Figure 3. Flt3L injections dramatically expand CD103⁺ DC progenitors in the bone marrow and promote their accumulation and expansion in the tumor mass.

(A) CyTOF analysis of tumor-infiltrating immune cells on day 13 after B16 tumor challenge in mice treated i.p. for 9 consecutive days starting at day 3 post tumor injection with 30μg FL or PBS. SPADE trees were colored by relative population frequency out of total live CD45⁺ cells. Five mice were pooled to build each SPADE tree. Trees are representative of 2 experiments with similar results.

(B) Fold changes in relative frequency of immune cell populations (identified by SPADE clustering performed in A) between FL- and PBS-treated mice.

(C) Representative confocal images of DCs infiltrating tomato⁺ Braf-mutant tumors (red) and B16-YFP tumors (green) treated with 9 daily injections of FL or PBS starting on day 18 after 4-HT topical application (Braf-mutant) or 3 days after tumor challenge (B16). Tumor biopsies were isolated and frozen 30 days (Braf-mutant) and 15 days (B16) after tumor challenge. In Braf-mutant tumors, DCs were identified as CD11c⁺ cells (green) whereas in B16 tumor DCs were identified as F4/80⁻CD11c⁺ (red). Scale bar: 50μm.

(D) Mice were treated as in A. Histograms show the expression of cell surface markers or transcription factors in CD103⁺ DCs (dashed line), CD11b⁺ DCs (grey line) and MHCII^hiCD11c⁺Ly6C⁻CD64⁻CD103⁻CD11b⁻ double negative cells (DN: full grey) infiltrating B16 tumors on day 13 after tumor challenge. Results shown are representative histograms of 2 experiments.

(E) Frequency of myeloid cell populations infiltrating B16 tumors in mice treated with FL or PBS as in A. Results shown are the mean ± SEM of 3 independent experiments with a total of 8 mice.

(F) Frequency of MHCII^hiCD11c⁺Ly6C⁻CD64⁻CD103⁻CD11b⁻IRF8⁺ (IRF8⁺ DN) cells in bone marrow (BM), blood and tumors of B16 tumor-bearing mice analyzed 2 days after 9 daily injections of FL or PBS. Shown is the mean ± SEM of one experiment (n=5).

(G) Frequency of Ki67⁺ cells among IRF8⁺ DN cells, CD103⁺ DCs and CD11b⁺ DCs infiltrating B16 tumors site analyzed 2 days after 9 daily injections of FL or PBS. Shown is the mean ± SEM of 2 independent experiments (n=4).

See also Figure S3.