Fig 2: Gating strategy for identification respiratory DC subsets. Respiratory dendritic cells (DC) were identified according to CD11c+Siglec-Fneg CD49bneg expression to exclude autofluorescent macrophages and NK cells and further dissected into plasmacytoid DC (pDC) according to 120g8 expression. The pDC negative DC fraction was gated for CD103 to identify CD103+ DC. Out of the pDC and CD103+ DC negative fraction, MoDC and CD11bhigh DC were separated according to MHC-class-II (I-A/I-E) and CD11b expression. MoDC are CD11bhigh and MHC-class-IImed-low whereas CD11bhigh DC are CD11bhigh and MHC-class-IIhigh.

Fig 3: Marked strain heterogeneity of respiratory DC subsets. Dissection of respiratory DC subsets with flow cytometry indicates significantly elevated plasmacytoid DC (A) numbers in 129SV mice. Respiratory CD103+ DC numbers are significantly elevated in C3H mice when compared to C57BL/6 (BL6), 129SV and DBA mice (B). Monocytic DC (MoDC) numbers are significantly elevated in C3H mice when compared to any other strain (C). CD11bhigh DC numbers are significantly elevated in 129SV mice compared to any other strain. Results are representative for n= 8 independent animals per strain (mean ± SEM; *p<0.05; **p<0.01; ***p<0.001).

Fig 4: mCALR inhibits lung cancer progression by facilitating DC infiltration in tumor tissues. (A) A tumor formation assay was performed by injecting LLC-NC and LLC-mCALR cells, n = 5 mice. (B, C) Quantification of the volume (B) and weight (C) of tumors generated in mice, ***P < 0.001. (D–F) The relative mRNA expression levels of TLR4, TNFa, and CCL19 were detected in tumors of LLC-NC and LLC-mCALR groups using real-time PCR; *P < 0.05. (G) The relative percentages of CD11c+, CD40+, CD80+, CD86+, and MHCII+ DCs in tumors were detected using flow cytometry; ns, P > 0.05, *P < 0.05. (H) The relative percentages of CD11c+CD8a+ and CD11c+CD8a- DCs in tumors were detected using flow cytometry; ns, P > 0.05, *P < 0.05. (I) The relative percentages of CD11c+CD103+, CD11c+CD207+, and CD11c+CD317+ DCs in tumors were detected using flow cytometry; ns, P > 0.05, *P < 0.05.

Fig 5: 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.