Immune Checkpoints in Circulating and Tumor-Infiltrating CD4+ T Cell Subsets in Colorectal Cancer Patients

Abstract

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.

Differential gene expression of tumor-infiltrating CD4 + T cells in advanced versus early stage colorectal cancer and identification of a gene signature of poor prognosis

“…21 We have previously reported that CD4 + T cell infiltration was significantly higher in the CRC microenvironment, compared with normal tissue with no significant differences between early and advanced stages. 12 In this study, we investigated the transcriptomes and potential functional networks of CD4 + TILs from various stages of CRC. Our analyses led to developing a gene signature based on the transcriptome of CD4 + TILs with a prognostic potential for CRC.…”

<p>Programmed Death-1 Receptor (PD-1) as a Potential Prognosis Biomarker for Ovarian Cancer Patients</p>

“…10 Similarly, an elevated percentage of CD3 + and CD8 + T cells in colorectal cancer was found to be related to improved OS, whereas a decreased percentage of T-cells was associated with poor prognosis. 41 As shown by Darb-Esfahani and colleagues, the presence of different subpopulations of TILs, including CD4 + and CD8 + T cells, is related to a better prognosis for OC patients. 32 Similar findings have been reported by Webb and co-workers.…”

Transcriptomic Analyses of Myeloid-Derived Suppressor Cell Subsets in the Circulation of Colorectal Cancer Patients

“…All patients provided written informed consent prior to sample collection. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood using Histopaque gradient centrifugation and stored, as previously described ( 17 ). This study was performed under ethics approvals from Hamad Medical Corporation, Doha, Qatar (protocol no.…”

“…Isolated PBMCs were stained as previously described ( 17 , 18 ). Briefly, cells were stained with antibodies against CD33–fluorescein isothiocyanate (clone HIM3-4; BD Biosciences, Oxford, UK), HLA DR–phycoerythrin (clone G46-6; BD Biosciences), CD14–phycoerythrin–Cy7 (clone M5E2; BD Biosciences), and CD15–allophycocyanin (clone HI98; BioLegend, San Diego, CA, USA).…”

“…7-AAD viability dye (eBioscience, San Diego, CA, USA) was used to identify live cells. The cells were prepared for analyses and sorting as previously described ( 17 , 18 ) Minimal sorter-induced cell stress was ensured following applicable measures, as previously described ( 19 ). FlowJo V10 software (FlowJo, Ashland, OR, USA) was used to perform data analyses.…”