Fig 1.
Antigen presenting cells are increased and of altered phenotype in K14.E7 transgenic mouse epidermis.
Single cell suspensions of C57BL/6 (C57) and K14.E7 epidermis were analyzed for different CD45+ (myeloid) antigen presenting cell subsets. (A) Gating strategy to identify LCs, CD11b+ and CD103+ DCs in C57BL/6 and K14.E7 epidermis. Plots were pre-gated on live CD45+ cell singlets. (B) Relative proportions of LCs, CD103+ and CD11b+ DC subsets from CD11c+ DCs in K14.E7 and C57BL/6 epidermis. (C) Relative (as % of CD45+ live cells) and absolute number of LCs in the epidermis of two K14.E7 and C57BL/6 mouse ears (D) Expression of cell surface markers MHCII, CD86, CD80, CD207 and CD11b on epidermal LCs from C57BL/6 and K14.E7 ear skin. Typical histograms (C57 = black, K14.E7 = white) and pooled median fluorescent intensity (MFI) data (n = 6, two independent determinations) are shown.
Fig 2.
LCs, CD11b+ and CD103+ dDCs are increased in K14.E7 transgenic mouse dermis.
Single cell suspensions of C57BL/6 and K14.E7 dermis were analyzed for different CD45+ (myeloid) antigen presenting cell subsets. (A) Gating strategy to identify LCs, CD11b+ and CD103+ DCs in C57BL/6 and K14.E7 dermis. Plots were pre-gated on live CD45+ cell singlets. (B) Relative proportions of LCs, CD103+ and CD11b+ DC subsets from CD11c+ DCs in K14.E7 and C57BL/6 dermis. (C) Absolute number of LCs, CD11b+ and CD103+ DCs in the dermis of two ears. (D) Expression of cell surface markers MHCII, CD86, CD80, CD207 and CD11b on dermal LCs from C57BL/6 and K14.E7 ear skin. Typical histograms (C57 = black, K14.E7 = white) and pooled median fluorescent intensity MFI data (n = 6, two independent determinations) are shown.
Fig 3.
Epidermal LCs of K14.E7 mice express immune-modulatory cytokines and enzymes.
(A) Single cell suspensions of C57BL/6 and K14.E7 epidermis were prepared. LCs (singlets, live CD45+ CD11c+ MHCII+ CD11b+ Epcam+ CD103-) of K14.E7 and C57BL/6 epidermis were sorted with purity above 96%. Relative gene expression of IDO-1, Arg-1, IL-10, IL-6 and IL12/23p40 messenger RNA was determined by real-time qPCR and normalized against housekeeping gene RPL32. Data are presented as means ±SEM of four independent experiments in which ear epidermis of either 4 C57BL/6 or 2 K14.E7 mice was pooled prior to LC isolation. (B) Single cell suspensions of C57BL/6 and K14.E7 dermis and epidermis were analyzed for the expression of PD-L1 on different CD45+ antigen presenting cell subsets. Gating strategy to identify LCs, CD11b+ and CD103+ DCs in C57BL/6 and K14.E7 skin was performed as shown in Fig 1A and Fig 2A. Expression of cell surface PD-L1 on dermal and epidermal DCs was analyzed. Representative histograms (C57 = black, K14.E7 = white) of CD11b+ DCs, LCs and matching isotype controls, and pooled median fluorescent intensity MFI data (n = 5) are shown.
Fig 4.
Skin-resident DCs of K14.E7 mice take up antigen and migrate to the draining lymph nodes.
C57BL/6 and K14.E7 mice were painted with 100μl of 5mg/ml FITC dissolved in 1:1 acetone:dibutylphalate on shaved flanks and ears. 24 hours later, axillary and inguinal lymph nodes were analyzed by flow cytometry for presence of FITC+ DC subsets. (A) FITC uptake by DC subsets in lymph nodes pre-gated according to Table 1 and S1 Fig. (B) MFI of FITC+ LCs in the ear epidermis pre-gated according to Fig 1A. (C) MFI and total number of FITC+ DCs in lymph node. Shown is one of two independent experiments, n = 4.
Table 1.
DC subsets analyzed in lymph nodes and skin and their identification by surface maker stainings.
Fig 5.
Isolated skin-derived DCs of K14.E7 mice present antigen in vitro, and induce CD8+ T cell proliferation.
DCs (CD45+ CD11c+ MHCII+) were sorted from ear epidermis and dermis of 10 pooled C57BL/6 and 8 pooled K14.E7 mice and co-cultured together with sorted splenic OT-I CD8+ T cells and SIINFEKL at a 1:10 ratio with 5x104 total cells per well. After 72 hours cells were incubated overnight with 3H-thymidine and uptake was determined. Shown are triplicates of two pooled independent experiments.
Fig 6.
Skin-resident DCs of K14.E7 mice are impaired in their ability to process antigen in vivo.
(A) Whole dermal and epidermal single cell suspensions of C57BL/6 and K14.E7 ear skin were incubated with 1ug/ml DQ-OVA for 1 hour and analyzed for photolytic degradation by flow cytometry. DC subsets were gated according to Table 1. The green median fluorescent intensity of whole LCs, DQ-OVA+ CD103+ DCs and DQ-OVA+ CD11b+ DCs was compared. (B) C57BL/6 and K14.E7 mice were injected intradermally into the ear with 20μl of 1mg/ml labeled DQ-OVA. 24 hours later, single cell suspensions of treated and untreated ear dermis and epidermis were prepared and analyzed by flow cytometry for photolytic degradation of applied DQ-OVA, as recognized by fluorochrome unmasking. Histogram overlays of fluorescence in LCs, CD103+ dDCs and CD11b+ dDCs determined according to Table 1 in one representative C57BL/6 (grey line) and K14.E7 (black line) mouse ear treated with DQ-OVA. The change in green median fluorescent intensity from untreated to DQ-OVA treated in dermal and epidermal LCs, CD103+ dDCs and CD11b+ dDCs from K14E7 (●) and C57BL/6 (■) mouse ears (n = 4) was compared. (C) C57BL/6 and K14.E7 mice were injected intravenously with OT-I CD8+ T cells and immunized intradermally into the ear pinnae with 20μl of 1mg/ml OVA. One week later, IFNγ secretion by OT-I T cells was analyzed by recalling ear-draining lymph node cells with SIINFEKL antigen in an ELISpot assay. Depicted are spots per 2x105 cells. Each data point represents means of triplicates of one individual animal (n = 5).