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.

Sex is an important biological variable in human biology with consequences for manifestations of various inflammatory diseases. Sex-associated difference is found in circulating T cell immunity as females are more prone to have autoimmune disease, but it is not clear whether tissue residing T cells are specific to sex. To investigate the effects of sex on the pool of resident T cells in human skin, the sex difference on skin T cell profiles were assessed. The effects of testosterone/estradiol on human CD8 T cell differentiation were also addressed. Both bulk and single RNA sequencing data from human skin from public repositories exhibited that female and male have distinct transcriptional signatures. Protein analyses revealed that female skin harbors around twice more cytotoxic and IFNγ producing CD8 resident memory T cells (Trm) in epidermis compared to that in males or testosterone-treated females. Furthermore, in vitro analyses displayed that testosterone hampers development of cytotoxic CD8 Trm from circulating T cells. These differences were not prominent in dermal skin compartment. Collectively, these results lead to regard sex as one of the variables to consider in the composition of epidermal skin T cells.

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.

The human skin harbors a heterogenous pool of cytotoxic tissue-resident memory T (TRM) cells seeded during infections and inflammation. TRM cells are retained at the site of viral infections and provide local immunity. These cells persist for years in the skin after bone marrow transplantation but circulating ex-TRM cells indicate several potential fates. Here, we explored the clonal relationships between human T cells in different skin compartments and circulation. 10x Genomics 5 prime immune profiling coupled with feature barcoding technology was employed to investigate the clonal relationship, transcriptomic and proteomic profiles of single T cells sorted from blood, subcutis, dermis, and epidermis. A few CD8+ TRM cell-clones dominate epidermis and cytotoxic CD8+CD103+ CD49a+ showed the least diverse T cell receptor (TCR) repertoire. Clonal overlap was found between all three compartments: circulating blood T cells, dermal and epidermal TRM cells. The highest degree of overlap was detected between epidermis and dermis. Clones overlapping was scarce between blood and highly abundant epidermal clones. Our results suggest that lowly abundant skin clonotypes have a circulatory counterpart, which can react to reinfection at secondary sides, whereas highly abundant epidermal clones might be skin specific.

Pathogenic memory T cells are implicated in the local relapse of allergic contact dermatitis (ACD), vitiligo and psoriasis. Here we investigate how resident T cells initiate relapses in response to the allergen Methylisothiazolinone (MI). MI–related ACD was sampled two months and two years after resolution of disease. T cells were activated in skin explants with MI or the pan T-cell agonist OKT-3. Epidermis was analysed by RNAseq, Nanostring and multiplex methods. Metalloproteinase 12 (MMP-12)–induction by ACD–related cytokines was assessed in primary keratinocytes and fibroblasts and a humanized xenograft mouse model was used. Ex vivo activation of T cells in resolved ACD induced chemokines and cytokines involved in immune cell recruitment and tissue remodeling, including the collagenase MMP-12. In response to cytokine stimulation, human primary fibroblasts and keratinocytes had the capacity to overexpress and release MMP-12. Local injections of MMP-12 and ACD–associated cytokines in vivo resulted in degradation of the Collagen IV–rich basement membrane separating the epidermis and dermis and a redistribution of T cells within the skin. Resident T cells in resolved ACD induce MMP-12–driven tissue remodeling that facilitates recruitment of local T cells. Our findings emphasize the potential for topical T cell eradication to promote deep remission of relapsing inflammatory dermatoses.

