It has become increasingly clear that a delicate balance of pathogenic and protective immune responses controls the development and clinical manifestation of type I diabetes. Although CD28-B7 and CD40L-CD40 interactions have been long recognized as the two major costimulatory pathways of T cell responses, the potential control of immunoregulatory mechanisms by costimulatory molecules is only starting to be unraveled. In this study, we showed that B7-2 was necessary for the optimal activation of diabetogenic T cells in the pancreatic LNs but not for the development of a functional Treg compartment. In addition, affecting the Treg compartment by complete disruption of CD28 signals or depletion of CD4+
T cells restored pathogenic T cell activation and diabetes. Finally, Treg’s were reduced and insulitis and diabetes were restored in NOD-CD40L–/–
mice as compared with NOD-CD40L–/–
mice. These results suggest that CD28–B7-2 and CD40L-CD40 interactions play a predominant role in diabetogenic T cell activation, whereas complete blockade of CD28 costimulation severely impacts the Treg compartment. Thus, a deficient Treg compartment circumvents the need for the two major costimulatory pathways to achieve autoimmunity against pancreatic islets. In this setting, costimulation requirements could be modified by chronic presentation of autoantigen (29
). Alternatively, costimulatory molecules such as inducible costimulator (ICOS), OX40 (CD134), or other as-yet-unidentified costimulatory molecules could play a role in T cell activation in the absence of CD28 and CD40L. In sum, diabetes is controlled by a balance of pathogenic and regulatory T cells, and although costimulatory signals regulate both arms of the balance, CD28-dependent Treg’s appear to be the dominant factor in the development of the autoimmune process (Figure ).
Figure 6 Autoimmune diabetes is controlled by a costimulation-dependent balance of pathogenic and regulatory T cells. In NOD mice, which are genetically prone to autoimmunity, autoreactive T cells dominate Treg’s, leading to development of autoimmune diabetes. (more ...)
The number of Treg’s in NOD–B7-2–/–
mice was slightly reduced as compared with NOD mice, and blocking B7-1 interactions with CD28 in NOD–B7-2–/–
mice decreased the number of Treg’s to levels similar to those in NOD-CD28–/–
mice. These results suggest that B7-1 and B7-2 additively signal through CD28 to maintain Treg homeostasis and that B7-1 is sufficient to maintain a functional Treg compartment in NOD mice. In addition, the development of diabetes in NOD–B7-2–/–
mice treated with anti–B7-1 mAb’s demonstrates that low levels of constitutive B7 expression play a prominent role in maintaining peripheral tolerance and preventing autoimmunity. This result confirms our previous report that blocking the CD28-B7 pathway in NOD breaks peripheral tolerance and results in exacerbated diabetes (14
). Recently, Lohr et al. showed that basal B7 expression on APCs reduced T cell responses in vivo and that CD4+
Treg’s were responsible for this effect (30
). In addition, low levels of B7-1 expression in the pancreatic LNs compared with the spleen of NOD-B7-2–/–
mice suggest that CD28 signals provided in other tissues is enough to maintain a functional Treg compartment in the LNs.
An alternative explanation for the pro-diabetogenic effects of blocking B7-1 in NOD–B7-2–/–
mice is that this treatment prevents CTLA-4 ligation and results in increased activation of autoreactive T cells. However, treatment of NOD–B7-2–/–
mice with anti–CTLA-4 failed to reproduce the increase in diabetes secondary to anti–B7-1 blockade, which suggests that the effects of this treatment are not limited to disruption of CTLA-4 engagement. Moreover, CTLA-4 has been shown to be most effective in controlling secondary rather than naive responses (31
) that are clearly defective in this model. In this regard, our data showed that anti–B7-1 treatment was mainly effective when administered early in disease development (at 2–4 weeks of age; i.e., before the development of insulitis), whereas Mathis and colleagues demonstrated in the NOD-BDC2.5 TCR Tg model that CTLA-4 controlled autoimmune diabetes principally after insulitis was established (32
). Finally, NOD mice deficient in CD40L, B7-1, and B7-2 do not develop exacerbated disease as compared with NOD-CD40L–/–
mice (data not shown), which suggests that additional disruption of CTLA-4 did not dramatically affect the balance of activation and regulation in that setting. In this regard, it has been suggested previously that B7 molecules could costimulate T cell responses by interacting with ligands other than CD28 and CTLA-4 on T cells (33
), and this could explain the increased autoimmune response in mice deficient for CD28 and CD40L compared with mice lacking CD40L and both B7-1 and B7-2.
