Page 1 of 47 Diabetes
DCIR2+ cDC2 DCs and Zbtb32 restore CD4+ T cell tolerance and inhibit diabetes.
Jeffrey D. Price1,2*, Chie Hotta Iwamura1*, Yongge Zhao1, Nicole M. Beauchamp1, and Kristin V. Tarbell1
1Immune Tolerance Section, Diabetes, Endocrinology and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 2Current address: Nebraska Center for Virology, University of Nebraska Lincoln, Lincoln, NE, 68583 * Equal contribution
Short title: cDC2s restore tolerance and inhibit diabetes
Corresponding author: Kristin V. Tarbell, Ph.D. DEOB, NIDDK, NIH P +1 (301) 451 9360 Bldg 10, CRC, West Labs, 5 5940 F +1 (301) 480 4518 Bethesda, MD 20892 [email protected]
K.V.T. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Conflict of interest statement: The authors have declared that no conflict of interest exists.
Related interest categories: Autoimmune diseases, Immunology
Related Keywords: Dendritic cells, Diabetes, Tolerance
Character count: 4587 words + 8 Figures + 5 Supplemental Figures + 1 Supplemental Table
1
Diabetes Publish Ahead of Print, published online June 12, 2015 Diabetes Page 2 of 47
Abstract
During autoimmunity, the normal ability of dendritic cells (DCs) to induce T cell tolerance is
disrupted, therefore autoimmune disease therapies based on cell types and molecular pathways
that elicit tolerance in the steady state may not be effective. To determine which DC subsets
induce tolerance in the context of chronic autoimmunity, we used chimeric antibodies specific
for DCIR2 or DEC 205 to target self antigen to CD11b+ (cDC2) DCs and CD8+ (cDC1) DCs,
respectively, in autoimmune prone non obese diabetic (NOD) mice. Antigen presentation by
DCIR2+ DCs but not DEC 205+ DCs elicited tolerogenic CD4+ T cell responses in NOD mice.
Beta cell antigen delivered to DCIR2+ DCs delayed diabetes induction and induced increased T
cell apoptosis without interferon (IFN) γ or sustained expansion of autoreactive CD4+ T cells.
These divergent responses were preceded by differential gene expression in T cells early after in vivo stimulation. Zbtb32 was higher in T cells stimulated with DCIR2+ DCs, and overexpression
of Zbtb32 in T cells inhibited diabetes development, T cell expansion and IFN γ production.
Therefore, we have identified DCIR2+ DCs as capable of inducing antigen specific tolerance in
the face of ongoing autoimmunity, and have also identified Zbtb32 as a suppressive transcription
factor that controls T cell mediated autoimmunity.
2 Page 3 of 47 Diabetes
Introduction
Antigen specific induction of T cell tolerance is a desired therapeutic outcome for type 1
diabetes because of the potential to stop undesirable pathogenic responses while minimizing
nonspecific immune inhibition. To date, little clinical efficacy has been observed for this
approach (1, 2). Autoimmune individuals elicit immune responses in an inflammatory context,
and are therefore refractory to tolerance induction, yet most studies of T cell tolerance have been
performed in either a steady state context or in models of autoimmunity requiring immunization
with autoantigen that best model the effector phase (3). Therefore, to move beyond therapies that
non specifically block effector functions, it is important to learn what conditions are needed to
enable antigen specific T cell tolerance induction in a chronic inflammatory autoimmune
environment, which can be modeled using autoimmune prone non obese diabetic (NOD) mice
that show spontaneous loss of self tolerance due to genetic and environmental factors (4).
These factors leading to autoimmune diabetes alter the capacity of antigen presenting cell
populations to induce tolerance. In NOD mice, dendritic cells (DCs) are in the pancreas prior to
T cell infiltration and are important for diabetes pathogenesis and regulation (5 7). DCs are
central for both induction of immunity and tolerance (8), and conventional DCs (cDCs) can be
divided into two broad subsets with similar function in both mouse and human (9). The cross
presenting cDC1 express XCR1 in both human and mouse, and can be identified by CD8 or
CD103 expression in mice (10, 11). cDC2 are CD11b+ in both mouse and human, CD1c+ in
human, and are dendritic cell inhibitory receptor 2 (DCIR2)+ in mice (12). CD11b+ cDC2 are
strong stimulators of antibody production and CD4+ T cell effector responses, and induce
regulatory T cell (Treg) proliferation, whereas CD8+ cDC1 endocytose apoptotic blebs and can
result in T cell tolerance directed against self antigens (13, 14). cDC1 are dependent on the
3 Diabetes Page 4 of 47
transcription factor Batf3, and loss of Batf3 in NOD mice leads to a block in diabetes pathogenesis (11, 15).
Patients with type 1 diabetes and NOD mice carry diabetes susceptibility alleles, some of which affect antigen presenting cells such as DCs, that lead to a loss of tolerance and development of autoimmune diabetes (16). The normal generation and maintenance of DCs may be altered in autoimmune diabetes and affect T cell tolerance induction (17 19). T cells appear in the pancreas of NOD mice as early as 4 weeks of age, but hyperglycemia does not occur until 12 weeks or later. This can be modeled by CD4+ autoreactive BDC2.5 T cell receptor (TCR)
transgenic T cells that respond to the beta cell granule protein chromogranin A as well as a series
of mimetope peptides (20 22). Prediabetic mice and humans show islet specific T cells and
antibody responses indicating active autoimmunity, but simultaneous immune regulation can
slow beta cell destruction (23 25). Unlike some autoimmune diseases, the early phases of
autoimmune diabetes are clinically silent because sufficient beta cell destruction for
hyperglycemia doesn’t occur until late. Autoantibodies and MRI signal present in prediabetic
mice and humans correlate with immune infiltrate in the pancreatic islets (26, 27), and
individuals with high risk can now be identified prior to hyperglycemia (28). Therefore, this prediabetic phase represents ongoing autoimmunity and is of interest as a target of
immunotherapy.
Targeting antigen to DCs without adjuvant can induce T cell tolerance (29 31). Chimeric
antibodies against lectin antigen uptake receptors efficiently targets antigen to specific DC
subsets, including DCIR2 expressed by CD11b+ cDC2 and DEC 205 expressed by CD8+ cDC1 and some migratory DCs (32). This allows characterization of in vivo presentation of relevant antigens by specific DC subsets and has therapeutic potential for induction of both immunity and
4 Page 5 of 47 Diabetes
tolerance (33). Interestingly, in mice without spontaneous autoimmunity, DEC 205+ migratory
DCs are important for tolerance via Treg induction (34). Antigen delivered to CD11b+ DCs via
anti DCIR2 can also be tolerogenic in non autoimmune prone mice, but little is known about the
differential programs these DC subsets elicit in CD4+ T cells (31, 35).
In contrast to these findings of DC mediated tolerance induction, we have shown,
surprisingly, that DEC 205+ cDC1 are unable to induce CD4+ T cell tolerance in NOD mice (18).
In this autoimmune context, DEC 205+ DCs elicit expansion and effector function, even when
starting with naïve T cells. Non targeted antigen delivery will likely have to overcome this
effector response to induce tolerance.
Therefore, we now asked if other DC subpopulations could elicit a more tolerogenic
response from autoreactive CD4+ T cells in NOD mice. Comparing T cells stimulated by DC
subpopulations divergent in tolerogenic ability allows identification of pathways critical for
tolerance induction in this context. Here we demonstrate that delivery of antigen to DCIR2+ DCs
in NOD mice induces a tolerant phenotype in diabetogenic BDC2.5 CD4+ T cells and inhibits
diabetes development in a NOD.scid transfer system. T cells stimulated by DCIR2+ DCs do not
produce interferon (IFN) γ and undergo significant deletion. Early after stimulation with DCIR2+
DCs, T cells displayed distinct gene expression including higher levels of zinc finger and BTB
domain containing 32 (Zbtb32), a transcription factor that can inhibit T cell differentiation (36).
We found that overexpression of Zbtb32 in BDC2.5 T cells increases deletion and inhibits their
ability to induce diabetes. These results demonstrate that, although DC mediated tolerance is
impaired in the context of chronic autoimmunity, specific DC subsets such as DCIR2+ DCs can
still elicit diabetes inhibiting T cell responses, in part by increased expression of Zbtb32.
5 Diabetes Page 6 of 47
Research Design and Methods
Mice. Original breeding pairs of NOD, NOD.BDC2.5, NOD.Thy1.1, NOD.scid, and C57Bl/6 mice were obtained from Jackson Labs (Bar Harbor, ME). NOD.BDC2.5.FoxP3 GFP mice were a kind gift of Vijay Kuchroo (Harvard University). Six to eleven week old mice were used. All mice were bred and housed under specific pathogen free conditions at the National Institutes of
Health according to protocols approved by the Institutional Animal Care and Use Committee.
