Flt3L dependence helps define an uncharacterized subset of murine cutaneous dendritic cells

Abstract

Skin-derived dendritic cells (DCs) are potent antigen-presenting cells with critical roles in both adaptive immunity and tolerance to self. Skin DCs carry antigens and constitutively migrate to the skin-draining lymph nodes (LNs). In mice, Langerin-CD11b- dermal DCs are a low-frequency, heterogeneous, migratory DC subset that traffics to LNs (Langerin-CD11b- migDCs). Here, we build on the observation that Langerin-CD11b- migDCs are Fms-like tyrosine kinase 3 ligand (Flt3L) dependent and strongly Flt3L responsive, which may relate them to classical DCs. Examination of DC capture of FITC from painted skin, DC isolation from skin explant culture, and from the skin of CCR7 knockout mice, which accumulate migDCs, demonstrate these cells are cutaneous residents. Langerin-CD11b- Flt3L-responsive DCs are largely CD24(+) and CX3CR1(low) and can be depleted from Zbtb46-DTR mice, suggesting classical DC lineage. Langerin-CD11b- migDCs present antigen with equal efficiency to other DC subsets ex vivo, including classical CD8α cDCs and Langerin+CD103+ dermal DCs. Finally, transcriptome analysis suggests a close relationship with other skin DCs, and a lineage relationship with other classical DCs. This work demonstrates that Langerin- CD11b- dermal DCs, a previously overlooked cell subset, may be an important contributor to the cutaneous immune environment.

Figure 1. Flt3L dependence of migratory DC subsets, including Langerin-CD11b− DC

A) Schema of LN DC from Flt3L-tumor treated vs. C57Bl/6 (B6) control mice at day 13. PDC (plasmacytoid DC), cDC (classical LN resident DC), migDC (migratory DC) are labeled. (B) LN DC subsets Flt3L-treated (upper) vs. untreated Langerin-GFP mice. (C) Quantitation of migratory DC from Flt3L-treated, control, Flt3L−/− mice (quantitated per skin draining LN; error bars show mean +/− SD of 3 individual mice per group, analyzed by an unpaired t-test). (D) Flt3L-induced expansion of skin resident DC subsets from "crawl-out” skin explants of doxycycline-inducible Flt3L mice given 8 days of doxycycline in their drinking water (top) vs. untreated controls (bottom) (one representative experiment of two). (E) Quantitation of DC isolated from skin explants “crawlouts” of Flt3L-treated vs. control Langerin GFP reporter mice (pooled from the ears of 3 mice per experiment, one representative experiment of three). (F) α CD205+ staining of skin DC subsets from explant cultures of doxycycline treated (Flt3L+) vs. untreated (control) mice (pooled from n=2 mice per condition).

Figure 2. CD11b− DC are skin-resident and migrate to the skin draining LN

(A) Upper: Representative skin draining LN DC gating from CCR7−/− mice treated with Flt3L (left), CCR7−/− untreated controls (middle), and B6 controls (right). Lower: CCR7−/− skin resident DC subsets from “crawl-out” explants (Flt3L treated vs. untreated controls). One of three representative experiments are shown. (B) FITC painting assay with gating on FITC+ migratory DC vs. total migratory DC in Flt3L treated (upper) and control mice (lower). CD11b− migDC (red) directly capture FITC in the skin and traffic to the skin draining LN at 16 hour and 24 hours in C57BL6/WT mice (n=3 mice per time point). (C) Immunofluorescence of flank sections of Flt3L-treated (upper) and control (lower) mice co-stained with langerin (green), CD11b (red) and CD11c (magenta) and counterstained with DAPI to visualize cell nuclei. Positions of Langerhans cells (LC: langerin+, CD11b+, CD11c+, #). Dermal DC (CD11c+) were further distinguished as langerin-CD11b− (↑), langerin-CD11b+ (+) and langerin+CD11b− (*) were marked in the merged images. Scale bar =50 µm for all images.

Figure 3. Flt3L responsive CD11b− migDC include CD24^high CX₃CR1^low subsets

(A) In skin and skin draining LN a subset of CD11b− migDC are CD24+ and expand with Flt3L. Gated pre (open) and post (closed) Flt3L in LN and skin explant (lower) as compared to classical CD8α + DC and other migDC (n=3 mice per group, one representative experiment of three). (B) Lower: Flt3L expanded CD24+ cells are CX₃CR1^low within the CD11b− migDC subset (red) as compared to migratory CD11b+ cells (blue). Upper: Comparison of LN resident CD11b+ CD8α- cDC (blue) and CD8α+ cDC which are CD24+ CX₃CR1^low (red) (n=2 Flt3L-treated or control CX₃CR1 mice per group, one representative experiment of two).

Figure 4. CD11b− migDC are Zbtb46 dependent

(A) LN classical vs. total migratory DC and (B) migratory DC subset counts from skin draining LNs after single dose DT vs. PBS administration 24 or 48 hours prior to harvest (n=3 mice per time point, one representative experiment of three). (C) Skin explants pooled from three Zbtb46DTR vs. C57BL/6 controls 24 hours after DT treatment (n=3 mice pooled per group, one representative experiment of two).

Figure 5. CD11b− migDC present antigen

CD11b− migDC present antigen CD11b− migDC present allo-antigens in the mixed leukocyte reaction (A–B) and present OVA protein (C–D) to CD8+ V α 2 OT-1 T cells wit equal efficiency to other DC subsets ex vivo. One of two duplicate experiments are shown with triplicate wells per sample.

Figure 6. Langerin- CD11b− DC relate closely to classical DC

(A) Hierarchical clustering of subsets based on 2 fold change or greater of all genes (n=8601) (B) Heat map comparison of DC signature gene expression across DC groups as compared to macrophages (yellow indicates the core DC signature). (C) Heat map comparison of normalized intensity values from selected DC genes across all subsets.