scholarly journals Single-cell T-cell receptor-β analysis of HLA-A*2402-restricted CMV- pp65-specific cytotoxic T-cells in allogeneic hematopoietic SCT

2013 ◽  
Vol 49 (1) ◽  
pp. 87-94 ◽  
Author(s):  
H Nakasone ◽  
Y Tanaka ◽  
R Yamazaki ◽  
K Terasako ◽  
M Sato ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Receptor ◽  
Cytotoxic T Cells ◽  
Cell Receptor

scholarly journals 192 Pooled T cell receptor screening (POTS) provides unbiased, high-throughput method for TCR discovery

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A204-A204
Author(s):  
Jack Reid ◽  
Shihong Zhang ◽  
Ariunaa Munkhbat ◽  
Matyas Ecsedi ◽  
Megan McAfee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Receptor ◽  
High Throughput ◽  
Ex Vivo ◽  
Cell Receptor ◽  
High Avidity ◽  
Reporter System ◽  
High Throughput Method

BackgroundT Cell Receptor (TCR)-T cell therapies have shown some promising results in cancer clinical trials, however the efficacy of treatment remains suboptimal. Outcomes could potentially be improved by utilizing highly functional TCRs for future trials. Current TCR discovery methods are relatively low throughput and rely on synthesis and screening of individual TCRs based on tetramer binding and peptide specificity, which is costly and labor intensive. We have developed and validated a pooled approach relying on directly cloned TCRs transduced into a fluorescent Jurkat reporter system (figure 1). This approach provides an unbiased, high-throughput method for TCR discovery.MethodsAs a model for POTS, T cells specific for a peptide derived adenovirus structural protein were sorted on tetramer and subjected to 10x single cell VDJ analysis. Pools of randomly paired TCR alpha and beta chains were cloned from the 10x cDNA into a lentiviral vector and transduced into a Jurkat reporter cells. Consecutive stimulations with cognate antigen followed by cell sorts were performed to enrich for functional TCRs. Full length TCRab pools were sequenced by Oxford Nanopore Technologies (ONT) and compared to a 10x dataset to find naturally paired TCRs.ResultsComparison between the ex vivo single cell VDJ sequencing and ONT sequencing of the transduced antigen specific TCRs showed more than 99% of the TCR pairs found in reporter positive Jurkat cells were naturally paired TCRs. The functionality of 8 TCR clonotypes discovered using POTS were compared and clone #2 showed the strongest response. Of the selected clonotypes, clone #2 showed a low frequency of 0.9% in the ex vivo single cell VDJ sequencing. After the first round of stimulation and sequencing, clone #2 takes up of 5% of all reporter-positive clones. The abundance of clone #2 further increased to 17% after another round of stimulation, sorting and sequencing, suggesting this method can retrieve and enrich for highly functional antigen specific TCRs.Abstract 192 Figure 1Outline of the POTS workflow.ConclusionsPOTS provides a high-throughput method for discovery of naturally paired, high-avidity T cell receptors. This method mitigates bias introduced by T cell differentiation state by screening TCRs in a clonal reporter system. Additionally, POTS allows for screening of low abundance clones when compared with traditional TCR discovery techniques. Pooled TCRs could also be screened in vivo with primary T cells in a mouse model to screen for the most functional and physiologically fit TCR for cancer treatment.

Expression of T Cell Receptor γ-Chain in Murine Cytotoxic T Cells: Analysis of a Highly Transcribed Nonrearranged Gene Cluster

Pathobiology ◽  
1988 ◽  
Vol 56 (4) ◽  
pp. 181-189
Author(s):  
Manfred Schulz ◽  
Gabi Lienhard ◽  
Fabio Rupp ◽  
Rolf M. Zinkernagel ◽  
Hans Hengartner
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Cluster ◽  
T Cell Receptor ◽  
Cytotoxic T Cells ◽  
Cell Receptor

Natural variants of cytotoxic epitopes are T-cell receptor antagonists for antiviral cytotoxic T cells

Nature ◽  
1994 ◽  
Vol 369 (6479) ◽  
pp. 407-410 ◽  
Author(s):  
Antonio Bertoletti ◽  
Alessandro Sette ◽  
Francis V. Chisari ◽  
Amalia Penna ◽  
Massimo Levrero ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cytotoxic T Cells ◽  
Cell Receptor ◽  
Receptor Antagonists ◽  
Natural Variants

scholarly journals Target-Cell Contact Activates a Highly Selective Capacitative Calcium Entry Pathway in Cytotoxic T Lymphocytes

2000 ◽  
Vol 148 (3) ◽  
pp. 603-614 ◽  
Author(s):  
Adam Zweifach
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
T Cell Receptor ◽  
Target Cell ◽  
Calcium Influx ◽  
Cytotoxic T Cells ◽  
Cell Receptor ◽  
Cell Contact ◽  
Crac Channels

