Cure of the declared autoimmune diabetic condition at a time early enough to preserve the remaining β-cells is still the best therapeutic approach for patients with type I diabetes. Furthermore, prevention of recurrent autoimmunity in islet transplantation in recipients with type I diabetes may be critical to achieve long-term graft function and survival. Immunomodulatory strategies that prevent autoimmune destruction of β-cells are highly sought.
In murine models of autoimmune diabetes, namely, the nonobese diabetic (NOD) mouse (1), several studies have suggested that immunomodulatory strategies given at an early prediabetic stage prevent disease occurrence (2–5). However, only two strategies targeting the T-cell compartment have been reported to be effective in reversing established diabetes in the NOD mouse. One consists of the “modulating,” nondepleting anti-CD3 monoclonal antibody (mAb) and the other consists of the nonspecific T-cell–depleting polyclonal antibody antilymphocyte serum (3, 6). Despite the success of anti-CD3 mAb OKT3 and antilymphocyte serum in transplantation, their applicability for the treatment of diabetes is hampered by their toxicity, including increased infectious and oncogenic risks and the initial cytokine “storm” that follows the first injections of these antibodies (7, 8). A multicenter trial using a humanized form of the anti-CD3 mAb, designed to minimize its toxicity, is currently under way to treat patients with new-onset diabetes. The outcome of this study is eagerly awaited. However, the field would still benefit from the development of an alternative, effective immunosuppressive regimen for the treatment of patients with early-onset diabetes and prevention of disease recurrence on islet grafts.
It is well established that CD4+ T cells play an important role in the autoimmune destruction of pancreatic islets. Thus, targeting CD4+ T cells represents a good alternative strategy for the cure of autoimmune diabetes. Compelling experimental evidence demonstrates that depletion of CD4+ T cells for a short period of time at a prediabetic stage of the disease (peri-insulitic stage) prevents the occurrence of diabetes in NOD mice (9). In addition, recurrence of autoimmunity in syngeneic islet grafts has been shown to be strictly dependent on CD4+ T cells (9, 10). The autoimmune destructive process of β-cells initially depends on CD8+ T cells and then progresses to CD4+ T-cell–mediated destruction independent of the action of cytotoxic CD8+ T cells. This further stresses the central role of CD4+ T cells in both the early and late phases of diabetes (11–13).
Blockade of B7/CD28 and CD154L/CD154 costimulation signaling is also effective in preventing autoimmune diabetes in prediabetic NOD mice (14–16). Treatment with cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4Ig) and anti-B7–2 mAb blocks the CD28/B7 pathway and prevents autoimmune diabetes when given to prediabetic NOD mice at the onset of insulitis (4). Similar treatment with CTLA4Ig/anti-B7–2 mAb and blockade of CD154/CD154L costimulation also prevents the occurrence of diabetes if NOD mice are treated at the peri-insulitic stage of the disease and we reproduce these data (2). Blockade of B7 or CD154 costimulatory pathways in experimental models has proven highly effective for the prevention of both solid-organ and islet allograft rejection without causing toxicity to the animals (17–19). In contrast, blockade of these pathways cannot prevent recurrent autoimmunity in NOD islet isografts (3). Their efficacy in reversing early-onset diabetes in NOD mice has not been tested.
Given these data, we reasoned that a short-term depletion of CD4+ T cells or blockade of costimulation could represent two potential clinical therapies for the treatment of new-onset insulin-dependent diabetes mellitus. In this study, we investigated the effects of the depleting anti-CD4 mAb GK1.5 on reversal of new-onset diabetes in the NOD mouse and on disease recurrence in severe combined immunodeficient (SCID)/NOD mice and its effect on allogeneic islet transplantation in diabetic NOD mice (3, 5, 20, 21). We next investigated whether costimulation blockade using a combined regimen of CTLA4Ig and anti-CD154 mAb therapy would affect the progression of diabetes after the onset of insulitis and new-onset diabetes (14, 22).
