Interleukin-10 increases Th1 cytokine production and cytotoxic potential in human papillomavirus-specific CD8(+) cytotoxic T lymphocytes

Abstract

Interleukin-10 (IL-10) is widely known as an immunosuppressive cytokine by virtue of its ability to inhibit macrophage-dependent antigen presentation, T-cell proliferation, and Th1 cytokine secretion. However, several studies have challenged the perception of IL-10 solely as an immunosuppressive cytokine. As part of an investigation on potentiation of the cytotoxic activity of human papillomavirus E7-specific CD8(+) cytotoxic T lymphocytes (CTL) for adoptive transfusions to cervical cancer patients, we found that IL-10 in combination with IL-2, unlike several other combinations, including IL-2 with IL-12, gamma interferon (IFN-gamma), tumor necrosis factor alpha, and transforming growth factor beta, was able to consistently increase cytotoxicity. This augmentation in cytotoxic activity correlated with a significant increase in the cytoplasmic accumulation of perforin as detected by fluorescence-activated cell sorter. Surface expression of both the alpha and beta chains of the CD8 heterodimeric coreceptor and CD56 molecules was increased by exposure of CTL to IL-10. More importantly, we found that administration of IL-10 in combination with IL-2 after antigen stimulation consistently increased the intracellular expression of Th1 cytokines (i.e., IFN-gamma and IL-2) compared to results for control CD8(+) T cells cultured in IL-2 alone. In kinetic studies, proliferation, intracellular perforin levels, cytotoxic activity, and IFN-gamma expression were consistently elevated in CTL cultures containing IL-10 compared to control cultures, both at early and late time points following stimulation. In contrast, intracellular IL-2 expression was consistently increased only at early time points following stimulation with autologous tumor cells or solid-phase anti-CD3 antibody. Taken together, these data support the use of IL-10 in combination with IL-2 for the in vitro expansion and potentiation of tumor-specific CTL for clinical use in the therapy of cancer.

FIG. 1

(A) Effect of various concentrations of IL-10 on IL-2-induced proliferation of E7-specific CD8⁺ T cells at 6 to 7 days after antigen stimulation with autologous tumor cells. Pure populations of CD8⁺ T cells were cultured in CM (open bar) or in CM supplemented with increasing concentrations of IL-10 (solid bars). Cells were assayed for [³H]thymidine incorporation during the final 16 h of a 96-h culture. Results represent the means of triplicate wells ± standard deviations (SD). Thymidine incorporation in the presence of 1 to 20 ng of IL-10/ml plus IL-2, compared to that for control CTL cultured in IL-2 alone, was significant at P values <0.01 by Student's t test. No significant differences were noted when thymidine incorporation in the presence of 5 ng of IL-10/ml plus IL-2 was compared to levels in the presence of 10 and 20 ng of IL-10/ml plus IL-2. (B) Effect of 5 ng of IL-10/ml on IL-2-induced proliferation of E7-specific CD8⁺ T cells at 4 to 6, 8 to 10, and 14 to 16 days after antigen stimulation with autologous tumor cells. Pure populations of CD8⁺ T cells were cultured in CM (open bars) or in CM supplemented with 5 ng of IL-10/ml (solid bars). Cells were assayed for [³H]thymidine incorporation during the final 16 h of a 96-h culture. Results represent the means of triplicate wells ± SD. Thymidine incorporation in the presence of IL-10 plus IL-2, compared to that in control CTL cultured in IL-2 alone, was significant at P values <0.01 at all time points tested.

FIG. 2

Effect of IL-10 on the expression of CD8β (A) and CD8α (B) by HPV-specific CTL as analyzed by flow cytometry. CD8⁺ T cells at 6 to 7 days after antigen stimulation with autologous tumor cells were stained with different MAb after incubation in CM alone (light lines) or in the presence of 5 ng of IL-10/ml for 72 to 96 h (heavy lines). Dashed lines, histograms from cells stained with control MAb.

FIG. 3

Effect of IL-10 exposure on the percentage of CD56⁺ CD8⁺ T cells, as assessed by two-color flow cytometric analysis. The results from one experiment are shown and are representative of five separate experiments.

