14-3-3z sequesters cytosolic T-bet, upregulating IL-13 levels in T_C2 and CD8⁺ lymphocytes from patients with scleroderma.

Abstract

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 (SSc; scleroderma). However, the molecular mechanisms underlying production of IL-13 and other type 2 cytokines by CD8⁺ T cells remain unclear. We sought to establish the molecular basis of IL-13 overproduction by CD8⁺ T cells from patients with SSc, focusing on T-bet modulation of GATA-3 activity, which we showed to underlie IL-13 overproduction in CD8⁺IL-13⁺ cells from patients with SSc. Biochemical and biophysical methods were used to determine the expression and association of T-bet, GATA-3, and regulatory factors in CD8⁺ T cells isolated from the blood and lesional skin of patients with SSc with severe skin thickening. Chromatin immunoprecipitation 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. Interaction of T-bet with the adaptor protein 14-3-3z in the cytosol of CD8⁺ T cells from patients with SSc 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 upregulation. Strikingly, we show that this mechanism is also found during type 2 polarization of CD8⁺ T cells (T_C2) from healthy donors. 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.