BIOCHEMISTRY NOTE: The flow of this trans is based

on Dr. Echavez’s lecture incorporated

Immunochemistry Part 2 with information from the Manual 
Dr. Echavez

CASE: DIFFERENCE BETWEEN HUMORAL AND CELLULAR IMMUNITY OF THE

ACQUIRED IMMUNITY RESPONSE
An apparently healthy medical student came in due to cough, cold, and on
and off fever for 3 days
Humoral Immunity Cell-mediated Immunity
CASE QUESTION: B Lymphocytes T Lymphocytes
Major defense against infection due
What phase of immune response is the patient having?
Good at killing parasites, identifying to viruses, fungi and a few bacteria
Answer: In between ACTIVATION & EFFECTOR PHASE. Why? Activation
foreign proteins and bacteria such as the tubercle bacillus and
phase because the patient already had on & off fever which is due to
listeria monocytogenes
cytokines that were released, changing the thermoregulatory set point. It is Length of time to react is SHORTER Length of time to react is LONGER
also in the early Effector phase wherein the body tries to eliminate the T Lymphocytes can either be:
nonself antigen o Cytotoxic T Cell (CD8) or
o T Helper Cell (CD4)
COMPONENTS OF ACQUIRED IMMUNITY
 Pluripotent stem cells from the bone marrow give rise to myeloid B Lymphocytes can differentiate T Helper Cell releases Cytokines
and lymphoid precursor cells into Plasma cell (for antibodies which helps in the:
 The lymphoid precursors, in turn, give rise to T and B lymphocytes production) and Memory B cell 1.) Activation of CD8
and NK cells 2.) Differentiation of B cells into
 In mammals, the transformation to B lymphocytes occurs in fetal Plasma cells
3.) Macrophage activation
liver and after birth, the bone marrow
4.) Inflammation
 Those that populate the thymus become transformed by the
environment in this organ into lymphocytes responsible for cellular
CHARACTERISTICS OF ANTIBODIES AND T LYMPHOCYTES
immunity (T lymphocytes)
 The cells that will differentiate into T lymphocytes (TL) leave the
Antibodies
bone marrow and migrate to the thymus, where the entire process Characteristics T Lymphocytes
(from B lymphocytes)
of selection and maturation occurs
Timing of Immediate response to Appears 24-48 hrs. after
 Only mature T lymphocytes leave the thymus and enter the response antigens antigen challenge
circulation Delayed hypersensitivity
 The cells that will differentiate into B lymphocytes (BL) remain in the reactions:
bone marrow and, at the end of the maturation stage, leave the bone
Immediate hypersensitivity
marrow and enter the circulation, migration to the secondary Purified Protein
reactions:
lymphoid organs Derivative (PDD) test;
Example
rejection of transplanted
Allergy or Anaphylactic
tissue grafts and
hypersensitivity
containment of the
growth of neoplastic
cells
TLs that initially
recognize the antigen
secrete factors that
Binding leads to release of recruit and activate
histamines and other other cells (e.g.
Mechanism mediators of allergic macrophages) and
response from intracellular release factors that
granules damage the antigen,
cells possessing the
antigen, or the
surrounding tissue

B LYMPHOCYTES
 Are initially produced in the vitelline sac and subsequently, during
fetal life, in the liver and finally, in the bone marrow
 The cells that will differentiate into BL remain in the bone marrow
during the maturation process and the mature BL leave the bone
marrow and enter the circulation, migrating to the secondary
lymphoid organs
 The molecules responsible for the recognition of antigens in the BL
are the membrane immunoglobulins, IgM and IgD
 These are the counterpart of T-cell receptors and are called B-cell
receptors (BCR)

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 BIOCHEMISTRY
Immunochemistry Part 2
Dr. Echavez
ROLE OF B LYMPHOCYTES  The Igα & Igß molecules contain activation motifs (ITAMs) that are
 Some 20-30% of circulating lymphocytes are B cells, which have phosphorylated after the binding of the antigen to the BCR complex
immunoglobulin or antibody on their surface and activate factors that promote the transcription of genes involved
 The BLs are responsible for the synthesis of circulating humoral in the proliferation and differentiation of the BL
antibodies (immunoglobulins)
 BLs differentiate into plasma cells and memory BLS
 When immunologically activated, BLs transform into plasma cells
 Plasma cells are specialized cells that synthesize and secrete
immunoglobulins into the plasma in response to exposure to a variety
of antigens
 Memory BLs are cells that have been exposed to an antigen and are
readily converted to effector cells by a later encounter with the same
antigen
 B cells persist in the body for months or even years

How are B lymphocytes activated?

