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October 7, 2004
IMMUNITY
ADAPTIVE
CELL MEDIATED
INNATE
EFFECTOR SYSTEMS
Fc Receptors
Complement
HUMORAL
ANTIBODIES
Antibodies are produced by B lymphocytes
and plasma cells but perform their effector
functions at sites distant from their
production
Many of the effector functions of antibodies are mediated by
the heavy chain constant regions and different Ig heavy chain
isotypes serve distinct effector functions
Activation of the classical pathway of complement
Effective in lysing bacteria
Antigen receptor on naïve B lymphocytes
First antibody made during the immune response
Opsonization of Ags for phagocytosis by
macrophages and neutrophils
Activation of classical pathway of complement
Antibody-dependent cell-mediated cytotoxicity
(ADCC) by NK and macrophages
Neonatal immunity - transferred across the
placenta and gut
Secreted into the lumens of the GI and
respiratory tracts
Provides protection against pathogens
that attack at the mucosal surfaces
Antibody-dependent cell-mediated cytotoxicity
involving eosinophils - parasite infection
Mast cell degranulation - immediate
hypersensitivity or allergy
Antigen receptor of naïve B lymphocytes
Although most effector functions are mediated by
the heavy chain, the functions are triggered
by the binding of antigen to the variable region
Immunofluorescence
Takes advantage of the fact that certain
dyes such as fluorescein and rhodamine
absorb light at one wavelenght and emit
it at another characteristic wavelength
Fluorescent dyes can be attached to Abs
without altering their affinity. By using
different filters and dyes it is possible to
look simultaneously at multiple antigens.
Direct and Indirect Detection of Surface Antigens
Fluorescent activated cell sorter
(FACS)
Measures the fluorescent intensity
of single cells
The simplest form of the
instrucment
instrument counts each cell and
records the level of fluorescence analysis or flow cytometry -FLOW
Can also be used to sort cells FACS
Analysis in one dimension
Analysis in two dimensions uses two different fluorchromes
attached to Abs specific for different antigens
Flow cytometry can also be adapted to
look at the presence of intracellular
proteins such as cytokines. Non-ionic
detergents at low concentrations are
used to permeabilize the cells before
the addition of antibody.
Cells were first stained with an antibody specific for the
cell surface protein CD4, then permeabilized and stained
for the intracellular cytokine IFN-g
Antibodies are effective in clearing pathogens
and activating an inflammatory response in
part because they bind to Fc receptors
present on cells
Fc
There are three families of Fcg receptors
FcgRI or CD64
FcgRII or CD32
FcgRIII or CD16
FcgRI or CD64
Expressed on macrophages and neutrophils
Requires an associated g (or ) chain for expression and function;
in g chain knock-out mice there is no expression of FcgRI.
g chain has ITAM (YXXL) motifs for signaling.
Can mediate ADCC and phagocytosis in response to X-linking
Ligation also leads to macrophage activation and cytokine production
Only receptor that binds monomeric IgG with high affinity
FcgRII or CD32
Has signaling motifs in its cytoplasmic tail
FcgRIIA the motif is an ITAM (activation)
FcgRIIB the motif is an ITIM (inhibition)
FcgRII or CD32
FcgRIIA
Expressed on macrophages, neutrophils, eosinophils.
Ligation leads to uptake.
Present in the human, but not the mouse.
FcgRII or CD32
FcgRIIB -- an inhibitory receptor
Acts as an inhibitory receptor on B cells; crosslinking of surface IgM
leads to proliferation only when F(ab)’2 is used. FcgRIIB bind the Fc of
the intact Ab and provides an inhibitory signal.
FcgRIIB also plays an inhibitory role in mast cell degranulation
through FcRI.
FcgRIII or CD16
FcgRIIIA, a transmembrane receptor is found on monocytes,
macrophages, NK (the only Fc receptor) and T cells. g and/or 
chains are required for surface expression.
FcgRIIIB is present on human neutrophils. It is secured in the
membrane by a glycosyl phosphatidyl inositol (GPI) anchor.
Antibody-Dependent Cell Mediated Cytotoxicity
ADCC
Leukocytes with Fc receptors bind to antibody-coated cells
and destroy them
Fc receptors are required
for antibody-mediated
tumor protection
Mice were injected weekly
with 2 ug/ml of therapeutic
antibody
Fcg-/- lack FcgRI and FcgRIII
4D5 - murine anti-HER2/neu
Herceptin - humanized
anti-HER2/neu
HER2/neu is a protein present
on breast and other cancers
Rituxan - chimeric anti-CD20
CD20 is present on lymphoma
cells
Inhibitory FcgRIIB receptors also modulate the in
vivo cytotoxicity of antibodies against tumors
Mice were injected with a sub-therapeutic dose (0.2ug weekly)
4D5 is a murine anti-HER2/neu; Trastuzumab is the same as Herceptin,
humanized anti-HER2/neu
There is a balance between activating and inhibitory receptors
From Ann. Rev. Immunol. 19:275, 2001.
Uptake of antigen by macrophages (J. Immunol. 168:3697, 2002)
Fc receptors influence the efficiency of antigen presentation
Cell number
Complement activation by antibodies also
is important for effective clearing of
pathogens and activation of an inflammatory
response
How was complement
discovered?
Lysis is dependent on
two factors: Ag-spcific
agent that is heat
stable and a separate
agent that is heat labile
and complements Ab
and causes lysis.
Complement - an important effector system has important
functions related to immune defense
1. Lysis of cells. This is the original function identified
and causes hypotonic cell death by making hole. It is
not effective against organisms with cell walls such as fungi
and Gram positive bacteria
2. Opsonization. Macrophage and PMNs have FcRs and
at least two different kinds of complement receptors that
aid in phagocytosis. C3b, a cleavage product formed during
activation is the major player. Antigen coated with C3b binds
to cells bearing complement receptors and if the cell is
a phagocyte the antigen will be phagocytosed.
