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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.

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.

Colorectal cancer (CRC) is influenced by infiltration of immune cell populations in the tumor microenvironment. While elevated levels of cytotoxic T cells are associated with improved prognosis, limited studies have reported associations between CD4 +  T cells and disease outcomes. We recently performed transcriptomic profiling and comparative analyses of sorted CD4 + and CD8 + tumor-infiltrating lymphocytes (TILs) from bulk tumors of CRC patients with varying disease stages. In this study, we compared the transcriptomes of CD4 + with CD8 + TILs. Functional annotation pathway analyses revealed the downregulation of inflammatory response-related genes, while T cell activation and angiogenesis-related genes were upregulated in CD4 + TILs. The top 200 deregulated genes in CD4 + TILs were aligned with the cancer genome atlas (TCGA) CRC dataset to identify a unique gene signature associated with poor prognosis. Moreover, 69 upregulated and 20 downregulated genes showed similar trends of up/downregulation in the TCGA dataset and were used to calculate "poor prognosis score" (ppScore), which was significantly associated with disease-specific survival. High ppScore patients showed lower expression of Treg-, Th1-, and Th17-related genes, and higher expression of Th2-related genes. Our data highlight the significance of T cells within the TME and identify a unique candidate prognostic gene signature for CD4 + TILs in CRC patients.

T cell immunoglobulin mucin-3 (TIM-3) is an immune checkpoint identified as one of the key players in regulating T-cell responses. Studies have shown that TIM-3 is upregulated in the tumor microenvironment (TME). However, the precise role of TIM-3 in colorectal cancer (CRC) TME is yet to be elucidated. We performed phenotypic and molecular characterization of TIM-3 + T cells in the TME and circulation of CRC patients by analyzing tumor tissues (TT, TILs), normal tissues (NT, NILs), and peripheral blood mononuclear cells (PBMC). TIM-3 was upregulated on both CD4 + and CD3 + CD4 - (CD8 + ) TILs. CD4 + TIM-3 + TILs expressed higher levels of T regulatory cell (Tregs)-signature genes, including FoxP3 and Helios, compared with their TIM-3 - counterparts. Transcriptomic and ingenuity pathway analyses showed that TIM-3 potentially activates inflammatory and tumor metastatic pathways. Moreover, NF-κB-mediated transcription factors were upregulated in CD4 + TIM-3 + TILs, which could favor proliferation/invasion and induce inflammatory and T-cell exhaustion pathways. In addition, we found that CD4 + TIM-3 + TILs potentially support tumor invasion and metastasis, compared with conventional CD4 + CD25 + Tregs in the CRC TME. However, functional studies are warranted to support these findings. In conclusion, this study discloses some of the functional pathways of TIM-3 + TILs, which could improve their targeting in more specific therapeutic approaches in CRC patients., Competing Interests: The authors declare no conflicts of interest.

: Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype, and it exhibits resistance to common breast cancer therapies. Immune checkpoint inhibitors (ICIs) targeting programmed cell death 1 (PD-1) and its ligand, PD-L1, have been approved to treat various cancers. However, the therapeutic efficacy of targeting PD-1/PD-L1 axis in breast cancer is under clinical investigation. In addition, the mechanisms of action of drugs targeting PD-1 and PD-L1 have not been fully elucidated. In this study, we investigated the effect of human TNBC cell lines, MDA-MB-231 and MDA-MB-468, and the non-TNBC cell line, MCF-7, on the expression of immune checkpoints (ICs) on CD4 + T cell subsets, including regulatory T cells (Tregs), using a co-culture system. We also examined the effect of blocking PD-1 or PD-L1 separately and in combination on IC expression by CD4 + T cell subsets. We found that breast cancer cells upregulate the expression of ICs including PD-1, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) and lymphocyte activation gene-3 (LAG-3) in CD4 + T cell subsets. We also found that the co-blockade of PD-1 and PD-L1 further upregulates the co-expression of TIM-3 and LAG-3 on CD4 + CD25 + T cells and CD4 + CD25 + FoxP3 + Helios + Tregs in the presence of TNBC cells, but not in non-TNBC cells. Our results indicate the emergence of compensatory inhibitory mechanisms, most likely mediated by Tregs and activated non-Tregs, which could lead to the development of TNBC resistance against PD-1/PD-L1 blockade.