October 7, 2004

IMMUNITY ADAPTIVEINNATE CELL MEDIATEDHUMORAL ANTIBODIES EFFECTOR SYSTEMS Fc Receptors Complement

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

Direct and Indirect Detection of Surface Antigens

instrument -FLOW

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- 

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 Fc  receptors Fc  RI or CD64 Fc  RII or CD32 Fc  RIII or CD16

Fc  RI or CD64 Requires an associated  (or  ) chain for expression and function; in  chain knock-out mice there is no expression of Fc  RI.  chain has ITAM (YXXL) motifs for signaling. Expressed on macrophages and neutrophils 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 ADCC = antibody dependent cellular cytotoxicity

Fc  RII or CD32 Has signaling motifs in its cytoplasmic tail Fc  RIIA the motif is an ITAM (activation) Fc  RIIB the motif is an ITIM (inhibition)

Fc  RII or CD32 Fc  RIIA Expressed on macrophages, neutrophils, eosinophils. Ligation leads to uptake. Present in the human, but not the mouse.

Fc  RII or CD32 Fc  RIIB -- 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. Fc  RIIB bind the Fc of the intact Ab and provides an inhibitory signal. Fc  RIIB also plays an inhibitory role in mast cell degranulation through Fc  RI.

Fc  RIII or CD16 Fc  RIIIA, a transmembrane receptor is found on monocytes, macrophages, NK (the only Fc receptor) and T cells.  and/or  chains are required for surface expression. Fc  RIIIB 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 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 Fc  -/- lack Fc  RI and Fc  RIII

Inhibitory Fc  RIIB 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 Complement - an important effector system has important functions related to immune defense 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 smooth- muscle 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 co- receptor 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.

Classical pathway Requires Ab Mannose binding lectin pathway Alternative pathway There are three pathways through which complement can be activated ADAPTIVE INNATE

The central player is C3 and all three pathways focus on generating activated C3

C 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 NH 2 or OH groups

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.

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.

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

F Formation of the membrane attack (MAC) complex

Immune complex clearance Opsonization Enhances immune response Mediator Release

Functions of complement

Complement and Fc receptors synergize in promoting phagocytosis