Phosphoproteomics reveals GITR agonism differentially regulates inflammatory vs. regulatory T cell subsets.

Abstract

Abstract Triggering the co-stimulatory receptor glucocorticoid-induced TNFR-related protein (GITR) on T cells has been identified as a promising cancer immunotherapeutic strategy, yet the signaling events by which GITR agonism induces antitumor immunity are not well understood. GITR is expressed on activated CD4+ and CD8+ T cells, and is constitutively expressed on regulatory T cells (Tregs). While GITR appears to act as a conventional co-stimulatory receptor in effector CD4+ and CD8+ T cells, the role of GITR on Tregs remains controversial. The goal of this research was to utilize a phosphoproteomics approach to identify and compare GITR-mediated intracellular signaling events in induced-Tregs (iTregs), CD4+ T cells, and CD8+ cytotoxic T cells. We used the GITR agonist monoclonal antibody DTA-1 to stimulate the T cell subsets and confirmed GITR signaling via NFκB pathway activation and phospho-JNK induction. The phosphoproteomics data revealed different protein phosphorylation patterns among T cell subsets. For example, we observed phospho-p38 MAPK induction in the iTreg subset but not in the CD4+ T cells or CD8+ cytotoxic T cells. We also observed additional phospho-proteins induced by GITR triggering in the p38 pathway in the iTreg subset including phospho-MKK3, phospho-MKK6, phospho-MAPKAPK-2, and phospho-ATF-2. Induction of p38 MAPK signaling is functionally important as inhibition of p38 abrogated GITR-induced proliferation of iTregs. These data demonstrate that GITR agonism induces distinct phospho-proteome profiles between T cell subsets, and this differential signaling leads to unique responses that will need to be considered when targeting GITR with immunotherapeutic strategies.