Female NOD/Lt and NOD/MrkTac mice were purchased from Jackson Laboratory (Bar Harbor, ME) and TACONIC (Germantown, NY). The NOD mice developed insulitis at 3 to 4 weeks of age and overt diabetes by 11 to 12 weeks of age. NOD/MrkTac animals were used for studies examining the effect of costimulation blockade in prediabetic mice. Procedures performed on mice were approved by the institutional animal care committee and performed according to National Institutes of Health recommendations for humane use of animals in research.
Anti-CD4 mAb (GK1.5; anti-mouse L3T4 rat IgG2b) was purchased as ascites from the American Type Culture Collection (Rockville, MD). The anti-mouse CD154 mAb (MR1), a gift from Dr. Randy Noelle (Dartmouth Medical School, Lebanon, NH), was produced commercially by Bioexpress Inc. (West Lebanon, NH). CTLA4Ig was a gift of Dr. Robert Peach (Bristol-Meyers Squibb, Princeton, NJ).
CD4 +T-Cell Depletion Experiments. GK1.5 was administered to three groups of NOD mice:
- Female NOD mice with new-onset diabetes (blood glucose >200 mg/dL for no more than 3 days; n=10). In this group, 0.2 mL of GK1.5 ascites was administered intraperitoneally (IP) per mouse on days 3, 4, and 5 after the onset of diabetes and then every second day for a total period of 14 days.
- Female NOD mice with established diabetes (blood glucose >200 mg/dL for >1 week; n=7). In this group, 0.2 mL of GK1.5 ascites was administered IP per mouse for 3 days consecutively and then every second day for a total period of 14 days.
- Female NOD mice with established diabetes (blood glucose > 350 mg/dL) and recipients of C57BL/6 islet allografts or NOD/SCID isografts. NOD recipients of allogeneic islets were treated with GK1.5 on days 0, 1, and 2, and then every second day until day 14 posttransplant, whereas NOD recipients of syngeneic isografts were treated until day 21 posttransplant.
Costimulation Blockade Experiments
CTLA4Ig and anti-CD154 mAb were administered to two groups of NOD mice:
- Prediabetic female NOD mice with insulitis were treated on days 0, 2, 4, and 6 of their eighth week with CTLA4Ig (250 μg/mouse IP; n=6) or anti-CD154 mAb (250 μg/mouse IP; n=6) alone or in combination (n=13).
- Female NOD mice with new-onset diabetes (blood glucose >200 mg/dL for no more than 3 consecutive days; n=5) were treated with combined costimulation blockade (CTLA4Ig+anti-CD154, 250 μg/ mouse each IP; n=5) on days 0, 2, 4, and 6 relative to the first day of hyperglycemia.
For controls, a cohort of female, nontreated, age-matched NOD mice were followed by blood glucose measurements.
Fluorescence-Activated Cell Sorter Analysis
Peripheral blood was obtained from NOD mice at diabetes onset, under depletion regimen with GK1.5, and 3 to 4 weeks after the end of treatment. Lymphocytes in single-cell suspension were directly labeled using rat anti-mouse GK1.5 conjugated with fluorescein isothiocyanate (PharMingen, San Diego, CA), rinsed, fixed in 1% paraformaldehyde, and analyzed on a FACScan (Becton Dickinson, San Jose, CA). Data were analyzed using Cellquest software (Becton Dickinson Immunocytometry Systems).
Adoptive Transfer of Diabetes into Nonobese Diabetic Mice
Nondiabetic NOD male mice (6–8 weeks of age) were irradiated at 650 rads and 12 hr later received an intravenous injection through the tail vein of 5.107 spleen cells retrieved from overtly diabetic NOD mice with or without the equal number of splenocytes retrieved from the anti-CD4 mAb–treated and cured NOD mice. Diabetes occurrence was monitored daily by measurement of blood glucose levels (>250 mg/dL for 2 consecutive days).
Transplantation of Allogeneic and Syngeneic Islets
Pancreatic islets were isolated using collagenase digestion followed by Histopaque 1077 (Sigma, Saint Louis, MO) density gradient separation and then hand-picking as described (23). A total of 700 C57BL/6 or NOD/SCID islets were transplanted under the kidney capsule of diabetic female NOD mice. This number of islets was found to be necessary to consistently achieve euglycemia. Rejection of the graft was defined according to blood glucose measurement (blood glucose >250 mg/dL for at least 2 consecutive days after restoration of euglycemia for at least 2 consecutive days).
