Date Published: May 7, 2012
Publisher: Public Library of Science
Author(s): Federico Simonetta, Nicolas Gestermann, Kim Zita Martinet, Michele Boniotto, Pierre Tissières, Benedict Seddon, Christine Bourgeois, Ana Claudia Zenclussen. http://doi.org/10.1371/journal.pone.0036596
Mechanisms governing peripheral CD4+ FOXP3+ regulatory T cells (Treg) survival and homeostasis are multiple suggesting tight and complex regulation of regulatory T cells homeostasis. Some specific factors, such as TGF-β, interleukin-2 (IL-2) and B7 costimulatory molecules have been identified as essentials for maintenance of the peripheral Treg compartment. Conversely, Treg dependency upon classical T cell homeostatic factors such as IL-7 is still unclear. In this work, we formally investigated the role of IL-7 in Treg homeostasis in vivo in murine models. We demonstrated that IL-7 availability regulated the size of peripheral Treg cell pool and thus paralleled the impact of IL-7 on conventional T cell pool. Moreover, we showed that IL-7 administration increased Treg cell numbers by inducing thymic-independent Treg peripheral expansion. Importantly the impact of IL-7 on Treg expansion was detected whether conventional T cells were present or absent as IL-7 directly participates to the peripheral expansion of Treg after adoptive transfer into lymphopenic hosts. Our results definitively identify IL-7 as a central factor contributing to Treg peripheral homeostasis, thus reassembling Treg to other T cell subsets in respect of their need for IL-7 for their peripheral maintenance.
CD4+ CD25+ FOXP3+ regulatory T cells are a population of CD4 T cells crucial for the regulation of immune responses and in preventing autoimmunity and chronic inflammation . Mice and humans genetically deficient in Treg cells as a result of mutations in Foxp3, a critical transcription factor for Treg development and function, typically present severe lympho-proliferation and immune pathology –. Treg exert their major role in the maintenance of immune-tolerance through several mechanisms including secretion of inhibitory molecules, suppression of antigen-presenting cells function, cytolysis and effector cells metabolic disruption . Despite the importance of the Treg compartment for the maintenance of immune tolerance and the intensive investigation focusing on Treg cell biology during the last few years, uncertainties remain about the factors controlling peripheral Treg survival and homeostasis. While Treg homeostasis appears to rely on some subset specific factors such as TGF-β –, IL-2 – and B7 costimulatory molecules , , their dependency upon factors classically involved in conventional T cells homeostasis such as IL-7 signaling still remains controversial. IL-7 plays a critical role in T cell development and peripheral homeostasis . All major CD4 T cell subsets, including naïve, memory and Th17 CD4 T cells, strictly rely on IL-7 for their peripheral homeostasis –. The only important exception seems to be represented by CD4+ FOXP3+ Treg which have been reported to express low levels of the IL-7 receptor alpha chain (CD127) –. Accordingly, peripheral Treg biology is believed to be essentially independent of IL-7/IL-7Rα signaling , – although Bayer et al suggested that IL-7 could contribute to Treg homeostasis when IL-2 signaling is disrupted . However, we and others have previously shown that in vitro IL-7 treatment induces significant STAT5 phosphorylation in Tregs , – and that IL-7 is able to increase in vitro Treg survival , , . These in vitro data revealing a potential role of IL-7 on Treg homeostasis need to be substantiated by in vivo analysis. We first considered Treg homeostasis in mice exhibiting altered IL-7 signaling pathway using IL-7Rα−/−, IL-7−/− or IL-7 Tg mice. Regarding the increasing development of IL-7 based therapies we also investigated how in vivo Treg homeostasis was affected following IL-7 injection. We demonstrated that IL-7 availability regulated the size of the peripheral Treg cell pool and that Treg and Tconv were equally affected following IL-7 injection. Moreover, we showed that IL-7 administration increased Treg cell numbers by inducing a thymic-independent peripheral expansion. Importantly the impact of IL-7 on Treg expansion was detected whether conventional T cells were present or absent as IL-7 directly participates to the peripheral expansion of Treg after adoptive transfer into lymphopenic hosts. Collectively, our data identify IL-7 as a central factor contributing to Treg peripheral homeostasis. Such a conclusion has major implications for the development of IL-7 based strategies to ameliorate immune-reconstitution in lymphopenic settings while preventing immune pathology.
