The interaction between murine melanoma and the immune system reveals that prolonged responses predispose for autoimmunity

Abstract

An assessment of antitumor immunity versus autoimmunity as provoked by the specific depletion of Foxp3⁺ Tregs is now possible with the development of Foxp3-diphtheria toxin receptor-like transgenic mouse models. We have used the poorly immunogenic B16F10 melanoma model to characterize a very heterogeneous antitumor effect of the immune response induced by Treg depletion. Depletion and neutralization studies demonstrated the importance of host T cells and interferon γ (IFNγ) in mediating the antitumor response developing in Treg-depleted mice. Such a response correlated with increased proliferation of granzyme B- and IFNγ-producing T cells in the tumor. Furthermore, enhanced antitumor immunity modulated the expression of MHC Class I molecules by B16F10 melanoma cells in Treg-depleted mice. Since Foxp3⁺ Treg depletion induced a significantly heterogeneous antitumor response, for the first time we were able to assess antitumor immunity and autoimmunity across different groups of responding mice. Strikingly, the duration of the tumor-immune system interaction provoked in individual Treg-depleted mice positively correlated with their propensity to develop vitiligo. A rapid complete tumor rejection was not associated with the development of autoimmunity, however, a proportion of mice that suppressed, but did not effectively clear, B16F10 melanoma did develop vitiligo. The significant implication is that approaches that combine with Treg depletion to rapidly reject tumors may also diminish autoimmune toxicities.

Keywords: Treg; autoimmunity; effector; immune modulation; suppression; tumor immunity.

Figure 1. Foxp3⁺ Treg depletion suppresses tumor growth. (A and B) Groups of C57BL/6 DEREG mice (n = 4–10) were inoculated s.c. with 5 × 10⁴ B16F10 melanoma cells or 5 × 10⁵AT3 mammary tumor cells on day 0. On days -2, 5, 12 and 19, mice were injected i.p. with either 500 ng diphtheria toxin A (DTA) or PBS. Tumor sizes are shown as means ± SEM. Statistically significant differences in tumor size between mice treated with PBS or DTA were determined by Mann-Whitney tests (**p < 0.01; ***p < 0.001).

Figure 2. Foxp3⁺ Treg depletion enhances immune cell infiltration in B16F10 tumors. (A–F) Groups of C57BL/6 DEREG mice (n = 4–7) were inoculated s.c. with 5 × 10⁴ B16F10 melanoma cells on day 0. On days -2, 5 and 12, mice were injected i.p. with either 500 ng diphtheria toxin A (DTA) or PBS. On day 18–20, tumors were excised and tumor-infiltrating leukocytes were analyzed by flow cytometry. Frequencies of CD45.2⁺ (A), NK cells (B), total T (C), CD4⁺ T (D), CD8⁺ T (E) and Treg cells (F), upon gating on CD45.2⁺ (B–E) or CD4⁺ T cells (F) from PBS- or DTA-treated mice are shown. Statistical differences in the frequency of the indicated cell subsets between mice treated with PBS or DTA were determined by unpaired Student’s t-tests (*p < 0.05; **p < 0.01; ***p < 0.001). Each symbol represents a single mouse. Data shown are pooled from three independent analyses.

Figure 3. Host T cells and interferon γ mediate antitumor effect following Treg depletion. (A–E) Groups of C57BL/6 DEREG mice (n = 5–7) were inoculated s.c. with 5 × 10⁴ B16F10 melanoma cells on day 0. On days -2, 5 and 12, mice were injected i.p. with either 500 ng diphtheria toxin A (DTA) or PBS. Some mice additionally received 250 μg anti-CD4 (A), (E) and/or 100 μg anti-CD8β (B and E) anti-CD8β monoclonal antibodies on days -1, 0, 4 and 7. Some mice additionally received anti-IFNγ monoclonal antibodies on days -1 (750 μg) and 7 (250 μg) (C and E), or 100 μg anti-asialo-GM1 monoclonal antibodies on days -1, 0, 4 and 7 (D). Tumor sizes are reported as means ± SEM. Data are representative of two independent experiments.

