Vitiligo is an autoimmune skin disease characterized by focal depigmentation due to melanocyte loss attributed to autoreactive T cells. Recent single cell studies highlight activation of IFNg pathways in both lesional and non-lesional skin and stressed keratinocytes with metabolic switch towards oxidative phosphorylation in lesional skin. In this integrative study we combine existing single cell datasets and put it into spatial context with 10x Visium spatial sequencing of 25 samples from non-, peri- and lesional vitiligo and healthy skin. We utilized bulk RNAseq to assess tissue responses to pan T cell stimulation and measure quantitatively deconvolution-derived cell types changes in health and vitiligo. Pigmented peri-lesional vitiligo was found to be a site of active IFNγ inflammation and oxidative phosphorylation, with IFNγ responses highly associated with Treg cell signal. In contrast, depigmented lesions displayed dormant IFNγ inflammation, that was revealed upon T cell stimulation. Besides IFNg responses, vitiligo epidermis reacted to T cell stimulation with local T cell proliferation. Lesional melanocytes had increased expression of the damage marker S100B and selenoamino acid metabolism, both previously proposed biomarkers of disease activity. Moreover, S100B protein was specifically upregulated in T cells-melanocytes colocalizations. The combined modality of the study revealed novel insights into spatially restricted disruptions of cellular environments in vitiligo.

Tissue-resident memory T (Trm) cells provide local protection against recurrent infections and are implicated in recurrent inflammatory skin diseases such as psoriasis. Once seeded, individual clones of Trm cells can persist in homeostatic skin up to a decade, despite the expected life-spans of memory T cells ranging between 150 days -2 years. In this study, we investigate local precursors capable of replenishing and therefore retaining site-specific T cell populations in steady state and disease affected skin. Employing single cell sequencing with T cell receptor (TCR) and CITEseq technology, we identified a subset of lower differentiated Trm cells marked by the expression of selectin CD62L in healthy skin, psoriasis lesions and cutaneous T cell lymphoma (CTCL). These lower differentiated CD62L+ Trm cells showed clonal sharing with highly enriched clones of CD69+ CD103+ CD49a+ cytotoxic T cells in the skin but were less abundant in the blood. Additionally, these had high proliferative potential and differentiated into cytotoxic CD103+CD49a+ Trm cells following activation. Taken together, our study reveals a source of local replenishment of Trm cells in healthy and diseased skin which could explain the longevity of localized disease memories.

Tissue-resident memory T cells (TRM) are seeded and persist for years at the site of inflammatory responses in non-lymphoid tissues. In human skin, enrichment of oligoclonal cytotoxic CD103+CD49a+ TRM in disequilibrium with circulating T cells indicates local renewal of epidermal T cells. Indeed, a subset of less differentiated skin resident CD62L+CD49a- CD103-CD69+ TRM cells was identified by single cell CITE-seq and T cell receptor sequencing. Resident CD62L+ CD69+ T cells showed clonal sharing with epidermal CD49a+ CD103+ CD69+ cytotoxic T cells. Following stimulation, CD62L+ CD69+ T cells displayed high proliferation potential and differentiated into cytotoxic CD103+ CD49a+ TRM cells. Furthermore, in cutaneous T cell lymphoma (CTCL), driven by one expanded clone of malignant CD8+ T cells, both lowly differentiated CD69+CD62L+ T cells and highly differentiated CD49a+CD103+ TRM cells phenotypes were detected in the malignant clone. In conclusion, we revealed a skin resident precursor population to CD49a+CD103+ TRM cells marked by the expression of CD69 and CD62L, capable of maintaining the local pool of skin resident memory T cells in health and skin diseases.

Vitiligo is an inflammatory disease that presents as well-demarcated, depigmented skin lesions. While different mechanisms are implicated in the development and maintenance of vitiligo lesions, immune-mediated destruction of melanocytes has been ascribed the major mechanism of depigmentation. We have previously observed enrichment of cytotoxic resident CD49a+ CD103+ T-cells in the skin of vitiligo patients and recent single cell data highlights crosstalk between CD8+ effector and Treg cells within the lesion, as well as a subclinical state of activation in the non-lesional skin. Here, T cell composition and how these cells interact in situ with melanocytes was assessed in lesional, peri-lesional, and non-lesional skin with Nanostring GeoMX spatial proteomics and 10x Genomics Visium spatial transcriptomic systems. We observed increased levels of S100B protein in the T cell and melanocyte colocalisations in vitiligo skin, previously proposed as a marker for disease activity. Spatial transcriptomic analysis corroborated these finding by revealing increased expression of S100 family genes in the basal epidermal layer of vitiligo lesion, combined with decreased expression of several collagen related genes. Pan- T cell activation in vitiligo skin activated IFNg pathway related genes and genes responsible for proteasome assembly proteins. While some of the genes were also upregulated in activated non-lesional and healthy skin, the levels of expression were several-fold higher in lesional vitiligo. Overall our study shows that the vitiligo skin is a site of quiet inflammation and melanocyte injury, with quiescent IFNg dominated pathophysiology that is induced following T cell activation.