Here we demonstrate that both thymic Treg cell development and their peripheral homeostasis effectively occur by IL-2Rs that harbor mutations of key tyrosine residues in the cytoplasmic tail of IL-2Rβ that are required for optimal signal transduction. Mutation of all three of these tyrosine residues still led to weak transient STAT5 activation, indicating that IL-2R signaling was not completely abrogated. Thus, these data demonstrate that key IL-2-dependent targets in Treg cells are index to a low IL-2R signaling threshold. One of these targets is likely Foxp3 because Foxp3 expression depends in part on STAT5 activation (Burchill et al., 2007
; Yao et al., 2007
; Zorn et al., 2006
) and we show that Foxp3 was upregulated to normal levels in thymic and peripheral Treg cells bearing mutant IL-2Rβ chains.
The indexing of Treg cells to a low IL-2R signaling threshold provides an explanation concerning how Treg cells effectively utilize an essential cytokine that is transiently and perhaps minimally expressed. For example, there are very few IL-2 producing cells in the thymus and such cells are not abundant in peripheral immune tissues (D’Souza and Lefrancois, 2004
; Sojka et al., 2004
; Yang-Snyder and Rothenberg, 1998
). More specifically to Treg cells, one of their suppressive mechanisms is to inhibit IL-2 production by autoreactive or effector T cells (Thornton and Shevach, 2000
). Thus, Treg cells likely exist in an IL-2 poor environment, yet IL-2 is essential for thymic maturation and subsequent homeostasis in the periphery. Treg cells, therefore, appear to adapt to low IL-2 through fully inducing and maintaining Foxp3 by a minimal requirement for IL-2-dependent pSTAT5 activation. As such, this mechanism links Foxp3, which enforces and maintains the Treg cell suppressive program (Gavin et al., 2007
; Lin et al., 2007
), to a low threshold for IL-2R signal transduction.
The effectiveness of a low IL-2R signaling threshold for Treg cells has important implications in immunotherapy that targets the IL-2/IL-2R. First, immunotherapy based on anti-IL-2R blockade is unlikely to fully inhibit IL-2 binding to the IL-2R or even approach the degree of IL-2R signaling inhibition associated with our IL-2Rβ mutants that readily support and maintain Treg cells in vivo. Thus, Treg cells are likely to be substantially resistant to approaches that aim to inhibit T cell immunity by directly blocking the IL-2R. Indeed, many clinical protocols have utilized humanized anti-IL-2R mAb (anti-Tac, Daclizumab, Zenapac) (Waldmann, 2007
) without noting side effects consistent with impaired Treg cells, e.g. T cell activation or autoimmunity. Second, Treg cells are predicted to be relatively responsive to IL-2-based therapy and administering low dose IL-2 might represent a means to enhance Treg cell function in vivo. In this regard, treatment of NOD mice with a low concentration of an agonist IL-2/anti-IL-2 complex prevented autoimmune diabetes by enhancing Treg cell survival and function (Tang et al., 2008
Our study shows that chronic low IL-2R signaling is remarkably effective in producing an effective population of Treg cells. Nevertheless, there are important biological consequences of such impaired IL-2R function. Notably, we consistently observed an increase in an activated T cell phenotype and inflammatory infiltrates, most often in the lung and salivary gland, in progeny of older mice that expressed mutant IL-2R β-chains. These types of abnormality were also reported for IL-2−/−
mice (Sharma et al., 2006
) and were also seen in several younger IL-2Rβ−/−
mice, as an apparent early consequence of their systemic autoimmunity. Thus, impaired chronic IL-2R signaling over a long time-frame leads to symptoms consistent with organ-specific autoimmunity, which in our model appears analogous to Sjögren’s syndrome. In a related manner, decreased IL-2 production has been associated with the Idd3 locus of NOD mice that impairs Treg cell function and contributes to autoimmune diabetes (Yamanouchi et al., 2007
). These findings also raise the possibility that the recent association of polymorphisms in human CD25 with susceptibility to several autoimmune diseases might ultimately be reflected by chronic lower IL-2R signaling.
