Tiffany Brocke

Immunology 2017 Outline

IMMUNOGLOBULINS I

I. Remember the innate vs. adaptive dichotomy of the immune system –

innate responses are encoded in the genome, whereas adaptive responses
are particular to individual hosts and plastic to the particular attack.
a. Antibodies form within days of infection and last weeks; a few may
persist for a lifetime.
II. Clonal selection theory: one progenitor produces many different
lymphocytes, those autoreactive are eliminated and those that encounter
a foreign antigen proliferate.
a. Each cell is specific for only one molecule on its surface.
b. Immunoglobulin molecules may be these surface molecules (B cells);
often requires cross-linking of 2+ antibodies to stimulate cell
response.
c. B cells may then present these activating antigens to T cells, changing
the activity of both cell types.
i. B cells can differentiate into plasma cells, which no longer
express membrane-bound Ig and rather produce large
quantities of antibody for release into serum.
d. The offspring of the clones stimulated by antigen are identical to the
parent in T cells; in B cells they are selected for even higher affinity to
the antigen.
e. Thus immunologic memory: response to second antigen exposure is
faster than first antigen exposure.
III. Antibodies (immunoglobulins) are composed of two identical heavy
chains and two identical light chains, linked by disulfide bonds, made of
mostly constant regions and with some variable sequence.
a. Constant region is Fc, keeps body from recycling antibodies too
quickly and denotes different effector functions.
b. Variable regions recognize epitope shapes, themselves are shapes
produced by tertiary rather than primary sequence
i. Means they can recognize molecules other than just proteins
c. Flexible hinges between chains allow antibody to accommodate
diverse arrangements of antigens on pathogen surfaces.
d. Papain cleaves to produce two Fab fragments (arms of the Y) and one
Fc fragment (the body); gut pepsin produces F(ab’)2, the arms
connected, and Fc’ fragments of varying size.
e. Three hypervariable loci in each heavy chain and light chain variable
region located at the complementarity-determining region (binding
surface)
f. Types of antibodies: all with either kappa or lambda light chain and
then a gamma, mu, alpha, delta, or epsilon heavy chain
 i. IgM may form a pentamer via J chain, helps with early
responses
ii. IgA does allergies and immunity under mucosal surfaces, also
joined by J chain
iii. IgG does immunity in the blood, most common
g. Isotypes – different chain loci on the chromosome (IgG vs. IgA);
allotype – different alleles in constant regions (polymorphisms in
gamma chain gene between individuals); idiotypes – coding not
explainable by classical genetics, differences at binding surfaces
i. Note there are 2 alpha isotypes, 4 gamma isotypes, and 2 light
chain isotypes – encode Fc effector function of antibody

INNATE IMMUNITY I

I. The best protection against infection is the epithelial barrier between the
body and the world – tight junctions, acid pH, mucus and cilia
II. Remember pathogens and the innate immune system co-evolve; mount a
rapid stereotyped defense against an attack based on common patterns of
pathogens.
a. Mast cells, dendritic cells, macrophages, natural killer cells,
complement proteins, basophils, eosinophils, neutrophils
III. Innate immune system surveys for PAMPs, DAMPs, missing/altered self,
and Fc recognition
a. PAMPs (pathogen-associated molecular patterns) are characteristic of
microbes not found in the host, and are necessary for survival of the
pathogen (can’t be quickly mutated)
i. May be as detailed as DNA in cytoplasm, or lots of
unmethylated CpG sequences in bacterial and viral genomes
ii. Important: lipopolysaccharide (LPS) from outer membrane of
gram(-) cell wall
b. DAMPs (damage-associated molecular patterns) are displayed on
stressed, injured, infected, or transformed human cells: heat shock
proteins, phospholipids in the wrong places
i. Along with PAMPs, recognized by PRRs (pattern recognition
receptors) encoded in the genome; many PRRs cooperate to
recognize pathogens
ii. These can be circulating, especially like mannose-binding
lectins and ficolins, and directly kill pathogens or recruit other
host defenses like complement or phagocytosis
iii. Can also be anti-microbial peptides like defensins carried in
neutrophilic granules or released upon epithelial damage
iv. Can also be pentraxins, like C-reactive protein and serum
amyloid P, which are acute-phase reactants (secreted during
inflammatory states); enhance complement cascade, clinical
marker of inflammatory response.
 v. Cell-associated PRRs include Toll-like receptors (TLRs) in the
plasma membrane and endosomes, and Nod-like receptors
(NLRs) in the cytoplasm
vi. TLRs may modify their affinity based on dimerization or
adapter proteins and their signaling by cytoplasmic molecules
like MyD88
1. Most important: TLR4 recognizes LPS
2. Activating signal moves the transcription factor NFkB
into the nucleus, promoting txn of cytokines (especially
IK-3 for the type 1 interferon pathway)
vii. NLRs survey intracellular environment and may activate
transcriptional responses against bacteria or apoptosis
1. Bind NOD1 and antigen to trigger similar signaling
cascade with NFkB to TLRs
c. Immune system recognizes cells without MHC, with MHC different
from one’s own, or altered self in the case of some tumors
d. Macrophages have Fc receptors that respond to things antibodies
have decorated – extends spectrum of recognizable molecules
IV. Once pathogens are recognized, they must be removed
a. Neutrophils, macrophages, dendritic cells (DCs), and B lymphocytes
can phagocytize microorganisms via actin reorganization and digest
them via chemicals like oxygen radicals and proteases
b. Neutrophils, eosinophils, and mast cells release their DNA in nets to
capture extracellular microorganisms in response to pro-
inflammatory stimuli and pathogens
c. Infected cells can send out interferons to tell nearby cells to produce
anti-infection proteins and other cell changes to protect themselves
from expansion
d. Many antibodies bound to a pathogen surface will be recognized by
FcgammaRIII (CD16) receptors on NK cells, resulting in degranulation
and pathogen apoptosis: antibody-dependent cell-mediated
cytotoxicity
i. Killing mechanism of monoclonal antibodies like Rituximab

INNATE IMMUNITY II

I. Inflammation is a multisystem, multistep program designed to protect

against injury, including roles by the immune system.
a. Acute inflammation: mast cells in epithelium recognize DAMPs via
PRRs, degranulate and histamine increases vascular permeability,
other factors recruit other cells to area to further healing process
i. Early response intended to limit further damage
b. Characterized by arteriolar vasodilation and increased vascular
permeability – transudate, then exudate; calor, rubor, tumor
c. Leukocyte emigration from bloodstream: begin by marginating from
central laminar flow, then begin to roll along endothelium, induces
 selectin expression, induces chemokine expression, induces integrin
expression, leads to adhesion and cells push through porous
epithelium (diapedesis) and follow chemokines to site of injury
(chemotaxis)
d. Begin to remove damaged and infected tissue, which releases further
DAMPs and further impacts systemic response to insult; macrophages
begin to create cursory tissue to be remodeled to normal tissue
II. Remember interferons are molecules that are secreted by infected cells in
response to IRF3 from recognition of viral genome that signal through
IFN-R on neighboring cells through the JAK-STAT signaling cascade and
cause production of interferon-related gene products to resist infection
a. Fair amount of redundancy in system – type I, II, and III, each with
several subtypes and similar functions and signaling
b. Impact various stages of viral life cycles, pathogens have evolved
defenses specific to the interferon-stimulated genes
III. Inflammasome is a scaffolded protein complex that delivers components
of the immune system; excess activity contributes to chronic
inflammatory diseases
a. Cytoplasmic receptors like NLRP1 respond to intracellular LPS
b. Defects contribute to well-known disease like Blau syndrome, Muckle-
Wells syndrome, and familial Mediterranean fever.
c. AIM 2 (related to cancer), NLRC4, NLRP1 (LPS), and NLRP3 (from
allergic disease) stimulate production of inflammasome, activating
caspase-1, up-regulating cytokines IL1-beta and IL18 – pro-
inflammatory.
d. Can be activated by TLRs through NFkB, but believed to require a
second signal to make an inflammasome, PAMPs or DAMPs
e. Pathogens have also evolved defenses to inflammasome processes
IV. Deficiencies in the innate immune system may be gain of function or loss
of function
a. Natural killer cell deficiency results in susceptibility to Herpesvirus
and Papillomavirus infections
b. TLR deficiencies (perhaps through MyD88 deficiency) manifest in
recurrent and severe infections; redundancy in system means these
aren’t so severe as far as immunodeficiencies go
c. Type I interferopathies are excessive output of the interferon system,
may lead to excessive inflammatory disease

IMMUNOGLOBULINS II

I. The human genome doesn’t contain enough base pairs to encode each
antibody distinctly: diversity accomplished by V(D)J recombination and
somatic hypermutation
a. Antibodies are the products of multiple gene segments arranged in
tandem arrays
 b. Three banks (V, D, J) joined for heavy chain and two banks (V, J) for
light chain
i. Heavy chain: D joins randomly with J, then V joins onto DJ at
DNA level, transcribed into RNA and processed; light chain: V
and J, then transcribed and processed
c. Each segment is flanked by a conserved bar code of 7 bp – 12 OR 23
bp – 9 bp, and 12 bp-spaced segments only join with 23-bp segments,
ensures only get DJ and not DD or JJ
i. Accomplished by dimer of dimers, RAG, the VDJ recombinase,
with swinging effector domain that requires one 12 and one 23
segment: brings segments next to one another and cleaves off
intervening DNA as a loop, NHEJ repairs the dsDNA break
ii. Cleavage carried out at recombination signal sequences (RSS)
d. Additional sources of genetic diversity: when hairpin forms, the ends
are filled in sloppily – addition of P element
i. Can also trim back the unpaired ends via Artemis and add back
completely random nucleotides via TdT (terminal transferase)
– generates an N element
ii. These both act to diversify hypervariable region 3, which
contributes most surface area to CDR; diversity in HV1 and
HV2 are germline encoded
e. Abolished activity of RAG leads to severe combined immunodeficiency
(SCID): deficiency of both B and T cells
II. Before loci may be rearranged, they must be transcribed a little first: the
start of transcription deposits a trimethyl on K9 of histone 3 (H3K4me3
marker): mark of active chromatin and critical for RAG activation
a. Class switching allows B cells to express different classes of antibodies
in the bone marrow vs. the periphery – same VDJ, different Fc
b. Different mechanism of recombination – each Fc locus has an
upstream switch sequence and interaction of these to place VDJ next
to the desired Fc leads to DNA looping and excision
i. Controlled by particular interleukins and other cellular factors
c. Antibody affinity for their antigens increases over time – somatic
hypermutation
i. Mutations occur randomly (clustered in HV regions) and
antibodies that bind antigen more strongly are selected for
ii. Clustered in HV regions because mutations in framework are
not positively selected on whereas in CDRs can improve
affinity for antigen and tolerate more structural variation
d. Both somatic hypermutation and class switch recombination initiated
by nicking DNA: activation-induced deaminase (AID) mutates cytidine
to uridine, removed, abasic site turned to ss break
i. One strand break = somatic hypermutation, ss breaks across
from one another = class switch recombination
 e. VDJ recombination and frequency of NHEJ is a risk to genomic
integrity – failure to occur properly induces apoptosis via p53, failure
of this pathway gives tumorigenesis
i. Aberrant VDJ recombination associated with 40-60% of acute
lymphoblastic leukemias
ii. Burkitt lymphoma: accidental joining of heavy chain gene to
Myc, occurs because of AID activity

