IMMUNOGLOBULINS STRUCTURE AND FUNCTION BSc Public Health 5th week, 2014

IMMUNOGLOBULINS Definition Glycoprotein molecules that are present on B cells (BCR) or produced by plasma cells (usually referred to as antibodies) in response to an immunogen (antigen that provokes immune response)

B CELL ACTIVATION Gene rearrangement in an immature B cell leads to the expression of membrane-bound IgM and IgD on the mature B cell surface. After encounter with an antigen this isotype is produced in a secreted form by the plasma cell (IgM largely while IgD only is small amounts). All isotypes (IgA, IgD, IgE, IgG and IgM) can be made in two forms: the bounded immunoglobulins serving as BCRs, and soluble secreted antibodies by the plasma cells. (The difference b/w membrane bound and secreted is a hydrophobic part and hydrophilic parts at the carboxy terminus, respectively).

Immunoglobulin STRUCTURE 2x Heavy chain (light blue) 2x light chain (dark blue) Variable regions  antigen binding Constant regions disulfide bond carbohydrate CL VL CH2 CH3 CH1 hinge region VH

mIg sIg BCR (B cell receptor) Antibody Transmembrane domain Associated chains for signaling Cytoplasmic domain SOLUBLE (freely circulating) MEMBRANE BOUND! Antigen binding  effector functions Produced by plasma cells Antigen recognition  B cell activation

ANTIBODY DOMAINS AND THEIR FUNCTIONS Antigen recognition Variable domain Ag Ag Constant domains Effector functions

B CELL ACTIVATION B cell BCR oligomerization results in B cell activation, proliferation and differentiation

ANTIGEN BINDING Antigen Binding Fragment (Fab) Complement binding site Placental transfer Constant fragment (Fc) Binding to Fc receptors on phagocytic cells

HYPERVARIABLE REGIONS B cell development in the red bone marrow  DNA recombination (somatic gene rearrangement) of gene segments encoding variable domains of heavy and light polypeptide chains is responsible for generation of B cells with highly variable specificity Epitope CDR1 CDR2 CDR3 Light chain Heavy chain CDR = complementarity determining region = hypervariable region

DIFFERENT VARIABLE REGIONS  DIFFERENT ANTIGEN-BINDING SITES  DIFFERENT SPECIFICITIES the same applies to TCRs!

Sequence variability of H/L-chain constant regions ISOTYPE (CLASS) Sequence variability of H/L-chain constant regions IgG - gamma (γ) heavy chains IgM - mu (μ) heavy chains IgA - alpha (α) heavy chains IgD - delta (δ) heavy chains IgE - epsilon (ε) heavy chains

PHASES OF B CELL RESPONSE Gene rearrangement in an immature B cell leads to the expression of membrane-bound IgM and IgD on the mature B cell surface. After encounter with an antigen this isotype is produced in a secreted form by the plasma cell (IgM largely while IgD only is small amounts). All isotypes (IgA, IgD, IgE, IgG and IgM) can be made in two forms: the bounded immunoglobulins serving as BCRs, and soluble secreted antibodies by the plasma cells. (The difference b/w membrane bound and secreted is a hydrophobic part and hydrophilic parts at the carboxy terminus, respectively).

ISOTYPE SWITCHING Isotype Switching during B Cell Development During the initial stages of a B cell’s primary response to antigen, it produces and secretes IgM. Later in the primary response or during subsequent responses, different heavy chain isotypes may be expressed by the progeny of the original IgM-producing clone. Such “switching” occurs at the DNA level, resulting in the production of an Ig protein with the same V region but a different C region. Thus, over the lifetime of a B cell clone, it may produce antibodies of the same specificity but different isotypes.

