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Changes in Antibodies Class switch recombination Membrane vs secreted mutation, selection Low affinity High affinity Affinity maturation IgE Constant region.

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Presentation on theme: "Changes in Antibodies Class switch recombination Membrane vs secreted mutation, selection Low affinity High affinity Affinity maturation IgE Constant region."— Presentation transcript:

1 Changes in Antibodies Class switch recombination Membrane vs secreted mutation, selection Low affinity High affinity Affinity maturation IgE Constant region (function) Variable region (antigen-binding) Transmembrane & Intracellular Domains B cell Plasma cell IgG1 IgE IgM IgA AID

2 B Cell Differentiation Pathways Germinal Center Activated B Cell Short-Lived Plasma Cell Affinity Maturation Hypermutation, Selection Long-Lived Plasma Cell Memory B Cell

3 Normal Functions of Antibodies Neutralization toxins viruses Mast cell Parasitic worm inflammation bacteria Complement activation Macrophage bacteria Opsonization Toxic granules Foreign/ infected cell Antibody-dependent cytotoxicity IgG1 IgE IgM IgA

4 Questions Why do IgE and IgG1 differ in abundance? How do IgE vs IgG1 B cells behave in germinal centers? What are possible models leading to the loss of IgE germinal center B cells?

5 Questions Why do IgE and IgG1 differ in abundance? How do IgE vs IgG1 B cells behave in germinal centers? What are possible models leading to the loss of IgE germinal center B cells?

6 Helminth parasite model: Nippostrongylus brasiliensis infection

7 Serum IgG1 vs IgE Infection N. brasiliensis L3 larvae Erazo A. et al. Immunity 2007, 26: Cheng L.E. et al. J Immunol 2010, 185:

8 Questions Why do IgE and IgG1 differ in abundance? How do IgE vs IgG1 B cells behave in germinal centers? What are possible models leading to the loss of IgE germinal center B cells?

9 IgE vs IgG1 B cells GFP (IgE) IgG1 NP-KLH Alum adjuvant TNP-OVA Alum adjuvant N. brasiliensis infection Talay O. et al. Nat Immunol 2012, 13: Yang Z. et al. Immunity 2012, 36:

10 Kinetics of IgE vs IgG1 B cells in GCs N. brasiliensis infection Talay O. et al. Nat Immunol 2012, 13: Yang Z. et al. Immunity 2012, 36: He J.S. et al. J Exp Med 2013, 210:

11 Questions Why do IgE and IgG1 differ in abundance? How do IgE vs IgG1 B cells behave in germinal centers? What are possible models leading to the loss of IgE germinal center B cells? – BCR signaling

12 BCR expression in IgE vs IgG1 Germinal Center B Cells He J.S. et al. J Exp Med 2013, 210: HA-specific B cells

13 Questions Why do IgE and IgG1 differ in abundance? How do IgE vs IgG1 B cells behave in germinal centers? What are possible models leading to the loss of IgE germinal center B cells? – BCR signaling – Plasma cell differentiation

14 IgE + B cells are Biased Toward Plasma Cell Differentiation in vivo NP-KLH Alum adjuvant subQ 8 days in vitro Naïve B cells Anti-CD40 IL-4 & spleen + 4 days Draining LNs (FACS) Yang Z. et al. Immunity 2012, 36:

15 Cell-Intrinsic Increase in IgE + GC B Cells Caused by Blimp-1 Deficienc y CD45.2 GC B / CD45.1 GC B CD45.2 naive B / CD45.1 naive B Yang Z. et al. Immunity 2012, 36: Blimp-1 is a transcription factor needed for plasma cell differentiation

16 Shorter Lifespan Reduced Affinity Yang Z. et al. Immunity 2012, 36:

17 Why IgE B cell lifespan and affinity may be limited Neutralization toxins viruses Mast cell Parasitic worm inflammation IgG1 IgE IgA Systemic IgE activation

18 IgA Background IgA is secreted in mucosal tissue and is transported across mucosal epithelial barriers by the poly-Ig receptor In the gut, class switch to IgA occurs in Peyer’s patches and Isolated Lymphoid Follicles IgA plasma cells traffic via the blood back to mucosal sites IgA deficiency is very common (~1 in 500) but is asymptomatic in a majority of individuals

19 IgA: Questions 1.What about gut bacteria induces an IgA response? 2.Are IgA-secreting plasma cells long-lived or short-lived? 3.Do IgA responses exhibit immunological memory? 4. What effect does IgA have on bacteria in the gut?

