Fig. 4. The IL-17-associated presence of neutrophils plays a role in the anti-tumor response to dual ICI.
Tumor growth kinetics of CM (BRAF-WT ICI-sensitive) (a) and YUMMER1.7 (BRAF-mutant ICI-sensitive) (b) tumors treated with IgG or H2O (control, CM, n = 5; YUMMER1.7, n = 4), anti-CTLA-4 + anti-PD-1 antibodies (CM, n = 4; YUMMER1.7, n = 4) or anti-CTLA-4 + anti-PD-1 + anti-Ly6G antibodies (CM, n = 5; YUMMER1.7, n = 4) according to the depicted treatment schedule. Data points show mean + s.e.m.; P values are from one-way ANOVA with Holm–Sidak’s multiple-comparison test. c, Violin plots showing tumor immune cell frequencies by flow cytometry from a (CM model). P values are from one-way ANOVA with Holm–Sidak’s multiple-comparison test. d, Schematic workflow for in vitro culture, isolation of BM neutrophils and splenic CD8+ T cells followed by the migration assay. e, Top, qPCR analysis of transcripts encoding T cell chemokines, adhesion molecules and TH17 signaling components in the control or rm-IL-17A-treated CM mouse cell line. Bar plots represent mean + s.e.m. from n = 3 biological replicates; P values are from the unpaired t-test. Shown is one representative of two independently performed experiments. Bottom, corresponding cytokine and chemokine levels in cell culture supernatants of CM cells treated with rm-IL-17A. Data points show mean + s.e.m. from n = 2 biologically independent samples; P values are from the unpaired t-test. f, Bar plot showing the percentage of migrated CD8+ T cells in the Boyden chamber assay. The different medium conditions used as chemoattractant in the bottom chamber are depicted below the horizontal line. Serum-free medium was used as the negative control, and recombinant mouse CXCL10 (200 ng ml−1) was used as the positive control. α-IL-17A (5 µg ml−1) was added to the top chamber (depicted above the horizontal line) as indicated. Individual data points represent n = 3–4 biological replicates per group; P values are from one-way ANOVA with Sidak’s multiple-comparison test. Shown is one representative of three independently performed experiments. All P values are two tailed. Tu, tumor; Neu, neutrophil; Grz, granzyme.