B Cell Generation, Activation, And Differentiation

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. B Cell Generation

4. 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. B Cell Generation

5. 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

6. B Cell Generation

7. Sequential Expression of Membrane Immunoglobulin

8. 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.

9. 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.

10. B Cell Activation by Antigen

11. 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

12. 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.

13. 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.

15. 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)

16. 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 an ITAM motif (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.

17. 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.

18. 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.

19. B Cell Activation by T Cell

20. B Cell Activation by T Cell • 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.

21. 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 antibody 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.

22. Primary vs. Secondary Immune Response

23. Comparison of Primary vs. Secondary Immune Response

24. Lymph Node

25. 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.

26. Coordinant Movement of Bound B and T Cells

27. 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.

28. 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 • IgG 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

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

30. 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.