B CELL DEVELOPMENT

I. Remember B cells can develop into plasma cells, producing high levels of
high-specificity antibody for an encountered pathogen, and memory B
cells, retaining specificity for infections over a lifetime
II. BONE MARROW: Starts as stem cell; then DJ recombination (pro-B cell),
then VDJ recombination, then functional heavy chain expressed on cell
surface with surrogate light chain (pre-B cell), VJ recombination and
expression of IgM on cell surface (immature B cell), then co-expresses IgD
and IgM and moves to spleen, can further mature there and/or in lymph
nodes
a. Starts as multipotent cell turning into a common lymphoid progenitor,
expression of IL-7alpha receptor drives maturation of B cells
i. Also expresses surface CD19 beginning as pro-B cell and after
b. Pax 5 is a transcription factor that drives production of Ig-alpha,
which associates with the pre-B cell receptor to signal and commit cell
to B cell lineage
c. IL-7 from stromal cells of bone marrow is picked up by IL-7R on pro-B
cells and drives further maturation; also CXCL12 on stromal cells
binds CSCL4 on immature B cells to keep them from leaving
prematurely
d. Signaling through pre-B cell receptor downregulates expression of IL-
7R, pauses RAG between production of heavy and light chain
i. Surrogate light chains crosslink to one another, activates
nearby membrane-bound Ig-alpha and Ig-beta with
cytoplasmic ITAMs, tyrosine kinase cascade through Btk and
SLP65 to downregulate IL-7R
III. Allelic exclusion: principle that each B cell expresses antibodies with only
one specificity, critical for useful +/- selection
a. Mutation in Btk leads to X-linked agammaglobulinemia: very few B
cells, frozen in pro-B stage
b. Compare to SLP-65 deficiency, B cells proliferate excessively but
never mature, predisposition to leukemia
c. Only two tries to produce a functional heavy chain (only two alleles,
property of triplicate recombination and splicing)
d. However cell will try many times to produce a successful light chain
rearrangement at each locus (J-kappa 1-5, other kappa allele, then
lambda genes)
 e. Autoreactive B cells will cross-link their receptors (excessive
stimulation in bone marrow) and will be deleted; can respond to a
smaller soluble self molecule and become anergic; positive selection is
not a contributing factor, if not self-reactive moves to periphery
IV. SPLEEN: B cells switch from surface expression of IgM to IgG and IgD
(signaled by BAFF released from spleen stromal cells) and differentiate
into further cell types (marginal zone or follicular type B cells)
V. LYMPH NODES: lymph nodes and white pulp of spleen are secondary
lymph organs
a. B cells pick up whole antigens presented by macrophages or follicular
dendritic cells, process them, and display on their surface in MHC2
receptors for interaction with follicular helper T cells
b. Interaction between helper T cells and B cells in germinal center
causes proliferation of these B cells as well as somatic hypermutation
and class switching: maturing into plasma cells or memory B cells
i. Occurs at immunological synapse, including CD40 on B cell and
CD40L on T follicular helper cell
ii. Inherited defects in AID can lead to inability to class-switch
and hyper-IgM syndrome with lack of other isotypes, prone to
bacterial infections of mucosal surfaces
c. B cells may develop autoreactivity in the periphery – after recognizing
an antigen, if it doesn’t interact with a T cell also recognizing that
antigen, it becomes anergic and dies
d. Many lymphoid tumors are B cells early in development

ANTIBODY EFFECTOR FUNCTION

I. Remember B cells become plasma cells, which produce great quantities of

antibodies, which have different functions based on their constant tail
region and can be switched based on cytokines secreted by helper T cells
a. The predominant immunoglobulin in fluids that are supposed to stay
in your body is IgG; fluid that is supposed to leave, where the body
interfaces with the environment, the secretory immune system, has
IgA
b. IgM is secreted as a pentamer bound by a J chain; multiple binding
sites means it can’t undergo affinity maturation but has high avidity
for a substrate; somewhat transported across mucosal surfaces, can
do neutralization and blocking adherence, and strongly activates
complement cascade
c. Four subclasses of IgG differ by the hinge regions and amount of
glycosylation; transported across placenta, does neutralization and
blocking adherence, does complement activation and only antibody to
do opsonization and antibody-dependent cellular cytotoxicity
d. IgA in serum is monomeric, but when secreted is dimeric held
together by a J chain; transported across mucosal surfaces, does
neutralization and blocking adherence strongly
 e. IgE is heavily glycosylated and in humans mostly causes allergies;
only antibody type to activate mast cells
II. Transport of antibodies: need to have antibody on the surface of
epithelium
a. Lymphoid follicles just under epithelium, receive sampled contents of
lumen from phagocytic M cells on the surface to analyze
i. Thus large quantities of IgA must be produced and lost daily
b. IgA has extra disulfide bonds to interact with secretory component
made by mucosal epithelium, and a region to bind J chain, synthesized
by B cells in mucosa
i. Two extra elements and dimerization offer physical protection
against proteases
c. Babies are protected by their mother’s IgG for 4 months after birth:
the Brambell receptor (an FcR) actively transports IgG from the
mother to the baby
i. Also expressed on vascular epithelial cells so IgG is moved into
tissue, protecting it from blood-based proteases
III. Non-inflammatory functions: remember the influenza virus uses
hemagglutinin to attach to sialic acid on host cells, and neuraminidase to
cleave itself free and escape after reproduction inside
a. Flu infection damages respiratory epithelium, leaving it open to
dangerous secondary infections
b. Want to prevent this damage, so want to prevent virus from entering
cells at all – antibodies bind hemagglutinin and prevent viral
adherence to cells, called neutralization
i. Ex. antibody to poliovirus locks it in one of two oscillating
isoforms, one binding event precludes infectivity
c. Similarly blocking adherence, antibody action against a toxin released
by a pathogen that causes the undesirable symptoms, like cholera
d. Remember IgA is most important for these functions, don’t want
intestines to get inflamed every time you’re fighting bacteria there
IV. Inflammatory functions: white cells can use fMetLeuPhe as a marker for
phagocytosis, but bacterial capsules may be difficult to grasp
a. Enter opsonization: antibodies bind bacteria, antibody Fc-gamma
regions bind Fc-gammaRs on macrophages and allow cross-linking of
these receptors to stimulate phagocytosis
b. IgM and IgG antibodies binding to antigens can stimulate the classical
pathway of the complement cascade and lead to formation of the
membrane attack complex and pathogen death
i. Note that C3b is also opsonic and phagocytic cells have C3b-Rs
c. Mast cell activation by IgE (usually in the US, an unproductive allergy
response): very little IgE in serum, yet very potent effect: Fc-
epsilonR1 on mast cells and basophils has very high affinity for its
ligand
i. Thus IgE binding to Fc-eR1 turns the nonspecific mast cell into
a detector for specific substances
 ii. Cross-linking of Fc-eR1s leads to degranulation
iii. In third-world countries, thought that IgE acts to cluster
eosinophils around parasites like worms to be destroyed
d. Antibody-dependent cellular cytotoxicity: IgG specific for altered self
cells will coat the surface of infected or transformed self cells and
attract natural killer cells via Fc-gammaR3 to lyse them via perforin
i. Rituximab recognizes CD20 on B cells, thus kills mature B cells
to respond to lymphoma or autoimmune diseases of
overproduction of antibodies

COMPLEMENT

I. Complement pathway is a system of proteins, some with high resting

plasma concentrations, that can be activated in three ways and is
amplified through cleavage and regulated by soluble and membrane
factors.
a. All three pathways generate a C3 convertase, which leads to
downstream recruitment of phagocytic cells to the site of infection,
formation of the membrane attack complex, and inflammation
II. Classical pathway: IgM or IgG bind to antigens, then C1q binds to the
pentamer Fc-mu (tails exposed only when bound to a surface) or to
clustered Fc-gamma
a. Cross-proteolysis and activation of C1r and C1s, which cleaves C4; C4a
diffuses away and C4b binds to bacterial surface
i. Cleavage of C4 exposes a reactive thioester bond on C4b, which
allows it to link with proteins
b. Complex binds and cleaves C2, releasing C2b, and C2a remains,
forming a C3 convertase; C3 binds and is cleaved and C3a is released,
functions as an anaphylotoxin
i. C1q has to bind long enough for C4 and C2 to bind (not
abundant) but C3 is incredibly abundant, so if the convertase
stays active it will highly amplify the signal
c. C3b can also bind the C4bC2a complex adjacently and create a C3/C5
convertase; each C3b can become a convertase deposited on the
bacterial surface and opsonize for phagocytosis
i. C5a is intensely inflammatory, major recruiter of neutrophils
for making pus
ii. C5b assembles with C6 and C7 to insert onto the bacterial
membrane; C8 binds, inserts into the membrane, and catalyzes
assembly of several molecules of C9, creating a large pore and
lysing the cell: membrane attack complex
III. Lectin-binding pathway: bacterial cell walls have lots of terminal
mannose sugars, whereas vertebral cell sugars are capped by sialic acid:
this is exploited by mannose-binding lectin
a. Similarly ficolin binds oligosaccharides with acetylated sugars
 b. MASP1 and MASP2 associate with MBL on the bacterial surface and
cleave C4, then cleaving C2, then producing the same C3 convertase as
the classical pathway
i. In particular, accomplished by MASP2
c. Can think of C1q as a link between the classical pathway and this
existing innate immune pathway
IV. Alternative pathway: serves as the amplification cascade for all
complement pathways; all sources of C3b feed into this.
a. C3 is spontaneously hydrolyzed and binds to factor B, which is
cleaved by factor D to generate C3(H2O)Bb, a C3 convertase
b. C3b deposits on cell surfaces and can bind factor B again and be
cleaved by D again, producing more C3bBb
i. Each C3 cleaved can produce more C3 convertase to produce
more C3 cleaved, and become a C3/C5 convertase to drive
downstream effects
c. Binding of factor P (properdin) stabilizes this complex, allowing
deposition of much C3b on pathogen surface; complex can also
become a C5 convertase when a second C3b is deposited
i. Factor P is a protective factor because it has high affinity for
Neisseria organisms (cause bacterial meningitis and
gonorrhea)
ii. People deficient in any component of complement cascade are
generally susceptible to Neisseria infection
d. Bb is very homologous to C2a – suggests the classical pathway
developed out of gene duplication
V. Regulated by C1INH, a suicide inhibitor for the C1r and C1s serine
proteases: covalently binds and inactivates them, and present at a high
enough concentration to quench most early activation of the complement
cascade, but local depletion can outstrip diffusion to produce activation
a. Our cell membranes have DAF (decay accelerating factor), C4BP (C4
binding protein), and CR1 to protect ourselves from complement
i. All three displace C2a from C4b2a (remember the C3
convertase); when C4b is bound by C4BP, MCP, or CR1 it is
cleaved by a soluble protease I to inactive C4d and C4c
b. CR1 can also displace C3b in the C3/C5 convertase, and CR1 and
factor H are cofactors for protease I to cleave C3b
i. Factor H binds sialic acid; RBCs have a bit of sialic acid
removed every time they pass through the spleen, so over time
become less able to protect themselves from complement,
leads to clear RBC life cycle timing
c. Our cells have CD59 to disrupt formation of the membrane attack
complex
d. CR1 is displayed on the membrane of PMNs, monocytes, macrophages,
and B and T lymphocytes – when membrane-bound CR1 binds a
surface decorated with C3b it stimulates adhesion, phagocytosis, IL-1
 production (inflammasome), and proliferation (recruitment and
division)
e. I-cleaved C3b (iC3b) is a ligand for CR3, expressed on PMNs,
monocytes, macrophages, and NK cells; binding causes adhesion,
chemotaxis, phagocytosis, and antibody-dependent cellular
cytotoxicity
i. NK cells are turned off from this by class 1 MHC expression
f. Factor I further cleaves iC3b into C3dg, a ligand for CR2 present on B
lymphocytes and follicular dendritic cells, which digest the product
and present it to T cells for help and eventual antibody proliferation
i. Antigen that binds an antibody and that is decorated with C3dg
causes high activation of B lymphocytes
VI. Acute-phase actions are mostly mediated by C5a; the C5a-R is a
chemotaxant
a. If C1q-R is cross-linked macrophages will be highly activated for
phagocytosis and antigen presentation
b. TNFalpha causes expression of IL-1beta and the inflammasome
c. IL-8 causes production of cachexin, which causes you to lose weight
when you’re sick
d. In the liver, bacteria induce macrophages to produce IL-1B, IL-6, and
TNFa, which mobilize acute-phase proteins from the liver, neutrohpils
from the bone marrow, dendritic cells to move to the lymph nodes
and mature, protein and energy to be released, and in the
hypothalamus cause increased body temperature
e. Deficiencies in the C1 esterase inhibitor can result in increased C2b
and severe edema when challenged by ACE inhibitors/other drugs:
hereditary angioedema
f. CH50 being abnormally low indicates a low level of a factor
somewhere in cascade; diminished C3, C4 serum level in someone
with systemic lupus erythematosus suggests currently in a lupus flare

