Because of the embryonic lethal phenotype of Foxo1-deficient mice, the function of Foxo1 in T cells has not been studied in vivo. We have developed a novel mouse strain that enabled cell-type specific deletion of Foxo1 gene using the cre-loxP system. In this report, we used CD4-Cre transgenic mice to delete Foxo1 gene in T cells and explored its role in thymic T cell development and peripheral T cell activity. We found that Foxo1 was not essential for the positive selection of CD4+ and CD8+ T cells, but was required for the expression of IL-7R and CD62L in mature thymocytes. Foxo1 deficiency also led to the compromised IL-7R and CD62L expression in naïve T cells in the peripheral lymphoid organs. Diminished expression of IL-7R was associated with failed IL-7 signaling in Foxo1 knockout T cells, which resulted in the compromised IL-7-induced T cell survival in vitro and reduced IL-7-dependent homeostatic proliferation in vivo. Using a strain of IL-7R transgenic mouse, we showed that reduced IL-7R expression was responsible for the homeostasis defects of naïve Foxo1-deficient OT-II T cells. In addition, Foxo1 deficiency caused spontaneous T cell activation, effector T cell differentiation, and the production of autoantibodies in mice. In a bone marrow transfer model, lack of Foxo1 expression in T cells resulted in colitis. These observations reveal previously undefined potent and pleiotropic roles for Foxo1 in the control of T cell homeostasis and tolerance in vivo.
A major finding of the present study was that Foxo1 controlled naïve T cell homeostasis via its regulation of IL-7R expression. As a transcription factor, Foxo1 can bind to regulatory DNA sequences on target genes (Obsil and Obsilova, 2008
). Indeed, using rVista program, we identified consensus Foxo1-binding sites in the promoter region of Il7r
gene. We further found direct Foxo1 association with the proximal Il7r
promoter and an evolutionarily conserved non-coding region 3.7 kb upstream of the translation start site. Future studies will be needed to test the importance of these Foxo1 binding site in control of IL-7R expression in T cells. In addition, it has been reported that Foxo1 can regulate gene expression independent of its DNA-binding domain (Ramaswamy et al., 2002
). In this case, Foxo1 may interact with other nuclear factors involved in the control of IL-7R expression. Previous studies have revealed that IL-7R transcription in T cells is positively regulated through proximal promoter region that contains binding motifs for the transcription factor GABP (Xue et al., 2004
). IL-7R transcription is also subjected to repression by the transcription repressor Gfi-1 (Park et al., 2004
), which binds to an intronic region of Il7r
gene. How Foxo1 interacts with these transcription factors in control of IL-7R transcription will be an interesting area for future exploration.
The expression of IL-7R is dynamically regulated at multiple stages of T cell differentiation (Mazzucchelli and Durum, 2007
). When naïve T cells encounter antigen during infection, they undergo expansion and differentiation. This is associated with the down-regulation of IL-7R expression on most effector T cells (Huster et al., 2004
; Kaech et al., 2003
). Stimulation of T cells via the TCR, co-stimulatory receptor, and cytokine signaling pathways also inactivates Foxo1 via PKB-induced phosphorylation (Peng, 2008
). It remains to be determined whether the down-regulation of IL-7R expression on effector T cells is a consequence of Foxo1 inactivation. It has been shown that a small subset of the effecor CD8+
T cells express high amounts of IL-7R, and differentiate into long-lived memory CD8+
T cells (Huster et al., 2004
; Kaech et al., 2003
). The function of Foxo1 in control of IL-7R expression in memory T cells warrants further investigation.
