GITR Ligation Improves Anti-PD1-Mediated Restoration of Human MMR-Proficient Colorectal Carcinoma Tumor-Derived T Cells

BACKGROUND & AIMS: In contrast to mismatch repair deficient colorectal carcinoma (CRC), MMR proficient (pMMR) CRC does not respond to immune checkpoint blockade. We studied immune checkpoint stimulation via glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) on ex vivo functionality of human tumor-infiltrating lymphocytes (TIL) isolated from pMMR primary CRC and liver metastases (CRLM). METHODS: Using lymphocytes from resected tumor, adjacent tissues, and peripheral blood mononuclear cells (PBMC) of 132 pMMR primary CRC or CRLM patients, we determined GITR expression and the in vitro T-cell agonistic activity of recombinant GITR ligation. RESULTS: Here, we show that GITR was overexpressed on TIL when compared with other stimulatory immune checkpoints (4-1BB, OX40). Its expression was enhanced in TIL compared with PBMC and adjacent tissues. Among CD4(+) TIL, GITR expression was primarily expressed by CD45RA- FoxP3hi activated regulatory T cells. Within CD8(+) TIL, GITR was predominantly expressed on functionally exhausted and putative tumorreactive CD103(+) CD39(+) TIL. Strikingly, recombinant GITRL reinvigorated ex vivo TIL responses by significantly enhancing CD4(+) and CD8(+) TIL numbers. Dual treatment with GITRL and nivolumab (anti-PD1) enhanced CD8(+) TIL expansion compared with GITRL monotherapy. Moreover, GITRL/ anti-PD1 dual therapy further improved anti-PD1-mediated reinvigoration of interferon gamma secretion by exhausted CD8 TIL from primary CRC. CONCLUSIONS: GITR is overexpressed on CD4(+) and CD8(+) TIL from pMMR CRC and CRLM. Agonistic targeting of GITR en-hances ex vivo human TIL functionality and may therefore be a promising approach for novel monotherapy or combined im-munotherapies in primary pMRR CRC and CRLM. (Cell Mol Gastroenterol Hepatol 2023;15:77-97; https://doi.org/10.1016/ j.jcmgh.2022.09.007)