In this study, we demonstrated that T1D progression in the NOD mouse was associated with a progressive loss of Treg:Teff balance in the inflamed islets, but not in the PLN. Intra-islet Treg cells expressed reduced amount of CD25 and Bcl-2 relative to the Treg cells in the PLN, suggesting that the Treg:Teff imbalance was due a defect in intra-islet Treg survival. We further demonstrated that IL-2 treatment of NOD mice restored CD25 expression on intra-islet Treg cells and led to diabetes prevention.
It is well established that Treg cells control the progression of T1D in the NOD mouse model (Chen et al., 2005
; Salomon et al., 2000
). However, the precise temporal and anatomical basis of Treg cell control is not clear, especially in regards to the relevant role of the PLN versus inflamed islets as the major site of immune regulation (Bour-Jordan et al., 2004
; Chen et al., 2005
; Tang and Bluestone, 2006
). In this study, we demonstrate that NOD Treg cells mount an appropriate response to tissue destruction in the PLN by expanding, acquiring an activated phenotype, and infiltrating inflamed islets. This attempt to “control” disease was also manifested within the islets, as a high fraction of the Treg cells continue to divide within the pancreatic islet tissue, suggesting that further activation occurred at the site of inflammation. In fact, quantitative in vivo
analysis of the Treg:Teff balance in individual islets revealed a high frequency of Treg cells in early islet infiltration in young prediabetic mice similar to what has been observed in other settings including models of tumor, infectious disease and other autoimmune diseases (Aluvihare and Betz, 2006
; Belkaid et al., 2006
; Munn and Mellor, 2006
; Rouse et al., 2006
; Sakaguchi et al., 2006
; Waldmann et al., 2006
). However, in NOD mice, Treg cells failed to survive in the tissue long term likely due to the limited availability of IL-2.
The importance of IL-2 in the maintenance of Treg cell homeostasis and suppression of T1D has been suggested by IL-2 neutralization studies (Setoguchi et al., 2005
). Genetic mapping studies have demonstrated the NOD Idd3
allele contributes to IL-2 defect in the NOD mice (Denny et al., 1997
). More recently, an elegant study further demonstrated the NOD idd3
allele led to systemic reduction in Treg frequency and higher mortality due to diabetes in a CD8+
T cell receptor transgenic NOD model (Yamanouchi et al., 2007
). Our findings in this study further illustrate that in untreated NOD mice, there is a selective defect in Treg cell survival in inflamed tissues. The survival defect was observed in mice as young as 6 weeks of age, supporting the notion that this phenotype is most likely genetically encoded. The reduced expression of IL-2 and IL-2-regulated genes such as CD25 and Bcl-2 on Treg cells suggests that the Treg cell survival defect was secondary to a IL-2 deficiency. The local inflammatory milieu in the islets may exacerbate the IL-2 shortage by further inhibiting IL-2 expression (Villarino et al., 2007
), competing for IL-2 by activated Teff cells, and cleaving of CD25 by matrix metalloproteases induced by local inflammation (Sheu et al., 2001
). Thus, the genetically-encoded inborn IL-2 deficiency in the NOD mice may be more pronounced in inflamed tissues compromising Treg cell survival locally. Common γ chain binding cytokines such as IL-4, IL-7, and IL-15 can help to sustain Treg cell survival in vitro
through provision of anti-apoptosis signals (Pandiyan et al., 2007
). It is thus possible that deficiency in these cytokines may also contribute to the demise of Treg cells in the inflamed islets. Finally, it should be noted that the deficiency in Treg survival may be compounded by an independent genetic defect in Bcl-2 expression in NOD mice (Garchon et al., 1994
). In fact, over-expression of Bcl-2 in T cells and B cells alleviated insulitis and conferred diabetes protection (Rietz et al., 2003
Similar to intra-islet Treg cells, Treg cells isolated from inflamed lachrymal and salivary glands in the NOD mice also expressed markedly reduced CD25. Several published reports in various disease models demonstrated that tissue infiltrating Treg cells could be readily identified by their surface expression of CD25 and the cells were functional in controlling local inflammation (Belkaid et al., 2002
; Yu et al., 2005
). Thus, loss of CD25 is not a general characteristic of Treg cells in inflamed tissues. It remains to be determined whether the local IL-2 deficiency and Treg cell imbalance observed in this study were restricted to sites of autoimmune inflammation or reflected a more general defect of Treg cells in the NOD mouse. Examining CD25 expression on Treg cells in other inflammatory settings in the NOD mice such as microbial infection would help to clarify this issue.
