretory fluid,
active as dimer
on epithelial
surfaces
68
COMPLEMENT
Rick A. Wetsel, Ph.D.
Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc,
New York, NY. 6th edition, 2009. Chapter 13; Geha and Notarangelo. Case Studies in
Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 32. Factor I
Deficiency; Case 33. Deficiency of C8.
Web Resource: https://med.uth.edu/pathology/courses/immunology/links-forlectures/complement/
WHAT IS COMPLEMENT?
Complement is a system of more than 30 serum and cell surface proteins that is
involved in numerous functions in inflammation and immunity. In conjunction with
specific antibodies, it acts as the primary humoral defense system against bacterial and
viral infections. Complement activity is heat labile and can be destroyed by heating
serum to 56O C 30 minutes (which inactivates the C3 and C4 proteins as well as other
complement components).
Most of the complement proteins in the serum are
produced by liver hepatocytes. C3 is the most abundant serum complement protein
with a normal range of 1.0 to 1.5 mg/ml in healthy individuals. Some of the complement
components (e.g. C3, factor B) are acute phase proteins and can increase in
concentration two to three fold. Many of the complement proteins have shared
sequences, indicating that they evolved by gene duplication and recombination. The
complement genes are scattered throughout the human genome; the genes for the
proteins C4, C2, and factor B are located within the Major Histocompatibility Complex
on chromosome 6.
COMPLEMENT FUNCTIONS
Activation of the complement system results in the production of several different
polypeptide cleavage fragments that are involved in five primary biological functions of
inflammation and immunity.
1. Cytolysis of foreign organisms (e.g. bacteria)
2. Opsonization and phagocytosis of foreign organisms
3. Activation of inflammation and directed migration of leukocytes
4. Solubilization and clearance of immune complexes
5. Enhancement of humoral immune response
69
70
71
Activated C1s can cleave C4 and C2 into large (C4b and C2b) and small (C4a and C2a)
fragments. C4 is cleaved first, and approximately 1% binds to a nearby surface via a
covalent linkage. C2 can complex with surface bound C4b and can be cleaved by C1s.
The resulting C4bC2b complex is the classical pathway C3 convertase, and has the
ability to specifically cleave C3 into large (C3b) and small (C3a anapylatoxin) fragments.
C3 is the most abundant complement protein and plays a pivotal role in complement
activation. Many molecules can be cleaved into C3b and C3a. Cleavage results in
exposure of the labile thiolester bond in C3b, permitting some to bind covalently to
proteins and carbohydrates on cell surfaces. The C3a anaphylatoxin is released into
the blood and mediates many important inflammatory activities that will be discussed
72
later. Some of the C3b binds to C4bC2b to form C4b2b3b, which is the C5 convertase.
This complex by the C2b protease subunit will cleave C5 into big (C5b) and small (C5a
anaphylatoxin) subunits. C5a is released into the blood and as C3a mediates many
important inflammatory activities. C5a on a molar basis is 100 times more potent than
C3a.
FIGURE 3. The classical pathway of complement activation generates a C3
convertase that deposits large numbers of C3b molecules on the surface of the
pathogen.
The classical pathway of complement can also be activated by the serum mannose
binding lectin complex (MBL-MASP). This complex is structurally similar to the C1
complex. However, instead of binding to immune complexes it binds to directly to
polysaccharides on gram-negative bacteria. The mannose binding lectin is C1q-like in
structure and the MBL associated proteases (MASP) are similar to C1r and C1s. MBLMASP on binding bacterial surfaces can cleave C4 and C2 thereby activating the
remainder of the classical pathway.
73
74
75
This does not normally happen because there exist several complement inhibitors in
serum as well as on the surface of host cells.
C1-inhibitor (C1INH)
CD59 (Protectin)
S Protein (Vitronectin)
Clusterin (SP-40-40)
Complement Receptors
There are several characterized complement receptors that are involved in binding
complement activation and degradation products. They are expressed on various cell
76
types and are involved in mediating many of the biological functions attributed to
complement.
TABLE II DISTRIBUTION AND FUNCTION OF RECEPTORS FOR COMPLEMENT
PROTEINS ON SURFACE OF CELLS
77
78
All three peptides mediate: 1. smooth muscle contraction 2. histamine release from
mast cells and 3. increase vascular permeability.
C5a on binding C5aR mediates chemoattraction of neutrophils, monocytes,
macrophages, and eosinophils. C3a is a chemoattractant for eosinophils but not
neutrophils.
79
80
81
82
SUMMARY
1. The complement system is a group of <30 serum proteins and cell surface
molecules that act as important part of the overall immune system.
2. The activities of complement include: 1) cytolysis of foreign organisms, 2)
opsonization and phagocytosis of foreign organisms, 3) activation of
inflammation and directed migration of leukocytes, 4) solubilization and clearance
of immune complexes, and 5) enhancement of humoral immune response.
3. The complement cascade is a series of reactions involving complement proteins.
It can be divided into two phases: activation and lysis (MAC formation).
Activation involves protein-protein interactions and proteolytic
cleavage,
whereas the lytic pathway involves protein-protein interactions.
4. There are three activation pathways: the classical, lectin, and alternative.
Classical pathway activation requires antigen-antibody complexes (containing
IgM or certain IgG subclasses). The Lectin pathway is a newly described
pathway that activates the classical pathway independent of antibody. The
Mannose Binding Lectin complex is substituted in place of C1 and recognizes
polysaccharides on bacterial surfaces. The alternative pathway is an innate
system in which complement components react directly with foreign substances.
Classical pathway activation involves the proteins C1,C4,C2, and C3, and the
alternative pathway utilizes the proteins C3, B, D, and P.
5. Complement activation results in the release of anaphylatoxins (C3a,C4a, and
C5a). They are important mediators of inflammation, causing recruitment and
activation of neutrophils, macrophages, and other cell types. Activation also
produces cleavage products (C3b, C3bi, and C4b) which serve as opsonins,
enhancing phagocytosis. The C3d cleavage fragment is involved in enhancing
the immune response.
6. MAC formation produces a channel in the cytoplasmic membrane of bacteria or
other cells, leading to cell lysis. It requires prior activation by either the classical
or alternative pathways, and utilizes the proteins C5b, C6, C7, C8, and C9.
7. The complement system is regulated by several inhibitors, including C1 inhibitor,
Factor H, C4 binding protein, CD 59, and Decay Accelerating Factor.
83
STUDY PROBLEMS (you should feel confident about the topic if you can answer
the following):
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
On the figure shown on the next page, assign the activation fragments
responsible for that function.
84
85
86
Actor. 2012. Figure 6.6. Activation of complement through the classic pathway (antigen-antibody
complexes), the alternative pathway (recognition of foreign cell surfaces), or the lectin pathway (or
mannose-binding pathway) promotes activation of C3 and C5, leading to construction of the membrane
attack complex..
87
I. General principles
1. Ag-binding diversity
results from differences in
the Variable domains due to
Multiple V gene segments
V(D)J joining
Random assortment of H, L
chains
Junctional/insertional diversity
Somatic mutation
2. Functional diversity
is due to differences in
the Constant domains
IgM C fixation
IgG C fixation, opsonization
through Fc receptors
IgA secreted Ig
IgE allergic reactions
Changes in these Ig isotypes occur through isotype switching
3. Generation of Ag-binding diversity occurs during B or T cell development before
exposure to antigens; isotype switching occurs in B cells after antigen exposure.
4. Both the generation of Ag-binding diversity and isotype switching involve DNA
rearrangement.
5. Each B cell and all of its progeny produce only one type of heavy and light chain V
region, and thus have a single antigen specificity. This specificity can be fine tuned by
point mutations (so-called somatic hypermutation) that occurs after antigen exposure.
Development of Ag-binding
Diversity (VDJ rearrangement)
(IgM/IgD producing B cells)
Exposure to Ag + T cell
cytokines results in
isotype switching (B cells
change expression from IgM
to other isotypes)
88
Meaning:
Variable
Diversity
Joining
Constant
Number:
~50
~20
~6
9*
Size:
Function:
~95 aa
~3-6 aa
Form part of Variable Domain
~13 aa
~110 aa
Form the Constant Regions
per Domain
*One for each heavy chain isotype; each constant region contains 3 or 4 C domains
(see figure on proceeding page)
3. Stem cells in the bone marrow or fetal liver are stimulated to differentiate into B cells.
These B cell precursors (pro-B cells) are then stimulated to undergo VDJ
rearrangement. The pro-B cells begin to express Rag-1 and Rag-2, which direct the
recombination of the Ig genes in B cell precursors and the TCR genes in T cell
precursors. In B cells, the first step is D-J joining, in which the DNA between
randomly selected D and J regions is looped out and the intervening sequence is
deleted (see diagram). (This process involves the recognition of 7-bp and 9-bp
sequences next to the D and J regions; the same type of recognition occurs in all Ig
and TCR VDJ rearrangements.)
4. Following D-J joining, a similar looping out and deletion mechanism occurs between
the V and D regions, resulting in V-D joining. The resulting contiguous V,D, and J
gene segments have no intervening introns and form the Variable Region Exon. If the
89
rearrangement is successful (i.e. does not contain any stop codons or frameshifts),
chains (IgM heavy chains) are produced. Further rearrangement of the Ig heavy chain
loci stops, a process called allelic exclusion. However, many VDJ rearrangements
are unsuccessful, in which case the second copy of chromosome 14 undergoes
rearrangement. If this recombination is unsuccessful, the cell undergoes
apoptosis and dies.
Coico et al., 2009 Fig. 6.3. V(D)J rearrangement. V-J joining in the V locus is shown
6. In mature B cells, the (IgM) and (IgD) heavy chains can both be expressed on the
same cell by the alternative splicing of RNA as shown in Fig. 6.4 (preceding page).
Clinical vignette B cell maturation, from Geha and Notarangelo, Case Studies in Immunology.
Case 1
III.
1. In humans and most other mammals, there are two light chain loci called kappa () and
lambda (). These are located on two different chromosomes. The germline
arrangement is similar to that of the heavy chain locus, except there are no D gene
segments. Also, for the kappa locus there is only one constant region, whereas the
lambda locus has multiple constant regions, each with its own J gene segment.
2. Once successful heavy chain rearrangement occurs, the pre-B cell proceeds with
kappa gene rearrangement. In this case, randomly selected V and J segments in one
chromosome join together to form the Variable Region Exon; no D segments are
involved.
3. If a functional kappa chain is produced, V(D)J rearrangement stops and the cell
becomes a immature B cell that expresses only IgM on its surface. The surface IgM
will be anchored by a hydrophobic tail, and will look like the molecule shown in Fig.
90
7.2 (see above). Later, the cell can coexpress both IgM and IgD and thus become a
mature B cell.
4. If the first rearrangement does not produce a kappa chain, then the second sister
chromosome will undergo rearrangement. If that is unsuccessful, then the two lambda
loci will rearrange one after the other. If those are nonproductive, the cell will undergo
apoptosis and die, as was the case for the heavy chain locus.
5. The end result of this process is an immature B cell expressing only IgM with either
kappa or lambda light chains. As the cell leaves the bone marrow, it begins to express
both IgM and IgD and thus becomes a mature B cell. This difference is important
because immature B cells are more readily tolerized (made nonresponsive to
antigen) than are mature B cells.
6. The steps of B cell development are summarized in the following figure (Coico, 2009,
Fig. 7.1). B cell tumors may be frozen at different stages of this maturation process;
the less mature tumor types tend to be more aggressive and to have a poorer
prognosis.
91
IV.
It is estimated that each individual is capable of producing B and T cells with 1015 to 1018
different antigen binding sites, each with a different (but perhaps overlapping) antigen
specificity! As a result, there are very few compounds that will not induce an immune
response, even if they are synthetic and have never been found in nature previously.
Equally amazing is that most of the diversity is generated during V(D)J rearrangement,
prior to antigen exposure. Thus each individual randomly produces a huge repertoire of
B and T cells, only a small proportion of which (<1%) will respond to any one antigen or
infectious agent.
How is this Ag-binding diversity created?
As described in the Antibody Structure and Function lecture, the portions of the Variable
Region that participate in antigen binding are called Complementarity Determining
Regions or CDRs for short. All differences in antigen binding are thus due to differences
in these sequences. Two of the CDRs (CDR1 and CDR2) are hard-wired into the V gene
segment, and thus depend upon the V segment selected during rearrangement. CDR3
consists of the junction of the V, D, and J gene segments and hence has a high degree of
variability. The CDRs of both the heavy and light chain participate in the formation of the
antigen binding pocket or paratope.
V
D J
3. Assortment of heavy and light chains. Because the heavy and light chain loci
recombine independently, each B cell will contain a different combination of H and L
chains. This raises the total number of possible V, D, and J combinations to ~2 x 106.
4. Junctional and insertional diversity. The recombination between, say, D and J
segments is not precise, i.e. may occur a few base pairs in one direction or the other.
This sloppiness causes differences in the amino acid sequence and leads to
junctional diversity.
Insertional diversity results from the activity of terminal deoxynucleotide
transferase (TDT), an enzyme that is expressed during heavy chain rearrangement.
TDT adds nucleotides randomly at the V-D and D-J junctions.
Both junctional and insertional diversity affect CDR3.
All of the above mechanisms occur during B cell development, before antigen
exposure.
5. Somatic hypermutation the V regions of the antibody heavy and light chain genes
undergo a >10,000 higher rate of mutation than regular DNA. This somatic
hypermutation occurs only after antigen stimulation. Some of these mutations
increase the affinity of antibody for antigen, and those B cells expressing antibody with
higher affinity will be selectively stimulated, increasing the proportion of high affinity
antibody in secondary responses. This process is called affinity maturation.
Altogether, these mechanisms produce almost an endless variety of antibody
specificities.
V.
Isotype switching
Isotype switching (also called class switching) results in the changing of the isotype of
antibody expressed by a given B cell, e.g. from IgM to IgG3. Here are some features of
isotype switching.
1. The constant region gene expressed is always the one immediately downstream of the
V region (exception: both IgM and IgD can be expressed by niave B cells that have
not been stimulated by antigen).
2. The V region does not change during isotype switching; therefore the same antigenic
specificity is retained.
3. Isotype switching results when antigen-stimulated B cells receive a cytokine signal from
T helper cells. For example, IL-4 stimulates B cells to switch to IgE or IgG1.
4. Switching involves the deletion of intervening DNA between specific recombination
sites called switch regions (see figure below). Because the intervening DNA is lost,
the B cell cannot switch back to an isotype that has already been deleted.
5. The V region and C regions are transcribed together, and RNA splicing and translation
results in expression of the new isotype.
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VI.
VII.
Regulation of Ig expression.
1. After VDJ joining, the promoter 5 to the V region is brought within a few thousand base
pairs of the enhancer element between the J region and the constant region. The
enhancer greatly increases the rate of transcription, increasing Ig production.
2. Ig production is further increased in the differentiation of B cells into plasma cells.
2. Domain structure. All members of the immunoglobulin (Ig) gene superfamily contain
structures called Ig domains. An example of the two domains found in Ig light chains is
shown below. Domains have the following features:
Primary amino acid sequence similarity (often only 20-30 percent)
About 100-110 amino acids in length
Beta-pleated sheet structure
Commonly have an intrachain disulfide bond
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96
the presence of cytokines to CD4+ lymphocytes. Class III molecules encoded by genes located
between those that encode class I and class II molecules include complement components.
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Class II molecules have 2 transmembrane polypeptide chains ( and , 30-34 and 26-29 kDa
respectively); the peptide-binding site is shared by the two domains furthest from the cell
membrane. The overall structure of the peptide-binding site is very similar for both class I
and class II MHC molecules; the base is made of -pleated sheet, as in an immunoglobulin
domain the sides of the groove that holds the peptide are -helices. Peptides bind within
the allele specific pockets defined by the 2 transmembrane polypeptide chains, where they
are presented to the TCR for recognition. The extracellular domain shows variability in
amino acid sequences, yielding grooves with different shapes. These grooves cradle the
processed antigen for interaction with the T cell receptor. The CD4 molecule assists in the
recognition process, and binds to the invariant portion of the MHC class II molecules.
Like class I genes, class II genes also exhibit polymorphism with multiple allelic forms
expressed. In humans, allelic forms are designated different from the mouse. For examples,
human class II genes are given numbers such as HLA-D4 or HLA-D7.
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MHC Class III Molecules: Class III HLA genes encode complement components that
show no structural similarity to either class I or class II molecules. These genes, along
with genes encoding tumor necrosis factor (TNF), separate HLA class II and class I genes
on the chromosome.
CD8+ T Cell recognize Ag-MHC class I and CD4+ T Cell recognize Ag-MHC
class II.
Antigen is recognized in conjunction with proteins of the major
histocompatibility complex (MHC). Different antigen degradation and
processing pathways produce MHC-peptide complexes where "endogenous"
peptides associate with class I molecules and "exogenous" peptides associate
with class II molecules.
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102
FIGURE 8.5. Processing of exogenous antigen in MHC class II pathway, (Ii = invariant
chain; CLIP = fragment of Ii bound to MHC class II groove.)
Coico and Sunshine, 2009. Figure 8.5.
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104
Step 1: Initially, immature thymocytes within the thymic cortex express low
levels of TCR, but high levels of both CD4 and CD8 (double-positive cells).
They interact with thymic epithelial cells that express high levels of both class I
and class II MHC molecules. Thymocytes with moderate affinities for these selfMHC molecules are allowed to develop further, while thymocytes with affinities
too high or too low for self-MHC are induced to die by apoptosis. The
thymocytes that survive are said to have been "positively selected" through their
interaction with self-MHC.
