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

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

3. 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

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

5. 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

6. Helminth parasite model: Nippostrongylus brasiliensis infection

7. Differences in Kinetics of Antibody Responses
Infection
N. brasiliensis
L3 larvae
Erazo A. et al. Immunity 2007, 26:
Cheng L.E. et al. J Immunol 2010, 185:

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

9. 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

10. 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:
Similar data in:
Talay O. et al. Nat Immunol 2012, 13:
He J.S. et al. J Exp Med 2013, 210:

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

12. 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

13. 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

14. 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

15. 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

16. 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

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

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

19. The gut has a complex microbial community (microbiome)

20. 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.

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

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

23. 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”

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

25. 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

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

27. 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: , 2014
Conclusion: bugs differ in what proportion is coated with IgA or not coated

28. 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

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

30. Most IgA plasma cells in gut are dependent on gut bacteria (B6 mice)
Hapfelmeier et al. Science 328: , 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)

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

32. 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

33. 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)

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

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

36. 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

37. 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.

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

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

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

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

42. Microbiota: an important variable in animal research

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