In-Vitro Inhibition of P38 Abrogates GITR-Induced Proliferation of Murine iTregs

Abstract

Abstract Triggering the co-stimulatory receptor glucocorticoid-induced TNFR-related protein (GITR) on T cells has been identified as a promising cancer immunotherapy, yet the signaling events by which GITR agonism induces antitumor immunity are not well understood (Knee et al., 2016). GITR is expressed on activated CD4+ and CD8+ T cells (Ward-Kavanagh, 2016), and is constitutively expressed on CD4+/CD25+/FoxP3+ regulatory T cells (Tregs) (Shimizu et al., 2002). While GITR appears to act as a canonical co-stimulatory receptor in effector CD4+ and CD8+ T cells, the role of GITR on CD4+/CD25+ Tregs remains controversial. Joetham et al. found that GITR stimulation resulted in loss of Tregs suppressive phenotype, while Liao et al., observed that GITR ligation enhanced natural Tregs proliferation and maintained their suppressive phenotype. These contradictory findings demonstrate a need to understand more about the signaling events occurring in Tregs as a result of triggering GITR. The goal of this research was to create a model system for elucidating and comparing GITR signaling events in induced-Tregs (iTregs), CD4+ T effector (Teff) cells, and CD8+ cytotoxic T cells. Phospho-proteomics was then leveraged to identify differential signaling events among the T cell subsets. We used the GITR agonist monoclonal antibody (mAb) DTA-1 to stimulate the T cell subsets and confirmed GITR signaling via NFκB pathway activation and phospho-JNK induction. The phospho-proteomics data identified many more unique phosphorylated protein sites than those shared among the subsets. We observed phospho-p38 MAPK induction, as well as additional upstream and downstream pathway members, in the iTreg subset but not in the CD4+ Teff cells or CD8+ cytotoxic T cells. In addition, inhibition of p38 abrogated GITR-induced proliferation of iTregs. These data demonstrate that GITR agonism induces unique phospho-proteome profiles between T cell subsets, and this differential signaling leads to unique phenotypic responses that will need to be considered when targeting GITR receptor with immunotherapeutic strategies.