Complement - an important effector system has important
functions related to immune defense
3. Inflammation. Peptides generated during activation
play a role in inflammation. The anaphylatoxins of which
C5a is the most potent bind receptors on mast cells and
basophils and cause degranulation with the release of
pharmacologically active mediators which induce smoothmuscle contraction and increases in vascular permeability.
C3a, C5a and C5b67 act as chemoattractants and induce
monocytes and neutrophils to adhere to vascular endothelial
cells, extravasate through the endothelial lining of the
capillaries and migrate to the site of complement activation
in the tissue.
Complement - an important effector system has important
functions related to immune defense
4. Immune clearance. Removes immune complexes from
the circulation and deposits them in the liver where they
are degraded. C3b facilitates immune complex binding to CR1
on RBCs. In the liver and spleen the complexes are stripped from
the RBC and phagocytosed. Complement also helps to solubilize
immune complexes.
5. Enhanced immune response. CD21, part of the coreceptor on the B cell, binds cleaved C3. Recently it has
also been shown that C3 is required for optimal expansion
of T cells during a systemic viral infection.
6. Virus neutralization. Complement mediates viral
neutralization by facilitating viral aggregation and by coating
the viral surface.
There are three pathways through which
complement can be activated
ADAPTIVE
INNATE
Classical pathway
Requires Ab
Mannose binding lectin pathway
Alternative pathway
The central player is C3 and all three
pathways focus on generating activated
C3
CLASSICAL PATHWAY
Antigen-antibody
C1qr2s2
(C1)
Activated C1
C2
C4
C4b
C2b
C4b2a
(C3 convertase)
C4a
C3
C3b
MBL
Activated C1-like
MASP complex
LECTIN PATHWAY
C3a
C4b2a3b
(C5 convertase)
C5
Ba
B
C3a
C3
C3b
C3bBb
(C3 convertase)
Microbial Surfaces
Factor D
ALTERNATIVE PATHWAY
MAC
C5b
C6 C7 C8 C9
C5a
C3bBb3b
(C5 convertase)
Cleavage of a labile thioester bond in C3, an
abundant serum protein, creates an active group
that allows C3 to attach to other proteins through
NH2 or OH groups
C
Clq has 6 legs. When C1q binds the associated enzymes C1r and
C1s are activated. The affinity of C1q for Fc is weak so that at least
two Fcs must be bound to have activation.
YY
Activated proteases cleave C4 and C2. The cleavage products
(C4b2a) form a new protease that cleaves C3 into C3a and C3b
Association of C3b with the protease (C4b2aC3b) alters its
specificity so that it now cleaves C5. C5b is the first protein in
the membrane attack (MAC) complex
IgM is a very rigid molecule
Binding of Ag by IgM leads to a conformational
change exposing the C1q binding sites.
There are multiple sites in each IgM molecule so
one IgM can bind C1q and activate the
complement cascade
IgG has only two sites per molecule. In order to achieve effective
C1q binding and complement activation two IgG molecules must
bind close to each other.
YY
Mannose Binding Protein
Mannose Binding Protein
Recognizes carbohydrate antigens common to pathogens
Like C1q it has two associated proteases that become activated upon
binding. These proteases cleave C4 and C2 and so the Lectin pathway
is identical to the Classical pathway except for the first activation
step
C3 is always being spontaneously hydrolyzed. Usually it decays.
However if it is in the proper environment such as a microbial surface
it will complex with Factor B which will be cleaved by Factor D and will
form a C3 convertase.
The cleavage products produced during complement activation are
very important effector molecules
Anaphylotoxins: C3a, C4a, C5a
C3a , C4a and C5a are anaphylatoxins
Small peptides that causes smooth muscle contraction,
increases vascular permeability and mast cell and
basophil degranulation.
C5a is also a chemoattractant and activator of WBC
Anaphylatoxins
Also amplify the inflammatory response by inducing
the synthesis of pro-inflammatory cytokines.
Their receptors are present on many cell types
including leukocytes, mast cells, macrophages,
endothelial cells, astrocytes and microglial cells
Anaphylatoxins
Anaphylatoxins trigger a cascade of events that
contributes to the pathogenesis of a number of
disease and conditions including hypersensitivity
reactions, endotoxin shock, multiple organ
failure and respiratory distress syndrome.
Neutralization of the effects of anaphylatoxin can
be of substantial clinical significance
Cleavage products of C3b are opsonins
They bind to receptors present on the surface of
phagocytes
Formation of the membrane attack (MAC)
complex
F
Regulation of complement
DAF: prevents assembly of and accelerates
disassembly of C3 convertase
displaces Bb from C3b
displaces C2b from C4b
Homologous restriction factor (HRF)
and CD59: bind C5b678 preventing C9
binding and MAC formation
Reactive species quickly decay
Complement Receptors
Type 1: CR1 or CD35
expressed on RBCs, m
B cell and some T cells
, neutrophils, eosinophils,
cells expressing CR1 can bind immune
complexes and particulate Ags to which C3b is
attached, facilitating Agclearance
Type 2: CR2 or CD21
expressed on B cells and some T cells
bind degradation products of C3b
potentiates response to Ags with C3b
degradation products
Type 3 and 4: CR3 and CR4
found on monocytes, m , neutrophils, NK
and some T cells
bind C3b degradation product
binding of complement coated particles
triggers phagocytosis by phagocytic cells
C3a/C4a and C5a
present on mast cells, basophils and
granulocytes and can be used for their
degranulation
Immune complex clearance
Opsonization
Enhances immune response
Mediator Release
Functions of complement
Complement and Fc receptors synergize in promoting phagocytosis