Lec 13: B Cell Ontogeny and Activation (Medical Immunology COM5850) PDF

B Cell Ontogeny Randal Gregg, PhD and Activation Medical Immunology COM5850 LECTURE 13 harry potter symbol - Bing images defense against the dark arts - Bing images 1 Learning Objectives 1) Recognize how the immunoglobulin gene...

B Cell Ontogeny Randal Gregg, PhD and Activation Medical Immunology COM5850 LECTURE 13 harry potter symbol - Bing images defense against the dark arts - Bing images 1 Learning Objectives 1) Recognize how the immunoglobulin gene rearrangements are tied to B lymphocyte ontogeny. 2) Identify the steps in B cell ontogeny, including checkpoint mechanisms, enzymes, and molecules involved at each stage. 3) Recognize the process of clonal proliferation and differentiation. 2 Process of lymphocyte development results in antigen receptor expression 3 B Cell Receptor (BCR) 4  B cell development occurs on the surface of bone marrow (BM) stromal cells SDF-1 (stromal cell- derived factor 1 or CXCL12) retains stem IL-7 produced by BM cells and lymphoid stromal cells progenitors at the promotes survival surface of bone signals in the marrow stromal cells progenitor cells to protect them during Cells that are each step of receiving stimulatory differentiation signals (growth, proliferation, IL-7 also induces differentiation, etc) expression of Kit are also programmed (receptor tyrosine to undergo apoptosis kinase or CD117) on the pro-B cell which Thus there must be a binds to SCF (stem mechanism to block cell factor) induces apoptosis for the cells proliferation of the to continue through pro-B cells the process 5 BCR generation and B cell development Now that the genetics for Ag receptor generation has been discussed we need to correlate gene rearrangement with the differentiation of naïve mature B cells from pro-B cells 6 H chain rearrangement activated in early pro- B cells IL-7 produced by BM stromal cells promotes survival signals in the developing B cells as they progress through BCR gene rearrangement – failure to successfully rearrange H chains leads to a reduction of IL-7 receptor (IL-7R) and a loss of survival signaling (mitochondria become porous due to Bax/Bak and cytochrome c released to induce caspase activity and apoptosis) IL-7 signaling important through formation of the pre-BCR 7 Successful D-J rearrangement = late pro-B cell 8  Pre-B cells express the pre-BCR (H chain) Pre-BCR signals drive proliferation of large pre-B cells independent of antigen binding Also triggers L chain gene rearrangements Pre-BCR signaling activates Btk (Bruton’s tyrosine kinase) which promotes survival, proliferation and maturation processes 9  X-linked Agammaglobulinemia (XLA) An X-linked disease in which there is a mutant version of Btk that results in a lack of mature B cell production and antibodies thus making the patients at high risk for infection (T cells are not affected) It is one of the most common pediatric primary immunodeficiencies and requires lifelong immunoglobulin (Ig) replacement therapy for survival 10 L chain rearrangement initiates characterizing a small pre-B cell 11  Successful V-J rearrangement results in BCR expression Immature B cells express the fully assembled H+L rearranged IgM structure and Ig and Ig proteins on the surface Ig and Ig serve as the signaling molecules after antigen has bound to the IgM on the B cell surface Signaling through this BCR complex in immature B cells induces PI3K that provokes cell survival and NOT proliferation 12 Summary of BCR gene rearrangement 13 IgM+ immature B cells circulate Immature (IgM+) B cells leave the bone marrow and circulate to the spleen Must interact with cells in the follicle to become a mature B cell 14  B cell maturation in the spleen Most immature B cells leave the bone marrow and reach the spleen and migrate into the T cell zone of the white pulp (PALS) where they become T1 transitional B cells 15 Site of negative selection of B cells in spleen T1 transitional B cells undergo negative selection at this point Negative selection involves the exposure to self-Ag to eliminate self-reactive B cells (discussed later) Those B cells that survive negative selection become T2 transitional B cells 16 T1 transitional B cells recognizing self-Ag are eliminated Most T2 transitional B cells migrate into the follicle to interact with follicular DC (FDC) and become naïve mature BAFF (B cell B cells activating factor) provided by FDC promotes survival and maturation of the B cells  Interaction of the surviving B cells with FDC completes maturation = naïve mature B cells Immature B cell half-life = 100 d Mature B cell half-life = 40 d after maturation 18  Marginal zone B cells generated Other T2 transitional B cells migrate to the marginal zone and develop into marginal zone B cells (rapid IgM response to blood Ag) 19 How do B cells engage Ag and where? 20  Most Ag flows into LN via the lymphatics Small Ag arrives to the LN alone, modified by complement fragments, or associated with APC B cells recognize native Ags Small Ags enter the LN from afferent lymphatic vessels through a system of conduits and can be captured by follicular dendritic cells (FDC) Macrophages in the subcapsular sinus and dendritic cells in the medulla capture large Ags These cells can release the larger Ags for uptake by FDC 21 FDC capture, concentrate and slowly release Ags 22  BCR engagement drives B cell activation Initial BCR contact with an Ag epitope induces movement of BCR to the site of this contact to increase Ag-BCR encounter As a cluster of BCR engage Ag and many Ags contain repeats of the epitope (multimeric Ag) – BCR crosslinking Signaling molecules of the BCR = Ig and Ig Ig and Ig contain cytoplasmic tails with ITAMs 23  BCR engagement drives B cell activation Activation of the BCR triggers phosphorylation of these ITAMs by Src kinases (Lyn, Fyn, Blk) Syk (spleen tyrosine kinase) binds to the P-ITAMs and acts similar to ZAP-70 in T cells PLC and Ras/Rac pathways are activated Syk Transcription factors bind to accessible promoters in the B cell DNA and induce an activation signal 24 B cell activation also requires additional signals B cell co-receptor signaling is required for B cell 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 25 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 26  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 27  Types of B cells B-1 B cells Arise early in development from fetal liver progenitor cells Located primarily in the peritoneal and pleural cavities (can’t enter lymphoid follicles) Produce only IgM in response to Ag Do NOT receive help from T cells to make antibody Recognize microbial carbohydrate epitopes (T-independent response) B-2 B cells Arise from the bone marrow and differentiate into two populations:  Marginal Zone B cells (MZB) o Localized in the marginal zone of the spleen o Rapid IgM response to Ags (include carbohydrate)  Follicular B cells (FOB) o Localized in the follicles of the LN and spleen o Require interaction with CD4+ T cells o Engage Ag and can produce different isotypes of Ig 28 Comparison of B cell subtypes and their activity Characteristics B-1 B cells Follicular B cells Marginal zone B cells Major location Pleural and peritoneal Secondary lymphoid tissue Spleen marginal zone cavities Production of new B Self-renewing B-1 cells Bone marrow Possible self-renewing cells Diversity Low High Moderate Need for T cell help No Yes Unclear Major isotypes produced IgM IgM, IgG, IgA, IgD, IgE IgM Binding to carbohydrate Yes Possible Yes Ag Binding to protein Ag Possible Yes Yes Production of memory Little to none Yes Unclear Presence of surface IgD Little to none Naïve B cells Little to none 29 What are the outcomes of FOB BCR recognition of Ag? 30 Activation outcome 1 = Clonal expansion Proliferation  Anti-apoptotic factors 31 Activation outcome 2 =  Ag presentation  MHC I + MHC II  B7 (CD80/CD86) 32 Activation outcome 3 =  Cytokine receptors IL-2R, IL-4R, IL-5R, IL-21R 33 Activation outcome 4 = CCR7 CXCR5 CCR7+  CXCR5 34