Circulating and Tumor-Infiltrating Immune Checkpoint-Expressing CD8+ Treg/T Cell Subsets and Their Associations with Disease-Free Survival in Colorectal Cancer Patients

Abstract

T cells in the tumor microenvironment (TME) have diverse roles in anti-tumor immunity, including orchestration of immune responses and anti-tumor cytotoxic attack. However, different T cell subsets may have opposing roles in tumor progression, especially in inflammation-related cancers such as colorectal cancer (CRC). In this study, we phenotypically characterized CD3⁺CD4^- (CD8⁺) T cells in colorectal tumor tissues (TT), normal colon tissues (NT) and in circulation of CRC patients. We investigated the expression levels of key immune checkpoints (ICs) and Treg-related markers in CD8⁺ T cells. Importantly, we investigated associations between different tumor-infiltrating CD8⁺ T cell subpopulations and disease-free survival (DFS) in CRC patients. We found that FoxP3 expression and ICs including PD-1, CTLA-4, TIM-3, and LAG-3 were significantly increased in tumor-infiltrating CD8⁺ T cells compared with NT and peripheral blood. In the TME, we found that TIM-3 expression was significantly increased in patients with early stages and absent lymphovascular invasion (LVI) compared to patients with advanced stages and LVI. Importantly, we report that high levels of certain circulating CD8⁺ T cell subsets (TIM-3-expressing, FoxP3^-Helios^-TIM-3⁺ and FoxP3^-Helios⁺TIM-3⁺ cells) in CRC patients were associated with better DFS. Moreover, in the TME, we report that elevated levels of CD25⁺ and TIM-3⁺ T cells, and FoxP3⁺Helios^-TIM-3⁺ Tregs were associated with better DFS.

Keywords: CD8+ T cells; PBMCs; Progression-Free Survival; TILs; Tregs; immune checkpoints.

Figure 1

Flow cytometric plots, scatter plots and Kaplan–Meier survival curves for DFS based on the levels of CD25-, FoxP3-, and Helios-expressing CD3⁺CD4⁻ T cells. Representative flow cytometric plots and scatter plots present CD25 (A), FoxP3 (B) and Helios (C) expression in CD8⁺ T cells in PBMCs, NILs and TILs. CRC patients with high levels of CD25 (D), FoxP3 (E) and Helios (F) expressing CD8⁺ T cells were compared with patients with low levels of these cells to determine DFS. The number of patients included in each comparison are specified on each survival curve. Statistical analysis are shown with significance levels indicated at * p < 0.05, ** p < 0.01, and **** p < 0.0001.

Figure 2

Flow cytometric plots, scatter plots, and Kaplan–Meier survival curves for DFS based on the levels of PD-1, CTLA-4, TIM-3, and LAG-3-expressing CD3⁺CD4⁻ T cells. Representative flow cytometric and scatter plots present the levels of PD-1(A), CTLA-4 (B), TIM-3 (C), and LAG-3 (D) expressing CD8⁺ T cells in PBMCs, NILs, and TILs. CRC patients with high levels of PD-1 (E), CTLA-4 (F), TIM-3 (G), and LAG-3 (H)-expressing cells were compared with patients with low levels of these cells to determine DFS. The number of patients included in each comparison are specified on each survival curve. Statistical analysis are shown with significance levels indicated at * p < 0.05 and **** p < 0.0001.

Figure 3

Comparison of TIM-3 and PD-1 expression on CD3⁺CD4⁻ T cells in PBMCs, TILs and NILs. CRC patients were grouped based on disease stages (early; stage I/II and advanced; stage III/IV), anatomical location (right-sided and left-sided) and absent/present LVI. Scatter plots present the differences in the levels of TIM-3-expressing cells in early and advanced stages (A), absent/present LVI (B), right-sided and left-sided tumors (C), and levels of PD-1 in early and advanced stages (D), absent/present LVI (E), and right-sided and left-sided tumors (F) in PBMCs, NILs, and TILs.

Figure 4

Comparison of different immune checkpoint expression on FoxP3⁻Helios⁻ and FoxP3⁻Helios⁺ in CD3⁺CD4⁻ T cells. Patients were divided based on the expression of Helios. Scatter plots show the differences in levels of immune checkpoint expression on CD8⁺FoxP3⁻Helios⁻ and CD8⁺FoxP3⁻Helios⁺ T cells in PBMCs (A), NILs (B), and TILs (C). Statistical analysis are shown with significance levels indicated at * p < 0.05, *** p < 0.001 and **** p < 0.0001.

Figure 5

Kaplan–Meier survival curves for DFS based on the levels of FoxP3⁻Helios⁻ and FoxP3⁻Helios⁺ CD3⁺CD4⁻ T cells expressing PD-1, CTLA-4, and TIM-3. CRC patients with high levels of FoxP3⁻Helios⁻ PD-1⁺ (A), CTLA-4⁺ (B), TIM-3⁺ (C), and FoxP3⁻Helios⁺ PD-1⁺ (D), CTLA-4⁺ (E), and TIM-3⁺ (F) cells were compared with CRC patients with low levels of these cells to determine DFS. The number of patients included in each comparison are specified on each survival curve.

Figure 6

Kaplan–Meier survival curves for DFS based on the levels of FoxP3⁺Helios⁺ and CD3⁺CD4⁻FoxP3⁺Helios⁻ CD3⁺CD4⁻ T cells expressing PD-1, CTLA-4, and TIM-3 in TILs. FoxP3⁺Helios⁺PD-1⁺ (A), FoxP3⁺Helios⁻PD-1⁺ (B), FoxP3⁺Helios⁺CTLA-4⁺ (C), FoxP3⁺Helios⁻CTLA-4⁺ (D), FoxP3⁺Helios⁺TIM-3⁺ (E), and FoxP3⁺Helios⁻TIM-3⁺ (F) cell subsets were grouped into low and high groups, and DFS determined. The number of patients included in each comparison are specified on each survival curve.

Figure 7

Kaplan–Meier curves of DFS based on levels of CD3⁺CD4⁻TIM-3⁺ in PBMCs, TILs, and NILs in the group of patients with LVI. Patients in the presence of LVI were divided into high and low groups for TIM-3 expression in PBMCs (A), TILs (B), and NILs (C), and DFS was determined for these groups. The number of patients included in each comparison are specified on each survival curve.