14-3-3z sequesters cytosolic T-bet, up-regulating IL-13 in Tc2 and scleroderma CD8+ lymphocytes.

Interleukin(IL)-13-producing CD8+ T cells have been implicated in the pathogenesis of type-2 driven inflammatory human conditions. We have shown that CD8+IL-13+ cells play a critical role in cutaneous fibrosis, the most characteristic feature of systemic sclerosis (scleroderma; SSc). However, the molecular mechanisms underlying IL-13 and other type-2 cytokine production by CD8+ T cells remain unclear.Establish the molecular basis of IL-13 over-production by SSc CD8+ T cells, focusing on T-bet modulation of GATA-3 activity, which we showed to underlie IL-13 over-production in SSc CD8+IL-13+ cells.Biochemical and biophysical methods were employed to determine expression and association of T-bet, GATA-3 and regulatory factors in CD8+ T cells isolated from the blood and lesional skin of SSc patients with severe skin thickening. ChIP analysis determined GATA-3 binding to the IL-13 promoter. ImageStream analysis and confocal microscopy visualized the subcellular localization of T-bet and GATA-3. Transcript levels were decreased by small interfering RNAs.The interaction of T-bet with the adaptor protein 14-3-3z in the cytosol of SSc CD8+ T cells reduces T-bet translocation into the nucleus and its ability to associate with GATA-3, allowing more GATA-3 to bind to the IL-13 promoter and inducing IL-13 up-regulation. Strikingly, we show that this mechanism is also found during type-2 polarization of healthy donor CD8+ T cells (Tc2).We identified a novel molecular mechanism underlying type-2 cytokine production by CD8+ T cells revealing a more complete picture of the complex pathway leading to SSc disease pathogenesis.