 The BL also act as antigen-presenting cells, after internalizing and

processing the antigen bound to the surface receptor (BCR)
 The peptides generated by antigen processing are expressed in the
membrane of the BL bound to the molecules of the major
histocompatibility complex (MHC) class II and presented to LTCD4+
(helper)
 Proteins of the complement system also offer secondary activation
signals through the receptor for the C3d fragment, called CR2 and
CD21, expressed on the BL surface
 The CD21 forms a complex with two other membrane proteins, CD19
and CD81, in BL, allowing the simultaneous recognition of C3d and of
the antigen, by BCR

STRUCTURAL COMPONENTS OF IMMUNOGLOBULINS

 The B Cell Receptor (BCR) is made up of an IgM monomer and 2 short
peptides (Igα & Igß). IgD can also appear on the surface of the B lymphocyte
and can serve as a tissue marker. It will be activated when there is binding
between the antigen and the IgM. The two short peptides (Igα & Igß) will
then cause the activation of the signaling pathway. When the signaling
pathway is activated, there will be transcription of the genes that is
responsible for the proliferation and differentiation of B lymphocytes.
So in order for the B lymphocytes to be activated, there must be BINDING
OF ANTIGEN TO THE IgM monomer
 Antibodies are the workhorses of the humoral response, providing
both target recognition and the signal for effector functions
Overview of Basic Structure:
 They have a common core structure of two identical light (L) chains
(23 kDa) and two identical heavy (H) chains (53-75 kDa), held
together as a tetramer (L2H2) by disulfide bonds
 The basic structure is Y shape, with binding of antigen occurring at
both tips of the Y
 Each chain can be divided conceptually into specific domains or
regions, that have structural and functional significance

Additional Information from Manual:

 BL are responsible for the humoral immunity, characterized by the
production and release of antibodies capable of neutralizing, or even
destroying, the antigens (Ag) against which they were generated
 In order to do so, the BL must be activated, which results in a process
of proliferation and differentiation that culminates in the generation
of plasma cells with the production of immunoglobulins with high-
affinity for the antigen epitope that originated the response
 For the activation to occur, it is necessary for the BCR to bind to an
antigen epitope, which triggers a sequence of intracellular events
 In addition to the antigen recognition, the activation of the BL also
depends on a second activation signal
 The BL receptor complex (BCR) includes, in addition to the
membrane immunoglobulin, two peptide chains, Igα & Igß, of which
function is to initiate the intracellular signaling after the encounter
with the antigen