Calcium influx is critical for T cell activation. Evidence has been presented that T cell receptor–stimulated calcium influx in helper T lymphocytes occurs via channels activated as a consequence of depletion of intracellular calcium stores, a mechanism known as capacitative Ca2+ entry (CCE). However, two key questions have not been addressed. First, the mechanism of calcium influx in cytotoxic T cells has not been examined. While the T cell receptor–mediated early signals in helper and cytotoxic T cells are similar, the physiology of the cells is strikingly different, raising the possibility that the mechanism of calcium influx is also different. Second, contact of T cells with antigen-presenting cells or targets involves a host of intercellular interactions in addition to those between antigen–MHC and the T cell receptor. The possibility that calcium influx pathways in addition to those activated via the T cell receptor may be activated by contact with relevant cells has not been addressed. We have used imaging techniques to show that target-cell–stimulated calcium influx in CTLs occurs primarily through CCE. We investigated the permeability of the CTL influx pathway for divalent cations, and compared it to the permeability of CCE in Jurkat human leukemic T cells. CCE in CTLs shows a similar ability to discriminate between calcium, barium, and strontium as CCE in Jurkat human leukemic T lymphocytes, where CCE is likely to mediated by Ca2+ release–activated Ca2+ current (CRAC) channels, suggesting that CRAC channels also underlie CCE in CTLs. These results are the first determination of the mechanism of calcium influx in cytotoxic T cells and the first demonstration that cell contact–mediated calcium signals in T cells occur via depletion-activated channels.

Cytotoxic T-Cells From T-Cell Receptor Transgenic NOD8.3 Mice Destroy β-Cells via the Perforin and Fas Pathways

Diabetes ◽  
2006 ◽  
Vol 55 (9) ◽  
pp. 2412-2418 ◽  
Author(s):  
Nadine L. Dudek ◽  
Helen E. Thomas ◽  
Lina Mariana ◽  
Robyn M. Sutherland ◽  
Janette Allison ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cytotoxic T Cells ◽  
Cell Receptor ◽  
Β Cells

scholarly journals Differential expression profile of gluten-specific T cells identified by single-cell RNA-seq

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258029
Author(s):  
Ying Yao ◽  
Łukasz Wyrozżemski ◽  
Knut E. A. Lundin ◽  
Geir Kjetil Sandve ◽  
Shuo-Wang Qiao
Keyword(s):  
T Cells ◽  
Celiac Disease ◽  
T Cell ◽  
Single Cell ◽  
Differential Expression ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Effector Memory ◽  
Fatty Acid Transport ◽  
Rna Seq

Gluten-specific CD4+ T cells drive the pathogenesis of celiac disease and circulating gluten-specific T cells can be identified by staining with HLA-DQ:gluten tetramers. In this first single-cell RNA-seq study of tetramer-sorted T cells from untreated celiac disease patients blood, we found that gluten-specific T cells showed distinct transcriptomic profiles consistent with activated effector memory T cells that shared features with Th1 and follicular helper T cells. Compared to non-specific cells, gluten-specific T cells showed differential expression of several genes involved in T-cell receptor signaling, translational processes, apoptosis, fatty acid transport, and redox potentials. Many of the gluten-specific T cells studied shared T-cell receptor with each other, indicating that circulating gluten-specific T cells belong to a limited number of clones. Moreover, the transcriptional profiles of cells that shared the same clonal origin were transcriptionally more similar compared with between clonally unrelated gluten-specific cells.

scholarly journals Linking T cell receptor sequence to transcriptional profiles with clonotype neighbor graph analysis (CoNGA)

2020 ◽  
Author(s):  
Stefan A. Schattgen ◽  
Kate Guion ◽  
Jeremy Chase Crawford ◽  
Aisha Souquette ◽  
Alvaro Martinez Barrio ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
Graph Analysis ◽  
Theoretic Approach ◽  
Neighbor Graph

AbstractMulti-modal single-cell technologies capable of simultaneously assaying gene expression and surface phenotype across large numbers of immune cells have described extensive heterogeneity within these complex populations, in healthy and diseased states. In the case of T cells, these technologies have made it possible to profile clonotype, defined by T cell receptor (TCR) sequence, and phenotype, as reflected in gene expression (GEX) profile, surface protein expression, and peptide:MHC (pMHC) binding, across large and diverse cell populations. These rich, high-dimensional datasets have the potential to reveal new relationships between TCR sequence and T cell phenotype that go beyond identification of features shared by clonally related cells. In order to uncover these connections in an unbiased way, we developed a graph-theoretic approach---clonotype neighbor-graph analysis or “CoNGA”---that identifies correlations between GEX profile and TCR sequence through statistical analysis of a pair of T cell similarity graphs, one in which cells are linked based on gene expression similarity and another in which cells are linked by similarity of TCR sequence. Applying CoNGA across diverse human and mouse T cell datasets uncovered known and novel associations between TCR sequence features and cellular phenotype including the classical invariant T cell subsets; a novel defined population of human blood CD8+ T cells expressing the transcription factors HOBIT and HELIOS, NK-associated receptors, and a biased TCR repertoire, representing a potential previously undescribed lineage of “natural lymphocytes”; a striking association between usage of a specific V-beta gene segment and expression of the EPHB6 gene that is conserved between mouse and human; and TCR sequence determinants of differentiation in developing thymocytes. As the size and scale of single-cell datasets continue to grow, we expect that CoNGA will prove to be a useful tool for deconvolving complex relationships between TCR sequence and cellular state in single-cell applications.

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