In addition to the control of naïve OT-II T cell homeostasis, Foxo1 was required for the inhibition of T cell activation and differentiation on T cell polyclonal background. It has been proposed that T cell activation and development of autoimmune diseases can be caused by T cell lymphopenia (King et al., 2004
; Marleau and Sarvetnick, 2005
), which is associated with IL-7-driven homeostatic T cell proliferation (Calzascia et al., 2008
). Foxo1-deficient naïve T cells were depleted, and expressed significantly lower levels of IL-7R than Foxo1-deficient T cells with the activated phenotype, raising the possibility that T cell activation was a consequence of enhanced IL-7 stimulation. Overexpression of IL-7R via an IL-7R transgene in Foxo1-deficient T cells largely nullified IL-7R expression difference between naïve and activated T cells, but did not correct the T cell activation phenotype. These observations suggest that T cell activation in the absence of Foxo1 was not caused by defective IL-7R expression. Treg cell number was not reduced in un-manipulated Foxo1-deficient mice, which is consistent with a dispensable role for the IL-7R signaling pathway in control of Treg cell homeostasis (Bayer et al., 2008
; Mazzucchelli et al., 2008
; Vang et al., 2008
). These findings imply that Foxo1 functions as a T cell intrinsic regulator of tolerance in these mice. The mechanisms by which Foxo1 regulates T cell activation remain to be determined. Gene expression profiling experiment revealed hundreds of putative Foxo1 target genes in naïve T cells. However, it is still an open question whether Foxo1 controls another master regulator of T cell tolerance, or alternatively Foxo1 regulates multiple signaling pathways that collectively ensure naïve T cell quiescence.
Reconstitution of sublethally irradiated Rag1−/−
mice with Foxo1-deficinet bone marrow cells resulted in severe colitis that was not observed in un-manipulated KO mice aged for 5–6 months. Whole body irradiation induces tissue damage, and triggers the release of microbes and microbial products that cause systemic inflammation (Paulos et al., 2007
). It remains to be determined whether the heightened inflammatory response associated with irradiation contributes to the development of colitis in the KO chimeras. In the KO chimeric mice, the number of CD4+
cells was diminished compared to that in the WT chimeras. Reduced percentage of KO Treg cells was also observed in the mixed chimeric mice that had received both wild-type and knockout bone marrows. These findings reveal a cell-intrinsic role for Foxo1 in control of Treg cell homeostasis in irradiated mice. Active immune suppression by Treg cells is essential for T cell tolerance (Sakaguchi et al., 2008
). How Foxo1 cross-talks with Treg cells in control of T cell responses will be an interesting area for future study. In contrast to T cells from the KO chimeras, KO T cell populations from the mixed chimeric mice exhibited a naïve T cell phenotype. Replenishment of WT Treg cell in the mixed chimeras might suppress KO T cell activation. KO T cells expressed low levels of IL-7R, and were not competitive to WT T cells in the periphery. Therefore, it is also possible that KO T cells were rapidly depleted upon release from the thymus, before they could be activated by peripheral antigens. Since T cell activation in un-manipulated Foxo1-deficient mice was not associated with observable Treg cell defects, Foxo1 likely played an autonomous role in control of T cell activation.
The nature of the antigens that drive the expansion and differentiation of effector T cells in T cell-specific Foxo1-deficient mice remains to be fully characterized. Interestingly, Foxo1-deficient OT-II T cells on the Rag1−/− background were not activated. Because OT-II T cells are specific for the foreign ovalbumin antigen, these results imply that cognate antigen stimulation is needed for the activation of Foxo1-deficient T cells. Increased production of nuclear and dsDNA antibodies in Foxo1-deficient mice further suggested that self-antigens might be involved in the activation of T cells. Although we did not observe spontaneous colitis in Foxo1-deficient mice aged up to 6 months, T cells isolated from the gut-draining mesenteric lymph node exhibited more pronounced T cell activation than T cells from the other peripheral lymph nodes. Importantly, transfer of bone marrow cells isolated from T cell-specific Foxo1-deficient mice into irradiated Rag1−/− mice led to the development of colitis in recipient mice. These observations imply that Foxo1 is also critical to prevent the activation of T cells reactive to commensal bacterium antigens.
In conclusion, in this report, we have uncovered critical functions for Foxo1 in regulation of T cell homeostasis and tolerance. IL-7R was identified as a novel Foxo1 target gene involved in Foxo1 maintenance of naïve T cells. These findings will advance our knowledge on the function of Foxo family proteins in the immune system and might, on the long term, be exploited for finding cures for autoimmune diseases and cancer.