103 Dissecting autoimmune signaling networks in vitiligo using single-cell RNA-sequencing of cells isolated directly from lesional skin

Abstract

Vitiligo is an autoimmune disease characterized by patchy, depigmented lesions of the skin. The disease is mediated by autoreactive CD8 + T cells that destroy melanocytes. To investigate signaling networks in the skin that lead to this patchy melanocyte destruction, we used a modified suction blistering technique to isolate cells directly from lesional skin and subjected them to single-cell RNA-sequencing. We sequenced cells from both lesional and non-lesional skin in six vitiligo subjects with active disease as well as six healthy controls. We identified multiple cell populations, including melanocytes, keratinocytes, Langerhans cells and immune cells using unbiased differential gene analysis to cluster over 18,000 cell transcriptomes. Moreover, keratinocytes clustered based on differential expression of keratin family members, identifying basal and suprabasal subpopulations. We found an IFN-g signature and elevated CD8 + T cell number in vitiligo lesions, which is consistent with previous findings by our group and others. We also identified CXCR6 and CXCL16, which were recently hypothesized to play a role in vitiligo, in addition to 87 other ligand-receptor pairs that may also help to promote disease progression. Intriguingly, Langerhans cells may play a more active role in vitiligo as they actively express many genes that have known risk alleles. Overall, single-cell RNA-sequencing of cells from blister fluid provides robust data that confirms many previous findings, and adds additional layers of information missed by conventional studies. This level of resolution will yield powerful insight into the identification of novel drug targets in previously unknown pathological signaling pathways in vitiligo and other diseases of the skin. 104IFNg and fas ligand are required for melanocyte destruction in a mouse model of vitiligo Autoreactive CD8 + T cells are responsible for the selective destruction of melanocytes in vitiligo. Previous studies by our group and others demonstrated that IFNg-induced chemokines are required for T cell recruitment into the skin and progression of vitiligo. To determine the mechanism of melanocyte killing by autoreactive T cells in our mouse model of vitiligo, we decided to test the role of known CD8 effector molecules. We found that disease severity and T cell accumulation in the skin was reduced in mice receiving adoptive transfer of either IFNg -/or Fas ligand (FasL) -/autoreactive T cells; however, perforin was dispensable. We did not observe defects in expansion or migration any of the transferred T cells. Co-injections of 50:50 mixed KO T cells with WT T cells rescued vitiligo scores, but did not achieve the same level of disease as mice injected only with WT T cells. Intriguingly, FasL -/and IFNg -/-T cells accumulated in greater numbers in the skin than co-injected WT T cells, suggesting that IFNg and Fas Ligand are required locally in the skin to cause disease. To determine if IFNg has a direct cytotoxic effect on melanocytes, we selectively deleted IFNgR from melanocytes using...

Single-cell sequencing reveals increased LAMB3-positive basal keratinocytes and ZNF90-positive fibroblasts in autologous cultured epithelium

“…Here, we provide a molecular characterization of the different cell types observed in cultured epithelial sheets derived from healthy skin in vitiligo patients using scRNA-seq. We identified 16 cell types, almost all of which had previously been described 13 , 25 . Considering that cellular transcriptomes can be affected under different culture conditions with different compositions of medium, we compared the cell compositions of cultured epithelium sheets with those of healthy skin.…”

“…Single-cell RNA sequencing, as an emerging technology, may provide an opportunity to uncover the cell heterogeneity of epithelial sheets. This method has revealed cell heterogeneity in vitiligo lesions, representing cell populations that promote disease progression, determine autoimmune disorders, and indicate therapeutic targets [12][13][14] . To identify potential molecular benefits for repigmentation, we conducted single-cell transcriptomics of epithelial grafts under SFF and SFD conditions to assess the similarities and variations in cell composition.…”

Single-cell sequencing reveals increased LAMB3+ basal keratinocytes and ZNF90+ fibroblasts in autologous cultured epithelium under serum- and feeder-free conditions

“…Here, we provide a molecular characterization of the different cell types seen in cultured epithelial sheets derived from healthy skin in vitiligo patients using scRNA-seq. We identi ed 16 cell types, almost all of which had previously been described 24,25 . The further discovery is that keratinocytes and broblasts were further distinguished by quantitative gene expression, with keratinocytes consisting of 7 clusters and broblasts consisting of 2 clusters.…”

“…Single cell RNA sequencing, as an emerging technology, may provide an opportunity to uncover the cell heterogeneity of epithelial sheets. It has revealed cell heterogeneity in vitiligo lesions, representing cell populations that promote disease progression, determine autoimmune disorders, and indicate therapeutic targets [12][13][14] . To identify potential molecule bene ts for repigmentation, single cell transcriptomics of epithelial grafts under SFF and SFD conditions were conducted to assess the similarities and variations in cell composition.…”