Figure 3. Inhibiting HRH1 on macrophages enhances T cell anti-tumor immunity.

(A) Flow cytometry analysis of M1-like (MHC II⁺) versus M2-like (CD206⁺) populations in bone marrow-derived macrophages (BMDMs) that were generated from wild-type (WT) or HRH1^−/− mice and treated with vehicle or fexofenadine (FEXO) (10 μM) in the presence of EO771 tumor cell-conditioned medium (TCM) for 48 hours.

(B) Analysis of IFN-γ⁺CD8⁺ T cells in splenocytes co-cultured with vehicle- or FEXO (10 μM)-treated WT or HRH1^−/− BMDMs (TCM-educated).

(C and D) Relative MHC II:CD206 MFI ratios of tumor-associated macrophages (TAMs) (C) and percentage of IFN-γ⁺CD8⁺ T cells (D) in EO771 tumors (left) or B16-GM tumors (right) growing in WT versus HRH1^−/− mice, and vehicle-treated versus FEXO-treated WT mice (n=5-6, t-test).

(E) EO771 (left) and B16-GM (right) tumor growth in WT versus HRH1^−/− mice, and vehicle-treated versus FEXO-treated WT mice (n= 5-8 mice/group, two-way ANOVA).

(F) B16-GM tumor growth with indicated treatment. CD8⁺ T cells were depleted by anti-CD8 antibodies (n=6-7 mice/group, two-way ANOVA).

(G) Growth of B16-GM tumor cells co-implanted with WT or HRH1^−/− BMDMs in HRH1^−/− or WT recipient mice, respectively (n=6-9 mice/group, two-way ANOVA).

(H) Percentages of IFN-γ⁺ and PRF1⁺ CD8⁺ T cells in primary tumors from WT and HRH1^−/− mice transplanted with B16-GM tumor cells alone, or both tumor cells and BMDMs (HRH1^−/− or WT respectively) (n=6, one-way ANOVA).

(I) t-distributed stochastic neighbor embedding (tSNE) plot of tumor-infiltrating leukocytes overlaid with color-coded clusters in EO771 tumors from WT or HRH1^−/− mice. Dotted ellipses highlight clusters with significant differences between two groups.

Mean ± SEM, *P<0.05, **P<0.01, ***P<0.001.

See also Figures S3, S4 and S5.