Human colorectal cancer: upregulation of the adaptor protein Rai in TILs leads to cell dysfunction by sustaining GSK-3 activation and PD-1 expression

Abstract

BackgroundThe immunosuppressive tumor microenvironment (TME) of colorectal cancer (CRC) is a major hurdle for immune checkpoint inhibitor-based therapies. Hence characterization of the signaling pathways driving T cell exhaustion within TME is a critical need for the discovery of novel therapeutic targets and the development of effective therapies. We previously showed that i) the adaptor protein Rai is a negative regulator of T cell receptor signaling and T helper 1 (Th1)/Th17 cell differentiation; and ii) Rai deficiency is implicated in the hyperactive phenotype of T cells in autoimmune diseases. MethodsThe expression level of Rai was measured by qRT-PCR in paired peripheral blood T cells and T cells infiltrating tumor tissue and the normal adjacent tissue in CRC patients. The impact of HIF-1 on Rai expression was evaluated in T cells exposed to hypoxia and by performing chromatin immunoprecipitation assays and RNA interference assays. The mechanism by which upregulation of Rai in T cells promotes T cell exhaustion were evaluated by flow cytometric, qRT-PCR and western blot analyses. ResultsWe show that Rai is a novel HIF-1-responsive gene that is upregulated in tumor infiltrating lymphocytes of CRC patients compared to patient-matched circulating T cells. Rai upregulation in T cells promoted PD-1 expression and impaired antigen-dependent degranulation of CD8+ T cells by inhibiting phospho-inactivation of glycogen synthase kinase (GSK)-3, a central regulator of PD- 1 expression and T cell-mediated anti-tumor immunity. ConclusionsOur data identify Rai as a hitherto unknown regulator of the TME-induced exhausted phenotype of human T cells. What is already known on this topicO_LITumor hypoxia contributes to establish an immunosuppressive microenvironment that unleashes T cell function and limits the efficacy of immunotherapy in CRC. C_LIO_LIThe molecular mechanisms underlying the impact of hypoxia on the signaling pathways controlling PD-1 expression are unknown. C_LI What this study addsO_LIA Rai/Akt/GSK-3 axis regulates PD-1 expression following TCR/CD28 co-stimulation C_LIO_LIThis study uncovers the signaling pathway controlling hypoxia-dependent T cell exhaustion. C_LI How this study might affect research, practice or policyRai expression levels in TILs of CRC patients might be explored as a potential new biomarker of T cell exhaustion and a predictive biomarker for anti-PD-1 response.