Session 13 - B Cell Activation and Differentiation (1).pptx

B Cell Activation And Differentiatio n PBC 9700 Session 13 Randal K. Gregg, PhD Learning Objectives 1) Identify the localization of naïve and activated B cells in the lymphoid tissue. 2) Identify the three signals required for B cell activation. 3) Recall the importance of chemokine receptor modulat...

B Cell Activation And Differentiatio n PBC 9700 Session 13 Randal K. Gregg, PhD Learning Objectives 1) Identify the localization of naïve and activated B cells in the lymphoid tissue. 2) Identify the three signals required for B cell activation. 3) Recall the importance of chemokine receptor modulation during activation of naïve B cells. 4) Identify the roles of follicular dendritic cells, cytokines, CD40-CD40 ligand interactions, activated induced deaminase in B cell activation. 5) Define centroblast, centrocyte, somatic hypermutation and affinity maturation. 6) Identify the signaling molecules downstream of the BCR leading to activation. 7) Recognize the cell markers for B cells during activation by antigen. What is the role of these surface markers? Humoral immune response The production of immunoglobulins or antibodies is the sole function of the humoral immune response Antibodies are present in the blood, lymph and extracellular fluids Bind to protein or carbohydrate epitopes on the surface of a microbe or infected host cell There are 5 different classes of antibodies – IgM, IgD, IgA, IgE, IgG – and the differences enable antibody-coated pathogens to interact with specific types of effector cells In other cases (as shown to the right) antibodies can prevent infection by binding to the pathogen thereby limiting its capacity to invade cells – neutralize the pathogen (object of most vaccines) How do B cells engage antigen and where does this occur? Most Ag flows into lymph node via the lymphatics See notes below for more information FDC capture, concentrate and slowly release Ags Follicular dendritic cell BCR signaling (crosslinked BCR) Initial BCR contact with an antigen (Ag) epitope induces movement of BCR to the site of this contact so as to increase Ag-BCR encounter As a cluster of BCR engage Ag and many antigens contain repeats of the epitope (multimeric Ag) – BCR crosslink Signaling molecules of the BCR = Igand Ig Igand Igcontain cytoplasmic tails with ITAMs (immunoreceptor tyrosine-based activation motif) Activation of the BCR triggers phosphorylation of these ITAMs by Src kinases (Lyn, Fyn, Blk) Syk (spleen tyrosine kinase) binds to the PITAMs and acts similar to ZAP-70 in T cells PLC and Ras/Rac pathways are activated Transcription factors bind to accessible promoters in the B cell DNA and induce an activation signal Signal 1 for activation Syk corresponds to ZAP-70 in T cells B cell activation also requires additional B cell co-receptor signaling is requiredsignals for B cell activation Signal 2 for activation B cell co-receptor components: o CR2 (CD21) = recognizes iC3b and C3d derivatives of C3b fragments o CD19 = signaling chain of the co-receptor o CD81 = binds CD19 and aggregates the co-receptor and BCR This occurs following BCR ligation with Ag B cell activation also requires additional signals Src kinases like Lyn phosphorylate the cytoplasmic tail of CD19 P-CD19 can enhance signaling pathways associated with BCR crosslinking Co-receptor activation increases the BCR signaling events up to 10,000-fold Patients lacking functional B cell co-receptor components, CD19 or CD81, have low levels of serum antibodies, limited isotype switching and poor responses to infections and vaccinations FDC store and display intact Ags FDC have an extensive dendrite surface area to display large quantities of Ags (even intact virion) and lack phagocytic activity Ags captured by FDC are preserved along the cell surface for months (to even years!) During complement activation C3b (CR1 ligand) attaches to pathogen/Ag degradation to C3d (CR2 ligand) FDC express both CR1 and CR2 to capture Ags coming into the LN or delivered by DC FDC tether the Ags on CR1 and CR2 (shown in image to the right) to be screened by naïve B cells Sinus macrophages aid FDC by similarly displaying Ags on CR2 See notes below for more information What are the outcomes of BCR recognition of antigen? Type of B cell responses Follicular B cells can produce all types of antibodies (require T cell help) Follicular B cells are found in the follicles of the spleen and lymph nodes Marginal zone (MZ) B cells respond to nonprotein antigens and rapidly produce IgM Marginal zone (MZ) B cells are localized to the marginal zone and upon activation rapidly produce IgM B-1 B cells in the body cavities respond to non-protein antigen and rapidly produce IgM B-1 B cells are unique cells in the fetal liver as well as the fetal/adult bone marrow T cell-dependent B cell activation Outcome of BCR signaling for each B cell Clonal expansion Proliferation Anti-apoptotic factors Receptors for IL-2, IL-4, IL-5 Interaction with TFH cells MHC I + MHC II CD80 + CD86 CCR7 CXCR5 Unique T helper cells reside near follicles Paracortex is the site containing CD4+ and CD8+ T cells Follicular B cells reside within follicles These unique T cells reside within the green area called the interfollicular zone Pre-TFH cells in the interfollicular zone CCR7+ naïve CD4+ T cells CXCR5+ naïve CD4+ T cells (pre-TFH) CXCL13 produced by FDC to recruit B cells to the follicle CXCL13 CXCL13 TFH – T follicular helper CCL19/CCL21 CCL19/CCL21 produced by DC to recruit T cells to the paracortex Pre-TFH cell activation in the paracortex Outcome of TFH cell activation Proliferation Anti-apoptotic factors CD40 ligand IL-2, IL-4, IL-5 (Th2like) Signal 3 Other cytokines dictated by the signal 