Intratumoral FoxP3+Helios+ Regulatory T Cells Upregulating Immunosuppressive Molecules Are Expanded in Human Colorectal Cancer

Abstract

Regulatory T cells (Tregs) can be antitumorigenic or pro-tumorigenic in colorectal cancer (CRC) depending on the presence of different Treg subsets with various immunosuppressive molecules. Some studies reported the phenotypic characteristics of tumor-infiltrating immune cells in CRC, but limited studies have focused on the co-expression of suppressive molecules on immune cells. The aim of this study was to characterize immune cells in the tumor microenvironment (TME), compared to paired adjacent non-tumor colon tissue of CRC patients. Additionally, we investigated co-expression of immunosuppressive molecules on different Treg subsets in the TME, normal colon tissue, and peripheral blood of CRC patients and healthy donors. In this preliminary study, we report that the majority of CD3+ T cells in the TME are CD4+ T cells with high co-expression of programmed death 1 (PD-1)/cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and PD-1/CD39 molecules. Levels of CD4+FoxP3+Helios+ Tregs were significantly increased in the TME. Furthermore, we observed increased levels of PD-1/CTLA-4 and PD-1/CD39 co-expressing cells within FoxP3+Helios+ and FoxP3+Helios- Treg subsets, indicative of their potent immunosuppressive potential. These results suggest synergistic associations between PD-1/CTLA-4 and PD-1/CD39 in dampening T-cell activation and function along with suppressing tumor-specific immune responses, suggesting that dual blockade of these molecules could be a more effective strategy for inducing antitumor immune responses in CRC.

Keywords: Forkhead box protein 3; Helios; colorectal cancer; immune checkpoint receptors; regulatory T cells; tumor microenvironment.

Figures

Figure 1

Phenotypic characterization of CD4+ T cells in non-tumor-infiltrating lymphocytes (NILs) and tumor-infiltrating lymphocytes (TILs). Freshly isolated NILs and TILs were stained for CD25, LAP, CD39, and PD-1 surface markers and their frequencies were calculated in CD4+ T cells. (A) Representative flow cytometric plots for these markers in NILs and TILs from a cancer patient. (B) Scatter plots showing the differences between NILs and TILs.

Figure 2

Co-expression of PD-1/CTLA-4 and PD-1/CD39 in CD4+ T cells in healthy donor (HD) peripheral blood mononuclear cells (PBMCs), colorectal cancer (CRC) patients PBMCs, non-tumor-infiltrating lymphocytes (NILs), and tumor-infiltrating lymphocytes (TILs). PBMCs from HD and CRC patients, NILs, and TILs were stained for CD3, CD4, PD-1, and CD39. After fixation and permeabilization, cells were stained for CTLA-4 expression. Live cells were gated using Fixable Viability Dye 660. Representative flow cytometric plots showing PD-1 and CTLA-4 in CD4+ T cells (A) and whisker plots (B) showing differences in their expression in HD-PBMC, CRC-PBMC, NILs, and TILs. (C) Pie charts show the relative percentages of PD-1 and CTLA-4 in CD4+ T cells. Representative flow cytometric plots showing different subpopulations of PD-1 and CD39 in CD4+ T cells (D) and whisker plots (E) showing differences in their expression in different samples.

Figure 3

FoxP3 and Helios expression in CD4+ T cells. Peripheral blood mononuclear cells (PBMCs) from healthy donor and colorectal cancer (CRC) patients, non-tumor-infiltrating lymphocytes (NILs) and tumor-infiltrating lymphocytes (TILs) were stained for CD3 and CD4 followed by FoxP3 and Helios intracellular staining. Live cells were first gated using Fixable Viability Dye 660. Representative flow cytometric plots of FoxP3 staining are shown in (A). Adjacent Scatter plot shows the differences between frequencies of regulatory T cells (Tregs) based on FoxP3 expression between different samples. (B) Non-parametric Spearman’s test was performed to investigate correlations between FoxP3, Helios, and PD-1 expressions in CRC-PBMCs and TILs. (C) Flow cytometric plots of FoxP3 and Helios co-expression in Tregs from different samples, and the adjacent whisker plot showing differences in various subsets of FoxP3 and Helios between them. (D) Pie charts show the relative percentages of three different FoxP3 and Helios Treg subsets.

Figure 4

PD-1 and CTLA-4 co-expression in different FoxP3 and Helios regulatory T cell (Treg) subsets. (A) Representative flow cytometric plots showing PD-1 and CTLA-4 co-expression in different FoxP3 and Helios Treg subsets from healthy donor (HD)-peripheral blood mononuclear cell (PBMC), colorectal cancer (CRC)-PBMC, non-tumor-infiltrating lymphocytes (NILs), and tumor-infiltrating lymphocytes (TILs). Whisker plots comparing the relative levels of PD-1+CTLA-4+ cells (B) and PD-1+CTLA-4− cells (C) in FoxP3−Helios+, FoxP3+Helios+, and FoxP3+Helios− Treg subsets. (D) Absolute percentages of PD-1+CTLA-4+ and PD-1+CTLA-4− in different FoxP3 (F) and Helios (H) Treg subsets in TILs.

Figure 5

PD-1 and CD39 co-expression in different FoxP3 and Helios regulatory T cell (Treg) subsets. Representative flow cytometric plots showing PD-1 and CD39 expression in different FoxP3 and Helios Treg subsets from healthy donor (HD)-peripheral blood mononuclear cell (PBMC), colorectal cancer (CRC)-PBMC, non-tumor-infiltrating lymphocytes (NILs), and tumor-infiltrating lymphocytes (TILs) are shown in panel (A). Whisker plots comparing the levels of PD-1+CD39+ cells (B) and PD-1−CD39+ cells (C) in FoxP3−Helios+, FoxP3+Helios+, and FoxP3+Helios− Treg subsets.