Fig 2: Differential contribution of Langerhans cell (LC) and LClike in skin immune responses.(A) Diphteria toxin (DT)-treated WT and DC-SIGN DTR mice were sensitized with 0.5% 2,4-dinitrofluorobenzene (DNFB) (applied to the shaved back skin) and ear-challenged 5 days later with 0.1% DNFB. (B) Ear swelling response of challenged WT and DC-SIGN DTR mice was determined over a 12-day period post challenge. (C) Quantitative PCR analysis of distinct inflammatory cytokines and growth factors in ears collected at day 3 post challenge. The error bars represent the SEM (n = 3 mice). *p<0.05; ***p<0.001; ****p<0.0001; two-tailed Student’s t-test. (D) Percentages of dermal IFN-?-producing CD8+ T-cells and dermal CD4+Foxp3+ICOS+ regulatory T-cells at day 3 post challenge. Single-cell suspensions were generated from the dermis and the cells were re-stimulated (5 hr) with PMA/Ionomycin to detect IFN-? production or directly stained for determination of activated Tregs. The error bars represent the SEM (n = 4 mice). *p<0.05; ns, non-significant; two-tailed Student’s t-test. Gating strategy is shown in Figure 8—figure supplement 1. (E) DT-treated WT, CD207-DTR, Cleac9A-DTR, and DC-SIGN DTR mice were tolerized with 1% 2,4-dinitrothiocyanobenzene hapten (DNTB) (applied to the shaved abdomen skin). After 7 days, the mice were sensitized with 0.5% DNFB (applied to the shaved back skin) and ear-challenged 5 days later with 0.1% DNFB. (F) The ear swelling response of painted mice was determined over a 6-day period post challenge. (G) Percentage of activated CD4+Foxp3+ICOS+ regulatory T-cells in the draining lymph nodes (LNs) of mice, 5 days after vehicle or DNTB painting. The data were pre-gated on singlet, live CD3+CD4+Foxp3+ Treg cells. The error bars represent the SEM (n = 6 mice). ****p<0.0001; ns, non-significant; two-way ANOVA. (H) Percentages of IFN-?-producing CD8+ T-cells in the draining LNs of vehicle or DNTB-painted mice followed by DNFB sensitization. Single-cell suspensions were generated from the LNs and the cells were re-stimulated (5 hr) with PMA/Ionomycin to detect IFN-? production. The data were pre-gated on singlet, live CD3+ CD8+ T-cells. The error bars represent the SEM (n = 6 mice). ****p<0.0001; ns, non-significant; two-way ANOVA. Figure 8—source data 1.Percentages of IFNgamma+ CD8+ T cells and ICOS+ FOXP3+ T regs measured in distinct skin immune repsonses.

Fig 3: Characterization of cutaneous Langerhans cell (LC) and dendritic cell (DC) subpopulations.(A) Representative flow cytometry dot plots for LC characterization in the epidermis. Cells from epidermis were first gated for FSC, SSC, and CD45 (not shown). Then, CD45+ cells were analysed for CD11b and F4/80 expression. The CD11bhiF4/80hi cell fraction was further analysed for CD207 and CD326 expression to identify classical bona fide LCs. (B) Representative flow cytometry dot plots for dermal LC and DC subpopulations. Isolated dermis cells were first gated for FSC, SSC, and CD45 (not shown). CD45+ cells were then analysed for CD11b and F4/80 expression. The CD11bhi F4/80hi fraction contained classical CD207+CD326+ LCs. The remaining cells were gated for CD11c+MHC II+ DCs and separated into three subsets based on CD103 and CD11b expression: CD103+CD11b- cells (labelled cDC1), CD103-CD11bhi DCs (labelled CD11bhi), and CD11blow/neg. CD207 and CD326 expression was detectable on cDC1 but not CD11bhi DCs, whereas CD11blow cells were further separated into CD207-CD326- (labelled triple negative [TN]) and CD207+CD326+ (labelled LClike). (C) Representative flow cytometry dot plots for cutaneous DC subpopulations in auricular skin-draining LNs. LN cells were first gated for FSC and SSC to exclude small lymphocytes before F4/80 and CD11b staining. The cell fraction excluding F4/80hi/CD11bhi cells was separated by CD11c and MHCII. CD11chiMHCIIhi migratory DCs were gated and analysed for CD103, CD11b, CD207, and CD326 expression. Four subsets were detected: CD103+CD11b-CD207+CD326+ (cDC1), CD103-CD11blowCD207-CD326- (TN), CD103-CD11blowCD207+CD326+ (LClike), and CD103-CD11bhiCD207-CD326-/+(CD11bhi). (D) Frequency of each DC subpopulation (LC, cDC1, LClike, TN, and CD11bhi) present in epidermis, dermis, and cutaneous lymph node (LN), respectively.

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.