Pathology and Immunohistochemistry
Tissue samples (whole pancreata and islet grafts from nephrectomy specimen) were recovered, snap-frozen in 2-methylbutane (Fisher Scientific, Pittsburgh, PA), cooled in liquid nitrogen, and stored at −80°C until processing. Five-micron sections were stained with hematoxylin-eosin for morphologic examination under light microscopy. Cryostat sections were stained with a polyclonal guinea pig anti-insulin antibody used at a dilution of 1/10 (Dako, Carpenteria, CA) followed by a biotin-conjugated goat anti-guinea pig polyclonal antibody used at 2 μg/mL (Vector, Burlingame, CA). The CD4+ and CD8+ T-cell infiltrate was evaluated in pancreata and islet grafts using rat anti-mouse CD4+ and CD8+ mAbs (3 μg/mL, PharMingen) followed by a secondary biotin-conjugated rabbit anti-rat polyclonal antibody used at 2 μg/mL (Vector). Appropriate isotype negative controls were used for each antibody.
Significance between groups was evaluated by comparing group median survival time (MST) using the two-tailed t test. Statistical significance was defined as a P value less than 0.05. Survival data were plotted using the Kaplan-Meier method.
Cure of Established Autoimmune Diabetes with a Short Course of Treatment Using a Depleting Anti-CD4+ T-Cell Monoclonal Antibody
We investigated the effect of a short course of CD4+ T-cell depletion on reversal of hyperglycemia in female NOD mice with new-onset or established diabetes. Ninety percent of mice (9/10 mice) with new-onset diabetes were cured and remained euglycemic for more than 100 days after treatment with anti-CD4 mAb (n=10) (Fig. 1A). One animal died suddenly on day 23 after the beginning of the therapy; it was still euglycemic and showed no sign of disease or wasting. However, we cannot rule out an unforeseen toxicity of the anti-CD4 mAb as a cause of this death. In all other animals, we did not observe any clinical signs of toxicity including weight loss, diarrhea, or infection.
With this result in hand, we investigated whether this therapy would be effective in NOD mice with established diabetes (mice with hyperglycemia for >1 week; n=7). In this context, treatment with the depleting anti-CD4 mAb was unable to reverse the diabetic condition (Fig. 1B). All mice remained hyperglycemic and were progressively affected by the metabolic dysfunction. This result reflects the total destruction of the β-cell mass by that time or the toxicity of higher noncontrolled hyperglycemia levels, rendering impossible any rescue therapy with anti-CD4 mAb. Control of hyperglycemia was demonstrated to be required for the success of other therapies aimed at reversing diabetes (24).
The CD4+ T-cell depletion after treatment with GK1.5 was monitored by fluorescence-activated cell sorter analysis of peripheral blood CD4+ T-cell number prior to, 2 weeks after, and 6 weeks after initiation of the anti-CD4 mAb. These dates correspond to the beginning and end of the depleting regimen and 4 weeks after its termination. Our data demonstrated that CD4+ T-cell depletion was nearly complete (>99% depletion) by the end of the treatment period with a return to pretreatment values 4 weeks after the end of anti-CD4 treatment (Fig. 1C). The percentage of CD4+ T cells was 34%±2% before anti-CD4 mAb therapy, decreased to 0.5%±0.3% 2 weeks after the initiation of the treatment, and returned to 29%±5% 4 weeks after stopping the anti-CD4 mAb treatment (n=4).
