B Cell Activation and the Humoral Immune Response

Each B cell after stimulation with Ag, proliferate and produces 4000 daughter cells and these cells produced 10 12 Ab molecules.

Humoral (Antibody-Mediated) Immunity Involves production of antibodies against foreign antigens. Antibodies are produced by a subset of lymphocytes called B cells. B cells that are stimulated will actively secrete antibodies and are called plasma cells. Antibodies are found in extracellular fluids (blood plasma, lymph, mucus, etc.) and the surface of B cells. Defense against bacteria, bacterial toxins, and viruses that circulate freely in body fluids, before they enter cells. Also cause certain reactions against transplanted tissue.

Antibody Protection of the Host

Humoral immune response properties Humoral immune response against non-protein Ag dose not need to helper T cells. Activation of B cells resulted in memory or plasma cells. Isotypes switching and affinity maturation are helper T cell dependent. The quantity and quality of primary and secondary humoral immune response is different.

Dynamics of Antibody Production Initial antibody produced in IgM Lasts 10-12 days Followed by production of IgG Lasts 4-5 days Without continued antigenic challenge antibody levels drop off, although IgG may continue to be produced.

Comparison of Primary and Secondary Responses Time course Antibody titer Antibody class Antibody affinity Study Guide Compare and contrast the primary and secondary immune responses.

Immunologic Memory Virgin lymphocyte pool PRIMARY RESPONSE effector cells memory cell pool Study Guide What is the significance of immunologic memory? SECONDARY RESPONSE effector cells memory cell pool

ITAM, immuno receptor tyrosin- based activation motif

CR2,CD19,CD81= BCR Complex, in this model of activation using complement receptor CD19 is phosphorylated and enhance B cell activation. In this way antigen is 1000 times more immunogen

Types of B cell Antigens T-independent (TI) T-cell dependent (TD) present T cell Ag T-independent antigens bacterial polysaccharides low dose LPS repeating surface molecules on viruses TLR signaling can synergize with this type of signal T-dependent antigens usually require a protein component, but B cell may recognize a non-protein part of the complex (e.g. a surface sugar or lipid of a virus) most pathogens contain both T-independent and T-dependent antigens mitogenic 'activation' signal mitogenesis BcR signal but not mitogenic differentiation

Types of antigen T-independent (TI) antigens T-dependent (TD) antigens induce division/differentiation by BCR signaling alone bacterial polysaccharides, repeating surface molecules on viruses T-dependent (TD) antigens activate via BCR but depend on additional signals from helper T cells to cause division/differentiation any antigen containing protein Most pathogens contain both T-I and T-D antigens Only TD antigens can induce Germinal Center responses

B cell activation Some responses require T help whereas other do not Thymus-dependent because T cells are required Thymus-independent because T cells are not needed T-independent antibody response generally have 1. no memory 2. no isotype switching 3. no somatic mutations

T-Independent (type II) Responses Old Paradigm: Multivalent Antigen B cell B cell plasma cells B cell plasma cells DC/Mø New Model: (BAFF)

T-Dependent Responses Antigen DC T cell T cell B cell plasma cells Dendritic Cell (DC) internalizes antigen (Ag), processes into peptides, presents peptides together with MHC molecules to T cells B cell binds Ag via surface Ig, transmits BCR signals and presents peptides to T cells, receives T cell help (growth and differentiation factors) Secretes Antibody (Ab)

B cells are antigen-presenting cells BCR cross-linking induces antigen internalization to endosomes antigen is proteolysed to peptides peptides associate with MHC class II MHC class II-peptide complexes traffic to surface of B cell B cells present antigen recognized by their BCR ~105 x more efficiently than other antigens ( BCR needs 104-106 lower dose)

B cells are antigen-presenting cells

B cell antigen presentation and the concept of linked help protein sugar T Protein Specific T cell CD40L Cytokines BCR endosome MHC II Polysaccharide Specific B cell

Cardinal features of B - T interaction

Role of CD40 in B cell activation TCR triggering up-regulates CD40L on T cell CD40 signaling promotes B cell activation CD40L-deficiency = 'hyper-IgM syndrome' (no isotype switching, no Germinal Centers) CD40 signaling also important in DC, MØ function Germinal center formation is CD40-CD40L interaction dependent increased expression of cell cycle molecules, survival molecules, cytokines, promotes isotype switching

B-T interaction summary BCR signals induce costimulatory molecules (B7.2/CD86, ICAM1 etc), prosurvival molecules (e.g. Bclxl), entry to G1, antigen presentation CD40L triggers CD40 -> synergizes with BCR signals to promote mitosis T cell derived cytokines promote proliferation, differentiation, isotype switching: IL2, IL6 promote differentiation IL4 -> IgG1, IgE IFNg -> IgG2a, IgG3 TGFb and IL5 -> IgA Many other molecules involved in T-B interactions ICOS/ICOSL (essential for GC response) SLAMM/SAP (promote prolonged B-T interaction essential for GC response) ICAM1/LFA1, CD30/CD30L, CD27L/CD27, OX40L/OX40, ...