Antibodies and flow cytometry. FoxP3 (FJK 16s) APC antibodies were purchased from eBioscience (San Diego, CA). CD40 (3/23) FITC and IFN γ (XMG1.2) APC antibodies were purchased from Becton Dickinson (San Jose, CA). CD4 (GK1.5) PacBlue, Thy1.1 (OX7) APC,
Thy1.1 PerCP, Thy1.2 (30 H12) APC, Thy1.2 PerCP, IL 2 (JES6 5H4) Alexa488, CD11c
(N418) PerCP, CD11b (M1/70) PE, IL 3 (MP2 8F8) PE, and CD8 (53 6.7) PacificBlue were purchased from BioLegend (San Diego, CA). Viability was assessed by Fixable Live/Dead Aqua staining (Life Technologies, Grand Island, NY). All DC stains were done in the presence of
TruStain FcX (BioLegend). Flow cytometry was performed on a CyAn (Beckman Coulter, Brea,
CA) or LSRII (Becton Dickinson). Western Blotting was performed using Flag (F3165) and β
actin (A1978) antibodies (Sigma Aldrich, St. Lois, MO), and Amersham ECL Western Blotting
Kit (GE Healthcare Bio Sciences, Pittsburgh, PA) as their detection reagent.
Production of chimeric antibodies. αDEC 205 and αDCIR2 antibodies generated from their
original hybridomas (NLDC 145 and 33D1, respectively) were obtained from Ralph Steinman
and Michel Nussenzweig, Rockefeller University. Antibodies contain an altered IgG1 heavy
chain constant region that ablates binding by Fc receptors (45), followed by a linker sequence
6 Page 7 of 47 Diabetes
and the antigenic peptides. αDEC BDC, and αDCIR2 BDC (1040 55, (20)) were expressed and
purified as previously reported (18, 29). Antibody binding to CHO lines expressing DEC 205 or
DCIR2 (Juliana Idoyaga, Steinman lab, Rockefeller University) was analyzed using a polyclonal
goat anti mouse IgG PE (Jackson ImmunoResearch, West Grove, PA) and binding to spleen
DCs was analyzed by an anti mouse IgG1 Alexa488 (Life Technologies). All batches were
tested for functional activity by BDC2.5 T cell proliferation in vivo and assessed for low
endotoxin levels (< 0.2 EU/ml) by LAL assay (Lonza, Basel, Switzerland).
Purification and stimulation of T cells. CD4+ T cells were recovered from spleens of
NOD.BDC2.5 mice by negative selection as previously reported (18). T cells were labeled with 5
M CFSE (Life Technologies) and 1 2 x 106 cells were injected intravenously. One day after T
cell injection, 100 ng of chimeric antibodies or controls were injected intraperitoneally, unless
otherwise indicated.
Regulation of diabetes by DC targeting antibodies. CD4+CD25 BDC2.5 T cells were isolated
from spleen by negative selection as above, with the addition of biotinylated αCD25
(BioLegend). 5 x 104 T cells were injected intravenously to NOD.scid mice. These NOD.scid
mice were treated one day prior, and concurrently with T cells, with either PBS or 500 ng αDEC
BDC or αDCIR2 BDC. Mice were considered diabetic on the first day of two consecutive days
with blood glucose levels over 250 mg/dL.
Cytokine production and apoptosis. Lymphoid cells were stimulated and intracellular cytokine
staining was performed as previously described (18). Total cell numbers in lymphoid tissues
7 Diabetes Page 8 of 47
were counted by gathering the total number of BDC2.5 T cells within the flow sample and the percentage of the total cell count in the tissue assessed by trypan blue exclusion. For apoptosis
measurement, lymphoid cells stimulated for four days in vivo were examined by CD4 and Thy1
antibodies followed by a one hour incubation with Mitocasp reagents (Cell Technology, Inc.,
Mountain View, CA) and immediate detection by FACS analysis.
Sorting of Treg and Teff. CD4+ T cells were purified as described from spleens of
BDC2.5.FoxP3 GFP mice. Cells were then stained for CD4 and sorted on a BD FACSAria for
CD4+GFP+ (Treg) and CD4+GFP (Teff) populations. Cells were then transferred intravenously
to NOD.Thy1.1 mice as previously described.
T cell rechallenge. Transferred BDC2.5 CD4+ T cells were stimulated for 10 days with PBS or
100 ng αDCIR2 BDC, followed by challenge by PBS or 100 ng αDCIR2 BDC with 20 ug
αCD40 plus 50 g poly(I:C) plus 5 g LPS. Spleen and lymph nodes were collected after four
days and BDC2.5 T cell numbers were assessed by flow cytometry.
Gene expression analysis. BDC2.5.Thy1.1 T cells were purified, transferred to NOD mice and
stimulated with chimeric antibodies as described. Fourteen hours after injection of αDEC BDC
or αDCIR2 BDC, BDC2.5 T cells were sorted from spleen and lymph nodes by Thy1.1 positivity on a BD FACSAria. RNA was isolated by the PicoPure kit (Life Technologies)
according to the manufacturer’s instructions. RNA amount and quality was checked by
Bioanalyzer analysis. RNA was provided to the NIDDK Genomics Core for cDNA synthesis and
array analysis. Gene expression was interrogated using Mouse Gene ST1.0 arrays (Affymetrix,
8 Page 9 of 47 Diabetes
Santa Clara, CA), followed by analysis by Partek Genomics Suite. The mRNA levels of Il2, Il3,
Irf1 and Zbtb32 were quantitatively confirmed using NanoString nCounter analysis.
Transfection of BDC2.5 T cells and diabetes transfer. CD4+ BDC2.5 T cells were isolated
from spleen and lymph nodes of 6 12 week old NOD.BDC2.5 mice using the CD4+ T cell
isolation kit (Miltenyi) and transfected with either the pcDNA3.1 plasmid or Zbtb32 plasmid
using the Amaxa Nucleofection kit for mouse T cells (Lonza). 5 x 105 transfected cntrl BDC2.5
T cells or Zbtb32 BDC2.5 T cells were injected intravenously to 6 week old NOD.scid mice.
The incidence of diabetes was monitored as described. Cell viability after transfection was
measured by FACS, and transfected Zbtb32 expression was determined by Western blotting.
Insulitis score. Pancreata from treated NOD.scid mice were fixed in formalin. Paraffin
embedded tissues were sectioned and stained with hematoxylin and eosin (H&E). The degree of
insulitis was scored in a blinded fashion using the following scale: 0 = intact islet; 1 = peri
insulitis; 2 = moderate insulitis (< 50% of the islet infiltrated); 3 = severe insulitis (≥ 50% of the
islet infiltrated); 4 = destructive insulitis. At least 20 islets per pancreas were analyzed by two
independent examiners.
Statistics. Differences between groups in diabetes experiments (Figs 1A and 8E) were analyzed
by the Log rank test. Analysis of all other data was done using an ANOVA analysis with
Bonferroni post tests or an unpaired, two tailed Student’s t test with a 95% confidence interval
(GraphPad Prism, San Diego, CA). P values less than 0.05 were considered significant.
9 Diabetes Page 10 of 47
Results
To study DC mediated tolerance induction in autoimmune NOD mice, we generated chimeric antibodies that target a BDC2.5 stimulatory mimetope peptide to antibodies against the lectin DEC 205 (αDEC BDC) or DCIR2 (αDCIR2 BDC) found on cDC1 CD8+ and cDC2
CD11b+ DCs, respectively (Supplementary Fig. 1A and B). These antibodies specifically bind to
their target lectins (Supplementary Fig. 1B), and they functionally targeted antigenic peptide to
DCs for efficient stimulation of beta cell reactive BDC2.5 T cells in vivo, while the same dose of
antigen given via a non specific antibody did not significantly stimulate BDC2.5 T cells (18).
To determine how autoantigen presentation by DEC 205+ or DCIR2+ DCs affected
diabetes pathogenesis, CD4+CD25 T cells from NOD.BDC2.5 mice were injected into
NOD.scid mice to induce diabetes. The NOD.scid mice were treated with PBS, αDEC BDC, or
αDCIR2 BDC and followed for hyperglycemia. αDEC BDC did not alter the time to diabetes, as previously reported (Fig. 1A and (18, 37)). αDCIR2 BDC, on the other hand, significantly
delayed diabetes progression as compared to PBS or αDEC BDC (Fig. 1, P < 0.001 for each).
Next, T cell expansion and Treg induction were measured at day 5 in NOD.scid mice transferred
with BDC2.5 T cells. The net expansion of islet specific cells was significantly higher in mice
given αDEC BDC (Fig. 1B), but no differences in the proportion of Foxp3+ cells was observed
(Fig. 1C), suggesting that the differential diabetes induction was not due to changes in Treg
levels, but perhaps differences in proliferation or cell death. Therefore, these two DC subsets
induce distinct pathogenic outcomes in autoreactive T cells.
To understand the differential effects of antigen presentation by these 2 subsets, BDC2.5
T cell responses were examined in lymphoreplete NOD mice. Because initial TCR engagement
and subsequent CD69 upregulation are necessary for antigen specific T cell activation or
10 Page 11 of 47 Diabetes
tolerance induction (38), early T cell responses after in vivo DC stimulation were measured.
Treatment with a dose range from 10 ng to 1 g of αDEC BDC and αDCIR2 BDC led to an
increase of T cell activation markers after 24 hours of stimulation with DEC 205+ or DCIR2+
DCs (Fig. 2A). Activation was observed at a low dose of chimeric antibody, indicating that the
targeting of the antigenic peptide was highly efficient. As expected due to the greater presence of
DEC 205+ DCs in lymph nodes and DCIR2+ DCs in spleen (31), the activation of BDC2.5 T
cells was more efficient for αDCIR2 BDC in spleen and more efficient for αDEC BDC in lymph
nodes (Fig. 2A). Antigen targeted to either DEC 205+ or DCIR2+ DCs induced proliferation and
initial expansion in BDC2.5 T cells in both the spleen and in lymph nodes after 3 days of
stimulation (Fig. 2B and C). After ten days of stimulation, cells continued proliferating when
stimulated by αDEC BDC (Fig. 2B). However, a significant decrease in the number of BDC2.5
T cells was observed in spleen and lymph nodes at day 10 after stimulation with αDCIR2 BDC
as compared to αDEC BDC (Fig. 2C). Cell numbers after ten days of stimulation by αDCIR2
BDC were similar to the number of cells remaining in mice given no antigenic stimulation
(PBS). Thus, while αDEC BDC induced proliferative responses that led to cell accumulation,
αDCIR2 BDC induced initial proliferation that was followed by contraction of the T cell
population in lymphoid tissues.
