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Single cell analysis of murine Dendritic Epidermal T Cells (DETC)

ABSTRACT: Purpose: To establish the steady state heterogeneity of murine Dendritic Epidermal T Cells (DETC) and to investigate the function of their constitutive interaction with neighbouring keratinocytes via Skint1 Methods: CD3+ cells were FACS purified from ear epidermal suspensions prepared from FVB mice administered isotype control or anti-Skint1 antibody 7d prior, then loaded into the 10x Genomics Chromium Platform and sequenced using Illumina HiSeq 4000. Conclusions: This analysis identifies four states of DETC in unperturbed epidermis, and reveals that DETC TCR sensing of keratinocytes via Skint1 potentially enables natural immune surveillance and maintains their thymic maturation signature in the periphery

ORGANISM(S): Mus musculus

PROVIDER: GSE160476 | GEO | 2021/12/12

REPOSITORIES: GEO

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Project description:Dendritic epidermal T cells (DETC) reside in murine skin and participate in homeostasis and wound repair. Upon wounding, DETC become activated through the recognition of an unidentified ligand expressed by keratinocytes proximal to sites of injury. Such DETC activation is mediated through a monoclonal T cell receptor (TCR). Using a soluble form of this monoclonal TCR, we have shown that keratinocytes upregulate DETC TCR ligands in wounded tissue within 2 hours following wounding. Down-modulation of the ligand is seen 3 hours following wounding, and no expression is evident in non-wounded skin. In vitro studies on cell lines which express this unknown ligand indicate that antigen recognition by the DETC TCR is dependent upon N-linked glycosylation of the ligand. Given the glycosylation sensitivity of the ligand and the restricted expression following wounding, we are interested in pursuing microarray analysis to identify genes that are modulated in keratinocytes in response to wounding. Keratinocytes represent 90% of the cells in the epidermis (DETC and Langerhan’s cells make up the remaining 10%). As such, we propose to isolate RNA from whole epidermis under either wounded or resting conditions. In addition to comparing RNA from wounded and non-wounded epidermis, we would like to compare RNA from tissue that has been wounded for different times. Initially, we would like to analyze 4 sampes (non-wounded epidermis, and epidermal cells isolated 30 minutes, 2 hours, and 4 hours following wounding). These time points would correlate to a period prior to cell surface expression of ligand (30 minutes), during cell surface expression (2 hours), and following down regulation of cell surface expression (4 hours). In addition to providing possible identification of the unknown DETC TCR ligand, such analysis would provide novel information about early responses by keratinocytes in response to physical wounding in vivo. We propose to isolate RNA from whole epidermis under either wounded or resting conditions. In addition to comparing RNA from wounded and non-wounded epidermis, we would like to compare RNA from tissues that has been wounded for different times.