B-Lymphocytes and Antibodies

• B lymphocytes originate and mature in the bone marrow.
• The stages to a mature naive B cell are: pluripotent haemoatopoietic stem cell, common lymphoid progenitor, pro-B cell, and finally, a mature naive B cell.
• Antibodies are also known as immunoglobulins.
• Antibodies are soluble proteins produced and secreted by B lymphocytes to bind to infectious microorganisms and prevent infection.
• Antibodies prevent infection by inhibiting attachment to host tissue, activating complement, enhancing phagocytosis, and inducing granulation of mast cells, eosinophils, and neutrophils.

Antibody Regions

• The Fab region binds antigen.
• The Fc region does not directly bind antigen but mediates antibody function.
• An antibody is comprised of 2 identical heavy chains (Mr 50K) and 2 identical light chains (Mr 25K), joined by di-sulphide bonds.
• Adjacent N-terminal VL and VH domains form the binding site for antigens, which is the Fab part of the antibody.
• The hinge region increases the ability of the antibody to bind to 2 sites of the same antigen, strengthening the affinity of binding.
• The heavy chain mediates functions such as activation of complement and enhancement of phagocytosis.

Antigens and Epitopes

• An antigen is any molecule that binds specifically to an antibody.
• An epitope is a specific site on the antigen that binds to the antibody.
• Individual antigens can bind to different antibodies of different specificities because an antigen has several different epitopes.
• An immune complex is an antibody bound to an antigen.
• Antibodies bind to antigens via the V region (VH and VL domains) through 3 loops between beta-strands called Complementarity Determining Regions (CDRs).

Antibody Effector Functions

• Effector functions are mediated by the Fc region.
• Activation of complement occurs when C1q is activated by antibodies in immune complexes or bound to the surface of a pathogen, leading to lysis or phagocytosis via complement receptors (CR1, CR3).
• Phagocytosis of immune complexes occurs via Fc receptors on phagocytic cells.
• Antibodies transport across epithelium to mucosal surfaces, blocking pathogen entry.
• Maternal antibodies transfer to the foetus through the placenta for protection.

Antibody Classes

• IgM has 5 antibodies with a J chain, a serum level of 1.5mg/ml, and functions in complement activation.
• IgD has 1 antibody, a serum level of 0.04mg/ml, and signals B cells to be activated.
• IgG has 1 antibody, a serum level of 13-14mg/ml, and functions in complement activation, Fc receptor binding for phagocytosis, and transplacental transport.
• IgA has 2 antibodies with a J chain, a serum level of 2mg/ml, and transports across mucosal epithelium to prevent pathogen entry.
• IgE has 1 antibody, a low serum level, and causes degranulation of immune cells to kill pathogens.
• IgG and IgM are highest in concentration in the serum.
• In complement activation, C9 pore formation causes lysis, and the C3b receptor causes phagocytosis of the pathogen involving antibodies IgM, IgG1, IgG2, IgG3.
• Fc gamma receptor binding results in phagocytosis involving IgG1, IgG3.
• IgE antibodies bound to high affinity Fc-Epsilon receptors on mast cells and eosinophils trigger degranulation, releasing histamine, serotonin, proteases, cytokines, and leukotrienes when an antigen binds to the IgE/Fc receptor complex.
• IgA is largely present on mucosal surfaces, preventing pathogen entry.

Antibody Diversity

• Antibody diversity results from random recombination of VDJ (heavy chain) and VJ (light chain) segments, and addition of nucleotides at VD, DJ, and VJ junctions.
• B cells assemble cell surface forms of IgM monomer (sIgM) and IgD (sIgD) with the same antigen binding site in the bone marrow, but different classes of heavy chain.
• B cells that express sIgM + sIgD that recognise self antigens undergo apoptosis or further DNA rearrangement, while those that do not recognise self antigens or bind weakly migrate to the spleen and lymph nodes.
• IgD and IgM are the first two antibodies produced in an antibody response.
• B lymphocytes in the spleen or lymph nodes are activated when antigens are recognised by B cells on macrophages or follicular dendritic cells (FDCs) via Fc receptors (immune complexes) and complement receptors (C3b, C4b modified).
• These antigens bind sIgM or sIgD to deliver signal 1.
• Antigen is internalised by the B cell and associated with peptides and MHC II in antigen processing.
• The Th cell recognises MHC II and sends signal 2 to the B cell (via CD154 binding to receptor CD40).
• Th secretes cytokines that induce B cell proliferation, differentiation, and production of antibodies (soluble form of IgM and IgD).
• B cell proliferation can result in class switching, short-lived plasma cells (2-3 days), and long-lived B memory cells.
• Class switching in B cells, determined by Th cell cytokines, leads to Th1 and Th2 responses.
• A Th1 response to intracellular pathogens, IL-4 and IL-21 activate IgG1 and IgG3, effective in removal via complement activation and Fc receptor binding.
• A Th2 response to multicellular parasites causes degranulation of mast cells, eosinophils mediated by IgE which is activated by IL-4.
• IgA, activated by IL-21, is transported to mucosal surfaces to block entry of most pathogens.
• IL4 can block IL-21, therefore blocking IgA activation.
• Class switching occurs simultaneously with somatic hypermutation, both initiated by Activation Induced Cytidine Deaminase (AID), which converts cytosine to uracil and therefore breaks correct base pairing.

Somatic Hypermutation

• Somatic hypermutation of B cells initiates diversity, competition, apoptosis, and evolution.
• More mutated forms of antigen receptors differ in affinity to the antigen.
• Competition results in B cells with antigen receptors with higher affinities surviving and proliferating by binding to the Fc receptor of FDC.
• Apoptosis occurs to B cells with lower affinity antigen receptors.
• Evolution of memory B cells now produces antibodies with higher affinity.
• The secondary response is stronger than the primary response due to affinity maturation caused by class switching and somatic hypermutation.
• Class switching causes an antibody released that is most appropriate for the pathogen.
• Somatic hypermutation results in high affinity plasma and memory cells the 2nd time around.