Because autoimmune pathogenesis in NOD mice is associated with a T helper type 1
response (39), we examined the cytokines that are secreted after stimulation by αDEC BDC or
αDCIR2 BDC. As we previously observed (18), stimulation with αDEC BDC led to secretion of
interleukin (IL) 2 and IFN γ by BDC2.5 T cells at both day 3 and day 10 (Fig. 3). Stimulation
with αDCIR2 BDC, however, led to increased IL 2 secretion by BDC2.5 T cells in the spleen at
day 3, but not in the remaining T cells at day 10, and did not induce IFN γ secretion at any time
11 Diabetes Page 12 of 47
point tested (Fig. 3). IL 4, IL 10, and IL 17 were not observed after stimulation with either DC subset (data not shown). Therefore, antigen delivered to DEC 205+ DCs, but not DCIR2+ DCs induce sustained expansion and effector responses in NOD mice.
Tolerance can be broken by maturation of DC populations, and mature DEC 205+ and
DCIR2+ DCs will stimulate effector T cells rather than tolerance (29, 31). NOD CD8+ DCs express higher CD40 compared to C57Bl/6 mice, and blocking this pathway abrogated expansion of BDC2.5 T cells after stimulation with DEC 205+ DCs, suggesting that increased
CD40 may contribute to the inability of NOD CD8+ DCs to induce tolerance (18). Therefore, we measured the level of CD40 expression on CD8+ and CD11b+ DCs. CD40 expression was higher
on CD11b+ DCs from NOD mice compared to C57Bl/6 mice, consistent with the ongoing
autoimmunity in prediabetic NOD mice. But CD11b+ DCs from both strains expressed lower
CD40 levels than CD8+ DCs (Fig. 4). This lower CD40 expression on CD11b+ DCs could
contribute to their lower immunogenicity in NOD mice.
To rule out the possibility that the reduction in T cells following treatment with αDCIR2
BDC was due to increased trafficking to sites of endogenous antigen after stimulation, the
number of T cells in the pancreatic draining lymph node was measured. As expected for beta
cell specific T cells, some proliferation of BDC2.5 T cells could be observed in pancreas
draining lymph nodes, even without exogenous stimulation (Supplementary Fig. 2A). BDC2.5 T
cell numbers in the pancreatic draining lymph nodes were similar after stimulation by DCIR2+ or
DEC 205+ DCs (Supplementary Fig. 2B). Therefore, the T cell reduction in spleen and peripheral lymph nodes after αDCIR2 BDC stimulation is not due to migration of the T cells to sites of endogenous antigen presentation.
12 Page 13 of 47 Diabetes
DEC 205+ and DCIR2+ DC subsets have been reported to be important for Treg induction
or expansion, respectively (34, 40). To separately test induction or expansion of Tregs, GFP+
(Foxp3+) and GFP CD4+ cells were sorted from NOD.BDC.Foxp3 GFP mice and injected into
NOD mice along with DC targeted antigen (Fig. 5A). Although αDEC BDC and αDCIR2 BDC
both caused proliferation and expansion of injected Foxp3+ Tregs at day 3, the expansion elicited
by the two DC subsets was not significantly different, and this expansion was similar to the
expansion of GFP cells (Fig. 5B and C). Likewise, little conversion of GFP to GFP+ cells was
observed after stimulation by either DC subset (Fig. 5C). No significant increase in the
percentage of Foxp3+ BDC2.5 T cells after stimulation of total CD4+ BDC2.5 T cells by DEC
205+ or DCIR2+ DCs was observed at antibody doses ranging from 10 1000 ng (Fig. 5D). This
suggests that Treg expansion is likely occurring at similar rates as parallel effector T cell (Teff)
responses. Therefore, Tregs do not likely contribute significantly to the differences in responses
induced by DEC 205+ and DCIR2+ DCs, and neither DC subset in NOD mice is capable of
increasing the Treg:Teff ratio to skew towards tolerance.
As the loss of T cells after stimulation by DCIR2+ DCs is not due to altered trafficking to
sites of endogenous antigen or due to suppression by Tregs, we hypothesized that DCIR2+ DC
stimulation may induce T cell deletion. As measured by caspase activation and mitochondrial
potential, DCIR2+ DCs induced more apoptosis in BDC2.5 T cells than DEC 205+ DCs (Fig. 6A
and B). Therefore, the reduction in T cell number after DCIR2+ DC stimulation is likely due to
an increase in apoptosis. To strengthen this conclusion, we tested whether antigen delivered to
DCIR2+ DCs rendered BDC2.5 T cells unable to respond to subsequent stimulus. NOD mice
were injected with BDC2.5 CD4+ cells and treated first with either PBS or αDCIR2 BDC. After
10 days, the mice were treated with either αDCIR2 BDC, αCD40, polyinosinic polycytidylic
13 Diabetes Page 14 of 47
acid (poly(I:C)), and lipopolysaccharide (LPS) to elicit a strong immunogenic response, or PBS
as a control. Initial treatment of mice with αDCIR2 BDC led to decreased responsiveness to the
challenge; T cell numbers were lower after the challenge in mice pre treated with αDCIR2 BDC,
especially in the lymph node (Fig. 6C). Therefore, targeting of antigen to DCIR2+ DCs in NOD
mice leads to decreased responsiveness that cannot be fully rescued by immunogenic challenge
and further suggests increased deletion of T cells after stimulation by αDCIR2 BDC.
To determine what distinct genetic programs are elicited in the T cells early after
stimulation by the two DC subsets, we sorted BDC2.5 T cells for gene expression analysis 14
hours after stimulation in vivo with either αDEC BDC or αDCIR2 BDC. We found a set of genes
with significant differential expression in BDC2.5 T cells stimulated by the two DC subsets in
NOD mice (Fig. 7A and Supplementary Table 1). Although both DCs stimulated the T cells,
larger gene expression changes occurred with DCIR2+ DC stimulation compared with DEC 205+
DC stimulation (Supplementary Fig. 3). Pathway analysis determined a preferential expression of gene sets related with leukocyte activation (gene ontology ID; 45321) in immune system process related genes (gene ontology ID; 2376) in T cells with αDCIR2 BDC stimulation over
αDEC BDC stimulation at this early timepoint (enrichment score = 8.22, Fig. 7B). The specific leukocyte activation genes with fold change greater than 2 are shown in Fig. 7C. These data give further evidence that the lack of T cell expansion at later time points is not due to an inability of
DCIR2+ DCs to elicit a response via the TCR, and suggest instead active tolerance induction.
IFN response genes, including Irf1 are downregulated more (P = 9.13 x 10 3, fold change 1.70)
in T cells after DCIR2+ DC stimulation (Fig. 7C), which correlates with the lack of IFN γ production in DCIR2 stimulated T cells. Matching the observed increased IL 2 protein secretion
at day 3 (Fig. 3), T cells stimulated by DCIR2+ DCs expressed more Il2 message early. In
14 Page 15 of 47 Diabetes
addition, Il3 message was much higher in DCIR2+ DC stimulated BDC2.5 T cells compared to
DEC 205+ DC stimulated T cells (Fig. 7A and Supplementary Table 1). Quantitative
measurement of mRNA levels for Il2, Il3, and Irf1 confirmed the different expression observed
by microarray (Supplementary Fig. 4A). IL 3 protein expression was observed on day 1 after
stimulation by DCIR2+ but not DEC 205+ DCs, but is gone by day 3 (Supplementary Fig. 4B and
C).
Importantly, expression of the transcription factor Zbtb32 is upregulated in T cells after
αDCIR2 BDC stimulation significantly more compared with αDEC BDC stimulation (P = 4.54 x
10 3, fold change 3.66, Fig. 7A and Supplementary Table 1). Quantitative measurement of Zbtb32
mRNA levels across a wide range of antibody doses confirmed this preferential induction in
DCIR2 stimulated T cells (Fig. 8A). Higher Zbtb32 induction was also observed in vitro when
BDC2.5 T cells were directly cultured with a BDC2.5 specific peptide and sorted splenic
CD11b+ DCs, compared with CD8+ DCs. After 14 hours of culture, Zbtb32 expression was 1.47
fold higher (P < 0.05) in sorted T cells stimulated with CD11b+ DCs compared to CD8+ DCs.
Zbtb32 is a zinc finger protein that is induced with activation and has been shown to inhibit
CD4+ T cell differentiation and cytokine production (41 44), which might contribute to the lack
of cytokine production observed in αDCIR2 BDC stimultated T cells. To determine the effect of
Zbtb32 on T cell responses, CD4+ T cells from NOD.BDC2.5 mice were transfected with a
plasmid encoding Zbtb32 or a control plasmid. Transfection efficiency was tested using co
transfection with GFP and Western blotting (Fig. 8B and Supplementary Fig. 5A and B).
