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.