Page 1 of 35 Diabetes · 2014. 2. 6. · combined with islet autoantigen-specific approaches to...

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Combination therapy with an anti-IL-1β antibody and GAD65 DNA vaccine can reverse recent-

onset diabetes in the RIP-GP mouse model

Philippe P. Pagnia, Damien Bresson

a,b, Teresa Rodriguez-Calvo

a, Amira Bel Hani

a, Yulia

Manenkovaa, Natalie Amirian

a, Alecia Blaszczak

a, Sina Faton

a , Sowbarnika Sachithanantham

a,

Matthias G. von Herratha, #

Affiliations: aType 1 Diabetes Center, La Jolla Institute for Allergy and Immunology, 9420

Athena Circle, La Jolla, California, United States, 92037.

bCurrent address: Ambrx, Inc. 10975 N Torrey Pines Rd, San Diego, CA 92121.

Short running title : Diabetes reversal on IL-1β blockade/GAD65 DNA vaccine.

Word count: 3,915 words; 6 figures; 4 online supplementary figures.

Abbreviations: GAD65, Glutamic acid decarboxylase of 65kDa; T1D, type 1 diabetes; NOD,

nonobese diabetic; LCMV, lymphocytic choriomeningitis virus; RIP-GP, rat insulin promoter-

glycoprotein; BGV, blood glucose value; TNF, tumor necrosis factor; IFN-γ, interferon-γ; IL,

interleukin; Th, T-helper; PBMC, peripheral blood mononuclear cells; IL-1Ra, interleukin-1

receptor antagonist.

#Corresponding author: Type 1 Diabetes Center, La Jolla Institute for Allergy and Immunology,

9420 Athena Circle, La Jolla, California, United States, 92037. Tel: 001-858-205-0646. Fax: 001-

858-752-6993. Email: [email protected]

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Diabetes Publish Ahead of Print, published online February 11, 2014

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Type 1 diabetes is thought to be an autoimmune condition in which self-reactive T cells attack

insulin-secreting pancreatic β-cells. As a proinflammatory cytokine produced by β-cells or

macrophages, interleukin (IL)-1β represents a potential therapeutic target in diabetes. We

reasoned IL-1β blockade could be combined with islet antigen-specific approaches involving

glutamic acid decarboxylase of 65kDa (GAD65)-expressing plasmids, as previously shown in

combination therapies (CT) with anti-CD3. Thus, we investigated whether anti-IL-1β antibody

alone or combined with GAD65 vaccine could reverse diabetes development in a virus-induced

mouse model. Given alone, anti-IL-1β had no effect on diabetes while GAD65 plasmid resulted

in 33% disease reversal after 5-week observation. However, CT cured 53% of animals and

prevented worsening of glycemic control in non-protected individuals for up to 12 weeks. While

the GAD65 vaccine arm of the CT was associated with increased Foxp3+ regulatory T-cell

frequency in pancreatic lymph nodes, islet infiltration by CD11b+/high

cells was less frequent upon

CT and its extent correlated with treatment success or failure. Altogether, our CT provided

prolonged improvement of clinical and immunological features. Despite unsuccessful clinical

trials using anti-IL-1β monotherapy, these data hold promise for treatment of type 1 diabetic

patients with IL-1β blockade combined with antigen-specific vaccines.

200 words.

Keywords

Type 1 diabetes; IL-1β; GAD65 DNA vaccine; Immunotherapy; RIP-LCMV

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INTRODUCTION

Inflammation of the pancreatic islets is observed in patients with type 1 and type 2 diabetes (1-3).

Islets are sensitive to proinflammatory cytokines and the combination of IFN-γ, TNF and IL-1β

can act synergistically to lead to β-cell demise (4-6). IL-1β can be produced by cells such as

monocytes, macrophages, dendritic cells or neurons (1; 7). In β-cells, IL-1β secretion in

conditions of high glucose concentrations can have cytotoxic effects such as altered insulin

secretion and β-cell apoptosis (8-10). Therefore, IL-1β blockade as a means to prevent or reverse

type 1 and type 2 diabetes onset has become a potential therapeutic target.

In type 2 diabetes, neutralization of IL-1β signaling has resulted in improved glycemic control

and insulin secretion in murine (11) and human studies (12-13). Microarray analyses have shown

that compared to healthy controls, IL-1β gene expression was increased 3-fold in peripheral

blood mononuclear cells (PBMCs) from both type 1 and type 2 recent-onset diabetic patients but

significantly declined upon a 4-month course of insulin therapy and adjusted glycemic control

(14). In type 1 diabetic patients, Pfleger et al. found a positive association at 1- and 6-month post-

diagnosis, between levels of C-peptide and that of interleukin-1 receptor antagonist (IL-1Ra), the

natural antagonist to pro-inflammatory cytokines IL-1α/β and the foot-print of earlier IL-1 action

(15). Moreover, short-term neutralization of IL-1β resulted in reduced chemokine receptor

expression on CD11b+ circulating monocytes (16) in people with type 1 diabetes. More recently,

two randomized, placebo-controlled Phase 2a trials both enrolling 69 type 1 diabetic patients

have evaluated whether IL-1 blockade could improve β-cell function in recent-onset type 1

diabetes (17). One used a human monoclonal anti-IL-1β antibody (Canakinumab) whereas the

other used a recombinant human IL-1Ra (Anakinra). Despite no severe safety issues, neither trial

met its primary or secondary endpoints. Thus, despite good scientific rationale, the effect of IL-

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1β blockade alone in overt type 1 diabetes is, if any, minor and combination with other agents

will be needed. Antigen-specific therapies, due to their lack of inducing systemic side effects, are

preferable to using additional systemic immunosuppressants.

Along these lines, while IL-1 receptor-deficient nonobese diabetic (NOD) mice display slowed

progression to diabetes but normal incidence (18), Ablamunits et al. have recently shown

synergistic reversal of type 1 diabetes after combined anti-IL-1β (using blocking antibody or IL-

1Ra) and anti-CD3 antibody treatments (19). Furthermore, preclinical studies in our laboratory

also demonstrated synergistic reversal of type 1 diabetes using similar anti-CD3 antibody

combined with islet autoantigen-specific approaches to induce and maintain tolerance to β-cell

antigens (20-22). Oral and nasal (pro)insulin were proved efficacious as part of the CT in the

NOD model (20; 21) whereas pCMV-encoded human glutamic acid decarboxylase of 65kDa

(pCMV-hGAD65) DNA plasmid was efficacious in the RIP-LCMV-GP model (22). RIP-LCMV-

GP mice express the glycoprotein (GP) from lymphocytic choriomeningitis virus (LCMV) as a

transgene under the rat insulin promoter (RIP) and turn diabetic within 2 weeks post-LCMV

infection (23). Other plasmid DNA vaccines encoding mouse proinsulin II or GAD65-IgFc and

IL-4 have also been shown to prevent and/or reverse hyperglycemia in NOD mice (24; 25).

Herein, we investigated whether an IL1-β neutralizing antibody alone or combined with an islet

antigen GAD65-specific vaccine could reverse recent-onset diabetes development in the RIP-GP

model. For this, we chose two main time-points for our studies: (i) 5 weeks post-treatment, i.e.,

one week after the last administration of anti-IL-1β antibody, and (ii) 12 weeks post-treatment

i.e., up to 2 months after the treatments have been stopped.

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RESEARCH DESIGN AND METHODS

Mice. Transgenic rat insulin promoter (RIP)-lymphocytic choriomeningitis virus (LCMV)-

glycoprotein (GP) mice on the C57Bl/6 background have been described (26). Mice were bred in-

house under specific pathogen free conditions at the La Jolla Institute for Allergy and

Immunology (LIAI). A dose of 1x104 plaque-forming units of LCMV Armstrong was injected

intraperitoneally. Blood glucose values (BGVs) were recorded with an AccuCheck Glucometer

(LifeScan Inc., Milpitas, CA).

Treatments. From day 7 post-infection, BGV was assessed for all mice and when above

250mg/dl, mice were included in one treatment group. Novartis (Basel, Switzerland) kindly

provided us with a monoclonal anti-mouse IL-1β antibody and its corresponding IgG2a isotype

(anti-cyclosporin B) control antibody. Both kinds of antibodies were administered

intraperitoneally (200µg) similar to Osborn et al. (11), once a week for 4 weeks and GAD65

DNA vaccine was given as described previously in our laboratory (22), i.e., intramuscularly

(100µg per leg) at day 0, 4 and 11 post-onset. At the end of different periods of observation, mice

were sacrificed and blood, spleen, pancreatic lymph nodes (PLNs) as well as pancreata were

collected and processed for analyses (27).

Histology. Mouse pancreata were embedded in optimum cutting temperature compound (VWR)

and stored at -80C. Six-micron-thick sections were stained for insulin and surface markers.

Staining for CD4, CD8 and CD11b was performed using biotinylated primary antibodies (1/100,

BD) followed by the addition of streptavidin coupled-horseradish peroxidase (1/1,000, Vector)

and developed using NovaRed Peroxidase substrate kit (Vector). Sections were simultaneously

stained with guinea pig anti-insulin primary antibody (Dako), followed by staining with goat anti-

guinea pig alkaline phosphatase antibody (Sigma) and developed using Vector Blue AP III

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(Vector). Sections were mounted with aqueous mounting solution (Lerner Lab.). Pictures were

taken with a Nikon 80i fluorescence microscope. The criteria for scoring islet infiltration were as

follows: (i) no insulitis, (ii) peri-insulitis with no islet destruction or a few (less than 10)

infiltrating cells, and (iii) infiltrating insulitis with extensive islet destruction (or islet destroyed).

The readings were performed in a blinded fashion by three different co-authors, S.F., A.B. and

N.A. at 1, 5 and 12 weeks post-onset, respectively.

Flow cytometry staining. After various periods of observations post-treatment onset, blood,

spleen and PLNs were collected from euthanized mice and processed for further staining as

previously described (27). Db/GP33, and Db/NP396 pentamers were purchased from Proimmune

(Sarasota, FL). Isolated cells were submitted to a Fc block incubation for 10 minutes at +4C,

followed by pentamer staining (3ul per 50ul reaction) at room temperature for at least 20 minutes.

