Abstract
Colorectal carcinoma (CRC) is the second leading cause of cancer mortality worldwide. CRC is stratified into two major groups: microsatellite stable (MSS) and microsatellite instability-high (MSI-H). MSS CRC constitutes the majority of cases, has worse overall prognosis, and thus far has failed to respond to immunotherapies targeting the immune checkpoint receptors PD-1, PD-L1, and CTLA-4. Here we examined the alternate immunotherapy targets Tim-3 and Lag-3, as well as PD-1, on immune cells in a cohort of MSS CRC using immunohistochemistry and flow cytometry together with mutational analysis and clinical data. We found that PD-1 was variably expressed across CD4+ tumor-infiltrating lymphocyte (TIL) subtypes, and Tim-3 was mostly restricted to CD4+ regulatory T cells. Lag-3, when detected by flow cytometry, was largely coexpressed with Tim-3 and PD-1 in CD4+ TILs. Furthermore, Tim-3+ PD-1+ CD8+ TILs accumulated in the tumor and exhibited a dysfunctional or 'exhausted' phenotype. Notably, we observed a subset of patients with a high proportion of Tim-3- PD-1- CD8+ TILs and, conversely, a low proportion of Tim-3+ PD-1+ CD8+ TILs, thus stratifying MSS CRC patients based on a feature of immune exhaustion (MSS-ImmEx). MSS-ImmExhi patients had abundant Tim-3+ PD-1+ CD8+ TILs, PD-1+ CD4+ effector, and regulatory T cells, and were enriched for left-sided colon tumors and mutations in the APC tumor-suppressor gene. We further investigated the spatial organization of Tim-3, Lag-3, PD-1, and PD-L1 by immunohistochemistry and found higher levels in the tumor margin; however, MSS-ImmExhi tumors exhibited a higher density of Tim-3+ cells in the tumor center over MSS-ImmExlow tumors. Immunofluorescence revealed a higher density of PD-1+ /CD8+ cells in the tumor center in this group. Our findings identify a subset of MSS CRC that exhibits evidence of higher prior immune activation (MSS-ImmExhi ) in which therapies targeting Tim-3 in conjunction with anti-PD-1 or other immunotherapies may provide clinical benefit. © 2022 The Pathological Society of Great Britain and Ireland.