L’eczéma de contact allergique est une dermatose inflammatoire récidivante dont la physiopathologie pourrait reposer sur la présence de lymphocytes T résidents mémoires (TRM) cutanés dans la peau précédemment exposée et sensibilisée. Ici nous souhaitions activer les TRM d’un allergène cutané donné, à différents stades de la maladie pour caractériser les réponses tissulaires précoces lors d’une élicitation. Des biopsies de patients avec une allergie cutanée au méthylisothiazolinone ont été prélevées à J7 de patch-tests sur peau lésionnelle et non-lésionnelle (n = 3), puis à deux mois et dans une autre cohorte de patients deux ans après la résolution des symptômes (n = 4). Les biopsies ont ensuite été sectionnées et exposées au méthylisothiazolinone (100 ppm), à OKT-3 (1 mg/mL) afin d’activer les TRM d’une manière non-antigène-spécifique, et à un contrôle. L’ARNm a été purifié de l’épiderme puis séquencé (Illumina HiSeq 2000) et les résultats ont été validés par Nanostring technologies. Les concentrations en médiateurs de l’inflammation ont été analysés dans le surnageant des biopsies stimulées. L’analyse transcriptomique épidermique retrouvait une signature moléculaires dans la peau guérie jusqu’à deux ans après l’arrêt des symptômes, avec une majoration des transcrits S100A7-9 dans la peau guérie comparée à la peau exempte de lésion. La simulation ex vivo de poussée, par l’exposition au méthylisothiazolinone, réalisée deux mois après la résolution des symptômes, entraînait une augmentation de l’expression et de la libération protéique des médiateurs IL-13 IL-22 et IL-9, alors que la simulation ex vivo de récidive deux ans après la résolution des symptômes n’entraînait que des modifications transcriptionnelles, sans détection de libération protéique du tissu cutané. L’exposition des biopsies à OKT-3 montrait des réponses tissulaires partiellement superposables à l’exposition à l’haptène méthylisothiazolinone, ce qui indique que le recrutement des TRM bystanders, non spécifiques du méthylisothiazolinone a un effet tissulaire distinct. En conclusion, la mémoire locale de l’eczéma de contact allergique persiste au long terme au niveau transcriptomique mais au long terme le tissu modifie le contrôle des manifestations post-transcriptionnelles qui interviennent lors d’une nouvelle exposition cutanée. Cela permet un arrêt de l’amplification du signal, et donc des dommages tissulaires. Les mécanismes de ce contrôle post-transcriptionnel et la cinétique de sa mise en place restent à décrire précisément dans la peau humaine et pourraient reposer sur des modifications épigénétiques ou encore sur le contrôle des populations de TRM exprimant des inhibiteurs de checkpoint, récemment décrites chez la souris dans cette maladie.

Human skin contains a large number of T cells many of which display markers associated with resident memory T cells (TRM). TRM cells stay in the same sites for long periods and are superior to recirculating T cells in their ability to initiate local barrier protection against pathogens. The skin barrier is challenged through life and T cell functionality may change with time. Here, T cell density, diversity and function was assessed in 91 skin specimens and 79 blood samples from Japanese and Swedish subjects of different ages. Aging did not affect the density of T cells both in blood and skin but the frequency of epidermal T cells, particularly CD49a+ CD8 TRM, increased in elderly individuals (CD49a+ in CD8+CD69+CD103+: p=0.0062 in epidermis). To investigate the functional anti-pathogen activity of T cells in blood and skin, heat-killed Staphylococcus aureus and Candida Albicans antigens were either co-cultured with PBMCs or injected to the resected skin specimens. Pro-inflammatory cytokine production from T cells were maintained in skin but declined in the blood from elderly individuals (e.g. % IL-17A production in CD4 T cells in blood: p=0.0154, in skin: p=0.3319). T-cell diversity was also evaluated by sequencing of genomic CDR3 in T cells. Over 60 % of skin TCR clones were found as single copies and this ratio did not decline as individuals age (p=0.5756). The occupancy of top 10 most frequently-detected TCR clones in the total clones was high in blood, not in skin, of elder individuals (% occupancy of top 10 clones; 20-49 y.o. vs 50-79 y.o.; p=0.0299 in blood, p=0.1252 in skin), suggesting that T cells in blood, but not in skin, of elderly subjects become oligoclonal with expansion of limited clones. Our findings demonstrate that skin T cells maintain diversity and protective cytokine production in elderly individuals despite reduced T-cell diversity and function in blood.