Adoptive Transfer of Splenocytes from Anti-CD4–Treated and Overtly Diabetic Nonobese Diabetic Mice Does Not Prevent Occurrence of Diabetes in Irradiated Nonobese Diabetic Mice
To investigate whether cured mice developed regulatory T cells, we adoptively transferred 5.107 splenocytes from overtly diabetic NOD mice alone (n=6) or in combination with an equal number of splenocytes from NOD mice that had been cured by anti-CD4 mAb and were euglycemic for more than 100 days (n=3) into sublethally irradiated 7-week-old male NOD mice. Diabetes occurrence was subsequently monitored. All mice were equally diabetic within 3 weeks of transfer of splenocytes whether or not they had received splenocytes from cured anti-CD4–treated NOD mice (Fig. 1C). Mice in the control group that were sublethally irradiated and left untreated were also monitored for the occurrence of diabetes. None developed diabetes by 6 weeks after irradiation. These experiments indicate that anti-CD4–mediated cure of new-onset diabetes does not relate to the development and expansion of regulatory T cells. Other mechanisms such as anergy or clonal deletion cannot be excluded at this time. These hypotheses are being tested.
Immunopathology of Pancreata from Cured Anti-CD4–Treated Female Nonobese Diabetic Mice
Pancreata of cured diabetic female NOD mice were retrieved at 100 (n=2) and 150 (n=2) days after treatment with anti-CD4 mAb. All mice were euglycemic and healthy at the time of the retrieval. Hematoxylin-eosin staining demonstrated normal pancreatic architecture with healthy islets devoid of any mononuclear cell infiltrate together with islets surrounded by a peri-insulitic mononuclear cell infiltrate (Fig. 2). Immunostaining with insulin showed well-granulated insulin positive cells in both the intact islets and those with peri-insulitic mononuclear cell infiltrate (Fig. 2). Immunohistologic staining with anti-CD4 and anti-CD8 mAbs demonstrated that the T-cell infiltrate comprised both T-cell subsets (Fig. 2). Notably, similar pathology was seen in 8-week-old prediabetic female NOD mice during the peri-insulitic stage of the diabetic disease before the onset of hyperglycemia, whereas all islets were destroyed in age-matched, nontreated NOD mice whose pancreata were retrieved at 28 weeks of age (Fig. 2). In the latter, almost no insulin was detected and the islet remnants were invaded with CD4+ and CD8+ T cells (Fig. 2). These results indicate that anti-CD4 mAb-mediated cure of early-onset diabetes occurs despite the persistence of a positive CD4+ and CD8+ T-cell infiltrate surrounding the islets as seen in the prediabetic stage of the disease. CD25 staining was weak and showed no difference with that detected in 8-week-old prediabetic female NOD (data not shown).
Depleting Anti-CD4 Monoclonal Antibody Significantly Prolongs Islet Allograft Survival in Diabetic Nonobese Diabetic Mice and Prevents Recurrence of Diabetes in Syngeneic Islet Grafts
Having established that a short course of depleting anti-CD4 mAb therapy successfully cures new-onset diabetes in NOD mice, we questioned its efficacy in another clinically relevant model, that is, allogeneic islet transplantation into diabetic autoimmune recipients. Diabetic NOD mice received transplantations of 700 C57BL/6 islets under the kidney capsule and were treated with GK1.5 starting day 0 of the transplant as described in Materials and Methods. CD4+ T-cell depletion was complete at the end of the treatment with a subsequent normalization of the CD4+ T-cell numbers 3 to 4 weeks after cessation of the treatment (data not shown). The anti-CD4 mAb treatment led to a significant prolongation of islet allograft survival (MST=37±6 days, n=4) but without achieving any long-term survival (Fig. 3A). This result is in agreement with the recent data of Guo et al. (3) demonstrating similar results using a nondepleting anti-CD4 mAb. In contrast with allogeneic islet allografts, a short-term anti-CD4 mAb therapy (treatment given on days 0, 1, and 2, and then every other day until day 21) prevented the recurrence of diabetes in NOD/SCID islet grafts (Fig. 3B). Female NOD recipients of NOD/SCID islet grafts remained euglycemic during the study period (>75 days, n=4).
Treatment with Combined Costimulation Blockade Prevents the Progression to Hyperglycemia in Prediabetic Female Nonobese Diabetic Mice with Peri-Insulitis but Does Not Affect the Progression of Early-Onset Diabetes
We then checked whether combined CTLA4Ig and anti-CD154 mAb (MR1) would affect the incidence of diabetes at an earlier stage of disease progression, that is, at the prediabetic, peri-insulitic stage (1). Eight-week-old, prediabetic, female NOD mice were treated with CTLA4Ig and anti-CD154 (see Materials and Methods) and subsequently monitored for occurrence of hyperglycemia (n=13). Our results demonstrated that by 28 weeks of age, 100% of female NOD mice were normoglycemic, whereas more than 60% of non-treated female NOD littermates (n=10) developed diabetes as evaluated by blood glucose monitoring (Fig. 4A).