20 Question 1: What about gut bacteria induces an IgA response?

21 Approach 1: Controlled antigen delivery to the mucosal immune system HA107 is an E coli auxotroph for m-diaminopimelic acid (DAP) and for D-alanine (required for peptidoglycan biosynthesis and not made by host)

22 Reversible microbial colonization of germ-free mice Hapfelmeier et al. Science 328: , 2010

23 Most IgA plasma cells in gut are dependent on gut bacteria HA107 gavaged 6 times over 14 days; IgA plasma cells (green) evaluated after 4 weeks total (blue: DAPI staining of nuclei) (ASF: altered Schaedler flora, a mixture of 8 mouse gut bacteria that creates a relatively stable community) Hapfelmeier et al. Science 328: , 2010

24 Threshold for IgA response to E coli K-12 Hapfelmeier et al. Science 328: , 2010

25 Question 1: What about gut bacteria induces an IgA response? Hapfelmeier et al. Science 328: , 2010 Live bugs induce IgA >10X better than heat-killed bugs

26 Question 2: Are IgA-secreting plasma cells short-lived or long-lived?

27 Hapfelmeier et al. Science 328: , 2010 Question 2: Are IgA-secreting plasma cells short-lived or long-lived? What does this result imply for the longevity of IgA plasma cells?

28 Hapfelmeier et al. Science 328: , 2010 Question 2: Are IgA-secreting plasma cells short-lived or long-lived? What does this result imply for the longevity of IgA plasma cells?

29 Question 3: Do IgA responses exhibit immunological memory? Approach 2: Deep sequencing of gut IgA+ plasma cells

30 Deep sequencing of gut IgA+ plasma cells Lindner et al. J Exp Med 209: , Gut IgA plasma cells are highly polyclonal with a subset of highly expanded clones -The frequency of somatic mutations goes up with age

31 Question 3: Do IgA responses exhibit immunological memory? What can be done to look at a memory response in this system?

32 Question 3: Do IgA responses exhibit immunological memory? Lindner et al. J Exp Med 209: , 2012 Plasma cells are killed off with Bortezomib, a proteasome inhibitor (used in multiple myeloma patients)

33 Question 3: Do IgA responses exhibit immunological memory? Lindner et al. J Exp Med 209: , 2012 Gut IgA plasma cells show considerable clonal overlap before and after killing plasma cells with Bortezomib Lindner et al. J Exp Med 209: , 2012

34 Question 4: What effect does IgA have on bacteria in the gut? Approach 3: Genetic defect in the IgA response (Activation-induced cytidine deaminase, AID, is required for somatic mutation AND class switch)

35 AID-/- mice exhibit a dramatic expansion of Peyer’s patches and ILFs AID +/- AID -/- 3 wks of age 20 wks of age Fagarasan et al. Science 298: , 2002.

36 AID-/- mice exhibit a dramatic expansion of Peyer’s patches and ILFs AID +/- AID -/- 3 wks of age 20 wks of age Fagarasan et al. Science 298: , Why is there a large expansion of germinal centers in the mucosal lymphoid tissues?

37 Changes in representation of different gut bacteria in AID -/- mice Fagarasan et al. Science 298: , 2002.

38 Somatically mutated IgA restricts the numbers of bacteria in the gut Wei et al. Nature Immunology, 2011 AID G23S: 9-15x lower somatic mutation; equivalent fraction of plasma cells that are IgA

39 IgA restricts the numbers of Segmented Filamentous Bacteria in the small intestine Suzuki et al. PNAS 101: , 2004 Upper Small Intestine Lower Small Intestine

40 Question 1: What about gut bacteria induces an IgA response?

41 Segmented filamentous bacterium Ivanov et al. Cell 139: , 2009

42 Question 1: What about gut bacteria induces an IgA response? Different components of the gut microbiome may differ greatly in their localization within gut, invasiveness, ability to induce inflammation, etc. (“pathobionts”)

43 Question 1: What about gut bacteria induces an IgA response? Approach 4: Separate bacteria that are coated with IgA from those that are not by flow cytometry

44 Sorting IgA-coated bacteria Palm et al. Cell 158: , 2014 MICE

45 Sorting IgA-coated bacteria Palm et al. Cell 158: , 2014 ICI: relative abundance IgA+/ relative abundance IgA-

46 Sorting IgA-coated bacteria from people with Inflammatory Bowel Disease Palm et al. Cell 158: , 2014

47 Consortia of IgA+ vs. IgA- microbes Palm et al. Cell 158: , 2014

48 Consortia of IgA+ vs. IgA- microbes: stable short-term colonization of mice Palm et al. Cell 158: , 2014

49 Consortia of IgA+ vs. IgA- microbes: different degrees of IgA induction Palm et al. Cell 158: , 2014

50 Consortia of IgA+ vs. IgA- microbes: invasion of the mucus layer by the IgA+ bugs Palm et al. Cell 158: , 2014

51 Consortia of IgA+ vs. IgA- microbes: inflammatory action Palm et al. Cell 158: , 2014

52

53 Somatically mutated IgA limits number of pathogenic bacteria that reach MLN Wei et al. Nature Immunology, 2011 Yersinia enterocolitica numbers detected in mesenteric LN


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