Step 2: The positively selected thymocytes then begin to express high levels of
TCR, some of which recognize self components other than self-MHC. These
cells must be deleted to prevent autoimmune destruction of healthy host tissues.
Negative selection is the elimination of T cells reactive with self components
other than the MHC. Negative selection occurs in the deeper cortex, at the
corticomedullary junction, and in the medulla of the thymus. The thymocytes
interact with antigen processed and presented by interdigitating cells and
macrophages. Only thymocytes that fail to recognize self antigens are allowed to
survive and proceed along the maturation process, with the remainder undergoing
apoptosis. Eventually, T cells that survive the negative selection process lose
either CD4 or CD8, becoming "single positive" cells. Fewer than 5% of
thymocytes survive selection and leave the thymus to take up residence in the
secondary lymphoid organs.
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Preformed perforins are released at the target cell surface which generate
transmembrane pores in the target cell, through which a second protein,
granzyme, can gain entry to the cytosol and induce the apoptotic series of events.
Apoptitic signaling via membrane-bound molecules can occur via Fas on the
target cell surface and Fas ligand on the CTL surface. The processes of antigen
recognition, CTL activation and delivery of apoptotic signals to the target cell can
be accomplished within 10 minutes. The apoptotic process in the targeted cell
may take 4 hours or more and continues long after the CTL has moved on to
interact with other tissue cells.
T helper lymphocytes: The initial interaction between T lymphocytes and the APC is
mediated by adhesion molecules. Interactions occur between LFA-1, CD-2 and ICAM-3
on the T-cell, and with ICAM-1, ICAM-2, LFA-1 and LFA-3 on the APC. These
molecules synergize in binding of lymphocytes to the APCs. This transient binding
allows the T-cell to sample the large numbers of MHC molecules on the surface of the
APCs for their specific peptide. If a T-cell recognizes its peptide ligand bound to MHC,
signaling via the T-cell receptor complex is induces more conformational changes,
eventually leading to the production of T-cell cytokines. T cells require co-stimulation
through binding of the CD-28 ligand with the CD-80/CD-86 ligand of the APC.
T-dependent B cell activation: B cells can also specifically take up antigen via binding
through their surface Ig. This is internalized, broken down to peptides and the peptides
are presented on the B cell surface held in the peptide binding grooves of MHC class II
molecules. If this B cell interacts with a primed T cell that recognizes the peptide/class II
complex on the B cell then the T cell may transiently express accessory ligands. This
results in cells going into cell cycle and the secretion of cytokines. For the B cell, this
action, in concert with correctly released T cell cytokines, will drive isotype switching as
well as maturation of the B lymphocyte into a plasma cell.
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Clinical Vignette - The case of Helen Burns (Case 8 in Geha and Notarangelo): Helen Burns
was 6 months old when she developed pneumonia, caused by the opportunistic pathogen
Pneumocystis carinii. Helen was tested for severe combined immunodeficiency; it was found that
Helen's T cells could be stimulated with mitogen (phytohemagglutinin), but could not respond to
specific antigenic stimuli. It was further established that Helen had low overall immunoglobulin
levels and decreased CD4 cells. Her CD8 cell counts were within normal range. Helen's white blood
cells were examined for expression of MHC Class I and Class II molecules. She was diagnosed with
MHC Class II deficiency. A bone marrow transplant was performed using Helen's mother as a
donor. The graft was successful and immune function was restored.
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109
T CELL RECEPTOR:
Structure and Genetic Basis
Jeffrey K. Actor, Ph.D.
713-500-5344
Objectives: (1) Present an overview of the T receptor structure and organization of the gene loci
encoding for the T cell receptor chains; (2) explain mechanisms underlying generation of T cell
receptor diversity; (3) examine the stages in thymic selection of T lymphocytes; and (4) compare
and contrast the T cell receptor with the B cell receptor.
Keywords: T cell receptor (TCR).
Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York,
NY. 6th edition, 2009. Chapter 9; Geha and Notarangelo. Case Studies in Immunology. Garland
Publishing, New York, NY. 6th edition, 2012. Case 7: Omenn Syndrome.
Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/the-t-cellreceptor-structure-and-genetic-basis/
The acquired immune response is subdivided, based on participation of two major cell types. B
lymphocytes originate in the bone marrow, and synthesize/secrete antibodies. This is termed
humoral immunity. T lymphocytes mature in the thymus, and secrete immunoregulatory factors
following interaction with antigen presenting cells; this is termed cellular immunity (CMI).
Lymphocyte Biology
Lymphoid cells provide efficient, specific and long-lasting immunity against microbes/pathogens
and are responsible for acquired immunity. This lecture will primarily examine the biology of two
classes of lymphocytes: (1) thymic-dependent cells or T lymphocytes that operate in cellular and
humoral immunity; and (2) B lymphocytes that differentiate into plasma cells to secrete
antibodies. T and B lymphocytes produce and express specific receptors for antigens.
The major properties of the acquired immune response are specificity, memory, adaptiveness,
and discrimination between self and non-self. All of these properties are related to the random
selection of variable region components during the development of both B cells and T cells.
The lymphatic organs are tissues in which lymphocytes mature, differentiate and proliferate. The
primary (central) lymphoid organs are those in which B and T lymphocytes mature into antigen
recognizing cells. In embryonic life, B cells mature and differentiate from hematopoietic stem
cells in the fetal liver. After birth, B cells differentiate in the bone marrow. Maturation of T cells
occurs in a different manner. Progenitor cells from the bone marrow migrate to the thymus where
they differentiate into T lymphocytes. The T lymphocytes continue to differentiate after leaving
the thymus, and are driven to do so by encounter with specific antigen in the secondary lymphoid
organs.
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The secondary lymphoid organs are those tissues in which antigen-driven proliferation and
differentiation take place. The spleen and lymph nodes are the major secondary lymphoid organs.
Additional secondary lymphoid organs include the tonsils, appendix, and Peyers patches.
Aggregates of cells in the lamina propria of the digestive tract lining may also be included in this
category, as well as any tissue described as MALT (mucosa-associated lymphoid tissue), GALT
(gut-associated lymphoid tissue) or BALT bronchus-associated lymphoid tissue).
T Lymphocytes: T lymphocytes are involved in regulation of immune response and cell mediated
immunity. They provide necessary factors to help B cells produce antibody. Mature T cells
express antigen-specific T cell receptors (TCR). Every mature T cell expresses the CD3 molecule,
which is associated with the TCR. The TCR/CD3 complex recognizes antigens associated with the
major histocompatibility complex (MHC) molecules on target cells (e.g. virus-infected cell). The
TCR is also expressed on the cell surface in association with co-receptor or accessory molecules
(CD4 or CD8).
The T cell receptor genes are closely related members of the immunoglobulin gene superfamily.
Each chain consists of a constant (C) and a variable (V) region, and is formed by a gene-sorting
mechanism similar to that found in antibody formation. The repertoire is generated by
combinatorial joining of variable (V), joining (J), and diversity (D) genes, and by N region
diversification (nucleotides inserted by the enzyme deoxynucleotidyl-transferase). Unlike
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immunoglobulin genes, genes encoding TCR do not undergo somatic mutation. Thus there is no
change in the affinity of the TCR during activation, differentiation, and expansion.
TCR-CD3-complex: The TCR heterodimer is tightly associated with six independently encoded
CD3 subunits (, , , , and ) required for efficient transport to the cell surface. CD3 subunits
possess long intracellular tails and are responsible for transducing signals upon TCR engagement.
Genes Coding for T-Cell Receptors: Genes which code for the T cell receptor and the
mechanisms used to generate TCR diversity are similar to those of immunoglobulins.
The TCR V, D, and J genes are mixed together in a more complicated manner than found for
immunoglobulin genes.
and uses only V and J gene segments.
and use V, D, and J gene segments.
There are many more V and V genes (50-100) than V and V genes (5-10) present in germ
line.
The and chain genes are mixed together in one locus. The genes encoding the chain are
entirely located between the cluster of V and J gene segments.
The top and bottom
rows show germline
arrangement of the
variable (V), diversity
(D), joining (J), and
constant (C) gene
segments at the T-cell
receptor and loci.
During T- cell development, a V-region
sequence for each chain
is assembled by DNA
recombination. For the
chain (top), a V gene
segment rearranges to a
J gene segment to
create a functional gene
encoding the V domain.
For the chain
(bottom), rearrangement of a D, a
J, and a V gene
segment creates the
functional V-domain
exon.
Geha and Notarangelo, 2012. Figure 7.1.
If and/or rearrangements are not functional, then gene rearrangement continues followed
by gene rearrangement. In this manner, a product appears, and the cell becomes an T
cell.
The Process of Recombination: Recombination of V, D, and J gene segments is coordinated by
recombinase-activating genes RAG-1 and RAG-2. The enzymes recognize specific DNA signal
sequences consisting of a heptamer, followed a spacer of 12 or 23 bases, and then a nonamer. If
either RAG gene is impaired or missing, homologous recombination events are abolished. This
gives rise to severe combined immunodeficiency (SCID). Mutations which result in partial
enzymatic activity can also occur, and can give rise to immunodeficiency diseases. An example of
such disorder is Omenn Syndrome, discussed in detail in the Case Studies in Immunology (Geha
and Notarangelo, chapter 7) text.
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Immunoglobulins
~2 - 3.4 x 106
~3 x 107
~1014
T cell : Receptors
5.8 x 106
~2 x 1011
~1018
Development of T lymphocytes
During differentiation in the thymus, immature T cells undergo
rearrangement of their TCR and genes to generate a diverse
set of clonotypic TCRs. Immature thymocytes are selected for
further maturation only if their TCRs do not interact with selfpeptides presented in the context of self-major histocompatibility
complex (MHC) molecules on antigen presenting cells. Different
signals lead to the alternate developmental outcomes of maturation
or apoptosis (positive versus negative selection). Positively
selected thymocytes undergo alternate commitment to either the T
killer or T helper lineages, which correlate precisely with a cell's
TCR specificity towards MHC class I or II molecules,
respectively. Lineage commitment is marked phenotypically by
the loss of expression of one of the co-receptor molecules, CD8 or
CD4. Immature thymocytes express both co-receptors (double
positive), while T killer or T helper cells express only CD8 or
CD4, respectively (single positive CD8+ or CD4+).
Figure. Changes in surface molecules of thymocytes at different
stages of maturation.
The majority of peripheral blood T lymphocytes express the and form of the TCR. In healthy
adults, less than 5% express a heterodimer comprised of the and chains. Virtually all the cells
that express the TCR- are CD4+CD8- (T helper) or CD4-CD8+ (T cytotoxic or T suppressor).
Almost all cells expressing TCR- are CD4-CD8- (double negative). While the TCR-
expressing lymphocytes are known to function as helper and cytotoxic cells, the function of the
TCR- cells is not well understood.
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No interaction =
MHC + self
MHC + non-self
++, CD4+CD8+ cell
interacts with interdigitating cell
High affinity interaction =
DELETION
SURVIVAL
Commitment CD4+ or CD8+
T Helper Cells: T helper cells (Th) are the primary regulators of T cell- and B cell-mediated
responses. They 1) aid antigen-stimulated subsets of B lymphocytes to proliferate and differentiate
toward antibody-producing cells; 2) express the CD4 molecule; 3) recognize foreign antigen
complexed with MHC class II molecules on B cells, macrophages or other antigen-presenting
cells; and 4) aid effector T lymphocytes in cell-mediated immunity.
Currently, it is believed that there are two main functional subsets of Th cells, plus other helper
subsets of importance. T helper 1 (Th1) cells aid in the regulation of cellular immunity, and T
helper 2 (Th2) cells aid B cells to produce certain classes of antibodies (e.g., IgA and IgE). The
functions of these subsets of Th cells depend upon the specific types of cytokines that are
generated, for example interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by Th1 cells; IL4, IL-6 and IL-10 by Th2 cells. Two other classes of T helper cells are thought to be involved in
oral tolerance and serve as regulators for immune function. Th3 cells secrete IL-4 and TGF- and
provide help for IgA production, and have suppressive properties for Th1 and Th2 cells. Th17
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cells, characterized by IL-17 secretion, are thought to be involved as effector cells for autoimmune
disease progression.
T Cytotoxic Cells: T cytotoxic cells (CTLs) are cytotoxic against tumor cells and host cells
infected with intracellular pathogens. These cells 1) usually express CD8, and, 2) destroy infected
cells in an antigen-specific manner that is dependent upon the expression of MHC class I
molecules on antigen presenting cells.
T Suppressor/ T Regulatory Cells: T suppressor cells suppress the T and B cell responses and
express CD8 molecules. T regulatory cells also affect T cell response, with many cells
characterized as CD4+CD25+, TGF- secretors.
T Cells: Not all T cells express TCRs. An alternative is to express chains of the TCR.
Generally, cells lack CD4, although some cells do express CD8. The functions of cells are
not well understood. T cells can function in the absence of MHC molecules. They home to the
lamina propria of the gut, and are thought to assist in protection against microorganisms entering
through epithelium at mucosal surfaces. Their range of response to antigens is limited.
expressing cells have been found to be active towards mycobacterial antigens and heat shock
proteins. They have the ability to secrete cytokines like their counterparts.
Natural Killer T Cells: Natural killer T cells (NKT) are a heterogeneous group of T cells that
share properties of both T cells and natural killer (NK) cells. The majority of these cells recognize
an antigen-presenting molecule (CD1d) that binds self- and foreign lipids and glycolipids. They
constitute only 0.2% of all peripheral blood T cells. The term NK T cells was first used in mice
to define a subset of T cells that expressed the natural killer (NK) cell-associated marker NK1.1
(CD161). It is now generally accepted that the term NKT cells refers primarily to CD1drestricted T cells coexpressing a heavily biased, semi-invariant T cell receptor (TCR) and NK cell
markers. Natural killer T (NKT) cells should not be confused with natural killer (NK) cells.
-------------------------Comparison of the B cell and T cell receptors:
Both BCRs and TCRs share these properties:
they are integral membrane proteins
they are present in thousands of identical copies exposed at the cell surface
they are made before the cell ever encounters an antigen
they are encoded by genes assembled by the recombination of segments of DNA
allelic exclusion ensures only one receptor with a single antigenic specificity
they demonstrate N region addition during gene rearrangement
they have a unique binding site
this site binds to a portion of the antigen called an antigenic determinant or epitope
the binding, like that between an enzyme and its substrate depends on complementarity of
the surface of the receptor and the surface of the epitope
the binding occurs by non-covalent forces (again, like an enzyme binding to its substrate)
successful binding of the antigen receptor to the epitope, if accompanied by additional
"signals", results in:
1. stimulation of the cell to leave G0 and enter the cell cycle
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2. repeated mitosis leads to the development of a clone of cells bearing the same
antigen receptor; that is, a clone of cells of the identical specificity.
Clinical Vignette - Omenn Syndrome (Case 7 in Geha and Notarangelo): Patients with Omenn
syndrome demonstrate severe immunodeficiency characterized by the presence of activated, anergic,
oligoclonal T cells, hypereosinophilia, and high IgE levels. There is a body of evidence to indicate that
the immunodeficiency manifested in patients with Omenn syndrome arises from mutations that decrease
the efficiency of V(D)J recombination. These individuals bear missense mutations in either the RAG-1 or
RAG-2 genes that result in partial activity of the two proteins. In many cases, amino acid substitutions
map within the RAG-1 homeodomain and decrease DNA binding activity, while others lower the
efficiency of RAG-1/RAG-2 interaction.
Summary: T Lymphocytes
T lymphocytes are involved in regulation of immune response and in cell mediated immunity.
Every mature T cell expresses CD3, which is associated with the TCR. During thymic
differentiation, immature T cells undergo rearrangement of their TCR and genes to generate a
diverse set of clonotypic TCRs. Immature thymocytes are selected for further maturation only if
they recognize foreign antigens in the context of MHC molecules. Mature T cells usually display
one of two accessory molecules. CD4+ T helper cells are the primary regulators of T cell- and B
cell-mediated responses, and are further subdivided into functional subsets dependent upon
cytokines secreted. CD8+ T cytotoxic cells (CTLs) are cytotoxic against tumor cells and host cells
infected with intracellular pathogens. T suppressor cells suppress the T and B cell responses and
express CD8 molecules.
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T cell receptor genes are closely related members of the immunoglobulin gene superfamily and
derive part of their structural diversity form recombination of different V, D, and J gene segments.
The mechanisms for T cell receptor gene switching are similar to those of immunoglobulin genes,
but T cell receptor genes do not have somatic mutations. chains of the TCR have only V and J
segments, and join to chains. chains of the TCR have genes for V, D, and J segments. The
process of recombination is coordinated by recombinase-activating genes RAG-1 and RAG-2.
If rearrangements are unsuccessful on both chromosomes, chains join to chains to give
phenotypic T cells. chains have only V and J segments; chains have V, D, and J segments.
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OBJECTIVES
1. Distinguish between innate and adaptive immune responses on the basis of
antigen specificity, HLA restriction and memory.
2. Understand role of immune mediators including cytokines, chemokines,
costimulatory and adhesion molecules in the development of adaptive immune
responses.
3. To describe the various effector and regulatory functions of T and B cells.
4. To demonstrate the molecular events associated with T cell and B cell activation.
5. Compare and contrast effector cells in cytotoxic mediated immunity.
a. Describe the concepts of tumor antigens.
b. Describe the effectors mechanisms in tumor immunity.
6. To develop a practical understanding of mechanisms and clinical relevance of T dependent and - independent antibody responses.
LEARNING OBJECTIVES:
KEY WORDS
C.
D.
E.
The major properties of the acquired immune response are specificity, memory,
adaptiveness, and discrimination between self and non-self. All of these properties
are related to the random selection of variable region components during the
development of B cells and T cells. The essential features for clonal selection of these
cells include:
B and T lymphocytes of all antigenic specificities exist prior to contact with antigen.
