Figure 5.

PTX in combination with PLX397 induces antitumor T-cell response. A and B, tumor infiltration by T lymphocytes enhanced by combined PTX and CSF1 or CSF1R blockade. Flow cytometric analyses of tumor infiltrating CD3⁺CD8⁺ and CD3⁺CD4⁺ T lymphocytes depicted as the mean number of positive cells, assessed as a percentage of total cells following treatment of MMTV-PyMT mice with PTX or PLX3397 or both (A), or treatment of mice having orthotopic PyMT-derived tumors with combined PTX/αCSF1 or PTX/PLX3397 (B), compared with controls. Mean values ±SEM are depicted. C, cytokine mRNA expression assessed in orthotopic PyMT-derived tumors from mice treated with PTX alone or in combination with PLX3397. Graph depicts mean fold change in mRNA expression compared with PTX treatment group (5 animals/group). SEM is depicted. D, tumor infiltration by DCs enhanced by combined PTX/PLX3397. Flow cytometric analysis of tumor-infiltrating CD45⁺Ly6G⁻Ly6C^lowCD11b^low/−CD11c^highMHCII^high DCs depicted as mean percentage of positive cells as a percentage of total cells from MMTV-PyMT mice treated with PTX or PLX3397 or both, versus controls. E, CD8⁺ T-lymphocyte activation repressed by TAMs. Purified T cells were loaded with CFSE and activated in vitro by plate-bound CD3/28 and cocultured with the indicated ratio of CD45⁺Ly6G⁻Ly6C^Low CD11b⁺F4/80⁺ TAMs isolated from late-stage mammary tumors of MMTV-PyMT mice. Data are depicted as the percentage of live CD8⁺ T lymphocytes exhibiting CFSE dilution after 60 hours. Data are representative of 2 independent experiments run in triplicate. Error bars represent SEM. F, analysis of the ratio of tumor-infiltrating CD45⁺Ly6G⁻Ly6C^lowCD11b⁺F4/80⁺ TAMs to CD3⁺CD8⁺ T lymphocytes depicted as mean ratio (TAM/CD8 CTL) ±SEM from MMTV-PyMT mice treated with vehicle or with PTX and/or PLX3397. *, statistically significant differences (P < 0.05, Mann-Whitney) in A–F. **, statistically significant differences (P < 0.05, Mann-Whitney) between PLX3397-treated groups in F.