Correlations between Circulating and Tumor-Infiltrating CD4⁺ Treg Subsets with Immune Checkpoints in Colorectal Cancer Patients with Early and Advanced Stages

The existence of various T regulatory cell (Treg) subsets in colorectal cancer (CRC) could play a variety of functions in the regulation of anti-cancer immunity. We studied correlations between CD4⁺ Treg subsets with the expression of immunological checkpoints on CD4⁺ T cells, including PD-1, TIM-3, LAG-3, and CTLA-4 in CRC patients with early and advanced TNM staging. Strong positive correlations were found between frequencies of FoxP3⁺ Tregs and FoxP3⁺Helios⁺ Tregs with frequencies of various immune checkpoint-expressing CD4⁺ T cells in the tumor microenvironment (TME). However, there were strong negative correlations between frequencies of FoxP3⁻Helios⁻ T cells and these immune checkpoint-expressing CD4⁺ T cells. Specifically, in the TME, we found that the correlations between FoxP3⁺ Tregs, FoxP3⁺Helios⁺ Tregs, FoxP3⁺Helios⁻ Tregs, and FoxP3⁻Helios⁻ T cells with CD4⁺LAG-3⁺ T cells and CD4⁺CTLA-4⁺ T cells were higher in patients with early stages, suggesting the potential of these highly immunosuppressive cells in inhibiting inflammatory responses in the TME. However, the correlations between FoxP3⁺ Tregs, FoxP3⁺Helios⁺ Tregs, and FoxP3⁻Helios⁻ T cells with CD4⁺TIM-3⁺ T cells were higher in patients with advanced stages. This is the first study to explore correlations of Treg subpopulations with immune checkpoint-expressing CD4⁺ T cells in CRC based on clinicopathological features of CRC patients. The findings of our study provide a justification for focusing on these cells that possess highly immunosuppressive features. Understanding the correlations between different immune checkpoints and Treg subsets in CRC patients has the potential to enhance our understanding of core mechanisms of Treg-mediated immunosuppression in cancer.