Antitumor T-cell responses contribute to the effects of dasatinib on c-KIT mutant murine mastocytoma and are potentiated by anti-OX40

Abstract

Targeted and immune-based therapies are thought to eradicate cancer cells by different mechanisms, and these approaches could possibly complement each other when used in combination. In this study, we report that the in vivo antitumor effects of the c-KIT inhibitor, dasatinib, on the c-KIT mutant P815 mastocytoma tumor were substantially dependent on T cell-mediated immunity. We found that dasatinib treatment significantly decreased levels of Tregs while specifically enhancing tumor antigen-specific T-cell responses. We sought to further enhance this therapy with the addition of anti-OX40 antibody, which is known to provide a potent costimulatory signal to T cells. The combination of dasatinib and anti-OX40 antibody resulted in substantially better therapeutic efficacy compared with either drug alone, and this was associated with enhanced accumulation of tumor antigen-specific T cells in the tumor microenvironment. Furthermore, the combination regimen inhibited the function of Tregs and also resulted in significantly up-regulated expression of the IFN-γ-induced chemokines CXCL9, 10, and 11 in the tumor microenvironment, which provides a feasible mechanism for the enhanced intratumoral CTL infiltration. These studies delineate a strategy by which targeted therapy and immunotherapy may be combined to achieve superior antitumor responses in cancer patients.

Figure 1

In vivo antitumor effect of dasatinib on P815 mastocytoma. DBA/2 mice were subcutaneously inoculated with 2 × 10⁶ P815 tumor cells on day 0. Tumor-bearing mice were treated with vehicle or dasatinib (150 mg/kg) on day 8, 9, and 10 by gavage. For depletion experiment, mice were injected IP with 200 μg of rat IgG or CD8 depletion antibodies starting from day 8 once a week. (A) Tumor sizes of mice treated with vehicle or dasatinib (*P = .0354). (B) Kaplan-Meier survival curve of the tumor-bearing mice for 2 different groups (*P = .0447). (C) Representative H&E-stained slides of control tumor and dasatinib-treated tumor. (D) Tumor sizes of CD4⁺/CD8⁺ T-cell depletion experiment (vehicle + IgG vs dasatinib + IgG: *P = .0427). (E) Kaplan-Meier survival curve of CD4⁺/CD8⁺ T-cell depletion experiment (vehicle + IgG vs dasatinib + IgG: *P = .0503).

Figure 2

Effect of dasatinib treatment on tumor antigen-specific T-cell response. (A) Representative dot plots for P1A tetramer staining in the periphery blood of vehicle- or dasatinib-treated mice. (B) Percentages of P1A tetramer⁺ T cells in total CD8⁺ T cells (*P = .0235). (C) Representative dot plots for intracellular IFN-γ staining. (D) Percentages of IFN-γ⁺ T cells in total CD8⁺ T cells (P1A: *P = .0449, P1E: *P = .0498). (E) IFN-γ secretion by peripheral blood T cells in response to P1A or P1E peptides detected by ELISA (P1A: *P = .0428, P1E: **P = .0017). (F) P1A tetramer staining of the CD4⁺ T-cell depletion experiment.

Figure 3

Effect of 3-day dasatinib treatment on levels of regulatory T cells and vaccine-mediated antigen-specific CD8⁺ T-cell responses. (A) Representative dot plots for Treg staining in the peripheral blood of tumor-bearing mice. (B) Percentages of Treg cells in CD4⁺ T cells in the peripheral blood of tumor-bearing mice on day 11 (***P < .0001). (C) Treg/ T effector ratio in the peripheral blood of tumor-bearing mice on day 11 (***P < .0001). (D) Percentages of Treg cells in CD4⁺ T cells in tumors on day 11 (*P = .0473). (E) Effect of dasatinib on proportions of Treg cells and Treg/T effector ratios in tumor-free mice (**P = .0026, *P = .0432). (F) Representative dot plots for P1A tetramer staining in the peripheral blood on day 7 after vaccination. (G) Effect of 3-day dasatinib treatment on vaccine-mediated P1A-specific CD8⁺ T-cell responses (***P < .0001).

Figure 4

Therapeutic effect of combining dasatinib and anti-OX40 antibody. Mice received daily gavage of vehicle or dasatinib (150 mg/kg) on day 8-10 and IP injection of IgG control antibody or anti-OX40 antibody (200 μg/mice) on day 10 and 13. (A) Schema of treatment. (B) Tumor sizes of mice for 4 different groups (vehicle + IgG vs dasatinib + anti-OX40: ***P < .0001, dasatinib + IgG vs dasatinib + anti-OX40: **P = .0042, vehicle + anti-OX40 vs dasatinib + anti-OX40: *P = .0257). (C) Kaplan-Meier survival curve of the tumor-bearing mice for 3 different groups (vehicle+IgG vs dasatinib + anti-OX40: ***P < .0001, dasatinib + IgG vs dasatinib + anti-OX40: **P = .0074, vehicle + anti-OX40 versus dasatinib + anti-OX40: *P = .0151). (D) Plots represent the tumor size of individual mice for each group. (E) Kaplan-Meier survival curve of the CD4⁺/CD8⁺ T-cell depletion experiment.