Each lymphocyte carries specific surface molecules (immunoglobulin or T cell
receptor) of only a single specificity.
Lymphocytes can be stimulated by antigen under appropriate conditions to give rise
to progeny with identical antigenic specificity.
Lymphocytes potentially reactive with self are deleted or inactivated to ensure that
no immune response is mounted against self components.
Cytokines
When confronted with above challenge, the host immune response will determine
appropriate degree of antigen-specific cell mediated vs. humoral response. In order to
accomplish this, various regulatory networks controlled by cytokines are activated.
1.
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2.
AND
III. T LYMPHOCYTES
T lymphocytes are involved in the regulation of the immune response and in cell
mediated immunity, and help B cells to produce antibody. Mature T cells express
antigen-specific T cell receptors (TCR). Every mature T cell expresses the CD3
molecule, which is associated with the TCR. In addition mature T cells usually display
one of two accessory molecules, CD4 or CD8. The TCR/CD3 complex recognizes
antigens associated with the major histocompatibility complex (MHC) molecules on
target cells (e.g. virus-infected cell).
Important T cell markers
Surface Markers of T cells. Additional markers include: CD45RO, Leukocyte common antigen for
memory T cells (activated). CD45RA, Leukocyte common antigen for naive T cells (resting). Coico and
Sunshine, 2009. Figure 9.4.
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TCR-CD3-complex: The TCR heterodimer is tightly associated with the CD3 coreceptor made up of independently encoded subunits (, , , and two chains). The
CD3 complex is required for efficient transport of the TCR to the cell surface. CD3
subunits possess long intracellular tails and are responsible for transducing signals
upon TCR engagement with MHC presented antigen.
A. T Helper cells
T helper cells (Th) are the primary regulators of T cell- and B cell-mediated responses.
They 1) aid antigen-stimulated subsets of B lymphocytes to proliferate and differentiate
toward antibody-producing cells; 2) express the CD4 molecule; 3) recognize foreign
antigen complexed with MHC class II molecules on B cells, macrophages or other
antigen-presenting cells; and 4) aid effector T lymphocytes in cell-mediated immunity.
1.
Antigen receptor
MHC II : TCR - antigen binding
MHC II : CD4 molecule - the co-receptor
Costimulatory pairs - second signals
CD40:CD40L (CD154)
CD28/CTLA-4:B7 (CD80, CD86)
Adhesion molecules
CD58(LFA-3):CD2
CD54(ICAM-1):CD11a/CD18 (LFA-1)
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2.
The TH1/TH2 paradigm was first proposed by Mossman and Coffman to explain the
differential effects of T cell help i.e. T cells helping B cells and T cells helping other T
cells. These cells were distinguished functionally rather than morphologically by the
differences in cytokine patterns that they produced.
TH2 help B cells (and other WBC) develop immunity against extracellular
pathogens (mostly through IgE, mast cells and eosinophils
Dendritic Cell
Currently, it is believed that there are multiple functional subsets of Th cells. In addition
to the ones mentioned above: Th17 cells, characterized by IL-17 secretion, are thought
to be involved as effector cells for autoimmune disease progression, and protect
surfaces (skin, gut) from extracellular bacteria. Tfh cells (follicular helper T cells) provide
help to B cells in germinal areas enabling them to develop into antibody-secreting
plasma cells; they function inside of follicular areas of lymph nodes.
The functions of all the subsets of Th cells depend upon the specific types of cytokines
that are generated, for example interferon-gamma (IFN-gamma) by Th1 cells and IL-4
by Th2 cells, and IL-17 by Th17 cells.
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125
126
3.
Intracellular Events receptor mediated transcription of cytokine
genes by a sequence of molecular events
FIGURE. Intracellular
events in CD4+ T-cell
activation. The result of
activation events is
enhanced transcription and
increased stabilization of IL2 mRNA. Coico, 2009.
127
128
4.
129
Generation of CD8+ T
cells effector cells and
target cell killing. (A)
dendritic cells activate
CD8+ T cells directly.
(B) One pathway for
CD4+ T cells to activate
CD8+ T cells. (C) Target
cell killing by a CD8+
effector T cell. Coico
and Sunshine, 2009. Fig
10.10.
130
Effector cells in Cytotoxic Cell Mediated Immunity. Both innate and adaptive cells play a
role in cytotoxic cell mediated immunity. The major cell players and their properties are
listed and summarized in the table below.
NK-ADCC
i. antibody-dependent cellular cytoxicity (ADCC).
ii. Have Fc receptors (CD16) that recognize Fc portion of IgG.
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Antigen
recognition
specific TCR
nonspecific
specific IgG
nonspecific
nonspecific
Cytotoxic cells (CTLs) directly kill tumor cells and host cells infected with intracellular
pathogens. These cells 1) usually express CD8, and, 2) destroy infected cells in an
antigen-specific manner that is dependent upon the expression of MHC class I
molecules on antigen presenting cells.
1.
2.
3.
General Considerations
Adaptive host defense against intracellular pathogens
CD8+ CTL is MHC I restricted
Is affected by TH1 cells which are also antigen-specific but MHC II
restricted
Development of CTL
TCR interacts with MHC I antigen complex
o In association with CD8
o Also involves costimulatory molecules
IL-2R upregulated
o IL-2 from Th cells cause clonal proliferation
o IFN causes activation of CTL
Killing of Target cells by CTL
IFNupregulates perforin formation
o Perforins form transmembrane channels that kill target
o Similar to complement-mediated lysis
IFNupregulates granzyme formation
o Serine proteases
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TNF-
Fas ligand
Nucleases
Serine
proteases
133
Comment
134
135
Overall, the CD1 molecules bind antigen in a deep, narrow hydrophobic pocket, with
ligands interacting via hydrophobic interactions rather than hydrogen bonding.The
role of CD1 in pathogenesis has not yet been fully determined.
2. Superantigens
Activate T cells expressing a specific Vsegment as part of TCR
Presented by Class II molecules on MHC but not in peptide groove
Several organisms have components that function as superantigens
o Staphylococcus
o Rabies virus
Activate large numbers of T cells (possible mechanism for toxic shock
syndrome)
SUPERANTIGENS
Superantigens bind
directly to T-cell
receptors and MHC,
without processing.
Usually involves
direct interaction to
V region of TCR.
V VD
J
V VD
J
3. Mitogens
Polyclonal activators of T cells by activating widespread mitosis
Derived from plant lectins
Phytohemagglutinin (PHA)
concanavalin A (conA)
pokeweed mitogen (PWM)
Other mitogens
Endotoxin (lipopolysaccharide) mouse B cells,
monocytes/macrophages
AntiCD3 polyclonal T cell activator
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human
VI.
B Lymphocytes: The genesis of and delta chainpositive, mature B cells from pre-B cells is antigenindependent. B cell development is characterized
by recombinations of immunoglobulin H and L chain
genes and expression of specific surface
monomeric IgM molecules. At this stage of
development, B cells are highly susceptible to the
induction of tolerance. Cells bearing only
monomeric IgM are referred to as immature. These
cells may undergo deletion (death by apoptosis),
anergy (long term inactivation, or receptor editing
(reactivation via V-D-J gene recombination).
Once these cells acquire IgD molecules on their
surface, they become mature B cells that are able
to differentiate after exposure to antigen into
antibody-producing plasma cells. Mature B cells
can have 1-1.5 x 105 receptors for antigen
embedded within their plasma membrane.
The activation of B cells into antibody producing/secreting cells (plasma cells) is
antigen-dependent. Once specific antigen binds to surface Ig molecule, the B cells
differentiate into plasma cells that produce and secrete antibodies of the same antigenbinding specificity. If B cells also interact with T helper cells, they proliferate and switch
the isotype (class) of immunoglobulin that is produced, while retaining the same
antigen-binding specificity. This occurs as a result of recombination of the same Ig VDJ
genes (the variable region of the Ig) with a different constant (C) region gene such as
IgG. T helper 2 cells are thought to be required for switching from IgM to IgG, IgA, or
IgE isotypes. The generation of memory B cells is associated with class switching; this
process occurs in the spleen or lymph node.
In addition to antibody formation, B cells also process and present protein antigens.
After the antigen is internalized it is digested into fragments, some of which are
complexed with MHC class II molecules and then presented on the cell surface to CD4+
T cells.
B cells secrete antibody upon antigenic stimulation, a multi-step process involving
interactions with T cells. B cells express many surface molecules which assist in the
process of antibody production through delivery of various activation signals. Some of
these costimulatory molecules are depicted in the figure below. Fc receptors are
important in "feedback" mechanisms to deliver negative signals to the cell.
137
Surface Markers of human and murine peripheral B cells. Remember that B cells carry the HLA-D
(and I-A/I-E), class II restricted major histocompatability marker, as well as have specific receptors for
complement receptors. Coico and Sunshine, 2009. Figure 7.7.
T dependent antigens
Require CD4+ help for B cells to make antibody
Must be to same antigen but different epitopes (linked recognition)
B cell epitope - hapten
T cell epitope - carrier
T B interactions
For primary response, requires APC (dendritic cell the best)
For secondary response, no APC necessary
Requires cytokines for B cell growth (IL-4), proliferation (IL-6)
Isotype switch from IgM to
o IgG (IFN
o IgA (IL-5)
o IgE (IL-4,13)
External Ag on B cell bound by surface IgM
Internalized, processed and presented via MCH II to TCR in
association with CD4 molecule
Costimulation (CD40:CD154; CD28:CD80/86)
Adhesion (CD58:CD2; ICAM-1:LFA-1; CD72:CD5)
Result is cytokine production by T cell that binds via receptor to B cell
138
T independent Responses
139
C.
Activated Syk activates PLC-which splits PIP2 into DAG and IP3
DAG>>PKC>>>multiple kinases
IP3 >>calcineurin
Both pathways activate transcription factors NF-, NF-AT
Result in nucleus is upregulation of cytokine receptor and Ig genes
140
SUMMARY
The major properties of the acquired immune response are specificity, memory,
adaptiveness, and discrimination between self and non-self.
Helper T cells (TH) provide assistance to B cells to make antibody and other T
cells to become cytotoxic by production of specific cytokines, expression of co
stimulatory and adhesion molecules molecular mechanisms involving
transcription factors
B cells make antibodies, the quantity and isotype of which is dependent upon
whether the T cell is involved (T dependent) or not (T-independent) and relates
to both the nature of the antigen and the underlying immunological capabilities of
the host
141
INTRODUCTION
It is clear that antibodies play a major role in protection from a variety of
diseases, toxins, viruses, parasites, etc. In addition, once antibodies have been made,
they can be used for a variety of diagnostic assays in the laboratory to detect the presence
of absence of a particular antigen or bacterium or virus in a sample. For instance, the use
of antibodies specific for red blood cell antigens has made routine transfusions possible.
The reaction of antigen with its homologous antibody is a two-stage phenomenon.
The initial or primary binding reaction can occur invisibly. The secondary manifestation
of that interaction is dependent on several factors such as:
a) Isotype of the antibody
b) Valence of antigen
c) Form (particulate or soluble) of the antigen
The type of assay used depends vitally on these factors. For example, determination of a
patients red blood cell type is done using intact red cells and so the assay called
agglutination is used. The kinds of assays used to detect soluble antigens such as growth
hormone cannot be used for red cell typing because of the particulate nature of the red
cell.
Review of Figure 5.1 on page 60 in the textbook will demonstrate many of the
features of antigens and of antibodies and fragments of antibodies that can dictate design
of specific assays. It is clear that valency of both antigen and antibody can be important.
Please review this figure thoroughly.
142
143
145
PRECIPITATION REACTIONS
Precipitation Reaction in Solution (Fluid Phase Reactions)
Antigen-Antibody reactions that result in the formation of visible precipitation of
the reactants are classed as secondary manifestations of Ag-Ab reactions. In general, this
reaction can be utilized to determine if there is a yes/no response for antibody binding to
an antigen (in other words, it is qualitative, rather than quantitative). Understanding the
Ab-Ag interactions that lead to this reaction is important, as the immune complexes
formed are also found in vivo and can contribute significantly to pathology.
In the precipitation reaction, various amounts of
soluble antigen are added to a fixed amount of serum
containing antibody. As illustrated in the figure to the
left, when small amounts of Ag are added, Ab-Ag
complexes are formed with excess Ab, and each
molecule of Ag is bound by Ab and cross-linked to
other Ab molecules. When enough Ag is added, ALL
of the antigen and antibody complex and fall out as
precipitate (the zone of equivalence). When an excess
of Ag is added only small Ag-Ab complexes form (no
crosslinking) and the precipitate is reduced.
NEPHELOMETRY
Nephelometry is a widely used methodology for accurately measuring quantities
of the Ig classes in serum. Obviously, dramatic increases or decreases in quantities of
these could contribute to diagnosis of numerous diseases.
In this assay, proteins in the sample react with specific antibody (e.g. an anti-IgE
antibody). The mixture is placed in a tube and inserted into the Nephelometer. When
light passes through the suspension that contains aggregated particles, a portion of the
light is scattered. The scattered light is measured and compared with stored standards.
Thus, this is a quantitative method using liquid-phase precipitation principles. It
can be applied to measuring any soluble substance provided specific antisera are
available.
146
147
be symmetrical, extending the same distance either side of the line connecting the well
centers. The presence of a line is a qualitative assay for the presence of either antibody in
the antiserum (using a standard antigen solution) or for the presence of antigen (using a
standard antiserum). See Figure 5.5 in the Textbook.
IMMUNOELECTROPHORESIS
Immunoelectrophoresis is a variation of the Ouchterlony double diffusion in gel
technique. It is designed to analyze complex protein mixtures containing many different
antigens. Electrophoresis separates proteins according to size (which corresponds to
their mobility in the electric field) within the gel matrix. A mix of antibodies specific for
the proteins is then added to a trough cut in the agar. The individual proteins and their
specific antibodies will diffuse toward one another, and lines of precipitate form for each
Ab-Ag interaction. This method and typical results are shown in Fig. 5.7, pg. 67 in the
textbook.
Clinical Relevance: The medical diagnostic use of immunoelectrophoresis is
for diagnosis of conditions where certain proteins are suspected of being
absent (e.g. hypogammaglobulinemia) or of being overproduced (e.g. Multiple
Myeloma). It is usually used as a first screening test, followed by quantitative
tests.
Immunoelectrophoresis is a qualitative assay. It is also used in medical research for
following the different steps of a purification protocol to show the disappearance of
unwanted proteins when purification of one component from a mixture is desired.
WESTERN BLOTS
The mechanisms that underlie immunoelectrophoresis form the basis for the more
commonly used Western Blot. Instead of relying on a precipitation reaction that is
manifested as a line in a gel, this assay uses labeled antibodies to visualize binding. Also
148
SOLID-PHASE IMMUNOASSAYS
There are a group of assays in which the antigen or the antibody is coated on the
surface of a plastic microplate and sensitive indicators such as radioactivity or enzymatic
action are used to detect the presence of Ag or of Ab. There are 5 of these assays
classified in two groups according to the types of antigens being analyzed: soluble or
cellular.
A. SOLUBLE ANTIGENS
RADIOIMMUNOASSAY--There are many different formats for doing
radioimmunoassay (RIA). The example below describes a clinical test to detect Hepatitis
B virus protein in patient plasma.
a) Antibody specific for Hepatitis B antigen (HBsAg) is first coated onto the
surface of plastic plates and the excess is removed by rinsing out the wells
with buffer solutions.
b) the remaining plastic surface is then blocked by adding an irrelevant protein
solution and washing
c) A mix of patient serum and a known quantity of radiolabeled HBsAg is
incubated in the wells. This leaves the plate with Ab bound to the Ag that is,
in turn, bound (noncovalently) to the plastic.
d) Detection of the amount of radiolabeled antigen that binds in the presence of
patient serum compared to a known control allows us to quantify the amount
of HBsAg in the blood sample. This is a binding competition assay less
signal means that more unlabeled (patient) HBsAg is present and bound to the
antibody. If no HBsAg is contained in the patient sample, only radiolabeled
Ag binds and the signal is higher.
149
Clinical Relevance:
Current Laboratory RIA Assays
The specific assays are to quantitate the amounts of specified antigens in body fluids such as
blood or urine.
Assays are available for measuring Renin, Gastrin, Parathyroid Hormone, Growth Hormone,
Urine Microalbumin, Vitamin B12 and Folate. Memorial-Hermann Hospital currently is phasing
out the radioimmunoassay laboratory.
150
Clinical Relevance: There are a large number of commercial ELISA kits available
for diagnostic purposes. Currently the Memorial-Hermann Hospital Laboratories
offer ELISA assays for Hepatitis antigen, HIV and HTLV antigens. Specific assays
are also available for detection of antibodies in patients serum for Hepatitis A virus,
Hepatitis B surface antigen, Hepatitis B core antigen, Hepatitis C virus,
cardiolipin, H. pylori, and for HIV types 1 and 2. Another ELISA assay is
available for detection of antibodies to Human T-lymphotropic virus type I.
Clinical Vignette
This is the case of a woman who contracted the AIDS virus from a blood transfusion
and transmitted it to her fetus later. Antibodies specific for the gp120 HIV antigen
were measured in the infant using an ELISA. The mother and father were also
tested and were found to have anti-gp120 antibodies by ELISA.
ELISPOT assays
Variation of the ELISA method. Incubate
with cells instead of soluble antibody. The #
of spots after addition of detection antibody
and precipitable substrate = the number of
cells secreting a specific antibody, thus can be
used to determine the frequency of antigen
specific B cells. Also used for T cell assays
(e.g. the number of T cells producing a
cytokine, as illustrated to the left). Often used
in biomedical research.
