Type 1 diabetes (T1D) is one of the most common autoimmune diseases, affecting almost 20 million people worldwide. During pathogenesis, insulin-producing pancreatic β cells are progressively destroyed by autoreactive CD4+
T cells. A destruction of approximately 80% of β cells occurs before type 1 patients become symptomatic. Importantly, insulin has been shown to be a major autoantigen in NOD mice as well as in humans (1
). In the past 2 decades, immunomodulatory approaches to prevent or cure T1D have been developed and tested, with some encouraging recent results. Development of a cure for T1D has been particularly difficult, because insulin substitution affords a reasonable life quality and expectancy, the disease frequently affects young adults and children, and therefore, the ethical window for any treatment is rather small, and long-term side effects have to be avoided. Thus, the risk-benefit ratio for future clinical trials has to be carefully weighed. On the other hand, insulin cannot prevent all of the late complications of diabetes, and life expectancy can be reduced by 10 to 15 years due to serious complications including retinopathy, nephropathy, cardiovascular diseases, or neuropathy (3
It is known that systemic immunosuppression, for example with cyclosporin, can halt β cell destruction (4
). However, the protection only lasted as long as the drug was present; long-term immunological tolerance to β cell antigens was not achieved, and extended therapy was not feasible due to side effects. In contrast, one much more promising intervention tested clinically during the past 5 years is the application of non–Fc-binding anti-CD3ε Ab, engineered as an F(ab′)2
fragment of hamster anti-CD3ε (145-2C11) for preclinical studies (5
) or as a fully humanized IgG1 (hOKT3γ1[Ala-Ala]) for human trials (6
). Although, its mechanism of action is not fully known yet, a decrease in the number of autoaggressive T cells together with an expansion of a CD4+
Treg population expressing the α-chain of the IL-2 receptor (CD25) relying on TGF-β have both been demonstrated following short-course treatment with anti-CD3 in NOD mice (7
). Thus, the increase in the number of such Tregs might explain the long-term protection observed in mice treated with anti-CD3 after recent onset, as well as the slowing of the progressive decline in C-peptide levels over an 18-month period following short-course treatment in 2 independent trials in humans (8
). However, the level of C-peptide began to decline after 18 months, indicating that permanent tolerance to β cell antigens had not been achieved. Therefore, efficacy needs to be enhanced. Safety concerns will prevent us from increasing the human anti-CD3 dose, since temporary EBV reactivation was seen in most individuals in the recent European anti-CD3 trial, and other options will need to be explored. One promising avenue is the β cell antigen–specific induction of Tregs.
In the 1990s, several groups including ours reported that immunization with islet autoantigens by various means and routes can induce islet antigen–specific Tregs and prevent T1D (10
). Those autoreactive Tregs can act as bystander suppressors and suppress site-specifically heterologous autoreactive immune responses (16
). For example, transferred insulin B-chain–induced (insB-induced) Tregs selectively proliferated in the pancreatic draining LNs (PLNs), where their cognate antigen is being presented by APCs during development of diabetes. There, they were capable of dampening autoaggressive CD8 responses (16
). This suppressive effect was associated with IL-4 and IL-10 production by the Tregs. Thus, antigen-specific induction of Tregs can result in long-lasting tolerance to β cell antigens mediated by local immune modulation in the PLNs, which makes this intervention safe, with low potential for side effects. However, from many tests in animal models, we know that the efficacy is limited, because prevention of T1D is only seen when the immunization is given during the prediabetic phase. Therefore, antigen-specific interventions will need “help” to be used successfully in humans, especially in recent-onset diabetics (17
We hypothesized that anti-CD3 would create a systemic immunomodulatory milieu to facilitate the islet antigen–specific induction of Tregs. As described previously, anti-CD3 treatment induces a shift in the cytokine profile (mainly from Th1 toward Th2) as well as an expansion of T cells with regulatory properties in mice (7
) and in humans (6
). Thus, one could envision that upon immunization with islet autoantigen, a combination therapy with anti-CD3 will expand islet-specific Tregs more forcefully. In addition, depletion of autoaggressive T cells following anti-CD3 administration would allow us to create a suitable window in recent-onset diabetes that would give newly activated Tregs enough time to proliferate and traffic to the PLNs to suppress further generation of autoaggressive cells.
To test this idea, we administered islet antigens and peptides through various routes in conjunction with anti-CD3. Here we report that a peptide derived from the human proinsulin II exhibited the best synergy and strongly enhanced Treg induction. Efficacy of this combination treatment was found in 2 animal models, and mechanistic analyses are presented herein.