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Naturally occurring CD4+CD25highFOXP3+ regulatory T cells (Tregs) constitute a powerful mechanism of immune regulation and therefore, have important therapeutic potential for disorders such as autoimmune diseases, allograft rejection and graft-versus-host disease. Disruption of the IL-2R signalling pathway by genetic defects of the interleukin (IL)-2 gene or components of the IL-2 receptor (R) complex results in severe T cell-mediated autoimmunity rather than immunodeficiency, indicating a crucial role for IL-2R signalling for Treg development and function. Signalling downstream of the IL-2R can act through the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway, the Janus kinase (JAK)/Signal transducers and Activators of Transcription (STAT) pathway and the mitogen-activated protein kinase (MAPK) pathway. In this report we focus on the relevance of these pathways as well as the impact of immunosuppressive drugs that may affect or enhance Treg function by targeting IL-2R signalling.
The high expression of the IL-2Rα chain (CD25) in naturally occurring CD4+CD25highFOXP3+ regulatory T cells (Treg)1 suggests that Tregs are more dependent on IL-2 for survival and function than CD4+CD25− T cells. This notion was later supported by several lines of evidence.2–4 When the IL-2 receptor (IL-2R) is engaged, heterodimerization of CD122 and CD132 follows, which activates the associated tyrosine kinases JAK1 and JAK3. The finding that a covalently linked JAK3 molecule can functionally replace the cytoplasmic domain of CD132 indicates that the role of this molecule in IL-2R signal transduction is the recruitment of JAK3.5 Conversely, CD122 provides essential docking sites for at least two major downstream mediators, the adaptor protein Shc and the transcription factor STAT5.5–7 In contrast to CD122, the role of CD25 appears to be confined to increasing binding affinity and it is postulated that the short cytoplasmic domain is unlikely to contribute to signal transduction.5–7 In line with the concept of IL-2 dependence of Treg, there are no Treg in IL-2-deficient mice,2,8,9 IL-2Rα (CD25)-deficient mice10 and IL-2Rβ-deficient mice.9,11 These studies in the murine system indicate that intact IL-2 signalling is requisite for the development and survival of Treg. Mechanistically it was shown that IL-2 directly regulates FOXP3 expression in human Treg and enhances the expansion of these cells in vivo.12,13 FOXP3 expression regulation involves the binding of STAT3 and STAT5 proteins to a highly conserved STAT binding site located in the first intron of the human FOXP3 gene.12 These data were complemented by studies in the murine system showing that Stat5a/b directly regulate FOXP3 expression.14 Consistent with this concept, recent data indicate an essential role for IL-2Rβ dependent JAK/STAT5 signaling for the development of Treg.15 These data reveal a critical link between IL-2 mediated JAK/STAT5 signaling and the maintenance of FOXP3 expression in Treg in mice as well as humans.16
In T cells, signalling downstream of the IL-2R can act through the PI3K/Akt/mTOR, JAK/STAT and the MAPK pathways. In Treg as a subset of T cells, engagement of the IL-2R fails to activate downstream targets of PI-3K signalling pathway, including Akt, mTOR or p70s6kinase.17–19 This observation has important therapeutic implications since the immunosuppressive drug rapamycin (RAPA) targets the mTOR pathway. RAPA was shown to be useful for the expansion of Treg in vitro20–22 and preserves the in vivo suppressive effects of Treg.23 These altered IL-2R PI-3 kinase downstream events may account for the hypoproliferative response of Tregs to IL-2 and are mediated through the inhibitory effect of PTEN (phosphatase and tensin homolog deleted on chromosome 10).24 Elegant studies by Walsh and colleges have demonstrated that targeted deletion of the lipid phosphatase PTEN using the Cre/Lox system allowed for expansion of Treg ex vivo in response to IL-2 without other stimuli.24 It was noteworthy that PTEN deficiency did not interfere with the thymic development or the function of Treg as evidenced in a colitis model.24 Based on these findings, we analyzed the relevance of PTEN for the relative resistance of Treg towards mTOR inhibition and found that expression levels of PTEN were rapidly downregulated in conventional CD4+CD25− (Tconv) T cells but not in Treg after stimulation.18 The physiological relevance of this finding may be that Tconv downregulate PTEN, as a negative regulator of T cell expansion, when they are activated which is supported by the observation that PTEN was shown to impose a requirement for CD28 costimulation in the presence of T cell receptor (TCR) stimulation, defining an important mechanism for its role in self-tolerance.25 Our findings that PTEN deficiency was associated with a reversal of the relative resistance of Treg towards mTOR inhibition is compatible with data from different tumor models in which PTEN knockdown sensitizes established tumors to RAPA in vitro and in vivo.26 Mechanistically we identified a relative resistance towards RAPA in Treg based on reduced usage of the mTOR pathway and on functional PTEN in vivo.18
