Correlations between Circulating and Tumor-Infiltrating CD4⁺ T Cell Subsets with Immune Checkpoints in Colorectal Cancer

T regulatory cells (Tregs) play different roles in the regulation of anti-tumor immunity in colorectal cancer (CRC), depending on the presence of different Treg subsets. We investigated correlations between different CD4⁺ Treg/T cell subsets in CRC patients with immune checkpoint-expressing CD4⁺ T cells. Positive correlations were observed between levels of different immune checkpoint-expressing CD4⁺ T cells, including PD-1, TIM-3, LAG-3, and CTLA-4 with FoxP3⁺ Tregs, Helios⁺ T cells, FoxP3⁺Helios⁺ Tregs, and FoxP3⁺Helios⁻ Tregs in the tumor microenvironment (TME). However, negative correlations were observed between levels of these immune checkpoint-expressing CD4⁺ T with FoxP3⁻Helios⁻ T cells in the TME. These correlations in the TME highlight the role of cancer cells in the upregulation of IC-expressing Tregs. Additionally, positive correlations were observed between levels of FoxP3⁺ Tregs, Helios⁺ T cells, FoxP3⁺Helios⁺ Tregs, and FoxP3⁺Helios⁻ Tregs and levels of CD4⁺CTLA-4⁺ T cells and CD4⁺PD-1⁺ T cells in peripheral blood mononuclear cells (PBMCs) and normal tissue-infiltrating lymphocytes (NILs). These observations suggest that CTLA-4 and PD-1 expressions on CD4⁺ T cell subsets are not induced only by the TME. This is the first study to investigate the correlations of different FoxP3^+/−Helios^+/− T cell subsets with immune checkpoint-expressing CD4⁺ T cells in CRC patients. Our data demonstrated strong correlations between FoxP3^+/Helios^+/− Tregs but not FoxP3⁻Helios^+/− non-Tregs and multiple immune checkpoints, especially in the TME, providing a rationale for targeting these cells with highly immunosuppressive characteristics. Understanding the correlations between different immune checkpoints and Treg/T cell subsets in cancer patients could improve our knowledge of the underlying mechanisms of Treg-mediated immunosuppression in cancer.