Flipped Classroom Session Micro 204 Flipped Classroom Session Antibodies IgE and IgA Chris Allen Tony DeFranco

IgE Antibody Function Parasitic worm mediators including histamine chemokines cytokines IgE FceRI inflammation mast cell or basophil

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

Differences in Fc Receptors bacteria IgG IgE Mast Cell FceRI Macrophage FcgR

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

Helminth parasite model: Nippostrongylus brasiliensis infection

Differences in Kinetics of Antibody Responses 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.

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

Germinal Center Selection Antigen Apoptosis BCR signal Proliferation highest affinity Differentiation Memory B Cell T cell T cell help GC B Cells highest affinity Plasma Cell

Differences in IgG1 vs IgE Germinal Center B Cell Responses N. brasiliensis infection Differences in IgG1 vs IgE Germinal Center B Cell Responses IgG1+ Germinal Center B Cells IgE+ Germinal Center B Cells Yang Z. et al. Immunity 2012, 36:857-872. Similar data in: Talay O. et al. Nat Immunol 2012, 13:396-404. He J.S. et al. J Exp Med 2013, 210:2755-2771.

Questions Why do IgE+ B cells disappear from the germinal center? What are the implications for the IgE antibody response?

IgE+ B cells are biased toward plasma cell differentiation in cell culture without antigen B cell culture IgG1+ IgE+ No antigen a-CD40 IL-4 CD138 IgM IgG1 IgE Blimp-1-GFP Yang, Z. et al. (2016) eLife 5:e21238 Similar to Haniuda, K. et al. (2016) Nat Immunol 17:1109 B cell Plasma cell Blimp-1 CD138 12

Ectopic expression of IgG1 vs IgE Retroviral transduction IgG1 IgE IgM B cell B1-8flox/+ Cg1-Cre Haniuda, K. et al. (2016) Nat Immunol 17:1109 Yang, Z. et al. (2016) eLife 5:e21238

IgE Induces Plasma Cell Differentiation without antigen (Plasma cell marker) Yang, Z. et al. (2016) eLife 5:e21238 Similar to Haniuda, K. et al. (2016) Nat Immunol 17:1109

Antigen-dependent vs. independent B cell receptor signaling Antigen-independent signaling ? B cell Apoptosis “Tonic” signaling needed for B cell survival B Cell Receptor Antigen B Cell Receptor B cell IgG1 IgE Plasma cell differentiation B cell Plasma cell

IgE GC B cells maintained with BCR signaling mutant Haniuda et al. (2016) Nat Immunol 17:1109 Similar to: Yang, Z. et al. (2016) eLife 5:e21238

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

The gut has a complex microbial community (microbiome) Donaldson, Lee and Mazmanian, Nature Rev Microbiol. 14: 20, 2016

The gut has a complex microbial community (microbiome)

The gut has a complex microbial community (microbiome) Mice from Taconic but not mice from Jackson have Segmented Filamentous Bacterium (SFB), which attaches itself to the epithelium in the small intestines and grows as a chain. Ivanov et al, Cell 139: 485, 2009.

Immune control of gut microbes Hooper and MacPherson, Nature Rev Immunol. 10: 159, 2010

Immune control of gut microbes Donaldson, Lee and Mazmanian, Nature Rev Microbiol. 14: 20, 2016

IgA Background IgA is secreted in mucosal tissue and is transported across mucosal epithelial barriers by the poly-Ig receptor Poly-Ig receptor is cleaved; the part that stays bound to IgA is “secretory component”

IgA in small intestines IgA plasma cells disperse to small intestines and colon Hooper and MacPherson, Nature Rev Immunol. 10: 159, 2010

IgA Background Some IgA is T cell-dependent; some IgA is T cell- independent Some IgA is produced in germinal center responses and exhibits somatic mutation Deep sequencing of IgA reveals a diverse response that includes a few specificities produced abundantly Gut bacteria that have IgA on them can be revealed by staining with a fluorescent anti-IgA and using flow cytometry and cell sorting followed by sequencing of 16S rRNA

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

Question 1: What about gut bacteria induces an IgA response? ICI: relative abundance IgA+/ relative abundance IgA- Mice: 4 taxons enriched in IgA+ coated; 22 taxons enriched in IgA- Palm et al. Cell 158: 1000-10, 2014 Conclusion: bugs differ in what proportion is coated with IgA or not coated

IgA Question 1: What about gut bacteria induces an IgA response? Formulate two or three hypotheses and come up with one experimental approach to address one or more of these hypotheses Tools to consider: -Flow cytometry to identify bugs that are coated with IgA or not coated (and relative proportions) -Introduction of individual species or sets of species into germ free mice -Mice or people with diseases, immunodeficiencies, and/or different diets

Question 1: What about gut bacteria induces an IgA response? Hypothesis one: amount of antigen available to the immune system.

Most IgA plasma cells in gut are dependent on gut bacteria (B6 mice) 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 1: What about gut bacteria induces an IgA response? live E. coli are 10x better stimulus than heat killed E. coli Hypothesis two: location of bugs relative to gut epithelium is important (bug must penetrate mucus layer and/or invade tissue to induce IgA response)

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

Question 1: What about gut bacteria induces an IgA response? Hypothesis three: bugs found in lumen of colon AND not in small intestines do not induce much IgA

Question 1: What about gut bacteria induces an IgA response? Conclusion: most bugs in small intestines induce specific IgA; most bugs in colon may not induce IgA Bunker et al. Immunity 43: 541, 2015.

Question 1: What about gut bacteria induces an IgA response? Hypothesis four: more “pathogenic” bugs preferentially induce IgA

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: inflammatory action Palm et al. Cell 158: 1000-10, 2014

Microbiota: an important variable in animal research

Sorting IgA-coated bacteria: role of location in IgA induction Bunker et al. Immunity 43: 541-553, 2015