Last, surface antibodies were added and incubated for 20 minutes at 4C. After acquisition of data

on a LSRII cytometer (Beckton Dickinson), events were analyzed with FlowJo software (Tree

Star), gating on Aqua live/dead dye (Life Tech) negative cells as well as CD19-CD8

+ T cells.

Statistics. Data are expressed as mean ± standard error of the mean. Diabetes incidences were

assessed using the Gehan-Breslow and Log-rank tests. Graphs were plotted and statistics

calculated with GraphPad Prism® (versions 4 and 5). Comparisons between the different groups

were performed with two-tailed, unpaired Mann-Whitneytests. A 2-way ANOVA test was used

for hisotological data analyses. The Wilcoxon signed rank test was used to determine statistical

significance for paired values. *P < 0.05, **P < 0.01, ***P < 0.001.

Ethics. These studies were performed at LIAI upon approval of Institutional Animal Care and

Use Committee.

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RESULTS

Anti-IL1β/GAD65 DNA vaccine combination, but not anti-IL1β treatment alone, can

reverse hyperglycemia in diabetic RIP-GP mice. To determine the efficacy of IL-1β

neutralization as a monotherapy in the RIP-LCMV-GP diabetes model, we treated diabetic mice

once weekly for 4 weeks with an anti-IL-1β antibody or an isotype control. After 5 weeks, all

isotype-treated animals remained hyperglycemic whereas 16.7% (1/6) of anti-IL-1β-treated mice

reverted to euglycemia (Fig. 1A left panels, 1B). This difference did not reach statistical

significance, suggesting that monotherapy based upon IL-1β blockade was inefficacious in the

rapid-onset RIP-GP mouse model, paralleling the recent clinical trial experience (17).

Previous studies by our laboratory had shown success in curing diabetic RIP-GP mice combining

anti-CD3 and pCMV-GAD65 DNA vaccines (22). Thus, we questioned whether the same DNA

vaccine could be associated with IL-1β blocking antibody to induce immunological tolerance to

GAD65. In accordance with Bresson et al. (22) and reflecting the initial success of DNA vaccines

in preserving β-cell mass in a trial in recent-onset diabetes (28), 33% (4/12) of mice treated with

GAD65 DNA plasmid and isotype antibody (below referred to as the isot/GAD group) reverted

to euglycemia after 5 weeks, providing some limited protection compared to isotype-treated

counterparts. However, when using a combination therapy (CT) with both the GAD65 DNA

vaccine and anti-IL-1β antibody (also referred to as the a-IL-1β/GAD group), we found a

significantly higher proportion of cured mice (9/17, 53%) (Fig. 1A right panels, 1B). In addition,

we observed a significantly faster reversal in the a-IL-1β/GAD group compared to the isot/GAD

group (Fig. 1C), as reflected by the Gehan-Breslow statistical test that gives more weight to

earlier events (Fig. 1B).

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Overall, the average glycemia levels at entry were comparable among all groups, therefore

excluding a bias in this regard (supplementary Fig. 1B). Moreover, the combination of anti-IL-

1β antibody with either empty pCMV plasmid or pCMV expressing the B chain of insulin

resulted in reversal rates similar to anti-IL-1β-treated mice (supplementary Fig. 1C), excluding

non-specific effects as observed previously (20-21) and indicating MHC restriction of GAD65-

based induction of tolerance. Supporting the point that antigenic vaccines will operate in an

MHC-restricted manner, earlier studies have shown that DNA vaccines expressing proinsulin but

not GAD65 can protect NOD and Balb/c, but not B6, mice from diabetes due to differences in

insulin B chain presentation on the H-2b background (20; 22; 29).

Anti-IL1β/GAD65 DNA vaccine combination prevents worsening of glycemic control in

non-protected mice. Glycemic control is evaluated in diabetic individuals by measuring the

percentage of glycated haemoglobin (% HbA1c). Importantly, uncontrolled, severe,

hyperglycemia (HG) steeply increases the risk to develop complications such as retinopathy or

neuropathy (30). Moreover, there is evidence for decreased responsiveness to anti-CD3-induced

decline in C-peptide after 2 years in type 1 diabetic patients with poorly controlled glycemia

(Protégé Trial) (31). Therefore, we examined closely the glycemic status of non-protected mice,

distinguishsing mice with mild and severe hyperglycemia, i.e., with final BGVs comprised

between 251-400mg/dl and 401-600mg/dl, respectively. The isot/GAD and CT groups both

presented 8 non-protected mice, out of which 12.5% (1/8) versus 75% (6/8) mice had mild

hyperglycemia, respectively (Fig. 1D, 1E). These data demonstrate that in cases where IL-1β

blockade as an add-on to GAD65-based tolerance induction does not cure mice from diabetes, it

can nevertheless stabilizethe extent of hyperglycemia in RIP-GP mice.

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Effector CD8 T-cell responses are reduced and regulatory T-cell responses are improved

after GAD65 DNA vaccine and combination therapy. Next, we assessed immunological

parameters systemically (in the spleen) and locally (in PLNs) in mice. Cells were isolated and

restimulated with LCMV immunodominant CD8 T-cell epitope, GP33-41 peptide, in order to

evaluate cytokine production. No significant difference was observed between isotype- and anti-

IL-1β-treated animals, consistent with the absence of efficacy of IL-1β monotherapy (Fig. 2A,

2B). In comparison to isotype antibody treatment, the isot/GAD group presented diminished

frequencies of IFN-γ and TNF-secreting cells in the spleen (Fig. 2A), but not in PLNs (Fig. 2B).

Likewise, compared to anti-IL-1β treatment, we observed reduced fractions of cytokine-secreting

cells in the spleen of the CT group (Fig. 2A), and also in PLNs regarding TNF (Fig. 2B).

The effect of IL-1β on the development and/or function of Foxp3-expressing regulatory T cells

(Tregs) is not completely understood. Indeed, while some argued for a role of IL-1β in breaking

immunological tolerance via expansion of CD25+ effector T cells (32) and in differentiating Th17

cells at the expense of Tregs (33), others argued for a costimulatory effect of IL-1β on the

expansion of Tregs and their maintenance of Foxp3 expression (34; 35). Here, the frequencies of

CD4+CD25

+Foxp3

+ Tregs were comparable between isotype- and anti-IL-1β-treated animals in

the spleen and PLNs (Fig. 2C, D). Of note, the CT group displayed a higher percentage of

Foxp3-expressing Tregs in PLNs (Fig. 2D), but not in the spleen (Fig. 2C). Interestingly, effector

(Fig. 2A, B) and regulatory (Fig. 2C, D) T-cell responses were similar upon GAD65 plasmid

treatment with or without anti-IL-1β antibody. This indicates that the beneficial neutralization of

IL-1β in the CT does not act through the inhibition of cytokine production by GP33-specific

(‘autoreactive’ in the RIP-LCMV model) CD8 T cells nor increased Foxp3+ Treg fractions.

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Anti-IL1β/GAD65 DNA combination therapy can reverse hyperglycemia up to 8 weeks

after the treatment has been stopped. From a translational point of view, one important issue is

to know whether the benefitial effect provided by the CT is durable on extended periods of time,

notably several weeks after the treatment has been stopped. Thus, we followed BGVs in mice up

to 12 weeks post-onset, i.e., up to 8 weeks after the last antibody administration. Given our data

at 5 weeks post-onset, monotherapies with anti-IL-1β or isotype antibody alone were not tested

here. We observed a higher frequency of mice cured by the CT (6/14, 43%) than by the isot/GAD

treatment (2/11, 18%). This difference was not significant by Log-rank test analysis (p=0.059)

(Fig. 3A, supplementary Fig. 1E) but mean BGVs in CT-treated mice were significantly lower

than that in their isot/GAD-treated counterparts (p<0.001) (Fig. 3B). Then, we performed

analyses on non-protected mice and found that CT reduced the frequencies of mice with severe

hyperglycemia at the end of the study (Fig. 3C), reminiscent of our findings at 5 weeks post-

onset. Likewise, 75% (6/8) of mice that were not cured by the CT at 12 weeks post-onset

nonetheless maintained glycemia levels comparable to those measured at entry (Fig. 3D). In

isot/GAD-treated mice, this proportion was lower (33%, 3/9) and BGVs at euthanasia were

significantly higher than at entry (Fig. 3D). Finally, we assessed how many non-protected mice

presented one or several episodes of transient reversal to euglycemia, defined by one BGV

reading lower than 250mg/dl during the course of our observation. Compared to the isot/GAD

group, CT induced transient reversal to euglycemia more frequently (6/8, 75% versus 3/9, 33%)

and more durably (4.16 versus 1.67 weeks in average, p=0.049) (Fig. 3E).

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Effector CD8 and CD4 T-cell responses at 12 weeks post-onset are largely unaffected by the

CT in spleen, PLNs and blood. In the perspective of unveiling potential immune biomarkers

that would be relevant for human clinical trials, the assessment of the presence of CD8 T cells

specific for islet antigens via multimer (e.g., Quantum-dot) flow cytometry staining has gained

importance, is now technically achievable and could thus be considered as possible secondary

end-point in the future (28; 36; 37). Here, we took advantage of the RIP-GP model to monitor

frequencies of CD8 T cells specific for LCMV GP33-41 (Fig. 4A) and NP396-404 (Fig. 4B) epitopes

via pentamer staining in the blood, as this is the main accessible compartment in human patients.

Our results after 12-week follow up did not reveal any differences in terms of LCMV-specific

CD8 T-cell frequencies for either epitope in the blood (Fig. 4) or spleen (supplementary Fig. 2).

Furthermore, intracellular cytokine staining in CT-treated animals showed decreased frequencies

of GP33-41-specific CD8 effector T cells producing TNF in PLNs (Fig. 4B, left panels). However,

this effect was limited as this was not observed regarding IFN-γ (Fig. 4B, right panels) nor was

it observed in the spleen. Effector and regulatory CD4 T-cell responses in the spleen and PLNs of

isot/GAD- and CT-treated mice were also comparable (supplementary Fig. 3). Altogether, these

results suggest that some, but not all, CD8 and CD4 T-cell responses in the spleen and PLNs are

consitent with the improved mean glycemia levels and diabetes incidence in CT-treated mice.