T CELL ANTIGEN RECOGNITION I AND II

I. T cells and B cells are indistinguishable by light microscope, and both are
key for producing a full antibody response.
a. T cells develop in the thymus, where autoreactive cells are eliminated;
most prominent in children and becomes fattier with age but some T
cell development continues lifelong
b. T cells recognize short linear primary sequences of protein only, and
only when the antigen is on the surface of an antigen-presenting cell
(macrophage, dendritic cell, B cell)
i. In particular, the antigen is displayed in a major
histocompatibility complex protein; T cell specific for both
antigen and self-MHC
c. Class 1 MHC is expressed on all cells except neurons and RBCs,
antigen-presenting region composed of two loops a1 and a2 of H
 chain; class 2 MHC is expressed on B cells, macrophages, and dendritic
cells, antigen-presenting region composed of the N-terminal regions
of alpha and beta chains
i. MHCs are highly polymorphic in the population with no “wild-
type”; that is, nature has selected for diversity in the MHC.
Called the “HLA” in humans, try to match in transplants.
ii. Unlike antibodies, cells express multiple isotypes of MHCs – ex.
each cell expresses 2 alleles of each of 3 loci for MHC class 1,
but each T cell recognizes only one MHC
II. T cells bind antigen-MHC complexes via plasma membrane T cell
receptors (TCRs); like B cell receptors, these have constant and variable
regions
a. Generated with same recombination and RAG proteins as antibodies
b. All TCRs are paired with identical plasma membrane CD3 proteins
critical for TCR signaling after antigen recognition
c. Unlike antibodies, TCRs are not secreted, do not undergo somatic
mutation, and recognized processed antigen + MHC, not the native
folded antigen
d. For antigen to be recognized, it must be degraded into peptides inside
an APC and displayed on the surface of the cell through an MHC
i. Thus if MHCs can’t bind a given protein sequence, they can’t
present it, but microbes are complex and can usually break
down into something your MHC can grasp
ii. MHC binds 9 aa-long protein; binding pocket may be specific
for particular amino acids in particular places
e. In the absence of antigen, MHC molecules just display self peptides, so
T cells that bind these self-MHC complexes with high affinity must be
eliminated
f. Mature T cells also display either CD4 or CD8; normally twice as many
CD4+ cells. These proteins are not polymorphic within an individual.
i. CD4+ cells recognize antigen presented with MHC2, and CD8+
recognize antigen+MHC1
ii. CD4 not normally near the TCR, but they cluster near antigen
recognition and the Lck tyrosine kinase bound to the
cytoplasmic domain of CD4 initiates intra-T cell signaling
iii. CD4 recognizes antigens in the extracellular space, what you
would want antibodies to, thus CD4+ cells use cytokines to
stimulate B cells and macrophages; CD8 recognizes antigens in
the intracellular space and lyses infected cells, thus MHC1 not
expressed on neurons (non-proliferative) or RBCs (can’t be
productively infected by viruses)
g. The MHC is produced with the extracellular domain inside the lumen
of the ER
i. Class II peptide binding site is plugged by invariant chain in the
ER, trafficked to vesicles with extracellular contents; proteases
 degrade antigens and invariant chain and antigens bind in cleft,
trafficked to surface
ii. Proteasome turns over viral proteins in the cytoplasm and
TAP1 and TAP2 transporters move these into the ER where
they bind MHC1 and are trafficked to the cell surface
h. Note that B cells recognize antigen shapes with antibodies, internalize
them for processing, and display small primary sequences in MHC2 to
be recognized by T cells – T and B cells seeing the same antigen in
completely different ways
i. B cells present only antigens for which their one BCR has high
affinity; other APCs present many antigens
ii. Many other proteins used to improve adhesion between T cells
and the target cells they’re looking at
i. Superantigens are bacterial/viral proteins that bind both class 2
MHCs and a particular variant of the B chain of TCRs outside of the
antigen-binding pocket: thus they bind a relatively large fraction of T
cells (~2%) and stimulate massive cytokine release and major
systemic fallout (e.g. toxic shock syndrome)
j. Case study: abacavir (nucleoside analog, HIV treatment)
hypersensitivity in pts with HLA B57 allele – why?
i. Abacavir binds to HLA B57 pocket too deeply to be accessed by
T cell, alters binding site and different self peptides can bind
and create a new unnatural self-MHC profile to which
autoreactive T cells haven’t been purged

ANTIGEN-PRESENTING CELLS

I. Lymphocytes may be very distant from entering pathogens, yet need to

be able to respond to antigens at low concentrations and quickly; APCs
survey the intracellular and extracellular environments for PAMPs and
DAMPs and shuttle antigens from tissues to lymphoid organs as well as
present antigens and accessory signals to T lymphocytes
a. Non-professional/atypical APCs include mast cells, basophils,
eosinophils, and ILC3
b. Professional APCs (what we rely on for immune function) are
dendritic cells, macrophages, and B lymphocytes
II. Dendritic cells are so named for their many branching processes; we
focus on myeloid dendritic cells, but others are plasmacytoid DCs
(produce interferons, not really APCs) and follicular DCs (maintain
library of antigens to facilitate B cell development)
a. Critical for primary immune response – only APC that can activate
naïve T lymphocytes
b. Immature DCs live in a near-continuous network under the skin;
when they take up and process antigen for surface MHC, they mature
and migrate to lymph nodes to be surveyed by T cells; functional
 maturation is expression of additional factors that support developing
lymphocytes
i. Very high-capacity macropinocytosis
ii. In immature state, most MHC2 kept inside cell
c. When DC recognizes something potentially foreign, shuts off
macropinocytosis and MHC2 production and shuttles MHC2-antigen
complexes to surface and moves into afferent lymph flow
d. In lymph nodes, expresses CCR7 (binds CCL19 and CCL21 expressed
in T zones of spleen, lymph nodes, and Peyer’s patches) and DC-CK
(chemokine to attract naïve T cells)
i. Stay in lymph nodes for several days; T cell zones are
surrounding vasculature but not in follicles
ii. Puts out lots of lumps of plasma membrane to expand surface
area and thus efficiency of T cell inspection
iii. Immunologic synapse where the T cell and DC interact; point of
contact is the SMAC (supramolecular activation cluster)
1. Activation molecules at center, adhesion at periphery
e. Binding of complementary TCR and MHC-antigen complex sends
Signal 1 into the T cell, activating it
i. Other critical factors, such as binding of CD28 from T cell to its
ligand on APC, give Signal 2, allowing T cell to survive
f. Other APC signals depending on what PAMP it was are Signal 3
allowing the T cell to functionally differentiate
i. IL-12 and IFN-gamma produce Th1 cells, themselves release
IL-2 and IFN-gamma
ii. IL-4 produces Th2 cells, themselves release IL-4 and IL-5
iii. TGF-beta and IL-6 produce Th17 cells, themselves release IL-6
and IL-17
iv. IL-6 produces T follicular helper cells, themselves produce IL-
21 and ICOS
v. TGF-beta produces T regulatory cells, themselves produce
TGF-beta and IL-10
g. Then licensing: once DC and CD4+ T cell have had a productive
interaction, survival signals from the DC and cytokines (IL-2) from
CD4+ support maturation of CD8+ cells
h. Follicular B cells activated by antigen express CCR7, T cells activated
by antigen express CXCR5, migrate toward each other at border of
follicle, and the helper T cell activates the B cell further
i. Note DCs also capture and display apoptotic cell fragments well and
migrate to the lymph nodes with them, but don’t upregulate co-
stimulatory molecules, delivering signal 1 alone and driving T cells
recognizing them into quiescence – tolerizing
III. Secondary immune response involves macrophages and B cells, both
using antibodies for high-affinity antigen capture and interacting with
memory T cells with less stringent requirements for co-stimulation
LYMPHOCYTE ACTIVATION

I. T cell activation is key for any major concerted immune response. Begins
with antigen recognition, then transcription and expression of IL-2 and
IL-2R, which signal as autocrine factors to promote proliferation
a. Without IL-2, cell will become anergized
b. SIGNAL 1: The TCR is a tyrosine kinase that activates both the Ras-
Raf-MAPK signaling pathway, and the PLC-PIP2-calcium influx –
calcineurin pathway
c. But TCRs lack their own self-coded kinase domains; complexed with
separate membrane-bound mostly intracellular zeta chains, which
produce the intracellular effects after the TCR binds its ligand
i. Contain repeated YXXL motifs called ITAMs: immunoreceptor
tyrosine-based activation motifs, which bind SH2 protein
domains and enable assembly of protein complexes
d. ITAMs link zeta chain to Zap70, a tyrosine kinase with high avidity for
paired phosphotyrosines
e. Zap70 phosphorylates downstream adapters like LAT, which links
signaling to RasRaf and PLCgamma pathways
f. Cross-linking of TCR and CD4/CD8 depending on which MHC it bound
augments this intracellular T cell response
i. CD4/8 are associated with intracellular Lck via zinc finger
bridges
ii. Lck phosphorylates the zeta chain so that it can bind Zap70
(remember CD4/8 and TCR are not associated unless bound
successfully to MHC-antigen complex), also phosphorylates
Zap70 to lock it on
g. Remember that the T cell response to signal 1 alone is anergy, a
protective mechanism against self recognition
II. Signal 2: CD28 on the T cell binds B7.1/B7.2 (CD80/CD86) on an APC,
serves as docking site for other molecules inside T cell that we don’t
know
a. But known that signal 1 activates NFAT whereas signal 2 from CD28
activates the transcription factors AP1 and NFkB as well as NFAT, key
for differentiation
i. Thought perhaps a distinct set of genes responds to NFAT
alone vs. NFAT + AP1 + NFkB to cause anergy, but unknown
b. All signals lead to the IL-2/IFN-gamma promoter
c. CTLA-4 is the body’s brake – deposits on T cell surface after a certain
number of cell divisions and outcompetes CD28 to bind B7.1/7.2 and
deliver cell cycle arrest signal
i. Knockout and get lymphocyte hyperproliferation; anti-CTLA4
antibody can enhance immune response and anti-tumor
response and improve survival in metastatic melanoma
d. CARs are chimeric antigen receptors: antibody against tumor antigen
linked to zeta chain, introduce to T cells, promising in clinical trials
 e. Note B cell receptor also associates with co-stimulatory molecules
with ITAMs; similar recruitment of primary and relay tyrosine kinases
and activation of NFAT, NFkB, and MAPK
i. Key co-stimulatory molecule is CD19 and turned off by
FcgammaRIIB, accessory chains are Ig-alpha/beta

T CELL DEVELOPMENT

I. Remember clonal theory: lymphocytes that recognize self are deleted,

lymphocytes responsive to foreign bodies proliferate, and this learning
happens during development
a. Central tolerance: induction of tolerance to antigen present during T
cell development in the thymus, primarily by clonal deletion
b. Peripheral tolerance: inactivation of antigen-reactive lymphocytes
from the pool of mature T cells, critical for tissue-specific self not
found in the thymus
c. Common lymphocyte progenitor breaks into the pro-B stem or the
pro-T stem; in the thymus, can become CD4, CD8, or gamma-delta T
cells
d. Thymus has an outer capsule and then inside has a dense outer cortex
surrounding interdigitations of medulla, where more mature T cells
are located
II. T cell development begins with generation of the primary repertoire of
TCRs, then positive and negative selection, then functional maturity
a. TCR genes and recombination are similar to Ig, but constant regions
alpha and delta are interspersed among the variable region genes, and
another chain has the genes for TCR-gamma
b. Immature T cells simultaneously rearrange gamma, delta, and beta
genes; signaling through gamma-deltaTCR turns off the beta-chain
gene and commits cell to become an innate sensor at epithelium;
rearrangement of B locus has cell stay in thymus and become CD4 or
CD8, more common pathway in adults
c. TCR: expression of beta chain inhibits its own gene recombination,
stimulation of alpha chain recombination, and CD4+CD8
d. When CD4 and CD8 are co-expressed, ready to test fitness against
thymic stromal cells (central tolerance): binding MHC1 or 2 too avidly
receive pro-apoptotic signals
i. Then cells binding either MHC well receive pro-survival signals
and stop expressing whichever CD4/8 didn’t bind; if it binds
MHC+self weakly, it won’t self-react but chance it may bind
MHC+antigen better
ii. And cells that don’t bind either MHC well don’t receive survival
stimuli and undergo apoptosis
iii. 95% of cells generated in the thymus die there
e. Cortex cells start to interrogate thymus cells, then migrate to medulla,
where AIRE (autoimmune regulatory element) gene is expressed:
 stimulates expression of non-thymic self antigens like insulin for the
purpose of eliminating autoreactive cells
i. Deficiency in AIRE = organ-specific autoimmune disease
ii. Thus negative selection happens in both cortex and medulla,
positive selection in cortex only

