Regulation of calcium signaling and cellular localization of NFAT in CD8+ anergic T cells.

Abstract

Anergy is an important mechanism of maintaining peripheral immune tolerance. T cells rendered anergic, are refractory to further stimulation and are characterized by defective proliferation and IL-2 production. I have used a model of in vivo anergy induction in murine CD8+ T cells to analyze the initial signaling events in anergic T cells. Tolerant T cells displayed reduced PLCγ activation and calcium mobilization, indicating a defect in calcium signaling. This defect could be overcome by facilitating complete release of calcium from the ER. Reduced calcium signaling in CD8+ anergic T cells is therefore predominantly regulated by modulations in signaling events upstream of ER store release of calcium. Impaired calcium mobilization correlated with a block in nuclear localization of NFAT1 in anergic cells, upon stimulation. However, I found that stimulation of anergic, but not naïve T cells induced nuclear translocation of NFAT2. This suggested that NFAT2 is activated preferentially by reduced calcium signaling, and I confirmed this hypothesis by stimulating naïve T cells under conditions of calcium limitation (by addition of the calcium chelator EGTA), or partial calcineurin inhibition. This phenomenon was independent of the phosphorylation activity of the NFAT1 and 2 kinases p38 and JNK, and resultant nuclear exit, as cellular translocation assays using specific kinase and nuclear export inhibitors caused no change in pattern of NFAT1 and NFAT2 nuclear localization. Proliferative and transcriptional changes were also observed under conditions of calcium limitation or sub optimal calcineurin activity. Thus, my work provides new insight into how T cell stimulation conditions might dictate activation of a specific member of the NFAT family and aid in differential transcriptional activation. My results have also led to the hypothesis that NFAT1 and NFAT2 transcriptional activation profiles in anergic T cells are different from responsive T cells. The preferential nuclear accumulation of NFAT2 in anergic T cells might be important for the regulation of genes related to anergy maintenance.