Islet infiltration by CD8+ and CD11b

+ cells is reduced after CT at 5 and/or 12 weeks post-

onset. Immune cell infiltrates in the pancreas of RIP-LCMV mice contain (CD4 and CD8) T cells

as well as innate immune cells such as CD11b+ macrophages and CD11c

+ dendritic cells. It is

noteworthy that diabetes onset is preceded by an early MHC class II upregulation and infiltration

of macrophages in mouse islets (38) while macrophages also count among the different

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inflammatory cell infiltrates in human type 1 diabetic patients (39). Thus, we assessed the extent

of overall insulitis and islet infiltration by CD11b+ cells (presumably macrophages) together with

CD4+ and CD8

+ T cells. We found that in accordance with diabetes incidence curves at 5-week

follow up, anti-IL-1β/GAD-treated animals presented significantly reduced insulitis compared to

isot/GAD- and anti-IL-1β-treated groups (supplementary Fig. 1D). In isot/GAD- and CT-treated

groups, we found that overall CD4+ and CD8

+ cell infiltration was similar at 1 and 5 weeks post-

treatment onset (Fig. 5A, middle and lower panels). However, mice treated with anti-

IL1β/GAD65 DNA vaccine displayed less severe infiltration by CD11b+ cells compared to

isot/GAD65-treated mice at 1, 5 and 12 weeks post-treatment onset (Fig. 5A, upper panels). In

accordance with histological data, flow cytometry data (Fig. 5B) revealed that islet CD8+

and

CD11bhigh

cells were decreased upon CT compared to isot/GAD treatment, although this was not

statistically significant for CD11bhigh

cells (p=0.052) but may be explained by the stringency of

our gating strategy (Fig. 5B, upper panel).

The extent of islet infiltration by CD11bhigh

but not T cells correlates with efficacy of the

CT. We then assessed the extent of different immune cell infiltrations distinguishing mice that

were, or failed to be, cured by their respective treatment. Doing so, we found that CT-treated

mice that reverted to euglycemia presented a significantly lower fraction of islet CD11bhigh

cells

than mice that were also treated with the CT but stayed hyperglycemic (Fig. 6A). Of note, this

dichotomy was not observed in the case of isot/GAD treatment, suggesting that this phenotype

was likely due to the IL-1β blockade component of the CT. Moreover, we assessed on a per-

mouse basis the correlation between pancreas-derived CD11bhigh

macrophage infiltration in

relation to mouse glycemia. In accordance with our findings in Figure 6A, the positive linear

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relationship between these two parameters was strong (Pearson r=+0.78) and significant (p=0.01)

in the CT group, but not in the isot/GAD group (Pearson r=+0.27; p=0.55) (Fig. 6B). In contrast,

no significant association was found when correlating mouse glycemia to pancreas infiltration by

CD8 T cells (Fig. 6C, D), CD4 T cells (data not shown) or Foxp3+ Tregs (Fig. 6E, F). We

conclude that on a per-mouse basis, the extent of islet infiltration specifically by CD11bhigh

macrophages, but not T cells, might serve as a biological marker for success or failure of the anti-

IL-1β/GAD65 CT.

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DISCUSSION

In accordance with the minor effect of IL-1R deficiency on diabetes development in the RIP-

LCMV and NOD models (18; 40), our present results showed no protective effect of anti-IL-1β

therapy alone. This lack of efficacy is in line with the negative outcome of two recently published

placebo-controlled Phase IIa trials in recent-onset type 1 diabetes patients (17). Similarly to a

recently published trial using DNA vaccines (28), we found that GAD65 plasmid alone provided

some efficacy, albeit limited. Thus, the enhanced effect of the CT indicates utility of anti-IL-1β

antibody as an induction agent rather than a monotherapy.

In human trials, it appeared that higher C-peptide levels at study enrollment, reflective of better

preserved β-cell function, was associated with increased responsiveness to drugs such as anti-

CD3 (31; 41). Following the same reasoning, we stratified glycemia levels at treatment onset and

found that CT induced disease reversal in more than half of the treated mice if glycemia at

enrollment was comprised between 250-400mg/dl (Fig. 1, 3) but not any more if above 400mg/dl

(supplementary Fig. 1A). This suggests that a minimal residual β-cell mass is necessary for

protection. Subsequently, we closely analyzed the clinical outcome of non-protected mice,

creating subsets with mild (BGV 251-400mg/dl) versus severe (BGV 401-600mg/dl)

hyperglycemia. This refined analysis allowed us to discover that the CT maintained BGVs to a

milder HG status compared to the isot/GAD group. It is noteworthy that maintenance of glycemic

control was not observed upon monotherapy with anti-IL-1β antibody alone (Fig. 1A). We

showed that compared to anti-IL-1β-treated mice, CT acted by increasing Treg frequencies in

PLNs (Fig. 2). However, in comparison to GAD65 DNA treatment alone, CT did not further

increase Treg fractions. We cannot exclude however, that the Treg pool presented better

regulatory properties or a higher fraction of cells with specificity to GAD65. Of note, we have

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previsouly shown that when given with anti-CD3, the same pCMV-hGAD65 DNA plasmid

promoted the expansion of T cells specific for the C-terminal domain of the GAD65 protein (22).

Consistent with this, we also observed a similar reactivity to this domain upon treatment of RIP-

GP mice with Alum-formulated GAD65 protein (27). Depending on the context, regulatory cells

displayed an IL-10 producing or Th2-biased, IL-5-secreting phenotype. Here, we analyzed by

ELISpot whether protection induced by the CT could be associated with increased

immunomodulatory cytokines levels (IL-10, TGF-β or IL-4), whose elevation can be protective

in animal models of diabetes (42-44). However, we did not detect significant differences in the

spleen (supplementary Fig. 3C) or PLNs (supplementary Fig. 3D).

Upon treatment with anti-IL-1β/anti-CD3, Ablamunits et al. have observed an augmentation in

splenic M2-type CD11b+ and CD11c

+ cells with increased arginase-1, and decreased nitric oxide,

production (19). In our case, fewer CD11bhigh

cells were observed in the pancreas of CT-treated

mice but further phenotypic characterization of these cells will be warranted. Since myeloid

dendritic cells can also under some circumstances express the CD11b integrin marker, the exact

nature of these CD11bhigh

(presumably) macrophages needs to be defined. Sanda et al. have

shown that short-term neutralization of IL-1β in type 1 diabetic patients resulted in a decrease of

CD11b expression on monocytes isolated from PBMCs, which is consistent with our

observations (16). Although the phenotype of CD11bhigh

cells present in anti-IL-1β/GAD65-

treated mice is still unclear, our data rather argue for a diminished cell infiltration degree.

We found a significant correlation between mouse glycemia and the extent of islet infiltration by

CD11bhigh

cells, but not T-cell populations (Fig. 6). We believe such findings could be of

importance in regard to establishing biomarkers tailored for a given treatment. In addition to

simultaneous correlations between an individual’s glycemic status and a defined immune marker,

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predictive correlations will yield a major advantage in that these would allow to predict

responsiveness to (a) tested drug(s) either before enrollment or early post-treatment. Because of

the inaccessibility of the target organ in type 1 diabetic patients, it will be invaluable to identify

biomarkers that can be measured in the peripheral blood. In our case, although the mechanism(s)

underlying such an impaired recruitment/infiltration of CD11b+/high

and CD8+ cells to the target

organ are unclear, it will be interesting to track the behavior of those cells at, or early after, onset

in PBMCs. Preliminary data 4 days post-treatment onset in the blood tend to exclude a role for

CXCR3 or costimulatory molecule CD40, CD80 and CD86 expression on CD11bhigh

cells

(supplementary Fig. 4).

Four recent Phase II/III clinical trials, two aiming at IL-1β blockade (17) and two promoting

induction of tolerance to GAD65 (45; 46) have failed to meet their primary endpoints. In all

cases, later studies in mouse models indicated that there would be no effect (19; 27). We have

shown here that compared to isot/GAD-treated mice, CT increased the frequency of diabetes

reversal albeit more modestly than other CTs involving synergistic effects of anti-CD3 treatments

(19-22). Additionally however, most of non-protected mice still maintained comparable glycemic

levels compared to baseline, suggesting a subtle but discernable effect of the IL-1β blockade arm

of the CT. Altogether, this suggests that two such therapies that target IL-1β blockade and

tolerance induction to GAD65, yielding sub-optimal outcome when given separately, might be

promising candidates for combination in future human trials. We believe that such trials could

begin soon, since it has been established that DNA vaccines encoding islet antigens such as

proinsulin, can preserve C-peptide secretion and are well tolerated by type 1 diabetic patients

(28).

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ACKNOWLEDGEMENTS

P.P.P., D.B. and M.G.v.H. designed study. P.P.P. and M.G.v.H. are the guarantors of this work

and, as such, had full access to all the data in the study and take responsibility for the integrity of

the data and the accuracy of the data analysis. P.P.P., A.B.H., Y.M., N.A., A.B., T.R.C, S.S. and

S.F. researched data. P.P.P. and M.G.v.H. wrote the manuscript. D.B. generated the

pCMV/hGAD65, contributed to discussion and reviewed/edited manuscript.

The authors are thankful to Priscilla Colby and Malina McClure from the von Herrath lab for the

administrative and mouse work, respectively. We thank Carmen Baca Jones, Sowbarnika

Sachithanantham and Jackie Miller from the von Herrath lab for the preparation of LCMV

Armstrong stocks. We also thank Ambima Buzhyason from the von Herrath lab for his technical

help. We are thankful to Dr. Hermann Gram (Novartis, Basel, Switzerland) for kindly providing

us with anti-IL-1β and isotype (anti-cyclosporin B) monoclonal antibodies.

Preliminary results from this manuscript were presented at the American Diabetes Association’s

71st Scientific Sessions in 2011 in San Diego (Abstract Number: 331-OR).

P.P.P. received funding from the American Diabetes Association (Mentor-Based Postdoctoral

Fellowship 7-09-MN-54) and The Philippe Foundation, Inc. D.B. was the recipient of a funding

from the Juvenile Diabetes Research Foundation (Grant 36-2008-921). This work was funded by

U prevention center grant (U01 DK078013-05S1). The funding sources had no involvement in

the study design, interpretation of data, or decision to submit the article for publication.