VIRAL DYNAMICS

I. Remember viruses are obligate intracellular parasites that seem to have

arisen independently by convergent evolution
a. Envelope viruses bud off host’s plasma membrane with embedded
glycoproteins virus encoded; determines tropism (which cells virus
can infect)
b. Accessibility problem for the immune system: viruses are living inside
cells and envelope viruses can be released without killing cells
i. Class I MHC can present viral proteins for antibody-dependent
cellular cytotoxicity, classical pathway of complement cascade
may recognize viral membrane glycoproteins, may neutralize
viruses after release with antibodies
c. Viruses may have large genomes with genes devoted to evading
immune system (herpesvirus) or retroviruses (HIV) with error-prone
polymerases to mutate and out-adapt immune system
i. Macromolecules come from host cell, thus harder for immune
system to see than bacterial macromolecules
ii. TLR-3 recognizes dsRNA in endocytic compartment and ssDNA
in cytoplasm as a PAMP
d. Remember infected cells can release interferons to trigger other cells
to protect themselves with inflammasome, restriction factors (host
antiviral proteins), and apoptotic pathways
i. Ex. APOBEC3G, a cytosine deaminase incorporated into viral
particles so when new cells are infected ssDNA cytidines are
converted to uridine, cell degrades it or produces high-error
nonsense mutation codons, destroying viral genome by lethal
mutation
ii. HIV has evolved vif gene to promote degradation of
APOBEC3G; vpu gene counteracts host protein tetherin also
blocking spread of infection
1. Tetherin is a transmembrane protein embedded in two
places, so when envelope viruses bud they stay tethered
to infected cells and can’t spread
II. Of uninfected host cells, virus particles, and infected host cells, rate of
change in quantity = production rate – clearance rate
a. Uninfected cells are infected by virus at a rate proportional to number
of infected cells
b. Can determine number of infected cells if you can reach steady state
where production rate = clearance rate
 c. Or calculate basic reproductive ratio, the number of new infected cells
produced by each infected cell – must be greater than 1 for infection
to take off
i. Various immune mechanisms fight different parameters of this
equation, like release of viral particles or half-life of infected
cells
ii. Thus for vaccines to work, must decrease R0 below 1
d. Acute infections, we clear virus completely; chronic infections, viral
load levels off at steady state
e. Usefulness: can figure out free HIV decays very quickly yet has high
mutation rate, so drugs that clear quickly give virus time to select for
resistance
f. Combination therapy was the breakthrough in HIV treatment, allowed
decay of viremia to below limit of detection of very sensitive clinical
assays
i. However latent state of infection persists, viruses with a very
long half-life and reversibly nonproductive life cycle can hide
ii. Virus infects T cells, some of which become memory cells, so
genome is not expressed but is also not affected by drugs and
persists stably for decades until that memory T cell re-
encounters its antigen
iii. Thus: lifetime of antiretroviral therapy

T CELL EFFECTOR FUNCTION I

I. After recognition of antigen+MHC complex in the lymph nodes or spleen,

T cells expand into various effector T cell types and migrate to the site of
infection; these are all CD4+ cells.
a. Recognition event induces synthesis of cytokines; those that act on
leukocytes are called interleukins, function as growth factors
b. Remember Th1 cells synthesize IL-2, TNF-a, and gamma interferon
and activate macrophages; Th2 cells synthesize IL-4, IL-5, and IL-13
and activate B cells, especially IgE responses. T follicular helper cells
live in lymphocyte follicles and help with antigen-specific B cells via
IL-4, IL-21, and interferon-gamma; Th17 synthesizes IL-6 , IL-22,, and
IL-17, which promote inflammation.
c. Remember IL-2 drives clonal expansion of recognition-stimulated T
cells via autocrine signaling
i. Resting cells express low-affinity IL-2R; signal 1 and 2 produce
IL-2R alpha chain, which confers high affinity
ii. Mutated IL-2R gamma chain can produce SCID in humans, yet
knockout of IL-2 allows viability of host – gamma chain shared
among many other IL growth factor receptors and key for B
and T cell development
d. When cytokines bind their receptors, JAK-STAT signaling leads to
altered transcription patterns
 i. Often interacting with Jak3 but different STATs depending on
the particular cytokine receptor
II. Th1 cells known to be critical for clearing infections from intracellular
bacteria, yet the killing is done by activated macrophages
a. Th1 cell recognizes infected macrophage and activates it via IFN-
gamma and CD40L binding to macrophage CD40, then macrophage
killing by proteases, glycases, nitric oxide, more
i. Once activated to kill what they have inside, macrophages are
not antigen-specific: specific recognition, non-specific response
III. Remember B cells can take up specific antigens through binding of their
BCRs, process them and present them to T cells, and be activated to
produce more antibodies
a. Interaction of CD40 on B cell and CD40L on T cell is critical
b. T follicular helper cells drive class switching and differentiation to
plasma cells or memory B cells, strategic location in lymph node
c. Switching factors: IL-4 drives production of IgE, IL-5 and TGFbeta
drive IgA secretion
d. Hyper-IgM syndrome is X-linked, no functional CD40L and can’t class-
switch from IgM, lymph nodes lack germinal centers
IV. Th17 cytokines turn on antimicrobial peptides in epithelial cells, drive
epithelial turnover and homeostasis of that barrier, and neutrophil
production and recruitment to site of infection
V. Whatever cytokines are in high concentrations around activated CD4 cells
drive them into particular differentiated effectors
a. May contribute to human disease – whether infection with leprosy
leads to the tuberculoid form (highly survivable) or the lepromatous
form (life-threatening) seems to depend on whether body
predominantly generates Th1 or Th2, dictated by body’s cytokine
fingerprint
b. Other cells like NKT cells make cytokines like IL-4 to drive T cells
along particular paths; once differentiation starts, cytokines
downregulate other differentiate pathways
c. Ex. defect in STAT3 leads to inability to generate Th17 and hyperIgE:
Jobs syndrome

T CELL EFFECTOR FUNCTION II

I. Cytotoxic T lymphocytes (CTLs) are CD8+ and therefore respond to

peptides derived from inside the cell and presented on MHC1 molecules
a. Thus these cells are key for eliminating virus-infected cells, bacteria-
infected cells, tumor cells, and mediating graft rejection
b. Known to be key in fighting off cytomegalovirus infection and in
keeping HIV quiescent
c. When virus replicating inside lymph node cells is displayed to naïve T
cell, it differentiates into this killer T cell (driven by IL-2) and
migrates into tissue to kill infected cells
 i. CD4 cells are critical for sustained effectiveness of CD8 cells:
dendritic cells display antigens through MHC1 and MHC2, and
CD4+ cells license dendritic cells via CD40-CD40L interactions,
making them more effective stimulators of CD8 T cells
d. How do CTLs get signaled when the virus infects cells that are not
dendritic cells? Cross-priming: when non-APCs are infected and die,
they release fragments with viral PAMPs, picked up by PRRs on
dendritic cells, move to lymph node to activate CD8 into CTLs
i. Cross-presentation: fragments that were intracellular in
another cell can access class I processing pathway inside DCs
e. Once activated, CTLs in tissue start to crawl over cells and engage
when their TCRs+CD8 bind MHC1+antigen complexes on infected
cells
f. Release their cytotoxic granules containing perforin, granzymes, and
granulysin to kill that cell before moving on to the next
i. Perforin: like C9 of MAC and makes a pore, granzymes are
serine proteases that initiate apoptosis, granulysin breaks
down lipids, all taking advantage of programmed cell death
1. Perforin isn’t part of T cell development, but without it
CTLs have greatly reduced ability to clear infection:
most important mechanism of CTL killing
ii. If TCR engages, whole cell architecture rearranges to point the
granules to the cell-cell interface
iii. Hermansky-Pudlak Syndrome: lack of AP-3 means granules
can’t fuse and CTLs are unable to kill
g. Can also kill by Fas ligand on CTLs binding Fas on target cells, Fas
interacts with Fad which has a death domain that activates caspase 3
i. May be involved in contraction of clonal population after
antigen has been cleared
II. Viruses have evolved mechanisms to evade CTL killing
a. Evading Class I pathway: viruses may encode proteins that block TAP,
or bind class I and prevent them from leaving ER, or CMV pushes new
class I molecules into cytosol for degradation
b. To manage this, immune system has NK (natural killer) cells
especially good at recognizing infected or tumor cells
i. Part of the innate immune system, from common lymphoid
progenitor but not B/T cells, have an activating and inhibiting
receptor controlling killing
ii. Keep virus from killing host while adaptive immune response
organizes – blunt infection
c. Surface activating receptors have ITAMs that follow a signaling
cascade to produce cytotoxicity and kill cells expressing the ligand to
these receptors (first noted in tumor cells)
d. Or when antibodies cluster on targets, NK cells have Fc receptors
(called CD16) that bind them and empty granules: antibody-
dependent cellular cytotoxicity
 i. Rituximab: anti-CD20 antibody so that NK cells eliminate B
cells
e. Or NKG2D is a receptor that recognizes ligands expressed on stressed
or tumor-type transformed cells and triggers killing
f. But any/many of these proteins may be part of normal differentiating
tissue – NK cells recognize normal levels of MHC1, inhibits from
killing
i. Inhibitory receptors have intracellular ITIMs, protein tyrosine
phosphatases that stop activation faster than ITAMs can
function
g. New taxonomy of non-B, non-T lymphocytes is innate lymphoid cells:
NK cells are ILC1, use IFN-gamma to defend against viruses; ILC2 use
IL-5 and IL-13 and do allergic inflammation; ILC3 use IL-17 and IL-22
and do intestinal barrier function and lymphoid organogenesis

T CELL TOLERANCE

I. The immune system doesn’t have a concept of good vs. evil, or even self
vs. non-self exactly - why don’t we get autoimmunity?
a. We know we generate receptors at random to deal with any/all
pathogens, but need tolerance from recognizing host organism
b. Self vs. non-self can be learned in the neonatal period, but central
tolerance (deletion of autoreactive T cells in thymus) is not enough to
protect self
c. Emerging model has immune response driven by the context in which
an antigen is presented, infection vs. normal homeostasis
d. If the APC is activated by concurrent infection, T cell will get signal 1
and signal 2; without it, get tolerance and anergy of the T cell
i. APCs are activated by PAMPs already in the environment;
innate immune system is key for activation of adaptive!
ii. Note “signal 2” is misleading, really the confluence of many
positive and negative signals
II. Peripheral tolerance may be induced in several ways
a. Peripheral self antigen may be present at low levels, or may be
sequestered from the immune system, so autoreactive T cells are
present but don’t find their antigen to act
b. Or clonal deletion, when peripheral self antigen is abundant and
autoreactive T cells receive signal 1 only and die
c. Note signal 1 stimulation alone may lead to either deletion or anergy!
But anergic T cells can be reversed to proliferate again in response to
IL-2 if the environment changes
d. What keeps autoreactive T cells from staying anergic and wreaking
autoimmune havoc when an antigen is present? Enter CD4+CD25+
regulatory T cells
i. Suppress wide range of immune responses, useful and harmful
 ii. Known to express Foxp3; deficiency leads to IPEX where
immune system reacts systemically against organs
iii. Formation can be induced by growth factors – tumors will
express TGF-beta to turn B cells into T-regs and inhibit
immune response
e. IL-10 contributes to plasma cell differentiation, but can also inhibit
autoimmune responses; TGF-beta in the gut promotes IgA synthesis,
but can also inhibit autoimmune responses
f. Clinically relevant: defects in tolerance lead to autoimmunity, but can
perhaps be exploited – try to induce tolerance to donor grafts to stop
chronic immunosuppression
i. Tolerance induction has led to persistent chimerism and cure
ii. Can we stop tumors from inducing T-regs so the immune
system can fight them properly?

