Blockade of inhibitory immune checkpoints (ICs) is a promising therapeutic approach; however, it has shown limited success in some cancers including colorectal cancer (CRC). The tumor microenvironment (TME) is largely responsible for response to therapy, and its constituents may provide robust biomarkers for successful immunotherapeutic approaches. In this study, we performed phenotypical characterization and critical analyses of key inhibitory ICs and T regulatory cell (Treg)-related markers on CD4⁺ T cell subsets in CRC patients, and compared with normal colon tissues and peripheral blood from the same patients. We also investigated correlations between the levels of different CD4⁺ T cell subsets and the clinicopathologic features including disease stage and tumor budding. We found a significant increase in the levels of CD4⁺FoxP3⁺Helios⁺ T cells, which represent potentially highly immunosuppressive Tregs, in the CRC TME. Additionally, tumor-infiltrating CD4⁺ T cells upregulated programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), T cell immunoglobulin and mucin domain-3 (TIM-3) and lymphocyte-activation gene 3 (LAG-3). We also characterized the expression of PD-1, CTLA-4, TIM-3, and LAG-3 on different CD4⁺FoxP3^−/+Helios^−/+ T cell subsets. Interestingly, we found that CTLA-4, TIM-3, and LAG-3 were mainly co-expressed on FoxP3⁺Helios⁺ Tregs in the TME. Additionally, FoxP3^high Tregs expressed higher levels of Helios, CTLA-4 and TIM-3 than FoxP3^low T cells. These results highlight the significance of Tregs in the CRC TME and suggest that Tregs may hamper response to IC blockade in CRC patients, but effects of different IC inhibition regimes on Treg levels or activity warrants further investigations. We also found that CD4⁺CTLA-4⁺ T cells in circulation are increased in patients with advanced disease stage. This study simultaneously provides important insights into the differential levels of CD4⁺ T cell subpopulations and IC expression in CRC TME, compared to periphery and associations with clinicopathologic features, which could be used as potential biomarkers for CRC progression and response to therapy.

Despite recent advances in colorectal cancer (CRC) treatment, a large proportion of patients show limited responses to therapies, especially in advanced stages. There is an urgent need to identify prognostic biomarkers and/or therapeutic targets in advanced stages, aiming to improve the efficacy of current treatments. We aimed to determine prognostic biomarkers in tumor tissue and circulation of CRC patients, with a special focus on T cell exhaustion markers. We found that mRNA levels of PD-1, TIM-3, CTLA-4, TIGIT, CD160, CD244, KLRG1, TOX2, TOX3, Ki-67, and PRDM1 were elevated in CRC tumor tissues. We also investigated differences in gene expression between early and advanced disease stages. We found that TOX and potentially TIM-3, CTLA-4, VISTA, TIGIT, KLRG1, TOX2, SIRT1, Ki-67, and Helios mRNA levels in tumor tissue were elevated in advanced disease stages, suggesting their potential roles in CRC progression. In contrast, PD-1 and CD160 levels in tumor tissue were downregulated in advanced stages. In the circulation of CRC patients, mRNA levels of PD-1, VISTA and LAG-3 were higher than those of healthy individuals. Moreover, in circulation, PD-1, CTLA-4 and TIGIT mRNA levels were reduced in advanced stages. Interestingly, levels of PD-1 in both tumor tissue and circulation were reduced in advanced stages, suggesting that targeting PD-1 in patients with advanced stages could be less effective. Altogether, these findings suggest some potential T cell exhaustion markers that could be utilized as prognostic biomarkers and/or therapeutic targets for CRC. However, further investigations and validations in larger cohorts are required to confirm these findings.