IL-2R signalling is primarily mediated through activation of JAK1 and JAK3 with subsequent phosphorylation and activation of STAT5.27 Interestingly, we found that Treg activation by alloantigen and IL-2 led to a preferential activity of the STAT5 pathway and promoted the preferential expansion of Treg and FOXP3 expression in the presence of the mTOR inhibitor RAPA whereas conventional CD4+CD25− T cells were much more impacted by RAPA. This notion is consistent with previous data indicating that RAPA-resistant proliferation of CD8+ T cell clones could be blocked by anti-IL-2 Abs, suggesting that some of the pathways parallel to the PI3K/mTOR pathway, in this case the STAT5 pathway, triggered by IL-2R signaling account for the antigen driven RAPA resistance.28 In addition, these findings are consistent with a previous report indicating that the ability of CD28 to promote efficient Treg generation requires neither an intact PI-3K-binding motif nor an intact Itk kinase-binding motif but does require an intact Lck-binding motif in the CD28 cytosolic tail.29 In concert, the in vitro and in vivo studies indicate that Treg survival is dependent on IL-2R-STAT5 pathway but not PI-3K pathway. Based on the presence of several STAT5B consensus sequences in the FOXP3 promotor, it was hypothesized that there is a critical linkage between IL-2 signaling/STAT5B and FOXP3 expression in Treg. Recent data demonstrated that IL-2 activated long-form (LF) STAT5 and sustained FOXP3 expression in Treg but not in Tconv.16 Importantly, blocking LF STAT5 activation with the JAK inhibitor (AG-490) significantly reduced FOXP3 expression in Treg.16 The concept of mTOR as compared to JAK3/STAT5 pathway usage is illustrated in Figure 1.
Regarding the relevance of the third pathway downstream of IL-2R, including activation of mitogen activated protein kinase 1/2 (MEK1/2), and extracellular signal-regulated kinase 1/2 (Erk1/2) in vitro studies demonstrated that CD3/CD28—mediated activation of Ras, MEK1/2 and Erk1/2 was impaired in Treg.30 This study also demonstrated that Tregs are blocked from cell cycle progression due to decrease of cyclin E and cyclin A and increase of p27kip1 (cyclin dependent kinase inhibitor).30 IL-2 induced sustained increase of cyclin E and cyclin A and prevented upregulation of p27kip1.30 Murine CD4+CD25+ Treg cells displayed reduced capacity to activate JNK but not extracellular signal-related kinase (ERK) or p38 MAPK following stimulation with PMA/ionomycin.31 Conversely, in vitro—expanded CD4+CD25+ Treg cell lines derived from human cord blood showed normal activation of AKT but significantly reduced activation of Ras, MAP kinase kinase (MEK)1/2 and ERK1/2 upon TCR-mediated activation.30
It was shown in the mouse model that one critical requirement for Treg function and expansion is calcineurin dependent IL-2 production, which is impacted by the calcineurin inhibitor cyclosporine A (CSA).23 In light of the finding that the interaction between NFATc and FOXP3 is required for the suppressive effects of Treg cells32,33 interference with NFAT by CSA may also contribute to the observed effects of CSA on Treg biology. Mechanistically it was demonstrated that CSA affects Treg development, expansion and graft-versus-host disease (GvHD) suppressor function in mice.23,34,35 In a murine skin graft model, CSA interfered with the de novo conversion of Tconv into alloantigen-specific Treg.36 In vitro studies on human cells suggested that CSA affects the highly suppressive subpopulation of human CD27+ Treg.37 Clinical data indicated that CSA treatment of patients undergoing renal allograft transplantation, reduced Treg frequencies as compared to other immunosuppressive regimens.38–40 In the setting of pulmonary allotransplantation immunosuppression with tacrolimus (FK506) was superior to CSA with respect to Treg frequencies.41 The studies on the impact of CSA on Treg are detailed in Table 1.