CELLULAR INTERACTIONS

I. For the immune system to work, lymphocytes with particular specificities

need to interact with the antigens they recognize – cell movement is
highly organized and cells move carefully in tissues
a. Occurs in three physical places – primary lymph organs where
lymphocytes are produced, secondary lymph organs where antigen
and lymphocytes are co-localized for activation, and delivery system
of activated cells to infection
i. Thymus and bone marrow; spleen and lymph nodes; adenoids,
tonsils, appendix, Peyer’s patches, and large intestine all have
germinal centers as well
b. Bone marrow is key for most blood development – has reticular and
stromal cells for stimulating particular stem cell differentiation
pathways, and macrophages to clean up unsuccessful cells
c. Thymus is an outgrowth of the third pharyngeal pouch; remember has
cortex with CD4+CD8+ developing T cells and MHC1 and 2 cells to be
interrogated by developing T cells, and medulla with more mature
cells
i. Includes characteristic Hassall’s Corpuscles in medulla,
concentric circles of reticular cells, may help produce T regs
d. Secondary lymphoid organs have special means to recruit circulating
lymphocytes and for antigen or APCs to access the necessary
microenvironment
i. Lymph nodes are termination points of lymphatics draining
lymph from most body tissues, and spleen filters blood and
collects antigens from vascular system
e. Each lymph node has its own artery, vein, and capillary bed; several
afferent and one efferent vessel force directionality so that lymph
contents pass near immune cells
 i. Presence of germinal centers indicate T cell-dependent B cell
activation is in process, germinal centers are B cell-rich and
surrounded by T cells
ii. Circulating lymphocytes enter lymph nodes through high
endothelial venules with homing receptors and chemokines to
attract T and B cells into lymphoid tissues
1. Then follicular dendritic cells and stromal cells secrete
chemokines to pull B cells into B cell area, and HEVs and
stromal cells secrete chemokines to pull T cells to T cell
area
iii. Lymph nodes contain fibroblast conduits for organized
migration
f. Where afferent lymphatics open up under the capsule macrophages
are present to pick up antigens and display to B cells nearby
g. When DCs pick up peripheral antigen, they express the chemokine
receptor CCR7 and use it to follow SLC into the T cell zones of nodes
i. T cells crawl by scanning the surface of DCs; when they receive
signal 1 and 2, make out with each other
II. After T cells are activated, they do clonal expansion and differentiate into
their effector types, all inside T cell zone, then move to B cell zone or into
tissue via different chemokine gradients
a. When activated T cells find their matching B cell, they activate them to
form germinal centers: outer dark zone crescent with replicating B
cells and inner mantle zone with B cells not activated by antigen
b. Germinal center functions to increase antibody affinity and select for
clones with high affinity
c. Clonal expansion occurs with centroblasts in dark zone, with Ig gene
somatic hypermutation; selection occurs with centrocytes in the light
zone, key role of follicular dendritic cells, differentiate to plasma or
memory B cells
III. The spleen filters blood, delivered by the splenic artery; open circulation
means arteries dump into trabecular framework holding T cell rich region
and then B cell rich follicles
a. Blood moves through splenic artery to trabecular and central arteries;
T cell rich tissue surrounds vessels (periarteriolar lymphoid sheath)
and then B cell marginal zone with germinal centers: white pulp
b. Blood then dumped into open red pulp, exits via splenic vein; this is
where antigen gets out of blood
IV. Also special mucosa-associated lymphoid tissue in extracellular matrix
under skin, lungs, and mucosa; think tonsils, intestinal Peyer’s patches
a. M cells bring in contents from lumen to be interrogated by lymph cells
just underneath
b. Think similar organization with germinal centers; dominant
immunoglobulin produced is IgA
c. However these germinal centers can be induced in any organ, in
response to chronic inflammation, may be transient
IMMUNOLOGICAL MEMORY

I. We know that immunological memory for antigens persists over decades

in both CD4 and CD8 cells, but in very small numbers – 10 per million
naïve T cells
a. When an infection begins, T cells divide and proliferate at an
astonishing rate; afterward, get a contraction phase that plateaus with
new frequency approx. 100x what it was before infection
b. Memory cells go to the lymph node where they’ll find antigens, and
some stay out in the tissue where another infection will happen
c. So upon second infection, tissue resident effector memory cells
proliferate and secrete cytokines, by 24 hrs the central memory T cells
from the lymph nodes have proliferated and started to enhance tissue
i. Central memory T cells express CD26L to bind GlyCAM-1 and
stay in lymph nodes; effector cells have more lytic activity and
central memory cells secrete more IL-2
d. Not consensus where memory cells come from – either linear model
(all T cells become effector T cells and some are converted further
into central memory T cells) or bifurcative model (factors near
dendritic cell do not divide evenly, more chemokines produces
effectors)
e. CD8 T cells that express IL-7R allow a secondary immune response
(have immune memory generated and maintained)
f. IL-7 and IL-15 maintain populations of memory cells, survival and
low-level proliferation respectively
i. Antigen is NOT required for memory cells to persist
g. In fact, persistent infection and exposure to an antigen causes loss of
those specific T cells
i. Thus when tumors persist in non-inflammatory settings, those
T cells are turned off – PD1 as a negative activation marker in
chronic exposure, vs. normal response in acute exposure
h. CD4 T cell help is key for establishing long-term memory
i. Amount of antigen present (ex. viral load, ex. antibiotic use early in
infection vs. not) does not impact quantity of T cells
j. Re-exposure to the same antigen generally leads to enhanced memory
response, particularly antigen-specific memory CD8 cells
II. Practical implications: timing of vaccine booster is optimally 1-3 months
after initial primer
a. Unlike T cell memory, 2 or more vaccinations results in significantly
greater long-term B cell memory
b. B cell memory is sustained in plasma cells and memory B cells
i. Plasma cells lose BCRs and no longer respond to antigen, just
produce 2000 antibody molecules per second
 ii. Memory B cells are responsive to antigen so that when re-
exposed they can make more plasma cells; also do not require
persistent antigen for survival

AUTOIMMUNITY

I. Autoimmunity is an immune response to self-proteins facilitating

maintenance of tolerance with no damage to self-tissues; autoimmune
disease is sustained immune attack and damage to specific targeted
tissues.
a. But for T cells to develop at all, they need to have some low affinity for
self in the context of MHC
b. Natural autoantibodies help clear debris from cell death – protected
by low to moderate affinity and minimal somatic hypermutation
II. Autoimmune diseases require immunologic memory developed over a
broad span of the immune system
a. Autoimmune diseases have been getting more common as infectious
diseases become less common – hygiene hypothesis
i. Affects 23.5 million Americans, extremely disproportionate
burden on women
b. May be organ-specific antigens, like type I diabetes from knockout of
B-islet cells of pancreas, with organ-specific pathology; or may be
systemic with ubiquitous antigens, like dsDNA for lupus
c. Ex. myasthenia gravis – auto-antibodies to acetylcholine receptor at
neuromuscular junction, leading to depletion of surface receptors and
widening and flattening of synaptic cleft, manifests in systemic muscle
weakness
d. Ex. multiple sclerosis – auto-antibodies and T cells target myelin
sheath of axons in the CNS, focal lesions in white matter dictating
symptoms of strength, cognition, and sensory deficiency
e. Ex. systemic lupus erythematosus – antibodies bind nuclear antigen
and deposit in tissues, especially kidneys, and fix complement to get
major inflammation – glomerulonephritis; skin sensitive to UV light,
characteristic butterfly rash on face
f. Ex. rheumatoid arthritis – antibodies to citrullinated proteins and to
other antibodies, large complexes deposit in joints, classic
presentation is swelling in small joints of hands and feet
g. May result from incomplete induction of tolerance in the thymus,
impaired clearance of apoptotic cells, defective production of T regs,
or altered immune signaling thresholds
III. Development of autoimmune disease begins with susceptibility – may be
particular genetic variants, like AIRE deficiency that helps train T cells
against peripheral self antigens in the thymus
a. Most autoimmune diseases are polygenic with susceptibility loci all
over the genome; remember monogenic disorders are rare and most
autoimmune diseases develop in adulthood
 b. CD4+ T cells are the primary drivers of autoimmune disease, since
when MHC2 cells present self-antigens they trigger B cells,
macrophages, and CD8+ T cells
i. One particular MHC2 allele associated with susceptibility
ii. However of course VDJ and somatic hypermutation are
stochastically driven, so luck involved in developing
autoimmune disease within context of genome
c. Other environmental factors contribute to development of
autoimmune disease – drugs, microbiome, particularly infection with
pathogens
d. At some point, tolerance breaks and auto-antibodies begin to
circulate, but may be years before disease develops
e. Perhaps an abnormal increase in concentration of auto-antigen, a pro-
inflammatory context, a non-tolerized structure created, any or all
contribute to the initiation of autoimmune disease
i. Like sympathetic ophthalmia: eyes are immune-privileged
sites, penetrating injury makes eye antigens available and then
tolerance is broken and immune system attacks other eye
ii. Or if dendritic cell eats an auto antigen while eating a virus,
may present it in MHC2 in context of stimulatory molecules,
like Coxsackie virus infection of heart and then myocarditis
iii. Or pathogens may look like self peptide (molecular mimicry):
antibodies to M protein of group A streptococcus can cross-
react and attack heart proteins, giving rheumatic fever
iv. Note that only a few self-antigens are “dominant” and readily
presented by MHC to purge self-reactivity in thymus; cryptic
peptides are poorly processed or presented and so
autoreactive T cells to these may escape into periphery
v. Or post-translational modification may change epitope in
periphery to one unseen in the thymus
f. Close relationship between cancer immunity and autoimmunity –
patients with best survival outcomes for melanoma get vitiligo
because they’re having anti-melanocyte immune response
g. Once autoimmune disease develops, get feed-forward loop of
immune-mediated damage of target tissue and proinflammatory
release of auto-antigen and recruitment of more effector pathways
i. Must disrupt this loop to address disease!

VACCINES

I. Thanks to vaccines, smallpox was eradicated in 1979; today, 80% of

American children are immunized against targeted diseases
a. Vaccination rates are somewhat struggling – measles rates have been
increasing, 2014 outbreak in Disneyland
b. May give people immunoglobulins against particular diseases to
provide short-term passive immunity, or vaccines/toxoids
 (chemically inactivated forms of microbial effector toxins) provoke
durable immunity
c. Ex. poliovirus – prior to 1900 had high endemic levels in the US so
most people were infected as young babies when still protected by
maternal antibodies, so asymptomatic infection and protected later in
life; improved sanitation made polio epidemics a big problem
II. An effective vaccine must be safe, protective over a long time, low-cost
and biologically stable with few side effects, and must induce both a
neutralizing antibody to prevent infection of irreplaceable cells and
protective T cells
a. Ideal vaccine has antigen persist long enough for many arms of
immune system to be primed against it
b. Can have whole killed vaccines, pathogen dead and non-replicating:
inexpensive to make but without cytosolic entry don’t make viral
protein and thus don’t present through MHC1 and miss that part of
the immune response
c. Live attenuated vaccine: infect people with avirulent strain to get
effective protective immunity
i. Can be generated by passaging organism in another species so
that it adapts to that species and is no longer very effective in
us, or now by genetic engineering
d. Subunit vaccines are injecting a component of the microorganism –
good for diseases caused by bacterial toxins, but not as immunogenic
and without PAMPs don’t activate dendritic cells
i. Bacteria with polysaccharide capsule like H. influenzae – get
antibodies against the capsule but T cells recognize peptides
ii. So no T cell help means no isotype switching, affinity
maturation, or memory response
iii. So try conjugating the polysaccharide to an antigen protein – T
cell recognizing the conjugated protein will still act as a helper
for the B cell recognizing the flu polysaccharide part
e. Can add danger signals to the vaccine to make the protein more
interesting to the immune system – adjuvants
i. May act as delivery systems, increase uptake of antigen by
APCs, delay clearance, increase antigen activation of signaling
pathways
ii. Commonly aluminum salts (alum), especially in subunit
vaccines where you need to induce inflammation and
recruitment of APCs
iii. But biased toward Th2, new adjuvants developed to activate
more specific arms of immune system, like CTLs and Th1
iv. For Gardasil (vaccine against HPV strains that cause most
cervical cancers), used ASO4 as an adjuvant: alum conjugated
to detoxified LPS to induce TLR4 pathway, gave higher
antibody titers and persistent increase over time
 III. These approaches may fail if it’s difficult to culture the pathogen or too
risky to give the pathogen in live attenuated or whole killed form and it
evades human immune response well
a. Take Hepatitis C Virus (HCV) and HIV – very variable, difficult to find
an antigen common among the strains
i. Many many strains of HIV with varying envelope construction
b. Have tried inserting genes for proteins characteristic of your
pathogen into a weak safe pathogen – induces robust effective
response, mechanism for Ebola vaccine
c. Managed to produce promising SIV vaccine by giving first and second
vaccine with the same antigen in different vectors – still in trials

TUMOR IMMUNOTHERAPY

I. The immune system is so proliferative and can be so specific that we’d

think it would be an excellent anti-cancer defense – last hundred years
trying to figure out immune checkpoints to use this for our own ends
a. Weirdly evidence that some elements of immune system may be
anticarcinogenic, and some may be procarcinogenic, based on
different pathways of differentiation
b. Th2 normally combats helminth infections; when cancers are
established, also get Th2 environment driving M2 macrophages
making growth factors for tumor
i. Vs. Th1 makes gamma interferon, activates macrophages,
promotes CTLs as immune response we generate to try to kill
cancers
ii. Stat3 shifts the balance between Th1 to Th17, cancer inhibition
vs. promotion
c. Cancer vaccines: may inject viruses into patient’s tumor so that
immune system will react and develop antigens against the cancer,
promising when used with checkpoint inhibitors
d. Exploring various ways to take the “brakes” off the immune system –
CTLA4 normally does feedback inhibition when a naïve T cell
encounters APC+antigen to modulate the response in the lymph node,
similarly PD1 modulates response in tissue
i. Anti-CTLA4 therapy had a modest effect for most people with
stage 4 melanoma but 20% with very long tail on Kaplan-Meier
ii. Or tumors will upregulate surface PD-L1 to fight the extant
immune response against them, and co-opt nearby non-
transformed cells to protect them
iii. So anti-PD-1 lets T cells infiltrate tumors better, many cancers
respond well to this therapy
e. Amazingly, the effect of immunotherapy persists after treatment is
stopped – novel relative to chemotherapy or radiation
f. If we can find biomarkers for tumors (like which ones are expressing
PD-L1) we can better predict who will respond to therapies
 i. Found that cancers with a high mutational load (microsatellite
instability) tend to respond well to anti-PD1 therapy