Figure 5

Influence of dasatinib and anti-OX40 treatment on the tumor-infiltrating lymphocytes. (A) Effect of dasatinib and anti-OX40 on levels of the P1A-specific T cells in the blood. (B) Total numbers of CD8⁺ T cells and the ratios of CD8⁺ T-cell number/tumor weight (for CD8⁺ T-cell total number, vehicle + IgG vs vehicle + anti-OX40: ***P = .0007, dasatinib + IgG vs dasatinib + anti-OX40: *P = .0250, vehicle + IgG vs dasatinib + anti-OX40: *P = .0165; for CD8⁺ T-cell number/gram, vehicle + IgG vs vehicle + anti-OX40: ***P = .0002, dasatinib + IgG vs dasatinib + anti-OX40: *P = .0234, vehicle + IgG vs dasatinib + anti-OX40: **P = .0076). (C) Total numbers of P1A tetramer⁺ CD8⁺ T cells and the ratios of P1A tetramer⁺CD8⁺ T-cell number/weight (for P1A tetramer⁺ T-cell total number, vehicle + anti-OX40 vs dasatinib + anti-OX40: *P = .0110, dasatinib + IgG vs dasatinib + anti-OX40: **P = .0078, vehicle + IgG vs dasatinib + anti-OX40: **P = .0024; for P1A tetramer⁺ T-cell number/gram, vehicle + anti-OX40 vs dasatinib + anti-OX40: *P = .0265, dasatinib + IgG vs dasatinib + anti-OX40: *P = .0507, vehicle + IgG vs dasatinib + anti-OX40: *P = .0229). (D) Total numbers of IFN-γ⁺ CD8⁺ T cells and the ratios of IFN-γ⁺ CD8⁺ T-cell number/tumor weight (for IFN-γ⁺ T-cell total number, vehicle + anti-OX40 vs dasatinib + anti-OX40: ***P < .0001, dasatinib + IgG vs dasatinib + anti-OX40: ***P < .0001, vehicle + IgG vs dasatinib + anti-OX40: ***P < .0001; for IFN-γ⁺ T-cell number/gram, vehicle + anti-OX40 vs dasatinib + anti-OX40: *P = .0195, dasatinib + IgG vs dasatinib + anti-OX40: *P = .0505, vehicle + IgG vs dasatinib + anti-OX40: *P = .0220).

Figure 6

Levels of CXCL9, 10, and 11 in tumors from 4 different groups. Tumors were harvested on day 16 after tumor inoculation. Real-time PCR was performed to detect the chemokine levels in the tumor microenvironment. GAPDH was used as the normalization reference gene. Graph shows the normalized fold expression of CXCL9, 10, and 11 (CXCL9: ***P = .0007, CXCL10: *P = .0327, CXCL11: *P = .0359).

Figure 7

Effect of dasatinib and anti-OX40 treatment on levels and functions of regulatory T cells. (A) Percentages of Treg cells in CD4⁺ T cells in the peripheral blood on day 11 (vehicle + IgG vs dasatinib + IgG: ***P = .0003, vehicle + anti-OX40 vs dasatinib + anti-OX40: **P = .0051, vehicle + IgG vs dasatinib + anti-OX40: *P = .0208). (B) Influence of dasatinib and anti-OX40 on functions of Tregs isolated from 4 different groups (vehicle + IgG vs dasatinib + IgG: *P = .0144, vehicle + IgG vs vehicle + anti-OX40: *P = .0254, dasatinib + IgG vs dasatinib + anti-OX40: **P = .0019, vehicle + anti-OX40 vs dasatinib + anti-OX40: *P = .0488, vehicle + IgG vs dasatinib + anti-OX40: ***P = .0002). (C) Total numbers of Tregs in tumor infiltrating lymphocytes on day 16. (D) Ratios of CD8⁺ effector T cells/Treg cells in tumor-infiltrating lymphocytes on day 16 (vehicle + anti-OX40 vs dasatinib + anti-OX40: **P = .0065, dasatinib + IgG vs dasatinib + anti-OX40: *P = .0228, vehicle + IgG vs dasatinib + anti-OX40: ***P = .0004).