All authors declare no conflict of interest.

of 35 Diabetes

18

FIGURE LEGENDS

Figure 1: Anti-IL1β/GAD65 DNA combination, but not anti-IL1β treatment alone, can

reverse hyperglycemia in recent-onset diabetic RIP-GP mice. (A) Individual blood glucose

values (BGV) in mg/dl in groups of recent-onset diabetic mice upon different treatments: isotype

antibody (open circles), anti-IL-1β antibody (open squares), isotype antibody plus GAD65 DNA

vaccine (closed circles) or anti-IL-1β antibody plus GAD65 DNA vaccine (closed squares).

Dashed horizontal lines represent threshold for enrollment (250mg/dl). (B) Cumulative diabetes

incidence after 5-week follow up in mice enrolled with BGV between 250-400mg/dl.

Significance from Gehan-Breslow test, which gives more weight to early reversals, is depicted in

grey on the graph whereas that of Log-rank test is depicted in black. (C) Time to reversal to

normoglycemia. Only one mouse reverted in the anti-IL-1β treated group and was not depicted.

Mean ± SEM is represented. (D) Fractions of mice 5 weeks post-treatment that were cured (Final

BGV < 250mg/dl, white), mildly hyperglycemic (HG) (Final BGV 251-400mg/dl, grey) and

severely HG (Final BGV > 401mg/dl, black). (E) BGV at, and 5 weeks after, treatment onset in

those mice that were not cured by their respective treatment. The Wilcoxon signed rank test was

used to determine statistical significance for paired values. Data pooled from seven independent

experiments are shown. *p<0.05. **p<0.01.

Figure 2: Effector CD8 T-cell responses are reduced and regulatory T-cell responses are

improved after GAD65 DNA vaccine and combination therapy. (A, B) Frequencies of IFN-γ-

and TNF-producing GP33-41-specific CD8+ effector T cells in spleen (A) and PLNs (B) of treated

mice. After 5-week treatment, spleens and PLNs were mashed onto 70µm cell strainers to obtain

single-cell suspensions. Cells were then plated in round bottom 96-well plates and left

of 35Diabetes

19

unstimulated, or stimulated for 4 to 5 hours with 5µg/ml (CD8 T-cell specific) GP33 peptide in

the presence of brefeldin A (10µg/ml). Cells were then stained in surface with fluorochrome-

coupled CD8 antibodies, fixed, permeabilized and stained with fluorochrome-coupled IFN-γ and

TNF antibodies. Data are expressed as % of gated CD8+ cells. (C, D) Frequencies of Foxp3-

expressing CD4+CD25

+ T regulatory cells in spleen (C) and PLNs (D) of treated mice. In parallel

to the intracellular cytokine staining, cells were stained for CD4, CD25 and intracellular Foxp3.

Data are expressed as % of gated CD25+Foxp3

+ within CD4+ cells. Results from 2-3 pooled

experiments with 2-3 mice per group are shown. *p<0.05.

Figure 3: Anti-IL1β/GAD65 DNA combination therapy can reverse hyperglycemia until 8

weeks after the treatment has been stopped. (A) Individual blood glucose values (BGV) in

mg/dl in groups of recent-onset diabetic mice upon different treatments: isotype antibody plus

GAD65 DNA vaccine (closed circles) or anti-IL-1β antibody plus GAD65 DNA vaccine (closed

squares). (B) Mean BGV in mice depicted in (A). Mean ± SEM is represented. Data pooled from

four independent experiments are shown. ***p<0.0001 (2-tailed paired t-test). (C) Fractions of

mice 12 weeks post-treatment that were cured (Final BGV < 250mg/dl, white), mildly

hyperglycemic (HG) (Final BGV 251-400mg/dl, grey) and severely HG (Final BGV > 401mg/dl,

black). (D) BGV at, and 12 weeks after, treatment onset in those mice that were not cured by

their respective treatment. The Wilcoxon signed rank test was used to determine statistical

significance for paired values. Data pooled from five independent experiments are shown.

*p<0.05. (E) Duration assessment of transient disease reversal observed in those mice that were

not cured by isot/GAD treatment (3/9, 33%) and CT (6/8, 75%). *p<0.05.

�

of 35 Diabetes

20

Figure 4: Effector CD8 and CD4 T-cell responses at 12 weeks post-onset are largely

unaffected by the CT in spleen, PLNs and blood. (A) Left panel, frequencies of gp33/Db

pentamer-positive CD8 T cells in the blood of treated mice. Right panel, representative FACS

plots depicting frequencies of np396/Db pentamer-positive CD8 T cells in the blood of treated

mice. Cells were pre-gated on Live/dead Aqua-negative, and CD8+CD19

- cells. (B) Frequencies

of TNF-producing GP33-41-specific CD8+ effector T cells in spleen (upper left panel) and PLNs

(lower left panel) of treated mice after 12 weeks of observation, as well as representative FACS

plots for frequencies of IFN-γ-producing cells (right panels). Data are expressed as % of gated

CD8+ cells. Results from 2 pooled experiments with 2-4 mice per group are shown. *p<0.05.

Figure 5: Islet infiltration by CD8+ and CD11b

+ cells is reduced after CT at 5 and/or 12

weeks post-onset. (A, left panels) CD4+, CD8

+ and CD11b

+ cell infiltration in mice treated with

GAD65 DNA with isotype or anti-IL-1β antibody. Pancreas sections from treated mice were

stained for insulin as well as CD4, CD8 and CD11b in parallel. Classification was performed as

follows: (i) no insulitis (white), (ii) peri-insulitis with no islet destruction or a few (less than 10)

infiltrating cells (grey), and (iii) infiltrating insulitis with extensive islet destruction (or islet

destroyed) (black). (A, right panels) Representative pictures at 20X. Two-way ANOVA tests

determined statistically significant differences (*P<0.05). (B) At 12 weeks post-onset, the

pancreas of mice was processed and cells stained for FACS analyses to confirm histological

analyses (*P<0.05 upon Mann-Whitney test). The extent of CD4+, CD8

+ or CD11b

+ cell

infiltration was counted as follows: 1 week post-onset: 29-134 islets from 3 mice per group; 5

weeks post-onset: 16-111 islets from 2-5 mice per group; 12 weeks post-onset: 13-39 islets from

3 mice per group.

of 35Diabetes

21

Figure 6: The extent of islet infiltration by CD11bhigh

but not T cells correlates with efficacy

of the CT. (A, C, E) Frequencies of CD11bhigh

(A), CD8 T (C) and CD4+CD25

+Foxp3

+ Treg (E)

cells found in the pancreas of mice upon indicated treatment. (B, D, F) Correlation between

mouse glycemia at 12 weeks post-treatment and the percentage of CD11bhigh

(B), CD8 T (D) and

CD4+CD25

+Foxp3

+ Treg (F) cell infiltration in mouse pancreata. Note that using Pearson

correlation test, r values between 0.3 and 0.7 (0.3 and -0.7) indicate a moderate positive

(negative) linear relationship.

of 35 Diabetes

22

REFERENCES

1. Dinarello CA, Simon A, van der Meer JW: Treating inflammation by blocking

interleukin-1 in a broad spectrum of diseases. Nature reviews Drug discovery 2012;11:633-652

2. Mandrup-Poulsen T, Pickersgill L, Donath MY: Blockade of interleukin 1 in type 1

diabetes mellitus. Nature reviews Endocrinology 2010;6:158-166

3. Willcox A, Richardson SJ, Bone AJ, Foulis AK, Morgan NG: Analysis of islet

inflammation in human type 1 diabetes. Clinical and experimental immunology 2009;155:173-

181

4. Mandrup-Poulsen T, Bendtzen K, Dinarello CA, Nerup J: Human tumor necrosis factor

potentiates human interleukin 1 mediated rat pancreatic beta cell cytotoxicity. Journal of

immunology 1987;139: 4077-82

5. Thomas HE, Darwiche R, Corbett JA, Kay TW: Interleukin-1 plus gamma-interferon-

induced pancreatic beta-cell dysfunction is mediated by beta-cell nitric oxide production.

Diabetes 2002;51:311-316

6. Eizirik DL, Colli ML, Ortis F: The role of inflammation in insulitis and beta-cell loss in

type 1 diabetes. Nature reviews Endocrinology 2009;5:219-226

7. Shaftel SS, Griffin WS, O'Banion MK: The role of interleukin-1 in neuroinflammation

and Alzheimer disease: an evolving perspective. Journal of neuroinflammation 2008;5:7

8. Mandrup-Poulsen T, Bendtzen K, Nerup J, Dinarello CA, Svenson M, Nielsen JH:

Affinity-purified human interleukin I is cytotoxic to isolated islets of Langerhans. Diabetologia

1986;29:63-67

9. Maedler K, Sergeev P, Ris F, Oberholzer J, Joller-Jemelka HI, Spinas GA, Kaiser N,

Halban PA, Donath MY: Glucose-induced beta cell production of IL-1beta contributes to

glucotoxicity in human pancreatic islets. The Journal of clinical investigation 2002;110:851-860

10. Maedler K, Storling J, Sturis J, Zuellig RA, Spinas GA, Arkhammar PO, Mandrup-

Poulsen T, Donath MY: Glucose- and interleukin-1beta-induced beta-cell apoptosis requires

Ca2+ influx and extracellular signal-regulated kinase (ERK) 1/2 activation and is prevented by a

sulfonylurea receptor 1/inwardly rectifying K+ channel 6.2 (SUR/Kir6.2) selective potassium

channel opener in human islets. Diabetes 2004;53:1706-1713

11. Osborn O, Brownell SE, Sanchez-Alavez M, Salomon D, Gram H, Bartfai T: Treatment

with an Interleukin 1 beta antibody improves glycemic control in diet-induced obesity. Cytokine

2008;44:141-148

12. Cavelti-Weder C, Babians-Brunner A, Keller C, Stahel MA, Kurz-Levin M, Zayed H,

Solinger AM, Mandrup-Poulsen T, Dinarello CA, Donath MY: Effects of gevokizumab on

glycemia and inflammatory markers in type 2 diabetes. Diabetes care 2012;35:1654-1662