PRIMARY IMMUNODEFICIENCY

I. Primary immunodeficiency diseases are disorders of the immune system

with a defect intrinsic to the cells/tissues of the immune system;
characterized by increased susceptibility to infection and/or autoimmune
disease
a. Antibody disorders lead to sinopulmonary (pyogenic bacteria) and
gastrointestinal (enterovirus, giardia) infections, also often
autoimmune disease
b. Disorders of cell-mediated immunity lead to pneumonia and
gastrointestinal viral infections and fungal infections of the skin and
mucus membranes; SCID, AIDS, opportunistic infections, issues with
antibodies as well as T cells
c. Disorders of complement lead to sepsis and other bloodborne
infections and especially autoimmune disease like lupus and
glomerulonephritis
d. Disorders of phagocytes have infections of bacteria and fungi in skin,
lymph nodes, and spleen with abscesses, and often IBD
e. Overall not rare, affect 1/500 people
II. Chronic granulomatous disease is a metabolic defect that impairs the
killing function of phagocytes; untreated, fatal in first decade of life
a. Prone to adenitis and many infections without an apparent common
feature; in vitro studies showed normal levels of immune components
and that defect is in destruction by phagocytes
b. Infections were by staph epidermidis, candida, aspergillus, E. coli and
other gram negative gut bacteria – found all produce catalase, which
breaks down hydrogen peroxide
c. Further found four mutations in proteins that normally produce
superoxide or peroxide ions in phagocytes, one X-linked and three
autosomal recessive, explaining inheritance patterns: defective
NADPH oxidase system
d. Treat with prophylactic antibiotics and interferon-gamma, which
increases ability of phagocytes to kill
III. Severe combined immunodeficiency (SCID) is a lack of B or T cell
function, susceptibility to all infections, fatal in first year of life
a. Often X-linked immune proteins, like common gamma chain of IL-R;
infants begin to become susceptible when maternal antibodies begin
to fade away, 3-4 mos
b. Highly susceptible to opportunistic infections
c. A syndrome, has multiple causes; many causes affect intra-thymic
maturation or function and survival of T cells, thus no cytotoxicity
d. Rule of 2/3: Should always have 8000 WBC/cu mm, under 3 yo should
be 2/3 lymphocytes, 2/3 of those should be T lymphocytes, 2/3 of
 those should be CD4+; over 3 yo, should be 1/3 lymphocytes, 2/3 of
those should be T lymphocytes, 2/3 of those should be CD4+
e. Therapy is a bone marrow transplant, but may be difficult to find a
match; depending on the defect, may be able to treat with commercial
enzyme supplements, some gene therapy for X-linked SCID now
available
IV. Common variable immunodeficiency (CVID) is a clinical syndrome with
very low IgG level, low IgA/IgM level, and impaired antibody responses
with other causes of hypogammaglobulinemia excluded
a. Aka, this is the catch-all for when someone’s immune deficient and we
can’t figure out why
b. Common problem is that they can’t make antibodies and so they get
autoreactive T cells
c. May result from drugs – like rituximab killing off your B cells and
them not returning, anti-convulsants, long-term high-dose
corticosteroids
d. Onset is often in adulthood – like many things, thought to occur when
someone is susceptible (overall somewhat reduced immune function)
and then system is challenged by another event

ALLERGY BASIC IMMUNOLOGY

I. Allergic disease is very common – 55 million Americans, and becoming

more common over time
a. Begins with priming – antigen is presented, Th2 responds with IL4
(leads to IgE production, which is loaded onto mast cells), IL5
(activating eosinophils), and IL13
i. Unclear where the IL-4 comes from that stimulates production
of Th2 from naïve T helper cells
b. Major allergens in urban settings are cockroaches and mice dandruff;
some are enzymes and can do things like cleave occludin to get
through epithelia and stimulate DCs
c. IgE-mediated reactions are type 1 hypersensitivity – get urticaria
(hives) and rhinitis (hay fever, if allergen stays in upper respiratory
tract)
d. Mast cells have a receptor for IgE; when these are crosslinked by
antigen, get degranulation and histamine, leukotriene, prostaglandin,
Th2 cytokines, proteases release and inflammatory (allergic)
response
i. These are subcutaneous – can do a skin allergy test and look
for localized histamine release (hive); enough for systemic
anaphylaxis and mast cells will degranulate all over body
ii. These are also under mucosal epithelium and will produce
vomiting and diarrhea; reactions are dependent on how much
IgE and how many mast cells to that allergen you have
 iii. Can get acute asthma and very severe restriction of
bronchioles
e. Eosinophils are also allergy effector cells – upon activation,
degranulate and release toxins and leukotrienes, prostaglandins,
amplify allergic response of original mast cells
i. Elevated eosinophils (more than 5%) think NAACP: neoplasm,
asthma/allergy, Addison’s disease, collegen-vascular disease,
or parasites
ii. Have developed an antibody against IL-5 used to treat allergy
in people who have eosinophils present during their reactions
f. Impression of basophil role in allergy is evolving - makes less
histamine and tryptase than mast cells and more IL4 and IL13,
possibly contributes to differentiation of Th2
g. Made an antibody to try to block all IgE-mediated reactions – couldn’t
be against receptor or would cross-link and cause massive
anaphylaxis; made antibody to the binding site on IgE for the Fc-
epsilonR
h. Allergic reactions happen when IgE binds to the IgE-R on mast cells,
basophils, and eosinophils, causing to degranulation; these changes
happen within a few minutes, and then get a second response in the
same place a few hours later
i. Mediated by mast cells for acute response and then when
eosinophils and other cells recruited from circulation arrive,
get second response
II. Thought that these type 2 Th2 responses are for expelling helminthes, a
non-emergent threat that requires a moderate immune response
a. Mast cells and IgE also resist venoms of snakes and bees
b. Known now that a dirtier childhood environment (pets, siblings, living
on a farm or near farm animals) protects against asthma and allergy
epidemiologically but not causally yet; some known genetic risk
factors for asthma
c. Type II hypersensitivity is IgG mediated, like IgG allergy to penicillin,
rheumatic fever, myasthenia gravis: antibody binding something is
directly resulting in pathogenesis
d. Type III hypersensitivity is when antibodies complex and deposit in
tissues, resulting in pathogenesis – can be triggered by horse serum
for anti-venom, cause lupus, rheumatoid arthritis
e. Type IV hypersensitivity is mediated by memory T cells, gives poison
ivy reaction, chronic asthma, tuberculin reaction; these II-IV types are
all delayed, compared to type I is immediate

ALLERGY II (FOOD ALLERGY)

I. Remember allergic disease can be IgE-mediated or non-IgE-mediated;

asthma and rhinitis are more common in adulthood, but eczema and food
allergies improve with age
 a. Atopy: tendency toward allergic disease, likely genetic and
environmental predisposition
b. Sensitization means production of IgE and arming of IgE-bearing cells,
but not necessarily symptoms from allergen exposure
c. A thorough history and physical is most important for diagnosing
allergy; look for the following symptoms
i. Nose: congestion, sneezing, runny nose (rhinorrhea); eyes,
itching, red conjunctiva; lungs, shortness of breath, trouble
breathing, chest tightness, cough, wheezing; GI tract, vomiting,
diarrhea
ii. Can do a skin test (immediate hypersensitivity reaction) with a
tiny scratch to introduce allergen: get local raised spot (wheal)
and red around (flare), can read after 10-15 mins and do
several allergens at once
iii. Can also do ELISA and look for IgE to allergen in the serum
II. Specific allergic diseases: commonly includes eczema, also called atopic
dermatitis
a. Eczema is skin becoming sensitive to exposure even to air, often early
age of onset, very dry and chronic relapsing; in infants, typically
shows on face, neck, and extensor surfaces; any age group, flexor
surfaces
i. Treat with moisturizers, topical anti-inflammatory, and
allergen avoidance
ii. Often precedes development of allergies, thought sensitization
may happen through inflamed and impaired skin barrier
b. Food allergies in children are commonly milk, egg, peanut, soy, wheat,
tree nuts, fish; in adolescents and adults, peanuts, tree nuts, fish, and
shellfish. Prevalence appears to be rising.
i. Symptoms include hives, eczema, cough, wheezing,
hypotension/shock, vomiting/diarrhea, anaphylaxis
ii. Acute urticaria (remember hives) is most common symptom,
unknown how severe food allergies are so may be anaphylaxis
next time, should counsel to avoid completely to be safe
iii. Food is a major trigger for children with atopic dermatitis
iv. Respiratory reactions are a common component of anaphylaxis
but rarely present without other signs of food allergy
v. Can get GI food hypersensitivity, swelling of mouth, lips, throat,
when fresh produce reacts with specific pollens (birch pollen
in spring, grass pollen in early summer, ragweed pollen in
early fall, each reacting with particular foods)
1. Eosinophilic gastrointestinal disease: abnormal
accumulation of eosinophils in GI tract
vi. Fatal food-induced anaphylaxis is uncommon but preventable,
risk factors are peanut/tree nut allergy, asthma, and failure to
treat promptly with epinephrine
 c. Skin tests are not terribly reliable, especially at a small size! Should
think of them as the probability of true food allergy in the context of
patient’s other symptoms and history
d. Treat food allergies by avoidance and availability of self-injectable
epinephrine; found can be prevented for peanut allergies in some
children by introduction to peanuts early in life (4-11 mos)
e. Allergic rhinitis can be seasonal or perennial, more common in adults;
symptoms include sneezing, runny nose, congestion, itchy eyes
i. For avoidance, have to do things like keep windows closed and
wash bedding often
ii. H1 antihistamines as needed, leukotriene receptor antagonists
for control, steroids for disease modification (reduce mast cell
and eosinophil numbers in nasal mucosa, don’t alter IgE levels
or mast cell function)
iii. Allergen immunotherapy is administering slowly increasing
doses of allergen to ameliorate symptoms for subsequent
exposure
f. Venom allergies to various families of bee stings, can treat excess mast
cells with immunotherapy in adults with systemic reactions
g. Anaphylaxis is a clinical diagnosis, serum tryptase can confirm but not
rule out; most commonly in response to medication but commonly
food in children
i. Remember will be biphasic and get a second wave of
symptoms up to 8 hours later
ii. Should be treated with epinephrine, often multiple injections,
early and aggressively to maintain airway, blood pressure, and
cardiac output; elevating feet to increase blood flow back to
heart