MAIN CHARACTERISTICS OF ANTIBODY ISOTYPES IgG1-IgG4 IgA1-IgA2

FLEXIBILITY OF ANTIBODIES

ANTIBODY PRODUCTION DURING THE PRIMARY AND THE SECONDARY IMMUNE RESPONSES Level of antibodies secondary response against antigen A Primary response against antigen A primary response against antigen B Days napok Antigen A Antigen A and B

EFFECTOR FUNCTIONS OF ANTIBODIES Antibody-mediated immune responses Fab part: NEUTRALIZATION Fc part: OPSONIZATION followed by opsonized phagocytosis (macrophage; IgG) ADCC (NK cell; IgG) mast cell degranulation (parasite, allergy; IgE) COMPLEMENT ACTIVATION

NEUTRALIZATION

Complement binding site Binding to Fc receptors Antigen binding Complement binding site Placental transfer Binding to Fc receptors

OPSONIZED PHAGOCYTOSIS Flagging a pathogen Antigen binding fragment (Fab) binds the pathogen  the Fc region is accessible for Fc-receptors of phagocytic cells, facilitating (speeding up) the process of phagocytosis

Opsonization facilitate and accelerate the recognition of the pathogens by phagocytes Main opsonins: antibodies Complement molecules Acute-phase proteins (CRP, SAP) Phagocytes must express receptors for the opsonins: IgG  FcγRI C3b  CR1

Antibody Dependent Cellular Cytotoxicity (ADCC)

MAST CELL DEGRANULATION FcεRI + IgE Mast cells, basophils and activated eosinophils in mucosal surfaces play a role in the defense against parasites and express the FcεRI. FcεRI has such high affinity that the IgEs, specific for many different antigens- cannot dissociate. Upon antigen binding and FcεRI cross-linking the mast cell is activated (degranulation) inflammatory mediators released, acting on vessel permeability- swelling, pain etc, and acting on smooth muscle cells  Sneezing, coughing, vomiting, diarrhea. Directly killing the parasite by toxic granule content or Indirectly flushing it. An unnecessary response to an innocuous substance (pollens etc.) are an unfortunate side effect of the highly specialized and powerful antibodies. (A) High-affinity FcRs on the surface of the cell bind antibodies before it binds to antigen. (mast cell) (B) Low-affinity FcRs bind multiple Igs that have already bound to a multivalent antigen. (macrophage, NK cell)

Complement binding site Binding to Fc receptors Antigen binding Complement binding site Placental transfer Binding to Fc receptors

Complement binding site Binding to Fc receptors Antigen binding Complement binding site Placental transfer Binding to Fc receptors FcRn on the placenta facilitate the transfer of maternal IgG to the body of the fetus

PRODUCTION OF IMMUNOGLOBULINS IgG transport is so efficient that at birth babies have as high a level of IgG in their plasma as their mothers These transfers are a form of passive immunization. The babies protection by IgG and IgA is against those pathogen that the mother has mounted The children are most vulnerable during the first year of life (esp.3-12m) when maternal IgGs have disappeared but the de novo synthesis is at low level

Pathological consequences of placental transport of IgG (hemolytic disease of the newborn) Passive anti-D IgG anti-Rh IgM Rhesus incompatibility: In case a fetus is Rh+ (meaning he expresses the D antigen on his RBCs surface) and the mother is RH- (no D antigens and no anti-D antibodies) after the first delivery when some fetal RBCs mix with maternal circulation, the mother will initiate a primary immune response towards the D antigen. These antibodies as it is the first immune response will be of IgM isotype and therefore not able to pass the placenta. However, with time, isotype switching might take place that will result in the production of IgG antibodies against the D antigen. These are now able to pass through the placenta, thus, in the second pregnancy if the fetus is Rh+ his RBCs will be attacked by maternal anti-D IgG antibodies, causing the mild to severe ‘hemolytic disease of the newborn’ (destruction of red RBCs anemia) To avoid that, i.m. anti-D IgGs are administered to the mother. These will bind any D antigens on fetal RBCs that entered the mothers circulation and prevent her from developing anti-D antibodies. The IgGs eliminate the RBCs coated with D antigens through opsonisation and elimination by macrophages and granulocytes. … One might say, well IgGs pass the placenta, then these anti D antibodies can attack the fetus RBCs- true, but the clinical course of such an event is benign and requires no treatment.

SECRETORY IgA AND TRANSCYTOSIS J C S s ‘Stalk’ of the pIgR is degraded to release IgA containing part of the pIgR (the secretory component) J C S s J C S s MUC US J C S s J C S s IgA and pIgR are transported to the apical surface in vesicles J C S s Epithelial cell pIgR and IgA are internalised Polymeric Ig receptors are expressed on the basolateral surface of epithelial cells to capture IgA produced in the mucosa J C S s plasma cells located in the submucosa produce dimeric IgA