MICR 304 Immunology & Serology

1. MICR 304 Immunology & Serology Lecture 10 B Lymphocytes Chapter 6.8, 6.10, 6.17–6.18, 7.1–7.6, 7.23–7.28, 9.1 – 9.13 Fig. 6.18

2. Overview of Today’s Lecture • Cell signaling through BCR • Development of B cells • Positive and negative selection • Survival and maturation in peripheral lymphoid tissue

3. Key Players in Immunology

4. B Cell Receptor Complex • Membrane bound immunoglobulin has no intrinsic signaling capacity • Iga and Igb are invariant non-antigen specific transmembrane molecules that initiate signaling • Contain tyrosine residues that can be phosphorylated by src kinases upon activation • ITAM (Immunoreceptor Tyrosine-based Activation Motive) • Iga and Igb are also important for transport of Ig to cell surface * • *Also present in TCR, NK receptors, Fc receptors

5. Cross-Linking is Required for B Cell Activation • Binding of a monovalent antigen to a single BCR will not produce a signal. • Receptor clustering caused by cross linking generates an intracellular signal.

6. Src Kinases Associate with BCR • B cell receptor cross linking is required • Src kinases Blk, Fyn, or Lyn associate with and phosphorylate Iga and Igb • Tyrosine kinase Syk with 2 SH2 domains is recruited, binds to phosphorylated ITAM and is activated.

7. 1. C3d B Cell Co-Receptor Complex • Binding of antigen is not enough for activation of naïve lymphocytes • Simultaneous ligand binding to co-stimulatory receptors enhance antigen dependent signaling • Co-receptor complex consists of • CD21 = complement receptor CR2 for C3d fragment • CD19: becomes phosphorylated and phosphorylated CD19 activates src kinases • CD81 = TAPA-1 (target of antiproliferative antibody); role? 2. P 3. 3.

8. B Cell Activation through the BCR and Co-Receptors

9. B Cell Development

10. Main Phases of a B Cell’s Life History

11. Main Phases of a B Cell’s Life History

12. B Cell Maturation is Coupled to Immunoglobulin rearrangement • Heavy chain rearrangement • DJ • VDJ • Transient expression of heavy m chain with surrogate light chain • Important check point • Successful signaling involves Btk (Bruton’s tyrosine kinase) • 45% unsuccessful! • Switch to the other chromosome • Light chain rearrangement • VJ • IgM expressed on cell surface • IgD expressed on cell surface in addition to IgM

13. B Cell Maturation is Coupled to Immunoglobulin rearrangement Check point Ability to express Ig on cell surface

14. Steps in Ig Gene Rearrangement at which Developing B Cells can be lost

15. Development Dependent Expression of Surface Proteins in B Cells

16. Expression of Proteins Involved in Gene Rearrangement and Production of pre-BCR and BCR

17. Immature B Cells are Tested for Self Reactivity Check point Ability to express Ig on cell surface Check point Self reactivity

18. The Fate of Self Reactive B Cells • Once complete IgM molecule is successfully expressed on B cell surface (sIgM) B cell has reached the immature B cell stage • Ag receptor is now tested for tolerance to self antigen (central tolerance) • Fate of immature B cell depends on the signal delivered from sIgM • No strong reactivity • Strong reactivity Further maturation Anergy Ignorance Clonal deletion Receptor editing

19. Anergy • In response to soluble self molecules with weak cross linking of IgM • Down regulation of IgM and mainly expression of IgD • Permanent block in signal transduction and unresponsiveness • Migration to periphery • Anergic B cells cannot respond to antigen anymore even under optimalconditions.

20. Ignorance • Monovalent or low affinity solublenon cross-linking self antigens • No signal transduction and activation in response to self antigen • Migration to periphery • Potentially self reactive under certain circumstances • High concentration of antigen • In the context of inflammation • Pool for autoreactive cells in autoimmune diseases

21. Clonal Deletion • B Cells that respond to multivalent self antigens are removed from repertoire • Premature activation of B cell initiates apoptotic events. • The removed B cell cannot anymore undergo clonal proliferation, hence clonal deletion. • The antibody molecule expressed by this B cell is irreversibly lost.

