The pathogenesis of type 1 diabetes in both humans and NOD mice appears dependent upon an aberrant immune response that results in the destruction of insulin-producing β-cells. While prevention of type 1 diabetes in NOD mice can be accomplished through a wide variety of monotherapies, reversal of overt disease has considerably fewer reported efficacious therapies (30
). Of those that do show success, many combine two or more therapeutic agents to achieve this reversal (32
). Indeed, one of the earliest demonstrations for the ability of combination therapy to reverse hyperglycemia in NOD mice utilized a somewhat similar form of murine ATG, anti-lymphocyte serum, in combination with exendin-4, to effectively reverse disease in this animal model of type 1 diabetes (14
). Consequently, herein we have described an approach using two clinically relevant therapies, ATG and GCSF, for the purpose of immunomodulation that would provide benefit in terms of reversing type 1 diabetes, as demonstrated in the NOD mouse. Aside from the ability for combination therapy to provide improved reversal rates, we also questioned whether this combination would improve disease reversal in animals that would not be subject to disease remission were they provided monotherapy.
The observed enhancement of murine ATG's ability to reverse new-onset NOD mice with greater starting blood glucose levels when used in combination with GCSF not only demonstrated this latter notion, but it also likely reflects the ability of combination therapy to induce remissions in mice with greater loss in β-cells than possible with monotherapy; although this hypothesis is subject to debate (34
). The finding is especially important as previous studies using similar immune-depleting agents as monotherapy (e.g., anti-CD3 monoclonal antibody) note a diminished ability to reverse type 1 diabetes in NOD mice in this metabolic range (i.e., ≥350 mg/dl) (8
). It is conceivable that monotherapies such as murine ATG or anti-CD3 monoclonal antibody may induce immunoregulation yet still fail to remit diabetes due to a profound loss of β-cell mass prior to the induction of the therapeutic regimen. Future studies would be well served to measure C-peptide in response to glucose challenge at the onset of therapy, as well as to transplant islets into mice that fail to respond to therapy. This will help to address the impact of starting β-cell mass upon the efficacy of these therapies.
While several reports demonstrate an ability to induce euglycemia in new-onset NOD mice, there often remains some doubt regarding the long-term robustness of these therapies. In our reversal trial using suboptimal murine ATG in combination with GCSF, we attempted to alleviate this concern by performing an IPGTT time course study. We demonstrated that beginning at 60 days, by which time all therapy has ceased, and continuing out to 120 days postonset, the quality of glucose control significantly increases as measured by area-under-the-curve analysis. The exact reason for this improvement is uncertain but possibly due to the recovery of endogenous β-cells (35
). Previous reports (8
) have indicated that the efficacy of reversal therapies hinges upon the recovery of these cells rather than the generation of new β-cells. A time course analysis of the pancreas in future reversal studies may address this hypothesis.
In our pre-diabetic study, the greatest percentage of regulatory T-cells was observed in mice receiving combination therapy. This is not surprising given that both murine ATG and GCSF individually have been shown to induce a population of regulatory T-cells (16
). The fact that by combining the two therapies results in a greater percentage of regulatory T-cells after 8 weeks of therapy than either monotherapy lends additional support to the use of combination therapy. Further studies, such as the adoptive cotransfer of these regulatory T-cell populations with effector T-cells into NOD.SCID mice, may be warranted to more explicitly demonstrate their suppressive potential. In addition, future efforts must expand on the effects of this therapy on regulatory T-cell populations in anatomic compartments beyond the spleen, such as the pancreatic lymph nodes and the islet infiltrate.
The presence of anti-GCSF IgG1 and IgM antibodies may explain the reduction in macrophages and neutrophils after 2 weeks of GCSF therapy. It is also possible that a reduction in GCSFR mRNA (supplemental Table 1) may also play a role (38
). This response is not surprising given that the recombination GCSF is a human protein and, consequently, is recognized as foreign in the treated mice (41
). In spite of this apparent neutralization, the combination therapy of murine ATG and GCSF remained viable for both reversal of overt disease and for maintaining the health of islets when administered to pre-diabetic NOD mice. If this immune response against the GCSF could be overcome, it is conceivable that the efficacy of this treatment would be enhanced.
The apparent lack of β-cell durability in the murine ATG-treated pre-diabetic mice reflects a similar finding in a previous report in which 12-week-old pre-diabetic NOD mice exhibited only transient protection following anti-CD3 monoclonal antibody therapy (17
). The transient protection seen with GCSF monotherapy group reflects the reversal study (A
) in which GCSF only led to a delayed return to hyperglycemia compared with control-treated mice. By combining these two monotherapies, however, the health of the islets was maintained relative to control as measured by insulitis scoring and β-cell area.
These results indicate that combined treatment of murine ATG with GCSF offers a highly effective means for reversal of type 1 diabetes in NOD mice. This combination therapy provides for a series of beneficial mechanistic actions (e.g., increased regulatory T-cell frequency, reduced islet inflammation, improved β-cell area, etc.) and dramatically extends the range of β-cell dysfunction allowable for effective and durable disease remission. These studies also provide support for the performance of human type 1 diabetes trials with this combination of agents and suggest that this form of therapy may be amenable to treatment of other autoimmune disorders.
With that notion, what has been attempted with ATG in humans that might provide support for this potential application? Studies involving human transplantation and treatment of autoimmunity do, in fact, suggest that that ATG provides therapeutic benefit that may involve tolerance. Transplant recipients have seen successful management with ATG induction therapy followed only by limited maintenance immunosuppression by tacrolimus (42
), while ATG has also been used successfully in the treatment of refractory systemic autoimmune diseases such as systemic lupus erythematosus, progressive systemic sclerosis, and rheumatoid arthritis (43
). There has also been promise for the efficacy of ATG in the treatment of type 1 diabetes. Early studies of equine ATG in combination with prednisone in new-onset type 1 diabetic patients indicated a prolongation of the honeymoon phase (44
). As far as more contemporary efforts, in a randomized, placebo-controlled, single-blinded trial with RATG (ATG-Fresenius; Hoechst Marion Roussel, Frankfurt, Germany), type 1 diabetic participants aged 18–35 years received a total dose of 18 mg/kg of ATG, which was administered in four infusions. Of 17 study participants, 11 received the drug and 6 received placebo. Increased glucagon-stimulated C-peptide levels, a lower insulin requirement, and lower glycosylated hemoglobin levels were observed in the ATG group, but not in the placebo group, 12 months into the study (45
). Perhaps most promising were two ATG-treated subjects that achieved disease remission (i.e., no exogenous insulin for at least 1 month and a fasting glycemia <126 mg/dl). A pilot study is currently underway in humans with new-onset type 1 diabetes, funded by the Immune Tolerance Network, that seeks to determine whether ATG will preserve C-peptide. This study will test the notion that selective depletion of lymphocytes will reset the immunologic rheostat, induce dynamic immune regulation, and potentially induce and maintain tolerance in type 1 diabetes. Since this study will also help establish safety data for the use of ATG in humans in type 1 diabetes, the background adverse-event rate will be established in this population, allowing for the study of combination therapies including ATG and additional tolerance-inducing agents such as GCSF. With time, the equipoise for utilizing agents having the potential for imparting deleterious side effects must be carefully weighed against the benefits of preservation of C-peptide and/or insulin independence for those with type 1 diabetes. The answer to this equation is not simple to address. Clearly, additional research is required with this particular application, as well as others, to establish the parameters for safe and efficacious translation of therapies from mouse to humans.