![Tumor-derived GM-CSF drives the accumulation of CD301b+moDCs. (A) Relative expression of selected genes was overlaid onto the UMAP from Fig. 2 G; expression values represent log-normalized counts. (B) B16-bearing mice on days 0 and 5 after RT. Left: tumor Csf2 transcript levels, by qPCR (mean + SD; n = 5–6/group, 2–4 experiments [exp.]). Right: GM-CSF protein levels in tumor lysate, by ELISA (mean + SD; n = 6–9/group, 4 exp.). (C) Expression of CD301b by DC2s/moDCs, by flow cytometry. Left: frequency of CD301b on DC2s (day 0) and moDCs (day 5 after RT) (1 of 3 exp.). Right: frequency of CD301b+ expressing cells (mean + SD; n = 3–11/group, 2–5 exp.). (D) B16 and MC38 tumor cells were irradiated with 20 Gy RT and cultured for 4 days. Left: tumor cell Csf2 transcript levels, by qPCR (mean + SD; n = 7–9/group, 3–4 exp.). Right: GM-CSF protein levels in the culture supernatant, by ELISA (mean + SD; n = 4–9/group, 3–4 exp.). (E) B16 and B16∆Csf2 tumor cell lines were irradiated with 20 Gy and cultured for 4 days. GM-CSF protein levels in the culture supernatant (mean + SD; n = 3/group, 3 exp.). (F–J) B16 and B16∆Csf2 tumor–bearing mice treated with 20 Gy RT. (F) GM-CSF protein level in tumor lysate 5 days after RT, by ELISA (mean + SD; n = 7–9/group, four exp). (G) Cells per mg of tumor 5 days after RT, by flow cytometry (mean + SD; n = 6–22/group, 4–5 exp). (H) TIL numbers and TIL/Treg ratio 7 days after RT, by flow cytometry (mean + SD; n = 9–11/group, 4 exp). (I) TILs producing IFNγ, TNF, and GzmB 7 days after RT, after ex vivo restimulation, by flow cytometry (violin plots show data distribution; n = 9–11/group, 4 exp.). (J) Left: average tumor growth with a ratio of surviving mice in parentheses (mean + SEM). Right: tumor volume day 10 after RT (mean + SD; n = 14/group, 3 exp.). (K–N) As in F–J, but Csf2−/− mice were transplanted with B16 and B16∆Csf2 tumor cells. (K) Left: GM-CSF protein level in tumor lysate 5 days after RT, by ELISA (mean + SD; n = 8–16/group, 4–5 exp.). Right: moDC numbers 5 days after RT (+RT) normalized to day 0 (−RT), by flow cytometry (mean + SD; n = 8–9/group, 4–5 exp.). (L) Cells per mg of tumor and CD8+ TIL-to-Treg ratio 7 days after RT, normalized to day 0 (−RT) (mean + SD; n = 8–11/group, 4–5 exp.). (M) TILs producing IFNγ, TNF, and GzmB 7 days after RT, after ex vivo restimulation, normalized to day 0 (−RT) (violin plots show data distribution, n = 8–9/group, three to five exp). (N) Left: average tumor growth with a ratio of surviving mice in parentheses (mean + SEM). Right: tumor volume 10 days after RT (mean + SD; n = 6–14/group, 2–3 exp). Statistics: two-way ANOVA plus Tukey’s post hoc test for mean tumor growth (J and N); one-way ANOVA plus Tukey’s post hoc test (C–E); two-tailed t test for the rest. