Transcript PowerPoint - UCSF Immunology Program

Changes in
Antibodies
• Class
switch recombination
Variable region
(antigen-binding)
IgG1
Constant region
(function)
IgE
IgM
IgE
IgA
• Affinity maturation
Low
affinity
mutation,
selection
High
affinity
• Membrane vs secreted
Transmembrane
& Intracellular
Domains
B cell
Plasma cell
AID
B Cell Differentiation Pathways
Short-Lived
Plasma Cell
Memory
B Cell
Germinal
Center
Long-Lived
Plasma Cell
Activated B Cell
Affinity Maturation
Hypermutation, Selection
Normal Functions
of Antibodies
Neutralization
viruses
Parasitic
worm
toxins
IgG1
inflammation
IgA
Mast cell
IgE
Opsonization
bacteria
Macrophage
Antibody-dependent cytotoxicity
Foreign/
infected
cell
Toxic granules
Complement activation
bacteria
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
Infection
N. brasiliensis
Serum
IgG1 vs IgE
Erazo A. et al. Immunity 2007, 26:191-203.
L3 larvae
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
IgG1
TNP-OVA
Alum adjuvant
NP-KLH
Alum adjuvant
GFP (IgE)
Talay O. et al. Nat Immunol 2012, 13:396-404.
N. brasiliensis
infection
GFP (IgE)
Yang Z. et al. Immunity
2012, 36:857-872.
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.
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.
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
8 days
Draining LNs (FACS) in vivo
Yang Z. et al. Immunity
2012, 36:857-872.
spleen
Naïve
+ Anti-CD40
B cells
& IL-4
4 days
in vitro
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
Shorter Lifespan
Reduced Affinity
Yang Z. et al. Immunity
2012, 36:857-872.
Why IgE B cell lifespan and affinity may
be limited
Neutralization
viruses
Parasitic
worm
toxins
inflammation
Mast cell
IgE
Systemic IgE
activation
IgG1
IgA
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
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
AID+/-
AID-/-
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
AID+/-
AID-/-
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