3 cytokine Activated Pre-TFH cells engage activated B cells in the interfollicular zone CXCL13 CCL19/CCL21 CXCL13 CXCR5+ CCR7+ PreTFH Interfollicular Zone CCL19 and CCL21 are produced by DC in the paracortex – cells expressing CCR7 are recruited by these ligands CXCL13 is produced by FDC in the follicle – cells expressing CXCR5 are recruited by these ligands Pre-TFH cells induce B cell proliferation and AID Pre-TFH IL-2 IL-4 IL-5 CD40L TCR BCR Ag CD40 MHC II IL-2R IL-4R IL-5R FO B cell Proliferation Survival Proliferation Activation Induced Deaminase (AID) B cell Ag presentation here is just to get the two cells close together (Pre-TFH is already activated) Pre-TFH fully differentiate into TFH cells Pre-TFH cell TFH cell * * * IL-10 IL-21 IL-21R Bcl-6 induction in pre-TFH CD40 ligand and CXCR5 expression stabilized Induce production of IL-10, IL-21 and the IL-21 receptor (IL-21R) Differentiation from pre-TFH to TFH Primary focus of B cell clonal expansion (in the Medulla) From the interfollicular zone – Activated B cells and TFH cells migrate to the medulla Both B cells and TFH cells divide – occurs over several days o IgM Promoted by IL-2, IL-4, IL-5 B cells B lymphoblasts differentiate into Plasma cells (PC) secreting the initial IgM detected in the blood TFH produces IL-5, IL-6, and IL-10 to promote differentiation of some of the B lymphoblasts into PC interfollicul ar zone Secondary focus of B cell clonal expansion (in the Primary Follicle) Some of the B lymphoblasts leave the medulla still attached to their TFH cells and traffick back to the primary follicle The B lymphoblasts begin to rapidly proliferate creating a secondary follicle primary follicle IgM Secondary focus of B cell clonal expansion (in the Germinal Center) These rapidly dividing B cells are known as centroblasts TFH cells also undergo some proliferation but not to the extent of the centroblasts Rapidly proliferating centroblasts push out naïve B cells and many TFH cells leading to formation of a germinal center Germinal centers represent B cell responses to a specific Ag IgM Proliferative BCR-negative centroblasts generated Activated B cell convert into centroblasts TFH cytokines (IL-2, IL-4, IL-5) initiate proliferation FDC cytokines [IL-6, IL-15, BAFF (B cell activating factor)] sustain proliferation Surface Ig (ie. BCR), or sIg, is lost AID promotes somatic hypermutations Somatic Hypermutations Interactions of centroblasts with TFH cells in the follicle/germinal center induce more AID Mutations occur in the Variable (V) region of both Heavy and Light Ig chains High rate of mutation (hypermutation) Primarily the result of the action of AID Results in Igs with varying affinities for Ag Centrocytes are generated = centroblasts that have completed somatic hypermutation and now express a BCR (sIg) Centroblasts + BCR = Centrocytes At the completion of somatic hypermutation – cells re-express the BCR Following somatic hypermutation centrocytes express a BCR (or sIg+) Engage FDC (affinity maturation) Affinity maturation by FDC BCR are tested for affinity for Ag (affinity maturation) (CR2) IgM secreted earlier is captured by Fc receptors (FcR) on FDC and then the Ig are used to bind the Ag for display (No Ag processing involved) FDC capture and display Ags bound on complement receptors (CR1-3) and Fc receptor-Ig complexes FDC CD21 binds degraded C3 which is attached to the Ag – CD21 on the B cell also engages the Ag (interaction here is more about stabilization See notes below for more information Centrocytes with high affinity BCR for Ag survive + + Cells with the highest affinity BCR for the Ag continue interaction with the FDC and receive survival signals (the two “+” symbols) Centrocytes require BCR and CR signaling to survive (i.e. remain attached to FDC) – now termed a germinal center B cell (GC B cell) Surviving centrocytes (GC B cells) engage TFH cells and differentiate into plasma cells TFH cell IL-10 and IL-21 signaling promotes proliferation and differentiation of GC B cells into plasma cells IL-10 IL-21 IL-10R IL-21R During this interaction – the GC B cells undergo isotype switching (more part II) Centrocytes undergo apoptosis if they fail to interact with TFH and FDC, respectively, and are phagocytosed by tingible body macrophages Short-lived plasma cells are generated first and produce IgM Formed during initial TFH cell and B lymphoblasts interaction in the medulla Usually the first source of the initial IgM response (released in the lymphoid medulla) – several days after Ag encounter  If IgM detected in serum within two days, source is marginal zone B cells Undergo apoptosis as long-lived PC are generated Long-lived plasma cells are generated and localized to two sites 1) Inflamed/infected tissues: Secrete high levels of Ig at Ag source site Undergo apoptosis as Ag decreases 2) Bone marrow: Plasmablasts differentiate into long-lived PC Reside associated with bone marrow stromal cell – up to the lifetime of the person Non-migratory Memory plasma cell Constantly secrete low levels of Ig (Ag exposure maintains levels) Memory B cells Remain in blood circulation or reside transiently in lymphoid organs Do NOT produce Ig but express a BCR Serve as a reservoir for memory B cell responses How do memory B cells develop? TFH cytokines determine PC and memory B cell differentiation TFH cell TFH cell * * IL-10 IL-21 * PLASMA CELLS IL-10 drives GC B cell differentiation into PC * * IL-4 IL-21 * MEMORY B CELLS IL-4 drives GC B cell differentiation into memory B cells Regulation of Ig production Ig production ON Ig production OFF Secreted Ig can bind Ag to form immune complexes (IC) IC are captured on B cells by Fc receptor to activate phosphatase to remove phosphates from BCR signaling proteins