Stimulatory role of interleukin 10 in CD8+ T cells through STATs in gastric cancer

Abstract

CD8⁺ T cells are considered to be critical in tumor surveillance and elimination. Increased CD8⁺ T cell frequency and function is associated with better prognosis in cancer patients. Interleukin 10 is a cytokine with controversial roles in CD8⁺ T cell-mediated anti-tumor immunity. We therefore examined the interleukin 10 expression and consumption in CD8⁺ T cells harvested from the peripheral blood and resected tumors of gastric cancer patients of stages II-IV. We found that the gastric cancer patients presented significantly elevated frequencies of interleukin 10-expressing cells in both CD4⁺ and CD8⁺ T cells compared to healthy controls. But distinctive from the interleukin 10-expressing CD4⁺ T cells, which increased in frequency in advanced cancer, the interleukin 10-expressing CD8⁺ T cells did not increase with cancer stage in the peripheral blood and actually decreased with cancer stage in resected tumor. Interleukin 10 and interleukin 10 receptor expression was also enriched in interferon gamma-expressing activated CD8⁺ T cells. Compared to interleukin 10-nonexpressing CD8⁺ T cells, interleukin 10 receptor-expressing CD8⁺ T cells secreted significantly elevated interferon gamma levels. Treatment of anti-CD3/CD28-stimulated, purified CD8⁺ T cells with interleukin 10 alone could significantly enhance CD8⁺ T cell survival, an effect dependent on interleukin 10 receptor expression. Interleukin 10 also increased CD8⁺ T cell proliferation synergistically with interferon gamma but not alone. Analysis of downstream signal transducer and activator of transcription molecules showed that interleukin 10 treatment significantly increased the phosphorylation of signal transducer and activator of transcription 3 and signal transducer and activator of transcription 1 to lesser extent. Together, these results demonstrate that interleukin 10 possessed stimulatory roles in activated CD8⁺ T cells from gastric cancer patients.

Keywords: Interleukin 10; gastric cancer.

Figure 1.

IL-10-expressing CD4⁺ and CD8⁺ T cells in gastric cancer patients and controls. (a) Representative gating of IL-10-expressing cells in CD4⁺ and CD8⁺ T cells. PBMCs were stimulated with PMA + ionomycin for 5 h with brefeldin A before intracellular staining. Gating standard was set using an isotype control antibody. (b) Frequency of IL-10-expressing cells as a percentage in CD4⁺ T cells in controls and gastric cancer patients of stages II–IV. (c) Frequency of IL-10-expressing cells as a percentage in CD8⁺ T cells in controls and gastric cancer patients of stages II–IV (*p < 0.05; ***p < 0.001).

Figure 2.

IL-10R expression in CD4⁺ and CD8⁺ T cells in gastric cancer patients and controls. (a) Representative gating of IL-10R-expressing cells in CD4⁺ and CD8⁺ T cells. (b) Frequency of IL-10R-expressing cells as a percentage in CD4⁺ T cells in controls and gastric cancer patients of stages II–IV. (c) Frequency of IL-10R-expressing cells as a percentage in CD8⁺ T cells in controls and gastric cancer patients of stages II–IV. (d) Representative IL-10R expression by IL-10⁺ CD8⁺ T cells versus IL-10⁻ CD8⁺ T cells (left: IL-10 vs IL-10R contour plot in CD8⁺ T cells and right: histogram comparison of IL-10R expression by IL-10⁺ (black unfilled) vs IL-10⁻ (gray filled) CD8⁺ T cells). (e) Differences in IL-10R expression level between IL-10⁺ and IL-10⁻ cells in CD4⁺ and CD8⁺ T cells from healthy controls and gastric cancer patients (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 3.

Expression of IFN-γ by IL-10⁺ versus IL-10⁺ CD8⁺ T cells. (a) The frequencies of IFN-γ-expressing cells in IL-10⁺ versus IL-10⁻ CD8⁺ T cells in healthy control individuals and gastric cancer patients of stages II–IV. PBMCs were stimulated with PMA + ionomycin for 5 h with brefeldin A before intracellular staining (mean ± standard deviation). (b and c) IL-10R⁺ and IL-10R⁻ CD8⁺ T cells from gastric cancer patients were sorted using the gate setting in Figure 2(a) and cultured separately at 10⁵ cells per mL with plate-bound anti-CD3/CD28. Supernatant was harvested after 12 h. (b) Secreted IL-10 level and (c) secreted IFN-γ level were measured by ELISA (*p < 0.05; ***p < 0.001).

Figure 4.

Apoptosis and proliferation by CD8⁺ T cells after treatment with IL-10 and IFN-γ. Purified CD8⁺ T cells were incubated at 2 × 10⁵ cells per mL in the presence of plate-bound anti-CD3/CD28 (2 µg/mL each) for 72 h without additional cytokine (blank) or with 10 µg/mL IL-10 and/or IFN-γ. In some cases, the CD8⁺ T cells were pre-labeled with CFSE before incubation. (a) Representative gating of apoptotic cells (Annexin V⁺) in blank-treated CD8⁺ T cells. (b) Representative Annexin V binding in IL-10-treated IL-10R⁺ versus IL-10R⁻ CD8⁺ T cells in the same gastric cancer individual. (c) Summary of the percentage of apoptotic cells in CD8⁺ T cells cultured under various conditions. (d) Comparison between IL-10R⁺ and IL-10R⁻ CD8⁺ T cells in the percentage of apoptotic cells under IL-10 treatment. (f) Representative CFSE-low gating in blank-treated versus IFN-γ-treated CD8⁺ T cells. (g) Percentage of proliferating cells (CFSE-low) in CD8⁺ T cells under various conditions (n = 7 for all experiments; *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 5.

Phosphorylation pattern in STATs after IL-10 treatment. (a) Representative pSTAT1, pSTAT3, and pSTAT5 staining pattern in IL-10-treated versus blank-treated IL-10R⁺ CD8⁺ T cells. (b) Summary of pSTAT1, pSTAT3, and pSTAT5 expression levels in blank and IL-10-treated IL-10R⁻ versus IL-10R⁺ CD8⁺ T cells (n = 7 gastric cancer patients). (c) Summary of pSTAT1, pSTAT3, and pSTAT5 expression levels in blank and IL-10-treated IL-10R⁻ versus IL-10R⁺ CD8⁺ T cells (n = 7 healthy controls; mean ± standard deviation; *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 6.

IL-10 expression by tumor-infiltrating CD8⁺ T cells in gastric cancer patients. (a) Frequency of CD8⁺ T cells in tumor-infiltrating lymphocytes in resected gastric tumors. (b) Frequency of IL-10-expressing cells in tumor-infiltrating CD8⁺ T cells from resected gastric tumors. Tumor-infiltrating lymphocytes were stimulated with PMA + ionomycin for 5 h with brefeldin A before intracellular staining. (c) Isolated CD8⁺ T cells were cultured at 10⁵ cells per mL with plate-bound anti-CD3/CD28. Supernatant was harvested after 12 h, and secreted IL-10 level was measured by ELISA (*p < 0.05; **p < 0.01).