Investigation of the prognostic value of CD4 T cell subsets expanded from tumor-infiltrating lymphocytes of colorectal cancer liver metastases

Abstract

Background: The positive role of CD8+ tumor-infiltrating lymphocytes (TIL) in patients with colorectal cancer (CRC) has been well described but the prognostic value of CD4 T cell subsets remained to be investigated. In this study, we expanded TIL from surgically resected liver metastases of patients with CRC and characterized the phenotype and the prognostic value of expanded-CD4 T cells.

Methods: Liver metastases were surgically resected from 23 patients with CRC. Tumors were enzymatically digested and cultured in high dose of interleukin-2 for up to 5 weeks. T cell phenotype and reactivity of cultured-T cells were measured by flow cytometry and correlated with patients' clinical outcomes.

Results: We successfully expanded 21 over 23 TIL from liver metastases of patients with CRC. Interestingly, we distinguished two subsets of expanded T cells based on T cell immunoglobulin mucin domain-containing protein 3 (TIM-3) expression. Medians fold expansion of expanded T cells after rapid expansion protocol was higher in CD3+TIM-3low cultures. In an attempt to investigate the correlation between the phenotype of expanded CD4 T cells and clinical outcomes, we observed on one hand that the level of Tregs in culture as well as the expression of both PD1 and TIM-3 by expanded T cells was not correlated to the clinical outcomes. Interestingly, on the other hand, cultures containing high levels of Th17 cells were associated with a poor prognosis (p=0.0007).

Conclusions: Our data confirmed the presence of Th17 cells in expanded T cells from liver metastases. Among CD4 T cell characteristics investigated, TIM-3 but not programmed cell death protein 1 predicted the expansion capacity of TIL while only the Th17 phenotype showed correlation with patients' survival, suggesting a particular role of this T cell subset in CRC immune contexture.

Trial registration number: NCT02817178.

Keywords: CD4-positive t-lymphocytes; adaptive immunity; biomarkers; gastrointestinal neoplasms; lymphocytes; tumor; tumor-infiltrating.

Conflict of interest statement

Competing interests: None declared.

© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

Flow chart of available material and analysis plan. mCRC, metastatic colorectal cancer; REP, rapid expansion protocol; TIL, tumor-infiltrating lymphocytes.

Figure 2

Fold expansion and characteristics of expanded tumor-infiltrating lymphocytes (TIL). (A) Scheme of isolation and expansion of liver metastases-derived TIL. (B) Median fold expansion after rapid expansion protocol (REP) (3438 (451–11 118)). (C, D) Frequencies of CD4 and CD8 T cells at three major steps : isolation day (n=10), Pre-REP (n=21) and Post-REP (n=21). (E, F) Median frequencies of CD28, respectively, on CD4 and CD8 T cells before REP (94.9 (31.6–99.4), 93.7 (19.0–99.0)) and after (58.1 (5.4–98.3), 63.3 (7.3–87.5)). (G) Gating strategy of REP T-cells memory profile. (H, I) CD8 and CD4 T cells memory profile after REP. (J) Unsupervised hierarchical clustering of the percentages of CD4 and CD8 T cells expressing T cell immunoglobulin mucin domain-containing protein 3 and programmed cell death protein 1, CD4 T cells polarization and Treg level (CD4+CD25HighFoxP3+CD127−) from pre-REP T cells. Statistical differences were calculated using Mann-Whitney test. Median (min−max). ns, not significant.

Figure 3

T cell immunoglobulin mucin domain-containing protein 3 (TIM-3) expression discriminates T-cell cultures fold expansion. (A) Gating strategy of inhibitory receptor expression profile on pre-rapid expansion protocol (REP) T-cells. (B, C) CTLA4, TIM-3 and programmed cell death protein 1 (PD1) expression (%) on CD4 and CD8 T cells were, respectively, 1.7% (0.1–7.2) and 1.3% (0.0–13.7), 56.6% (3.2–96.2) and 45.8% (6.7–99.5), 47.8% (21.7–86.4) and 57.1% (11.2–87.0). (D, E) Medians for PD1 expression (%) before and after REP, respectively, on CD4 (47.3 (21.7–86.4) and 33.4 (10.7–63.8) (p=0.0026)) and CD8 T cells (56.6 (11.2–87.0) and 21.2 (4.6–44.7) (p

Figure 4

Functional potential of rapid expansion protocol (REP) T-cells. REP T cells were subjected to a 16 hours CD3/28 bead stimulation (1:1) and analyzed by flow cytometry using intracellular staining for Th1 cytokines (IFN-γ, TNF-α and interleukin-2 (IL-2)) gating on CD4 and CD8 T cell populations (n=11). (A, B) IFN-γ, TNF-α and IL-2 expression by (A) CD4 and CD8 REP T cells and (B) according to TIM-3 expression. (C) CD107a production by previously stimulated CD8 REP T cells (n=8). Median (%) with 95 CI or median (%) (min–max). Mann-Whitney tests: *p

Figure 5

Characterization of Treg within T-cell cultures. (A) Regulatory T cells (Treg) was investigated by intracellular staining for FoxP3 on the CD4+CD25HighCD127− population before and after the rapid expansion protocol (REP) (p=0.2921). (B) Median fold expansion in Treg low and in Treg high culture. There was no significant difference (p=0.5920) (C) Percentage of Treg before and after REP according to T cell immunoglobulin mucin domain-containing protein 3 (TIM-3) expression on CD8 T cells. (D) Glycoprotein-A repetitions predominant (GARP) expression (%) on Treg within peripheral blood mononuclear cells isolated from healthy donors, patients with metastatic colorectal cancer (mCRC) and match Pre-REP T cells. Plots are representative of at least five experiments. (E, F) Kaplan-Meier curve for relapse free survival () according to the percentage of (E) Treg and (F) CD8+TIM-3+ T cells (low vs high) in Pre-REP T cell cultures following resection of liver metastases from patients with mCRC. Log-rank test was used for the comparison of survival curves. Mann-Whitney tests: *p<0.05, ns, not significant; PBMC, peripheral blood mononuclear cells; TIL, tumor-infiltrating lymphocytes.

Figure 6

Characterization and prognostic value of expanded Th17 lymphocytes. (A) Gating strategy of CD4 T cells Th1, Th17 and Th1* subsets relied on cytokine production of interleukin (IL)-17A and IFN-γ in regard of their chemokine receptors expression. (B, C) Levels of each Th cells within pre-rapid expansion protocol (REP) culture were analyzed according to (B) CD4+TIM-3low/high populations and (C) CD8+TIM-3low/high population.(D) Median frequencies of Th1, Th17, Th1* CD4 T cells and double negative subsets were, respectively, 34.0% (0.2–81.5), 50.7% (10.5–99.4), 5.9% (0.2–33.0) and 4.2% (0.0–29.5).(E–G) Kaplan-Meier curve for rapid expansion protocol (RFS) according to the percentage of Th1, Th1* and Th17 T cell (low vs high) in Pre-REP T cell cultures following resection of liver metastases from patients with metastatic colorectal cancer. Log-rank test was used for the comparison of survival curves. Median (%). *P<0.05, **p<0.01; ***p<0.001, ****, p<0.0001; ns, not significant; TIM-3, T cell immunoglobulin mucin domain-containing protein 3.