Investigation of the Role of Nuclear Factor of Activated T Cells (NFAT2) in T Regulatory Cell-induced Immune Suppression in Feline Immunodeficiency Virus Infection

CD4+CD25+ T regulatory cells play an important role in maintaining the balance between protective immune responses and excessive inflammatory conditions. The molecular mechanisms by which Treg cells suppress the function of T helper and CD8+ cells remain to be elucidated. As Treg cells play a major role in lentivirus-induced immunodeficiency, the role of two transcription factors NFAT2 and FoxP3 as potential regulators in Treg-induced T helper cell suppression in FIV infection was examined. A human NFAT2 homologous protein was identified in the cat that plays a role in IL-2 induction by binding to the IL-2 promoter in activated lymphocytes. Further studies in FIV+ cats revealed that in the absence of in vitro stimulation, FoxP3, a suppressor of IL-2 transcription was expressed in CD4+CD25+ Treg but not in CD4+CD25- T helper cells. Moreover, PMA/ionomycin stimulation did not significantly influence FoxP3 expression, suggesting that it is constitutively expressed. In contrast, IL-2 mRNA was not expressed in unstimulated CD4+CD25- or CD4+CD25+ T cells but was expressed at high levels by both T cell subsets from FIV+ and control cats stimulated with PMA/ionomycin. NFAT2 mRNA was equally expressed in both subsets of unstimulated CD4+ T cells and was equally highly inducible by PMA/ionomycin. Co-culture of fresh CD4+CD25+ Treg cells isolated from FIV+ with T helper cells showed elevated expression of FoxP3 mRNA and inhibited IL-2 production in the T helper cells compared to T helper cells co-cultured with CD4+CD25- cells. However, while NFAT2 mRNA expression was increased in both co-cultures, there was no significant difference between the two co-cultures, suggesting that the decrease in IL-2 production in the Treg-suppressor co-culture is not related to NFAT2 mRNA expression. As TGF-beta has been implicated as a mediator of Treg suppression, the role of TGF-beta in FoxP3 and NFAT2 expression and Treg-mediated suppression of CD4+CD25- T helper cells was assessed. ConA-stimulated CD4+CD25- T helper cells cultured in the presence of soluble TGF-beta up-regulated FoxP3 and had a suppressed IL-2 response compared to cells cultured in the absence of TGF-beta. NFAT2 mRNA expression was increased to similar levels in both cultures, suggesting that NFAT2 transcription was not affected by TGF-beta mediated suppression. ChIP assay revealed that, while NFAT2 mRNA levels were similar in stimulated versus stimulated/suppressed cells, there was more NFAT2 bound to the IL-2 promoter in the nonsuppressed cells. These data suggest that FIV infection activates Treg cells. These activated Treg cells suppress T helper cell IL-2 production, by inducing FoxP3 expression which in turn interferes with NFAT2 binding to the IL-2 promoter. This Treg cell-induced T helper cell suppression is mediated via TGF-beta signaling. These studies support the concept that FoxP3 functions as a suppressor molecule in a number of IL-2-dependent responses.