INFLAMMATION

I. Inflammation is a defense mechanism to contain pathogens, limit tissue

damage, and set body up for repair process, initiated by tissue-resident
cells that detect PAMPs/DAMPs and use alarm signals to recruit
additional immune cells
a. Changes in blood flow, vascular permeability, endothelial adhesion,
and ECM composition enable transit of cells from blood to injury site
b. Remember hypersensitivity reactions include type 1 (IgE mediated,
immediate wheal and flare), type 2 (immediate to chronic, IgG,
cytotoxic and complement-mediated cell attacks), type 3 (IgG,
immune-complex deposition, arthus reaction), type 4 (delayed-type
hypersensitivity, poison ivy, metal hypersensitivity – mobilizing T
cells)
c. Acute inflammation can be against exogenous substances (infection,
toxins) or endogenous substances (tissue damage, cell death) and
leads to tissue repair; chronic inflammation can be against exogenous
 substances (persistent infection, chronic environmental exposure) or
endogenous (autoinflammation, autoimmunity) and leads to tissue
remodeling
d. Four characteristics of inflammation – rubor, calor, tumor, dolor
II. Acute inflammation begins with tissue damage and plugging damaged
vasculature, but bacteria introduced with PAMPs and damaged cells
releasing DAMPs start to pull in immune mediators
a. Several types of cell death: apoptosis is non-inflammatory, cell
contents released in apoptotic bodies with membranes flipped for
tissue-resident macrophages to recognize and take up for degradation
i. But necroptosis mediated by RIP and MLKL kinases, pyroptosis
by caspases 1 and 11, cell contents released more haphazardly
and act as DAMPs
ii. DAMPs may come from nucleus, cytosol, mitochondria, or ER;
recognized by TLRs and release into extracellular spaces cues
that cell is dying in an aberrant way, triggers inflammation
b. Interaction between DAMPs/PAMPs and tissue resident macrophages
and innate lymphoid cells leads to release of cytokines, chemokines,
vasoactive mediators to make local vessels dilated and leaky
i. Get edema with complement components and
immunoglobulin; endothelium starts to express selectins to
pull in neutrophils from circulation
ii. Lays down collagen for innate cells to traffic within area, local
hypoxia causes neoangiogenesis to keep oxygen flowing to
area for wound repair
c. To function properly, immune cells must be able to circulate in the
blood and lymph and then adhere at the right place and right time –
via constitutively expressed low-affinity integrins and selectin ligands
i. Remember neutrophils arrive first, then monocytes
ii. Integrins are aB heterodimers; ligands for P selectin and E
selectin are PSGL-1 and CD24, L selectin on many cells, called
CD62
d. Macrophages produce IL1, IL6, and TNFa depending on which PRRs
were engaged by PAMPs/DAMPs and interact with basolateral surface
of endothelial cells; start to express integrin ligands, P selectin, E
selectin; vessel dilates to exclude WBCs to periphery; they tether, start
engaging, slow down, begin to roll and become activated and
phosphorylate integrin to become high-affinity; then arrest and
rearrange cytoskeleton to begin diapedesis
i. Follow chemokine gradients, use integrins to crawl along
collagen scaffold in tissue
ii. Not exactly true – endothelial cells have basement membrane
maintained by pericytes, cells crawl out of vasculature at
places where pericytes leave basement membrane uncovered
 iii. Also perivascular macrophages and mast cells, cytokines and
chemokines interact with these to translate message to
endothelial cells to regulate adhesion and barrier function
iv. Inflammatory response is dynamic – endothelium first
expresses E selectin and P selectin to get neutrophils, then
ICAM1 (ligand is on surface of monocytes), then VCAM1 (ligand
is on lymphocyte surface)
v. Neutrophils are short lived, do NETtosis (DNA nets),
monocytes and T cells in about 48 hours, innate immune
system may be able to resolve infection in that time
e. Then fibroblasts arrive, differentiate into myofibroblasts to make
collagen and fibrotic tissue, after infection is resolved get re-
epithelialization (infection walls itself off) and start to reestablish
normal vascularization, fibrin scar replaced with normal tissue
f. IL25, IL33, and TSLP are DAMPs also called alarmins; interact with
basophils, nuocytes (also ILC2), Th2, and mast cells, produce IL4 and
IL13 to activate local macrophages into M2 (wound repair) phenotype
vs M1 (pro-inflammatory)
i. M2 macrophages produce proteins to remodel collagen matrix
and convert fibroblasts into myofibroblasts; regulatory
macrophages produce molecules that downregulate this
process, balance of these dictates return to homeostasis vs.
chronic inflammation
III. Chronic inflammation: chronic exposure to allergens leads to
hypertrophy of epithelium, airway muscle, overproduction of goblet cells
and mucus, airway smaller and hypersensitive to activation
a. Can get idiopathic pulmonary fibrosis (keep making fibrotic tissue in
lungs), cirrhosis, collagen deposition disease, fatty liver disease
b. All marked by infiltrate of macrophages and lymphocytes
c. Chronic insults like smoking or bacterial infections may chronically
activate NFkB and STAT3 to cause cancer, elevated IL5 and TNF of
inflammatory environment are exploited by cancers for further
growth and expansion, ROSs in environment enhance mutation rate
d. And cancer can cause inflammation itself, angiogenesis helps tumor
e. Fat cells themselves can hypertrophy in obese people and lead to
chemokine production and monocyte infiltration, polarized to M1
phenotype, supports insulin resistance and further lipolysis, disrupts
microbiome in intestine
i. Vs. healthy fat tissue has macrophages in alternatively
activated states to convert cells to beige fat; different
inflammatory profile in fat of obese vs. healthy individuals

ANTI-INFLAMMATORY DRUGS

I. Prostaglandin synthesis mediates pyresis (fever), hyperalgesia

(sensitivity to pain), and inflammation (vasodilatation, vascular
permeability, leukocyte migration); anti-inflammatory drugs block this
pathway for relief
a. But they have various undesirable effects, like GI bleeding, renal
insufficiency, and platelet inhibition, MI, and stroke
b. Today: acetylsalicylic acid is irreversible non-selective COX inhibitor;
ibuprofen, naproxen, and ketorolac (IV NSAID) are reversible non-
selective COX inhibitors; celecoxib is reversible COX-2 selective
c. Cyclooxygenase (COX) 1 is constitutively produced for housekeeping
function in tissue (prostanoids, precursor molecules), COX2 is induced
by inflammatory stimulus to cause effects in excess of normal
i. COX2 expressed at rest in brain in significant amounts, some
constitutive expression in kidneys and GI tract, thus inhibiting
COXs and/or COX2 affects these organs’ homeostasis
ii. Acetylsalicylic acid acetylates a Ser in COX1 and 2 active site so
arachidonic acid can’t fit to be converted to prostanoids,
irreversible covalent modification, and then salicylate can also
reversibly inhibit other enzymes
iii. COX1 has Ile, COX2 has Val, so whether sulfonamide moiety can
fit into active site confers specificity of COX-2 selective drugs
d. Drugs selective for COX1 have higher relative risk of GI perforation
and bleeding, which has no symptoms 80% of the time – concern
about GI bleeding = indication for COX-2 selective inhibitor
e. But thromboxane increases vascular tone via COX1 and arachidonic
acid is part of platelet and endothelium membranes, so COX2-
selective inhibitor doesn’t affect platelet aggregation
i. Vs. aspirin does, can be secondary prophylaxis for MI
ii. COX-2 selective inhibitors may actually leave normal
housekeeping unopposed and produce some pro-coagulant
and pro-thrombotic imbalance effect
f. Also prostaglandins vasodilate and other mediators vasoconstrict,
unbalanced may lead to relative vasoconstriction with impact on renal
function
i. Problematic for any kind of COX inhibitor
g. Most recent large randomized prospective clinical trial: celecoxib,
ibuprofen, naproxen are same for cardiovascular risk , celecoxib
better for GI problems, celecoxib and naproxen slightly better for
renal
h. Arachidonic acid can also go through lipoxygenase to make
leukotrienes, so through mass action in aspirin-sensitive patients get
bronchospasm, congestion, and mucus plugging: Samter’s Triad
i. Pharmacokinetics: NSAIDs are easily absorbed and distributed via
protein binding, metabolized by kidneys and liver; ibuprofen has half-
life under 5h, naproxen has half-life over 5h, aspirin is mixed-order
metabolism at anti-inflammatory doses but not heart health
prophylactic doses
 j. Celecoxib has half-life 11.2h, higher AUC in elderly and black pts, issue
in people with sulfonamide allergy
II. Acetaminophen is analgesic and anty-pyretic but not anti-inflammatory,
and we have ideas but not certainty on the mechanistic explanation
a. Reduced GI and renal adverse effects compared to NSAIDs, has liver
toxicity at overdose
b. Less effective when administered with food, half-life 2-4h
III. Corticosteroids circulate attached to globulins, diffuse into cytoplasm and
bind intermediate to give dissociated complex that can penetrate nucleus
and alter transcription
a. Binding inverted or direct repeats of 6 bp separated by a few bp
b. Normally managing homeostasis of much metabolism, fluid balance,
stress resistance, and most key body systems – disrupting to reduce
inflammation has widespread side effects!
c. Glucocorticoid agonists increase synthesis of lipocortin-1, which
inhibits production of inflammatory cytokines and prostaglandins
i. Thus anti-inflammatory effect closely related to
immunosuppressive effect, used in rheumatic disorders
d. Glucocorticoids have a range of anti-inflammatory activity and
generally have topical activity (not prednisone); mineralocorticoids
have high salt-retaining activity and no topical activity
e. Corticosteroids inhibit the hypothalamus-pituitary-adrenal axis and
thus must be tapered off if taking for a long (3 weeks+) time

PHARMACOLOGY OF IMMUNOSUPPRESSION

I. When foreign tissues are grafted into the body, MHC2 molecules from
donor activate CD4 T cells of host, secrete cytokines like IL2, and
stimulate CD8 T cells and B cells as well as own CD4 clonal expansion:
thus immunosuppressive drugs target CD4 activation and cytokine-
mediated clonal expansion
a. General cell signaling pathways: TCR binding leads to calcium influx
and IL2 and IL2R production, autocrine signaling leads to T cell
differentiation and proliferation
b. So immunosuppressive agents can act at any step of this pathway,
starting with antibodies to block TCR-MHC2 binding
i. Originally grown in animals, thus highly polyclonal, used to
prevent acute rejections but side effects like serum sickness
from immune response to animal proteins
ii. More recently, anti-CD3 antibodies can be made monoclonal
and with human Fc region to reduce host immune response;
however, can bind TCRs bivalently and cross-link to act as
agonists, cytokine release syndrome either mild or severe
c. Can next inhibit intracellular signal transduction
i. Began with cyclosporine A (CsA), a fungal metabolite, first
specific immunosuppressant and new era in graft rejection
 ii. Absorbed quickly and stored in RBCs, reservoir and carrier for
drug; mostly metabolized in liver, less side effects and higher
rates of graft retention but some toxicity to kidneys
iii. FK506 (Tacrolimus) is an orthologous bacterial peptide of
higher potency, similar liver metabolism and nephrotoxicity
but also stimulates hepatocyte growth
iv. Act by binding cyclophilin or FKBP respectively, then binds and
blocks calcineurin, critical signal transducer that
dephosphorylates NFAT; two complexes bind the same
calcineurin interface and block the active site despite different
chemical structures and mediating residues
d. Can also next use antibodies against the IL-2R to prevent T cell
proliferation
i. Daclizumab or basiliximab, humanized monoclonal antibody;
especially given for kidney transplants, avoids toxicity
e. Can then block the next intracellular signaling pathway that leads to
IL-2-dependent T cell proliferation
i. Rapamycin binds FKBP but with a different effector domain,
binds to mTOR (PI3 kinase homolog) and occludes binding site,
normally senses cellular signals like nutrients, growth factors,
to dictate downstream processes in proliferation
f. Can otherwise block components of T cell proliferation:
mycophenolate mofetil is a prodrug for mycophenolic acid, inhibits
inosine monophosphate dehydrogenase, enzyme in de novo synthesis
of GMP
i. T and B cells do mostly de novo synthesis, other cells use
purine salvage pathways, so rapamycin can be specific for
lymphocytes
g. Steroids and cytotoxic drugs are other alternate inhibitors, but much
more side effects; would be better to induce host tolerance of the
transplant
i. Achieved in rats with CXCR4 antagonist to mobilize
hematopoietic stem cells from bone marrow with small dose of
FK506: long-term graft acceptance

TRANSPLANTATION

I. Survival rate for solid organ transplants is improving over time, but while
we’ve had a good impact on acute rejection we’ve failed to do so for
chronic graft loss – think under 50% survival after 5 yrs for heart and
lung transplants
II. Transplants may be syngeneic (between genetically identical people),
allogeneic (between organisms of the same species; vast majority of
transplants, leads to alloreactivity), or xenogeneic (from other species,
mostly focused on pigs for human organs)
 a. Known that after one transplant, a second from the same donor to the
same recipient will reject much more quickly: memory is specific and
resides in lymphocytes
b. Based on multiple genes (P to F1 will succeed, but F1 to P will not; F2
and beyond to F1 will succeed; P to F2 will usually fail)
i. Frequency of P to F2 failure allowed to determine 30-50
histocompatibility loci
1. These can be major (HLA genes in humans) or minor,
like HY genes encoding sex-specific proteins; all minors
together = rejection rate similar to MHC genes
c. Can measure tritiated thymidine uptake as an indicator of cell
proliferation
d. Can get highly prolific alloreactive response because there are many
unprimed cells (1-10% of T cells) that recognize alloantigen and have
never been negatively selected against; may recognize alloantigenic
MHC without foreign peptide (cross-reactivity)
e. Transplants from donors incompatible at minor loci are rejected
much more slowly
f. Can get both recipient APCs processing peptides derived from the
graft for presentation, and donor APCs migrating to lymph nodes to
directly stimulate alloreactive T cells
III. Not all graft rejection is the same: mediated by PRR-dependent signals,
both infection and DAMPs
a. Can be hyperacute, happening in minutes to hours if immune system
has already been primed to respond to this alloantigen, but shouldn’t
happen if we’ve done the right tests ahead of time
i. Chronic DTH reaction in vessel wall causes chronic graft
rejection – vessel occlusion
b. Pathology: hyperacute has neutrophil infiltration, chronic has
accumulation of smooth muscle cells and connective tissue
c. Can test for hyper-reactivity by incubating sera from recipients with
antibodies from donor and looking for early response: complement-
dependent cytotoxicity crossmatch, flow cytometric crossmatch, and
luminex bead immunoassay, in order of increasing sensitivity
d. Remember cells need both signal 1 and signal 2 to avoid anergy –
experimentally can block C7/CD28 and CTLA4 to get long-term
survival

