Changes in Antibodies• Class switch recombination

• Membrane vs secreted

mutation, selection

Low affinity

Highaffinity

• Affinity maturation

IgEConstant region(function)

Variable region(antigen-binding)

Transmembrane& IntracellularDomains

B cell Plasma cell

IgG1

IgEIgM

IgAAID

B Cell Differentiation Pathways

Germinal Center

Activated B Cell

Short-LivedPlasma Cell

Affinity MaturationHypermutation, Selection

Long-LivedPlasma Cell

Memory B Cell

Normal Functions of AntibodiesNeutralization

toxins

viruses

Mast cell

Parasiticworm

inflammation

bacteria

Complement activation

Macrophagebacteria

Opsonization

Toxic granulesForeign/ infected cell

Antibody-dependent cytotoxicity

IgG1

IgE

IgM

IgA

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 IgEInfectionN. brasiliensisL3 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)Ig

G1

NP-KLHAlum adjuvantTNP-OVA

Alum adjuvant

N. brasiliensisinfection

Talay O. et al. Nat Immunol 2012, 13:396-404. Yang Z. et al. Immunity 2012, 36:857-872.

Kinetics of IgE vs IgG1 B cells in GCs

N. brasiliensisinfection

Talay O. et al. Nat Immunol 2012, 13:396-404.

Yang Z. et al. Immunity 2012, 36:857-872.

He J.S. et al. J Exp Med 2013, 210:2755-2771.

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

He J.S. et al. J Exp Med 2013, 210:2755-2771.

HA-specific 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?– BCR signaling– Plasma cell differentiation

IgE+ B cells are Biased Toward Plasma Cell Differentiation

in vivo

NP-KLHAlum adjuvant

subQ 8 days

in vitro

NaïveB cells

Anti-CD40

IL-4&spleen+

4 days

Draining LNs (FACS)

Yang Z. et al. Immunity 2012, 36:857-872.

Cell-Intrinsic Increase in IgE+ GC B Cells Caused by Blimp-1 Deficiency

CD45.2 GC B / CD45.1 GC B

CD45.2 naive B / CD45.1 naive B

Yang Z. et al. Immunity 2012, 36:857-872.

Blimp-1 is a transcription factor needed for plasma cell differentiation

Shorter LifespanReduced Affinity

Yang Z. et al. Immunity 2012, 36:857-872.

Why IgE B cell lifespan and affinity may be limited

Neutralization

toxins

viruses

Mast cell

Parasiticworm

inflammation

IgG1IgE IgA

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

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?

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

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: 1705-9, 2010

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?

Hapfelmeier et al. Science 328: 1705-9, 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?

Hapfelmeier et al. Science 328: 1705-9, 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?

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

Gut IgA plasma cells show considerable clonal overlap before and after killing plasma cells with Bortezomib

Lindner et al. J Exp Med 209: 365-77, 2012

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 ILFsAID+/- AID-/-

3 wksof age

20 wksof age

Fagarasan et al. Science 298: 1424-1427, 2002.

AID-/- mice exhibit a dramatic expansion of Peyer’s patches and ILFsAID+/- AID-/-

3 wksof age

20 wksof 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

Suzuki et al. PNAS 101: 1981-1986, 2004

Upper Small Intestine

Lower Small Intestine

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

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

MICE

Sorting IgA-coated bacteria

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

ICI: relative abundance IgA+/ relative abundance 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: 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