An interesting issue is whether immune stimulation of mice that contain Treg cells with impaired IL-2R signaling may predispose one to severe autoimmunity. In this regard, 2RβWT/Thymus
mice, whose Treg cells also exhibited weak transient STAT5 due to the very low expression of the IL-2Rβ chain, did not show obvious clinical symptoms when challenged with vaccinia virus, allografts, nominal antigen, or superantigen even though they mounted largely normal and effective immune responses (Jin et al., 2006
; Yu et al., 2003
). Whether other forms of immune challenges and/or inflammatory signals exacerbate the autoimmune symptoms associated with older mice harboring Treg cells with impaired IL-2R signaling remains to be determined.
Past work is consistent with a model where Treg cells depend primarily on IL-2-dependent activation of STAT5 and our study is consistent with that view. However, the basis by which IL-2R-dependent STAT5-activation successfully supports Treg cells production remains poorly understood. Another important aspect of this study is that we show that Treg cells utilize a unique mechanism to activate STAT5 for their development and homeostasis but this signaling is insufficient to support IL-2-dependent T cell growth by conventional T cells or development of iTreg cells in vitro. Weak transient STAT5 activation occurred after mutations of 3 critical tyrosine residues in the cytoplasmic tail of IL-2Rβ and this IL-2R readily supported Treg cell production in vivo. Deletion of the distal 123 amino acids of the cytoplasmic tail of IL-2Rβ, i.e. the H-region, including the two dominant tyrosine residues for STAT5 docking, also supported transient STAT5 activation and Treg cell production. These results demonstrates that initial STAT5 activation does not depend upon some other undefined interaction within the H-region of IL-2Rβ and provides a mechanistic explanation for past work that showed that domain deletions of the A- or H-regions of the IL-2Rβ cytoplasmic tail also prevented autoimmune symptoms after expression in IL-2Rβ−/−
mice (Fujii et al., 1998
). Overall these findings are consistent with a model where the initial IL-2R-dependent activation of STAT5 is independent of docking with these 3 tyrosine resides and may be independent of docking with the cytoplasmic tail of IL-2Rβ. Nevertheless, phosphorylation of these tyrosine residues ultimately promotes docking of STAT5 and other signaling molecules, such as the adapter Shc, to assembly a complex for sustained IL-2R signal transduction. The basis for IL-2Rβ tyrosine-independent activation of STAT5 remains to be determined. One possibility is a tyrosine-independent H-region-independent association of STAT5 with IL-2Rβ. Alternatively, past studies examining gp160 in cell lines that over-expressed receptor related components provided evidence for direct Jak-1/STAT5 activation (Fujitani et al., 1997
) and perhaps this is also operative for the IL-2R.
Gene expression profiling of WT vs. 2RβWT/Thymus
peripheral Treg cells demonstrated that a substantial number of targets remain IL-2 dependent. Thus, the capacity of weak transient STAT5 activation to readily support largely normal Treg cell development and homeostasis is likely due to the sensitivity of a few key targets, e.g. Foxp3 as discussed above, to this low IL-2R signal transduction rather than a global control of the IL-2 program in Treg cells by low threshold IL-2R signaling. The types of genes dependent upon IL-2 included those related to the immune system and cell cycle. This latter finding corresponds to past work where we showed that WT peripheral Treg cells exhibited substantially greater BrdU incorporation and proliferation than 2RβWT/Thymus
Treg cells (Bayer et al., 2007
) and is consistent with the view that IL-2 is normally an important mediator of Treg cell homeostasis. Past gene profiling of IL-2-dependent genes in Treg cells also reached a similar conclusion related to Treg cell growth and the cell cycle (Fontenot et al., 2005
). However, many of the key IL-2-dependent targets identified previously were not found in our gene array analysis. We believe that this is accounted for in the distinct protocols utilized. We examined Treg cells from autoimmune-free mice with chronic long-term impaired IL-2R signaling. Past work evaluated IL-2 dependent targets after autoimmune IL-2-deficient mice received a brief acute exposure to IL-2 (Fontenot et al., 2005
Direct assessment of IL-2R signal transduction in CD4+
Treg and T effector cells closely approximate each other. However, the IL-2-dependent targets in these cell populations were largely distinctive, but with a high significant overlap (10–15%). This latter finding is consistent with IL-2 controlling several common processes in these two cell types. Even more striking was the overlap (20%) of IL-2-dependent transcripts in Treg cells with the recently published Treg cell transcriptional signature (Hill et al., 2007
). Thus, our study places on firm ground the importance of IL-2 in regulating a significant portion of the Treg cell gene program.