scholarly journals Partitioning stable and unstable expression level variation in cell populations: a theoretical framework with application to the T cell receptor

2019 ◽  
Author(s):  
Thiago S. Guzella ◽  
Vasco M. Barreto ◽  
Jorge Carneiro
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Characteristic Time ◽  
Single Cells ◽  
Cell Receptor ◽  
Original Population ◽  
Expression Variation ◽  
Cellular Expression ◽  
The Difference ◽  
Expression Variance

AbstractPhenotypic variation in the copy number of gene products expressed by cells or tissues has been the focus of intense investigation. To what extent the observed differences in cellular expression levels are persistent or transient is an intriguing question. Here, we develop a quantitative framework that resolves the expression variation into stable and unstable components. The difference between the expression means in two cohorts isolated from any cell population is shown to converge to an asymptotic value, with a characteristic time, τT, that measures the timescale of the unstable dynamics. The asymptotic difference in the means, relative to the initial value, measures the stable proportion of the original population variance . Empowered by this insight, we analysed the T-cell receptor (TCR) expression variation in CD4 T cells. About 70% of TCR expression variance is stable in a diverse polyclonal population, while over 80% of the variance in an isogenic TCR transgenic population is volatile. In both populations the TCR levels fluctuate with a characteristic time of 32 hours. This systematic characterisation of the expression variation dynamics, relying on time series of cohorts’ means, can be combined with technologies that measure gene or protein expression in single cells or in bulk.

scholarly journals Reconstitution of paired T cell receptor  - and beta-chains from microdissected single cells of human inflammatory tissues

2006 ◽  
Vol 103 (32) ◽  
pp. 12057-12062 ◽  
Author(s):  
S. Seitz ◽  
C. K. Schneider ◽  
J. Malotka ◽  
X. Nong ◽  
A. G. Engel ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Single Cells ◽  
Cell Receptor

scholarly journals High-throughput determination of the antigen specificities of T cell receptors in single cells

2018 ◽  
Author(s):  
Shu-Qi Zhang ◽  
Ke-Yue Ma ◽  
Alexandra A. Schonnesen ◽  
Mingliang Zhang ◽  
Chenfeng He ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
High Throughput ◽  
Single Cells ◽  
Cell Receptor ◽  
Cross Reactivity ◽  
Wild Type ◽  
Type Antigen

We present tetramer-associated T-cell receptor sequencing (TetTCR-Seq), a method to link T cell receptor (TCR) sequences to their cognate antigens in single cells at high throughput. Binding is determined using a library of DNA-barcoded antigen tetramers that is rapidly generated by in vitro transcription and translation. We applied TetTCR-Seq to identify patterns in TCR cross-reactivity with cancer neo-antigens and to rapidly isolate neo-antigen-specific TCRs with no cross-reactivity to the wild-type antigen.

scholarly journals Identification and tracking of alloreactive T cell clones in Rhesus Macaques through the RM-scTCR-Seq platform.

2021 ◽  
Author(s):  
Ulrike Gerdemann ◽  
Ryan A. Fleming ◽  
James Kaminski ◽  
Connor McGuckin ◽  
Xianliang Rui ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Vaccine Development ◽  
Rhesus Macaques ◽  
Single Cells ◽  
Cell Receptor ◽  
Cytotoxic T Cell ◽  
Cell Clones ◽  
Alloreactive T Cell

T cell receptor clonotype tracking is a powerful tool for interrogating T cell mediated immune processes. New methods to pair a single cells transcriptional program with its T cell receptor (TCR) identity allow monitoring of T cell clonotype-specific transcriptional dynamics. While these technologies have been available for human and mouse T cells studies, they have not been developed for Rhesus Macaques, a critical translational organism for autoimmune diseases, vaccine development and transplantation. We describe a new pipeline, RM-scTCR-Seq, which, for the first time, enables RM specific single cell TCR amplification, reconstitution and pairing of RM TCRs with their transcriptional profiles. We apply this method to a RM model of GVHD, and identify and track in vitro detected alloreactive clonotypes in GVHD target organs and explore their GVHD driven cytotoxic T cell signature. This novel, state-of-the-art platform fundamentally advances the utility of RM to study protective and pathogenic T cell responses.

scholarly journals High throughput single cell sequencing of both T-cell-receptor-beta alleles

