Chapter 7

1. Chapter 7 Humoral Immune Response Activation of B Lymphocytes and Production of Antibodies

2. Antibodies • Part of the adaptive immune response functions to neutralize and eliminate extracellular microbes and microbial toxins • More useful against polysaccharide and lipids and these types of toxins – B cells respond to many Ag types • Naïve B cells recognize Ag but can’t make Ab until activation allows differentiation to Ab secreting plasma cells

3. Phases and Types of HIR • Naïve B-cells have 2 classes of membrane bound Ab – IgM and IgD • act as receptor for Ag • Activation of Ag specific cells leads to clonal expansion • Go from 1 to 4000 Ab secreting cells • Differentiation into effector cells called plasma cells • secreted Ab is same specificity of as naïve membrane bound receptor but during differentiation can cause Ab with different heavy chain and effector function – heavy chain class (isotype) switching • can handle different types of microbes • Repeated exposure to causes Ab with increasing affinity to Ag – affinity maturation • improved capacity to bind and neutralize Ag

4. Recognition and Activation

5. T-Cell Dependence?? • Ab response to different Ag may depend on the requirement of T-cell help or not • B-cells recognize many Ags – protein, polysaccharide, lipid and small chemicals • Protein Ag makes poor or no Ab response when T-cells are absent = T-dependent Ab response • T-helper cells play role in activation and powerful inducer of isotype switching • Polysaccharidde, lipid and non-protein Ag stimulate Ab production without T helper cells = T independent Ab response • little to no heavy chain switching or affinity maturation

6. B-Cell Subsets Respond Differently • Protein and non-protein Ag result in different responses • Majority are follicular B-cells (follicles of lymph organs) make the majority of T-dependent, class-switched ans high-affinity Ab to protein Ag • also long-lived plasma cells • Marginal zone B-cells (marginal zone of spleen white pulp) respond to blood bourne-polysaccharide Ag • B-1 B-cells respond to non-protein Ag in mucosa and peritoneum • Marginal zone and B-1 B-cells have Ag receptors of limited diversity and mainly IgM response

7. Responses • Ab responses differ qualitatively and quantitatively • 1 response is the first time exposed • small amounts of Ab produced • 2 response is the subsequent exposure • more Ab produced • if to a protein, see isotype switching and affinity maturation • increase in TH cells with repeated stimulation • Ag recognition ACTIVATES B-lymphocytes

8. Must be able to tell the difference between 1 and 2 responses

9. Stimulation of B-Cells by Ag • Initiated when Ag-specific B-cells in lymphoid follicles of the spleen, LN and mucosal lymphoid tissues recognize Ag • Ag of microbes in tissues or blood are transported and concentrated in B-cell rich follicles by possible method of: • macrophages in subcapsular sinus may capture Ag and display to B-cells in adjacent follicles • use BCR to recognize Ag in native conformation – triggers pathway for signal transduction • require additional signals from innate system • Ag without processing – NATIVE CONFORMATION

10. Ag-Induced Signaling in B-Cells • Membrane Ig receptors cluster (bind and move together) and trigger biochemical signals that are transduced by receptor-associated signal molecules • similar to T-cells activation • must cross-link 2 or more receptors for signal transduction – 2 or more Ag in an aggregate or repeating epitopes of one Ag molecule – binds adjacent Ig molecules • Polysaccharide, lipid and other non-protein Ag have multiple identical epitopes in each molecule (bind numerous Ig receptors on B-cell) • transduced by receptor-associated proteins

11. Signals • Receptor IgM and IgD – highly variable with short cytoplasmic domains • recognize Ag but do not transduce signals • IgM and IgD are non-covalently linked to Ig and Ig to make the B-cell receptor (BCR) complex • Ig and Ig contain conserved immunoreceptor tyrosine-based activation motifs (ITAM) – seen in other activation receptors • 2 or more Ag receptors cluster, Tyr in ITAM of Ig and Ig are phosphorylated by kinase associated with BCR complex • Tyr-PO4 is the docking sites for adaptor protein which gets phosphorylated and recruits a number of signal molecules • Signal cascade not as well understood as T-cells but essentially similar – activates trnscription factor that turn on genes whose proteins involved in B-cell proliferation and differentiation