Although Zbtb32 transfection did not directly affect viability (Supplementary Fig. 5A),
overexpression of Zbtb32 reduced expansion and IFN γ production in BDC2.5 T cells after in
vitro stimulation, similar to observed changes after stimulation with DCIR2+ DCs in vivo (Fig.
15 Diabetes Page 16 of 47
8C and D). We next determined the diabetogencity of BDC2.5 T cells overexpressing Zbtb32
using the NOD.scid transfer model. BDC2.5 CD4+ T cells transfected with either Zbtb32 or control plasmid were transferred to NOD.scid mice. Mice that received T cells transfected with
Zbtb32 had significantly delayed diabetes compared to mice that received T cells transfected with the control plasmid (Fig. 8E). Insulitis scores at day 6 (before the development of hyperglycemia) were lower in NOD.scid mice that received Zbtb32 expressing T cells and fewer of these cells were recovered in lymphoid tissues, including in the pancreas draining lymph nodes (Fig. 8F and G). Hence Zbtb32 is increased by T cell interactions with DCIR2+ DCs and this transcriptional regulator can inhibit expansion of islet specific CD4+ T cells and their
diabetogenic potential.
16 Page 17 of 47 Diabetes
Discussion
Taken together, these data demonstrate that DEC 205+ and DCIR2+ DCs in NOD mice
stimulate effector and tolerant responses, respectively, in autoreactive CD4+ T cells and have
differential ability to induce diabetes inhibiting T cell responses. The observed reduction in T
cell numbers after stimulation by DCIR2+ DCs has several potential mechanisms. First, T cells
could possibly expand less due to a lower level of stimulation, but we observed similar levels of
early activation marker protein expression and proliferation in BDC2.5 T cells after stimulation
with either DC subset. The gene expression signatures actually indicate a stronger activation and
proliferative signal in T cells stimulated by DCIR2+ DCs. Second, T cells could be migrating to
sites of endogenous antigen expression, yet DCIR2+ DC stimulated T cells are not preferentially
accumulating in the pancreas draining lymph nodes. Finally, after initial proliferation, T cells
stimulated with DCIR2+ DCs may undergo more apoptosis. This third mechanism is consistent
with our data that caspase activation is increased, and T cells have lower responsiveness to
secondary antigen challenge. Therefore, the increased tolerance induction in T cells after
DCIR2+ DC stimulation is likely due to increased deletion. Because DCIR2+ DCs are more
abundant in the spleen, and DEC 205+ DCs more abundant in the lymph node, these differences
could be due in part to the different initial sites of activation.
In contrast to wild type mice, neither DEC 205+ nor DCIR2+ DCs are able to induce a net
increase in regulatory T cells. Previously, we observed that bone marrow derived DCs from
NOD mice could expand functional Tregs in vitro with IL 2 (46, 47). However, our data here
shows the inability of both of these DC subsets to increase Tregs in vivo in NOD mice. This
indicates a second defect in DC mediated peripheral tolerance in NOD mice and highlights
possible differences in the environment in which these DCs act during chronic autoimmunity.
17 Diabetes Page 18 of 47
DCIR2+ DC stimulation in NOD, although more tolerogenic than NOD DEC 205+ DCs, may not optimally induce tolerance, as diabetes was delayed but not completely abrogated after treatment.
Because the DCIR2 induced deletion tolerance is intrinsic to the affected T cells and not dominant tolerance such as that mediated by regulatory T cells, this type of treatment may require delivery of antigen multiple times. Therefore, it will be important to identify signals that create the proper context for therapeutic tolerance induction via DCIR2+ DCs and to determine how to use DCs in vivo to induce dominant tolerance during autoimmunity
DCIR2+ and DEC 205+ DCs induce distinct genetic programs in autoreactive T cells
early after activation that correlate with the differential T cell phenotypes observed, including
decreased Irf1 expression that correlates with decreased IFN γ production. High IL 3 expression is also induced early after DCIR2+ DC stimulation. Interestingly, IL 3 therapy can block diabetes
development in NOD mice, suggesting that, in some contexts, IL 3 may be an important
regulator of autoimmunity (48, 49). It also presents the possibility that these two DC subsets may
induce T cell tolerance through different pathways, some of which are likely impaired in NOD
mice. At the transcriptional level, we observed higher Zbtb32 in T cells stimulated by DCIR2+
DCs. T cells overexpressing Zbtb32 displayed an inhibited ability to transfer diabetes,
demonstrating that Zbtb32 is an important cell intrinsic regulator of T cell tolerance. Zbtb32 is a
known negative regulator of cytokine production (33), and correlates with NFAT mediated T
cell non responsiveness (50). Our data suggests Zbtb32 may also regulate antigen induced cell
death.
Our data points to two possible therapeutic targets: utilizing cDC2 DCIR2+ DCs to
induce tolerance and generating tolerance in T cells by upregulating Zbtb32. For optimal in vivo tolerance induction the inflammatory environment in which DCs stimulate T cells during chronic
18 Page 19 of 47 Diabetes
autoimmunity also needs to be addressed. In conclusion, this study indicates that, to
preferentially elicit the desired tolerogenic response, antigen specific treatment of type 1 diabetes
and other autoimmune diseases will need to target antigen to the correct DC subset to present
antigen to autoreactive T cells.
19 Diabetes Page 20 of 47
Acknowledgements
Author Contributions: JDP designed the project, collected and analyzed data, and wrote the manuscript. CH I designed the project, collected and analyzed data, and revised the manuscript.
YZ provided reagents, and collected and analyzed data. NMB collected and analyzed data. KVT designed the project, analyzed data, and wrote the manuscript.
We would like to thank Mrs. Alice Franks (DEOB, NIDDK) for mouse husbandry, the
NHLBI/NIDDK flow core for sorting, Amiran K. Dzutsev of the National Cancer Institute for helping with design of NanoString analysis, Patricia Johnson and Steve Shema of the CCR
Genomics Core in NCI for technical support with NanoString analysis, and Weiping Chen and
Chithra Keembiyehetty of the NIDDK Genomics Core for gene array support. This work was supported by the Intramural Research Program of the National Institute of Diabetes and
Digestive and Kidney Diseases and the JDRF.
K.V.T. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Conflict of interest statement: The authors have declared that no conflict of interest exists.
20 Page 21 of 47 Diabetes
References
1. Bluestone JA, Herold K, and Eisenbarth G. Genetics, pathogenesis and clinical interventions in type 1 diabetes. Nature. 2010;464(7293):1293 300. 2. Clemente Casares X, Tsai S, Huang C, and Santamaria P. Antigen specific therapeutic approaches in Type 1 diabetes. Cold Spring Harb Perspect Med. 2012;2(2):a007773. 3. Ben Nun A, Kaushansky N, Kawakami N, Krishnamoorthy G, Berer K, Liblau R, Hohlfeld R, and Wekerle H. From classic to spontaneous and humanized models of multiple sclerosis: Impact on understanding pathogenesis and drug development. J Autoimmun. 2014;54C(33 50. 4. Jayasimhan A, Mansour KP, and Slattery RM. Advances in our understanding of the pathophysiology of Type 1 diabetes: lessons from the NOD mouse. Clinical science. 2014;126(1):1 18. 5. Welzen Coppens JM, van Helden Meeuwsen CG, Leenen PJ, Drexhage HA, and Versnel MA. Reduced numbers of dendritic cells with a tolerogenic phenotype in the prediabetic pancreas of NOD mice. J Leukoc Biol. 2012;92(6):1207 13. 6. Calderon B, Carrero JA, Miller MJ, and Unanue ER. Cellular and molecular events in the localization of diabetogenic T cells to islets of Langerhans. Proc Natl Acad Sci U S A. 2011;108(4):1561 6. 7. Morel PA. Dendritic cell subsets in type 1 diabetes: friend or foe? Frontiers in immunology. 2013;4(415. 8. Turley SJ. Dendritic cells: inciting and inhibiting autoimmunity. Curr Opin Immunol. 2002;14(6):765 70. 9. Guilliams M, Ginhoux F, Jakubzick C, Naik SH, Onai N, Schraml BU, Segura E, Tussiwand R, and Yona S. Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny. Nat Rev Immunol. 2014;14(8):571 8. 10. Bachem A, Guttler S, Hartung E, Ebstein F, Schaefer M, Tannert A, Salama A, Movassaghi K, Opitz C, Mages HW, et al. Superior antigen cross presentation and XCR1 expression define human CD11c+CD141+ cells as homologues of mouse CD8+ dendritic cells. J Exp Med. 2010;207(6):1273 81. 11. Hildner K, Edelson BT, Purtha WE, Diamond M, Matsushita H, Kohyama M, Calderon B, Schraml BU, Unanue ER, Diamond MS, et al. Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity. Science. 2008;322(5904):1097 100. 12. Guilliams M, Ginhoux F, Jakubzick C, Naik SH, Onai N, Schraml BU, Segura E, Tussiwand R, and Yona S. Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny. Nat Rev Immunol. 2014;14(8):571 8. 13. Iyoda T, Shimoyama S, Liu K, Omatsu Y, Akiyama Y, Maeda Y, Takahara K, Steinman RM, and Inaba K. The CD8+ dendritic cell subset selectively endocytoses dying cells in culture and in vivo. J Exp Med. 2002;195(10):1289 302. 14. Liu K, Iyoda T, Saternus M, Kimura Y, Inaba K, and Steinman RM. Immune tolerance after delivery of dying cells to dendritic cells in situ. J Exp Med. 2002;196(8):1091 7. 15. Ferris ST, Carrero JA, Mohan JF, Calderon B, Murphy KM, and Unanue ER. A minor subset of batf3 dependent antigen presenting cells in islets of langerhans is essential for the development of autoimmune diabetes. Immunity. 2014;41(4):657 69.