To discriminate whether this protective effect was related to a combined effect of CTLA4Ig and anti-CD154 mAb or could be achieved by either of the two, CTLA4Ig and anti-CD154 mAb were separately administered (four doses during a 6-day period) to prediabetic, 8-week-old, female NOD mice (n=6 per group). Mice were then followed for 20 additional weeks and compared with a group of nontreated control animals (n=10). Our results demonstrated that 100% of NOD mice receiving anti-CD154 mAb alone and 83% of mice receiving CTLA4Ig alone were protected from diabetes up to 28 weeks of age and remained euglycemic during the follow-up period (Fig. 4B and C). In contrast, 80% of control, non-treated, female NOD littermates had developed diabetes at that time. This result indicates that most of the beneficial effect of the combined costimulation blockade regimen could be achieved by the single use of anti-CD154 mAb alone.
To determine the impact of costimulation blockade on the incidence of insulitis, three 28-week-old, cured, female NOD mice treated with CTLA4Ig and anti-CD154 mAb or anti-CD154 mAb alone were killed, and their pancreata were evaluated for pathology and immunohistochemistry. Immunostaining with insulin showed small and scattered islets comprising well-granulated insulin positive cells in both the CTLA4Ig+anti-CD154 mAb–treated mice and the anti-CD154 mAb–treated mice (Fig. 5). No significant mononuclear cell infiltrate was detected. Normal-sized, insulin-positive, well-granulated islets were detected in nondiabetic, age-matched controls (Fig. 5). These data indicate that costimulation blockade at an early prediabetic stage of the disease leads to the disappearance of the T-cell infiltrate from the pancreata of cured animals.
We next evaluated the effect of combined costimulation blockade on new-onset diabetes. Female NOD mice with new-onset diabetes were treated with CTLA4Ig and anti-CD154 mAb (see Materials and Methods) (n=5). In contrast with anti-CD4 mAb, combined costimulation blockade had no effect on the progression of the disease once established. All mice with new-onset diabetes remained hyperglycemic and progressively weakened because of poor metabolic control (Fig. 6A). This result agrees with previous data obtained in NOD/Lt mice and was highly predictable, indicating that, once started, the destructive T-cell process is independent of costimulation (25).
This study defines a new, clinically applicable immunosuppressive regimen that would reverse early-onset diabetes and prevent established autoimmunity from recurring in islet grafts. We investigated the effect of a short-term depleting anti-CD4 mAb therapy or costimulation blockade, administered once diabetes has been diagnosed, on disease progression. We chose these therapies on the basis of their successful use in experimental studies for the prevention of autoimmune diabetes and treatment of allograft rejection and their relatively low toxicity (2, 4, 9, 22). Preventive therapies in rodent models are typically administered at a pre-insulitis or early insulitis stage of the disease, a time point that is difficult to target in humans. In our studies, we focused on therapies targeting new-onset diabetes, which could more easily translate to clinical practice.
Our results are encouraging. We demonstrate, for the first time, that a short-term treatment with a depleting anti-CD4 mAb of diabetic female NOD mice, at a time when the autoimmune attack of the β-cells has already been initiated, cured the disease. Euglycemia was permanently (>100 days) restored. However, we found that the timing of initiation of the therapy was critical. Diabetes was not cured if anti-CD4 mAb was administered later than 5 or 6 days after the onset of hyperglycemia, a finding consistent with earlier data (20). This may be related to the absence of viable β-cells that can be rescued at this point given the rapid destructive process in the NOD mouse model. Alternatively, the immune response might have evolved at this stage to be independent of CD4+T cells or other CD4+ cells. In humans, the destruction of β-cells during the course of insulin-dependent diabetes mellitus is a slower process evolving over many months or years (26). Indeed, a “honeymoon period” often follows the initial diagnosis of diabetes. This period represents an ideal therapeutic window to intervene in the disease process.
