This research aimed to understand the mechanisms that dictate differentiation, maintenance and functionality of skin Trm cells and it was carried out through two work packages (WP):
• WP1: Determining the chromatin landscape and transcriptional regulation of Trm cell subsets
• WP2: Unravelling the phenotypic and molecular properties of IFNg- and IL17-producing Trm cells
WP1. Establishment of immunological memory requires stable changes occurring at epigenetic level. Our laboratoratories have previously uncovered a functional dichotomy of epidermal Trm cells with respect to the expression of CD49a, where epidermal CD49a+ Trm cells mediated cellular cytotoxicity and excelled at IFNg production whereas IL-17-producing cells were contained within the CD49a– counterpart. Aiming to understanding the molecular circuitry controlling the differentiation of specialized populations of human skin CD8+ Trm cells, we leveraged chromatin accessibility information as a way to infer cell identity and lineage development. By mapping transposable-accessible chromatin using sequencing (ATAC-seq), we identified regions that are relevant for different Trm cell subsets both in the dermis and the epidermis. We revealed that CD49a expression marks CD8+ Trm cells endowed with a distinct chromatin landscape and a specific trajectory of differentiation, accompanied by pre-existing accessibility at key effector loci. We revealed the transcription factors (TF) able to bind to subset-specific accessible regions. In particular, we demonstrated that one TF is expressed at highest levels concomitant with acquisition of the Trm phenotype, displays the strongest binding in the epidermis CD49a+ cells and is responsible for acquisition of CD49a surface expression by circulating memory CD8+ T cells.
WP2. Phenotypic and functional heterogeneity is a feature of Trm cells located in the tissue. By combining chromatin accessibility with transcriptomic information, we revealed phenotypic and molecular determinants of CD8+ Trm cell subsets that correlated with their capacity to produce IL-17 or IFNg and to upregulated cytotoxic mediators upon IL-15 stimulation. In order to unveil human skin CD8+ Trm cell heterogeneity at single cell level and to couple information on clonal dynamics, we performed scRNAseq in collaboration with the Dr Eidsmo’s research team. This part of the proposal is still ongoing and the data will be used to resolve gene expression at single cell level in healthy or pathological tissues. The analysis of circulating and resident CD8+ memory T cells from the blood and skin, respectively, of individual healthy donors, revealed overlap of TCR clonotypes suggesting common origin of CD8+ T cells that display different phenotypes and localization. By in vitro cultures of blood-derived CD8+ T cells with skin-derived signals, we recapitulated the acquisition of phenotypic, functional and transcriptional determinants of epidermal CD49a+ Trm cells. Moreover, we revealed the subset of circulating CD8+ T cells that preferentially give rise to epidermal CD49a+ Trm-like cells.
