Associations of different immune checkpoints-expressing CD4(+) Treg/ T cell subsets with disease-free survival in colorectal cancer patients

There are different subsets of T regulatory cells (Tregs), orchestrating critical roles in the regulation of anti-tumor immunity in colorectal cancer (CRC). In this study, we report that a high frequency of circulating CD4(+)FoxP3(+) Tregs was associated with poorer disease-free survival (DFS), while their higher frequencies in tumor-infiltrating CD4(+) Tregs was associated with better DFS. We further investigated such associations with four Tregs/T cells expressing or lacking FoxP3 and Helios (FoxP3(±)Helios(±)). For the first time, we report that a high frequency of circulating CD4(+)FoxP3(+)Helios(+) Tregs was associated with poorer DFS, while a high frequency of tumor-infiltrating CD4(+)FoxP3(−)Helios(−) T cells was associated with poorer DFS. In the four FoxP3(±)Helios(±) T cell subsets expressing any of the immune checkpoints (ICs) investigated, we found that a high frequency of CD4(+)FoxP3(+)Helios(−)PD-1(+) Tregs in circulation was associated with worse DFS. We also found that high frequencies of FoxP3(+)Helios(+)CTLA-4(+) Tregs, FoxP3(+)Helios(−)CTLA-4(+) Tregs, and FoxP3(−)Helios(+)CTLA-4(+) CD4(+) T cells in circulation were associated with worse DFS. In contrast, high frequencies of CD4(+)TIM-3(+) T cells, FoxP3(+)Helios(+)TIM-3(+) Tregs, and FoxP3(−)Helios(+)TIM-3(+) CD4(+) T cells in circulation were associated with longer DFS. Our data show that certain CD4(+) Treg/T cell subsets could serve as independent predictive biomarkers in CRC patients. Identification of the exact subpopulations contributing to clinical outcomes is critical for prognoses and therapeutic targeting.