Islet Antigen-reactive B Cells as Participants
and Therapeutic Targets in T1D
John C. Cambier
Department of Immunology and Microbiology
University of Colorado School of Medicine
No relevant COI
Evidence that B cells play a role in Type 1 Diabetes
• Skewing the BCR repertoire toward an islet antigen, e.g. insulin, reactivity
promotes T1D, while skewing it away from insulin reactivity prevents T1D.
Hulbert et al. J Immunol. 2001 Nov 15;167(10):5535-8
% Diabetic
100
75
~3% of B cells insulin reactive (VH125tg)
~0.1% of B cells insulin reactive (non‐tg)
50
25
0
0 % of B cells insulin reactive (VH281tg)
10 13 16 19 22 25 28 31 34 37
AGE (weeks)
• Rituximab has shown efficacy in T1D.
Pescovitz et al N England J Med 2009 Nov 26;361(22):2143-52
Silencing of autoreactive B cells
Central Tolerance
Peripheral Tolerance
75% autoreactive
Receptor Editing 
(30%)
Anergy
(40%)
Deletion (apoptosis)
(5%)
Self antigen
Foreign antigen
mIgM
mIgD
ANERGY:
Chronic exposure of low
avidity antigen to autoreactive B cells leads to
unresponsiveness.
 can have range of
affinity for self-antigen
downregulation of
surface IgM (IgD is
retained)
can occur in immature
and mature B cells
 reversible (can lead to
autoimmunity)
Hypothesis:
We posit that potentially pathogenic insulin-reactive B are
normally silenced by anergy, but they become activated and
participate in development of T1D.
Questions:
Are potentially pathogenic insulin-reactive B cells found in anergic
compartments in healthy subjects?
Do these B cells show signs of activation and move to the
pancreas and PLN during disease development?
Anergic B cells in the healthy human
Andy Duty and Patrick Wilson (JEM, 2009):
• Described two anergic B cell subpopulations in healthy humans. These
express autoreactive antigen receptors
• Approximately 3% of peripheral blood B cells are IgD+IgM– Approximately 2.5% are naïve (BND cells) and 0.5% are memory
(Cδ-CS cells)
Tam Quach and Inaki Sanz (JI, 2011):
• Expanded definition to include cells with low surface levels of mIgM (IgMlo)
but relatively normal levels of IgD
Method of isolation of insulin-binding B cells in human
peripheral blood
PBMCs isolated by Ficoll‐Hypaque density gradient centrifugation
Fc block, biotinylated insulin, and surface marker antibodies
*
fix
Run over MACS anti‐A647 *
* column, positively *
magnetic SA‐A647
select for insulin beads
binding Human T1D CD19+ B Cells
* Wash
No enrichment
Enrichment of insulin binding cells
InsulinBystanders binding
99.5
0.49
70.7
Insulin binding 26.3
Gating strategy for identification of insulin-binding B cell
subpopulations in PBL
5
10
4
150K
150K
100K
10 3
100K
50K
31.9
Naive Memory
CD19
FSC-A
SSC-A
200K
99.8
10
50K
10
5
10
4
10 3
2
10
0
0
50K
100K
150K
FSC-A
200K
250K
2
5
10
4
10
3
10
2
0
Mature
Naive
BND
0
0
0
50K
100K
150K
FSC-H
200K
250K
0
10
2
10
3
CD27
10
4
10
5
0
10 2
10 3
10 4
Insulin binding
10 5
10
5
10
5
10
4
10
4
CD19
0
IBCs
10
IgM
200K
10
IgM
250K
CD19
250K
10 3
10
2
10
3
10
2
0
10 2
0
10
10 3
IgD
10 4
10 5
0
0
0
10
2
10
3
10
4
Insulin binding
10
5
2
10
3
IgD
10
4
10
5
BND cells have reduced Ca2+ flux and Syk phosphorylation
upon stimulation, suggesting they are anergic
Stim w/ 20μg/ml F(ab’)2 anti-human IgD
10
3
Mature
Naive
Naive
102
0 10
2
10
3
IgD
10
4
10
80
3
2
0
50
5
100
80
60
40
20
0
0 10
2
10
60
40
20
1
BND
Bnd
0
4
100
% of Max
4
[Ca2+]i
10
% of Max
IgM
105
3
pSyk
pSyk
10
4
10
5
100
Time
150
200
0
0 10 2
103
IgD
104
105
IBCs in the anergic BND fraction bear high affinity
auto/polyreactivity antigen receptors
Single cell sort MN and BND IBCscloned Ig variable regionsre-expressed as mAb
Insulin
Chromatin
LPS
VH region sequences of IBCs in the anergic BND fraction
are consistent with auto/polyreactivity
Serum antibodies from IAA+ new onsets show increased
reactivity to both insulin and chromatin
Conclusions Part I
•
•
•
•
The BND cells appear functionally anergic based on decreased BCR-mediated Ca2+
mobilization and Syk phosphorylation.
B cells bearing BCR with high affinity for insulin are found in the BND fraction
indicating normal silencing by anergy.