B. CELLULAR ANTIGENS
IMMUNOFLUORESCENCE
It is sometimes of diagnostic value to determine if a particular antigen is
found on or in the cells of a particular tissue. In this case, assays are needed that can be
151
performed directly on biopsies of tissue and seen using a microscope. The method
originally developed by Albert Coons and his colleagues at Harvard involves covalent
attachment of fluorescent organic compounds to specific antibodies that then can be used
to detect the antigen in question. The fluorescent compounds excite at different
wavelengths. This is a highly sensitive and specific assay, and cells individual cells can
be stained with up to 12 different compounds.
152
Clinical Relevance: FACS can measure CD4+ cell numbers in AIDS patients to
follow disease progression. FACS was used in Case 5MHC Class I Deficiency to
measure peripheral blood lymphocytes
MULTIPLEX ANALYSIS
A relatively new technology has been developed that combines aspects of an ELISA with
the sensitivity of flow cytometry. These Multiplex Bead Arrays rely on the engineering
of microspheres internally coded with two fluorescent dyes. Combinations of the dyes
can be used to generate up to 100 individual bead sets each of which can be coated
with specific antibody. Mutiple bead sets may be incubated with sample (plasma or cell
culture supernatant) in a well, and 96 well plates can be used for this analysis allowing
for the measurement of multiple parameters in multiple samples. The antibody-coated
beads bind to their specific antigen target (e.g. cytokines), then biotin-labeled secondary
detection antibodies are used to sandwhich the antigens bound on the beads. A reporter
molecule, streptavidin-phycoerythrin (SA-PE) then indicates each complex. In a method
similar to that used in flow cytometry, the beads are passed single file through a laser
beam, complexes are identified by the fluorescence of PE, and the internal fluorochromes
unique to each bead emit specific signals that are detected by digital processors. In this
manner, up to 100 different analytes can be detected in a single sample, making this a
powerful tool for use in screening patient responses in different disease conditions.
153
154
Attempts to develop human hybridoma technology have not been very successful.
To adapt the murine system for making human antibodies, recombinant DNA
methodologies have been developed to humanize murine antibodies. The methods
usually use murine V-region sequences coupled to human C-region sequences. There are
many variations on this theme and the methodologies are also applicable to engineering
receptors (for cytokines, etc.) into cell lines in which they are not normally expressed.
HIGH THROUGHPUT IMMUNOSEQUENCING
Recent advances in sequencing technology and computing algorithms have allowed for
the development of methods to rapidly screen and characterize polyclonal immune
responses to antigen. Collectively, these methods are referred to as high-throughput Ig
sequencing (Ig-Seq) technologies, and they take advantage of the unique manner in
which Ig genes recombine to specifically amplify heavy and light chain variable
sequences isolated from class-switched B cells (or TCR from memory T cells). Genomic
DNA isolated from a population of B cells is amplified using primers complementary to
rearranged VDJ. Alternatively cDNA can be amplified using a primer pool
complementary to leader peptides or framework regions of V gene segment combined
and specific CH-specific primers for heavy or light chains (5 RACE amplification can
also be used). The samples are then sequenced, and various bioinformatics approaches
are used to generate the output. Results from these analyses can be applied to many
experimental and clinical questions to understand the generation of the antibody response
and its role in human health and disease.
MICROARRAYS TO ASSESS GENE EXPRESSION
Levels of expression of thousands of genes can be measured simultaneously using a
technology called gene chips or microarrays. Briefly, thousands of short cDNA
representing genes from all parts of the genome are attached to a slide. Samples of
mRNA from cells in culture are used and reverse transcribed into cDNA and by labeling
this cDNA from different sources (i.e. normal cells and tumor cells) with different
fluorochromes, the differential expression of distinct sets of genes can be measured. By
scanning with a laser, different spots can have different colors depending on the success
of binding by the two different cDNAs.
This methodology has great potential in fields such as clinical diagnosis of lymphoid
tumors.
155
SUMMARY
1. In addition to functioning in vivo, antibodies are used in numerous diagnostic
formats in the clinical laboratory.
2. The primary binding reaction of antibody with antigen follows the rules of the
Law of Mass Action and an Association Constant (antibody affinity) can be
accurately measured while functional avidity is defined as the affinity
enhancement due to multivalency.
3. Secondary Ag-Ab reactions include the agglutination assay used in blood
banking. A prozone in agglutination assays is due to a huge excess of
antibody molecules. Zeta potential is an electrical repulsion of like-charged
particles. Coombs tests utilize anti-Ig reagents.
4. Precipitation reactions between antibodies and soluble antigens occur
regularly in vivo. The degree of precipitation depends on valency of antigen,
ratio of antibody to antigen and the classes/subclasses of antibodies that
predominate. Usually IgG is the only effective antibody class mediating
precipitation.
5. Several precipitation reactions in gel media are widely used for different
purposes. The Ouchterlony double diffusion assay is a qualitative assay for
measuring antigen presence and comparing antigens. Immunoelectrophoresis
is a qualitative method for measuring the numbers of components in mixtures
and Radial immunodiffusion is a quantitative method.
6. Nephelometry is a widely used method for measuring Ig concentrations.
7. Radioimmunoassays and ELISA (Enzyme Linked ImmunoSorbent Assay)
are the two most widely used immunoassays used in US clinical laboratories
although radioimmunoassays are slowly being phased out in favor of ELISA
156
157
I. INTRODUCTION
The immune system cannot be understood in isolation from infectious diseases. All living
organisms exist in a hostile environment and are continually used as hosts and energy sources by other
organisms. Since there are many different foreign invaders which can infect humans, we must have
many different ways to defend ourselves. To provide the versatility required, the two major effector
arms of specific immunity: antibody (humoral) and cellular, employ an incredible variety of
accessory mechanisms. These lectures will introduce you to some of these mechanisms.
In defense against infections, antibody is generally operative against extracellular bacteria or
bacterial products, whereas cell mediated immunity (CMI) primarily operates against intracellular
viral and bacterial infections, as well as fungal infections. The killing effects of immune reactions are
extremely efficient and, when specifically directed to a given infection, are able to eliminate large
number of organisms in a short period of time.
The immune response is a double-edged sword. In most cases, the immune system is
protective, providing life-saving defenses against infectious diseases and tumors. However, it can
also be destructive, causing immunopathology, defined as tissue damage resulting from the immune
response. These destructive responses result in some of the adverse effects of infections, in allergies
or hypersensitivity reactions (antibody- or T cell-mediated reactions to environmental or
administered antigens), and in distinct autoimmune disorders (antibody- or T-cell mediated reactions
to self-antigens). The seven immune mechanisms listed below are active in both immunoprotective
and immunopathologic reactions.
II. SEVEN IMMUNE MECHANISMS
Until the 1960s, immune reactions where not classified according to mechanism, but were
presented as a bewildering list of lesions with peculiar names. The first working classification of Type
I to Type IV immune mechanisms as introduced be Gell and Coombs was a major advance in
understanding immunopathologic reactions; seven mechanisms are presented in this handout. The
terms Type I - Type IV reactions, although out of date, are still used in some textbooks.
TABLE 1: Classification of Immune Mechanisms
Handout
Gell and
Coombs (1963)
General Properties
Antibody-Mediated
Inactivation or Activation
Cytotoxic or Cytolytic
Immune Complex
Atopic or Anaphylactic
-Type II
Type III
Type I
Cell-Mediated
T-cell Cytotoxic (TCTL)
Delayed Hypersensitivity (TDTH)
-Type IV
Either
Granulomatous Reactions
--
These immune mechanisms are similar in many ways to antibody- or cell-mediated reactions observed
in vitro. Primary reactions consist of the formation of Ag-Ab complexes or Ag-TCR reactions, secondary
reactions the effects of this interaction that can be demonstrated in vitro, and tertiary reactions the
corresponding in vivo manifestations (see figure).
Route of infection/exposure.
Genetic factors.
Page 159
B. Mechanisms
1. Binding of antibodies to a protein can stearically inhibit its binding to substrate, or
alter its conformation, resulting in loss of activity.
2. Antibody binding to viral receptor proteins can interfere with binding to cells, alter
viral structure, or mediate Ab- or C-mediated opsonization and clearance
3. In some cases, antibodies against hormone or neurotransmitter receptors can either
block or activate the receptor.
Page 160
Clinical Vignette Inactivation Reactions (Geha and Notarangelo, Case Studies in Immunology, 6th edition)
Case 42
2. CYTOTOXIC REACTIONS
A. Definition - reaction of antibodies with
cell surface antigens may result in
destruction of cells by opsonization,
complement activation, or AntibodyDependent Cellular Cytotoxicity (ADCC).
Also called Type II hypersensitivity.
B. Mechanisms
1. Complement activation may lyse
bacteria directly through formation of
the membrane attack complex (MAC).
A single IgM molecule or 2 or more
IgG molecules complexed to surface
antigens are sufficient to activate the
classical pathway.
2. Phagocytosis of infectious agents by
macrophages or neutrophils can be enhanced through antibody binding (interaction with
Fc receptors) or fixation of C3b
(interaction with complement receptors).
3. ADCC results from IgG-mediated binding
of null lymphocytes (and in some cases
macrophages) to target cells via Fc
receptors, and direct killing of the target
cell through cytolytic mechanisms (see
below).
4. In parasitic infections, IgE-mediated
binding of eosinophils to helminths results
in eosinophil degranulation and damage to
the worm tegument (surface).
C. Medical Aspects (Examples)
1. Protective
a. Many bacteria (particularly Gram
positive bacteria) are susceptible to Cmediated killing and/or opsonization.
This is particularly true of pyogenic
bacteria (such as Staph and Strep) that
result in massive accumulations of
neutrophils (see Immune Complex
reactions below).
b. Ab and C-mediated MAC formation
and opsonization are active against
some protozoal infections, including
Plasmodium and Trypanosoma.
Page 161
c. ADCC may be active against virally-infected cells, tumor cells, protozoa, and
helminths.
2. Immunopathologic
a. Transfusion reactions - ABO mismatches result in rapid lysis of transfused cells
due to anti-A or anti-B isohemagglutinins, naturally occurring IgM antibodies that
bind to the transfused erythrocytes and activate complement.
b. Rh reactions - birth of an Rh+ infant to a previously sensitized Rh- mother may
result in binding of maternal anti-Rh antibodies to the infants erythrocytes, causing
opsonization and phagocytosis hemolytic disease of the newborn.
c. Hemolytic anemia - autoantibodies can cause erythrocyte lysis, anemia.
d. Goodpastures syndrome - autoantibodies to basement membrane components and
complement are bound in an even, ribbon-like pattern to glomeruli and other
tissues. (Contrast with lumpy-bumpy appearance of immune complex disease; see
below).
Clinical Vignettes Cytotoxic Reactions (Geha and Notarangelo, Case Studies in Immunology, 6th ed., 2012)
Case 46
Case 41
Hemolytic Disease of Newborn maternal anti-Rh antibodies cause hemolysis in Rh+ newborn
(Cynthia Waymarsh)
Autoimmune Hemolytic Anemia patient Gwendolyn Fairfax develops hemolytic autoantibodies
following a mycoplasma infection
1. Large immune complexes are typically phagocytosed and destroyed by phagocytic cells
(such as resident macrophages of the reticuloendothelial system). Smaller complexes can
become lodged in the walls of venules, in joints, and in glomeruli. Deposition of immune
complexes causes complement activation,
attraction of neutrophils, and release of
lysosomal contents (frustrated phagocytosis),
resulting in vasculitis, reactive arthritis, and
glomerulonephritis.
2. The uneven distribution of immune complexes,
complement components, and lysosomal
contents results in the formation of lumpybumpy membrane deposits detectable by
binding the anti-Ig or anti-C3 antibodies.
3. Injection of an antigen in a previously
immunized individual can result in an Arthus
reaction due to deposition of Ag-Ab
complexes, complement activation, and
resulting erythema, edema, and attraction of
neutrophils. An Arthus reaction typically takes
2 to 6 hours to develop.
Page 163
C.
Clinical Vignettes Immune Complex Reactions (Geha and Notarangelo, Case Studies in Immunology, 6th
ed. 2012)
Case 37
Case 52
Systemic Lupus Erythematosus Nicole Chawner, age 16, butterfly rash after sun exposure. Immune
complexes due to antibodies against DNA and other nuclear components cause tissue damage
Drug-Induced Serum Sickness Gregory Barnes, antibodies against penicillin cause vasculitis,
hemorrhage
Page 164
B. Mechanisms
1. Requires the production of antigen-specific IgE, also called reagin or reaginic
antibody. Isotype switching to IgE during formation of memory cells requires Th2
expression of IL-4. IL-6 further enhances the production of IgE. An individual
having significant levels of IgE against a certain antigen is said to be sensitized.
Individuals vary greatly in levels of IgE production; those expressing high levels are
called atopic patients.
2. Very little IgE is found in the circulation. Rather, most is bound to the surface of
mast cells present in tissue around blood vessels, or basophils found in the
circulation or tissue. IgE binds specifically to the FcR1 receptor, and can persist
for weeks to months on the surface of mast cells.
3. Crosslinking of antigen-specific bound IgE by antigen causes a decrease in cyclic
AMP levels and mast cell activation, resulting in rapid degranulation and de novo
synthesis of arachidonic acid, which is subsequently converted to leukotrienes,
prostaglandins, and thromboxanes.
4. Within seconds to minutes, the preformed contents of mast cell granules act locally
to produce a typical wheal and flare reaction (in cutaneous exposures) or hayfever
symptoms (in
CROSS LINKING
respiratory tract
IgE
exposures).
ALLERGEN
Histamine
+
bind
MAST
s to
CELL
tissu
LEUKOTRIENES
PROSTAGLANDINS
DEGRANULATION
e
HISTAMINE RECEPTORS
hista AND ALLERGIC REACTIONS
INFLAMMATORY EFFECTS
min H2 RECEPTORS - DILATION
SMOOTH MUSCLE DILATION
(INCREASED BLOOD FLOW)
VASCULAR = SHOCK
e
NEUTROPHIL
rece H1 RECEPTORS - CONSTRICTION
EOSINOPHIL
INFILTRATE
LUNG = ASTHMA
ptor
ENDOTHELIAL CONTRACTION
GI = DIARRHEA
(INCREASED VASCULAR
s H1
GU = URINATION
PERMEABILITY)
(ind
VASCULAR ENDO = EDEMA
uces
smooth muscle contraction, endothelial cell separation and leakiness
vascular permeability) and H2 (mucus secretion, vasodilation).
Eosinophil chemotactic factor (ECF-A) - attracts eosinophils (present in latephase or chronic anaphylactic reactions)
Neutrophil-chemotactic factors (NCF) - attract neutrophils (late-phase)
Heparin - anti-coagulant, not directly involved in anaphylaxis
Wheal and flare - local erythema (due to vasodilation), edema (due to increased
vascular permeability
Hayfever - increased mucus secretion, mucosal swelling
Prausnitz-Kustner reaction - passive cutaneous anaphylaxis, caused by
experimental injection of IgE and antigen into skin.
5. In severe cases, systemic effects can cause shock (vascular collapse, loss of blood
pressure) and/or airway obstruction (laryngeal edema, bronchoconstriction and
mucus production resulting in suffocation).
6. Leukotrienes (formerly known as Slow-Reactive Substance A) cause long-term
smooth muscle contraction which is not alleviated by antihistamines. Cause some
Page 165
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SPOROZOITES
ENDOGENOUS
PROCESSING
CLASS I MHC
IL-1
INDUCTION
IFN-g
T-CTL
T-CTL
T-CTL
TL
T -C
CIRCUMSPORATE
ANTIGEN
EXPRESSION
TL
T -C
cytotoxicity and DTH, CTL almost certainly play a role in some autoimmune
diseases. An example is insulin-dependent diabetes mellitus, in which the cells in
the islets of Langerhans are destroyed by autoreactive immune responses. Cytolytic
T cells specific for cells can be found at the scene in IDDM experimental models.
Also, CTL are thought to be
A. TISSUE CULTURE MONOLAYER
responsible for thyroid cell
DYING CELLS
killing in Hashimotos thyroiditis
(see figure). Reactive
SPECIFIC T-CTL
lymphocytes also surround target
TISSUE CULTURE
cells and separate them from
TARGET CELLS
neighboring cells and basement
membranes, similar to what is
B. AUTOIMMUNE THYROIDITIS
THYROID
seen in cell cultures. This
DYING FOLLICULAR
FOLLICULAR
CELLS
disorientation also favors target
CELLS
cell death.
b) Contact dermatitis. Again,
T-CTL TO THYROID
FOLLICULAR CELLS
both CTL and TDTH are involved
in contact dermatitis (described
BASEMEMT MEMBRANE OF THYROID GLAND
in more detail below).
c) Viral exanthems. The eruptive lesions and fever characteristic of many viral
infections are partially due to the host immune response. Tissue damage due to
cytotoxic T cell responses may cause permanent loss of function.
d) Graft rejection. Cytotoxic T cell (and DTH) responses are involved in acute graft
rejection in transplant patients.
Page 171
Clinical Vignette T-Cell Cytotoxicity (Geha and Notarangelo, Case Studies in Immunology, 6th Ed., 2012)
Case 45
51
lysis, release
of 51Cr
No lysis, little
release of 51Cr
MHC
475 cpm
Page 172
IFN
IT
!
FO
R
IL-2
GO
DTH
ETC.
CTL
HELP!
ACTIVATION
5.
6.
7.
8.