BARRIERS TO HEMATOPOIETIC STEM CELL TRANSPLANT

I. HSCT may be indicated for treatment of hematologic malignancies or

damaged myeloid tissue, delivery of immunotherapy, treatment of
autoimmune disorders
II. Remember HLA is the word for the human major histocompatibility
complex; two classes, I (A, B, C) and II (DP, DQ, DR); high polymorphism
in these genes, but close matching helps complications
 a. Remember these molecules are very abundant thus provoke robust
immune response, primary driver of graft-versus-host and host-
versus-graft diseases we worry about
b. Stem cells can come from HLA-identical siblings, unrelated donors,
cord blood, and other related haploidentical donors; match at 7/8 of
the loci is good enough for an unrelated donor
i. Cord blood units and haplo-identical donors are more readily
available (good for patients with narrow time frame before
disease relapse) but have more HLA-mismatch
c. Recipient must be immunosuppressed so that donor cells are not
rejected; if donor infusion contains mature lymphocytes, graft-versus-
host disease results
d. Pre-transplant conditioning involves irradiation and chemotherapy to
create physical space in the bone marrow; full myeloablation
completely destroys normal hematopoiesis, non-myeloablative
regimens are better for older patients with more co-morbidities
i. Immunosuppression: standard methotrexate plus FK506 or
CsA, or mycophenolate mofetil plus Tac or CsA, may also try for
T cell depletion in unrelated donor or haplotype transplants
e. After transplant, innate immune system is first to repopulate; ranges
from NK cells in 3-4 weeks, IgG around 1 year; extremely susceptible
to infections until reconstitution, major source of mortality
f. Up to half of HSCT recipients develop GVHD, generally controlled by
current immunosuppression; happens when graft contains
immunocompetent donor cells and host has antigens not present in
donor, following antibody inflammatory cascade
i. Increases with HLA mismatch, patient age, sex mismatch,
weaker conditioning regimen, post-HSCT infections like CMV
ii. Begins with conditioning: damage to host tissues from
myeloablative regimens begins inflammatory cascade, donor T
cells recognize foreign host antigens, cytokine storm activates
massive immune response and target cell apoptosis
iii. Can be acute or chronic, chronic GVHD is the leading cause of
late nonrelapse mortality in patients treated with allogeneic
blood or bone-marrow transplants
III. Acute GVHD targets recipient epithelium (skin, liver, stomach, intestines);
chronic GVHD is more like an autoimmune process, tissue fibrosis from
chronic exposure to Th2 cytokines
a. However, donor T cells may also have a graft-versus-tumor response
that decreases rates of disease relapse
b. Cord blood can be extremely expensive
c. Use haploidentical donors because they’re nearly universally available
for patients; can address by trying to deplete cells with TCRs and
infusions of Tregs, or calcineurin inhibitors to cause clonal deletion of
alloreactive T cells
 d. Post-transplant cyclophosphamide can decrease incidence of chronic
and severe acute GVHD
e. Have begun to experiment with mini-alloHSCTs to treat sickle cell
disease

SECONDARY IMMUNODEFICIENCY

I. Secondary immunodeficiency may present in anyone, most commonly

from prescription medication as well as from infection and cancer;
management depends on elucidating the defect in host defense
a. Ex. premature babies may have not had a chance to take up maternal
immunoglobulin
b. May be from diseases of other organ systems – chronic loss of protein
in urine or stool causing hypogammaglobulinemia
c. Severe malnutrition causes neutropenia, sickle cell disease can cause
infarct of spleen
d. Radiation therapy kills rapidly dividing cells, many antibodies are
anti-cancer or immunosuppressive agents, anti-inflammatory agents
and infections suppress different parts of immune system
i. Ex. pathogens like meningococci secrete proteases specific for
hinge regions of IgA dimer
e. Diseases may get into bone marrow, extensive burns lead to protein
loss and lack of barrier function of skin
f. Epstein-Barr Virus is a member of the herpes family, infects
epithelium of pharynx and B cells, can cause infectious
mononucleosis; mostly presents as nondescript illness in young
children and lends to immunity, but infection in adolescence and
beyond is much more severe illness
i. Notable histologic finding: atypical lymphocytes, large with
excess cytoplasm deformed by surrounding red cells
ii. Also has BCRF1 gene similar to human IL-10 gene, thus causes
B cells to proliferate
iii. But IL10 is normally secreted by Th2 cells after CD4 activation,
turns off Th1 cells that do cell-mediated immunity: thus EBV
turns off this whole arm of immunity and can cause significant
opportunistic infections
g. Can look for sensitization to TB by delayed-type hypersensitivity skin
test: deposit antigen under skin, if recognized from previous infection
after 2-3 days will get influx of lymphocytes and cytokines creating
palpable lump in skin
i. But immune system can be “distracted”: known positives for
TB can be read as negative during EBV acute illness
ii. TB is such a strong driver of cell-mediated immunity that
almost completely turns off Th2 arm, no antibodies to
tuberculus organism are really made
 h. Remember HIV presents with acute mono-like illness upon first
infection, and then asymptomatic until CD4 count drops low enough
to start getting opportunistic infections
i. Or tumors can infiltrate bone marrow and physically crowd out
lymphocytes
II. Clinical indicators of immunodeficiency: more than one pneumonia in a
decade, 2+ sinus or ear infections in a year, or infections in multiple
anatomic sites, recurrent abscesses or opportunistic infections
a. Note foreign bodies inside skin normally cause inflammatory reaction
with neutrophils and macrophages; cancer patients see very little of
this response
b. Look at Rebuck skin test: make a scrape on skin and cover with cover
slip to see how many WBCs migrate to surface – can be deficiencies in
leukocyte adhesion, or people with cancer have a harder time getting
cells from capillaries to injury
c. Many tumors produce TGF-B, does class-switch to IgA and suppresses
pro-inflammatory cytokines and proliferation of T and B cells
i. Also suppresses IL-1 release and killing capacity of monocytes
for intracellular threats
d. Tumors also secrete VEGF (vascular endothelial growth factor) to
promote vessel growth and inhibit maturation of dendritic cells so
they can’t present tumor antigens
III. Most important is immunodeficiency caused by drugs – may directly
damage immune cells, promote autoimmune reactions, and
immunomodulate cell function
a. Phenytoin is an anti-convulsant, causes dramatic loss of serum IgA in
some individuals; some people’s levels return to normal after
stopping therapy, some never do
i. Because degraded by NADPH P450 system with intermediate
hapden that is toxic and binds lymphocytes to kill them; most
people metabolize quickly enough not to be a problem but
homozygous variant gets toxicity; shared by many anti-
convulsants
b. Interestingly, immunosuppression leads to increased incidence of
cancers, but not the same ones in the general population – thought
that suppressing T cells may decrease T reg control
i. Risk from solid organ transplant and long-term
immunosuppression; BMTs may only be suppressed for 6 mos
ii. Also note persistence of opportunistic infectious complications
in late-stage status-post heart transplant

ETHICAL ISSUES WITH VACCINES

I. Vaccines have had an incredible impact on the incidence of infectious

diseases we’ve developed them for
 a. But of course, the money for this development is located in first-world
countries, which focus on diseases that are problems for them – Ebola
was endemic in Africa for decades before it became pandemic and
affected the US
b. Ethics of doing controls in vaccine trials: can avoid direct placebo by
giving one arm the vaccine right away and one arm a few weeks later;
need a control to prove efficacy
c. Data support that children make strong immune responses to
vaccines early in life and that their ability to respond to other threats
is not diminished; delaying vaccine schedule delays protection at a
very vulnerable time
i. Most common reasons parents state for not vaccinating are
that they might cause harm or overload immune system, or
that the disease is a danger to their child
ii. Yet measles requires hospitalization in 1/4 cases and fatal
subacute sclerosing panencephalitis several years after
infection in 1/10,000
iii. Measles is spread by aerosol – extremely infectious in public
places
d. Much vaccine rejection comes from 1998 paper in the UK about a link
between MMR vaccine and autism with GI symptoms – basically
terrible research, invented results, ridiculous methods, very shady
funding sources, but still led to resurgence of measles in Europe
i. Huge body of further research all showing no association
e. Medical contraindications to vaccines are immunodeficiencies
(primary, HIV infection, cancer chemotherapy) making live attenuated
vaccines too dangerous, or severe allergy to vaccine/component
i. Immunosuppressed people aren’t likely to develop resistance
when vaccinated anyways, wait till they’re off treatment
ii. Can still go out in public by herd/community protection: if
enough people are immune, an epidemic can’t spread
f. Other objections include religious (prohibitions against taking life,
prohibitions against porcine origin) all firmly rejected by religious
authorities
g. HPV vaccine can prevent 75% of
cervical/vaginal/vulval/anal/rectal/oropharyngeal cancers in women
and nearly 70% of penile/anal/rectal/oropharyngeal cancers in men,
yet the penetration rate into population is not high and many health
care providers do not recommend to all their pts, especially boys
i. And must be given before first exposure to HPV, thus
recommended around 12
h. AAP does not recommend discharging patients for refusing
vaccinations, because can often change parents’ minds

MONOCLONAL ANTIBODIES AS DRUGS

I. The movement to use antibodies as drugs began with Rituximab, an
antibody against CD20 on the surface of B cells; by 2019 predicted that
biologics will be half of the monetary value of the pharmaceutical
industry, and up to half of that value will be in monoclonal antibodies
a. Make these antibodies by combining splenic cells that make
antibodies with myeloma cells that will live forever-ish – then just
have to select for the cells making your antibody with ELISA
b. Knock out HGPRT in the myeloma cell, so can’t do salvage pathway for
purines and only do de novo synthesis, and only when precursors are
available, so you control their survival
c. Can control antibody structure for your needs – make it gigantic to
keep half-life long and minimize number of treatments, or make it
smaller so it can clear toxins
d. Can be various fractions mouse antibody (“o”); our immune system
will start to recognize them after one or two treatments and destroy
them, so graft light chain to human back bone (chimeric, “xi”), or
mouse CDR onto human framework (humanized, “zu”) or
transgenically put human light and heavy chain loci into mouse
(human, “u”)
e. Can make the mice by either knock out and knock in, or more
precisely do targeted substitution in embryonic stem cells
II. Rituximab: chimeric, approved for CD20+ non-Hodgkins lymphoma and
now rheumatoid arthritis; can monitor CD20+ cell activity with CD19
marker; after a few doses, levels drop to zero and can persist there for
years if you’re elderly or otherwise compromised
a. Does so by binding to Fcgamma-R3 and bringing about antibody-
dependent cellular cytotoxicity; would need to use IgG3 isotype to get
CDCC (complement-dependent cellular cytotoxicity) but has a shorter
half-life
b. Can act by ADCC: bind FcgammaRs and bring about killing of target by
recruiting NK cells, macrophages, or neutrophils; can increase by
glycosylating Fc and thus receptor binding
c. Can act by CDCC, but rare because of instability
d. Can block a receptor or ligand (antagonist), usually use IgG4 and
eliminate ADCC function by decreasing glycosylation
e. Can be an agonist and increase signaling, activating antibodies like
anti-PD1, but risking autoimmune disorders by revving signaling
f. Four Fcgamma-Rs, and four sub-types of human IgG – IgG1 has
longest half-life and most provoking of ADCC, so most commonly
used; the stronger it binds to FcRN (recycling receptor) the longer the
half-life
g. Can creatively modify antibodies, like TRAP molecules with anti-VEGF
and FcR blocking angiogenesis; or bi-specific T cell engagers that
bring malignant targets to T cells for lysis by co-expressing CD19 and
CD3 binding regions – incredibly high fevers from cytokine storm; can
 combine antibodies and TCRs to make antibodies more able to kill
what you want
h. Most recently and promising: antibody-drug conjugates, antibodies
specific for your antigen with a toxic payload like chemotherapy,
allows very targeted delivery with reduced side effects
i. Ex. TDM1 binds target, is internalized, and causes cell lysis
i. Other examples: Trastuzumab is IgG1 and prevents dimerization, does
ADCC; urelumab is IgG4 and an agonist, still in trials; nivolumab is
anti-PD1, IgG4, antagonist, approved for melanoma, lung and kidney
cancer