1. 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

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

3. 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

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

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

11. 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

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

13. 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

14. 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: 14

15. Cell-Intrinsic Increase in IgE+ GC B Cells Caused by Blimp-1 Deficiency
Yang Z. et al. Immunity 2012, 36:
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

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
Parasitic
worm
inflammation
Mast cell
IgG1
IgA
IgE
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 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?

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

21. 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)

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
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)

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

28. Question 2: Are IgA-secreting plasma cells short-lived or long-lived?
Hapfelmeier et al. Science 328: , 2010
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: , 2012
-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
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
Lindner et al. J Exp Med 209: , 2012
Gut IgA plasma cells show considerable clonal overlap before and after killing plasma cells with Bortezomib

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
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
3 wks
of age
20 wks
of age
Fagarasan et al. Science 298: , 2002.
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
Upper Small Intestine
Lower Small Intestine
Suzuki et al. PNAS 101: , 2004

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
MICE
Palm et al. Cell 158: , 2014

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

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

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