The polarized effect of high and low dose IL-2 therapy on autoimmune response observed in this study is striking and highlights the pleiotropic effect of this cytokine. Although IL-2 has an indispensable role in Treg cell homeostasis, it was originally discovered as a T cell growth factor and activator of cytotoxic lymphocytes (Taniguchi et al., 1983
). IL-2 has been used in the clinic since mid-1980s as an immune-boosting cancer therapy with limited success. Part of the limitations in IL-2 cancer therapy be due to the expansion of Treg cells (Wei et al., 2007
). We observed that a regimen of multiple, low dose injections IL-2 favored Treg over Teff cells while a high dose regimen led to rapid Teff expansion and disease onset. In light of this result, it is interesting to point out that a high bolus dose of IL-2 was more efficacious than low dose in treating renal cell carcinomas (Fisher et al., 2000
; Yang et al., 2003
). Together, the results suggest that the in vivo
effect of IL-2 can vary widely depending on the dosing regimen, amount of endogenous IL-2, and the numbers of activated CD4 and CD8 Teff cells, NK cells, and Treg cells in the host. Thus, optimal IL-2 treatment regimen may be difficult to predict for a heterogeneous patient population and adjunct therapy will be needed to ensure desired outcome. For example, combining IL-2 with Teff cell-depleting treatments such as anti-CD3 or Rapamycin may prevent potential disease exacerbation and help to restore long-term self-tolerance in an autoimmune setting (Chatenoud, 2003
; Rabinovitch et al., 2002
). In contrast, in cancer setting, IL-2 treatment in conjunction with Treg cell depletion may be more effective than IL-2 monotherapy in inducing tumor regression.
Three independent studies in the NOD mice demonstrated that diabetes progression is associated with the acquisition of regulation resistance by Teff cells over time (Gregori et al., 2003
; Pop et al., 2005
; You et al., 2005
). Our results extend these findings suggesting that the development of diabetes in the face of increasing frequencies of Treg cells may reflect this increased Teff cell resistance. The protracted disease course in the NOD mice may reflect the time needed for regulation-resistant Teff cells to emerge and accumulate to sufficient numbers under the constant control of Treg cells. Furthermore, the regulation-resistant phenotype may be linked to reduced IL-2 production by these Teff cells. We previously demonstrated that Treg cells expanded in the presence of strong co-stimulation through CD28 and large doses of IL-2 can effectively prevent and even reverse diabetes (Tang et al., 2004
). These Treg cells survive long-term (greater than 50 days) in recipient mice and are less dependent on B7 co-stimulation and IL-2 from the hosts (QT and JAB unpublished observations). Thus, the heightened regulation resistance and unfavorable survival environment for Treg cells found in the NOD mice can be overcome by Treg cell therapies if appropriate Treg cell preparative regimens are used.
Immune deficiency is often associated with autoimmunity in mice and man (Arkwright et al., 2002
; Dupuis-Girod et al., 2003
; Horak et al., 1995
; Mombaerts et al., 1993
), and in the NOD mouse, immune stimulation can protect mice against diabetes (Qin and Singh, 1997
; Sharif et al., 2001
). Our findings provide one possible explanation for these paradoxical observations suggesting that normal Treg cell homeostasis and a healthy balanced immune system depend ultimately on a robust Teff cell response. IL-2 production by activated Teff cells expand and sustain Treg cells, which in turn feed back to suppress the Teff cell response and maintain normal immune homeostasis. Disruption of this cross-talk can lead to the dysregulation of the Treg:Teff balance and contribute to the development of autoimmune diseases in the NOD mice. The effective control of diabetes with low dose IL-2 treatment leads to an intriguing suggestion that, in some instances, immune stimulation rather than immunosuppression may be an effective approach for the treatment of autoimmune diseases.