Project description:By sequencing tens of millions of TCR? chain transcripts from naïve mouse CD8+ T cells, we observed a hugely diverse repertoire, comprising nearly all possible TRAV-TRAJ combinations. Our findings are not compatible with sequential coordinate gene recombination, but rather with a model in which contraction and DNA looping in the TCR?? locus provide equal access to TRAV and TRAJ gene segments, similar to that demonstrated for IgH gene recombination High-throughput sequencing of entire TCRa repertoire from C57Bl/6 mice
Project description:The data corresponds to the analysis of T cell receptor (TCR) repertoires of FACS-purified Tstem, Tpex and TEM cells of six individuals. The analysis of the TCR beta chain (TRB) demonstrated the differences between Tstem and Tpex repertoire properties. In total, 36 samples were analyzed using the Human TCR Profiling Kit (MiLaboratory LLC) for sequencing libraries preparation and Illumina NextSeq 550 sequencing (150+150bp) followed by the demultiplexing procedure using MIGEC software.
Project description:HLA-E molecules can present self and pathogen-derived peptides to both NK-cells and T-cells. T-cells that recognize HLA-E peptides via their T-cell receptor (TCR) are termed donor-unrestricted T-cells due to restricted allelic variation of HLA-E. The composition and repertoire of HLA-E TCRs is not known so far. We performed TCR sequencing on CD8+ T-cells from 21 individuals recognizing HLA-E tetramers (TM) folded with 2 Mtb HLA-E restricted peptides. We sorted HLA-E Mtb TM+ and TMCD8+ T-cells directly ex vivo and performed bulk RNA-sequencing and single cell TCR sequencing. The identified TCR repertoire was diverse and showed no conservation between and within individuals. TCRs selected from our single cell TCR sequencing data could be activated upon HLA-E/peptide stimulation, although not robust, reflecting potentially weak interactions between HLA-E peptide complexes and TCRs. Thus, HLA-E Mtb specific T-cells have a highly diverse TCR repertoire.
Project description:Considerable progress has recently been made in cancer immunotherapy, including immune checkpoint blockade, cancer vaccine, and adoptive T cell methods. The lack of effective targets is a major cause of the low immunotherapy response rate in colorectal cancer (CRC). Here, we used a proteogenomic strategy comprising immunopeptidomics, whole exome sequencing, and 16S ribosomal DNA sequencing analyses of 8 patients with CRC to identify neoantigens and bacterial immunopeptides that can serve as antitumor targets. This study directly identified several personalized neoantigens and bacterial immunopeptides. Immunoassays showed that all neoantigens and 5 of 8 bacterial immunopeptides could be recognized by autologous T cells. Additionally, T cell receptor (TCR) αβ sequencing revealed the TCR repertoire of epitope-reactive CD8+ T cells. Functional studies showed that T cell receptor -T (TCR-T) could be activated by epitope pulsed lymphoblastoid cells. Overall, this study comprehensively profiled the CRC immunopeptidome, revealing several neoantigens and bacterial immunopeptides with potential to serve as immunotherapy targets in CRC.
Project description:To explore the role of Satb1 in rearrangement and repertoire of TCR genes, we used Satb1contional knockout micwhich the Satb1 gene was deleted in long term hematopoietic stem cells. We applied 5ʹRACE (rapid amplification of cDNA ends) PCR technique to generate deep sequencing libraries of Tcrd, Tcrg, Tcrb, and Tcra cDNA by using a single set of primers for each TCR gene. We obtained millions of TCR sequences and unraveled the complexity of T-cell diversity. Our experimental evidence demonstrates that Satb1 has substantial influences on the TCR repertoire.
Project description:To track for T cell clones from donor memory T cell fraction infused after abT/CD19-depleted allogenic HSCT we performed TCR beta repertoire sequencing. Patient peripheral blood repertoire was sequenced in two timepoints (p3 and p4: d120-180 and d360-500). Reperotoires for bulk and CD4+ or CD8+ cells from CD45RA-depleted donor apheresis were obtained for most donor-recipient pairs. TCR beta cDNA libraries were prepared using previously published protocol (Zvyagin I.V. et al., Leukemia, 2017). Libraries were sequenced on Illumina NextSeq 500/550 and HiSeq2000/2500 in pair-end mode with read length 100-150 bp. MiGEC software were used for demultiplexing and unique molecular identifier sequence extraction software (https://github.com/mikessh/migec).
Project description:The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identified 642 RBPs in the thymus and focused on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 was downregulated in response to T cell receptor (TCR) and Ca2+ signals. Downregulation required Stim1/Stim2 and CaMK4 expression and involved Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly bound RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21-bound transcriptome revealed strong interactions with the Rag1 3'-UTR. Arpp21-deficient thymocytes showed reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype was recapitulated in Rag1 3'-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities.