SUMMARY The transcription factor Foxo3 plays a crucial role in myeloid cell function but its role in lymphoid cells remains poorly defined. Here, we have shown that Foxo3 expression was increased after T cell receptor engagement and played a specific role in the polarization of CD4+ T cells towards pathogenic T helper-1 (Th1) cells producing interferon-γ (IFN-γ) and granulocyte monocyte colony stimulating factor (GM-CSF). Consequently, Foxo3-deficient mice exhibited reduced susceptibility to experimental autoimmune encephalomyelitis. At the molecular level, we identified Eomes as a direct target gene for Foxo3 in CD4+ T cells and we have shown that lentiviral-based overexpression of Eomes in Foxo3-deficient CD4+ T cells restored both IFN-γ and GM-CSF production. Thus, the Foxo3-Eomes pathway is central to achieve the complete specialized gene program required for pathogenic Th1 cell differentiation and development of neuroinflammation.

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The Foxo3 transcription factor regulates cell cycle progression, survival and DNA repair pathways. In addition to its role as a tumor suppressor gene, studies have established the crucial role of Foxo3 in immune cells, notably in dendritic cells and CD8 T cells in the context of a viral infection. However, the role of Foxo3 in CD4 T cell function is still unknown. Here, we show that Foxo3 expression levels are increased in CD4 T cells after TCR engagement. We also show that Foxo3 deficiency in CD4 T cell leads to decreased secretion of IFN-γ and GM-CSF but not of IL-17. By dissecting the underlying molecular mechanisms, we have identified a new direct target gene of Foxo3 that participates to IFN-γ production and that was previously identified as a susceptibility gene for multiple sclerosis. This led us to hypothesize that Foxo3 could be involved in the susceptibility to central nervous system inflammation. To test this hypothesis, we used the well-established animal model of multiple sclerosis, the experimental autoimmune encephalomyelitis (EAE) induced by MOG immunization. We show that Foxo3 deficiency is associated with significantly decreased EAE severity and that Foxo3-deficient MOG-specific CD4 T cells fail to differentiate into pathogenic IFN-γ+GM-CSF+cells. Thus, our results show for the first time that Foxo3 plays a crucial role in the pathophysiology of neuroinflammation by directly controlling the expression of genes involved in CD4 T cell differentiation.

CD4 T cell differentiation is a process finely controlled by specific transcriptional programs leading to the acquisition of specific T helper effector functions. Up to now, the functions of Foxo3 in CD4 T cells have not been investigate. Here, we described for the first time the critical role of the transcription factors Foxo3 in Eomes expression and their involvement in pathogenic T Helper 1 differentiation. First, we showed that TCR triggering resulted in a dose-dependent upregulation of Foxo3 in CD4 T cells with an increased expression over time. We next addressed Foxo3 functions in CD4 T cells and we observed a decreased production of the Th1 related cytokines IFN-g and GM-CSF by Foxo3 KO CD4 T cells, with no impact survival, proliferation or other lineages differentiation. We then compared the transcriptome of WT and Foxo3 KO CD4 T cells and found that Foxo3 deficiency resulted in a decreased expression of genes related to the IFN- g/IFN-g response pathway and that the most downregulated gene in this pathway is Eomes. We showed, using ChIP and luciferase experiments, that Foxo3 binds and induces the expression of Eomes through direct DNA binding within the eomes locus. Using lentivirus experiments we showed overexpression of Eomes in Foxo3 KO CD4 T cell restored the proportion of IFN-g and GM-CSF producing cells, thus comforting Eomes role’s in the pathogenicity of CD4 T cells. In addition to this defect of differentiation, Foxo3-deficient mice developed a less severe Experimental Autoimmune Encephalomyelitis (EAE) as compared to WT mice, thus emphasizing the role of Foxo3 and Eomes in the development of autoimmunity. Altogether, we described here a new pathway whereby Foxo3 and Eomes drive CD4 T cells pathogenicity and neuro-inflammation.

Foxo proteins control gene expression during many cellular processes including cell cycle progression, reactive oxygen species detoxification, survival and death. During viral infection, we demonstrated that Foxo3 critically down-regulates the magnitude of the anti-viral T cell response by constraining the production of key inflammatory cytokines by dendritic cells (DCs). This impact on the innate immune response suggests that Foxo3 is likely to control the intensity of the adaptive immune response irrespective of its nature. We therefore analyze the implication of Foxo3 in the susceptibility to central nervous system inflammation. We showed that mice deficient in Foxo3 are remarkably resistant to experimental autoimmune encephalomyelitis (EAE), a common mouse model for multiple sclerosis. We showed that the reduced severity to EAE of Foxo3 deficient mice is not the consequence of an impaired priming of CD4 T cells in response to immunization. Rather, Foxo3 appears to play a role in the regulation of Th17 and Th1 differentiation. Thus, the reduced severity of EAE in Foxo3 deficient mice may be related to the inability of Foxo3-deficient CD4 T cells to differentiate into encephalogenic T cells. By dissecting the cellular mechanism involved in this phenotype, we showed that Foxo3 plays crucial role in both DC and T cells. Understanding Foxo3 function will bring new insights into the mechanisms that support immune cell homeostasis during normal and pathologic immune responses.

Mucosal immunity in the gut is achieved in part through cellular connections between lymphoid cells such as regulatory-T cells (Treg), T follicular helper cells (Tfh), T follicular regulatory cells (Tfr) and Germinal Center B cells (GC B). Btla (B and T Lymphocyte Attenuator) is a receptor mainly expressed on B and T cells that is activated by its ligand Hvem (Herpesvirus entry mediator; Tnfrsf14) to inhibit cellular activation. However, the role of these specific immune checkpoints in the regulation of mucosal immunity in the gut is still unknown. In mice containing genetic ablation of Btla in T cells, we observed an increased frequency of Tfh and GC B cells in Peyer’s Patches (PP), and a decreased frequency of Treg and Tfr cells in these tissues. Interestingly, in mice with a B-cell-specific deletion of the Btla ligand Tnfrsf14 (Hvem), we observe similar increases in GC B cells and Tfh cells and decreased in Treg. Moreover, these mice are characterized by an increase of mucosal IgA bound to bacteria in fecal pellets, indicating a functional outcome of Btla regulation of the GC. Finally, treating wild-type mice with an antibody agonist for Btla increases the frequency of regulatory T cells in PP demonstrating the potential therapeutic effect of activating Btla inhibitory signaling in mucosal immunity. Together, we show that T cell-expressed Btla interacts with B cell-expressed Hvem to regulate GC responses in Peyer’s Patches. A disruption of the balance effector T cell/regulatory T cell is responsible for many inflammatory autoimmune diseases. It will be crucial to understand the role of Btla and its ligand in these populations in mucosal tissues to understand how immunity is regulated, and to develop potential novel therapeutics to treat disease.