The initial step during the biological action of RAPA is the binding to the intracellular immunophilin FK506-binding protein (FKBP12) which resembles calcineurin inhibitors.42 In contrast to the calcineurin inhibitors, RAPA does not inhibit TCR-induced calcineurin activity but the RAPA-FKBP12 complex inhibits the serine/threonine protein kinase mTOR, the activation of which is required for protein synthesis and cell cycle progression (Fig. 1). Therefore, RAPA blocks signalling in response to cytokines/growth factors, whereas calcineurin inhibitors exert their inhibitory effects by blocking TCR-induced activation.42 In vitro studies demonstrated that RAPA enables in vitro expansion of functionally suppressive Treg of murine20 or human21,22,43 origin (Table 1). Mechanistically different groups have suggested the the beneficial effect of RAPA is through selective Treg expansion,20 conversion of CD4 T cells into Treg22 and improved survival of Treg as compared to Tconv.43 In vivo the presence of RAPA allowed for the expansion and effector function of Treg in a GvHD model18,23 and the combination of RAPA and IL-10 induced tolerance in a diabetes model.44 Furthermore, a synergistic effect between Treg and RAPA has been demonstrated in a model of bone marrow graft rejection45 and GvHD.18 Differential expansion kinetics of Treg and Tconv in the presence of RAPA were based on a relative resistance towards RAPA in Treg based on reduced usage of the mTOR pathway and on functional PTEN as a negative regulator of the mTOR pathway.18
Preclinical studies demonstrate that MMF contributes to the generation of Treg and enhances their activity.46 However, in these studies MMF was combined with 1α,25-dihydroxyvitamin D3 in a diabetes model. Therefore, no direct evidence that the expansion of CD4+CD25+ cells was due to either MMF or 1α,25-dihydroxyvitamin D3 was provided.46 Recent in vitro studies on murine Treg indicate that MMF did not interfere with expansion and suppressor function of Treg in the presence of alloantigen stimulation.47 In human renal allograft recipients, the use of MMF and calcineurin inhibitor sparing was associated with increased Treg frequencies.48
Dexamethasone (DEX) induces cell death in T cells and could have unfavorable effects on Treg. However, Treg were shown to express higher levels of glucocorticoid receptor and Bcl-2, which coincided with an increased resistance to DEX-mediated cell death as compared to CD4+CD25− T cells.49 In the murine system, the administration of DEX to BALB/c mice enhances the proportion of Treg and the ratio of Treg/CD4+CD25− cells in the lymphoid organs, especially in the thymus. This correlates with the in vitro observation that Tregs express higher levels of glucocorticoid receptor.49 Furthermore, IL-2 selectively protected Treg from DEX-induced cell death, while IL-7 and IL-15 did not exert such preferential protective effects.49 Consistent with this concept, the steroid fluticasone propionate increased Treg mediated suppression of allergen-stimulated T effector cells through an IL-10-dependent mechanism50 and inhaled or systemic glucocorticoids have been found to induce FOXP3 and IL-10 expression and generation of Tregs.51 Further evidence that steroid treatment is compatible with intact Treg functions comes from studies on myasthenia gravis patients showing that the number of Treg cells in the blood is significantly lower in untreated myasthenia gravis patients than in age-matched healthy subjects, whereas it is normal or elevated in patients receiving prednisone (+/−azathioprine).52
Increased secretion of TNF, along with IL-1 and IL-6, is important in the pathogenesis of GvHD and rheumatoid arthritis (RA). Increased apoptosis of Tregs in patients with active RA is reduced by anti-TNF mAb infliximab.53 This is consistent with data indicating that TNF downmodulates the in vivo function of Treg.54
Recent data from a human allogeneic in vitro model indicate that the immunosuppressive drug FK778, induces regulatory activity in stimulated human CD4+CD25− T cells.55 Treg induced by FK778 displayed potent suppressor activity in a cell-cell contact-dependent manner; were CD25high, CD45RO+, CD27−, CTLA-4+, GITR+ and FOXP3+.55
The sphingosine-1-phosphate analogue FTY720 has shown promising immunomodulatory properties based on its pronounced effects on lymphoid migration. One could speculate that FTY720 may interfere with Treg function since it abrogates S1P/S1P1-dependent egression of lymphoid cells from lymphoid organs and in light of the fact that Treg express S1P which appears to be required for optimal suppression of effector T cell activities.56 In contrast it has been shown that FTY720 does not block the sequestration of Treg and importantly, increases their functional activity in a murine model of suppression of OVA-induced airway inflammation.57 Furthermore, data from a different mouse model indicates that FTY720 does not impair Treg mediated suppression of GvHD.58
Multiple studies have shown that Treg play a critical role in the maintenance of self tolerance, control of autoimmune diseases, transplant rejection and cancer.59 In order to modify Treg activity depending on the disease setting, the choice of immunosuppression is critical. Different studies have contributed to delineate the relevance of the IL-2R downstream events, and it is increasingly recognized that in contrast to conventional T cells, Treg have reduced mTOR pathway activity. Conversely, signalling through the JAK3/STAT5 pathway enhances Treg FOXP3+ expression and is crucial for Treg development. In conclusion immunossuppressants that target the mTOR pathway such as sirolimus or everolimus seem favorable for sparing Treg function in vivo. However, corticosteroids and novel agents that act independent of the IL-2R pathway also hold promise for a combination with Treg mediated suppressor function.
This report was supported by grants from the National Institute of Health (NIH; RO1 CA0800065 and P01 HL075462 to R.S.N.) and by the Deutsche Krebshilfe (Dr. Mildred-Scheel-Stiftung), Bonn, Germany (grant#108034 to R.Z.). We apologize to those whose work was not cited due to space limitations.