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Immunochemistry Part 2
Dr. Echavez
Chains:
 Has a light and heavy chain. A pair of these makes 4 polypeptide
chains, two pairs of H and L chains in a Y-shaped arrangement
 The subunit composition is L2H2
 Each L chain is linked to an H chain by a disulfide bond
 The H chains are linked to each other by at least one disulfide bond
 The L and H chains are synthesized as separate molecules that are
subsequently assembled within the B cell or plasma cell into mature
antibody molecules, all of which are glycoproteins
 The two arms of the Y are created by the pairing of heavy and light
chains, forming the antigen-binding region which provides for target
recognition
 The unpaired sections of the longer heavy chains interact to form the
tail of the Y, linked to the arms by a flexible linker or “hinge”
Immunoglobulin Domains/Regions/Portions:
Fc and Fab Fragments:
o Fc fragment (fragment crystallisable region)
 Does not bind antigen but mediates in effector
functions. These functions include initiation of
the complement cascade, a process that leads to
the lysis of target cells
 Fc fragments can bind to cells such as
neutrophils, monocytes and mast cells through
their Fc receptors
 Fc receptor binding amplifies the biological
activity of antigen-bound antibody
o Fab fragment (fragment antigen-binding region)
 For antigen binding
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Light Chains:
o The half of the light chain towards the C-terminal is referred
to as the constant region (CL), while the N-terminal half is
the variable region of the light chain (VL)
o There are 2 general types of light chains: kappa (κ) and
lambda (λ), which can be distinguished on the basis of
structural differences in their CL regions
o A given immunoglobulin molecules always contain 2 κ and
2 λ light chains – NEVER A MIXTURE of κ and λ
Heavy Chains:
o Approximately ¼ of the heavy chain at the N-terminals is
referred to as the variable region (VH), and the other ¾ of
the heavy chain are referred to as the constant regions (CH1,
CH2, CH3) of the H chain
o The C-terminal end of the heavy chains is termed the Fc
region
o 5 classes of H chain have been found in humans,
distinguished by differences in their CH regions:
gamma (γ), alpha (α), mu (µ), delta (δ), epsilon (ε)
o The mu and epsilon chains each have 4 CH domains rather
than the usual three
o The type of H chain determines the class of
immunoglobulin and thus its effector function
o There are thus 5 immunoglobulin classes:
IgG, IgA, IgM, IgD & IgE
Hinge Region:
o The linkers between the stem and the two arms consist of
relatively extended polypeptide regions within the H chains
– the region between the CH1 and CH2 domains – is referred
to as the “hinge region” which confers flexibility and allows
both Fab arms to move independently, thus helping them to
bind to antigenic sites that may be variable distances apart
 There are designated end of each light and heavy chains, called
variable portion; the remainder of each chain is called the constant
portion
Constant Regions:
o Amino acid sequence is repeating
o They are C-terminal
o Located on the tail or Fc portion of the Ig
o Responsible for the class-specific effector functions of
different antibody molecules
(e.g. complement fixation, transplacental passage,
mediation of allergic responses, opsonization)
Variable Regions:
o Amino acid sequence is variable
o They are N-terminal
o Create the antigen-binding site (Fab region)
QUESTION:
What cells have receptors for the tails (Fc portion) of IgE?
Answer: BEM (Basophils, Eosinophils, Mast cells)
What cell have receptor for the tails (Fc portion) of IgG?
Answer: NK Cells
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Dr. Echavez
 An antibody digested by PEPSIN yields 2 fragments (1 Fab, 1 Fc)
 An antibody digested by PAPAIN yields 3 fragments (2 Fab, 1 Fc)
Notes from Recordings:
Most immunoglobulins have 3 constant regions. Only IgM and IgE have 4
constant regions
“Remember 4 ME – meaning 4 constant regions for IgM and IgE”
DESCRIPTION AND FUNCTIONS OF THE DIFFERENT TYPES OF IMMUNOGLOBULINS
Note: This table is just a summary. Complete information can be found on the
manual (If trip niyo pa basahin)
Notes from Recordings:
What is the first step in Classical Pathway?
Answer: Presence of at least 2 antibody, IgM & IgG.
IgM and IgG fix your complements, whereas IgA and IgE do not fix
complements.
Primary response to an antigen (Primary immune reaction)– IgM
Secondary response to an antigen (Secondary immune reaction)- IgG
*IgG can cross the placenta
*IgM is first to be formed
*IgA is in the secretory form and is found in the mucus membranes,
preventing the attachment of your viruses and bacteria
*IgE is has a receptor for BEM. It is for Esthma (Asthma), Ellergy (Allergy),
Eww (Parasites)
*IgD is a tissue marker found in the B cells
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T LYMPHOCYTES
 The Pre-T cell enter the thymic cortex through the arteries during the
process of selection and maturation, they migrate towards the bone
marrow, from where they go into the circulation
 The process of TL maturation involves the expression of a functional
T cell receptor (TCR) and the co-receptors CD4 and/or CD8
 The TL only recognize processed antigen, presented by MHC
molecules on the surface of an antigen-presenting cell
 The TCR is expressed in the membrane of the TL in association with
