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

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

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

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?

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?

Helminth parasite model: Nippostrongylus brasiliensis infection

Serum IgG1 vs IgE Infection N. brasiliensis L3 larvae Erazo A. et al. Immunity 2007, 26:191-203. Cheng L.E. et al. J Immunol 2010, 185:5040-5047.

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?

IgE vs IgG1 B cells GFP (IgE) GFP (IgE) IgG1 NP-KLH Alum adjuvant TNP-OVA Alum adjuvant IgG1 GFP (IgE) Talay O. et al. Nat Immunol 2012, 13:396-404. Yang Z. et al. Immunity 2012, 36:857-872. N. brasiliensis infection GFP (IgE)

Kinetics of IgE vs IgG1 B cells in GCs N. brasiliensis infection Kinetics of IgE vs IgG1 B cells in GCs Yang Z. et al. Immunity 2012, 36:857-872. Talay O. et al. Nat Immunol 2012, 13:396-404. He J.S. et al. J Exp Med 2013, 210:2755-2771.

Why do IgE and IgG1 differ in abundance? 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

BCR expression in IgE vs IgG1 Germinal Center B Cells HA-specific B cells He J.S. et al. J Exp Med 2013, 210:2755-2771.

Why do IgE and IgG1 differ in abundance? 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

IgE+ B cells are Biased Toward Plasma Cell Differentiation NP-KLH Alum adjuvant subQ in vitro Naïve B cells Anti-CD40 IL-4 & spleen + 4 days 8 days Draining LNs (FACS) in vivo We found that this was indeed true early after immunization in vivo. Interestingly, we observed that IgE+ B cells were also more likely to differentiate into plasma cells in vitro. This indicates that there is an intrinsic propensity of IgE+ B cells to differentiate into plasma cells. Yang Z. et al. Immunity 2012, 36:857-872. 14

Cell-Intrinsic Increase in IgE+ GC B Cells Caused by Blimp-1 Deficiency Yang Z. et al. Immunity 2012, 36:857-872. Blimp-1 is a transcription factor needed for plasma cell differentiation CD45.2 GC B / CD45.1 GC B CD45.2 naive B / CD45.1 naive B We wanted to test whether the elevated expression of Blimp-1 in IgE+ B cells drives the cells towards plasma cell differentiation and thus reduces the number of IgE+ B cells in the germinal center. To test whether Blimp-1 has an intrinsic role in IgE+ B cells in vivo, we took a mixed chimera approach, in which 50% of bone marrow will give rise to wt B cells and 50% of bone marrow will give rise to B cells deficient in Blimp-1, which can be identified by the congenic markers CD45.1 and CD45.2. After reconstitution and immunization, we compared the ratio of Blimp-1 deficient B cells to wild-type B cells in the germinal center, normalized by the same ratio of naïve B cells. Comparing the Blimp-1 deficient B cells with the control B cells, we found that Blimp-1 deficiency greatly increased the frequency of IgE+ B cells within germinal centers. This means that Blimp-1 expression normally limits the number of IgE+ germinal center B cells. 15

Shorter Lifespan Reduced Affinity Yang Z. et al. Immunity 2012, 36:857-872.

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

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

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

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

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

Reversible microbial colonization of germ-free mice Hapfelmeier et al. Science 328: 1705-9, 2010

Most IgA plasma cells in gut are dependent on gut bacteria Hapfelmeier et al. Science 328: 1705-9, 2010 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)

Threshold for IgA response to E coli K-12 Hapfelmeier et al. Science 328: 1705-9, 2010

Question 1: What about gut bacteria induces an IgA response? Hapfelmeier et al. Science 328: 1705-9, 2010 Live bugs induce IgA >10X better than heat-killed bugs

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

Question 2: Are IgA-secreting plasma cells short-lived or long-lived? Hapfelmeier et al. Science 328: 1705-9, 2010 What does this result imply for the longevity of IgA plasma cells?

Question 2: Are IgA-secreting plasma cells short-lived or long-lived? Hapfelmeier et al. Science 328: 1705-9, 2010 What does this result imply for the longevity of IgA plasma cells?

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

Deep sequencing of gut IgA+ plasma cells Lindner et al. J Exp Med 209: 365-77, 2012 -Gut IgA plasma cells are highly polyclonal with a subset of highly expanded clones -The frequency of somatic mutations goes up with age

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

Question 3: Do IgA responses exhibit immunological memory? Lindner et al. J Exp Med 209: 365-77, 2012 Lindner et al. J Exp Med 209: 365-77, 2012 Plasma cells are killed off with Bortezomib, a proteasome inhibitor (used in multiple myeloma patients)

Question 3: Do IgA responses exhibit immunological memory? Lindner et al. J Exp Med 209: 365-77, 2012 Lindner et al. J Exp Med 209: 365-77, 2012 Gut IgA plasma cells show considerable clonal overlap before and after killing plasma cells with Bortezomib

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)

AID-/- mice exhibit a dramatic expansion of Peyer’s patches and ILFs 3 wks of age 20 wks of age Fagarasan et al. Science 298: 1424-1427, 2002.

AID-/- mice exhibit a dramatic expansion of Peyer’s patches and ILFs 3 wks of age 20 wks of age Fagarasan et al. Science 298: 1424-1427, 2002. Why is there a large expansion of germinal centers in the mucosal lymphoid tissues?

Changes in representation of different gut bacteria in AID-/- mice Fagarasan et al. Science 298: 1424-1427, 2002.

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

IgA restricts the numbers of Segmented Filamentous Bacteria in the small intestine Upper Small Intestine Lower Small Intestine Suzuki et al. PNAS 101: 1981-1986, 2004

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

Segmented filamentous bacterium Ivanov et al. Cell 139: 485-498, 2009

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”)

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

Sorting IgA-coated bacteria MICE Palm et al. Cell 158: 1000-10, 2014

Sorting IgA-coated bacteria ICI: relative abundance IgA+/ relative abundance IgA- Palm et al. Cell 158: 1000-10, 2014

Sorting IgA-coated bacteria from people with Inflammatory Bowel Disease Palm et al. Cell 158: 1000-10, 2014

Consortia of IgA+ vs. IgA- microbes Palm et al. Cell 158: 1000-10, 2014

Consortia of IgA+ vs. IgA- microbes: stable short-term colonization of mice Palm et al. Cell 158: 1000-10, 2014

Consortia of IgA+ vs. IgA- microbes: different degrees of IgA induction Palm et al. Cell 158: 1000-10, 2014

Consortia of IgA+ vs. IgA- microbes: invasion of the mucus layer by the IgA+ bugs Palm et al. Cell 158: 1000-10, 2014

Consortia of IgA+ vs. IgA- microbes: inflammatory action Palm et al. Cell 158: 1000-10, 2014

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