TIM-3 expression characterizes regulatory T cells in tumor tissues and is associated with lung cancer progression

Abstract

Background: T cell immunoglobulin-3 (TIM-3) has been established as a negative regulatory molecule and plays a critical role in immune tolerance. TIM-3 is upregulated in exhausted CD8(+) T cells in both chronic infection and tumor. However, the nature of TIM-3(+)CD4(+) T cells in the tumor microenvironment is unclear. This study is to characterize TIM-3 expressing lymphocytes within human lung cancer tissues and establish clinical significance of TIM-3 expression in lung cancer progression.

Methodology: A total of 51 human lung cancer tissue specimens were obtained from pathologically confirmed and newly diagnosed non-small cell lung cancer (NSCLC) patients. Leukocytes from tumor tissues, distal normal lung tissues, and peripheral blood mononuclear cells (PBMC) were analyzed for TIM-3 surface expression by flow cytometry. TIM-3 expression on tumor-infiltrating lymphocytes (TILs) was correlated with clinicopathological parameters.

Conclusions: TIM-3 is highly upregulated on both CD4(+) and CD8(+) TILs from human lung cancer tissues but negligibly expressed on T cells from patients' peripheral blood. Frequencies of IFN-γ(+) cells were reduced in TIM-3(+)CD8(+) TILs compared to TIM-3(-)CD8(+) TILs. However, the level of TIM-3 expression on CD8(+) TILs failed to associate with any clinical pathological parameter. Interestingly, we found that approximately 70% of TIM-3(+)CD4(+) TILs expressed FOXP3 and about 60% of FOXP3(+) TILs were TIM-3(+). Importantly, TIM-3 expression on CD4(+) T cells correlated with poor clinicopathological parameters of NSCLC such as nodal metastasis and advanced cancer stages. Our study reveals a new role of TIM-3 as an important immune regulator in the tumor microenvironment via its predominant expression in regulatory T cells.

Figure 1. TIM-3 is highly expressed on both CD4⁺ and CD8⁺ tumor infiltrating T cells in lung cancer.

Tumor infiltrating lymphocytes (TILs) were harvested from lung cancer tissues, adjacent normal tissues, and peripheral blood monouclear cells (PBMCs) from whole blood of patients. Cells were then stained for CD4, CD8, TIM-3 and PD-1. A. Representative dot-plots show TIM-3 expression on CD4⁺ and CD8⁺ T cells in various tissues from lung cancer patients. Lymphocytes were gated for further analysis of CD4 and CD8 T cells. More than 90% CD4⁺ or CD8⁺ cells were CD3⁺. B. Summarized results of the percentage (%) of TIM-3 expression on CD4⁺ and CD8⁺ T cells from lung cancer patients are shown. Horizontal bars depict the mean percentage of TIM-3 expression on CD4⁺ and CD8⁺ T cells. Error bars: s.e.m. C. Dual expression of TIM-3 and PD-1 on gated CD4⁺ and CD8⁺ T cells is shown. The p-values were calculated using the one-way ANOVA.

Figure 2. IFN-γ production by TIM-3⁺ and TIM-3⁻ T cells in lung cancer tissues.

TILs were harvested from lung cancer tissue. Cells were then stimulated with plate-bound anti-CD3 mAbs (1 µg/ml) and plate-bound anti-CD28 mAbs (2 µg/ml) for 16 hours and incubated for the last 3 hours with Brefeldin A (10 µg/ml). Cells producing IFN-γ were examined with intracellular cytokine staining and flow cytometry. (A and B). Representative dot plots from one patient showed the percentage of TIM-3⁺IFN-γ⁺ and TIM-3⁻IFN-γ⁺ within CD8⁺ or CD4⁺ T cell compartment. Data shown are representative of six independent experiments. C. The average percentage of IFN-γ-producing CD8⁺ or CD4⁺ T cells among TIM-3⁺ and TIM-3⁻ fractions is shown.

Figure 3. TIM-3 expression on CD4⁺ TILs and Treg.

TILs were harvested from lung cancer tissue. Cells were then stained for CD4, TIM-3, and FOXP3 (A) or CD4, PD-1, and FOXP3 (B). Lymphocytes were gated for further analysis of CD4 and CD8 T cells. The percentage of each population within CD4⁺ T cell compartment was indicated. Data shown are representative of five independent experiments.

Figure 4. TIM-3 and PD-1 were up-regulated upon T cell activation.

(A and B). Human naïve T cells were purified from PBMCs of health donors. Cells were then stimulated with plate-bound anti-CD3 plus anti-CD28 mAb. 24, 48 and 72 hours later, cells were collected and stained for CD4, CD8, TIM-3 and PD-1. The expression of PD-1 and TIM-3 was analyzed by flow cytometry on gated CD4⁺ (A) or CD8⁺ (B) T cells. C. CD4⁺CD25^high T cells were isolated from peripheral blood by FACS. These cells were then stimulated with anti-CD3 and anti-CD28 in the presence of IL-2 (200 U/mL). 24, 48 and 72 hours later, cells were harvested and stained for CD4, TIM-3 and FOXP3. The expression of TIM-3 on CD4⁺FOXP3⁺ T cells is shown. Data are representative of three independent experiments.

Figure 5. Expression of Tim-3 on TILs in mouse model of transplantable tumor.

6–8-week-old C57BL/6 mice were inoculated with 2×10⁵ B16F0 cells i.d., tumor samples, spleens, and lymph nodes were removed when tumor sizes reached around 15 mm in diameters on day 20. TILs, splenocytes, and lymph node cells were isolated for analysis by flow cytometry. Tim-3 expression on mouse CD4⁺Foxp3⁺ or CD4⁺Foxp3⁻ T cells is shown. Data shown are representative of five independent experiments.