B. Medical aspects
1. Protective
Page 173
Page 174
*
*
2-5 days
*
*
ConA Added
* Thymidine Incorporated
Add mitogen
or antigen
Donor lymphocytes
added
Nothing added
Page 175
GRANULOMATOUS REACTIONS
INSOLUBLE ANTIGEN
C1->C3b
OPSONIZATION
MACROPHAGE
IgG ANTIBODY
+
LYMPHOKINES
T-DTH
ACTIVATED
MACROPHAGES
SENSITIZED
CELLS
CLINICAL CONDITIONS
TUBERCULOSIS
LEPROSY
PARASITIC INFECTIONS
SARCOIDOSIS
GRANULOMATOSES
C. Medical aspects
1. Mycobacterial infections - as described above, granulomas are important in
tuberculosis and leprosy. They can be detected in chest Xrays and are indicative
of past or present active TB.
2. Parasitic infections - attempts to destroy or wall off parasites (such as worms)
can result in granulomas. In extreme cases (e.g. Roundworm Wuchereria
bancrofti), these can occlude lymphatic vessels and cause elephantiasis.
3. Sarcoidosis - disease of unknown etiology that causes granulomas in multiple
sites, including the lungs and skin.
4. Crohns disease - inflammatory disease of the bowel, in which granulomatous
reactions can cause stricture (obstruction) and fistula formation. Etiology
unknown.
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Page 177
Lentiviruses
Virus
Human immunodeficiency virus
Simian immunodeficiency virus
Visna/maedi virus
Equine infectious anemia virus
Caprine arthritis/encephalitis virus
Disease
Cause of human AIDS
AIDS in monkeys
Neurologic and lung
disease in sheep
Horse anemia
Goat encephalitis
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179
180
181
Viral Load at Different Stages of HIV Infection. Viral load is the total number of
virions per ml, as determined by a quantitative competitive polymerase chain reaction
(QC-PCR) technique. It is estimated that the number of infectious virions is about
60,000-fold less than the total number of virions determined by QC-PCR and other
means. The noninfectious virions may have defective RNA or be missing other required
components.
Clinical Stage
Primary (acute) infection
Asymptomatic
Early symptomatic
AIDS
182
183
184
185
186
187
188
D. Animal models
Animal models of HIV infection provide a means of studying HIV pathogenesis
and immunity. However, none of these closely resemble the human disease. One of the
most effective models is the SCID mouse- human lymphoid cell chimera. In this case, a
mouse strain with severe combined immunodeficiency can be injected with human
lymphoid cells. The human cells persist, and can be infected with HIV upon subsequent
injection of the virus. This system permits the study of human cell infection in a
surrogate model.
Animal
Chimpanzee
Macaques
Virus
HIV-1
SIV
Rabbits
Mice- HIV genome
transgenic
Mice - tat transgenic
SCID-human lymphoid
cell chimera
Sheep
Goats
Horses
HIV-1
--
Result
Latent Infection
AIDS-like wasting
disease
Defective infection
AIDS-like illness
Used to study:
Vaccine efficacy
Vaccine efficacy,
therapy, pathogenesis
Latent infection
Pathogenesis
-HIV-1
Kaposis sarcoma
AIDS-like disease
Carcinogenesis
Pathogenesis, therapy
Visna
Chronic
neurodegeneration
arthritis, encephalitis
hemolytic anemia
Lentivirus pathogenesis
Caprine
EIAV
189
Lentivirus pathogenesis
Lentivirus pathogenesis
III. SUMMARY
1. HIV-1 and HIV-2 are lentiviruses that cause CD4+ T lymphocyte depletion and
immunodeficiency in humans.
2. Infection of CD4+ T cells, macrophages, and other cell types can lead to virus
production and cytolysis or long-term latent infection.
3. HIV infection progresses through primary infection, asymptomatic infection,
early symptomatic infection, and late symptomatic infection (AIDS).
4. Depletion of CD4+ cells leads to profound defects in DTH and T helper activity,
resulting in susceptibility to rampant HIV infection, opportunistic infections,
and tumors.
5. Antibody and cytotoxic lymphocyte activities combat virus production, but
eventually are overwhelmed by the virus ability to inactivate and destroy CD4+
cells, to change its antigenic structure, and to alternate between latent and active
infection.
6. In their current forms, immunotherapy and vaccination have not demonstrated
the ability to prevent or combat HIV infection. However, intensive research for
the development of an HIV vaccine is ongoing.
190
The course of response against typical acute infections can be subdivided into distinct stages.
Initially, the level of infectious agent is low, beginning with breach of a mechanical barrier (e.g.
skin, mucosal surface). Once inside the host, the pathogen encounters a microenvironment for
suitable replication. The agent replicates, releasing antigens that trigger innate immune function,
generally characterized as non-specific. This innate function assists in limiting expansion of the
organism. After 4 or 5 days, effector cells and molecules of the adaptive and specific immune
response enable control and eventual clearance of the infectious agent. Once the agent is cleared,
the host is left with residual effector cells and antibodies, as well as immunological memory to
provide lasting protection against reinfection.
The response to initial infection is therefore subdivided into 3 phases. The first is an early innate
and non-specific response. Preformed effector cells and molecules recognize microorganisms
within the first 4 hours of infection. Although this may be enough to clear the organism, typically
more help is necessary. The second phase lasts from 4 hours to 4 days. Again, this is primarily a
non-specific encounter with the organism. This phase is characterized by recruitment of effector
cells (professional phagocytes, NK cells) to the site of infection, and specific activation of these
effectors. The final phase is one where one where adaptive immunity occurs. Antigen specific
cells (B and T lymphocytes) undergo clonal expansion to become specific effectors. Some of these
effector cells remain even after clearance of the organism, and are able to provide a much more
rapid and specific memory response if the organism is re-encountered.
A wide variety of pathogenic microorganisms exist. They may be globally classified into groups:
Bacterial, Mycobacterial, Viral, Protozoal, Worms, and Fungal. The major immune defense
mechanisms are summarized in the following chart:
Type of Infection
Bacterial
Mycobacterial
Viral
Protozoal
Worms
Fungal
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The host defense is based upon availability of resources to combat a localized pathogen. Virtually
all pathogens have an extracellular phase where they are vulnerable to antibody-mediated effector
mechanisms. An extracellular agent may reside on epithelial cell surfaces, where antibodies (IgA)
and non-specific inflammatory cells may be sufficient for combating infection. If the agent resides
within interstitial spaces, in blood or in lymph, then protection may also include complement
components, macrophage phagocytosis and neutralization responses. Intracellular agents require a
different response to be effective. For cytoplasmic agents, T lymphocytes and NK cells, as well as
T-cell dependent macrophage activation, are usually necessary to kill the organism.
191
Pathogens can damage host tissue by direct and indirect mechanisms. Organisms may directly
damage tissue by release of exotoxins that act on the surface of host cells, or via released
endotoxins that trigger local production of damaging cytokines. Pathogens may also directly
destroy the cells they infect. Adaptive mechanisms can cause disease; formation of
antibody:antigen complexes can lead to the release of proteins and factors that both mediate
control of disease as well as cause tissue damage. Some of the representative infectious agents and
the common names of associated diseases are listed in the figure.
192
BACTERIAL INFECTIONS
Bacterial infections begin with a breach
of a mechanical barrier. Release of
bacterial factors upon replication
initiates a cascade of events. Initially,
infection may be resisted by
antibody-mediated immune
mechanisms, including neutralization of
bacterial toxins. With the help of
complement factors direct cytotoxic
lysis can occur. Release of C3a and C5a
in the complement cascade will cause
vasodilation and vasopermeability
resulting in an influx of professional
phagocytes and acute
polymorphonuclear infiltration (Arthus
reaction). Opsonization of bacteria leads
to increased phagocytosis and acute
anaphylactic vascular events permitting
exudation of inflammatory cells and fluids. Phagocytosis may also occur via specific surface
receptors for ligands such as mannose or sialic acids. During the chronic stage of the infection cell
mediated immunity is activated. TDTH-cells that react with bacterial antigens may infiltrate the site
of infection, become activated and release lymphokines that attract and activate macrophages. The
activated macrophages phagocytose and degrade necrotic bacteria and tissue, preparing the lesion
for healing.
The role of complement in response to bacterial infection must be stressed. There are three major
biological components of the complement system. They are (1) activation of phagocytes including
macrophages and neutrophils, (2) direct cytolysis of target cells, and (3) opsonization of
microorganisms and immune complexes for cells expressing complement receptors. The
polymorphonuclear cells, especially neutrophils, are an excellent example of the first line of innate
defense. Puss is composed of dead and dying polymorphonuclear and host cells, local fluids and
exudates, and dead and dying bacteria.
Important factors released by macrophages in response to bacterial antigens include cytokines that
exert both local and systemic. Locally, IL-1, TNF-, and IL-8 cause inflammation and activate
vascular endothelial cells to increase permeability and allow more immune cells to enter infected
area. TNF- will also destroy local tissue to limit growth of bacteria. In addition, IL-6 can
stimulate an increase in B cell maturation and antibody production, and IL-12 will lead to
activation of NK cells and priming of T cells towards a T Helper 1 (TH1) response. Systemically,
IL-1, IL-1, TNF-, IL-6 and IL-8 all contribute to elevated body temperature (fever) and
production of acute-phase protein production.
193
MYCOBACTERIAL INFECTIONS
Mycobacterial infections such as
tuberculosis and leprosy are extremely
complex. The Mycobacteria have evolved to
inhibit normal macrophage killing
mechanisms (e.g. phagosome-lysosome
fusion) and survive within the disarmed
professional phagocyte. These organisms
are resisted mainly by TDTH initiated cellular
mechanisms, including granulomatous
hypersensitive responses, but only after the
infection have become established. T cells
are mainly responsible for control and
containment of the infection. They
recognize mycobacterial antigens expressed
on the surface of infected cells and release
factors that recruit additional immune
effectors. A local environment is established
to contain infection. Healing of the infected
center may occur, with limited necrosis of
the infected tissue. However, if the infection
194
persists, an active caseous granuloma results. Here, an infected nidus is comprised of infected and
active macrophages, ringed by T cells. A host mediated destructive response occurs inside a
contained area. The infection itself is surrounded by activate epithelioid cells, with the presence of
Giant cells (activated syncytial multinucleated cells,).
At one time it was thought that the tissue lesions of the disease tuberculosis required the effect of
delayed hypersensitivity. The term hypersensitivity was coined because animals with cellular
immune reactivity to tubercle bacilli developed greater tissue lesions after re-inoculation of bacilli
than did animals injected for the first time. The granulomatous lesions seen in tuberculosis do
depend upon immune mechanisms for their formation. However these lesions are not really the
cause of the disease but an unfortunate effect of the protective mechanisms; the granulomatous
inflammatory reaction to the infective mycobacterium results in destruction of normal tissue. In
the lung, for instance, extensive damage done by the formation of large granulomas in response to
a tuberculosis infection can result in respiratory failure. The granulomatous immune response
produces the lesion, but the mycobacterium causes the disease.
VIRAL INFECTIONS
Immune resistance to viral
infections is mainly mediated by
cell-mediated-immunity, but
humoral (antibody) responses also
play a role by preventing virus from
attaching to cell receptors. The
antiviral response id dictated by
availability to be seen by the
immune system. Viruses live within
the host's cells and can spread from
cell to cell. To be effective in
attacking intracellular organisms, an
immune mechanism must have the
capacity to react with cells in solid
tissue. This is a property of
cell-mediated reactions, in
particular TCTL, but not of antibody
mediated reactions. Antibodies do
play a role during the extracellular
life cycle of the virus. Antibodies
can bind to virus forming
complexes to inactivate virions, and
allow them to be cleared effectively
by profession phagocytes. Humoral antibody can prevent the entry of virus particles into cells by
interfering with the ability of the virus to attach to a host cell, and secretory IgA can prevent the
establishment of viral infections of mucous membranes. However, once the virus is within cells, it
195
196
Antigen reacting with IgE antibody bound to intestinal mast cells stimulates release of
inflammatory mediators, such as histamine, proteases, leukotrienes, prostaglandins and serotonin.
These agents cause an increase in the vascular permeability of the mucosa, exposing worms to
serum immune components, stimulate increased mucous production and increase peristalsis. These
activities are associated with expulsion of parasitic worms from the gastrointestinal tract through
formation of a physical barrier to adherence and interactions with the mucosal surface.
Eosinophils contain granules containing basic proteins which are toxic to worms. Eosinophils may
be directed to attack helminths by cytophilic antibodies that attach to the eosinophil through the Fc
region and to the helminth by specific Fab binding (ADCC). Anaphylactic antibodies (IgE) are
also frequently associated with helminth infections, and intradermal injection of worm extracts
elicits and wheal-and-flare reaction. Children infested with Ascaris lumbricoides have attacks of
urticaria, asthma, and other anaphylactic or atopic types of reactions presumably associated with
dissemination of Ascaris antigens.
FUNGAL INFECTIONS
A great deal is not known concerning immune response to fungal agents. Cellular immunity
appears to be the most important immunologic factor in resistance to fungal infections, although
humoral antibody certainly may play a role. The importance of cellular reactions is indicated by
the intense mononuclear infiltrate and granulomatous reactions that occur in tissues infected with
fungi and by the fact that fungal infections are frequently associated with depressed immune
reactivity of the delayed type (opportunistic infections). Chronic mucocutaneous candidiasis refers
to persistent or recurrent infection by Candida albicans of mucous membranes, nails, and skin.
Patients with this disease generally have a form of immune deviation, i.e., a depression of cellular
immune reactions, with high levels of humoral antibody; similar to lepromatous leprosy. Fungi
appear to be resistant to the effects of antibody, so that CMI is needed for effective resistance.
ANTIBODY PRODUCTION
DELAYED HYPERSENSITIVITY
197
been implicated in the tendency for certain individuals to develop IgE (allergy) antibodies rather
than IgG antibodies. In addition, for reasons that are unclear, but may be genetically determined,
some individuals tend to make strong cellular immune responses, but weak antibody response to
certain antigens, whereas other individuals will have the opposite response. The course of leprosy
depends upon the immune reaction of the patient. Leprosy is classified into three major
overlapping groups: tuberculoid, borderline and lepromatous. In tuberculoid leprosy there are
prominent well-formed granulomatous lesions, many lymphocytes and few if any organisms.
Delayed hypersensitivity skin tests are intact and there is predominant hyperplasia of the diffuse
cortex (T-cell zone) of the lymph nodes. The level of antibodies is low. In lepromatous leprosy
granulomas are not formed, there are few or no lymphocytes and lesions consist of large
macrophages filled with viable organisms. Delayed hypersensitivity skin tests are depressed and
there is marked follicular hyperplasia in the lymph nodes with little or no diffuse cortex. The
levels of antibodies are high and vascular lesions due to immune complexes are seen (erythema
nodosum leprosum). Borderline leprosy has intermediate findings. The prognosis in tuberculoid
leprosy is good and the response to chemotherapy is excellent. In borderline leprosy a good
response to therapy is associated with a conversion to the tuberculoid form. The prognosis in
lepromatous leprosy and the response to chemotherapy is poor. The example of the forms of
leprosy illustrates the role of cellular immunity (delayed hypersensitivity) in controlling the
infection, and the lack of protective response provided by humoral antibodies. This concept is also
considered valid for immunity to Candida albicans (chronic mucocutaneous candidiasis).
Depressed cellular immunity is associated with chronic mucocutaneous candidiasis, a condition in
which the infected individual is unable to clear Candida infections.
A diagram illustrating the relationship of the degree of cellular and humoral immune response to
the stages of leprosy is shown. The overlapping triangles indicate the relative strength of delayed
hypersensitivity and antibody production. The cross-hatched triangle indicates delayed
hypersensitivity; the open triangle, antibody production. High levels of delayed hypersensitivity
(DTH) are associated with cure of tuberculoid leprosy; weak DTH is associated with progressive
disease; balanced DTH and antibody production with borderline leprosy and slowly progressive
disease. The cytokine patterns in the two polar forms of the disease are different. Typically T
Helper 2 (TH2) cytokines (IL-4, IL-5 and IL-10) dominate in the lepromatous form, while
cytokines produced by TH1 cells (IFN-, TNF and IL-2) predominate in tuberculoid leprosy. IFNg would be expected to activate macrophages to kill intracellular pathogens and control organism
expansion; high IL-4 may explain hypergammaglobulinemia in lepromatous patients.
198
host and its infectious organisms results in an eventual mutual co-existence with most
environmental organisms. No better evidence of this is the loss of this coexistence when the
immune mechanisms do not function properly. Then organisms which do not normally cause
disease become virulent. The lesson of AIDS demonstrates that new infectious organisms will
become dominant when introduced into a previously unexposed population. In a fully evolved,
mature relationship host and infectious agent initially co-exist without detrimental affects. Thus
the ultimate evolution of the host parasite relationship is not "cure" of an infection by complete
elimination of the parasite, but least mutual co-existence without deleterious effects of the parasite
on the host. In fact, in many human infections, the infectious agent is never fully destroyed and the
disease enters a latent state that can be reactivated under different conditions.
Bacteria have evolved to evade different aspects of the phagocyte-mediated killing. For example,
they may (1) secrete toxins to inhibit chemotaxis, (2) contain outer capsules that block attachment,
(3) block intracellular fusion with lysosomal compartments, and (4) escape from the phagosome to
multiply in the cytoplasm. Viral entities also subvert immune responses usually through the
presence of virally encoded proteins. Some of these proteins block effector functions of antibody
binding, block complement mediated pathways, and inhibit activation of infected cells. The
Herpes virus produces a factor that inhibit inflammatory responses by blocking effects of
cytokines through receptor mimicking, and another that blocks proper antigen presentation and
processing. Finally, Epstein-Barr virus encodes a cytokine homolog of IL-10 which leads to
immuno-suppression of the host by activating TH2 rather than TH1 responses.
