1. B Cell Generation, Activation, And Differentiation W. Robert Fleischmann, Ph.D. Department of Urologic Surgery University of Minnesota Medical School rfleisch@umn.edu (612) 626-5034

2. Objectives To understand B cell development To understand the rearrangement of the antibody variable region during B cell maturation To understand B cell activation and the signals involved in the activation process To understand the importance of somatic cell mutations To understand antibody class switching

3. Harry Hammels, age 18 months, is brought to the clinic by his parents. He has fever (103.3°F), dyspnea, and non- productive cough. Harry has a history of repeated bacterial upper respiratory infections. What do you think? What would you do next?

4. Total WBC count is 11,000 cells/µl Differential count: Neutrophils40%22-40% Lymphocytes45%49-73% Monocytes5%3-10% Eosinophils10%0-6% Basophils0%0-3% What do you think? What would you do next?

5. Oscultation: fine crepitant rales and rhonchi with focal decreased resonance and bronchial breathing. Bronchial breathing is indicative of Consolidation Cavitation Complete alveolar atelectasis (collapse) with patent airways Mass interposed between chest wall and large airways Tension pneumothorax Massive pleural effusion with complete atelectasis of lung Chest X-ray shows diffuse, bilateral perihilar infiltrates. What do you think? What would you do next?

6. The clinical picture is indicative of an infection with Pneumocystis jirovecii. Examination of material from bronchial lavage shows the presence of cysts, consistent with Pneumocystis jirovecii. What do you think? What would you do next?

7. Lymphocytes are differentially identified. CD4+ T cells: normal level CD8+ T cells: normal level NK cells: normal level B cells: normal level HIV tests: ELISA test for HIV: negative What do you think? What would you do next?

8. B Cell Generation

9. Progenitor B cells (pro-B cells) express c-Kit which binds to stem cell factor expressed on BM stromal cells, inducing them to proliferate and differentiate to Precursor B cells (pre-B cells). Pre-B cells express IL-7R and are stimulated to divide and differentiate. Pre-B cells rearrange antibody  chain genes first, then  chain genes.

10. B Cell Development Progenitor B cell –cKit expressed/binds stem cell factor –Ig-  /Ig-  expressed –Heavy chain gene rearrangement Precursor B cell –IL-7R expressed –Express pre-B cell receptor (heavy chain + surrogate light chain) –Light chain gene rearrangement Immature B cell –IgM mRNA –mIgM on cell surface Mature, naïve B cell –Pre-mRNA differentially spliced to IgM and IgD mRNAs –mIgM/mIgD on cell surface

11. B Cell Generation Surface Markers Intracellular Events Progenitor B cellcKitHeavy chain gene rearrangement Precursor B cellIL-7RLight chain gene rearrangement Immature B cellmIgMIgM mRNA Mature, naïve B cellmIgM/mIgDPre-mRNA differentially spliced to IgM and IgD mRNAs

12. Sequential Expression of Membrane Immunoglobulin

13. B-1 B cells A subset of B cells that is made before the major group of B cells (B-2 B cells) Comprise 5% of total B cell population –Mostly found in peritoneal and pleural cavities where they predominate B-1 B cells exhibit a limited repertoire of Ab variable regions and the variable regions bind antigen with lower affinity. –More likely to respond to carbohydrate than to protein Ag –Ab are somewhat multi-specific and can bind several Ags –Class switching is not common, mostly IgM –No hypermutation of Ig genes, so no affinity maturation Unlike B-2 B cells, B-1 B cells are self-renewing and can generate more naïve B-1 cells.

14. Negative Selection of Self- Reactive B Cells Negative selection of many self-reactive B cells occurs in the bone marrow (clonal deletion). –This limits development of antibody-mediated autoimmunity. –If immature B cells, expressing mIgM recognize self-antigen, Some of the immature B cells undergo apoptosis. Some of the immature B cells undergo editing of light chain genes to produce a different light chain that, when combined with the heavy chain does not recognize self-antigen. Negative selection of some self-reactive B cells occurs in the periphery. –Not all self-reactive B cells are eliminated in the bone marrow because not every self-antigen is expressed in the bone marrow. –Mechanism not really understood.

15. B Cell Activation by Antigen

16. Thymus-Dependent vs. Thymus-Independent Antigens Thymus-dependent antigens (TD Ags) require direct contact of B2 cells with Th2 cells to induce an antibody response. –Generally, a stronger response than a thymus- independent Ag response Thymus-independent antigens (TI Ags) induce B1 cells to produce antibodies without the need for Th2 cell activity. –Type 1 TI antigen = lipopolysaccharide and other bacterial cell wall components –Type 2 TI antigen = highly repetitious molecules such as polymeric proteins (flagellin) or bacterial cell wall polysaccharides with repeated subunits

17. B Cell Response to Type 1 TI Antigen B1 cells bind lipopolysaccharide via either toll-like receptor-4 (TLR4) or via the B cell receptor (specific antibody + Ig-  and Ig-  ). –TLR4 binding is non-specific, so B cells with many different antibodies will be activated (polyclonal activation). In this way, it acts as a B cell mitogen. –Antibody binding is specific, so only B cells with specificity for the type of LPS will be activated. Type 1 TI antigen can activate both immature and mature B cells because it doesn’t require a surface antibody molecule (can activate through TLR4). Only IgM is produced.

18. B Cell Response to Type 2 TI Antigen B1 cells bind Type 2 TI antigen by crosslinking of the B cell receptor (specific antibody + Ig-  and Ig-  ). –Antibody binding is specific, so only B1 cells with specificity for the type of antigen will be activated. –Only mature (not immature B1 cells) will be stimulated. Mostly IgM is produced. While Th2 activity is not required, cytokine production by Th2 cells are needed for full B cell response and for class switching to isotypes other than IgM.

