Fig 2: Depletion of CD11c+ cells results in a reduction of CD8 tissue resident memory cells.(A, B, C, D) Melanoma-associated vitiligo (MAV) was induced in CD11c.DTR+ and CD11c.DTRneg mice. 30 d after surgery, mice received three doses of 50 ng diphtheria toxin (DT) i.d. over 7 d at the surgical site before the skin was analyzed. (B) Expression of CD8 T cells and CD11c+ cells in skin from CD11c.DTRneg and CD11c.DTR+ MAV-affected mice; CD11c (green), CD8ß (red), and nuclei (blue). Scale bar, 50 µm. (C) Number of clusters found per cm2 of skin from CD11c.DTR+ and CD11c.DTRneg MAV-affected mice treated with DT. (D) Expression of CXCL16 and CD11c in skin from CD11c.DTRneg and CD11c.DTR+ MAV-affected mice treated with DT; CD11c (green), CXCL16 (red), and nuclei (blue). Scale bar, 50 µm. (E) Relative CXCL16 expression in skin from CD11c.DTR+ and CD11c.DTRneg MAV-affected mice treated with DT. (F, G) 6 wk after lethally irradiated WT congenic CD45.1 mice received CD11c.DTR bone marrow, MAV was induced and beginning d30 after surgery mice received 250 ng DT or PBS i.p. every 3 d until skin was harvested d60 post-surgery. (G) Percent of CD8 T cells or CD11c+ cells isolated from skin of MAV-affected mice treated with either DT or PBS. (C, G) Symbols represent individual clusters (C), individual mice (G); horizontal lines indicate mean. (B, C, D, E, G) n = 4–6 mice pooled from two independent experiments (B, C, D, E) n = 10–14 mice pooled from two independent experiments (G) Significance was determined by Mann–Whitney test; *P = 0.04, ***P = 0.0001.

Fig 3: Multiple CD11c+ subsets are found in skin from melanoma-associated vitiligo (MAV)–affected mice.(A) CD11c+ cells and CD11b+ cells in skin from MAV-affected mice. CD11c (green) and CD11b (red). Scale bar, 50 µm. White arrows indicate CD11c+CD11b+ cells. (B) Immunofluorescence microscopy of macrophages in unaffected and MAV-affected skin identifying. Stains identify CD11c (red), F4/80 (green), and nuclei (blue). Scale bar, 50 µm. (C) Representative flow cytometry plots of gating strategy to sort CD8+, CD11c+CD11bneg, and CD11c+CD11b+ from the surgical site of MAV-affected skin 50 d after surgery. (D, E) Cells used for RNAseq were sorted from three individual mice based on expression of CD11c and CD11b. (D) Heat map of top 100 differentially expressed transcripts in CD11c+CD11bneg and CD11c+CD11b+ sorted cell populations; presented as log2-normalized expression. (E) Heat map of myeloid transcripts in CD11c+CD11bneg and CD11c+CD11b+ sorted cell populations; presented as log2-normalized expression.

Fig 4: Clustering CD8 T cells have a tissue-resident memory phenotype.(A) Representative flow plots showing expression of CD103 and CD69 on CD8 T cells isolated from LN and skin from melanoma-associated vitiligo (MAV)–affected mice. Cells were isolated from experimental mice 35 post-surgery and were identified by CD8+CD62LnegCD44+. (B) Skin from MAV-affected mice, stains identify CD8ß (red), CD103 (green), and KLRG1 (white). Scale bar, 50 µm. (C) Number of CD103negKLRG1negCD8+, CD103+KLRG1negCD8+, and CD103negKLRG1+CD8+ T cells per cluster in a 10 µm thick skin section. Symbols represent individual clusters; horizontal lines indicate mean; n = 9 clusters. (D) Heat map of skin-resident memory transcripts comparing bulk endogenous CD8 T cells to Ag-specific Pmel CD8 T cells and naïve CD8 T cells. Bulk endogenous and Ag-specific Pmel CD8 T cells were isolated from MAV-affected skin, naïve CD8 T cells were isolated from spleen and LN of naïve Pmel mice.

Fig 5: RNA and antibody-based epitope expression in brain infiltrating leukocytes(A) Example of gating leukocyte populations using CITE antibodies. Briefly, CNS native myeloid cells (microglia and BAM) were gated based on both populations lowly expressing CD45 and then differentiated from each other based on higher I-A-I-E and CD38 expression in BAM. Peripheral leukocytes were gated based on high CD45 expression. Within the peripheral leukocyte population: B cells were defined as having CD19 and B220 expression. T cells were defined based on high CD3 expression and low NK1.1 expression, then subpopulations further distinguished on the basis of CD4 and CD8 expression. Myeloid cells were distinguished from adaptive peripheral immune cells by high CD11b expression, and then further subdivided on the basis of Ly6C expression levels and Ly6G expression.(B) Example of gating leukocyte populations using CyTOF antibodies.(C) Biaxial plots of CITE epitope expression on the x axis and its corresponding mRNA expression on the y axis. Discordant expression of mRNA and protein can be observed.(D) UMAPs derived from CITE-seq data clustered by RNA transcription cluster color coded by transcriptional cluster (left) or canonical cell ID determined by CITE-antibody gating (right). While canonically identified cells generally group within the same cluster, there is appreciable dispersion of canonical cell types among clusters, indicating the utility in using CITE antibody tags rather than mRNA alone to identify canonical cell types.