199
200
Clinical Vignette - The case of Ursula Iguaran (Case 48 in Geha and Notarangelo): Ursula Iguaran, a
native of Columbia, developed hypopigmented lesions on her hands and arms when she was 16, with
progressive lesions developing through the next two years. Blood tests revealed normal white blood
counts. Dermatological evaluation revealed numerous Virchow's cells (foamy macrophages) and few
lymphocytes within the lesions. Histological analysis of a forearm biopsy revealed clumps of acid-fact
bacilli. She was diagnosed as having Mycobacterium leprae.
Ursula was aggressively treated with a multiple drug regime (dapsone, clofazamine and rifampin). Her
skin lesions gradually flattened and improved.
The immune response in patients with Lepromatous Leprosy is skewed towards the production of T
helper 2 cytokines. On this basis, would Ursula be more susceptible to certain types of infections? Which
ones and why?
201
202
203
Summary
204
Many factors influence the nature, intensity and duration of an immune response
including age, neuroendocrine hormone levels, HLA allotypes, antigen dose,
antigen access, and cytokine milieu.
205
Objectives
1. Define and discuss autoimmunity.
2. Use autoimmune diseases to illustrate mechanisms of autoimmunity.
3. Provide you with clinical correlations and applications of the basic principles of
immunology.
Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc,
New York, NY. 6th edition, 2009. Chapter 12 (p190-204).
R. S. Geha and Notarangelo, L. Case Studies in Immunology: A Clinical Companion. (6th
Ed) Garland Publishing, New York, 2012. Chapter 36. Rheumatoid Arthritis; Chapter 40.
Multiple Sclerosis; Chapter 41. Autoimmune Hemolytic Anemia.
Web Resource: https://med.uth.edu/pathology/courses/immunology/links-forlectures/autoimmunity/
INTRODUCTION:
The regulation of immune function and overall immuno-homeostasis is under control of
multiple factors that include genetic and environmental components. HLA allotypes,
antigen dose, and existing cytokine milieu can all influence responses to both pathogenic
agents and self antigens.
206
Hyperthyroidism, opthalmopathy
Celiac Disease
207
HLA-DR4
HLA-DR3
HLA-DR3, DR2
HLA-DR3
HLA-DR3
HLA-DR3
HLA-DR2
208
Autoantibodies
Antibodies against to self-antigens
Can be found in normal, healthy individuals
Important effectors in autoimmune disease
Autoimmune Hemolytic Anemia
Autoantibodies against RBC antigens
Warm autoantibodies
IgG, react with Rh antigen on RBC at 37degC
Result in opsonization of RBCs and macrophage phagocytosis
Cold autoantibodies (cold agglutinins)
IgM, react with I or i antigen on RBC when <37degC
Activate complement and result in complement mediated lysis
Drug induced antibodies
Penicillin acts as a hapten, binds to RBC and form antibodies
against RBCs
Myasthenia Gravis
Target antigen is alpha chain of the nicotinic acetylcholine receptor in the
neuromuscular junction
Autoantibodies act as antagonist
Symptoms of muscle weakness, diplopia, dysarthria, dysphagia
May be associated with a thymoma
Can be transmitted to fetus through placental transmission of autoantibodies
Graves Disease
Symptoms of hyperthyroidism
Heat intolerance, Increased metabolism, weight loss
Palpitations, increased HR, Hair loss, Fatigue
Nervousness, Opthalmopathy
Autoantibodies against thyrotropin stimulating hormone receptor (TSHreceptor)
Autoantibodies act as an agonist
Symptoms of hyperthyroidism
Maternal antibodies can be transmitted to fetus through the placenta resulting
transient neonatal hyperthyroidism
Systemic Lupus Erythematosus
Autoimmune disease characterized by
systemic autoimmunity
multi-organ involvement
production of autoantibodies against nuclear components
immune complexes
Autoantibodies and immune complexes deposit in tissues including skin, joints,
blood vessels, kidneys, etc.
209
Rheumatoid Arthritis
Antibodies to citrullinated peptides (anti-CCP antibodies)
Antibodies to Fc portion of IgG (rheumatoid factor)
210
Targeted Therapeutics
As our understanding of the pathogenesis increases, targeted therapeutic
approaches are becoming available
TNF-alpha inhibitors for the treatment of rheumatoid arthritis,
ankylosing spondylitis, psoriasis, inflammatory bowel disaese
CTLA-4 Ig for the treatment of rheumatoid arthritis
antiCD20 antibody (targeting B-cell) for the treatment of rheumatoid
arthritis
Beta interferon for the treatment of multiple sclerosis
Anti-type I interferons for the treatment of systemic lupus
erythematosus (in development)
Many others in development
CONCLUSION:
. The mechanisms underlying all autoimmune diseases are not fully elucidated;
however, genetic polymorphisms of MHC class II genes (alleles of HLA-DR and/or HLADQ) are associated with increased susceptibility to autoimmune diseases. Possible
mechanisms for a loss of tolerance leading to autoimmune reactions include (1) a lack of
Fas-Fas ligandmediated deletion of autoreactive T cells in the thymus during
development, (2) loss of T-regulatory or T-cell suppressor function, (3) cross-reactivity
between exogenous and self-antigens (molecular mimicry), (4) excessive B-cell function
due to polyclonal activation by exogenous factors (of viral or bacterial origin), (5)
abnormal expression of MHC class II molecules by cells that normally do not express these
surface molecules, and (6) release of sequestered self-antigens from privileged sites, thus
priming for responses not previously seen by the immune system.
Autoimmune diseases can be classified as organ specific or systemic in nature . Three major
types of autoimmune reaction mechanisms are recognized as causing different autoimmune
disorders ... Two of these mechanisms involve autoantibodies directed against self-antigens; for
both, classical complement pathway activation exacerbates local damage and inflammatory
response. In the first case, autoantibodies may be directed against a specific self-component,
211
such as a surface molecule or receptor. Examples include antibodies against the acetylcholine
receptor producing myasthenia gravis, and antithyroid- stimulating hormone receptor antibodies
producing Graves disease. Autoantibodies may also bind with antigens present in the blood,
forming antigen-antibody (immune) complexes that later deposit in organs, thus inciting an
inflammatory response. An example is seen in lupus glomerulonephritis in which complexes of
anti-DNA antibodies and free DNA accumulate in the kidney. The third mechanism is that of
autoreactive T cells that recognize targeted self-antigens on organs, leading to direct damage to
tissue. In many cases, autoreactive T cells coexist with autoantibody responses, leading to
exacerbation of disease and organ damage. In the case of multiple sclerosis, T cells reactive to
myelin basic protein destroy the protective layer surrounding axons, thereby eliminating
effective transfer of signals through nerves." [adapted from Actor, J.K. Elseviers Integrated
Immunology and Microbiology, Mosby/Elsevier, Philadelphia, 2007.]
212
CLINICAL CORRELATIONS
Faculty Taught Class Correlations
Clinical Cases will be presented by faculty. Cases are taken from the Geha and Notarangelo. Case Studies in Immunology.
Garland Publishing, New York, NY. 6th edition, 2012.*
Clin. Corr.
Class
Date Time
2/12 11:00-11:50 AM
First Case
36. Rheumatoid Arthritis
Second Case
37. Systemic Lupus
Erythematosus
Clinical Correlation Cases will be presented by faculty. This is NOT an extra credit assignment, but rather a clinical
correlate that is part of the Immunology curriculum.
Review the study questions at the end of each chapter for the cases presented.
There is no formal requirement to complete the questions below. No extra credit points will be given for this Faculty
taught Clinical Correlate. Rather, it is recommended that you be prepared to answer the following during class discussion:
2.
(Hyperreactivity)
Health
Immunopathology
/\
How does this immune disorder directly or indirectly involve or impact each of the
following (answer all):
3.
Describe the underlying mechanism(s) (e.g. at the organ, cellular or molecular level) in these cases (brief paragraph).
4.
Give a short, succinct summary of the immunologic principle illustrated by these cases.
214
PRIMARY IMMUNODEFICIENCIES
WILLIAM T. SHEARER, M.D., PH.D.
Objectives
I.
II.
Definitions
The immunodeficiency disorders are a diverse group of illnesses that, as a
result of one or more abnormalities of the immune system, predispose a person
to infection. The abnormalities of the immune system can involve absence or
malfunction of blood cells (lymphocytes, granulocytes, monocytes) or soluble
molecules (antibodies, complement components) and can result from an
inherited genetic trait (primary) or from an unrelated illness or treatment
(secondary).
III.
B.
214
D.
E.
F.
[recommended reading] Shearer WT, Fischer A. Editorial. The last 80 years in primary
immunodeficiency: How far have we come, how far need we go? J Allergy Clin Immunol
2006;117:748-752.
[recommended reading] Notarangelo L, Casanova JL, Conley ME, Chapel H, Fischer A,
Puck J, Roifman C, Seger R, Geha RS; International Union of Immunological Societies
Primary Immunodeficiency Diseases Classification Committee. Primary immunodeficiency
diseases: an update from the International Union of Immunological Societies Primary
Immunodeficiency Diseases Classification Committee Meeting in Budapest, 2005. J
Allergy Clin Immunol 2006;117:883-896.
[recommended reading] Orange JS, Hossny EM, Weiler CR, Ballow M, Berger M,
Bonilla FA, Buckley R, Chinen J, El-Gamal Y, Mazer BD, Nelson RP Jr, Patel DD, Secord
E, Sorensen RU, Wasserman RL, Cunningham-Rundles C; Primary Immunodeficiency
Committee of the American Academy of Allergy, Asthma and Immunology. Use of
intravenous immunoglobulin in human disease: A review of evidence by members of the
Primary Immunodeficiency Committee of the American Academy of Allergy, Asthma and
Immunology. J Allergy Clin Immunol 2006;117:S525-S553.
[recommended reading] Puck JM, Malech HL. Gene therapy for immune disorders:
good news tempered by bad news. J Allergy Clin Immunol 2006;117:865-869.
IV.
General Considerations
There are greater than 120 types of primary immunodeficiency that have
been characterized and their incidence in the general population is 1:10,000
(aside from the extremely common selective IgA deficiency, 1:500). The most
serious forms of immunodeficiency, such as severe combined immunodeficiency
and X-linked agammaglobulinemia, occur in 1:100,000 individuals. At least 58%
of cases are diagnosed in children (less than 5 yr) and 83% of these patients are
male. Autosomal recessive, X-linked recessive, and familial inheritance patterns
are observed.
V.
Etiology
It is likely that genetic abnormalities underlie all primary states of
immunodeficiency. The simplest concept to remember is the scheme of the
developing immune system. T and B lymphocytes pass through unique
development stages as they mature and differentiate from the primordial stem
cells in the bone marrow. Probably due to an underlying genetic lesion causing
absent or altered enzymes, lymphocyte maturation may stop at a certain stage,
deemed an arrest point." Lymphocyte maturation arrest points may lead to
clinical immunodeficiency states; for example, in patients with X-linked
agammaglobulinemia (X-LA, B lymphocyte maturation is interrupted between the
pre-B and B cell stage. By virtue of their lack of mature B cells, patients with XLA have no circulation of antibody-producing plasma cells, and are very
susceptible to infection with multiple organisms. Patients with common variable
immunodeficiency possess B cells which are present but malfunctional, due to
intrinsic developmental block between B cells and plasma cells, failure to secrete
immunoglobulin, or lack of effective T helper cell function. Patients with X-linked
severe immunodeficiency lack maturation of stem cells into mature T cells.
215
VI.
Clinical Features
The principal manifestation of immunodeficiency is an increased susceptibility
to infection as documented by: a) increased frequency of infection, b) increased
severity of infection, c) prolonged duration of infection, d) development of an
unexpected complication or unusual manifestation, e) infection with organisms of low
pathogenicity. Certain types of cancer, e.g., Epstein-Barr virus-induced B-cell
lymphomas, appear in patients with certain immunodeficiencies. Autoimmune
conditions also appear in patients with immunodeficiencies.
VII.
Wiskott-Aldrich Syndrome
Cellular Disorders
A.
1.
Severe Combined Immunodeficiency (SCID)
There are many types of combined (B- and T-cell) immunodeficiency
which result in recurrent life-threatening infections, severe diarrhea, and failureto-thrive. The classical form of SCID is X-linked, but there are autosomal
recessive forms, as well as sporadic forms. The usual lymphocyte analysis
reveals an absence of lymphocytes bearing mature T-cell antigens (CD3, CD4,
CD8) and inability of lymphocytes to respond to mitogenic and antigenic
stimulation. Frequently, there are small numbers of B cells with mature antigens
(CD19, CD20), but most infants with SCID do not make serum immunoglobulins.
The genetic and molecular defect in X-linked SCID is a mutation in the gene that
codes for the gamma chain of the IL-2 receptor. Without a normal IL-2 receptor,
normal T-cell maturation and proliferation cannot take place. Consequently, all
normal T-cell functions are absent. The profound nature of the SCID defect is
due to the fact that the defective gamma chain renders not only the IL-2 receptor
dysfunctional but also the IL-4, IL-7, IL-15, and IL-21 receptor.
There are several other forms of SCID involving genetic lesions (all
inherited in autosomal fashion) in the: 1) T-cell receptor complex, CD3; 2) kinase
218
XP21.1). The white blood cell count (WBC) is high in an attempt to compensate
for lack of bacterial killing.
Laboratory tests used to diagnose CGD are the nitroblue tetrazolium dye test
(NBT), dihydro rhodamine test (DHR), and the chemiluminescence assay. All tests
measure H2O2 and subsequent superoxide production by the granulocyte. Gamma
interferon has been shown to augment the effectiveness of antimicrobial treatments in
CGD, but bone marrow transplantation offers the only permanent therapy.
B.
Toll-Like Receptors
Toll-like receptors (TLRs) are a series of 10 receptors in humans that
participate in detecting pathogens. TLRs recognize a small number of pathogenassociated molecular patterns (PAMPs). PAMPs represent molecules (LPS,
lipoproteins, flagellin, unmethylated DNA, double-stranded and single-stranded RNA)
that promote for the survival of a pathogen. Activation of TLRs leads to recruitment of
neutrophils and macrophages to sites of infection and augmentation of antimicrobial
activity. On engagement of TLRs, dendritic cells (DCs) undergo maturation and migrate
to draining lymph nodes, where they present antigen to T cells. PAMPs binding to all
known Toll-like receptors cause the production of inflammatory cytokines, including
TNF-. IRAK-4 (interleukin receptor-associated kinase-4) is a critical effector in
signaling by TLRs and the IL-1 receptor, which share homology in their intracellular
domain. Patients with IRAK-4 deficiency are susceptible to invasive bacterial infections
and to viral infections.
220
B.
Complement
The complement system is a complex of approximately 31 innate host defense
proteins that acts in three activation patterns (classical, alternate, and lectin-binding) to
augment adaptive host defense mechanisms and to exert a bacteriolytic effect of its
own. Genetic deficiencies or mutations of any one of these proteins leads to impaired
host defense.
The prototype complement component deficiency disease is that of C3
deficiency, a very rare disorder but one that allows us to assess the crucial importance
of the total complement pathway in host-defense mechanisms. Since C3 is the pivotal
complement component through which both classical and alternative pathways act, its
absence does not permit complete activation of complement. Because of the lack of C3
chemotactic factors, C3a and C5a are not released and phagocytic cells are not drawn
to the focus of infection. Moreover C3b serves as an opsonin of bacteria and by virtue
of a chemical affinity of C3b for a receptor on phagocytic cells (polymorphonuclear
leukocytes and monocytes) the engulfment of a C3b-coated bacterium by a phagocytic
cell is facilitated. Persons with complement component deficiencies of C1, C2, or C4
can still activate the complement cascade via the alternative pathway and persons with
complement component deficiencies of C5, C6, C7, C8, or C9 can still generate
chemotactic factors and have opsonin (C3b) function.
Pyogenic infection (streptococcal, staphylococcal) and autoimmune diseases are
associated with early complement component deficiencies (i.e., C1, C2, C4) and
meningococcal and gonococcal infections are associated with late complement
component deficiencies (i.e., C5-C9).
XI.
B.
C.
D.
Complement Deficiency
1.
General supportive care
221
XII.
Medical History
1.
Age of patient at onset of symptoms
2.
History of live microbial immunizations
3.
Severity of illness
4.
Family history
B.
Physical Examination
1.
Tonsils present
2.
Palpate lymph nodes
3.
Organomegaly
4.
Growth - measurements
C.
Laboratory Investigation
1.
Antibody function
a.
Immunoglobulin levels
b.
Specific antibody responses
- Isohemagglutinins (anti A, anti B)
- Anti-diphtheria and tetanus antigen antibodies
- Anti X 174 antibody
2.
B and T Lymphocyte, NK cell, and monocyte/macrophage function
a.
T cell
- Delayed hypersensitivity skin testing (e.g. SK-SD, monilia
antigens)
- T cell subsets (flow cytometry)
- Mononuclear cell phenotypes (subsets-monoclonal
antibodies)
- PHA reactivity and antigen reactivity
- Specific antigen stimulation in vitro
- T cell excision circles (TREC)
- V TCR spectratyping
b.
B cell (B cell subsets by flow cytometry)
- Memory (CD27+) B cells
c.
Nucleic acid enzyme assay
- Adenosine deaminase
- Nucleotide phosphorylase
d.
NK cell surface markers and functional assay
e.
Monocyte/macrophage receptor assays
3.
White blood cell function
a.
WBC
b.
NBT, DHR tests
c.
CD11a,b,c CD18 assay by flow cytometry
d.
Chemotaxis and opsonization assays
4.
