Fig 2: Tumor-Derived Exosomes (TDEs) Promote Lipid Accumulation in Dendritic Cells (DCs)In the MC38-OT I tumor model, DCs (CD45+MHC II+CD11c+ F4/80−) in spleen (sDCs) and in tumor (TIDCs) were analyzed on days 13 and 27 (n = 3 per group).(A) Intracellular lipid in sDCs and TIDCs.(B and C) The proliferation and function of OT I CD8+ T cells primed by sDCs or TIDCs were evaluated by CFSE dilution (B) and IFN-γ production (C).(D) Intracellular lipid levels in BMDCs co-cultured with or without TC-1 tumor cells.(E) Lipid droplets in BMDCs were photographed by transmission electron microscopy (TEM). Scale bar, 2 μm.(F) Tumor cell medium (TCM) was separated with an MWCO filter (30 and 100 kDa cutoff). Intracellular lipid levels in BMDCs after incubation with different fractions for 24 h.(G) TEM analysis of exosomes secreted by TC-1 cancer cells. Scale bar, 100 nm.(H) Western blot analysis of exosome markers.(I) Intracellular lipid levels in BMDCs treated with tumor-derived exosomes (400 μg/mL) or exosome-free supernatant for 24 h.(J) Scheme of analysis.(K) GFP+ cells/gram tumor tissue of TIDCs and TAMs were measured (n = 4 per group).(L) Intracellular lipid levels in TIDCs.(M) Isolated GFP+ and GFP− TIDCs were co-cultured with OT I CD8+ T cells for 3 days to detect IFN-γ production by ELISA. Each dot represents technical repeats.*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (A)–(D) were analyzed with 2-way ANOVA. (F) and (I) were analyzed with 1-way ANOVA. (K)–(M) were analyzed with 2-tailed t test. Error bars represent SEMs. The error bars represent SDs for (C) and (M), and SEM for all of the others. Each dot represents an individual mouse in (A)–(D), (F), (I), and (K)–(M). Representative of 3 independent experiments in (F) and 2 independent experiments in (I).See also Figures S1 and S6.

Fig 3: Loss of PD-L1 Leads to a Reduction in the Number of Dendritic Cells (DCs) in the Draining Lymph Node (dLN) following Poly(I:C)(A) Flow cytometry gating strategy for conventional DC subsets and myeloid cells in the LN from WT and Pdl1−/− mice. PD-L1 expression shown is from naive mice and mice 24 h following poly(I:C) injection.(B) Quantification of PD-L1 expression on WT conventional dendritic cell (cDC) 1 and cDC2 at baseline and 24 h following poly(I:C), as well as Pdl1−/− DCs.(C) Number of cDC1 and cDC2 in the dLN of WT and Pdl1−/− before and 24 h after intradermal poly(I:C) injection (10 μg/mouse). Data are shown from axillary and brachial LNs combined.(D) Number of CD11b+ CD11c− cells in the dLN of WT and Pdl1−/− mice. Data are shown from axillary and brachial LNs combined.(E) Number and percentage of CD11c-high cells in the skin from the site of injection of WT and Pdl1−/− mice based on weight.For (B–E), n = 3 mice per group. Data are shown from one experiment that was repeated four times with similar results. Statistical analysis was done using a one-way ANOVA. *p < 0.05; **p < 0.01; ****p < 0.0001; n.s., p > 0.05. Error bars indicate standard error of the mean. See also Figure S1.

Fig 4: LClike cells derived from embryonic and adult haematopoiesis have a similar transcriptomic signature.(A) Generation of BM chimeras: CD45.1+ WT BM cells (106) were transferred into lethally irradiated CD45.2+ recipient mice. The epidermis, dermis, and draining lymph nodes (LNs) obtained from the reconstituted chimeras were analysed 1 and 4 months later by flow cytometry. (B) Flow cytometry analysis of donor (CD45.1+) and host (CD45.2+) chimerism in different epidermal, dermal, and skin-draining LN LC and dendritic cell (DC) subpopulations, 1 and 4 months after reconstitution. LC, cDC1, triple negative (TN), LClike, and CD11bhi subsets were gated and analysed for CD45.1 (x-axis) and CD45.2 (y-axis) expression. (C) The percentage of CD45.1 donor cells detected in the epidermis, dermis, and skin-draining LNs of chimeras, 1 or 4 months after reconstitution. Data are represented as mean ± SEM; n = 6 single mice; **p<0.01; ***p<0.001; ****p<0.0001; ns, non-significant; two-tailed Student’s t-test. (D) Uniform manifold approximation and projection (UMAP) analysis of distinct LN DC subpopulations obtained from chimeras 4 months after reconstitution, based on the expression of different markers (CD11c, MHCII, CD103, CD11b, CD326, CD207, CD45.1, CD45.2). (E) Transcriptome analysis of LN CD45.1+ LClike cells (n = 3) and LN CD45.2+ LClike (n = 3) cells collected from 10 mice. The Venn diagram shows the percentage of overlapping genes expressed by CD45.1+ and CD45.2+ LClike cells. Figure 5—source data 1.Percentage of CD45.1+ donor cells detected in the epidermis, dermis and skin-draining LNs of mouse chimeras, 1 or4monthsafter reconstitution.

Fig 5: PPARα in DCs Is Required for Anti-tumor Immunity(A and B) MC38-OT I tumors were subcutaneously (s.c.) injected in CD11c-DTR chimeric mice and treated with the combination of PD-L1 mAb (200 μg/dose/mouse) and GW6471 (10 mg/kg). Diphtheria toxin (DT) was intraperitoneally (i.p.) injected every other day before antibody therapy. The percentage of CD45+MHCII+CD11c+F4/80− cells was measured (A). Each dot represents an individual mouse. The tumor growth curve was shown (B) (n = 10 per group).(C) MC38-OT I-bearing mice were treated with WT DC or PPARα−/− DC (5 × 106 per dose) s.c. 3 times (days 7, 14, and 21). The WT or PPARα−/− DC was pre-immunized with 10 μg/mLOVA250–264 overnight. The tumor growth curve was shown (C) (n = 9 per group).(D) Scheme of TDE-mediated DC immune dysfunction.*p < 0.05; **p < 0.01; ****p < 0.0001. Data were analyzed with 2-tailed t test. The error bars represent SEMs.