13. Larsen CM, Faulenbach M, Vaag A, Volund A, Ehses JA, Seifert B, Mandrup-Poulsen

T, Donath MY: Interleukin-1-receptor antagonist in type 2 diabetes mellitus. The New England

journal of medicine 2007;356:1517-1526

14. Kaizer EC, Glaser CL, Chaussabel D, Banchereau J, Pascual V, White PC: Gene

expression in peripheral blood mononuclear cells from children with diabetes. The Journal of

clinical endocrinology and metabolism 2007;92:3705-3711

15. Pfleger C, Mortensen HB, Hansen L, Herder C, Roep BO, Hoey H, Aanstoot HJ, Kocova

M, Schloot NC, Hvidore Study Group on Childhood D: Association of IL-1ra and adiponectin

with C-peptide and remission in patients with type 1 diabetes. Diabetes 2008;57:929-937

of 35Diabetes

23

16. Sanda S, Bollyky J, Standifer N, Nepom G, Hamerman JA, Greenbaum C: Short-term

IL-1beta blockade reduces monocyte CD11b integrin expression in an IL-8 dependent fashion in

patients with type 1 diabetes. Clinical immunology 2010;136:170-173

17. Moran A, Bundy B, Becker DJ, DiMeglio LA, Gitelman SE, Goland R, Greenbaum CJ,

Herold KC, Marks JB, Raskin P, Sanda S, Schatz D, Wherrett DK, Wilson DM, Krischer JP,

Skyler JS, Pickersgill L, de Koning E, Ziegler AG, Boehm B, Badenhoop K, Schloot N, Bak JF,

Pozzilli P, Mauricio D, Donath MY, Castano L, Wagner A, Lervang HH, Perrild H, Mandrup-

Poulsen T, for the Type 1 Diabetes TrialNet Canakinumab Study Group, for the AIDA Study

Group: Interleukin-1 antagonism in type 1 diabetes of recent onset: two multicentre, randomised,

double-blind, placebo-controlled trials. Lancet 2013;381:1905-1915

18. Thomas HE, Irawaty W, Darwiche R, Brodnicki TC, Santamaria P, Allison J, Kay TW:

IL-1 receptor deficiency slows progression to diabetes in the NOD mouse. Diabetes 2004;53:113-

121

19. Ablamunits V, Henegariu O, Hansen JB, Opare-Addo L, Preston-Hurlburt P, Santamaria

P, Mandrup-Poulsen T, Herold KC: Synergistic reversal of type 1 diabetes in NOD mice with

anti-CD3 and interleukin-1 blockade: evidence of improved immune regulation. Diabetes

2012;61:145-154

20. Bresson D, Togher L, Rodrigo E, Chen Y, Bluestone JA, Herold KC, von Herrath M:

Anti-CD3 and nasal proinsulin combination therapy enhances remission from recent-onset

autoimmune diabetes by inducing Tregs. The Journal of clinical investigation 2006;116:1371-

1381

21. Mamchak AA, Manenkova Y, Leconet W, Zheng Y, Chan JR, Stokes CL, Shoda LK,

von Herrath M, Bresson D: Preexisting autoantibodies predict efficacy of oral insulin to cure

autoimmune diabetes in combination with anti-CD3. Diabetes 2012;61:1490-1499

22. Bresson D, Fradkin M, Manenkova Y, Rottembourg D, von Herrath M: Genetic-induced

variations in the GAD65 T-cell repertoire governs efficacy of anti-CD3/GAD65 combination

therapy in new-onset type 1 diabetes. Molecular therapy : the journal of the American Society of

Gene Therapy 2010;18:307-316

23. Oldstone MB, Nerenberg M, Southern P, Price J, Lewicki H: Virus infection triggers

insulin-dependent diabetes mellitus in a transgenic model: role of anti-self (virus) immune

response. Cell 1991;65:319-331

24. Solvason N, Lou YP, Peters W, Evans E, Martinez J, Ramirez U, Ocampo A, Yun R,

Ahmad S, Liu E, Yu L, Eisenbarth G, Leviten M, Steinman L, Garren H: Improved efficacy of a

tolerizing DNA vaccine for reversal of hyperglycemia through enhancement of gene expression

and localization to intracellular sites. Journal of immunology 2008;181:8298-8307

25. Tisch R, Wang B, Weaver DJ, Liu B, Bui T, Arthos J, Serreze DV: Antigen-specific

mediated suppression of beta cell autoimmunity by plasmid DNA vaccination. Journal of

immunology 2001;166:2122-2132

26. von Herrath MG, Dockter J, Oldstone MB: How virus induces a rapid or slow onset

insulin-dependent diabetes mellitus in a transgenic model. Immunity 1994;1:231-242

27. Boettler T, Pagni PP, Jaffe R, Cheng Y, Zerhouni P, von Herrath M: The clinical and

immunological significance of GAD-specific autoantibody and T-cell responses in type 1

diabetes. Journal of autoimmunity 2013;

28. Roep BO, Solvason N, Gottlieb PA, Abreu JR, Harrison LC, Eisenbarth GS, Yu L,

Leviten M, Hagopian WA, Buse JB, von Herrath M, Quan J, King RS, Robinson WH, Utz PJ,

Garren H, Investigators BHT, Steinman L: Plasmid-Encoded Proinsulin Preserves C-Peptide

While Specifically Reducing Proinsulin-Specific CD8+ T Cells in Type 1 Diabetes. Science

translational medicine 2013;5:191ra182

of 35 Diabetes

24

29. Abiru N, Maniatis AK, Yu L, Miao D, Moriyama H, Wegmann D, Eisenbarth GS:

Peptide and major histocompatibility complex-specific breaking of humoral tolerance to native

insulin with the B9-23 peptide in diabetes-prone and normal mice. Diabetes 2001;50:1274-1281

30. Lind M, Oden A, Fahlen M, Eliasson B: The true value of HbA1c as a predictor of

diabetic complications: simulations of HbA1c variables. PloS one 2009;4:e4412

31. Hagopian W, Ferry RJ, Jr., Sherry N, Carlin D, Bonvini E, Johnson S, Stein KE, Koenig

S, Daifotis AG, Herold KC, Ludvigsson J, for the Protege Trial I: Teplizumab preserves C-

peptide in recent-onset type 1 diabetes: 2-year results from the randomized, placebo-controlled

Protege trial. Diabetes 2013;

32. O'Sullivan BJ, Thomas HE, Pai S, Santamaria P, Iwakura Y, Steptoe RJ, Kay TW,

Thomas R: IL-1 beta breaks tolerance through expansion of CD25+ effector T cells. Journal of

immunology 2006;176:7278-7287

33. Bertin-Maghit S, Pang D, O'Sullivan B, Best S, Duggan E, Paul S, Thomas H, Kay TW,

Harrison LC, Steptoe R, Thomas R: Interleukin-1beta produced in response to islet autoantigen

presentation differentiates T-helper 17 cells at the expense of regulatory T-cells: Implications for

the timing of tolerizing immunotherapy. Diabetes 2011;60:248-257

34. Brinster C, Shevach EM: Costimulatory effects of IL-1 on the expansion/differentiation

of CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- T cells. Journal of leukocyte biology

2008;84:480-487

35. Ganesh BB, Bhattacharya P, Gopisetty A, Sheng J, Vasu C, Prabhakar BS: IL-1beta

promotes TGF-beta1 and IL-2 dependent Foxp3 expression in regulatory T cells. PloS one

2011;6:e21949

36. Trudeau JD, Kelly-Smith C, Verchere CB, Elliott JF, Dutz JP, Finegood DT, Santamaria

P, Tan R: Prediction of spontaneous autoimmune diabetes in NOD mice by quantification of

autoreactive T cells in peripheral blood. The Journal of clinical investigation 2003;111:217-223

37. Velthuis JH, Unger WW, Abreu JR, Duinkerken G, Franken K, Peakman M, Bakker AH,

Reker-Hadrup S, Keymeulen B, Drijfhout JW, Schumacher TN, Roep BO: Simultaneous

detection of circulating autoreactive CD8+ T-cells specific for different islet cell-associated

epitopes using combinatorial MHC multimers. Diabetes 2010;59:1721-1730

38. von Herrath M, Holz A: Pathological changes in the islet milieu precede infiltration of

islets and destruction of beta-cells by autoreactive lymphocytes in a transgenic model of virus-

induced IDDM. Journal of autoimmunity 1997;10:231-238

39. Itoh N, Hanafusa T, Miyazaki A, Miyagawa J, Yamagata K, Yamamoto K, Waguri M,

Imagawa A, Tamura S, Inada M, et al.: Mononuclear cell infiltration and its relation to the

expression of major histocompatibility complex antigens and adhesion molecules in pancreas

biopsy specimens from newly diagnosed insulin-dependent diabetes mellitus patients. The

Journal of clinical investigation 1993;92:2313-2322

40. Barral AM, Thomas HE, Ling EM, Darwiche R, Rodrigo E, Christen U, Ejrnaes M,

Wolfe T, Kay TW, von Herrath MG: SOCS-1 protects from virally-induced CD8 T cell mediated

type 1 diabetes. Journal of autoimmunity 2006;27:166-173

41. Bresson D, von Herrath M: Immunotherapy for the prevention and treatment of type 1

diabetes: optimizing the path from bench to bedside. Diabetes care 2009;32:1753-1768

42. Bot A, Smith D, Bot S, Hughes A, Wolfe T, Wang L, Woods C, von Herrath M: Plasmid

vaccination with insulin B chain prevents autoimmune diabetes in nonobese diabetic mice.