21 Diabetes Page 22 of 47
16. Jeker LT, Bour Jordan H, and Bluestone JA. Breakdown in peripheral tolerance in type 1 diabetes in mice and humans. Cold Spring Harb Perspect Med. 2012;2(3):a007807. 17. Jansen A, van Hagen M, and Drexhage HA. Defective maturation and function of antigen presenting cells in type 1 diabetes. Lancet. 1995;345(8948):491 2. 18. Price JD, Beauchamp NM, Rahir G, Zhao Y, Rieger CC, Lau Kilby AW, and Tarbell KV. CD8+ dendritic cell mediated tolerance of autoreactive CD4+ T cells is deficient in NOD mice and can be corrected by blocking CD40L. J Leukoc Biol. 2013. 19. Steptoe RJ, Ritchie JM, and Harrison LC. Increased generation of dendritic cells from myeloid progenitors in autoimmune prone nonobese diabetic mice. J Immunol. 2002;168(10):5032 41. 20. Judkowski V, Pinilla C, Schroder K, Tucker L, Sarvetnick N, and Wilson DB. Identification of MHC class II restricted peptide ligands, including a glutamic acid decarboxylase 65 sequence, that stimulate diabetogenic T cells from transgenic BDC2.5 nonobese diabetic mice. J Immunol. 2001;166(2):908 17. 21. Haskins K, Portas M, Bergman B, Lafferty K, and Bradley B. Pancreatic islet specific T cell clones from nonobese diabetic mice. Proc Natl Acad Sci U S A. 1989;86(20):8000 4. 22. Stadinski BD, Delong T, Reisdorph N, Reisdorph R, Powell RL, Armstrong M, Piganelli JD, Barbour G, Bradley B, Crawford F, et al. Chromogranin A is an autoantigen in type 1 diabetes. Nat Immunol. 2010;11(3):225 31. 23. Feuerer M, Shen Y, Littman DR, Benoist C, and Mathis D. How punctual ablation of regulatory T cells unleashes an autoimmune lesion within the pancreatic islets. Immunity. 2009;31(4):654 64. 24. Tang Q, Adams JY, Penaranda C, Melli K, Piaggio E, Sgouroudis E, Piccirillo CA, Salomon BL, and Bluestone JA. Central role of defective interleukin 2 production in the triggering of islet autoimmune destruction. Immunity. 2008;28(5):687 97. 25. Spencer J, and Peakman M. Post mortem analysis of islet pathology in type 1 diabetes illuminates the life and death of the beta cell. Clin Exp Immunol. 2009;155(2):125 7. 26. Fu W, Wojtkiewicz G, Weissleder R, Benoist C, and Mathis D. Early window of diabetes determinism in NOD mice, dependent on the complement receptor CRIg, identified by noninvasive imaging. Nat Immunol. 2012;13(4):361 8. 27. Lebastchi J, and Herold KC. Immunologic and metabolic biomarkers of beta cell destruction in the diagnosis of type 1 diabetes. Cold Spring Harb Perspect Med. 2012;2(6):a007708. 28. Sosenko JM, Palmer JP, Greenbaum CJ, Mahon J, Cowie C, Krischer JP, Chase HP, White NH, Buckingham B, Herold KC, et al. Increasing the accuracy of oral glucose tolerance testing and extending its application to individuals with normal glucose tolerance for the prediction of type 1 diabetes: the Diabetes Prevention Trial Type 1. Diabetes care. 2007;30(1):38 42. 29. Hawiger D, Inaba K, Dorsett Y, Guo M, Mahnke K, Rivera M, Ravetch JV, Steinman RM, and Nussenzweig MC. Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. J Exp Med. 2001;194(6):769 79. 30. Bonifaz L, Bonnyay D, Mahnke K, Rivera M, Nussenzweig MC, and Steinman RM. Efficient targeting of protein antigen to the dendritic cell receptor DEC 205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance. J Exp Med. 2002;196(12):1627 38.
22 Page 23 of 47 Diabetes
31. Dudziak D, Kamphorst AO, Heidkamp GF, Buchholz VR, Trumpfheller C, Yamazaki S, Cheong C, Liu K, Lee HW, Park CG, et al. Differential antigen processing by dendritic cell subsets in vivo. Science. 2007;315(5808):107 11. 32. Kamphorst AO, Guermonprez P, Dudziak D, and Nussenzweig MC. Route of antigen uptake differentially impacts presentation by dendritic cells and activated monocytes. J Immunol. 2010;185(6):3426 35. 33. Bruder D, Westendorf AM, Hansen W, Prettin S, Gruber AD, Qian Y, von Boehmer H, Mahnke K, and Buer J. On the edge of autoimmunity: T cell stimulation by steady state dendritic cells prevents autoimmune diabetes. Diabetes. 2005;54(12):3395 401. 34. Idoyaga J, Fiorese C, Zbytnuik L, Lubkin A, Miller J, Malissen B, Mucida D, Merad M, and Steinman RM. Specialized role of migratory dendritic cells in peripheral tolerance induction. J Clin Invest. 2013;123(2):844 54. 35. Neubert K, Lehmann CH, Heger L, Baranska A, Staedtler AM, Buchholz VR, Yamazaki S, Heidkamp GF, Eissing N, Zebroski H, et al. Antigen delivery to CD11c+CD8 dendritic cells induces protective immune responses against experimental melanoma in mice in vivo. J Immunol. 2014;192(12):5830 8. 36. Zang YS, Fang Z, Liu YA, Li B, and Xiu QY. Repressor of GATA 3 can negatively regulate the expression of T cell cytokines through modulation on inducible costimulator. Chin Med J (Engl). 2012;125(12):2188 94. 37. Gardner JM, Metzger TC, McMahon EJ, Au Yeung BB, Krawisz AK, Lu W, Price JD, Johannes KP, Satpathy AT, Murphy KM, et al. Extrathymic Aire Expressing Cells Are a Distinct Bone Marrow Derived Population that Induce Functional Inactivation of CD4 T Cells. Immunity. 2013. 38. Skokos D, Shakhar G, Varma R, Waite JC, Cameron TO, Lindquist RL, Schwickert T, Nussenzweig MC, and Dustin ML. Peptide MHC potency governs dynamic interactions between T cells and dendritic cells in lymph nodes. Nat Immunol. 2007;8(8):835 44. 39. Haskins K, and Cooke A. CD4 T cells and their antigens in the pathogenesis of autoimmune diabetes. Curr Opin Immunol. 2011;23(6):739 45. 40. Yamazaki S, Dudziak D, Heidkamp GF, Fiorese C, Bonito AJ, Inaba K, Nussenzweig MC, and Steinman RM. CD8+ CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells. J Immunol. 2008;181(10):6923 33. 41. Hirahara K, Yamashita M, Iwamura C, Shinoda K, Hasegawa A, Yoshizawa H, Koseki H, Gejyo F, and Nakayama T. Repressor of GATA regulates TH2 driven allergic airway inflammation and airway hyperresponsiveness. J Allergy Clin Immunol. 2008;122(3):512 20 e11. 42. Yoon HS, Scharer CD, Majumder P, Davis CW, Butler R, Zinzow Kramer W, Skountzou I, Koutsonanos DG, Ahmed R, and Boss JM. ZBTB32 is an early repressor of the CIITA and MHC class II gene expression during B cell differentiation to plasma cells. J Immunol. 2012;189(5):2393 403. 43. Beaulieu AM, Zawislak CL, Nakayama T, and Sun JC. The transcription factor Zbtb32 controls the proliferative burst of virus specific natural killer cells responding to infection. Nat Immunol. 2014;15(6):546 53. 44. Miaw SC, Kang BY, White IA, and Ho IC. A repressor of GATA mediated negative feedback mechanism of T cell activation. J Immunol. 2004;172(1):170 7. 45. Clynes RA, Towers TL, Presta LG, and Ravetch JV. Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nature medicine. 2000;6(4):443 6.
23 Diabetes Page 24 of 47
46. Tarbell KV, Yamazaki S, Olson K, Toy P, and Steinman RM. CD25+ CD4+ T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes. J Exp Med. 2004;199(11):1467 77. 47. Tarbell KV, Petit L, Zuo X, Toy P, Luo X, Mqadmi A, Yang H, Suthanthiran M, Mojsov S, and Steinman RM. Dendritic cell expanded, islet specific CD4+ CD25+ CD62L+ regulatory T cells restore normoglycemia in diabetic NOD mice. J Exp Med. 2007;204(1):191 201. 48. Enzler T, Gillessen S, Dougan M, Allison JP, Neuberg D, Oble DA, Mihm M, and Dranoff G. Functional deficiencies of granulocyte macrophage colony stimulating factor and interleukin 3 contribute to insulitis and destruction of beta cells. Blood. 2007;110(3):954 61. 49. Ito A, Aoyanagi N, and Maki T. Regulation of autoimmune diabetes by interleukin 3 dependent bone marrow derived cells in NOD mice. J Autoimmun. 1997;10(4):331 8. 50. Martinez GJ, Pereira RM, Aijo T, Kim EY, Marangoni F, Pipkin ME, Togher S, Heissmeyer V, Zhang YC, Crotty S, et al. The transcription factor NFAT promotes exhaustion of activated CD8(+) T cells. Immunity. 2015;42(2):265 78.