Although it is well established that Treg’s suppress effector T cell proliferation in vitro, the mechanism of suppression by Treg’s in vivo is still controversial (30
). Our results suggest that Treg’s control diabetogenic T cells in NOD mice by suppressing their initial islet antigen–specific activation and expansion in the pancreatic LNs and thus before they could reach later stages of differentiation or effector function. This is in agreement with several studies showing suppression of proliferation by Treg’s in vitro and in vivo (34
). However, this result is in contrast to recent reports suggesting that Treg’s do not affect proliferation but rather production of IFN-γ in vivo (30
). In addition, Herman and colleagues recently showed that CD4+
T cells expressed the Treg-specific transcription factor Foxp3 and regulated diabetogenic T cells directly in the pancreas (40
). This discrepancy could be explained by the different experimental systems used. In spontaneous autoimmune models, low-affinity autoreactive T cells are activated by their natural autoantigen in the pancreatic LNs. This is in contrast with other TCR Tg models, where higher-precursor-frequency and high-affinity T cells are examined or where activation is maximized by dendritic cells loaded with foreign antigen (30
). Thus, our results suggest that Treg’s may suppress primary T cell activation in vivo, especially under conditions of low-avidity TCR interactions due to low-affinity T cell receptors and/or chronic presentation of low levels of antigens such as autoantigens.
Depletion of CD4+
Treg’s partially restored the proliferation of islet antigen–specific T cells in the pancreatic LNs of NOD–B7-2–/–
mice and promoted diabetes in an adoptive transfer setting. Similarly, the defect in Treg’s in NOD-CD28–/–
mice resulted in accelerated disease and increased number and expansion of autoreactive T cells in the pancreatic LNs compared with that in NOD mice. Finally, whereas it is known that NOD-CD40L–/–
mice fail to develop diabetes due to inefficient activation of autoreactive T cells (7
), our data showed that insulitis and diabetes were partially restored in NOD-CD40L–/–
mice as a result of decreased Treg numbers. Although it has been shown that the CD40L-CD40 interaction plays a role in Treg development (41
), our results strongly suggest that the Treg compartment in NOD mice is principally controlled by the CD28-B7 pathway. This is in agreement with the recent report that disruption of CD40L-CD40 interactions affected helper T cells but not Treg’s in NOD mice (42
). Moreover, enhanced autoimmunity in NOD-CD40L–/–
mice compared with NOD-CD40L–/–
mice reveals that CD28 and CD40L control autoimmunity via distinct pathways. This is in contrast to the hypothesis that defective CD40 ligation affects T cell responses indirectly due to impaired upregulation of B7 molecules leading to insufficient CD28 costimulation (10
). The observation that depletion of CD4+
Treg’s only partially restored the proliferation of BDC2.5 T cells and/or the development of diabetes could be due to limiting costimulation leading to reduced activation of pathogenic T cells. Similarly, the absence of BDC2.5 cell proliferation following anti–B7-1 treatment in adult NOD–B7-2–/–
mice could reflect the absence of CD28 costimulation, resulting in delayed T cell activation that could not take place within the 2-week period of our experiment. Alternatively, other Treg populations such as NKT cells (43
) and DX5+
T cells (45
) have been shown to protect NOD mice from diabetes and could be playing a regulatory role in the absence of CD4+
Treg’s. Finally, it is possible that costimulation-regulated Treg’s could function in an age-dependent manner in NOD mice, which would thus explain the differential efficiency of B7-1 blockade observed in 2- to 4-week-old versus adult mice.
Taken together, our results have several important implications. First, the development of diabetes in NOD mice deficient for both CD40L and CD28 demonstrates that Treg’s play a dominant role in the balance of autoreactivity versus regulation that determines the outcome of disease. Second, in the absence of a functional Treg compartment, the two major costimulatory pathways can be bypassed for pathogenic T cell activation in individuals prone to autoimmunity. Third, while both CD28–B7-2 and CD40L-CD40 interactions controlled the activation of pathogenic T cells, constitutive levels of CD28-B7 interactions played a unique role in maintaining an efficient Treg compartment. Therapeutic approaches could thus be designed to affect selectively CD28–B7-2 and CD40L-CD40 interactions while maintaining a residual level of CD28 costimulation in order not to eliminate Treg’s. In addition, it will be essential to establish a careful monitoring of Treg’s when patients are treated with drugs blocking costimulatory pathways. In conclusion, these results have important consequences for the design of immunotherapy aimed at selectively suppressing autoimmunity and suggest that it will be essential to maximize immune regulation in addition to targeting autoreactive T cells in autoimmune-prone individuals.