Journal of immunology 2001;167:2950-2955

43. Homann D, Holz A, Bot A, Coon B, Wolfe T, Petersen J, Dyrberg TP, Grusby MJ, von

Herrath MG: Autoreactive CD4+ T cells protect from autoimmune diabetes via bystander

suppression using the IL-4/Stat6 pathway. Immunity 1999;11:463-472

of 35Diabetes

25

44. King C, Mueller Hoenger R, Malo Cleary M, Murali-Krishna K, Ahmed R, King E,

Sarvetnick N: Interleukin-4 acts at the locus of the antigen-presenting dendritic cell to counter-

regulate cytotoxic CD8+ T-cell responses. Nature medicine 2001;7:206-214

45. Ludvigsson J, Krisky D, Casas R, Battelino T, Castano L, Greening J, Kordonouri O,

Otonkoski T, Pozzilli P, Robert JJ, Veeze HJ, Palmer J, Samuelsson U, Elding Larsson H, Aman

J, Kardell G, Neiderud Helsingborg J, Lundstrom G, Albinsson E, Carlsson A, Nordvall M, Fors

H, Arvidsson CG, Edvardson S, Hanas R, Larsson K, Rathsman B, Forsgren H, Desaix H,

Forsander G, Nilsson NO, Akesson CG, Keskinen P, Veijola R, Talvitie T, Raile K, Kapellen T,

Burger W, Neu A, Engelsberger I, Heidtmann B, Bechtold S, Leslie D, Chiarelli F, Cicognani A,

Chiumello G, Cerutti F, Zuccotti GV, Gomez Gila A, Rica I, Barrio R, Clemente M, Lopez

Garcia MJ, Rodriguez M, Gonzalez I, Lopez JP, Oyarzabal M, Reeser HM, Nuboer R, Stouthart

P, Bratina N, Bratanic N, de Kerdanet M, Weill J, Ser N, Barat P, Bertrand AM, Carel JC,

Reynaud R, Coutant R, Baron S: GAD65 antigen therapy in recently diagnosed type 1 diabetes

mellitus. The New England journal of medicine 2012;366:433-442

46. Wherrett DK, Bundy B, Becker DJ, DiMeglio LA, Gitelman SE, Goland R, Gottlieb PA,

Greenbaum CJ, Herold KC, Marks JB, Monzavi R, Moran A, Orban T, Palmer JP, Raskin P,

Rodriguez H, Schatz D, Wilson DM, Krischer JP, Skyler JS, Type 1 Diabetes TrialNet GADSG:

Antigen-based therapy with glutamic acid decarboxylase (GAD) vaccine in patients with recent-

onset type 1 diabetes: a randomised double-blind trial. Lancet 2011;378:319-327

of 35 Diabetes

*

Pagni et al. Fig 1.

B C

ns

* *

*

A Isotype Ab Anti-IL1β Ab Anti-IL1β/GAD Isot/GAD

Diabetes incidence for E2-8BGVs 250-400

0 1 2 3 4 50

20

40

60

80

100 anti-isot Ab (n=7)

anti-IL1b Ab (n=6)

anti-isot Ab / GAD (n=12)

anti-IL1b Ab / GAD (n=17)

Weeks post treatment onset

Dia

bete

s incid

ence (

%)


0 1 2 3 4 50

20

40

60

80


anti-IL1b Ab (n=6)




Dia

bete

s incid

ence (

%)


0 1 2 3 4 50

20

40

60

80


anti-IL1b Ab (n=6)




Dia

bete

s incid

ence (

%)


0 1 2 3 4 50

20

40

60

80


anti-IL1b Ab (n=6)




Dia

bete

s incid

ence (

%)

Time to reversion to normoglycemia (weeks)

anti-

isot

Ab

+ GAD (n

=4)

anti-

IL1b

Ab

+ GAD (

n=9)

0

2

4

6

0.0466

I also have a significant diff with Gehan-Breslow graph.This graph may not be needed to make my point then.

Tim

e to r

evers

ion

(weeks p

ost-

treatm

ent sta

rt)

*

5-week follow up

E2-9, BGVs 250-400

0 1 2 3 4 50

200

400

600

Weeks post treatment start

BG

V (

mg/d

l)

E2-9, BGVs 250-400

0 1 2 3 4 5Weeks post treatment start

0/6 (0%) 9/17 (53%) 4/12 (33%) 1/6 (17%)


0 1 2 3 4 50

20

40

60

80


anti-IL1b Ab (n=6)




Dia

bete

s incid

ence (

%)

E2-9, BGVs 250-400


E2-9, BGVs 250-400


% cured (Final BGV < 250mg/dl)

% mild HG (Final BGV 251-400mg/dl)

% severe HG (Final BGV > 401mg/dl)


0 1 2 3 4 50

20

40

60

80


anti-IL1b Ab (n=6)




Dia

bete

s incid

ence (

%)


0 1 2 3 4 50

20

40

60

80


anti-IL1b Ab (n=6)




Dia

bete

s incid

ence (

%)


0 1 2 3 4 50

20

40

60

80


anti-IL1b Ab (n=6)




Dia

bete

s incid

ence (

%)


0 1 2 3 4 50

20

40

60

80


anti-IL1b Ab (n=6)




Dia

bete

s incid

ence (

%)

D % mice cured vs mild vs high BG

anti-

isot

Ab

(n=6

)

anti-

IL1b

Ab

(n=6

)

anti-

isot

Ab

+ GAD (n

=12)

anti-

IL1b

Ab

+ GAD (n

=17)

0

20

40

60

80

100

% cured (Final BG < 250mg/dl)% mild HG (Final BG 251-400)% high HG (Final BG >401)

% e

nro

lled m

ice

% mice cured vs mild vs high BG

anti-

isot

Ab

(n=6

)

anti-

IL1b

Ab

(n=6

)

anti-

isot

Ab

+ GAD (n

=12)

anti-

IL1b

Ab

+ GAD (n

=17)

0

20

40

60

80

100


% e

nro

lled m

ice

* *

E Anti-IL1β/GAD Isot/GAD

At onset 5 wks At onset 5 wks


0 1 2 3 4 50

20

40

60

80


anti-IL1b Ab (n=6)




Dia

bete

s incid

ence (

%)


0 1 2 3 4 50

20

40

60

80


anti-IL1b Ab (n=6)




Dia

bete

s incid

ence (

%)

BGV at entry vs 5wks post-onset (ANTI-IL-1b/GAD)

Ant

i-IL-

1b/G

AD a

t ent

ry

Ant

i-IL-

1b 5

wks

pos

t-ons

et200

300

400

500

600

BG

V (

mg/d

l)

BGV at entry vs 5wks post-onset (ANTI-ISOT/GAD)

Ant

i-iso

t/GAD a

t ent

ry

Ant

i-iso

t/GAD 5

wks

pos

t-ons

et200

300

400

500

600

BG

V (

mg/d

l)


anti-

isot

Ab

(n=6

)

anti-

IL1b

Ab

(n=6

)

anti-

isot

Ab

+ GAD (n

=12)

anti-

IL1b

Ab

+ GAD (n

=17)

0

20

40

60

80

100


% e

nro

lled m

ice


anti-

isot

Ab

(n=6

)

anti-

IL1b

Ab

(n=6

)

anti-

isot

Ab

+ GAD (n

=12)

anti-

IL1b

Ab

+ GAD (n

=17)

0

20

40

60

80

100


% e

nro

lled m

ice


anti-

isot

Ab

(n=6

)

anti-

IL1b

Ab

(n=6

)

anti-

isot

Ab

+ GAD (n

=12)

anti-

IL1b

Ab

+ GAD (n

=17)

0

20

40

60

80

100


% e

nro

lled m

ice


anti-

isot

Ab

(n=6

)

anti-

IL1b

Ab

(n=6

)

anti-

isot

Ab

+ GAD (n

=12)

anti-

IL1b

Ab

+ GAD (n

=17)

0

20

40

60

80

100


% e

nro

lled m

ice


anti-

isot

Ab

(n=6

)

anti-

IL1b

Ab

(n=6

)

anti-

isot

Ab

+ GAD (n

=12)

anti-

IL1b

Ab

+ GAD (n

=17)

0

20

40

60

80

100


% e

nro

lled m

ice


anti-

isot

Ab

(n=6

)

anti-

IL1b

Ab

(n=6

)

anti-

isot

Ab

+ GAD (n

=12)

anti-

IL1b

Ab

+ GAD (n

=17)

0

20

40

60

80

100


% e

nro

lled m

ice

** ns

Anti-IL1β/GAD Isot/GAD

of 35Diabetes

% C

D8+

IFN

-γ+

% C

D8+

TNF+

GP33 peptide in vitro stimulation S

plee

n P

LNs

*

% C

D4+

CD

25+ F

oxp3

+

C

5-week follow up

% C

D8+

IFN

-γ+

% C

D8+

TNF+

% C

D4+

CD

25+ F

oxp3

+

Anti-IL1β Ab Anti-IL1β/GAD

A Sp- E2-4 Total IFNg+ in CD8+Sp-

anti-i

sot A

b (n=

6)

anti-I

L1b A

b (n=

7)

Anti-is

ot+GAD (n

=3)

anti-I

L1b A

b+GAD (n

=7)

0

5

10

15

0.0112%

IFN

g+ in

CD

8+

Sp- E2-4 Total IFNg+ in CD8+Sp-

*

Sp- E2-4 Total TNFa+ in CD8+Sp-

anti-i

sot A

b (n=

6)

anti-I

L1b A

b (n=

7)

Anti-is

ot+GAD (n

=3)

anti-I

L1b A

b+GAD (n

=7)

0

10

20

30

% T

NF+

in C

D8+

Sp- E2-4 Total TNFa+ in CD8+Sp-

* *

PLNs- Total IFNg+ in CD8+

anti-i

sot A

b (n=

6)

anti-I

L1b A

b (n=

7)

Anti-is

ot+GAD (n

=3)

anti-I

L1b A

b+GAD (n

=7)

0

2

4

6

% IF

Ng+

in C

D8+

PLNs- Total IFNg+ in CD8+

Sp- E2-4 CD4+CD25+Foxp3+

anti-i

sot A

b (n=

6)

anti-I

L1b A

b (n=

7)

Anti-is

ot+GAD (n

=3)

anti-I

L1b A

b+GAD (n

=7)

0

5

10

15

20

% C

D4+

CD

25+F

oxp3

+

Sp- E2-4 CD4+CD25+Foxp3+

PLNs- E2-4 CD4+CD25+Foxp3+

anti-i

sot A

b (n=

6)

anti-I

L1b A

b (n=

7)

Anti-is

ot+GAD (n

=3)

anti-I

L1b A

b+GAD (n

=7)

0

5

10

15

20

0.0358

% C

D4+

CD

25+F

oxp3

+

PLNs- E2-4 CD4+CD25+Foxp3+

PLNs- Total TNFa+ in CD8+

anti-i

sot A

b (n=

6)

anti-I

L1b A

b (n=

7)

Anti-is

ot+GAD (n

=3)

anti-I

L1b A

b+GAD (n

=7)

0

1

2%

TN

F+ in

CD

8+

PLNs- Total TNFa+ in CD8+

* *

d4pi bld- MFI CXCR3 in CD4+

a-iso

t (n=

7)

a-IL1

b (n=

7)

a-iso

t/GAD (n

=7)

a-IL1

b/GAD (n

=7)

