Tumor regulatory T cells potently abrogate antitumor immunity

Abstract

Regulatory T cell (Treg) from mice bearing a breast tumor were elevated (tumor Treg). In vitro, whereas tumor Treg ability to inhibit tumor-primed CD4(+) T cell activity is comparable to Treg from naive mice (naive Treg), only tumor Treg suppress naive CD8(+) T cell activation and DC function. Neither tumor Treg nor naive Treg can suppress antitumor immunity at the effector phase of the immune response induced by adoptively transferred tumor-primed CD4(+) T cells. This is consistent with the observation that, in this model, neither tumor Treg nor naive Treg can inhibit effectors in vitro or in vivo. However, tumor Treg abrogate tumor-specific CD8(+) T cell responses in tumor-draining lymph nodes and antitumor immunity at the early stage of the immune response induced by adoptively transferred tumor-primed CD4(+) T cells. These data indicate that, in this model, tumor Treg potently abrogate tumor-specific CD8(+) T cell responses in tumor-draining lymph nodes, thereby suppressing antitumor immunity at the early stage of the immune response induced by adoptively transferred tumor-primed CD4(+) T cells.

FIGURE 1

CD4⁺CD25⁺ T cells from splenocytes of tumor-bearing mice are elevated, express CTLA-4 and Foxp3, and exhibit suppressor function in vitro. A. Splenocytes from tumor-bearing or age-matched naïve mice were stained by anti-CD4-PETXRED and anti-CD25-PE, and analyzed by flow cytometry. One of two independent experiments with similar results is shown. B. The frequency of CD4⁺CD25⁺ T cells in CD4⁺ T cells in naïve (n=4) or tumor-bearing (n=7) mice is shown. Tumor vs. naïve: p<0.005. The surface (C) or intracellular (D) expression of CTLA-4 in purified tumor Treg or naïve Treg is shown in one of two independent experiments with similar results. E. The expression of Foxp3 in tumor Treg or tumor CD4⁺CD25⁻ T cells is shown in one of three independent experiments with similar results. F. Tumor-primed CD4⁺ T cells were cultured alone, with tumor Treg or tumor CD4⁺CD25⁻ T cells. IL-2 in the culture supernatant was determined by ELISA. CD4 vs. CD4 + tumor Treg: p<0.0005. CD4 vs. CD4 + tumor CD4⁺CD25⁻: NS. G. Tumor-primed CD4⁺ T cells, tumor Ag-loaded DC and naïve CD8⁺ T cells were cultured alone, with tumor Treg or tumor CD4⁺CD25⁻ T cells. IFN-γ in the culture supernatants was determined by ELISA. CD4/DC/CD8 vs. CD4/DC/CD8 + tumor Treg: p<0.0005; CD4/DC/CD8 vs. CD4/DC/CD8 + tumor CD4⁺CD25⁻: NS. The data represents three independent experiments.

FIGURE 2

Suppressor functions of tumor Treg and naïve Treg in vitro. A. Tumor-primed CD4⁺ T cells were cultured alone, with tumor Treg or naïve Treg at ratios of 1:1, 1:2 or 1:4 (CD4:Treg). IL-2 in the culture supernatants was determined by ELISA. CD4 vs. CD4 + tumor Treg or naïve Treg: p<0.0005. B. Tumor-primed CD4⁺ T cells, tumor Ag-loaded DC and naïve CD8⁺ T cells were cultured alone, with tumor Treg or naïve Treg at ratios of 1:1:1:1; 1:1:1:2 or 1:1:1:4 (CD4:DC:CD8:Treg). IFN-γ in the culture supernatant was determined by ELISA. CD4/DC/CD8 vs. CD4/DC/CD8 + tumor Treg: p<0.005. CD4/DC/CD8 vs. CD4/DC/CD8 + naïve Treg: NS. The data represents three independent experiments.

FIGURE 3

Tumor Treg suppress DC function in vitro. Splenic DC and tumor-primed CD4⁺ T cells were cultured alone, with tumor Treg or naïve Treg at a ratio of 1:1:1 (DC:CD4:Treg) in the presence of LPS. A. The data represents one of three independent experiments with similar results showing CD80 or CD86 expression on gated CD11c⁺ DC. B. IL-12 in the culture supernatant was determined by ELISA. DC/CD4 vs. DC/CD4 + tumor Treg: p<0.0005 (18h), p<0.005 (48h); DC/CD4 vs. DC/CD4 + naïve Treg: NS. The data represents three independent experiments.

FIGURE 4

Neither tumor Treg nor naïve Treg suppress antitumor immunity at the effector phase of the immune response induced by adoptively-transferred CD4⁺ T cells. A. Purified tumor-primed CD4⁺ T cells were adoptively transferred into naïve BALB/c mice on d −1. These mice were inoculated with tumor cells on d 0. Tumor Treg or naïve Treg were adoptively transferred on d 9. Mice adoptively transferred with tumor-primed CD4⁺ T cells alone served as a positive control. Mice without treatment served as a negative control. Animal survival is presented using Kaplan-Meier Survival Curves. The data represents two independent experiments with 4–5 mice per group. CD4 (n=8), CD4 + tumor Treg (n=9), CD4 + naïve Treg (n=8), non-treatment (n=8). B. Effectors from tumor-rejection mice were cultured with tumor Treg or naïve Treg in vitro. IFN-γ in the culture supernatant was determined by ELISA. Effectors vs. Effectors + tumor Treg or naïve Treg: NS. The data represents two independent experiments. C. Tumor Treg or naïve Treg were adoptively transferred into tumor-rejection mice, and effectors from these mice were cultured in vitro. IFN-γ in the culture supernatant was determined by ELISA. Effectors vs. Effectors + tumor Treg or naïve Treg: NS. The data represents two independent experiments.

FIGURE 5

Tumor Treg abrogate tumor-specific CD8⁺ T cell responses in TDLN and antitumor immunity at the early stage of the immune response induced by adoptively-transferred tumor-primed CD4⁺ T cells. A. Mice were adoptively cotransferred with tumor-primed CD4⁺ T cells and tumor Treg or naïve Treg on d −1, and inoculated with tumor cells on d 0. Mice adoptively transferred with tumor-primed CD4⁺ T cells alone served as a positive control. Mice without treatment served as a negative control. On d 5, CD8⁺ T cells purified from TDLN were restimulated by 4T1.2-Neu- or CT26-loaded DC in vitro. IFN-γ in the culture supernatants was determined by ELISA. CD4 vs. non-treatment: p<0.0005; CD4 vs. CD4 + tumor Treg: p<0.0005; CD4 vs. CD4 + naïve Treg: NS. The data represents two independent experiments with 3–4 mice per group. B. Mice were adoptively cotransferred with tumor-primed CD4⁺ T cells and tumor Treg or naïve Treg on d −1, and inoculated with tumor cells on d 0. Mice adoptively transferred with tumor-primed CD4⁺ T cells alone served as a positive control. Mice without treatment served as a negative control. Animal survival is presented using Kaplan-Meier Survival Curves. The data represents two independent experiments with 3–5 (with Treg) to 6–9 (without Treg) mice per group. CD4 (n=18), CD4 + tumor Treg (n=9), CD4 + naïve Treg (n=6), non-treatment (n=12).