Functional impairment of infiltrating T cells in human colorectal cancer

Abstract

T cells play a crucial role in preventing the growth and spread of colorectal cancer (CRC). However, immunotherapies against CRC have only shown limited success, which may be due to lack of understanding about the effect of the local tumor microenvironment (TME) on T cell function. The goal of this study was to determine whether T cells in tumor tissue were functionally impaired compared to T cells in non-tumor bowel (NTB) tissue from the same patients. We showed that T cell populations are affected differently by the TME. In the tumor, T cells produced more IL-17 and less IL-2 per cell than their counterparts from NTB tissue. T cells from tumor tissue also had impaired proliferative ability compared to T cells in NTB tissue. This impairment was not related to the frequency of IL-2 producing T cells or regulatory T cells, but T cells from the TME had a higher co-expression of inhibitory receptors than T cells from NTB. Overall, our data indicate that T cells in tumor tissue are functionally altered by the CRC TME, which is likely due to cell intrinsic factors. The TME is therefore an important consideration in predicting the effect of immune modulatory therapies.

Keywords: Colorectal cancer; IL-2; T cells; exhaustion; proliferation.

Figure 1.

Tumor tissue has a distinct T cell infiltrate compared to NTB tissue. Tumor and NTB tissue from patients with CRC was mechanically and enzymatically digested to extract single cells and analyzed by flow cytometry. (A) Gating strategy used to identify CD4⁺ and CD8⁺T cell populations: regulatory T cells, IFNγ producing T cells, inflammatory (IL-17⁺) T cells and IL-2-producing T cells. All gating was determined by fluorescence minus one control. The difference in frequency of (B) CD4⁺ and (C) CD8⁺ T cells populations between matched tumor and NTB tissue samples from individual patients. Statistical analyses were calculated using Wilcoxon matched-pairs signed rank test. Dotted line represents no difference in frequency of indicated subset between NTB and tumor tissue from individual patients. The bar represents the median (N = 23–88. *p < 0.05, ***p <0.001, ****p < 0.0001).

Figure 2.

Lower frequencies of IFNγ⁺IL-2⁺ T cells and higher frequencies of IFNγ⁺IL-17⁺T cells in tumor tissue compared to matched NTB tissue. Tumor and NTB tissue from patients with CRC was mechanically and enzymatically digested to extract single cells and analyzed by flow cytometry. (A) Representative plots of NTB and tumor tissue showing identification of CD4⁺IFNγ⁺IL-2⁺IL-17⁻ cells. (B) Difference in frequency of CD4⁺(left) and CD8⁺(right) IFNγ⁺IL-17⁻IL-2⁺ T cells between NTB tissue and tumor tissue. (C) Representative flow plots of NTB and tumor tissue showing demonstrating identification of CD4⁺IFNγ⁺IL-2⁻IL-17⁺ cells. (D) Difference in frequency of CD4⁺(left) and CD8⁺(right) IFNγ⁺IL-17⁺IL-2⁻ T cells between NTB tissue and tumor tissue. Statistical analyses were calculated using Wilcoxon-matched pairs signed rank test. Dotted line represents no difference in frequency of indicated subset between NTB and tumor tissue from individual patients. The bar represents the median (N = 23–88. **p < 0.01).

Figure 3.

Tumor-infiltrating T cells produce different amounts of IL-17 and IL-2 than T cells from NTB tissue. Tumor and NTB tissue from patients with CRC was mechanically and enzymatically digested to extract single cells and analyzed by flow cytometry. (A) Representative plot of NTB and tumor tissue showing difference in MFI of CD4⁺ IL-17 producing T cells. (B) Difference in MFI of IL-17⁺CD4⁺ and IL-17⁺CD8⁺ T cells from tumor tissue compared to matched NTB tissue. (C) Representative plot of NTB and tumor tissue showing difference in MFI of CD4⁺ IL-2-producing T cells. (D) Difference in MFI of IL-2⁺CD4⁺ and IL-2⁺CD8⁺ T cells from tumor tissue compared to matched NTB tissue. Statistical analyses were calculated using Wilcoxon-matched pairs signed rank test. Dotted line represents no difference in frequency of indicated subset between NTB and tumor tissue from individual patients. The bar represents the median (N = 23–88. *p < 0.05, **p < 0.01).

Figure 4.

T cells from tumor tissue have impaired proliferative ability. T cells were isolated from NTB and tumor tissue of Stage I–IV patients and stimulated with anti-CD3/CD28 beads. Ki67 expression was measured by flow cytometry after 72 h. (A) Frequency of Ki67⁺ T cells between matched pairs of NTB and tumor tissue. (B) Frequency of IL-2-producing cells of total T cells in matched NTB and tumor tissue. (C) Difference in MFI of IL-2-producing T cells from tumor and NTB tissue after 3 d of culture. (D, E) Frequency of Ki67⁺ T cells with and without removal of CD25^hi cells from NTB (D) and tumor tissue (E). Experiments were performed on matched pairs of tissue from 11 (A) and 5 (B–E) different patients. Statistical analyses were calculated using Wilcoxon-matched pairs signed rank test, *p < 0.05.

Figure 5.

Higher frequency of exhausted T cells ex vivo from tumor compared to NTB tissue. Expression of inhibitory receptors on T cells from Stage I–IV patients was examined ex vivo by flow cytometry. Frequency (A) and MFI (B) of T cells expressing inhibitory receptors. (C) Frequency of T cells expressing only LAG-3⁺PD-1⁺ or no inhibitory receptors as identified by Boolean gating. Data is shown from matched pairs of NTB and tumor tissue from six individual patients. Statistical analyses were calculated by two-way ANOVA, *p < 0.05.