12. B-Cell Activation • Syk is similar to ZAP-70

13. Complement and B-Cell Activation • B-cells express receptor for a complement protein which provides signals to activate B-cells when bound to ligand • Complement is activated by microbes and Ab attached to microbes – functions as effector mechanism of host defense (Chapter 8)

14. Complement Activation of B-Cells • Microbe has C3d (cleavage product of C3) coating the surface • B-cells has type 2 complement receptor (CR2 or CD21) and it binds to C3d • B-cells recognize microbe Ag by the Ig receptor and also recognizes C3d on CR2 receptors • C3d acts as the 2nd signal for B-cell activation • CR2 enhances Ag-dependent activation of B-cells • Ag recognition is 1st step and C3d-CR2 binding is the 2nd signal

15. Activation • Microbes themselves or innate immune response to microbes provide signals in addition to Ag recognition to activate lymphocytes or microbial products attaching to TLR on B-cell • C3d is similar to co-stimulators in T-cell activation

16. Consequences of Ag-Mediated B-Cell Activation • Initiate B-cell proliferated and differentiation and prepare B cells to interact with TH cells if protein Ag • B-cell enters cell cycle and proliferates – clonal expansion • Produces more IgM and secrete IgM – early phase immune response induced

17. Early Phase is Greatest • Response is greatest for polysaccharide and T-independent Ag • multivalent • cross-links many Ag-receptors • activates C’ strongly • Proteins can’t do the 1st 2 so make weak response without TH response • lack multiple, identical epitopes

18. Additional Steps • At least 3 other changes to the B-cell to enhance ability to interact with TH cells – acting as APC • increase the expression of B7 costimulator molecule (2nd signal for T-cells), may amplify helper T-cell response and expression of cytokine receptors – cytokine mediators of TH cell functions • decrease the expression of chemokine receptors – chemokines keep B-cell in follicles, without chemokines they can migrate out of follicle to where TH cells are concentrated

19. Function of T-Helper Cells • Required for strong response to protein Ag • B-cell and T-cell must recognize protein Ag and come together to stimulate B-cell proliferation and differentiation in the lymphoid tissue • takes 3-7 days for Ab response

20. Activation and Migration • Activated helper T-cells (effector cells) interact with Ag-stimulated B-cells at edge of follicles in lymphoid tissue

21. TH and B-Cell Interaction • Naïve CD4+ T-cell is stimulated to proliferate and differentiate into cytokine secreting effector cell • Ag recognition and co-stimulation • microbial Ag and protein Ag with adjuvants that cause expression of co-stimulators on APC • extracellular microbes and vesicular microbes on MHC II in T-cell rich zones in LN • depending on Ag – become TH1, TH2 or TH17 subset • portion of cells move out of LN to look for Ag • T-cells move out of LN to circulation – remove microbes CMI • some T-cells migrate to the edge of lymphoid follicles while B-cells are moving outward; dependent on changes in chemokine receptors on activated lymphocytes and the chemokines are also expressed to bind receptors

22. Ag Presentation by B-Cells • B-cells bind protein Ag (extracellular) by specific receptors and endocytose them – process in endosome vesicles and display on MHC II for recognition by helper T-cells • Membrane bound Ig can bind Ag with high affinity (low Ag concentration); very efficiently endocytosed and processed to be peptide on MHC II = act as APC • specific recognition of Ag by Ig on the surface • B-cells have ability to display multiple Ag for T-cell recognition – B and T cells can recognize different epitopes of same Ag • interactions remains Ag specific • Ag-activated B-cells also express co-stimulators such as B7 to stimulate T-cells that recognize Ag on B-cells • cannot activate naïve T-cells

23. B- and T-Cell Interaction

24. TH-Cell Mediated Activation • TH-cell recognize Ag on APC B-cell and activate it by expressing CD40L and secreting cytokines – similar to T-cell activation of macrophages in CMI • CD40L binds CD40 on B-cell – sends signals for B-cell expansion and for synthesis and secretion of Ab • Cytokines made in the T-cell bind to the B-cell and enhance proliferation and Ig production • CD40L:CD40 interaction insures that B-cells are activated only when they come in contact with T-cell • Helper T- cell signals trigger heavy chain switching and affinity maturation – as seen in T-dependent protein Ag recognition