20. Two Signals Are Required for B Cell Activation TI Antigen –Binding of antigen provides both signal 1 and signal 2 TD Antigen –Binding of antigen provides signal 1 –Binding of Th2 cell via CD40:CD40L provides signal 2 (analogous to B7 for T cells)

21. Role of Ig-  /Ig-  in Signaling mIgM and mIgD have short cytoplasmic tails that are too short to transduce a transmembrane signal. Ig-  /Ig-  has ITAMs (immunoreceptor tyrosine-based activation motif) on the cytoplasmic tails. The tyrosine based activation motif is activated by antigen cross-linking antibody on the surface of the B cell. This initiates a cascade of events.

22. Intracellular Activation Cascade Sequential phosphorylations activate a series of proteins that activate phospholipase C that in turn leads to activation of transcription factor NF-  B. Activation of B cell receptor activates G proteins that activate transcription factors Rho, Rac, and Ras.

23. Additional Regulation of Signaling Stimulation –B cell coreceptor complex –CR2 (CD21) binds to complement C3d bound to antigen to cause phosphorylation of cytoplasmic tail of CD19, initiating further activation. Inhibition –CD22 –Activates a phosphatase that cleaves phosphate from ITAM. –Blocks ITAM signaling. –CD22 KO mice develop autoimmunity.

24. B Cell Activation by T Cell

25. The activation of B cells by antigen binding to receptor and by coreceptor binding creates conditions for proliferation and differentiation of B cells. However, important T cell interactions and T cell produced cytokines are necessary to complete the activation of B cells for thymus-dependent antigens.

26. Activation of B Cell by T Cell Requires Two Steps The first signal is sent by antigen binding. The B cell takes up antigen by receptor-mediated endocytosis. –After about 30-60 minutes, the antigen reappears on the B cell surface bound to MHC class II. –The Th2 cell recognizes the MHC class II bound antigen and, when costimulated by B7 binding to CD28, is activated. The activated Th2 cell synthesizes several molecules. –CD40L = ligand for CD40 on B cell causes the second signal for proliferation to be sent –Cytokines = IL-2, IL-4, and IL-5 push B cell to proliferate and differentiate The B cell expresses cytokine receptors.

27. Primary vs. Secondary Immune Response

28. Comparison of Primary vs. Secondary Immune Response

29. Lymph Node

30. Germinal Centers Antigen flows through the lymph to the lymph node either as free antigen or as antigen bound to an antigen-presenting cell. Free antigen binds to follicular dendritic cells in follicles and germinal centers, macrophages throughout the lymph node, dendritic cells in the paracortex, or B cells in the follicles and germinal centers. Processed antigen is presented to T cells and to B cells. T and B cells recognize each other at the periphery of the medullary region, bind, and move to the germinal centers. In the germinal centers, the B cells known as centrocytes undergo affinity maturation that is mediated by somatic cell mutation. In the germinal centers, there is class switching. The B cells become plasma cells and exit the germinal centers.

31. Coordinant Movement of Bound B and T Cells

32. Importance of Somatic Cell Mutations Somatic hypermutation occurs within the VDJ region with each cell division an activated B cell. The hypermutations give rise to a slightly different antibody molecule that may or may not have greater affinity for the antigen (affinity maturation). –If less affinity, then antibody will not be able to remain bound to antigen and apoptosis will eliminate the B cell from the population. –If greater affinity, the stronger interaction of the antigen for the antibody drives proliferation, until a clone of B cells with the greatest antigen affinity is founded.

33. Class Switching An mIgM-bearing cell can produce secreted IgM or it can undergo class switching by recombining the DNA at class switch recombination sites. Class switching exchanges the constant region of the antibody molecule. –IgM becomes IgG –IgG becomes IgE (recent work suggests IgM becomes IgE) –IgG becomes IgA (recent work suggests IgM becomes IgA) Class switching is driven by B/T cell interactions via the CD40/CD40L interaction and by specific cytokines produced by the T cell. Class switching occurs in the germinal centers of lymph nodes. Individuals whose T cells lack CD40L cannot undergo class switch and express a condition called X-linked hyper-IgM syndrome. –IgM only –No memory cell generation

34. Harry Hammels Harry is diagnosed with pneumonia caused by Pneumocystis jirovecii. He does not appear to be infected with HIV. His T cells and B cells respond to mitogen stimulation. Antibody expression is evaluated: IgM: higher than normal levels IgG: absent IgA: absent What do you think is wrong with Harry?

35. Harry Hammels It appears that Harry may have X-linked hyper-IgM syndrome. X-linked hyper-IgM syndrome occurs because there is a failure of T cells to send an appropriate signal to B cells for them to class-switch.

36. Harry Hammels Harry is found to lack expression of CD40 ligand. Without the binding of CD40 ligand on T cells to CD40 on B cells, the B cells cannot undergo class-switch. Hence, hyper-IgM syndrome.

37. Differentiation and Class Switching Is Also Driven by Specific Cytokines

38. Immunoglobulin Production Down- Regulates the Immune Response As antibody is produced, it exerts a feedback inhibition of its own production. This occurs by two mechanisms –Soluble antibody binds antigen and prevents the antigen from reaching the mIgM/mIgD antibody on the surface of other naïve B cells. –Antibody/antigen complexes bind to Fc receptor molecules on the surface of the B cells making it ever more likely that the down-regulatory events mediated by CD22 will be triggered. There are few molecules of CD22 on a cell and many molecules of antibody. When few antibodies are bound to antigen, it is difficult for the antigen/antibody complex and CD22 to find each other to bind. When many antibodies are bound to antigen, the antigen/antibody complex and CD22 find each other easily.