22. Receptor Editing • Some B cells that respond to multivalent self antigen are rescued. • B cell obtains a second chance. • Surface IgM is decreased. • RAG enzymes continue rearrangement of light chain. • New light chain is coupled to existing heavy chain and IgM is expressed at the cell surface. • Retesting of self reactivity ensues.

23. Summary for Possible Outcomes of Self Reactivity at Check Point

24. Survival and Maturation of B Cells

25. Main Phases of a B Cell’s Life History

26. Homing of Naïve B Cells to Lymph Nodes • High endothelial venules (HEV) and stromal cells secrete chemokine CCL21 • Dendritic cells are attracted by CCL21 and secrete chemokines CCL18 and CCL19 • B lymphocytes are attracted and stimulate follicular dendritic cells to secrete CXCL13 which attracts more B cells

27. B cells are APC. Antigen bound sIgM is internalized. After fusion with lysosomes antigen is processed and presented via MHC II to T cells. B cells express also costimulatory molecule CD40. Activated T cells that bind to antigen on MHC II activate B cells via binding to CD40 and by releasing cytokines that stimulate B cells to develop into plasma cells and memory cells. Activation of B Cells by TH Cells CD40 Ligand CD40

28. Activated B Cells Differentiate into Memory Cells and Plasma Cells Cognate TH cell Under control of BLIMP-1 (B-ly induced maturation protein)

29. Somatic Hypermutation of B Cells Activated by TH Cells Quickly engulfed by macrophages

30. Comparison of B Cells and Plasma Cells • Plasma cells are terminally differentiated • Low expression of sIg • No MHC II, no more interaction with TH • High rate of Ig secretion • No more growth, somatic hypermutation or isotype switch

31. Cytokines Dictated Isotype Switch

32. Memory B Cells • Long lived descendants of cells that were once stimulated by antigen • > 70 years • Half time ~ 10 – 15 years • Slow divisions if any • Low or absent Ig secretion • High level of MHC II • Increased expression of costimulatory signals • High affinity for antigen

33. T Cell Independent B Cell Activation • Response to bacterial polysaccharides, polymeric proteins, lipopolysaccharides • Thymus independent antigens (TI) • TI-1 antigens • Intrinsic activity and polyclonal B cell activation at high concentrations • B-cell mitogens • E.g. LPS • Can activate immature and mature B cells • TI-2 antigens • Highly repetitive structure • E.g. bacterial capsular polysaccharide • Activates only mature B cells • Costimulatory signals provided by dendritic cells and macrophages

34. Innate B Lymphocytes

36. Today’s Take Home Message(Student Contribution) • B cell maturation involves heavy chain rearrangement in which upon successful completion, this chain will be expressed with a surrogate light chain. If this expression is successful, the heavy chain rearrangement will halt and the cell can transition to rearranging the light chain. IgM will be expressed on the surface to check if the immature B cell is reactive to self antigens. If it is not reactive then IgD will be expressed alongside with IgM. • When the B cell is tested for tolerance to self antigen then no strong reaction to cross linking self antigens leads to further maturation; a response to soluble self molecules with weak cross linking leads to anergy; a response to monovalent or low affinity soluble non cross-linking self antigens leads to ignorance; a strong response to self antigen leads to receptor editing and if this does not reduce self reactivity to clonal deletion. • B cells are activated by T cells after cognate T cells have bound via their TCR to MHC II presented antigen that B cells have captured with their surface immunoglobulin, and the costimulatory receptors CD40 (on the B cell) and CD40 L (on the T cell) have interacted which leads to further B cell maturation into plasma cells and memory cells. • Plasma cells originate from T cell activated B cells but have become independent from T cells and have directed their cell function to high antibody production and secretion which is accompanied by a reduction of surface Ig and MHC II expression. • T cell independent antigens like polysaccharides and LPS can activate B cells independent from T cells and innate B cells are fully functional without the aid by TH cells.