FIG. 4

(A) Effect of different cytokines in combination with IL-2 on the cytotoxic activity of E7-specific CTL. CD8⁺ T cells at 6 to 7 days from the last antigen stimulation with autologous tumor cells were cultured in CM (open bar; control) or in CM with different cytokines (solid bars), as described in Materials and Methods for 72 to 96 h before being tested in cytotoxicity assays against autologous tumor cells. Percent lysis (± standard deviation) at a 5:1 effector/target cell ratio is shown. The increase in cytotoxic activity, compared to that for control CTL cultured in IL-2 alone, was significant at P values <0.01 in the presence of IL-10 plus IL-2 and at P values <0.05 in the presence of IL-12 plus IL-2 by Student's t test. (B) Effect of various concentrations of IL-10 in combination with IL-2 on the cytotoxic activity of E7-specific CTL against autologous tumor targets. CD8⁺ T cells were cultured in CM (open bar; control) or in CM with the addition of various doses of IL-10 (solid bars) for 72 to 96 h before being tested in cytotoxicity assays against autologous tumor cells. Percent lysis (± standard deviation) at a 5:1 effector/target cell ratio is shown. The increase in cytotoxic activity, compared to that for control CTL cultured in IL-2 alone, was significant at P values <0.01 in the presence of 5 to 20 ng of IL-10/ml (C) Effect of 5 ng of IL-10/ml on the cytotoxic activity of E7-specific CD8⁺ T cells at 4 to 6, 8 to 10, and 14 to 16 days after antigen stimulation with autologous tumor cells. Pure populations of CD8⁺ T cells were cultured in CM (open bars) or in CM supplemented with 5 ng of IL-10/ml (solid bars) for 72 to 96 h before being tested in cytotoxicity assays against autologous tumor cells. Percent lysis (± standard deviation) at a 5:1 effector/target cell ratio is shown. Increased cytotoxic activity by CTL cultured in the presence of IL-10 plus IL-2, compared to that for control CTL cultured in IL-2 alone, was significant at P values <0.01 at all time points tested.

FIG. 5

Effect of 5 ng of IL-10/ml in combination with IL-2 on the cytotoxic activity of E7-specific CTL measured in a 6-h ⁵¹Cr release assay against autologous tumor cells, autologous tumor cells plus anti-HLA class I blocking MAb (W6/32), autologous LCL, and K562. CD8⁺ T cells at 6 to 7 days after antigen stimulation with autologous tumor cells were cultured in CM (open bar; control) or in CM with the addition of 5 ng of IL-10/ml (solid bars) for 72 to 96 h before being tested in cytotoxicity assays. Percent lysis (± standard deviation) at a 5:1 effector/target cell ratio is shown. Inhibition of CTL-mediated killing by anti-HLA class I MAb (50 μg/ml) was significant at P values <0.01 for CD8⁺ T cells cultured in the presence of 5 ng of IL-10/ml plus IL-2 as well as control CTL cultured in IL-2 alone.

FIG. 6

Effect of 72 to 96 h of exposure to IL-10 on the expression of intracellular perforin by HPV-specific CTL, as analyzed by flow cytometry. CD8⁺ T cells at 6 to 7 days (A) and 14 to 16 days (B) after antigen stimulation with autologous tumor cells were cultured in CM alone (light lines) or in the presence of 5 ng of IL-10/ml (heavy lines) before being stained with FITC-conjugated MAb against perforin, as described in Materials and Methods. Dashed lines, histograms from cells stained with isotype control MAb.

FIG. 7

Two-color flow cytometric analysis of intracellular IFN-γ and IL-4 expression by tumor-specific CD8⁺ T cells. CD8⁺ T cells at 2 to 4 days (A and B), 8 to 10 days (C and D), and 14 to 16 days (E and F) after antigen stimulation with autologous tumor cells were cultured in CM alone (A, C, and E) or in the presence of 5 ng of IL-10/ml for 72 to 96 h (B, D, and F) before being activated overnight with solid-phase anti-CD3 in the presence of brefeldin A, as described in Materials and Methods. A representative experiment is shown.

FIG. 8

Two-color flow cytometric analysis of intracellular IL-2 and IL-4 expression by tumor specific CD8⁺ T cells. CD8⁺ T cells at 2 to 4 days (A and B), 8 to 10 days (C and D), and 14 to 16 days (E and F) after antigen stimulation with autologous tumor cells were cultured in CM alone (A, C, and E) or in the presence of 5 ng of IL-10/ml for 72 to 96 h (B, D, and F) before being activated overnight with solid-phase anti-CD3 in the presence of brefeldin A, as described in Materials and Methods. A representative experiment is shown.