Tissue Resident Memory (Trm) CD8+ T cells: Genome-wide dissection of cellular differentiation and heterogeneity

Periodic Reporting for period 1 - SkinTrmDeep (Tissue Resident Memory (Trm) CD8+ T cells: Genome-wide dissection of cellular differentiation and heterogeneity)

Okres sprawozdawczy: 2019-03-01 do 2021-02-28

The action SkinTrmDeep 794827 was carried out by the researcher Dr. Beatrice Zitti under the supervision of Prof. Bryceson at the Centre of Hematology and Regenerative Medicine (HERM) at the Karolinska Institute in Stockholm. The main focus of the project was to study the mechanisms underlying the differentiation and functionality of cytotoxic lymphocyte subsets localized in the human skin. Such cells are named tissue-resident memory (Trm) cells given their ability to provide long-term protection at barriers tissue where continuous challenges require the presence of specialized cells that patrol the environment and are able to rapidly recognize and kill virus-infected and cancer cells. Emerging evidence indicates a beneficial role of CD8+ Trm cells in human solid cancers, where their densities among tumor infiltrating lymphocytes correlates with improved patient survival. In human skin, Trm cells represent the predominant lymphocyte population and they comprise subsets devoted to different protective functions contributing to local immune protection to distinct pathogens. However, Trm cells may also be associated with different T cell-mediated pathologies upon aberrant activation contributing to disease burden in psoriasis and vitiligo. Translation of these new findings to the clinic requires further studies seeking a deeper understanding of the molecular mechanisms that support the acquisition, maintenance and functionality of different Trm subsets. Capitalizing on access to unique tissue samples, strengths on high-throughput sequencing and bioinformatics the project aimed to decipher how tissue-derived signals instruct the differentiation of Trm cells with distinct phenotypes and functional capacities. The long-term goal of the project was to elucidate how cells can be controlled in settings of aberrant activation or harnessed for immunity to infections or cancer.

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.

Results from WP1 demonstrate that chromatin accessibility of memory subsets of CD8+ T cells in the human skin foreseen functional capacities and differentiation potential of CD8+ Trm subsets. We revealed that CD49a expression marks the acquisition of a specific epigenetic landscape associated with pre-existing accessibility at effector functions-coding genes. These data emphasize that resident memory CD8+ T cells are equipped with a specific regulatory program imprinted in the chromatin structure, that allows them to fulfill their effector functions at barrier sites. Acquisition of this particular epigenetic programs allows cells in the epidermis to rapidly perform cytotoxicity and release IFNg in the context of viral infection or cancer. Moreover, in WP1 we delineate the transcription factors involved in promoting or impairing CD8+ Trm differentiation. Highlighting the molecular mechanisms that are critical to drive the CD49a-differentiation program is of interest in settings of genetic manipulation of CD8+ T cells for adoptive cell therapy (ACT) in solid cancers. Indeed, translations of these findings will help to elucidate the molecular requirements needed to generate CD8+ T cells able to be maintained and to perform effector functions in environments where tumors occur. Having explored the phenotypic heterogeneity of CD8+ Trm cells in the skin in WP2, we can investigate surface receptors specifically expressed by cells primed for cytotoxicity or IL-17 production that can be engaged or blocked to aid immune responses at the skin barrier. We also revealed shared clonotypes between circulating and resident CD8+ T cell populations and which compartment in the blood more effectively give rise to CD103+CD49a+CD8+ T cells phenotypically, functionally and transcriptionally resembling CD49a+ cells in the epidermis. These findings are interesting in the context of reinfections at tissue sites and provide an opportunity to optimize CD49a+CD8+ Trm cell formation taking advantaged of circulating memory cells via vaccination. Finally, this MSCA allowed the researcher to strongly improve her scientific background, technical expertise and transferable skills providing a strong foundation for the following steps in her scientific career.

publishable-abstract-bz.jpeg

Periodic Reporting for period 1 - SkinTrmDeep (Tissue Resident Memory (Trm) CD8+ T cells: Genome-wide dissection of cellular differentiation and heterogeneity)

Udostępnij tę stronę

Pobierz