0

1000

2000

3000a-isot (n=7)a-IL1b (n=7)a-isot/GAD (n=7)a-IL1b/GAD (n=7)

MFI

CX

CR

3 in

CD

4+


a-iso

t (n=

7)

a-IL1

b (n=

7)

a-iso

t/GAD (n

=7)

a-IL1

b/GAD (n

=7)

0

1000

2000


MFI

CX

CR

3 in

CD

4+


a-iso

t (n=

7)

a-IL1

b (n=

7)

a-iso

t/GAD (n

=7)

a-IL1

b/GAD (n

=7)

0

1000

2000


MFI

CX

CR

3 in

CD

4+


a-iso

t (n=

7)

a-IL1

b (n=

7)

a-iso

t/GAD (n

=7)

a-IL1

b/GAD (n

=7)

0

1000

2000


MFI

CX

CR

3 in

CD

4+


a-iso

t (n=

7)

a-IL1

b (n=

7)

a-iso

t/GAD (n

=7)

a-IL1

b/GAD (n

=7)

0

1000

2000


MFI

CX

CR

3 in

CD

4+


a-iso

t (n=

7)

a-IL1

b (n=

7)

a-iso

t/GAD (n

=7)

a-IL1

b/GAD (n

=7)

0

1000

2000


MFI

CX

CR

3 in

CD

4+


a-iso

t (n=

7)

a-IL1

b (n=

7)

a-iso

t/GAD (n

=7)

a-IL1

b/GAD (n

=7)

0

1000

2000


MFI

CX

CR

3 in

CD

4+


a-iso

t (n=

7)

a-IL1

b (n=

7)

a-iso

t/GAD (n

=7)

a-IL1

b/GAD (n

=7)

0

1000

2000


MFI

CX

CR

3 in

CD

4+Pagni et al. Fig 2.

D B

Isot/GAD Isotype Ab

of 35 Diabetes


anti-

isot

Ab

+ GAD

(n=1

1)

anti-

IL1b

Ab

+ GAD (n

=14)

0

20

40

60

80

100


% e

nro

lled m

ice

B A

12-week follow up Anti-IL1β/GAD Isot/GAD


0 1 2 3 4 50

20

40

60

80


anti-IL1b Ab (n=6)




Dia

bete

s incid

ence (

%)


0 1 2 3 4 50

20

40

60

80


anti-IL1b Ab (n=6)




Dia

bete

s incid

ence (

%)

C

***

Pagni et al. Fig 3.

E5+6+7+8 GP- BGVs

0 2 4 6 8 10 120

200

400

600E5_Anti-IL1b/GAD #3570

E5_Anti-IL1b/GAD #3542





E7_a-IL1/GAD #5350

E7_a-IL1/GAD #5327

E8_a-IL1/GAD #5355

E8_a-IL1/GAD #5320

E8_a-IL1/GAD #5331

E8_a-IL1/GAD #5340

E9_a-IL1/GAD #6001

E9_a-IL1/GAD #6003

E9_a-IL1/GAD #6015

Weeks post-treatment

BG

V (

mg/d

l)

E5+6+7+8 GP- BGVs

0 2 4 6 8 10 120

200

400

600

E5_Anti-isot/GAD #3590




E7_a-isot/GAD #5329

E7_a-isot/GAD #5358

E8_a-isot/GAD #5363

E8_a-isot/GAD #5370

E8_a-isot/GAD #5371

E9_a-isot/GAD #6008

E9_a-isot/GAD #6009


BG

V (

mg/d

l)

E5+6+7+8+9 GP- BGVs

0 2 4 6 8 10 120

200

400

600

Anti-iso/GAD (n=9)+5

Anti-IL1b/GAD (n=14+5)


Mean B

GV

(m

g/d

l)

6/14 (43%) 2/11 (18%)

% cured (Final BGV < 250mg/dl)

% mild HG (Final BGV 251-400mg/dl)

% severe HG (Final BGV > 401mg/dl)

E


anti-

isot

Ab

(n=6

)

anti-

IL1b

Ab

(n=6

)

anti-

isot

Ab

+ GAD (n

=12)

anti-

IL1b

Ab

+ GAD (n

=17)

0

20

40

60

80

100


% e

nro

lled m

ice

*

Anti-IL1β/GAD

At onset 12 wks At onset 12 wks


0 1 2 3 4 50

20

40

60

80


anti-IL1b Ab (n=6)




Dia

bete

s incid

ence (

%)


0 1 2 3 4 50

20

40

60

80


anti-IL1b Ab (n=6)




Dia

bete

s incid

ence (

%)


anti-

isot

Ab

(n=6

)

anti-

IL1b

Ab

(n=6

)

anti-

isot

Ab

+ GAD (n

=12)

anti-

IL1b

Ab

+ GAD (n

=17)

0

20

40

60

80

100


% e

nro

lled m

ice


anti-

isot

Ab

(n=6

)

anti-

IL1b

Ab

(n=6

)

anti-

isot

Ab

+ GAD (n

=12)

anti-

IL1b

Ab

+ GAD (n

=17)

0

20

40

60

80

100


% e

nro

lled m

ice


anti-

isot

Ab

(n=6

)

anti-

IL1b

Ab

(n=6

)

anti-

isot

Ab

+ GAD (n

=12)

anti-

IL1b

Ab

+ GAD (n

=17)

0

20

40

60

80

100


% e

nro

lled m

ice


anti-

isot

Ab

(n=6

)

anti-

IL1b

Ab

(n=6

)

anti-

isot

Ab

+ GAD (n

=12)

anti-

IL1b

Ab

+ GAD (n

=17)

0

20

40

60

80

100


% e

nro

lled m

ice


anti-

isot

Ab

(n=6

)

anti-

IL1b

Ab

(n=6

)

anti-

isot

Ab

+ GAD (n

=12)

anti-

IL1b

Ab

+ GAD (n

=17)

0

20

40

60

80

100


% e

nro

lled m

ice


anti-

isot

Ab

(n=6

)

anti-

IL1b

Ab

(n=6

)

anti-

isot

Ab

+ GAD (n

=12)

anti-

IL1b

Ab

+ GAD (n

=17)

0

20

40

60

80

100


% e

nro

lled m

ice

* ns BGV at entry vs 12wks post-onset (ANTI-ISOT/GAD)

Ant

i-iso

t/GAD a

t ent

ry

Ant

i-iso

t/GAD 1

2wks

pos

t-ons

et

200

300

400

500

600

BG

V (

mg/d

l)

0.0391

BGV at entry vs 12wks post-onset (ANTI-ISOT/GAD)

Ant

i-IL-

1b/G

AD a

t ent

ry

Ant

i-IL-

1b 1

2wks

pos

t-ons

et

200

300

400

500

600

BG

V (

mg/d

l)

0.5469

D

# of weeks spent <250mg/dl

anti-

isot

Ab

+ GAD (n

=3)

anti-

IL1b

Ab

+ GAD (n

=6)

0

2

4

6

8

# o

f w

eeks s

pent

with B

GV

< 2

50m

g/d

l

*

Isot/GAD

Dura

tion o

f tr

ansie

nt

revers

ion (

weeks)

a-IL1β/GAD Isot/GAD

Anti-IL1β/GAD Isot/GAD

of 35Diabetes

% n

p396

/Db+

C

D8+

T c

ells

E5+6+8_spl_GP33stim- Total TNFa+

Anti-is

ot / G

AD65 (n

=7)

Anti-IL

-1b /

GAD65

(n=1

0)

0

2

4

6

8

% T

NF+

in C

D8+

E5+6+8_spl_GP33stim- Total TNFa+

Anti-is

ot / G

AD65 (n

=7)

Anti-IL

-1b /

GAD65

(n=1

0)*

A

B

12-week follow up %

CD

8+TN

F+

Spl

een

PLN

s

% C

D8+

TNF+

CD8

% g

p33-

31/D

b+

CD

8+ T

cel

ls

IFN

-γ+

CD8

PLN GP33- %CD8+TNF+

E8_Anti

-isot/

GAD (n=5

)

E8_Anti

-IL-1

b/GAD (n

=5)

0

1

2

3

4*

%TN

F+ in

CD

8+

PLN GP33- %CD8+TNF+

E8_Anti

-isot/

GAD (n=5

)

E8_Anti

-IL-1

b/GAD (n

=5)

*

GP33-41 peptide in vitro stimulation

E5+6_Bld_GP33 pentamer

Anti-is

ot / G

AD65 (n

=4)

Anti-IL

-1b /

GAD65

(n=6

)

0

1

2

3

4

% o

f gp3

3-31

/Db+

C

D8

T ce

lls

E5+6_Bld_GP33 pentamer

Blo

od

Isot/GAD Anti-IL-1β/GAD

0102 103 104 105

0102

103

104

105

<APC-A>: CD8

<PE

-A>:

NP

396

pent

amer 3.41

0102 103 104 105

0102

103

104

105

<APC-A>: CD8

<PE

-A>:

NP

396

pent

amer 4.41

Isot/GAD Anti-IL-1β/GAD

Anti-IL1β/GAD


a-iso

t (n=

7)

a-IL1

b (n=

7)

a-iso

t/GAD (n

=7)

a-IL1

b/GAD (n

=7)

0

1000

2000


MFI

CX

CR

3 in

CD

4+


a-iso

t (n=

7)

a-IL1

b (n=

7)

a-iso

t/GAD (n

=7)

a-IL1

b/GAD (n

=7)

0

1000

2000


MFI

CX

CR

3 in

CD

4+


a-iso

t (n=

7)

a-IL1

b (n=

7)

a-iso

t/GAD (n

=7)

a-IL1

b/GAD (n

=7)

0

1000

2000


MFI

CX

CR

3 in

CD

4+


a-iso

t (n=

7)

a-IL1

b (n=

7)

a-iso

t/GAD (n

=7)

a-IL1

b/GAD (n

=7)

0

1000

2000


MFI

CX

CR

3 in

CD

4+

0102 103 104 105

0102

103

104

105

<PE-Texas Red-A>: CD8

<Ale

xa F

luor

700

-A>:

IFN

g

0.705

0102 103 104 105

0102

103

104

105


<Ale

xa F

luor

700

-A>:

IFN

g

1.13

0102 103 104 105

0102

103

104

105

<PE-Texas Red-A>: CD8<A

lexa

Flu

or 7

00-A

>: IF

Ng

4.62

0102 103 104 105

0102

103

104

105


<Ale

xa F

luor

700

-A>:

IFN

g

6.37

Pagni et al. Fig 4.