BND IBCs have a higher affinity for insulin than their mature naïve IBC counterparts,
suggesting the latter are ignorant of their autoantigen.
The anergic BND IBCs are polyreactive.
– These BND cells could be initially activated by antigens other than insulin, such as host or pathogenderived DNA
•
“New onsets” who make insulin autoantibodies also make chromatin
autoantibodies.
– Suggests activation of the anergic, high affinity, polyreactive B cell population found in healthy
controls
Do IBCs show signs of activation and move to the
pancreas and PLN during disease development?
•
First degree relatives (FDR) (n=25)
– Autoantibody negative
•
Pre-diabetic groups (n=17)
– Autoantibody positive
•
New onset T1D (n=21)
– Time from onset < 12 mths
•
T1D long standing (n=21)
– Time from onset > 12 mths
•
Healthy controls (n=36)
– Age/sex matched
Mature
Naive
% Mature Naive
There is a decrease in the anergic BND IBC population in prediabetic and new onset patients, and in some first degree
relatives.
% BND
BND
“At risk”
for T1D?
Loss of BND in FDRs correlates with high risk HLA genotype
“Transient” loss of the total BND population in FDRs, prediabetics and new onsets
4
***
% BND
***
2
4
2
p = 0.003
0
To
ta
l
IB
C
0
H
/C
H
/C
T1
D
0
6
**
T1
D
2
6
FD
Pr R
eT1
D
N
/O
T1
D
Total BND
**
***
***
4
FD
Pr R
eT1
D
N
/O
T1
D
[% of IBCs]
IBC BND
6
Total
[% of total B cells]
Insulin-binding
Conclusions Part II
• There appears to be a transient loss of anergic BND IBCs and total
BND cells in some first degree relatives and all pre-diabetic, new
onset patients.
Suggests a possible early biomarker for T1D and/or other autoimmune
diseases
Loss of anergy may be a very early step in progression to T1D.
• The transient nature of this loss is consistent with initiation of
autoimmunity by acute injury or infection, the exact nature of which is
determined by genetic risk factors.
• Loss of BND cells correlates with risk HLA allele genotype.
T cells may promote activation of anergic B cells
Future directions: Can we target high affinity IBCs with
hormonally inactive insulin-toxin conjugates?
des-penta insulin
des-penta proinsulin
X38
des-penta
fragment
Cleavage sites
C-peptide fragment
Acknowledgements
Cambier Lab
Peter Gottlieb Lab
Mia Smith
Lisa Fitzgerald-Miller
Marynette Rihanek
Aimon Alkanani
Tom Packard
Shannon O’Neill
Rochelle Hinman
Andrew Getahun
Melissa Walker
Janie Akerlund
Elizabeth Franks
Soojin Kim
Patrick Wilson Lab
Carole Dunand
Future Directions
•
Is low IBC BND status in FDRs associated with high T1D risk genotype, e.g. PTPN22?
– Use ImmunoChip data from TrialNet
•
What is the stability of the BND phenotype and utility of low BND as a biomarker for risk of
progression to T1D?
– Longitudinal study currently underway
•
Are BND cells able to present antigen to insulin reactive T cells?
– ???
•
Is there a similar loss of BND cells in other autoimmune diseases?
– Current funding to look at autoimmune thyroiditis
•
Is loss of IBC BND associated localization in the pancreas and PLN?
– Use nPOD samples and repertoire analysis
•
Are high affinity, polyreactive B cells more pathogenic than low affinity, low polyreactive B
cells in NOD mice?
– Retrogenics/knockins
Human Type 1 Diabetes (T1D)
• Autoimmune disorder characterized by destruction of beta cells in
pancreas  decreased production of insulin  hyperglycemia
• Predicted by presence of genes and autoantibodies
– e.g. HLA genes (esp DR3/4-DQ2/8 haplotype)
– e.g. anti-insulin, GAD65, ICA, IA-2A, ZnT8
• Occurs more often in individuals < 30 years old; clinical symptoms
include polyuria, polydipsia
• Both genetics and environment thought to play a role
• Incidence is rising globally and as much as 4% annually in U.S.
Loss of BND correlates with high risk HLA alleles
Mature
Naive
% Mature Naive
There is a decrease in the anergic BND IBC population in prediabetic and new onset patients, and in some first degree
relatives.
% BND
BND
“At risk”
for T1D?
Evidence that B cells play a role in Type 1 Diabetes
• Serum auto-antibodies are a hallmark of T1D, but are not required for
disease. Wong et al. Diabetes. 2004
• NOD mice that lack B cells are resistant to T1D. B cell depletion also
has protective effects. Serreze et al. J Exp Med. 1996 and Noorchashm et al. Diabetes.
1997
• Skewing the BCR repertoire toward an islet Ag, e.g. insulin, reactivity
promotes T1D, while skewing it away from insulin reactivity prevents
T1D. Hulbert et al. J Immunol. 2001 Nov 15;167(10):5535-8
• Rituximab has shown efficacy in T1D.
Pescovitz et al N England J Med 2009 Nov 26;361(22):2143-52