complex called CD3, which consists of five different proteins from the
immunoglobulin family
 The TCR is responsible for the recognition of the peptide-MHC
molecule complex and the CD3, for the subsequent cell signaling
 The T cell receptor (TCR) is formed by two peptide chains of the
immunoglobulins superfamily, with a variable region and a constant
one, formed from gene segments which, during the maturation,
undergo a recombination process similar to the BCR
ROLE OF T LYMPHOCYTES
 The presentation of antigens to the TL starts with the antigen
processing by the APCs
How do we activate T lymphocytes?
 In activating T cell, you need 2 signals. The first signal is the binding of the
MHC Class II of B cell (Remember the B cell is also an APC – BMED) with the
T cell receptor (TCR). The second signal, which is coming from the co-
stimulatory receptors ( ex: CD28 of T cell and CD40 of B cell
CD40L of T cell and CD40 of B cell)
When there is presence of 2 signals, the co-stimulatory receptors gives the T
cell the “license to kill” or “respond.”
Additional Information from Manual:
 For the activation of TL to occur, after the recognition of the peptide
by the TCR, a second signal is necessary, which is mediated by the
interaction of several other co-stimulatory molecules present on the
surface of the TL and the APC
 Due to its importance in the regulation of the immune response, it is
worth mentioning the co-stimulatory molecules that participate in
the CD28-CD80 or CD28-CD86 interaction, which results in
stimulation signals and the CD28-CTLA4 interaction, which promotes
inhibitory signaling
 The majority of circulating lymphocytes are T lymphocytes or T cell,
which comprise 40 – 60% of the circulating pool of lymphocytes
 While BLs mediate immune responses by releasing antibodies, TLs
often exert their effects by synthesizing and releasing cytokines,
hormone-like proteins that act by binding specific receptors on their
target cells
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 2 main subtypes of TLs:
1. CD4 or Helper T cell (Th)
o Are responsible for the orchestration of other
cells of the immune response in the
eradication of pathogens and are also very
important in the activation of BL,
macrophages or even CD8
2. CD8 or Cytotoxic T cell
o Are involved mainly in antiviral responses
and also have anti-tumor activity
FOUR DIFFERENT VARIETIES OF T CELLS
1. Cytotoxic TLs
o Effector TLs or Killer cells
o Recognizes intracytoplasmic antigens presented by MHC
molecules class I
o They kill cells that display foreign motifs in their surfaces;
they destroy transplanted and other foreign cells
o Cells infected by viruses and tumor cells are normally
recognized by the CD8 TL
o They possess the ability to kill tumor cells without prior
exposure or priming
o After adhering to the target-cells presenting an antigen
associated with the MHC and adequate co-stimulation, the
CD8 TL proliferate and, at a subsequent contact, can
eliminate by cytotoxicity any cell that presents this specific
antigen, regardless of the presence of co-stimulatory
molecules
o The CD8 TL induce the apoptosis in the target cell through
the action of perforins and granzymes and can also lead to
apoptosis through the expression of the Fas L receptor
(CD95), which interact with the Fas molecule in the target
cells
2. Helper TLs
o Contribute to both humoral and the cellular immune
responses by stimulating the differentiation and
proliferation of appropriate BLs and cytotoxic TLs
o They possess the CD4 antigen cluster
o Patients with AIDS show decreased circulating CD4-
positive cells
o There are two subtypes of Helper TLs:
1) T helper 1 (Th1) which are concerned
primarily with cellular immunity
2) T helper 2 (Th2) which interact primarily with
BLs in relation to humoral immunity
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Th1 Lymphocytes
o Produce large quantities of IL-2, which induce the
proliferation of TL and also induce the proliferation
and increase the cytotoxic capacity of the CD8 TL
o The Th1 response is essential for the control of
intracellular pathogens and possibly contributes to the
pathogenesis of autoimmune diseases such as
rheumatoid arthritis (RA) and multiple sclerosis (MS)
Th2 Lymphocytes
o Produces IL-4, 5, 6 & 10, favoring the production of
antibodies
o The Th2 responses are associated with allergic
diseases and infection by helminths, as the IL-4
induces the change of immunoglobulin class in B
lymphocytes into IgE
o IL-5 induces the production and activation of
eosinophils
T Follicular helper cells
o Helps transform B cells into plasma cells
Th17
o Produce IL-22 and IL-26 cytokines and cytokines from
the IL-17 family
o IL-17 are potent inducers of inflammation, inducing
cell infiltration and the production of other pro-
inflammatory cytokines
o For fungal immunity
3. Suppressor TLs
o Are involved in the regulation of cellular and humoral
immunity
o They lack CD4 but possess CD8
o The cells with immunoregulatory function present as
basic characteristic, the capacity to produce
immunosuppressive cytokines, such as IL-4, IL-10 and
TGF-ß
o They act in a complex network of regulatory mechanisms
aimed at guaranteeing the modulation of immunological
responses in the presence of several antigens from
infectious agents, tumors, alloantigens, autoantigens and
allergens
o IL-10 is a powerful immunosuppressant
4. Memory TLs
o Memory T cells which have been previously exposed to