25. Isotype Switching • Heavy class chain is switched to a different isotype to broaden functional capabilities • Helper T-cell help with the switch from IgM/IgD to another isotype • allows humoral immune response to adapt in order to combat microbe optimally • IgG is used to opsonize microbes and extracellular viruses – phagocytosed by neutrophils and macrophages; usually IgG1 and IgG3 classes • bind the Fc receptor on the phagocyte with high affinity and specificity for  heavy chain • IgE is used to fight helminths by recruiting eosinophils which recognize the  heavy chain by way of Fc receptor

26. Effector Functions of Ab • Know cytokines and which Ab

27. Reasons for Switching • Need to make a response that can remove various microbes – needs to be more than IgM and IgD • Class switching is mediated by CD40L:CD40 mediated signals – different cytokines involved • cytokines influences which heavy chain class an individual B-cell and progeny will switch to • No CD40L or CD40 – B-cells secrete only IgM

28. X-Linked Hyper IgM Syndrome • Inactivating mutations in CD40L gene on the X-chromosome – no functioning forms of CD40L • Mostly IgM Ab produced due to the defective heavy chain switching • Also defective CMI because of the effect on T-cell activation (use of CD40L)

29. Class Switching (Switch Recombination) • Process is well understood • In IgM B-cells the VDJ region of variable portion is adjacent to C gene splice mRNA of VDJ RNA to CRNA = make  heavy chain • Cytokines and CD40 signals stimulate transcription thru a C gene segment to make something other than IgM – 5’ end intron has conserved sequence that is the switch region • activation causes 3’ switch region of C to link to 5’ switch region of downstream constant region and delete entire intron, removing the  gene RNA • switch region is not present for C gene • enzyme Activation-Induced Deaminase (AID) plays a role in switch recombination

30. Switching • VDJ region in contact with C regions downstream – Ab may have new heavy chain but the specificity in the variable region is the same as IgM • VDJ region stays the same (where Ab specificity resides)

31. Cytokine Impact • INF complements both B-cells and phagocytes • INF makes opsonizing Ab (from TH1 cells) • promotes phagocytosis – also INF is phagocyte activating • bacteria and viruses stimulate the formation of TH1 cells (secretes INF) • IgE class switching is ruled by IL-4 (TH2 cytokine) and eosinophil activation by IL-5 (also from TH2 cells) • Nature of helper cell activity guides the Ab response • optimized for the microbe • helper T-cells may act as “master controller” of the immune response • Also influenced by site of immune response • mucosal epithelium has IgA – B cells and unknown cytokine to switch to IgA

32. Affinity Maturation • Affinity of Ab produced to protein Ag increases with prolonged or repeated exposures to that Ag • Ab binding to microbe or Ag increases if infection is persistent or recurrent • due to point mutations in the V region – Ag binding hypervariable region • happens only in T-dependent protein Ag • mutation rate 1:1000 nucleotides – somatic hypermutation

33. Mutation Accumulation

34. Helper Cells Are Critical • Affinity maturation occurs in germinal center of lymphoid follicles – Somatic Hypermutation of Ig genes in dividing B-cells – followed by selection of high affinity B-cells by Ag displayed by follicular dendritic cells • As response goes on, increase in the level of Ab and decrease in the Ag so B-cells with high affinity Ab receptors will bind Ag on the FDC and therefore survive apoptosis • B cells are selected to survive must bind Ag at lower and lower Ag concentration • germinal center B-cells die unless rescued by Ag-recognition • Ag:Ab complex can activate complement

35. Affinity Maturation

36. Where?? • Occurs in different parts of lymphoid tissue • Mature naïve B-cell recognize Ag and move to peripheral edge of the follicle where it encounters the T helper cell and start to secrete Ab • Plasma cells move away from B-cell rich regions continue to secrete Ab even when Ag is gone • Fraction of cells after heavy class switching (occurs outside of follicle) will become memory cells and wander until contact the Ag again

37. T-Independent Ab Response • Know the bottom part of this table • Needs no T-cell help • Polysaccharides, lipids and non-protein molecules • no T-cell recognition / no MHC • response happens because these Ag are multi-valent (have multiple identical epitopes) and can activate B-cell • Two types of Ab differ

38. Ab Feedback • Most B-cells will die by apoptosis except for memory and small number of plasma cells • Removal comes from the B-cell having a Fc receptor, FcRII that will bind Fc part of Ab and receptor mediated signaling • terminates the humoral immune response when sufficient IgG present