Engineered cell entry links receptor biology with single-cell genomics [bulk RNA-Seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE212984 | GEO | 2022/12/12
REPOSITORIES: GEO
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Project description:Cells communicate with each other via receptor-ligand interactions. Here we describe lentiviral-mediated cell e¬ntry by engineered receptor-ligand interaction (ENTER) to display ligand proteins, deliver payloads, and record receptor specificity. We optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. A viral presentation strategy allows ENTER to capture interactions between B cell receptor and any antigen. We engineer ENTER to deliver genetic payloads to antigen-specific T or B cells to selectively modulate cellular behavior in mixed populations. Single-cell readout of ENTER by RNA-sequencing (ENTER-seq) enables multiplexed enumeration of antigen specificities, TCR clonality, cell-type and states of individual T cells. ENTER-seq of CMV-seropositive patient blood samples reveals the viral epitopes that drive effector memory T cell differentiation and inter- vs intra-clonal phenotypic diversity targeting the same epitope. ENTER technology enables systematic discovery of receptor specificity, linkage to cell fates, and antigen-specific cargo delivery.
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Project description:T cells bearing gamma delta T cell antigen receptors (TCRs) function in lymphoid stress surveillance. However, the contribution of gamma delta TCRs to such responses is unclear. Here we found that the TCR of a human V gamma4Vdelta5 clone directly bound endothelial protein C receptor (EPCR), which allowed gamma delta T cells to recognize both endothelial cells targeted by cytomegalovirus and epithelial tumors. EPCR is a major histocompatibility complex–like molecule that binds lipids analogously to the antigen-presenting molecule CD1d. However, the V gamma4Vdelta5 TCR bound EPCR independently of lipids, in an antibody-like way. Moreover, the recognition of target cells by gamma delta T cells required a multimolecular stress signature composed of EPCR and costimulatory ligand(s). Our results demonstrate how a gamma delta TCR mediates recognition of broadly stressed human cells by engaging a stress-regulated self antigen. 2E9 is an antibody that blocks gd-TCR recognition of human cells that are the targets of the clone LES. 2E9 also stains cells that are targeted by LES. Therefore, to identify the TCR ligand expressed by 2E9-positive cells, Gene Expression was compared in two cell lines that stain with 2E9 (K562 and U937) versus two cell lines that do not (Hutu80 and Huh7).
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Project description:<p>Diversity and size of the antigen-specific T cell receptor (TCR) repertoire are two critical determinants for successful control of chronic infection. Varicella zoster virus (VZV) that establishes latency during childhood is able to escape control mechanisms, in particular with increasing age. We examined the TCR diversity of VZV-specific CD4 T cells in individuals older than 50 years by studying three identical twin pairs and three unrelated individuals before and after vaccination with live attenuated VZV. While all individuals had a small number of dominant T cell clones, the breadth of the VZV-specific repertoire differed markedly among different individuals. A genetic influence was seen for the sharing of individual TCR sequences from antigen-specific cells, but not for repertoire richness or the selection of clonal dominance. VZV vaccination favored the expansion of infrequent VZV-specific TCRs including those from naïve T cells while leaving dominant T cell clones mostly unaffected.</p>
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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.
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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.
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Project description:<p>High-throughput linking of T cell receptor (TCR) sequences to their binding antigens is vital for immune profiling, yet challenging. We present Tetramer associated TCR Sequencing (TetTCR-Seq) to address this challenge. Binding is determined using a library of DNA-barcoded antigen tetramers that are rapidly and inexpensively generated using an in vitro transcription/translation platform. We included CMV+ donors (CMV seropositive donors who are infected with Cytomegalovirus) to screen for CMV specific TCRs.</p>
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Project description:T cells bearing gamma delta T cell antigen receptors (TCRs) function in lymphoid stress surveillance. However, the contribution of gamma delta TCRs to such responses is unclear. Here we found that the TCR of a human V gamma4Vdelta5 clone directly bound endothelial protein C receptor (EPCR), which allowed gamma delta T cells to recognize both endothelial cells targeted by cytomegalovirus and epithelial tumors. EPCR is a major histocompatibility complex–like molecule that binds lipids analogously to the antigen-presenting molecule CD1d. However, the V gamma4Vdelta5 TCR bound EPCR independently of lipids, in an antibody-like way. Moreover, the recognition of target cells by gamma delta T cells required a multimolecular stress signature composed of EPCR and costimulatory ligand(s). Our results demonstrate how a gamma delta TCR mediates recognition of broadly stressed human cells by engaging a stress-regulated self antigen.
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