Complement function
a.
Total hemolytic complement
b.
C3
c.
C4
222
5.
XIII.
223
OBJECTIVES:
1. To understand the application of the major immunological principles and
concepts to modification of immune response, immunoprophylaxis and
immunotherapy of human diseases.
2. To define the current approaches and future strategies to
immunoprophylaxis and immunotherapy of immune-mediated and nonimmune-mediated diseases
KEYWORDS:
Immunoprohylaxis,
vaccine,
immunization, immunotherapy
active
READING:
and
passive
WEB RESOURCE:
https://med.uth.edu/pathology/courses/immunology/links-for-lectures/immunoprophylaxis-vaccines/
I.
INTRODUCTION
II.
IMMUNIZATION BASICS
A. Types of Immunizations
1.
Active exposure to antigen with the host generating
protective immunity. Objective: provide long lasting immunity
against future exposures
2.
Passive administration of humoral and/or cellular factors
that provide immunity for the host. Objective: provide
temporary immediate protection against an imminent or
ongoing exposure
3.
Heard immunity in preventing spread of infection
225
How acquired
Infection
Artificial (deliberate)
Vaccination
Passive
Natural
Transfer of antibody
from mother to infant
in placental
circulation or though
breast-feeding
(colostrums)
Passive antibody
therapy (serum
therapy, immune
human globulin,
monoclonal
antibodies)
Artificial
III.
Active Immunization
1.
1a.
Age
Birth
12 months
2 months
4 months
6 months
1215 months
46 years
1112 years
2564 years
>65 years
Vaccine
Hepatitis B (Hep B)
Hep B
Diphtheria and tetanus toxoids and acellular
pertussis (DTP), Haemophilus influenzae
type b (Hib), inactivated polio (IPV)
DTP, Hib, IPV, rotavirus (Rv)
Hep B, DTP, Hib, IPV, Rv
Oral poliovirus vaccine (OPV), measles,
mumps, rubella (MMR), varicella vaccine for
susceptible children
DTP, OPV, MMR
Hep B, MMR, varicella
Measles, rubella
Influenza, pneumococcal disease
226
1b.
2.
2A.
227
2B.
Infants
a. generally do not respond well to polysaccharide antigens
at less than 2 yrs of age
b. antigenicity improves when conjugated to protein or
toxoid
2C.
2D.
228
2E.
Hazards
a. Live vaccines in immunocompromised
individuals and pregnant women
b. Reversion to wild type
c. Arthralgias/myalgias
d. Hypersensitivity reactions
1. Arthus phenomenon
2. arthritis and arthralgia
3. anaphylaxis
3.
d.
e.
f.
g.
h.
IV.
PASSIVE IMMUNIZATION
A.
Natural
Placental Antibody Transfer
1.
majority of IgG in neonates plasma is passive from mom
2.
protection wanes by 6mo as infant makes own immunoglobulin
3.
specific immunization of mother antenatal can protect
neonate (i.e. tetanus neonatorum)
Colostrum protection
1.
contains enzymes, cells, antibodies
2.
B cells migrate to breast from intestine (enteromammary)
3.
antigen-specific T cells also transmitted but role is unclear
B.
Artificial
Passive Antibodies Specific vs. Nonspecific
specific antigen raised in animal sera (e.g. horse)
result was serum sickness with repeated exposure
229
C.
230
D.
IgG
IgA
IgM
Whole serum
Immune serum globulin
Intravenous immunoglobulin
Placental immune serum globulin
1,200
16,500
30005,000
16,500
180
100500
trace
200700
200
25200
trace
150400
E.
Precautions
IM aggregates may cause anaphylactoid reaction
aseptic meningitis
noninfectious hepatitis
anaphylactoid in selective IgA deficiency
231
V.
IMMUNOTHERAPY
Use of immunological approaches for treatment of immune-based
and non-immune-based human diseases
A.
Immune-based diseases
1.
Mechanisms
Deficiency
Dysregulation
Dysfunction
2.
Clinical Manifestations
Infectious
Hypersensitivity
Cancers
Others
3.
232
C.
Clinical Examples with Potential for Using Cytokine Therapies (immunebased and non-immune-based diseases)
1.
Metabolic Diseases
Osteoporosis IL-6
Diabetes mellitus TH1
2.
CNS diseases
Multiple sclerosis TH1
ALS TH1
Alzheimers TH2 (?)
3.
Infectious Diseases
Opportunistic infection T cell deficit
HIV disease CD4 T cell deficit
4.
5.
6.
Hypersensitivity Diseases
Allergic/asthmatic diseases TH2
Autoimmune/inflammatory diseases TH1 and TH2
D.
a.
b.
c.
d.
e.
f.
g.
233
h.
SUMMARY
There are multiple vaccine preparation methods, each with their own
advantages and disadvantages
234
IMMUNOLOGY OF CANCER
Jeffrey K. Actor, Ph.D.
MSB 2.214, 713-500-5344
[Special thanks to Priya Weerasinghe, M.D., Ph.D.]
Objectives
(1) Discuss Tumor Antigens.
(2) Review effector mechanisms to combat tumors and tumor development.
(3) Discuss role of antibodies in diagnostics and in immunoprophylaxis and
immunotherapy.
Reading: Coico and Sunshine (2009), Chapter 19.
Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/cancerimmunology/
Cell-Mediated Responses to Tumor Cells
Many concepts discussed to date also apply to protection against tumor cell
development. Please refresh these concepts by visiting Chapter 19 of the Coico
and Sunshine text, beginning on page 303.
Introduction
What are tumor antigens/tumor specific transplantation antigens (TSTAs)?
Some tumor antigens consist of molecules that are unique to the tumor cell but not to the
normal cell. Some tumor antigens are qualitatively not different from those found on
normal cells but are over expressed on the tumor cell. Examples include the HER in some
breast and ovarian cancers- over expression of the HER-2/neu-1 oncogene, and the ras
oncogene on some human prostate cancer cells.
235
Embryonic
Type of Antigen
Name of
Antigen
Types of Cancer
Oncofetal
antigens
MAGE-1
MAGE-2
CEA
Several
Several
Lung, pancreas, breast,
colon, stomach
Liver, melanoma, carcinoma
of bladder, lung, testis
AFP
Differentiation
Mutant
cellular
gene
products
Normal
intracellular
enzymes
Oncoprotein
Carbohydrate
Prostate
specific
antigen, CT
antigen
tyrosinase
HER-2/neu
Lewis
Prostate
Clonal
amplification
Ig isotype
Specific
antibody of B
cell clone
Lymphoma
Point
mutations
Oncogene
product
Mutant RAS
protein
Mutant P53
Several
Mutant
CDK-4
Melanoma
E6 and E7
proteins of
HPV
Cervical
Several
Suppressor gene
product
CDK
Viral gene
products
Melanoma
Breast, ovary
Lymphoma
Transforming
viral gene
Nuclear protein
236
Activation
mechanism
Chromosomal change
Associte
cancer
C-myc
Genetic
rearrangement
Burkitts
lymphoma
C-abl
Genetic
rearrangement
Translocation 9-22
CML
C-H-ras
Point mutation
Bladder
carcinoma
C-K-ras
Point mutation
Lung and
colon
carcinoma
Comment
237
Immuno-diagnosis
1. ImmunohistochemistryAntibodies to specific antigens detected by amplified signals.
Applications: Diagnosis on surgical specimens.
to identify the original cancer
to classify the type of cancer
to predict the aggressiveness of the tumor
Tumor immunoprophylaxis
Cervical cancer vaccine:
-Gardasil (by Merck Pharmaceuticals)
virus (HPV )16, 18, 6, 11.
-Cervarix: (by GlaxoSmithKline)-
238
Tumor Immunotherapy
(1) Stimulate the immune system, reject and destroy tumors.
- BCG immunotherapy for early stage bladder cancer.
- Imiquimod: topical therapeutic to supplement local production of IFN-
(2) Monoclonal antibodies to target and destroy tumors.
Examples: Antibody Immunotherapy targeted towards cancers
Name
Trade name
Used to treat
Target
Year
approved
Rituximab
Rituxan
Non-Hodgkins
lymphoma
CD20
1997
Trastuzumab
Herceptin
Breast cancer
Erb b2
1998
Gemtuzumab
ozogamicin
Mylotarg
Acute myelogenous
leukemia (AML)
CD33
2000
Alemtuzumab
Campath
Chronic lymphocytic
leukemia (CLL)
CD52
2001
Ibritumomab
tiuxetan
Zevalin
Non-Hodgkins
lymphoma
CD20
2002
Panitumumab
Vectibix
Colorectal cancer
EGFR
2006
Cetuximab
Erbitux
Colorectal cancer,
Head and neck cancers
EGFR
2004
Bevacizumab
Avastin
Colorectal cancer
VEGF
2004
See also diagram in Appendix for additional mechanisms to regulate immune responses
to tumors.
239
Summary:
1. Tumor cells differ from normal counterparts by indefinite proliferation, changes in
growth regulation.
2. Normal cells can be transformed in vitro by chemical and physical carcinogens, or
by transforming viruses.
3. Tumor cells express TSTAs and TATAs.
4. Some tumor antigens are recognized by CTL cells: They are TSTAs, antigens that
are over expressed in various tumors, antigens that are normally expressed in
certain stage of differentiation and antigens from mutated proteins.
5. Proto-oncogenes encode proteins that control normal cellular growth. Key step in
induction of human cancer is conversion of proto-oncogenes to oncogenes. This
conversion may result from mutation, translocation or amplification of an
oncogene.
6. Immune responses to tumors include: CTL mediated cell lysis, NK cell killing,
ADCC and macrophage mediated cell killing. There are several cytotoxic factors
such as TNF- and TNF-.
7. Some tumors cells utilize immune response evading mechanisms.
8. Cancer immune-therapy includes monoclonal antibodies, antibodies coupled with
toxins, chemotherapeutic agents or radioactive elements.
9. There are new strategies for cancer immune therapy: identification of specific
tumor antigens, effective presentation of tumor antigens, generation of activated
CTLs and T helper cells.
240
February
26th
8:00-9:50 a.m.
Persons missing the session must provide written notice explaining circumstances
for not attending. Written approval must be obtained from the Office of
Educational/Student Affairs prior to consideration for any makeup session or
alternate assignment.
241
TRANSPLANT IMMUNOLOGY
Keri C. Smith, PhD
MSB 2.218
713-500-7235 Keri.C.Smith@uth.tmc.edu
Objectives: (1) Discuss the immunobiology of transplantation. (2) Appreciate the importance of
innate and adaptive functions in graft recognition. (4) Define molecular aspects of hyperacute,
acute and chronic rejection. (5) Recognize clinical consequences of transplantation.
Keywords: Histoincompatibility, Allorecognition, Rejection, GVHD, Tolerance
Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New
York, NY. 6th edition, 2009. Chapter 18; Geha and Notarangelo. Case Studies in Immunology.
Garland Publishing, New York, NY. 6th edition, 2012. Case 11. Graft-Versus-Host Disease.
Kidney Graft Complications (Blackboard file, case #46).
Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/transplantation/
The response to a transplant, or non-self, may involve nearly every facet of the immune system
that you have learned about thus far in this course. In this section, it is important to crossreference chapters in the Coico, as well as syllabus sections, to review the cellular and
molecular mechanisms at work in various forms of rejection.
Terms used in transplantation
Autologous = self
Syngeneic = genetically identical (same MHC). An autologous graft, such as a skin graft from
one area of the body to another as sometimes performed to treat burns, is a syngeneic graft. Also,
grafts from one individual to another who share the same MHC (as in the case of identical twins)
are also syngeneic. Syngeneic grafts (also called isografts) are histocompatible, that is, the
donor tissue does not induce an immune response in the recipient.
Allogeneic = genetically different (different MHC). Thus, a graft from one genetically distinct
individual to another is called an allograft. It is histoincompatible and induces an immune
response in the recipient. Another histoincompatible transplant is a xenograft, which is a graft
between a donor and a recipient from a different species.
The majority of transplant rejection is due to differences in MHC expression between donor and
recipient. If you need a refresher on class I and class II MHC expression, please review the
Role of the MHC in the Immune Response syllabus chapter as well as Chapter 8 in Coico.
242
243
tissue function. The specific mechanisms by which T cells mediate acute rejection are discussed
below. Acute rejection is currently managed by corticosteroids, cyclosporine, and other drugs.
Chronic rejection occurs months or years following the transplant. It is caused by both
antibody- and cell-mediated immunity. Multiple episodes of acute rejection, even if they were
eventually controlled, probably contribute to the development of chronic rejection as the
transplanted tissue remodels in response to injury from the immune attack. Mechanisms of
chronic rejection vary depending on the transplanted tissue: in general, pathology is associated
with fibrosis and thickening of arterioles in the transplant. There is no cure for chronic rejection;
once the process begins it is impossible to stop.
244
Tissue typing
As a rule, the fewer the MHC and mH mismatches, the less response to a transplant. Several tests
are employed to attempt to make the best match between donor and recipient.
These tests are often referred to as tissue typing because they determine the HLA allele
expression on donor and recipient.
Research has shown that some HLA mismatches are better than others in terms of transplant
survival. These data, combined with our increased knowledge of HLA antigen sequencing, have
been combined to form the basis for the commonly used panel reactive antibody test (PRA). The
PRA score is expressed as a percentage between 0% and 99%. It represents the proportion of the
population to which the person being tested will react via pre-existing antibodies.
http://optn.transplant.hrsa.gov/converge/resources/allocationcalculators.asp?index=78
The panel in this case is a well-characterized panel of lymphocytes expressing known HLA
subtypes. Peripheral blood cells from the recipient are incubated with these targets, and the
amount of complement dependent cell cytotoxicity that occurs in response to recognition of the
target cell is determined for each antigen. Recent advances in flow cytometry and multiplex
technology have also been employed for highly sensitive detection of recipient cells that can bind
to donor HLA.
Another old school, but very effective method to determine if a recipient will respond to donor
HLA is to measure the mixed leukocyte response (MLR).
245
If leukocytes from a donor and recipient are cultured together (for 72 hours or so), the CD4+
cells will respond to the foreign MHC and activate, secrete cytokines, and proliferate. CD8+
cells respond by CTL response
against their targets. Since both
the donor and recipient are
responding to each other (this is
known as a 2-way MLR), this
pretty much results in proliferation
and mutually assured destruction
of both populations. If we want to
analyze the response of just the
recipient, or just the donor
leukocytes, we can treat one
population to stop it from
proliferating (using mitomycin C
or irradiation), then only the
untreated population response will
be measured (this is a one way
MLR as shown at left)
246
Immunosuppressive Therapies
Since pretty much every facet of the immune system can play a role in rejection of a transplant,
multiple therapies are needed to control the anti-graft response.
The broadest acting agents are the corticosteroids, including prednisone, prednisolone, and
methylprednisolone. Corticosteroids exert their anti-inflammatory effects by binding to
intracellular steroid receptors. Most of the effects of the corticosteroids are linked to their downregulation of the inflammatory response. They can downregulate the genes that code for
inflammatory cytokines, inhibit leukocyte migration, and reduce the activity of and MHC
expression by APC.
For a review of inflammation, see Dr. Actors Innate Immunity and Inflammation syllabus
section and page 16-17 of Coico.
Since corticosteroids are so broad-acting, they have potent side effects (including edema, weight
gain, and diabetes) and thus are usually used sparingly and in combination with more targeted
immunotherapies.
As T cells are the major players in acute allograft rejection, agents which interfere with
lymphocyte signaling and activation are very effective in the transplant setting. There are several
agents with potent effects on T cells:
Calcineurin inhibitors, Cyclosporine and FK506 (tacrilomus) block TCR signal transduction
(signal 1) and prevent secretion of IL-2, IL-4, and IFN.
mTOR inhibitors (rapamycin, temsirolimus, everolimus) bind to mammalian target of
rapamycin (mTOR), which is crucial for the propagation of downstream signaling following
binding to co-stimulatory molecules (signal 2) and cytokines such as IL-2 (signal 3).
Inhibitors of DNA synthesis including cyclophosphamide azathioprine (a purine analog),
prevent activated T cells and B cells from rapidly proliferating.
Monoclonal antibodies are some of the newest immunosuppressives on the scene. OKT3 is a
mouse anti-CD3 antibody that blocks TCR signaling (probably by inducing CD3 to be
internalized from the cell surface). The humanized antibodies Daclizamab and Basiliximab bind
to the IL-2 receptor (CD25) and prevent T cell proliferation in response to this cytokine.
Rituximab is a B-cell depleting antibody that is sometimes used in cases where antibody
response to transplant is evident.
247
Tolerance
The ultimate goal of HLA matching efforts and immunosuppressive therapies is to induce
tolerance to the graft. Unfortunately, true tolerance of the immune system to a graft is nearly
impossible to achieve. However, there are records of remarkably long lasting grafts (the longest
known allogeneic kidney transplant survived longer than 30 years!) Factors that probably keep
the recipient immune system in check include the development of graft antigen-specific T
regulatory cells, induction of anergy in graft-reactive CD8+ cells, the secretion of
immunosuppressive cytokines such as TGF (this is a main player in the maintenance of the eye
as an immune privileged site), and finally, the development of microchimerism, that is, the
establishment of a very small percentage of donor cells in the bone marrow of the recipient.
Transplant biology is not the story of foreignness repulsed. It is the story of transplant tissues impeded
by the immune system in their struggle to return to functional homeostasis.*
Introduction, Immunobiology of Organ Transplantation 1st edition, Kluwer Academic, New York. 2004.
248
MODERN IMMUNOTHERAPY
TBA
Objectives: (1) Discuss the emergence of emergence of natural and engineered antibodies as a
tool in scientific discovery, and, (2) understand their potential for utility in both diagnostic and
therapeutic applications.