Isot/GAD

of 35 Diabetes

Weeks post-onset CD11b infiltration, 12wks p.t.s

Anti-iso

t / G

AD65

(n=3

; 39i)

Anti-IL-1b / G

AD65

(n=3

; 20i)

Anti-iso

t (n=

2; 56i)

Anti-IL-1b (n=1

; 23i)

0

20

40

60

80

100no infiltrationmild infiltrationheavy infiltration

ns

# mice: 3 and 3, resp. ; # counted islets: 39 and 20, resp.

% o

f counte

d isle

ts

CD4 infiltration, 12wks p.t.s

Anti-iso

t / G

AD65

(n=3

; 36i)

Anti-IL-1b / G

AD65

(n=3

; 13i)

Anti-iso

t (n=

2; 39i)

Anti-IL-1b (n=1

; 13i)

0

20

40

60

80


# mice:

% o

f counte

d isle

ts

CD8 infiltration, 12wks p.t.s

Anti-iso

t / G

AD65

(n=3

; 31i)

Anti-IL-1b / G

AD65

(n=3

; 15i)

Anti-iso

t (n=

2; 44i)

Anti-IL-1b (n=1

; 16i)

0

20

40

60

80


# mice:

% o

f counte

d isle

ts

%CD8+ Infiltration

anti-i

sotyp

e + G

AD (n=3

, 43 i

slets)

anti I

L1B +

GAD (n=3

, 42 i

slets)

0

20

40

60

80

100no infiltration mild infiltrationheavy infiltration

% o

f cou

nted

isle

ts

Cel

ls

CD

4+

CD

8+

CD11b insulin

No infiltration

Mild (peri) infiltration

Heavy (intra) infiltration

*

Ant

i-IL-

1β

Ab/

GA

D65

CD

11b+

5 12 CD11b infiltration

Anti-iso

t / G

AD65

Anti-IL-1b / G

AD65

Anti-iso

t

Anti-IL-1b

0

20

40

60

80


ns


% o

f counte

d isle

ts

CD8 infiltration

Anti-iso

t / G

AD65

Anti-IL-1b / G

AD65

Anti-iso

t

Anti-IL-1b

0

20

40

60

80



% o

f counte

d isle

ts

CD4 infiltration

Anti-iso

t / G

AD65

Anti-IL-1b / G

AD65

Anti-iso

t

Anti-IL-1b

0

20

40

60

80



% o

f counte

d isle

ts*

Ant

i-IL-

1β

Ab/

GA

D65

A B

*

Ant

i-IL-

1β

Ab/

GA

D65

*

CD11b infiltration

Anti-iso

t / G

AD65

Anti-IL-1b / G

AD65

Anti-iso

t

Anti-IL-1b

0

20

40

60

80


**

# mice: 3, resp. ; # counted islets: 43iso, 31IL1, 47isoGAD, 29IL1GAD

% o

f counte

d isle

ts

CD8 infiltration

Anti-iso

t / G

AD65

Anti-IL-1b / G

AD65

Anti-iso

t

Anti-IL-1b

0

20

40

60

80

100

no infiltrationmild infiltrationheavy infiltration


% o

f counte

d isle

ts

CD4 infiltration

Anti-iso

t / G

AD65

Anti-IL-1b / G

AD65

Anti-iso

t

Anti-IL-1b

0

20

40

60

80

100

no infiltrationmild infiltrationheavy infiltration


% o

f counte

d isle

ts

1

Isot

/ G

AD

65

Ant

i-IL-

1β

Ab/

GA

D65

Weeks post-onset

12

% C

D4+

CD

25+ F

oxp3

+

1

Pc- %CD11b+

E568_

Anti-is

ot/GAD (n

=7)

E568_

Anti-IL

-1b/G

AD (n=1

0)0

20

40

60

80

0.0520

%C

D11

bhigh

Pc- %CD11b+0.0520

Pc- % total CD8+

E6_Anti

-isot/

GAD (n=7

)

E6_Anti

-IL-1

b/GAD (n

=10)

0

10

20

30

40

E6_Anti-isot/GAD (n=7)E6_Anti-IL-1b/GAD (n=10)

*

% C

D8+

Pc- % total CD8+

Pc- %CD4+

E568_

Anti-is

ot/GAD (n

=7)

E568_

Anti-IL

-1b/G

AD (n=1

0)0

5

10

15

20

25

%C

D4+

Pc- %CD4+

% C

D4+

%

CD

8+

% C

D11

bhig

h

Pagni et al. Fig 5.

Isot

/ G

AD

65

Isot

/ G

AD

65

Isot

/ G

AD

65

E5+6+8_Pc- %CD25+Foxp3+

E5+6+

8_Anti

-isot/

GAD (n=6

)

E5+6+

8_Anti

-IL-1

b/GAD (n

=10)

0

10

20

30

Within these cells, similar MFI for CTLA-4, Helios, PD-1, Neuropilin-1 and LAP

%C

D25

+Fox

p3+

in C

D4+

E5+6+8_Pc- %CD25+Foxp3+

of 35Diabetes

A B *

1

Pagni et al. Fig 6.

Isot/GAD

12-week follow up

BGV at 12wks vs %CD11b in Pc (cured vs sick)

%CD8+

(isot/

GAD): Cur

ed

%CD8+

(isot/

GAD): Sick

%CD8+

(CT):

Cured

%CD8+

(CT):

Sick

0

10

20

30

% C

D8+

in p

ancr

eas

a-IL-1β/GAD

Data 1

200 300 400 500 6000

20

40

60

%CD11b+ in Pc (CT)%CD11b+ in Pc (isot/GAD)

BGV at 12 wks post-onset

% C

D11

bhigh

in

pan

crea

s

Pearson r95% confidence intervalP value (two-tailed)

%CD11b+ in Pc (CT)0.77990.2401 to 0.95130.0132

%CD11b+ in Pc (isot/GAD)0.2711-0.6057 to 0.85060.5564

Isot/GAD Pearson r = 0.27 P value = 0.55

aIL-1β/GAD Pearson r = 0.78 *P value = 0.01

Isot/GAD Pearson r = -0.36 P value = 0.43

aIL-1β/GAD Pearson r = 0.38 P value = 0.32

Isot/GAD Pearson r = -0.59 P value = 0.16

aIL-1β/GAD Pearson r = 0.27 P value = 0.48

Data 1

200 300 400 500 6000

20

40

60



% C

D11

bhigh

in

pan

crea

s


%CD11b+ in Pc (CT)0.77990.2401 to 0.95130.0132


Data 1

200 300 400 500 6000

20

40

60



% C

D11

bhigh

in

pan

crea

s


%CD11b+ in Pc (CT)0.77990.2401 to 0.95130.0132


BGV at 12wks vs %CD11b in Pc (cured vs sick)

%CD11

b+ (is

ot/GAD):

Cured

%CD11

b+ (is

ot/GAD):

Sick

%CD11

b+ (C

T): Cur

ed

%CD11

b+ (C

T): Sick

0

20

40

60

% C

D11

bhigh

in

pan

crea

s

C D

E F BGV at 12wks vs %CD11b in Pc (cured vs sick)

%CD25

+Fox

p3+ (

isot/G

AD): Cur

ed

%CD25

+Fox

p3+ (

isot/G

AD): Sick

%CD25

+Fox

p3+ (

CT): Cur

ed

%CD25

+Fox

p3+ (

CT): Sick

0

10

20

30

% C

D25

+ Fox

p3+

in

CD

4+ in

pan

crea

s

Data 1

200 300 400 500 6000

20

40

60



%CD11b+ in Pc (CT)0.77990.2401 to 0.95130.0132


BGV at 12 wks post-onset%

CD

11bhi

gh

in p

ancr

eas

Data 1

200 300 400 500 6000

20

40

60



%CD11b+ in Pc (CT)0.77990.2401 to 0.95130.0132



% C

D11

bhigh

in

pan

crea

s

Data 1

200 300 400 500 6000

20

40

60



%CD11b+ in Pc (CT)0.77990.2401 to 0.95130.0132



% C

D11

bhigh

in

pan

crea

sData 1

200 300 400 500 6000

10

20

30


%CD8+ in Pc (CT)-0.2713-0.7927 to 0.47930.4802

%CD8+ in Pc (isot/GAD)-0.5884-0.9296 to 0.29590.1646


% C

D8+

in p

ancr

eas

Data 1

200 300 400 500 6000

20

40

60



%CD11b+ in Pc (CT)0.77990.2401 to 0.95130.0132



% C

D11

bhigh

in

pan

crea

s

Data 1

200 300 400 500 6000

10

20

30

%CD4+25+Foxp3+ in Pc (CT)%CD4+25+Foxp3+ in Pc (isot/GAD


%CD4+25+Foxp3+ in Pc (CT)0.3783-0.3819 to 0.83320.3155

%CD4+25+Foxp3+ in Pc (isot/GAD-0.3609-0.8759 to 0.53870.4265


% C

D25

+ Fox

p3+

in C

D4+

in p

ancr

eas

of 35 Diabetes

of 35Diabetes

of 35 Diabetes

#Ghanashyam Sarikonda, Sowbarnika Sachithanantham, Yulia Manenkova, Tinalyn Kupfer, Amanda Posgai,

Clive Wasserfall, Philip Bernstein, Laura Straub, Philippe P. Pagni, Darius Schneider, Teresa Rodriguez Calvo,

Marilyne Coulombe, Kevan Herold, Ronald G. Gill, Mark Atkinson, Gerald Nepom, Mario Ehlers, Teodora

Staeva, Hideki Garren, Lawrence Steinman, Andrew C. Chan, Matthias von Herrath. Transient B-Cell

Depletion with Anti-CD20 in Combination with Proinsulin DNA Vaccine or Oral Insulin: Immunologic Effects

and Efficacy in NOD Mice. PLoS One. 2013;8(2):e54712. doi: 10.1371/journal.pone.0054712. Epub 2013 Feb

6.

of 35Diabetes

of 35 Diabetes

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