Innate features of pathogens act as B cell costimulators Pathogen multivalency provides a level of BCR crosslinking optimal for activation Many pathogens activate TLRs TLR signaling synergizes with BCR signal Some induce type I IFNs IFNa/bR signaling synergizes with BCR Many activate the complement cascade and become C3d coated - complement receptor (CR) crosslinking synergizes with BCR signal

After appropriate activation the B cell differentiates into an antibody secreting cell, also known as a Plasma Cell B cell Plasma Cell membrane Ig secretory Ig blimp1 XBP1 Electron microscopy Images from Molecular Biology of the Cell Alberts et al., 3rd Edition., 1994

Production of membrane vs secreted Ig

B cell antibody response - clonal replication enters into a higher order upon plasma cell differentation 3 days 12 divisions 1 day differentiation 1 day 104 Ab/cell/sec naive B cell activated B cells plasma cells antibodies 4,096 >1012 1 212 = 4,096 bacteria - dividing every ~60 min 5 days = 2120 divisions = 1.3x1036

Plasma Cells are antibody secreting cells Two types: 1. Plasma cells generated early in the primary response - short-lived (~ few days) - typically low affinity - form in T-independent and T-dependent responses - home to red pulp of spleen, medullary cords of lymph nodes - IgM but also IgG and other isotypes 2. Plasma cells generated later in the primary response and that predominate in secondary responses - arise predominantly from germinal centers (in primary) or from memory B cells (in secondary) - long-lived (possibly several months) - often home to bone marrow, gut, lactating mammary gland - predominantly isotype switched

BCR isotype switching AID dependent

Ig Heavy chain class (isotype) switching neutralization neutralization

Affinity Maturation Affinity maturation occurs in germinal centers and is the result of somatic hypermutation of Ig-genes in dividing B cells followed by selection of high affinity B cells by antigen displayed by FDCs The high affinity B cells emerging in germinal centers give rise to long-lived plasma cells and memory B cells

Antibody Affinity Maturation Germinal Center Low affinity B High affinity B mutation selection VH VL Ag VH VL VH VL Ag VH VL

Germinal Center Organization FDC High Affinity T T memory B cell Light Zone FDC CC CC FDC CC T plasma cell Selection, proliferation, differentiation CC FDC FDC CC CC MØ FDC MØ CB CB CB CB CB Dark Zone CB CB CB Certainly a lot of work has been done on fixed tissue sections and with isolated cells and this has led to a general model that proliferation and somatic hypermutation occur predominantly in the dark zone while selection occurs in the light zone. Strong experimental evidence has been obtained to indicate that there is a movement of cells from dark to light zone. In our own studies, work that was presented in workshop here 2 yrs ago by Chris Allen, we showed that CB positioning in the DZ is CXCR4 dependent whereas movement to the LZ involves CXCR5. But, we have known very little about the dynamics… CB T – T cell FDC – follicular dendritic cell MØ – macrophage CC - centrocyte CB - centroblast Proliferation, somatic hypermutation CB CB CB CB CB CB Low Affinity B CC, doubling time 6-12 hr , within 5 days each B cell produces 5000 cells, FDC:CR-1,2,3+, MHCII+

Germinal Centers Function: to generate B cells that produce antibodies with increased affinity for the inducing antigen => affinity maturation Germinal Center Reaction: Activated B cells give rise to Centroblasts - localize in follicle, undergo rapid cell division and turn on machinery that causes somatic mutation in V-regions Centroblasts give rise to Centrocytes - migrate to the FDC-rich region of the Germinal Center - survival is dependent on interaction with FDC-bound Ag and presentation of Ag to T cells - centrocytes that successfully compete to bind antigen (e.g. by having higher affinity BCR) and to receive T cell help are selected and may differentiate into long-lived plasma cells or memory B cells

Germinal Center mantle zone GC light zone GC dark zone T zone (CD23+ naive B cells) GC light zone (CD23++ FDC and centrocytes) GC dark zone (Ki67 cell cycle antigen+ centroblasts) T zone from Liu et al., Immunology Today 13, 17-21 (1992)

Affinity maturation is T-dependent CD3e LZ IgD DZ T cells are enriched in the light zone Represent ~10% of GC cells

Somatic hyper mutation occurred 10 3 in V H and VL gene and the neucleotide sequences of mutated IgG is 50% more than germinal gene and resulted in change of 10 a.a. in variable area.

Selection Model GC B cells that have improved affinity capture more antigen in a given amount of time, receiving a stronger BCR signal and presenting more MHC-peptide complexes to GC T cells, outcompeting surrounding B cells for T cell help

Memory B cells Generated during the primary response best characterized for T-dependent responses involving GC but some evidence exists for memory to TI antigens Small, recirculating cells Typically isotype switched (e.g. IgG+ or IgA+) Typically have higher affinity for the inducing Ag Longer lived than naïve B cells Persistence of memory B cells after an immune response ensures that we have increased numbers of B cells specific for the antigen and ready to respond on re-encounter May have intrinsic differences that promote greater clonal expansion and more rapid differentiation to plasma cells differences in cytoplasmic domains of IgG vs IgM/D upregulation of TLRs

Down-regulation of B cell response by pre-formed IgG

Antibody Dependent Cell Mediated Cytotoxicity (ADCC) Target cell is covered with antibodies, leaving Fc portion sticking outwards. Natural killer and other nonspecific cells that have receptors for Fc region are stimulated to kill targeted cells. Target organism is lysed by substances secreted by attacking cells. Used to destroy large organisms that cannot be phagocytosed.

FcgRIIIA

Receptor Cell type Effect of ligation FcgRI Macrophages Neutrophils, Eosinophils, Dendritic cells Uptake, Respiratory burst FcgRIIA Macrophages Neutrophils, Eosinophils, Platelets Langerhans cells Uptake, Granule release FcgRIIB1 B cells, Mast Cells No Uptake, Inhibition of stimulation FcgRIIB2 Macrophages Neutrophils, Eosinophils Uptake, Inhibition of stimulation FcgRIII NK cells, Eosinophils, Macrophages, Neutrophils Mast cells Induction of killing (NK cells)

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