Figure 4. Foxp3⁺ Treg depletion enhances immune effector cell activation. (A–D) Groups of C57BL/6 DEREG mice (n = 4–11) were inoculated s.c. with 5 × 104 B16F10 melanoma cells on day 0. On days -2, 5 and 12, mice were injected i.p. with either 500 ng diphtheria toxin A (DTA) or PBS. On day 18–20, tumors were excised and tumor-infiltrating leukocytes (TILs) were analyzed bv flow cytometry. Mean fluorescence intensity (MFI) of TCRβ gated on CD8⁺ [(A), left panel)] or CD4⁺ [(A), right panel)] B16F10 TILs from PBS- or DTA-treated mice. Each symbol represents a single mouse. Data shown are pooled from three independent analyses. Statistical differences between PBS- and DTA-treated mice were determined by unpaired Student’s t-tests (***p < 0.001). The frequency of the CD44⁺ and CD62L⁺ subsets of CD8⁺ [(B), left panel)] or CD4⁺ [(B), right panel)] B16F10 TILs from PBS- or DTA-treated mice is shown. Statistical differences between PBS and DTA-treated mice were determined by unpaired Student’s t-tests (*p < 0 0.05; ***p < 0.001). Frequencies of IFNγ⁺(C), granzyme B⁺(D) or Ki67⁺(E) cells, upon gating on CD8⁺ (left panel) or CD4⁺ (right panel) B16F10 TILs from PBS- or DTA-treated mice are shown. Each symbol represents an individual mouse. Data shown in (C) and (D) are from one independent analysis. Data shown in (E) are pooled from two independent analyses. Statistical differences between PBS- and DTA-treated mice were determined by an unpaired Student’s t-tests (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 5. Modulation of surface immunogenicity of tumor cells by interferon γ. (A) Groups of C57BL/6 DEREG mice were inoculated s.c. with 5 × 10⁴ B16F10 melanoma cells on day 0. On days -2, 5, 12 and 19, mice were injected i.p. with either 500 ng diphtheria toxin A (DTA) or PBS. Tumors from each group were excised when tumor size exceeded 120 mm², cell lines were generated and then stained for the expression of H-2K^b and H-2D^b. Representative histograms are shown (PBS, n = 5 cell lines; DTA, n = 10 cell lines). (B) Parental B16F10 tumor cells were incubated with increasing doses of interferon γ (IFNγ) for 48 h in triplicate wells and then analyzed by flow cytometry for the expression of H-2K^b and H-2D^b. Representative histogram plots are shown. (C and D) Groups of C57BL/6 DEREG mice (n = 4–7) were inoculated s.c. with 5 × 104 B16F10 melanoma cells on day 0. On days -2, 5 and 12, mice were injected i.p. with either 500 ng diphtheria toxin A (DTA) or PBS. On day 18–20, tumors were excised and tumor-infiltrating leukocytes (TILs) were analyzed by flow cytometry. The frequency of Vβ11⁺(C) or Vβ13⁺(D) CD8⁺ B16F10 TILs from PBS- or DTA-treated mice is shown. Statistical differences between PBS- and DTA-treated mice were determined by unpaired Student’s t-tests (***p < 0.001). Each symbol represents an individual mouse. Data are pooled from three (C) or two (D) independent analyses.

Figure 6. Depletion of Foxp3⁺ Treg provokes heterogeneous antitumor responses that correlate with development of vitiligo. (A–C) Groups of C57BL/6 DEREG mice (n = 4–25) were inoculated s.c. with 5 × 10⁴ B16F10 melanoma cells on day 0. On days -2, 5, 12 and 19, mice were injected i.p. with either 500 ng diphtheria toxin A (DTA) or PBS. Mice were then monitored and tumor growth was measured over 150 d. Tumor-bearing mice were categorized into 4 groups based on tumor growth pattern: Group A (PBS treated), Group B (DTA-treated, tumor outgrowth by 60 days post-inoculation), Group C (DTA-treated, tumor eliminated) and Group D (DTA-treated, tumor outgrowth 60 days post-inoculation). Tumor size (mm²) for each individual mouse was plotted and results were pooled from seven independent experiments (A). Representative pictures of mice from each group are shown in (B). The incidence of vitiligo in B16F10-bearing mice belonging to the indicated group is shown in (C). Statistical differences between mice groups were determined by Fisher’s exact tests (*p < 0.05; **p < 0.01; ***p < 0.001).