antigen and readily converted to effector cells when
exposed again to the same antigen
o A small population of peripheral TL, the TCR of these cells
can recognize antigens even in the absence of
presentation by the MHC molecule
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DEVELOPMENT OF LYMPHOCYTES
Additional information from Manual:
Explanation during lecture:
Yung B lymphocytes nanggaling sa bone marrow. Yung T lymphocyte
nanggaling sa thymus. When these “baby” lymphocytes are growing, they
are exposed to antigens. Sa baby lymphocyte tinitingnan kung sino yung may
“functional receptor.” Ang trabaho ng lymphocytes is to protect us from
infections or cancerous cells. So when you introduce an antigen to the
lymphocyte, the lymphocyte SHOULD bind to it. When a lymphocyte BINDS to
an antigen, it means that its RECEPTOR is FUNCTIONAL. Those “baby”
lymphocytes that has a functional receptor (yung kumakapit sa antigen) will
be allowed to MATURE. So those “baby” lymphocytes that did not bind to
antigens kapag inintroduce sa kanila, they will undergo RECEPTOR EDITION.
I-eedit yung amino acid sequence niya para maayos yung pagfunction ng
kanilang RECEPTOR (kakapit na sa antigens). The gene responsible for
receptor edition is RAG1 and RAG2 gene.
So those lymphocytes na nag undergo ng receptor edition, will be allowed to
be exposed again to antigens. Kapag hindi parin siya kumapit sa antigens, it
will undergo APOPTOSIS.
Positive selection – is a positive reaction to the antigen. There is binding of
the lymphocyte receptor to the antigen. These lymphocyte will be allowed to
mature
Negative selection – these are lymphocytes presented to self-antigens and
they DID NOT bind to these self-antigens. So kapag hindi kumapit ang
lymphocyte sa self-antigen, mag mamature yung lymphocyte
So basically dapat pumasa ang lymphocytes on both positive and negative
selection in order to mature
Continued….
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This is much more complicated in T lymphocytes because you have CD4 and
CD8
At the start, they do not have receptors pero as they go to the medulla of
thymus, they possess both CD4 and CD8, this is called double positive. Paano
malalaman kung magiging CD4 siya or CD8? It depends on where it will bind.
If it binds with CLASS I MHC then it will become CD8. If it binds with CLASS II
MHC then it will become CD4. Usually the signal is to create first CD8, if there
is enough number of CD8, the signal for the creation of CD8 will be lowered
and will in turn create CD4 instead
Maturation of B Lymphocytes
 The maturation of BL start with pro-B cells that express three genes,
TdT, RAG1 and RAG2, which command gene recombination
necessary for the production of immunoglobulins
 The assembly of the heavy chain starts with the random combination
of a D segment and a J segment, which subsequently bind to a V
segment
 Each BL presents a single type of light chain associated with the heavy
chain
 The successful expression of a complete IgM on the surface of the BL
leads to the maturation progression with subsequent production of
membrane IgD
 Every gene rearrangement occurs in the beginning of the maturation
of the BL in the pre-B stage, still in the bone marrow and is
completely independent from any contact with the antigen
Maturation of T Lymphocytes
 Initially, there is the rearrangement of the genes in the ß chain of the
TCR and subsequently, of the α chain
 The thymocytes, or immature lymphocytes, start to express low
levels of CD4 and CD8 on the surface and there, are double positive
 At this phase, the thymocytes migrate towards the thymic medulla
and enter in contact with the self-antigen presented by the epithelial
cells of the thymic stroma
 Only those that bind to the MHC/Ag complex with adequate affinity
receive stimulation to survive (positive selection)
 The thymocytes of which TCR do not present affinity for the self MHC
undergo apoptosis due to the lack of stimulation (death by neglect)
 The interaction with MHC molecules class I or II determines the
differentiation of these thymocyte into CD8+ or CD4+ TL,
respectively
 Continuing the maturation process, the αß thymocytes that survived
the positive selection and express only CD4 or CD8 enter in contact
in the medulla with dendritic cells and macrophages, extremely
efficient antigen-presenting cells (APCs), that present self-Ag
associated with MHC
 The immature thymocytes that interact with high affinity with these
complexes die due to apoptosis (negative selection)
 The cells that survive become mature TL, ready to leave the thymus
and exercise their action in the periphery
 Only around 5% of the cells that enter the thymus become mature
TL17
 This process of thymic education aims to guaranteeing that the
circulating TL are tolerant to the self-Ag, but capable of recognizing
AG that are foreign to the body, when presented by the self-MHC
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Immunochemistry Part 2
Dr. Echavez
CLONAL SELECTION HYPOTHESIS
1. Every individual contains numerous clonally derived lymphocytes,
each clone having arisen from a single precursor and being capable
of recognizing and responding to a specific antigenic determinant
2. Antigen selects a specific pre-existing clone and activates it, leading
to its proliferation and its differentiation into effector or memory
cells

REFERENCES
 Biochemistry Manual (2018)
 Dr. Echavez Recordings

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