Full Syllabus Chapter to be distributed via Blackboard prior to lecture presentation.
249
Timeline of Immunology
Sources: Wikipedia, Timeline of Immunology; Immunology History IV, History of Immunology
Time Line (Keratin.com); Stewart Sell and Scott L. Rodkey, A short history of
Immunopathology.
Also see: http://aai.org/timeline/digital-timeline/
Time Line 1
1888 - Identification of bacterial toxins (diphtheria bacillus) (Pierre Roux and Alexandre
Yersin)
1888 - Bactericidal action of blood (George Nuttall)
1890 - Demonstration of antibody activity against diphtheria and tetanus toxins. Beginning
of humoral theory of immunity. (Emil von Behring) and (Shibasaburo Kitasato). Attempt to
cure tetanus with passive immunotherapy (Behring)
1891 - Demonstration of cutaneous (delayed type) hypersensitivity (Robert Koch)
1893 - Use of live bacteria and bacterial lysates to treat tumors-"Coley's Toxins" (William
B. Coley)
1894 - Bacteriolysis (Richard Pfeiffer)
1896 - An antibacterial, heat-labile serum component (complement) is described (Jules
Bordet)
1900 - Antibody formation theory side chain theory horror autotoxicus (Paul Ehrlich)
1901 - blood groups (Karl Landsteiner)
1901-8 Carl Jensen & Leo Loeb, Transplantable tumors
1902 - Immediate hypersensitivity anaphylaxis (Paul Portier) and (Charles Richet)
1902 Paul Portier & Charles Richet, Anaphylaxis
1903 - Intermediate hypersensitivity, the "Arthus reaction" (Maurice Arthus)
1903 - Opsonization (Almroth Wright & Stewart Douglas)
1905 - "Serum sickness" allergy (Clemens von Pirquet and (Bela Schick)
1905 successful organ transplantation (Correl and Guthrie)
1906 Clemens von Priquet, coined word allergy
1907 - Svante Arrhenius, coined the term immunochemistry
1910 - Emil von Dungern, & Ludwik Hirszfeld, Inheritance of ABO blood groups
1910 - Peyton Rous, Viral immunology theory
1911 - 2nd demonstration of filterable agent that caused tumors (Peyton Rous)
1914 - Clarence Little, Genetics theory of tumor transplantation
1915-20 - Leonell Strong & Clarence Little, Inbred mouse strains
1917 - hapten (Karl Landsteiner)
1921 - Cutaneous allergic reactions (Carl Prausnitz and Heinz Kstner)
1922 Fleming found lysozyme and penicillin
1924 - Reticuloendothelial system (Aschoff)
1925 Chemical mediators of inflammation (Lewis)
1926 - Lloyd Felton & GH Bailey, Isolation of pure antibody preparation
1935 Quantitative precipitin reaction (Heidelberger)
1936 - Peter Gorer, Identification of the H-2 antigen in mice
1938 Gammaglobulin identified (Tiselius and Kabat)
1938 - Antigen-Antibody binding hypothesis (John Marrack)
1940 - Identification of the Rh antigens (Karl Landsteiner and Alexander Weiner)
1941 Hemolytic disease of the newborn (Rh antigens) (Levine)
1941 - Albert Coons, Immunofluorescence technique
1942 - Anaphylaxis (Karl Landsteiner and Merill Chase)
1942 - Adjuvants (Jules Freund and Katherine McDermott)
1944 - hypothesis of allograft rejection (Peter Medawar)
1945 - Passive transfer of cell mediated immunity (Chase)
1946 - identification of mouse MHC (H2) by George Snell and Peter A. Gorer
Time Line 2
Time Line 3
Time Line 4
Glossary
Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for
Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK.
Also see on-line Glossary: Roitt: Essential Immunology (WileyBlackwell, 12th ed)
Active Immunity: Present immunity acquired through the presence of protective antibodies and
memory T lymphocytes.
Accessory Molecule: Cell surface molecules participating in cellular interactions to modulate
strength and direction of specific immune response.
Acquired Immunodeficiency Syndrome (AIDs): An infectious disease caused by the human
immunodeficiency virus (HIV) characterized by loss of CD4+ T helper lymphocytes.
Acute Phase Proteins: Any of the non-antibody proteins found increased in serum during active
and immediate innate responses; includes complement factors, C-reactive protein and fibrinogen.
Acquired Immunity/Adaptive Immunity: Network of antigen specific specialized lymphocytes
that function to eliminate or prevent systemic infection. Responses take days or weeks to
develop, and results in immune readiness (memory) that may be sustained for long periods.
Adjuvant: Excipient added to an immunogen to direct immune response during vaccination.
Affinity: Binding strength of antibody for its cognate antigen.
Affinity Maturation: Process by which B lymphocytes mature response to increase specificity
of antibody for is cognate antigen.
Allele: Variants of a polymorphic gene at a genetic locus.
Allelic Exclusion: Expression of only one gene while the alternate copy (allele) remains silent.
Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary
Applications. Academic Press, Elsevier. July, 2014. Actor, JK.
Glossary 1
Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary
Applications. Academic Press, Elsevier. July, 2014. Actor, JK.
Glossary 2
against self.
Autologous: From the same individual.
Autoreactive: Describes immune cells mounting a response against self antigens.
Avidity: Combined strength of antibody-antigen interaction, taking into account multiple
binding sites between molecules.
2-microglobulin: A 12 kDa protein that interacts with MHC class I-encoded molecules.
B Cell/B Lymphocyte: Type of lymphoid cell produced in the bone marrow from lymphoid
progenitor stem cells that possesses specific antibody cell-surface antigen receptors; cell capable
to produce antibodies when activated.
Basophil: Polymorphonuclear granulocytic cell involved in allergic reactions during Type I
hypersensitivity.
Blood Group Antigens: Red blood cell surface molecules detectable with antibodies produced
by sensitization to environmental substances. Major blood group antigens include ABO and Rh
(Rhesus) markers used in routine blood screening to designate blood type.
CD3 Complex: Set of signal transduction molecules assisting in T cell activation once the
antigen receptor has been engaged.
CD4: Cell surface glycoprotein on helper T-cells that recognizes MHC class II molecules on
antigen-presenting cells.
CD8: Cell surface glycoprotein on cytotoxic T-cells that recognizes MHC class I molecules on
target cells.
Cell Mediated Immunity/Cellular Immunity: Adaptive immune responses initiated by antigen
specific T cells.
Chemokines: Family of related small polypeptide cytokines involved in directed migration and
Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary
Applications. Academic Press, Elsevier. July, 2014. Actor, JK.
Glossary 4
Cytotoxic T cell: T lymphocytes bearing CD8 cell surface molecules that respond to antigenic
stimulation through elicitation of toxic mediators. Critical for anti-viral responses.
Defensins: Natural molecules able to limit growth of microorganisms.
Degranulation: Process by which myeloid leukocytes release digestive proteins stored in
cytoplasmic vesicles.
Delayed Type Hypersensitivity (DTH): See: Hypersensitivity, Type IV.
Dendritic Cell: Primary phagocytic antigen-presenting cell capable of initiating immune
response and lymphocytic activation, accomplished by cytokine secretion.
Enzyme-Linked Immunoadsorbent Assay (ELISA): Assay used to detect antigens bound to
solid wells in a plate format. Labeled reagents are used for quantitation, by linking enzymes to an
antibody to allow substrate color change for recognition of antigenic detection.
Endocytosis: Mechanism utilizing receptors or pinocytosis whereby materials are up-taken from
solution into plasma membrane vesicles by cells.
Eosinophil: Polymorphonuclear granulocytic cell involved in the innate response to parasitic
infections.
Epitope: Antigenic determinant; portion of antigen capable of interacting with antibody or
eliciting a lymphocytic response.
Extravasation: Movement across blood endothelial barriers into tissue.
Fab fragment/F(ab)2: Portion of the antibody heavy and light chains which combine to make
up the antigen binding region.
Fas-FasL: Cell surface molecule interactions involved in activation of apoptosis.
Fc Fragment: Portion of the antibody heavy chain that comprise regions able to interact with
cellular receptors; confers biological function to the immunoglobulin.
Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary
Applications. Academic Press, Elsevier. July, 2014. Actor, JK.
Glossary 6
Foreign: Non-self.
Germ Line: Genetic material in original configuration, representing non-rearranged
chromosomes.
Germinal Center: Secondary lymphoid tissues sites where lymphocytic populations can
proliferate and mature in response to antigen.
Graft-versus-Host Disease (GVHD): Clinical state where donor immune cells develop
pathological reactions to recipient post transplantation.
Granulocyte: General term for phagocytic leukocyte containing granular particles.
Granzyme: Protein involved in cytotoxic reactions; involved in cell lysis.
Hapten: Small low-weight molecule that can only elicit immune responsiveness when attached
to a larger carrier molecular, thus rendering it immunogenic.
Heavy Chain: Larger protein associated with the antibody molecule; confers biological
functions, associated with the constant portion of the chain.
Helper T Cell: Class of CD4+ T lymphocytes that respond to antigens by secreting cytokine
subsets to give help to cells to become effectors of cellular immunity, or to stimulate B cells to
make antibodies.
Hematopoietic Stem Cell: Precursor cell found in bone marrow. Can give rise to leukocytes.
Herd Immunity: Social concept to preventing spread of infection within a community;
vaccination of a significant portion of a population provides a measure of protection for
individuals who have not developed immunity, due to limitation of infection spread.
Heterograft: Graft in which the donor and recipient are of different species. See: Xenograft.
Histamine: Compound released from neutrophils during immunological and allergic reactions
causing vasodilation and smooth muscle contraction.
Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary
Applications. Academic Press, Elsevier. July, 2014. Actor, JK.
Glossary 7
Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary
Applications. Academic Press, Elsevier. July, 2014. Actor, JK.
Glossary 13
Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary
Applications. Academic Press, Elsevier. July, 2014. Actor, JK.
Glossary 14
T-dependent Antigen: An antigen that requires helper T-cells to elicit an antibody response.
T-independent Antigen: An antigen able to elicit an antibody response in the absence of Tcells; usually not able to drive maturation of B cells to induce antibody class switching.
TAP-1/TAP-2: Transporters of antigen processing molecules that transfer antigenic peptides
from cytoplasm into lumen of the endoplasmic reticulum for incorporation onto MHC class I
molecules.
Thymocyte: Hematopoietic progenitor cell present in the thymus.
Titer: Method to express relative antibody concentration.
Tolerance: State of less responsiveness to a substance or a physiological insult; instrumental in
prevention of autoimmunity.
Toll-like Receptors: Subset of pattern recognition receptors recognizing conserved molecular
motifs associated with infectious agents; initiate strong innate immunity when triggered.
Toxoid: Chemically or physically modified toxin that retains immunogenicity without harmful
effects of native toxin molecule.
Transplantation: Grafting of tissue from one individual to another.
Tumor Necrosis Factor: Substance secreted by multiple cell phenotypes; member of a group of
cytokines that stimulate a proinflammatory response.
Vaccine: Immunogenic substance used to stimulate production of protective immunity
(antibodies or T cell based) to provide protection against clinical disease.
Vaccination: Artificial induction of adaptive immunity by pre-exposure of antigen or pathogen
to generate a memory lymphocytic response.
Variable Domain/Variable Region: End portion of the antibody or T cell receptor which
comprises the antigen binding region.
Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary
Applications. Academic Press, Elsevier. July, 2014. Actor, JK.
Glossary 15
Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary
Applications. Academic Press, Elsevier. July, 2014. Actor, JK.
Glossary 16
APPENDIX
Nomenclature of Immune System Cells.
1.3-3.5 109/L
B cell
Monocytic
Plasma cell
Monocytic
T cell
Monocytic
Monocytic
Innate
Effector Function
Neutrophil
Eosinophil
Basophil
Mast cell
Monocytes
Monocytic
0.2-0.8 10 /L
Macrophage
Monocytic
Tissue specific
Tissue specific
Appendix I
Antibodies
Isotype
IgM
IgD
Structure
Pentamer
Monomer
Monomer
Monomer
Monomer, dimer
Heavy chain
designation
184
146-165
188
160 2
Serum
concentration(mg/mL)
1.5
0.03
0.5-10.0
<0.0001
0.5-3.0
5-10
7-23
2.5
J chain
Yes
No
No
No
Yes
No
Yes, except
IgG4
No
No
Bacterial toxin
neutralization
Yes
No
Yes
No
Yes
Antiviral activity
No
No
Yes
No
Yes
No
No
No
Yes
No
Additional properties
Effective
agglutinator of
particulate antigens,
bacterial
opsonization
Found on surface
of mature B cells,
signaling via
cytoplasmic tail
AntibodyMediation of
dependent cell allergic response,
cytotoxicity
effective against
parasitic worms
Appendix II
IgA
Monomer in
secretory fluid,
active as dimer
on epithelial
surfaces
IgG2
IgG3
IgG4
70
20
Half-life (days)
23
23
23
Complement binding
Strong
No
Placental passage
++
++
++
Strong
Strong
Appendix III
Appendix IV
T Cells
Appendix V
Appendix VI
Cytotoxic T Cells
MHC
recognition
Antigen
recognition
required
Class I
no
specific TCR
nonspecific
no
specific IgG
no
nonspecific
no
nonspecific
Complement Cascade
Appendix VIII
Appendix IX
Interleukins are the cytokines that act specifically mediate activity between leukocytes.
Major Cell Source
Major Functions
Macrophages
Appendix X
Interferons were first recognized for their ability to confer resistance to viral infection.
Major Cell Source
IFN-;
24 distinct
species
identified
IFN-
IFN-
Major Functions
Anti-viral, Anti-tumor
Regulate differentiation
Leukocytes
Modulates lipid metabolism
Inhibits angiogenesis
Immunoregulates (monocyte/macrophage activation)
Fibroblasts
Enhances MHC expression
Class I: IFN- and IFN- beta
T cells (TH1), macrophages (rare)
Class II: IFN-
Increases cytotoxic T-cell activity, activates NK cell activity.
CCL5 (Rantes)
T cells, Endothelium
CCL11 (Eotaxin)
CXCL1
CXCL2
CXCL3
Monocytes, Fibroblasts,
Epithelium
CXCL8 (IL-8)
CCL2 (MCP-1)
CCL3 (MIP-1)
GranulocyteMacrophage
Colony
Stimulating
Factor
(GM-CSF)
Transforming
Growth Factorbeta (TGF-); 5
isoforms
Tumor Necrosis
Factor-alpha
(TNF-);
Lymphotoxin B,
Cachectin
Tumor Necrosis
Factor-beta
(TNF-)
Major Functions
Appendix XI
Appendix XII
Appendix XIII
Type II Hypersensitivity is due to antibody directed against cell membraneassociated antigen that results in cytolysis. The mechanism may involve complement
(cytotoxic antibody) or effector lymphocytes that bind to target cell-associated
antibody and effect cytolysis via a complement independent pathway (Antibody
Dependent Cellular Cytotoxicity, ADCC). Cytotoxic antibodies mediate many
immunologically-based hemolytic anemias while ADCC may be involved in the
pathophysiology of certain virus-induced immunological diseases.
Appendix XIV
Type III Hypersensitivity results from soluble antigen-antibody immune complexes that
activate complement. The antigens may be self or foreign (e.g. microbial). Such complexes are
deposited on membrane surfaces of various organs (e.g. kidney, lung, synovium, etc). The
byproducts of complement activation (C3a, C5a) are chemotaxins for acute inflammatory cells.
These result in the inflammatory injury seen in diseases such as rheumatoid arthritis, systemic
lupus erythematosus, postinfectious arthritis, etc).
Appendix XV
Appendix XVI
Appendix XVII
Pathology
Autoimmune thrombocytopenia
purpura
Myasthenia gravis
Blockage of neuromuscular
junction transmission and
muscle weakness
Graves' disease
Stimulation of increased
release of thyroid hormone
(hyperthyroidism)
Hashimoto's thyroiditis
Goodpasture's syndrome
Glomerulonephritis
Rheumatic fever
Pemphigus vulgaris
Acantholytic dermatosis,
skin blistering
Multiple sclerosis
Demyelination, marked by
patches of hardened tissue
in the brain or the spinal
cord; partial or complete
paralysis and jerking
muscle tremor
Rheumatoid arthritis
AUTOIMMUNE DISEASE
Joint inflammation,
destruction of cartilage and
bone
CLINCAL PHENOTYPE
Systemic Lupus Erythematosus Rash; inflammation of joints and serosal linings; glomerulonephritis;
hemolytic anemia, systemic symptoms
Rheumatoid Arthritis
Scleroderma
Ankylosing Spondylitis
Multiple Sclerosis
Myasthenia Gravis
Hashimotos Thyroiditis
Graves Disease
Hyperthyroidism, opthalmopathy
Celiac Disease
Sjorgrens Syndrome
Appendix XVIII
Figure 8-5 Primary immunodeficiencies. Manifestation of immunodeficiency is dependent upon the etiology of response. B-cell
deficiency is marked by recurrent infections with encapsulated bacteria. T-cell deficiency manifests as recurrent viral, fungal, or
protozoal infections. Phagocytic deficiency with associated inability to engulf and destroy pathogens usually appears with recurrent
bacterial infections. Complement disorders demonstrate defects in activation patterns of the classical, alternative, and/or lectin-binding
pathways, which augment adaptive host defense mechanisms.
Appendix XIX
Appendix XX
Mechanisms of Tolerance.
Appendix XXI
Appendix XXII
http://www.cdc.gov/vaccines/schedules/downloads/child/0-18yrs-child-combined-schedule.pdf
Appendix XXIII