24 Page 25 of 47 Diabetes
Figure Legends
Figure 1
Stimulation of BDC2.5 T cells by DCIR2+ DCs delay diabetes development in NOD.scid
mice.
A: Percent of NOD.scid mice that are diabetes free after injection with CD4+CD25 BDC2.5 T
cells and the indicated treatments. Statistical analysis was performed with Log rank test. P <
0.001 for αDEC BDC vs. αDCIR2 BDC, P = 0.452 for PBS vs. αDEC BDC. Summation of four
experiments. n = 28 mice treated with PBS, n = 20 mice were treated with αDEC BDC, n = 19
mice were treated with αDCIR2 BDC. B and C. 5 x 104 BDC2.5 T cells were transferred to
NOD.scid mice and treated with the indicated conditions. Graphs indicate the total BDC2.5 T
cell number (B) and the ratio of Foxp3+ BDC2.5 T cells (C) in NOD.scid mice on day 5. Average
of three independent experiments + SEM. Statistical analysis was performed with one way
ANOVA with Bonferroni post test. *P < 0.05.
Figure 2
DEC 205+ DCs, but not DCIR2+ DCs, elicit sustained expansion of BDC2.5 T cells in NOD
mice.
A: Activation markers (CD69 and CD25) on BDC2.5 T cells in spleen and lymph nodes (LN)
following stimulation of BDC2.5 T cells in NOD mice with the indicated dose of αDEC BDC or
αDCIR2 BDC (or PBS control, 0 ng). Average of two independent experiments + SEM. B:
Histograms of CFSE dilution for BDC2.5 T cells stimulated with the indicated antibodies at day
3 or day 10. Numbers indicate the percentage of divided T cells. C: Fold expansion (over PBS
controls, dotted line at fold expansion 1) of BDC2.5 T cells after the indicated treatments.
25 Diabetes Page 26 of 47
Statistical analysis was performed with one way ANOVA with Bonferroni post tests. *P < 0.05
for αDEC BDC vs. αDCIR2 BDC.
Figure 3
DEC 205+ DCs but not DCIR2+ DCs stimulate sustained IL 2 and IFN γ production by T
cells.
Percent of BDC2.5 T cells expressing IL 2 or IFN γ after the indicated treatments for 3 or 10
days in NOD mice following the indicated treatments. Average of four independent experiments
+ SEM. Statistical analysis was performed with one way ANOVA with Bonferroni post tests. *P
< 0.05, **P < 0.01, ***P < 0.001.
Figure 4
NOD CD8+ DCs express higher levels of CD40 than CD11b+ DCs.
Geometric mean fluorescence intensity (MFI) of CD40 on spleen CD8+ or CD11b+ DCs in
C57Bl/6 (B6) or NOD mice. Each dot represents a single mouse. Representative of three independent experiments. Statistical analysis was performed with two way ANOVA with
Bonferroni post tests. **P < 0.0001.
Figure 5
DEC 205+ and DCIR2+ DCs induce similar Treg proliferation, with little Treg induction
and no increase in the Treg:Teff ratio.
A: BDC2.5 Foxp3+ T cells and BDC2.5 Foxp3 T cells, sorted from BDC2.5.Foxp3 GFP mice, were stimulated in vivo with αDEC BDC or αDCIR2 BDC for 3 days in NOD mice. B: The
26 Page 27 of 47 Diabetes
number of GFP+ cells was assessed among transferred GFP+ cells after treatment with the
indicated antibodies. Average of three independent experiments + SEM. C: The number of GFP+
cells converted from transferred GFP cells (left) and the number of GFP cells expanded by the
indicated treatments (right) were assessed. Average of three independent experiments + SEM.
ND, not detected. D: NOD mice were injected with BDC2.5 T cells, and 5 days after treatment
with the indicated antibodies the total percentage of BDC2.5 T cells expressing Foxp3 was
measured. Average of two independent experiments + SEM. No significant differences were
observed by statistical analysis with one way ANOVA (B and C) or two way ANOVA (D).
Figure 6
αDCIR2 BDC increases BDC2.5 T cell apoptosis and decreases responsiveness to antigen
rechallenge.
A: BDC2.5 cells from spleen were assessed for caspase activation and mitochondrial membrane
potential after four days with the indicated treatments in NOD mice. B: The average percentage
of BDC2.5 cells within the apoptotic gate in spleen and LN. Average of three experiments +
SEM. ***P < 0.0005. C: NOD mice injected with BDC2.5 T cells were treated with either PBS
or αDCIR2 BDC intraperitoneally. Ten days following the initial treatment, mice were
challenged with αDCIR2 BDC + LPS + poly (I:C) + αCD40; or given PBS as a control. Five
days later (day 15 post initial treatment), numbers of transferred cells in spleen and LN were
assessed. Each dot represents cells from a single mouse. Representative of three individual
experiments. Statistical analysis was performed with one way ANOVA with Bonferroni post
tests. *P < 0.05.
27 Diabetes Page 28 of 47
Figure 7
In vivo stimulation with αααDCIR2 BDC induces distinct gene expression changes in BDC2.5
T cells.
BDC2.5 T cells were transferred to NOD mice, followed by treatment with αDEC BDC or
αDCIR2 BDC. Fourteen hours after treatment BDC2.5 T cells were sorted from spleen and LN
and RNA was obtained and then analyzed by microarray. Genes differentially regulated by
DCIR2+ and DEC 205+ DCs are analyzed by (A) volcano plot of genes (dark lines indicate a P value of 0.05 (horizontal) and a fold change of 2 (vertical)), (B) pathway analysis of immune
system process in gene ontology ID; 2376 (P < 0.05, FDR < 0.75), and (C) of leukocyte activation related genes (P < 0.05, FDR < 0.75).
Figure 8
Overexpression of Zbtb32 in BDC2.5 T cells inhibits T cell expansion, IFN γγγ production
and diabetes development.
A: BDC2.5 T cells were transferred to NOD mice, followed by the indicated treatments.
Fourteen hours after treatment, BDC2.5 T cells were sorted from spleen and RNA was obtained
and then analyzed by NanoString nCounter analysis. **P < 0.001, *P < 0.05. B: BDC2.5 T cells
were transfected with the indicated plasmids and allowed to rest for 14 hours before being lysed.
Western blotting was performed using anti Flag antibodies (top) with anti actin antibodies
(bottom) as a loading control. Transfected BDC2.5 T cells were stimulated with indicated
concentration of plate bound anti CD3 antibody and anti CD28 antibody in vitro for 3 days to
see the proliferation (C, **P < 0.001, *P < 0.01) and IFN γ secretion following the PMA and
ionomycin stimulation (D, *P < 0.05). E-G: BDC2.5 T cells were transfected with the indicated
28 Page 29 of 47 Diabetes
plasmids and transferred to NOD.scid mice. Percent of NOD.scid mice that were diabetes free at
the indicated time points after BDC2.5 T cell injection is shown (E). P < 0.0001 (n = 15 mice).
Six days after T cell injection, the pancreata were harvested and scored for the degree of insulitis
with at least 20 islets per pancreas analyzed by two independent examiners (F), and the numbers
of transferred cells in spleen, LN and pLN (pooled from 3 mice) were assessed (G). **P < 0.01,
***P = 0.0005 (n = 3 mice). Statistical analysis was performed with two way ANOVA with
Bonferroni post tests (A and C), Student’s t test (D and G), and Log rank test (E).
29 Diabetes Page 30 of 47
Supplementary Figure 1
Chimeric antibodies deliver antigen specifically to DEC 205+ or DCIR2+ DCs.
A: SDS PAGE and western blot analysis of αDEC BDC and αDCIR2 BDC. B: Binding of
αDEC BDC (red) and αDCIR2 BDC (blue) to CHO cell lines expressing DCIR2 (top left) or
DEC 205 (bottom left) or CD11b+ (top right) and CD8+ (bottom right) DCs in the spleen.
Secondary antibody only (filled gray histograms) was used as a negative control.
Supplementary Figure 2
BDC2.5 T cells display expansion in pancreatic lymph nodes after stimulation by αDEC
BDC or αDCIR2 BDC.
A: Histograms of CFSE levels in BDC2.5 T cells in the pancreatic draining lymph node (pLN) after ten days of stimulation with the indicated treatments. B: Expansion of BDC2.5 T cells in
the pLN over PBS treatment ten days after the indicated treatments. The data was pooled from 3
mice.
Supplementary Figure 3
In vivo stimulation of BDC2.5 T cells with αDCIR2 BDC drives larger gene expression
changes from PBS control compared to αDEC BDC.
BDC2.5 T cells were transferred to NOD mice followed by treatment with αDEC BDC or
αDCIR2 BDC. Fourteen hours after treatment, BDC2.5 T cells were sorted and RNA samples
were obtained for microarray analysis. The heat map shows the result of hierarchical clustering
analysis of all differentially expressed genes induced by αDCIR2 BDC (black in left panel),
αDEC BDC (gray in left panel), and PBS (white in left panel). The mean was set to 0 for all of
30 Page 31 of 47 Diabetes
duplicate array samples, and analysis performed using the Partek Genomics Suite software. The
upper clustering is from the correlation of each gene expression. FDR < 0.5.
