Extended Data Fig. 5: Modulation of the myeloid cell compartment restores immunotherapy response.

a, DC maturation score comparing gene expression profiles of R^TT vs. N^TT tumours (Braf/Pten N^TT, Braf N^TT, BRAF R^TT n = 3; Braf/Pten R^TT n = 4 tumours). b, Mean fluorescence intensity (MFI) of maturation markers on CD103⁺ DCs from N^TT and R^TT Braf/Pten melanoma (N^TT, n = 5; R^TT, n = 6 tumours), assessed by flow cytometry. Experiment performed twice with independent cell lines; representative example shown. P-value: **** 6.9E-5, ** 0.0043, * 0.0149. c, PCA plot displaying top 500 most variable genes for CD103⁺ DCs sorted from N^TT and R^TT ± Poly I:C Braf/Pten^OVA tumours. d, GSEA of IFN-alpha response in CD103⁺ DCs sorted from R^TT vs. N^TT Braf/Pten^OVA melanoma. e, Quantification of T cell proliferation based on CFSE dilution in DC co-culture assays displayed in Fig. 3i (n = 4 tumours per condition). P-value: * 0.029. f, Scheme outlining experiment to assess impact of depleting suppressive myeloid cells on ACT in Braf/Pten^OVA tumours. g, Depletion of Ly6C⁺CD11b⁺ and Ly6G⁺CD11b⁺ cells in blood 3 days post anti-GR-1 administration. h, Tumour infiltration of effector OT-1^Luc T cells measured by BLI at 24 h post ACT in Braf/Pten^OVA tumour bearing mice treated with Isotype CTRL or anti-GR-1 antibody (n = 9, 9, 8, 8 mice from left to right). Experiment performed 3 times; representative example shown. P-value: ns 0.53, * 0.0128. i, Treatment response of Braf/Pten^OVA tumours treated with ACT or anti-GR-1 plus ACT (N^TT ACT + ISO, n = 6; R^TT ACT + ISO, n = 4; N^TT ACT + anti-GR-1, n = 6; R^TT ACT + anti-GR-1, n = 4 mice). j, DC maturation in Poly I:C injected Braf/Pten^OVA tumours assessed by CD40 expression using flow cytometry. k, CD103⁺ DC influx in R^TT tumours overexpressing FLT3L, assessed by flow cytometry (n = 3 tumours). P-value: * 0.0118. l, Survival in response to ACT ± Poly I:C ± FLT3L in R^TT Braf/Pten^OVA tumours. (R^TT CTRL, R^TT + ACT, R^TT FLT3L + ACT, n = 3; R^TT + ACT + Poly I:C, R^TT FLT3L + ACT + Poly I:C, n = 4 mice). Experiment performed twice; representative example shown. P-value: ** 0.0100. m, Treatment response of R^TT Braf melanoma in WT mice (left) and BATF3^-/- mice (right) treated with indicated therapies; black arrows indicate anti-PD-1/CTLA-4 administration, red arrows indicate Poly I:C injection (CTRL, anti-PD-1/CTLA-4, Poly I:C, n = 6 tumours; Poly I:C + anti-PD-1/CTLA-4, n = 4 tumours). Experiment performed twice; representative example shown. P-value: ** 0.01, ns 0.1931. Data in (b, e, h, k, m) is displayed as mean ± SEM. Data analysis (b, h, k) two-tailed unpaired t-test with Welch correction for unequal variance or with Mann-Whitney-U-test if not normal distributed l two-sided log-rank test (Mantel-Cox) test (m) two-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = non-significant.