Although the therapeutic effect of GK1.5 on the cure of early-onset diabetes and recurrence of the disease in islet isografts is likely related to depletion of the effector CD4+ T-cell population, one cannot rule out an effect of this antibody on other CD4+ cell populations that may take part in the disease process. These include monocytes and macrophages, dendritic cells, and eosinophils. The implication of anti-CD4–mediated depletion of these cell populations in curing diabetes remains to be addressed.
Our analysis of pancreata from euglycemic anti-CD4–treated NOD mice demonstrated intact β-cells surrounded by both CD4+ and CD8+ T cells resembling the peri-insulitic infiltrate observed in young NOD mice. These data raise a number of interesting questions. They indicate that once auto-reactive CD4+ T cells are depleted from the periphery, newly generated CD4+ T cells home back to the islets but are not destructive. This could relate to a specific loss of diabetogenic clones, the absence of costimulatory signals required for the progression of these infiltrating T cells into cytotoxic effectors, or the change in the phenotype of these cells in becoming regulatory T cells (27, 28). These hypotheses warrant further investigations, including evaluation of the phenotype of the intrapancreatic T cells (e.g., CD25 and cytokine profile) and analysis of precursor frequency against alloantigens and autoantigens such as glutamic acid decarboxylase (GAD) and insulin in anti-CD4–treated NOD mice compared with newly diabetic NOD mice. On the basis of our pilot adoptive transfer studies, we do not believe that the induction of “regulatory” clones is involved in the cure from diabetes afforded by anti-CD4 mAb, although we recognize that additional studies are needed to confirm that.
The use of depleting anti-CD4 mAb in diabetic NOD mice receiving allogeneic islets prolonged graft survival up to 35 days but never resulted in long-term graft acceptance, whereas it protected NOD/SCID syngeneic islet grafts from diabetes recurrence. These data reproduce that observed by Guo et al. (3), who used a nondepleting anti-CD4 mAb, and to some extent data by Stegall et al. (29), who used a lighter GK1.5 regimen. They indicate that anti-CD4 therapies are more effective in protection from autoreactive T cells than from alloreactive T cells. Treatment of new-onset diabetes with costimulation blockade was not able to prevent disease progression, a finding that confirms earlier data in islet isografts in NOD mice (25). However, costimulation blockade could prevent disease development when administered to prediabetic NOD mice reproducing data previously published by other groups (2, 4). Histologic analysis of cured mice pancreata showed the absence of a significant peri-insulitic infiltrate. However, the architecture of the islets was altered in that the islets appeared scattered and smaller than usual (18, 30).
Earlier studies in our group have shown that short-term, combined costimulation blockade prolonged islet allograft survival for up to 8 weeks in diabetic NOD mice (25). We investigated whether simultaneous inhibition of allograft rejection (using costimulation blockade) and autoimmunity (using anti-CD4 mAb) leads to long-term survival of islet allografts in a diabetic autoimmune environment. Islets from C57BL/6 mice were transplanted into diabetic NOD mice. Recipients were treated with short-term anti-CD4 mAb and combined costimulation blockade starting the day of transplantation. Survival of islet allografts was prolonged, but all islet grafts were ultimately rejected at 40±3.6 days (n=4; data not shown). This MST was not significantly different from that achieved by islet allografts treated with anti-CD4 mAb alone (MST of 36±6; n=4). Taken together, these results confirm the relative resistance of NOD mice compared with other mice strains in achieving tolerance of allogeneic antigens (25). We surmise that an additional immunosuppressive regimen such as rapamycin may be required to achieve long-term survival of islet allografts in an autoimmune recipient. These experiments are currently under way.
Our data provide the first evidence that a short course of CD4+ T-cell–depleting antibody cured all diabetic NOD mice when it was administered early after disease onset. This result is relevant to clinical practice because it provides a potentially safe alternative therapy for patients with new-onset diabetes during the “honeymoon period.” Furthermore, an immunosuppressive regimen combining anti-CD4 mAb may be beneficial in preventing autoimmune recurrence in islet allografts.
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