Supplementary Figure 4
DCIR2+ DCs induce early IL 2 and IL 3 secretion and suppress Irf1 by BDC2.5 T cells.
A: BDC2.5 T cells were transferred to NOD mice, followed by treatment with the indicated
amount of αDEC BDC or αDCIR2 BDC. Fourteen hours after treatment, BDC2.5 T cells were
sorted from spleen and gene expression was analyzed by NanoString nCounter analysis. B:
Contour plots of IL 2 and IL 3 production by splenic BDC2.5 T cells after 24 hours of
stimulation with the indicated treatments. C: Percentage of BDC2.5 T cells secreting IL 3 after
PMA and ionomycin stimulation after the indicated times of in vivo treatment with PBS, αDEC
BDC or αDCIR2 BDC. Average of three independent experiments + SEM. Statistical analysis
was performed with two way ANOVA (A) and one way ANOVA (C) with Bonferroni post tests.
*P < 0.05, **P < 0.01, ***P < 0.001.
Supplementary Figure 5
Transfection of Zbtb32 does not affect cell viability.
BDC2.5 T cells were transfected with the indicated plasmids and allowed to incubate for the
indicated culture time in vitro. A, Cell viability. B, Cell number showing GFP expression as
indicator of transfection. Statistical analysis was performed with one way ANOVA (A) and
Student’s t test (B) at each time point.
31 Diabetes Page 32 of 47
Supplemental Table 1
Genes significantly different in T cells stimulated with DCIR2+ DCs vs DEC205+ DCs.
32 Page 33 of 47 Diabetes
Figure 1 Stimulation of BDC2.5 T cells by DCIR2+ DCs delay diabetes development in NOD.scid mice. A: Percent of NOD.scid mice that are diabetes free after injection with CD4+CD25 BDC2.5 T cells and the indicated treatments. Statistical analysis was performed with Log rank test. P < 0.001 for αDEC BDC vs. αDCIR2 BDC, P = 0.452 for PBS vs. αDEC BDC. Summation of four experiments. n = 28 mice treated with PBS, n = 20 mice were treated with αDEC BDC, n = 19 mice were treated with αDCIR2 BDC. B and C. 5 x 104 BDC2.5 T cells were transferred to NOD.scid mice and treated with the indicated conditions. Graphs indicate the total BDC2.5 T cell number (B) and the ratio of Foxp3+ BDC2.5 T cells (C) in NOD.scid mice on day 5. Average of three independent experiments + SEM. Statistical analysis was performed with one way ANOVA with Bonferroni posttests. *P < 0.05. 96x104mm (600 x 600 DPI)
Diabetes Page 34 of 47
Figure 2 DEC 205+ DCs, but not DCIR2+ DCs, elicit sustained expansion of BDC2.5 T cells in NOD mice. A: Activation markers (CD69 and CD25) on BDC2.5 T cells in spleen and lymph nodes (LN) following stimulation of BDC2.5 T cells in NOD mice with the indicated dose of αDEC BDC or αDCIR2 BDC (or PBS control, 0 ng). Average of two independent experiments + SEM. B: Histograms of CFSE dilution for BDC2.5 T cells stimulated with the indicated antibodies at day 3 or day 10. Numbers indicate the percentage of divided T cells. C: Fold expansion (over PBS controls, dotted line at fold expansion 1) of BDC2.5 T cells after the indicated treatments. Statistical analysis was performed with one way ANOVA with Bonferroni posttests. *P < 0.05 for αDEC BDC vs. αDCIR2 BDC. 171x331mm (600 x 600 DPI)
Page 35 of 47 Diabetes
Figure 3 DEC 205+ DCs but not DCIR2+ DCs stimulate sustained IL 2 and IFN γ production by T cells. Percent of BDC2.5 T cells expressing IL 2 or IFN γ after the indicated treatments for 3 or 10 days in NOD mice following the indicated treatments. Average of four independent experiments + SEM. Statistical analysis was performed with one way ANOVA with Bonferroni posttests. *P < 0.05, **P < 0.01, ***P < 0.001. 78x68mm (600 x 600 DPI)
Diabetes Page 36 of 47
Figure 4 NOD CD8+ DCs express higher levels of CD40 than CD11b+ DCs. Geometric mean fluorescence intensity (MFI) of CD40 on spleen CD8+ or CD11b+ DCs in C57Bl/6 (B6) or NOD mice. Each dot represents a single mouse. Representative of three independent experiments. Statistical analysis was performed with two-way ANOVA with Bonferroni posttests. **P < 0.0001. 40x18mm (600 x 600 DPI)
Page 37 of 47 Diabetes
Figure 5 DEC 205+ and DCIR2+ DCs induce similar Treg proliferation, with little Treg induction and no increase in the Treg:Teff ratio. A: BDC2.5 Foxp3+ T cells and BDC2.5 Foxp3 T cells, sorted from BDC2.5.Foxp3 GFP mice, were stimulated in vivo with αDEC BDC or αDCIR2 BDC for 3 days in NOD mice. B: The number of GFP+ cells was assessed among transferred GFP+ cells after treatment with the indicated antibodies. Average of three independent experiments + SEM. C: The number of GFP+ cells converted from transferred GFP cells (left) and the number of GFP cells expanded by the indicated treatments (right) were assessed. Average of three independent experiments + SEM. ND, not detected. D: NOD mice were injected with BDC2.5 T cells, and 5 days after treatment with the indicated antibodies the total percentage of BDC2.5 T cells expressing Foxp3 was measured. Average of two independent experiments + SEM. Statistical analysis with one way ANOVA (B and C) or two way ANOVA (D) showed non significance. 83x38mm (600 x 600 DPI)
Diabetes Page 38 of 47
Figure 6 αDCIR2 BDC increases BDC2.5 T cell apoptosis and decreases responsiveness to antigen rechallenge. A: BDC2.5 cells from spleen were assessed for caspase activation and mitochondrial membrane potential after four days with the indicated treatments in NOD mice. B: The average percentage of BDC2.5 cells within the apoptotic gate in spleen and LN. Average of three experiments + SEM. ***P < 0.0005. C: NOD mice injected with BDC2.5 T cells were treated with either PBS or αDCIR2 BDC intraperitoneally. Ten days following the initial treatment, mice were challenged with αDCIR2 BDC + LPS + poly (I:C) + αCD40; or given PBS as a control. Five days later (day 15 post initial treatment), numbers of transferred cells in spleen and LN were assessed. Each dot represents cells from a single mouse. Representative of three individual experiments. Statistical analysis was performed with one way ANOVA with Bonferroni posttests. *P < 0.05. 115x151mm (600 x 600 DPI)
Page 39 of 47 Diabetes
Figure 7 In vivo stimulation with αDCIR2 BDC induces distinct gene expression changes in BDC2.5 T cells. BDC2.5 T cells were transferred to NOD mice, followed by treatment with αDEC BDC or αDCIR2 BDC. Fourteen hours after treatment BDC2.5 T cells were sorted from spleen and LN and RNA was obtained and then analyzed by microarray. Genes differentially regulated by DCIR2+ and DEC 205+ DCs are analyzed by (A) volcano plot of genes (dark lines indicate a P value of 0.05 (horizontal) and a fold change of 2 (vertical)), (B) pathway analysis of immune system process in gene ontology ID; 2376 (P < 0.05, FDR < 0.75), and (C) of leukocyte activation related genes (P < 0.05, FDR < 0.75). 104x123mm (600 x 600 DPI)
Diabetes Page 40 of 47
Figure 8 Overexpression of Zbtb32 in BDC2.5 T cells inhibits T cell expansion, IFN γ production and diabetes development. A: BDC2.5 T cells were transferred to NOD mice, followed by the indicated treatments. Fourteen hours after treatment, BDC2.5 T cells were sorted from spleen and RNA was obtained and then analyzed by NanoString nCounter analysis. **P < 0.001, *P < 0.05. B: BDC2.5 T cells were transfected with the indicated plasmids and allowed to rest for 14 hours before being lysed. Western blotting was performed using anti Flag antibodies (top) with anti actin antibodies (bottom) as a loading control. Transfected BDC2.5 T cells were stimulated with indicated concentration of plate bound anti CD3 antibody and anti CD28 antibody in vitro for 3 days to see the proliferation (C, **P < 0.001, *P < 0.01) and IFN γ secretion following the PMA and ionomycin stimulation (D, *P < 0.05). E G: BDC2.5 T cells were transfected with the indicated plasmids and transferred to NOD.scid mice. Percent of NOD.scid mice that were diabetes free at the indicated time points after BDC2.5 T cell injection is shown (E). P < 0.0001 (n = 15 mice). Six days after T cell injection, the pancreata were harvested and scored for the degree of insulitis with at least 20 islets per pancreas analyzed by two independent examiners (F), and the numbers of transferred cells in spleen, LN and pLN (pooled from 3 mice) were assessed (G). **P < 0.01, ***P = 0.0005 (n = 3 mice). Statistical analysis was performed with two way ANOVA with Bonferroni posttests (A and C), Student’s t test (D and G), and Log rank test (E). 92x47mm (600 x 600 DPI)
Page 41 of 47 Diabetes Supplementary Figure 1
A B CHO Spleen 100 100
80 80 DCs
60 60 +
-DCIR2 -DCIR2 40 40 DEC-BDC DEC-BDC DCIR2-BDC DEC-BDC DCIR2-BDC 205 DEC- 20 20 CHO 0 CD11b 0
100 100
50 kDa 80 80
60 DCs 60
-DEC -DEC 25 kDa + 40 40
WB: Goat 20 CD8 20 CHO
anti-rat IgG 0 0 1050 102 103 104 1050 102 103 104
Supplementary Figure 1 Chimeric antibodies deliver antigen specifically to DEC-205+ or DCIR2+ DCs. A, SDS-PAGE and western blot analysis of aDEC-BDC and aDCIR2-BDC. B, Binding of aDEC-BDC (red) and aDCIR2-BDC (blue) to CHO cell lines expressing DCIR2 (top left) or DEC-205 (bottom left) or CD11b+ (top right) and CD8+ (bottom right) DCs in the spleen. Secondary antibody only (filled gray histograms) was used as a negative control.