All major CD4 T cell subsets, including naïve, memory and Th17 CD4 T cells, strictly rely on IL-7 for their peripheral homeostasis. The only important exception seems to be represented by CD4+ FOXP3+ Treg cells, which are believed to essentially rely on other factors such as IL-2, TGF-β and costimulatory molecules. Although several γc cytokines, including IL-2, IL-4, IL-7, IL-15, IL-21, have been reported to increase Treg survival in vitro, only IL-2 has been clearly established as a major factor controlling Treg cell homeostasis in vivo–, , . Several reports investigated the role of IL-7 on thymic Treg development , ,  and tried to extend the analysis to the peripheral effects of IL-7. Using adoptive transfer of total CD4 T cells into irradiated Rag−/− or Rag−/− IL-7−/− mice, Mazzuchelli et al, failed to detect any differences in Treg numbers recovered concluding that IL-7 availability does not affect Treg peripheral survival . Unfortunately, total lymph-node cells were transferred, impeding any conclusions on the direct effect of IL-7 on Treg cells in this setting. The present study aimed to further investigate the effects of IL-7 on the homeostasis of CD25+ FOXP3+ regulatory T cells. Importantly, we demonstrated that IL-7 availability controls the size of both Tconv and Treg pools. Mice in which IL-7 signaling was impaired presented a reduction in Treg cell numbers similar to those observed in conventional T cell numbers. Conversely, IL-7 Tg mice, in which IL-7 production is constantly increased, showed an expansion of the Treg compartment. However, one may question the relevance of studies performed in mice exhibiting disruption of IL-7/IL-7R signaling, which severely impact thymic differentiation and peripheral T cell homeostasis. We next used a more relevant model allowing us determining the impact of IL-7 on physiologically generated Treg. Using IL-7 injection protocol, we confirm the impact of IL-7 on Treg homeostasis. IL-7 treatment in normal mice induced an increase in conventional CD4 T cell numbers that was strictly paralleled by an increase in Treg cell numbers. Importantly demonstrating that IL-7 levels equally affect the size of conventional and Treg subsets perfectly corroborate results obtained in humans during clinical trials investigating the effects of IL-7 therapy on immune-reconstitution in lymphopenic settings. Indeed, three published reports analyzed the proportion of Treg cells in peripheral blood from patients treated with IL-7 – and detected either slight reduction or no difference in the percentage of Treg cells among CD4 T cells after IL-7 treatment. Because total CD4 T cell count increased in IL-7 treated patients, essentially preserved Treg percentages indicate that Treg changes in absolute numbers also paralleled those detected in conventional CD4 T cells in human studies. We next aimed to determine the mechanisms involved in IL-7 mediated effects on Treg pool. Because the impact of IL-7 on conventional T cell proliferation  and survival  has been extensively described, we aimed to determine whether IL-7 influenced Treg homeostasis by similar mechanisms. Interestingly, IL-7 differently impacted conventional and regulatory T cell compartment: the fraction of proliferating T cells were 3-fold increased in conventional T cells, but mildly increased in Treg fraction in IL-7 treated C57Bl/6 mice. Although significant, such low increases in Treg proliferative capacity is unlikely to account for the highly significant increase in Treg cellularity upon IL-7/αIL-7 complex treatment. These results thus suggest IL-7 treatment is likely to increase Treg cell numbers in the periphery by affecting both their survival and accumulation as previously suggested , ,  together with their proliferative capacity. Because the fraction of proliferating conventional T cells was significantly increased upon IL-7 treatment, we speculated that the beneficial impact of IL-7 on Treg homeostasis essentially rely on increased IL-2 availability due to the increased fraction of activated/effector conventional T cells. Indeed, IL-7 is highly heterogeneous in targets and mechanisms , favoring both conventional T cell homeostasis and effector differentiation , . To determine the impact of IL-7 on Tregs in the absence of conventional T cells, we performed adoptive transfer of purified Treg into Rag−/− or Rag−/− IL-7−/− mice. We showed that Treg cells transferred into Rag-deficient mice lacking IL-7 proliferated and accumulated less than Treg transferred into IL-7 competent Rag-deficient mice. To note, residual proliferation was still present in the absence of IL-7 indicating that other factors participate to the phenomenon. Although residual IL-2 production by DCs or “exFOXP3” converted Treg cannot be formally excluded , , other γc cytokines may also impact Treg peripheral homeostasis . We thus demonstrated the direct impact of IL-7 on Treg biology in the absence of conventional T cells. However, we cannot exclude that the impact of IL-7 on Treg homeostasis may combine both direct and indirect effects .