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.](https://cdn.rupress.org/rup/content_public/journal/jem/222/6/10.1084_jem.20231717/1/jem_20231717_fig6.png?Expires=1773657654&Signature=Np7ueV407lRDdNaBFOd-sAw5kdAEZMq2RDiylgRQFtQXlNH5BdAE3cMP45kvpIVkCLiqWLjhxFn6OIIbR5XcBRakFbGIID1xHOsCjrtcYvnpbxLupA~marfAFqpeLRN-3PQdT01btwKkTUY3OKIUhC9ITg7f9~zUUArTGNC-yZpfXreqD6ArYYNVJdGosPI-3zNVJTTW3JzElW3oZJwWX3eeMgJrSQVqRsf1Kl9CuQP86fKDbcgwXkPN3y8DKPafp-luYEwkDlzjMiF5RshXfOsvZRqs1keIdQFkiurXxx-Le0vun4TMum3jDl1IlHbbPNTBUXeRKvyiGPRWga0e7A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Tumor-derived GM-CSF drives the accumulation of CD301b + moDCs. (A) Relative expression of selected genes was overlaid onto the UMAP from Fig. 2 G; expression values represent log-normalized counts. (B) B16-bearing mice on days 0 and 5 after RT. Left: tumor Csf2 transcript levels, by qPCR (mean + SD; n = 5–6/group, 2–4 experiments [exp.]). Right: GM-CSF protein levels in tumor lysate, by ELISA (mean + SD; n = 6–9/group, 4 exp.). (C) Expression of CD301b by DC2s/moDCs, by flow cytometry. Left: frequency of CD301b on DC2s (day 0) and moDCs (day 5 after RT) (1 of 3 exp.). Right: frequency of CD301b+ expressing cells (mean + SD; n = 3–11/group, 2–5 exp.). (D) B16 and MC38 tumor cells were irradiated with 20 Gy RT and cultured for 4 days. Left: tumor cell Csf2 transcript levels, by qPCR (mean + SD; n = 7–9/group, 3–4 exp.). Right: GM-CSF protein levels in the culture supernatant, by ELISA (mean + SD; n = 4–9/group, 3–4 exp.). (E) B16 and B16∆Csf2 tumor cell lines were irradiated with 20 Gy and cultured for 4 days. GM-CSF protein levels in the culture supernatant (mean + SD; n = 3/group, 3 exp.). (F–J) B16 and B16∆Csf2 tumor–bearing mice treated with 20 Gy RT. (F) GM-CSF protein level in tumor lysate 5 days after RT, by ELISA (mean + SD; n = 7–9/group, four exp). (G) Cells per mg of tumor 5 days after RT, by flow cytometry (mean + SD; n = 6–22/group, 4–5 exp). (H) TIL numbers and TIL/Treg ratio 7 days after RT, by flow cytometry (mean + SD; n = 9–11/group, 4 exp). (I) TILs producing IFNγ, TNF, and GzmB 7 days after RT, after ex vivo restimulation, by flow cytometry (violin plots show data distribution; n = 9–11/group, 4 exp.). (J) Left: average tumor growth with a ratio of surviving mice in parentheses (mean + SEM). Right: tumor volume day 10 after RT (mean + SD; n = 14/group, 3 exp.). (K–N) As in F–J, but Csf2−/− mice were transplanted with B16 and B16∆Csf2 tumor cells. (K) Left: GM-CSF protein level in tumor lysate 5 days after RT, by ELISA (mean + SD; n = 8–16/group, 4–5 exp.). Right: moDC numbers 5 days after RT (+RT) normalized to day 0 (−RT), by flow cytometry (mean + SD; n = 8–9/group, 4–5 exp.). (L) Cells per mg of tumor and CD8+ TIL-to-Treg ratio 7 days after RT, normalized to day 0 (−RT) (mean + SD; n = 8–11/group, 4–5 exp.). (M) TILs producing IFNγ, TNF, and GzmB 7 days after RT, after ex vivo restimulation, normalized to day 0 (−RT) (violin plots show data distribution, n = 8–9/group, three to five exp). (N) Left: average tumor growth with a ratio of surviving mice in parentheses (mean + SEM). Right: tumor volume 10 days after RT (mean + SD; n = 6–14/group, 2–3 exp). Statistics: two-way ANOVA plus Tukey’s post hoc test for mean tumor growth (J and N); one-way ANOVA plus Tukey’s post hoc test (C–E); two-tailed t test for the rest. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.