Background: Increased numbers of myeloid-derived suppressor cells (MDSCs) are positively correlated with poor prognosis and reduced survivals of cancer patients. They play central roles in tumor immune evasion and tumor metastasis. However, limited data are available on phenotypic/transcriptomic characteristics of the different MDSCs subsets in cancer. These cells include immature (I-MDSCs), monocytic (M-MDSCs), and polymorphonuclear/granulocytic (PMN-MDSCs).Methods: Phenotypic characterization of myeloid subsets from 27 colorectal cancer (CRC) patients was assessed by flow cytometric analyses. RNA-sequencing of sorted I-MDSCs, PMN-MDSCs, and antigen-presenting cells (APCs) was also performed.Results: We found that the levels of I-MDSCs and PMN-MDSCs were increased in tumor tissues (TT), compared with normal tissues (NT) in colorectal cancer. Our functional annotation analyses showed that genes associated with histone deacetylase (HDAC) activation- and DNA methylation-mediated transcriptional silencing were upregulated, and histone acetyl transferase (HAT)-related genes were downregulated in tumor-infiltrating I-MDSCs. Moreover, pathways implicated in cell trafficking and immune suppression, including Wnt, interleukin-6 (IL-6), and mitogen-activated protein kinase (MAPK) signaling, were upregulated in I-MDSCs. Notably, PMN-MDSCs showed downregulation in genes related to DNA methylation and HDAC binding. Using an ex vivo model, we found that inhibition of HDAC activation or neutralization of IL-6 in CRC tumor tissues downregulates the expression of genes associated with immunosuppression and myeloid cell chemotaxis, confirming the importance of HDAC activation and IL-6 signaling pathway in MDSC function and chemotaxis.Conclusions: This study provides novel insights into the epigenetic regulations and other molecular pathways in different myeloid cell subsets within the CRC tumor microenvironment (TME), giving opportunities to potential targets for therapeutic benefits.

Background: Cytotoxic CD8⁺ T cell-mediated response is the most important arm of adaptive immunity, which dictates the capacity of the host immune response in eradicating tumor cells. Due to tumor intrinsic and/or extrinsic factors, the density and function of CD8⁺ tumor-infiltrating lymphocytes (TILs) could be compromised, leading to poor prognosis and survival. Methods: Using RNA-Seq, transcriptomes of sorted CD3⁺CD8⁺ TILs from treatment-naïve colorectal cancer (CRC) patients at advanced stages (III and IV) were compared with those from patients with early stages (I and II). A signature referred to as ‘poor prognosis CD8 gene signature (ppCD8sig)’ was identified and analyzed in The Cancer Genome Atlas CRC dataset. Scores for the ppCD8sig were calculated and classified as high, intermediate and low, and its prognostic significance was assessed using multivariate analysis and Cox proportional hazard model. Densities of CD3⁺ and CD8⁺ T cell infiltration in tumors from patients with high and low ppCD8sig scores were assessed by flow cytometry and immunostaining. Results: Genes related to epigenetic regulation and response to hypoxia were upregulated in CD8⁺ TILs from patients with advanced stages, while genes related to T cell activation, cell proliferation and cell cycle were downregulated. Patients with high ppCD8sig score had poorer disease-specific survival (DSS) and shorter progression-free interval (PFI). The ppCD8sig was an independent prognostic indicator for DSS (HR 1.83, 95% CI 1.40 to 2.38, p<0.0001) and PFI (HR 1.42, 95% CI 1.04 to 1.93, p=0.026). Additionally, patients with high ppCD8sig score were more likely to have advanced stages (χ² p<0.0001) and residual disease after primary therapy (χ² p=0.046). Patients with high ppCD8sig score had reduced levels of CD3⁺ and CD8⁺ TILs and low Immunoscores (IS), compared to patients with low ppCD8sig score. Conclusions: Our data provided insights into the altered regulation of biological mechanisms and signaling pathways in CD8⁺ TILs during CRC progression, and revealed a gene signature as an independent prognostic indicator. Patients with high ppCD8sig score had lower levels of TILs and low IS. These data further confirm the prognostic value of the identified ppCD8sig and potentially highlight its clinical relevance.

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.

Immune checkpoint inhibition is an effective anti-cancer therapeutic approach but has shown limited efficacy in treating colorectal cancer (CRC) patients. Importantly, immune constituents of the tumor microenvironment (TME) can influence therapy response and cancer progression. We investigated the expression of immune checkpoints (ICs) on lymphoid populations within the CRC TME and compared with cells from normal colon tissues using samples from 50 patients with varying disease stages. We found that the levels of B cells, T cells, and NK cells were similar, IC-expressing CD4⁺ and CD4⁺CD8⁺ double positive T cells were higher, while CD8⁺ T cells and CD4⁻CD8⁻ double negative T cells were significantly lower in CRC tumors. Notably, patients with mismatch-repair deficiency/microsatellite instability-high tumors had higher levels of IC-expressing CD4⁺ and CD8⁺ T cells than patients with proficient MMR and microsatellite stable tumors. Lastly, The Cancer Genome Atlas Colon Adenocarcinoma datasets showed associations between low expression of selective genes and poorer progression-free interval. Our findings highlight differential expression of ICs on lymphoid cells in CRC tumors in the era of cancer immunotherapy, which at present is solely approved for anti-PD-1 therapy in patients with dMMR/MSI-H tumors. Further investigations into their functionality have potentials for deciphering resistance mechanisms to IC inhibition.