2018 ◽  
Author(s):  
Tomonori Hosoya ◽  
Hongyang Li ◽  
Chia-Jui Ku ◽  
Qingqing Wu ◽  
Yuanfang Guan ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
High Throughput ◽  
Single Cells ◽  
Cell Receptor ◽  
Allelic Exclusion ◽  
Vdj Recombination ◽  
T Cell Receptor Beta ◽  
B And T Lymphocytes ◽  
Receptor Beta

ABSTRACTAllelic exclusion is a vital mechanism for the generation of monospecificity to foreign antigens in B- and T-lymphocytes. Here we developed a high-throughput barcoded method to simultaneously analyze the VDJ recombination status of both mouse T cell receptor beta alleles in hundreds of single cells using Next Generation Sequencing.

scholarly journals Hodgkin and Reed-Sternberg cells do not carry T-cell receptor gamma gene rearrangements: evidence from single-cell polymerase chain reaction examination

Blood ◽  
1995 ◽  
Vol 85 (6) ◽  
pp. 1590-1595 ◽  
Author(s):  
H Daus ◽  
L Trumper ◽  
J Roth ◽  
F von Bonin ◽  
P Moller ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Receptor ◽  
Single Cells ◽  
Cell Receptor ◽  
Chain Reaction ◽  
Gamma Chain ◽  
Polymerase Chain

Hodgkin and Reed-Sternberg (H&RS) cells are generally accepted to be the neoplastic cells of Hodgkin's disease (HD), even though they represent only a minority of the cellular infiltrate in affected tissues. Recent immunologic studies and Southern blot analyses of DNA extracted from whole lymph node tissue favored, but did not convincingly prove a lymphoid origin of H&RS cells. To detect rearrangements of the T-cell receptor gamma chain (TCR gamma) genes at the single-cell level as an indication of early T-cell lymphoid differentiation, we isolated H&RS cells by micromanipulation from cytospin preparations of fresh biopsy material. TCR gamma chain rearrangement was detected by polymerase chain reaction using four “forward primers” that were constructed corresponding to all four V families and two that were constructed corresponding to all four V families and two “reverse primers” corresponding to consensus sequences of J segments. Rearrangements of all V families in combination with the different J segments were detected in human peripheral blood and tonsillar T cells. Although rearrangements of TCR gamma chain genes were shown in single cells of 10 of 10 T-cell leukemias, no rearrangement of these genes was found in single H&RS cells from 13 consecutive patients with HD. Our results indicate that H&RS cells from the vast majority of cases are not derived from T cells. This finding may have implications for the pathogenesis of HD and the development of more effective treatment regimens.

scholarly journals The requirement for Notch signaling at the β-selection checkpoint in vivo is absolute and independent of the pre–T cell receptor

2006 ◽  
Vol 203 (10) ◽  
pp. 2239-2245 ◽  
Author(s):  
Ivan Maillard ◽  
LiLi Tu ◽  
Arivazhagan Sambandam ◽  
Yumi Yashiro-Ohtani ◽  
John Millholland ◽  
...  
Keyword(s):  
T Cell ◽  
Notch Signaling ◽  
T Cell Receptor ◽  
Transcriptional Activation ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Cell Receptor ◽  
Dominant Negative ◽  
Thymocyte Development

Genetic inactivation of Notch signaling in CD4−CD8− double-negative (DN) thymocytes was previously shown to impair T cell receptor (TCR) gene rearrangement and to cause a partial block in CD4+CD8+ double-positive (DP) thymocyte development in mice. In contrast, in vitro cultures suggested that Notch was absolutely required for the generation of DP thymocytes independent of pre-TCR expression and activity. To resolve the respective role of Notch and the pre-TCR, we inhibited Notch-mediated transcriptional activation in vivo with a green fluorescent protein–tagged dominant-negative Mastermind-like 1 (DNMAML) that allowed us to track single cells incapable of Notch signaling. DNMAML expression in DN cells led to decreased production of DP thymocytes but only to a modest decrease in intracellular TCRβ expression. DNMAML attenuated the pre-TCR–associated increase in cell size and CD27 expression. TCRβ or TCRαβ transgenes failed to rescue DNMAML-related defects. Intrathymic injections of DNMAML− or DNMAML+ DN thymocytes revealed a complete DN/DP transition block, with production of DNMAML+ DP thymocytes only from cells undergoing late Notch inactivation. These findings indicate that the Notch requirement during the β-selection checkpoint in vivo is absolute and independent of the pre-TCR, and it depends on transcriptional activation by Notch via the CSL/RBP-J–MAML complex.

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