Diabetes Page 42 of 47 Supplementary Figure 2
A PBS DEC-BDC DCIR2-BDC 30 10 30
56.8 8 79.6 69.0 20 20 6
4 10 10 2 Cell numberCell
0 0 0 1050 102 103 104 1050 102 103 104 1050 102 103 104 CFSE
B 4
3 DEC205-BDC DCIR2-BDC 2
1 Fold Expansion Expansion Fold 0
Supplementary Figure 2 BDC2.5 T cells display expansion in pancreatic lymph nodes after stimulation by αDEC- BDC or αDCIR2-BDC. A, Histograms of CFSE levels in BDC2.5 T cells in the pLN after ten days of stimulation with the indicated treatments. B, Expansion of BDC2.5 T cells in the pLN over PBS treatment ten days after the indicated treatments. The data indicated was pooled from 3 mice. Page 43 of 47 Diabetes Supplementary Figure 3
3.59 2.80 2.00 1.20 172.29 0.00 0.00 BDC BDC DCIR2- DEC- BDC BDC PBS
-2.0 0.0 2.0 Color Key
Supplementary Figure 3 In vivo stimulation of BDC2.5 T cells with αDCIR2-BDC drives larger gene expression changes from PBS control compared to αDEC-BDC. BDC2.5 T cells were transferred to NOD mice followed by treatment with αDEC-BDC or αDCIR2-BDC. Fourteen hours after treatment, BDC2.5 T cells were sorted and RNA samples were obtained for microarray analysis. The heat map shows the result of hierarchical clustering analysis of all differentially expressed gene expression induced by αDCIR2-BDC (black in left panel), αDEC-BDC (gray in left panel), and PBS (white in left panel). The mean was set to 0 for all of duplicate array samples, and analysis performed using the Partek Genomics Suite software. The upper clustering is from the correlation of each gene expression. FDR < 0.5. SupplementaryFigure 4 experiments + SEM. experiments way ANOVA ( with PBS, αDEC treatment treatments. indicated with the stimulation B and from sorted spleen were of αDEC amount A, after PMAafter PMA/Iono(+ ionomycinand DCIR2 SupplementaryFigure4 , Contour plots of IL-2 and IL-3 production by splenic BDC2.5 bysplenic 24 hours productionIL-3 Tofand after ofIL-2 plots , Contour cells BDC2.5 T cells were transferred to NOD mice followed by treatment with the indicated with the BDC2.5bytreatment followed to NOD miceTtransferred were cells A B + DCs induce early IL-3 Count 200 400 600 800 10 10 10 10 10 10 10 10 10 10 10 10 IL 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5
0 0 Unstimulated Unstimulated 98.7 98.7 1.09 99.4 99.4 0.61 96.9 96.9 2.44 -2 -2 C 0 10 0 0 0 ) 2 2 - with Bonferroni post tests. post Bonferroniwith BDC orBDC αDCIR2
10 * * 10
3 Statistical analysis was performed with two-waywasanalysis performed Statistical ANOVA (
Il2 0.52 0.52 0.17 0.00 0.00 0.00 0.00 0.08 100 10 ** 4
52.8 52.8 1.50 82.1 0.40 82.5 0.95 10 + PMA/+ 0 10 0 1000 5 - BDC orBDC αDCIR2 IL 2 gene expression wasexpression gene
-2 and -2 and IL-3 10 120 40 80 3
iono 0 0
Ab 10 8.29 8.29 0.24 0.17 17.3 17.3 37.6 37.6 16.3 -BDC. Fourteen hours after treatmenthours afterFourteen -BDC. 4 0 0
treatment ( treatment
10 PBS 5 10 DEC-BDC * * DCIR2-BDC
.) stimulation after the indicated times oftimes indicated the after stimulation .) Il3 * *** 100 C Diabetes secretion and suppress , Percentage of BDC2.5 , IL-3TPercentagesecreting cells /mouse) ng/mouse) * -BDC. Averageindependentthree of
1000 P * * C
< 0.05, ** < 15 10 analyzed by NanoString nCounter analysis.NanoStringnCounter by analyzed + 100 200 4 3 0 %5 IL-3 00 00 10 10 15 0 0 0 0 5
0 0 Day 1 Day 10 P
1
< 0.01, Irf1 ** * 100 1000 Irf1 ***
PBS DEC-BDC Day 3 Day DCIR2-BDC P by BDC2.5 T cells. , 2 < 0.01. BDC2.5 T cells DEC2 DCIR2-BDC 05-BDC A in vivo
) and one- and ) Page 44of47
Page 45 of 47 Diabetes Supplementary Figure 5
A B 60 8 Untransfected NS pcDNA3.1 pcDNA3.1 Zbtb32
NS 6 Zbtb32 ) 40 4 NS (x10
+ 4 NS
% viability % viability 20
# GFP # 2
0 0 14 24 14 24 Time (hours) Time (hours)
Supplementary Figure 5 Transfection of Zbtb32 does not affect cell viability. BDC2.5 T cells were transfected with the indicated plasmids and allowed to incubate for the indicated in vitro culture time. A, Cell viability. B, Cell number showing GFP expression as transfected indicator. Statistical analysis was performed with one-way ANOVA (A) and Student’s t-test (B) at each time point. Diabetes Page 46 of 47
Supplemental Table 1
Genes significantly different in T cells stimulated with DCIR2+ DCs vs DEC205+ DCs.
(P < 0.05, Fold-change > 2)
Transcript P-value Fold-Change Gene Symbol RefSeq Cluster ID (DCIR2 vs. DEC) (DCIR2 vs. DEC) 10385918 Il3 NM_010556 0.01340 21.13 10404422 Serpinb6b NM_011454 0.00625 3.88 10562044 Zbtb32 NM_021397 0.00454 3.66 10497878 Il2 NM_008366 0.04518 3.36 10493108 Crabp2 NM_007759 0.02790 3.26 10388591 Cpd NM_007754 0.00239 3.15 10389064 Ccl1 NM_011329 0.00534 3.05 10460585 Fosl1 NM_010235 0.02738 2.52 10558580 Utf1 NM_009482 0.00244 2.44 10359697 Xcl1 NM_008510 0.04519 2.42 10478967 Cstf1 NM_024199 0.02302 2.37 10571312 Dusp4 NM_176933 0.01169 2.34 10365933 Eea1 NM_001001932 0.00978 2.25 10549222 Bcat1 NM_001024468 0.03169 2.23 10511282 Tnfrsf4 NM_011659 0.01408 2.12 10433963 Ydjc NM_026940 0.00579 2.09 10445241 Tnfrsf21 NM_178589 0.02079 2.08 10469951 Rnf208 NM_176834 0.01916 2.07 10471715 Mrrf NM_026422 0.01209 2.05 10429957 Fbxl6 NM_013909 0.01954 2.03 10573008 Zfp827 NM_178267 0.02731 2.02 10543067 Asns NM_012055 0.01919 2.01 10352048 Exo1 NM_012012 0.00609 2.00 10366767 Ctdsp2 NM_146012 0.00260 -2.03 10454831 Paip2 NM_026420 0.00611 -2.04 10429564 Ly6a NM_010738 0.03268 -2.05 10456699 Acaa2 NM_177470 0.01645 -2.08 10414714 Gm16452 ENSMUST00000103572 0.01447 -2.08 10362676 Cdk19 NM_001168304 0.01358 -2.09 10385533 Tgtp1 NM_011579 0.02167 -2.11 10499431 Syt11 NM_018804 0.01020 -2.13 10531987 Gbp4 NM_008620 0.02277 -2.14 10385526 9930111J21Rik2 NM_173434 0.01981 -2.17 Page 47 of 47 Diabetes
10498599 Ift80 NM_026641 0.03178 -2.19 10479726 Pcmtd2 NM_153594 0.02905 -2.19 10385518 Tgtp1 NM_011579 0.01386 -2.21 10512480 Sit1 NM_019436 0.01592 -2.22 10360391 Ifi203 NM_001045481 0.00988 -2.28 10360684 Ephx1 NM_010145 0.01200 -2.30 10368199 Myb NM_010848 0.01024 -2.33 10497817 Anxa5 NM_009673 0.01270 -2.36 10385513 9930111J21Rik2 NM_173434 0.03111 -2.39 10361234 Hsd11b1 NM_008288 0.01083 -2.41 10460392 Pold4 NM_027196 0.04485 -2.45 10438445 Klhl6 NM_183390 0.01633 -2.52 10434302 Klhl24 NM_029436 0.00729 -2.62 10463997 Pdcd4 NM_011050 0.00923 -2.78