scholarly journals Decision letter: Combined transient ablation and single-cell RNA-sequencing reveals the development of medullary thymic epithelial cells

2020 ◽  
Keyword(s):  
Epithelial Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Thymic Epithelial Cells ◽  
Single Cell Rna Sequencing ◽  
Medullary Thymic Epithelial Cells

scholarly journals Combined transient ablation and single cell RNA sequencing reveals the development of medullary thymic epithelial cells

2020 ◽  
Author(s):  
Kristen L. Wells ◽  
Corey N. Miller ◽  
Andreas R. Gschwind ◽  
Wu Wei ◽  
Jonah D. Phipps ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Expression Patterns ◽  
Critical Role ◽  
Lineage Tracing ◽  
Thymic Epithelial Cells ◽  
Single Cell Rna Sequencing ◽  
Medullary Thymic Epithelial Cells

AbstractMedullary thymic epithelial cells (mTECs) play a critical role in central immune tolerance by mediating negative selection of autoreactive T cells through the collective expression of the peripheral self-antigen compartment, including tissue-specific antigens (TSAs). Recent work has shown that gene expression patterns within the mTEC compartment are remarkably heterogenous and include multiple differentiated cell states. To further define mTEC development and medullary epithelial lineage relationships, we combined lineage tracing and recovery from transient in vivo mTEC ablation with single cell RNA-sequencing. The combination of bioinformatic and experimental approaches revealed a non-stem transit-amplifying population of cycling mTECs that preceded Aire expression. Based on our findings, we propose a branching model of mTEC development wherein a heterogeneous pool of transit-amplifying cells gives rise to Aire- and Ccl21a-expressing mTEC subsets. We further use experimental techniques to show that within the Aire-expressing developmental branch, TSA expression peaked as Aire expression decreased, implying Aire expression must be established before TSA expression can occur. Collectively, these data provide a higher order roadmap of mTEC development and demonstrate the power of combinatorial approaches leveraging both in vivo models and high-dimensional datasets.

scholarly journals Combined transient ablation and single-cell RNA-sequencing reveals the development of medullary thymic epithelial cells

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Kristen L Wells ◽  
Corey N Miller ◽  
Andreas R Gschwind ◽  
Wu Wei ◽  
Jonah D Phipps ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Critical Role ◽  
Lineage Tracing ◽  
Thymic Epithelial Cells ◽  
In Vivo Models ◽  
Single Cell Rna Sequencing ◽  
Medullary Thymic Epithelial Cells

Medullary thymic epithelial cells (mTECs) play a critical role in central immune tolerance by mediating negative selection of autoreactive T cells through the collective expression of the peripheral self-antigen compartment, including tissue-specific antigens (TSAs). Recent work has shown that gene-expression patterns within the mTEC compartment are heterogenous and include multiple differentiated cell states. To further define mTEC development and medullary epithelial lineage relationships, we combined lineage tracing and recovery from transient in vivo mTEC ablation with single-cell RNA-sequencing in Mus musculus. The combination of bioinformatic and experimental approaches revealed a non-stem transit-amplifying population of cycling mTECs that preceded Aire expression. We propose a branching model of mTEC development wherein a heterogeneous pool of transit-amplifying cells gives rise to Aire- and Ccl21a-expressing mTEC subsets. We further use experimental techniques to show that within the Aire-expressing developmental branch, TSA expression peaked as Aire expression decreased, implying Aire expression must be established before TSA expression can occur. Collectively, these data provide a roadmap of mTEC development and demonstrate the power of combinatorial approaches leveraging both in vivo models and high-dimensional datasets.

scholarly journals Single-cell RNA sequencing confirms IgG transcription and limited diversity of VHDJH rearrangements in proximal tubular epithelial cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Zhenling Deng ◽  
Xinyao Wang ◽  
Yue Liu ◽  
Xinyu Tian ◽  
Shaohui Deng ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Mesangial Cells ◽  
Gene Segment ◽  
Tubular Epithelial Cells ◽  
Mesenchymal Transition ◽  
Single Cell Rna Sequencing ◽  
Proximal Tubular Epithelial Cells ◽  
Proximal Tubular

AbstractIncreasing evidence has confirmed that immunoglobulins (Igs) can be expressed in non-B cells. Our previous work demonstrated that mesangial cells and podocytes express IgA and IgG, respectively. The aim of this work was to reveal whether proximal tubular epithelial cells (PTECs) express Igs. High-throughput single-cell RNA sequencing (scRNA-seq) detected Igs in a small number of PTECs, and then we combined nested PCR with Sanger sequencing to detect the transcripts and characterize the repertoires of Igs in PTECs. We sorted PTECs from the normal renal cortex of two patients with renal cancer by FACS and further confirmed their identify by LRP2 gene expression. Only the transcripts of the IgG heavy chain were successfully amplified in 91/111 single PTECs. We cloned and sequenced 469 VHDJH transcripts from 91 single PTECs and found that PTEC-derived IgG exhibited classic VHDJH rearrangements with nucleotide additions at the junctions and somatic hypermutations. Compared with B cell-derived IgG, PTEC-derived IgG displayed less diversity of VHDJH rearrangements, predominant VH1-24/DH2-15/JH4 sequences, biased VH1 usage, centralized VH gene segment location at the 3′ end of the genome and non-Gaussian distribution of the CDR3 length. These results demonstrate that PTECs can express a distinct IgG repertoire that may have implications for their role in the renal tubular epithelial-mesenchymal transition.

Integrative analysis of scRNAs-seq and scATAC-seq revealed transit-amplifying thymic epithelial cells expressing autoimmune regulator

2021 ◽  
Author(s):  
Takahisa Miyao ◽  
Maki Miyauchi ◽  
S. Thomas Kelly ◽  
Tommy W. Terooatea ◽  
Tatsuya Ishikawa ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Single Cell ◽  
Tolerance Induction ◽  
Integrative Analysis ◽  
Thymic Epithelial Cells ◽  
Self Tolerance ◽  
Cell Assays ◽  
Medullary Thymic Epithelial Cells ◽  
Specific Antigens ◽  
Accessible Chromatin

SummaryMedullary thymic epithelial cells (mTECs) are critical for self-tolerance induction in T cells via promiscuous expression of tissue-specific antigens (TSAs), which are controlled by transcriptional regulator AIRE. Whereas AIRE-expressing (Aire+) mTECs undergo constant turnover in the adult thymus, mechanisms underlying differentiation of postnatal mTECs remain to be discovered. Integrative analysis of single-cell assays for transposase accessible chromatin (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) suggested the presence of proliferating mTECs with a specific chromatin structure, which express high levels of Aire and co-stimulatory molecules CD80 (Aire+CD80hi). Proliferating Aire+CD80hi mTECs detected by using Fucci technology express a minimal level of Aire-dependent TSAs and are converted into quiescent Aire+CD80hi mTECs expressing high levels of TSAs after a transit amplification. These data provide evidence for the existence of transit amplifying Aire+mTEC precursors during Aire+mTEC differentiation process of the postnatal thymus.

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