GRK2 mediates TCR-induced transactivation of CXCR4 and TCR-CXCR4 complex formation that drives PI3Kγ/PREX1 signaling and T cell cytokine secretion.

The immune system includes abundant examples of biologically-relevant cross-regulation of signaling pathways by the T cell antigen receptor (TCR) and the G protein-coupled chemokine receptor, CXCR4. TCR ligation induces transactivation of CXCR4 and TCR-CXCR4 complex formation, permitting the TCR to signal via CXCR4 to activate a phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein (PREX1)-dependent signaling pathway that drives robust cytokine secretion by T cells. To understand this receptor heterodimer and its regulation, we characterized the molecular mechanisms required for TCR-mediated TCR-CXCR4 complex formation. We found that the cytoplasmic C-terminal domain of CXCR4 and specifically phosphorylation of Ser-339 within this region were required for TCR-CXCR4 complex formation. Interestingly, siRNA-mediated depletion of G protein-coupled receptor kinase-2 (GRK2) or inhibition by the GRK2-specific inhibitor, paroxetine, inhibited TCR-induced phosphorylation of CXCR4-Ser-339 and TCR-CXCR4 complex formation. Either GRK2 siRNA or paroxetine treatment of human T cells significantly reduced T cell cytokine production. Upstream, TCR-activated tyrosine kinases caused inducible tyrosine phosphorylation of GRK2 and were required for the GRK2-dependent events of CXCR4-Ser-339 phosphorylation and TCR-CXCR4 complex formation. Downstream of TCR-CXCR4 complex formation, we found that GRK2 and phosphatidylinositol 3-kinase γ (PI3Kγ) were required for TCR-stimulated membrane recruitment of PREX1 and for stabilization of cytokine mRNAs and robust cytokine secretion. Together, our results identify a novel role for GRK2 as a target of TCR signaling that is responsible for TCR-induced transactivation of CXCR4 and TCR-CXCR4 complex formation that signals via PI3Kγ/PREX1 to mediate cytokine production. Therapeutic regulation of GRK2 or PI3Kγ may therefore be useful for limiting cytokines produced by T cell malignancies or autoimmune diseases.

TCR-CXCR4 signaling stabilizes cytokine mRNA transcripts via a PREX1-Rac1 pathway: implications for CTCL.

As with many immunopathologically driven diseases, the malignant T cells of cutaneous T-cell lymphomas (CTCLs), such as Sézary syndrome, display aberrant cytokine secretion patterns that contribute to pathology and disease progression. Targeting this disordered release of cytokines is complicated by the changing cytokine milieu that drives the phenotypic changes of CTCLs. Here, we characterize a novel signaling pathway that can be targeted to inhibit the secretion of cytokines by modulating either CXCR4 or CXCR4-mediated signaling. We demonstrate that upon ligation of the T-cell antigen receptor (TCR), the TCR associates with and transactivates CXCR4 via phosphorylation of S339-CXCR4 in order to activate a PREX1-Rac1-signaling pathway that stabilizes interleukin-2(IL-2), IL-4, and IL-10 messenger RNA (mRNA) transcripts. Pharmacologic inhibition of either TCR-CXCR4 complex formation or PREX1-Rac1 signaling in primary human T cells decreased mRNA stability and inhibited secretion of IL-2, IL-4, and IL-10. Applying this knowledge to Sézary syndrome, we demonstrate that targeting various aspects of this signaling pathway blocks both TCR-dependent and TCR-independent cytokine secretion from a Sézary syndrome-derived cell line and patient isolates. Together, these results identify multiple aspects of a novel TCR-CXCR4-signaling pathway that could be targeted to inhibit the aberrant cytokine secretion that drives the immunopathogenesis of Sézary syndrome and other immunopathological diseases.