Project description:The T cell compartment must contain diversity in both T cell receptor (TCR) repertoire and cell state to provide effective immunity against pathogens. However, it remains unclear how differences in the TCR contribute to heterogeneity in T cell state. Single cell RNA-sequencing (scRNA-seq) can allow simultaneous measurement of TCR sequence and global transcriptional profile from single cells. However, current methods for TCR inference from scRNA-seq are limited in their sensitivity and require long sequencing reads, thus increasing the cost and decreasing the number of cells that can be feasibly analyzed. We present TRAPeS, a publicly available tool that can efficiently extract TCR sequence information from short-read scRNA-seq libraries. We apply it to investigate heterogeneity in the CD8+ T cell response in humans and mice, and show that it is accurate and more sensitive than existing approaches. Coupling TRAPeS with transcriptome analysis of CD8+ T cells specific for a single epitope from Yellow Fever Virus (YFV), we show that the recently described “naive-like” memory population have significantly longer CDR3 regions and greater divergence from germline sequence than do effector-memory phenotype cells. This suggests that TCR usage is associated with the differentiation state of the CD8+ T cell response to YFV. Overall design: We applied and tested TRAPeS to scRNA-seq data from a range of CD8+ T cell responses. These data sets were selected to include both mouse and human CD8+ T cells as well as those expected to have a range of TCR complexities. In mice, we used the lymphocytic choriomeningitis virus (LCMV) infection model, and profiled CD8+ T cells responding to either acute or chronic infection (using the Armstrong and Clone 13 strains of LCMV, respectively). In healthy human subjects we profiled naive CD8+ T cells, effector memory CD8+ T cells, and antigen-specific CD8+ T cells elicited by CMV infection; vaccination with the live attenuated yellow fever virus infection (YFV-17D); or by vaccination with adenoviral and modified vaccinia Ankara vectors encoding HCV proteins. We sorted up to 128 single CD8+ T cells from each dataset to a total of 565 cells, and generated scRNA-seq libraries with short (25-30bp) paired-end reads. To evaluate the accuracy of TRAPeS, we compared its output with that of directly sequencing the TCR sequence using long reads (in which reconstruction is not required). To that end, we sequenced libraries of epitope-specific cells for Clone 13, Armstrong and CMV, and naive T cells from the CMV donor with both short (25-30bp) paired-end and 150bp single-end sequence reads.
Project description:Transcriptome analysis comparing naive, protective and non-protective spleen memory CD8 T lymphocytes were conducted to identify key functions associated with memory CD8-mediated immune protection. Memory CD8 T cells generated in response to influenza or vaccinia infection (Flu-memory and VV-memory) were compared to inflammatory memory cells (TIM) that were generated by peptide in inflammatory context. Gene expression analysis was performed on quiescent and re-stimulated CD8 T cells. Overall design: Influenza or Vaccinia virus induced memory cells (Flu-TM or VV-TM) were generated in C57BL/6J mice that received naive NP68-specific F5xLy5.1 CD8+ T cells one day before intranasal infection with Flu-NP68 or VV-NP68. Specific memory F5 CD8 T cells from spleens were FACS-sorted at least 6 weeks after immunization. T Inflammatory memory (TIM) cells were generated by i.p. injection of NP68 peptide in naive thymectomized F5 mice. Purified CD8 T cells were stimulated with NP68 peptide (restimulated condition, R) or not (homeostatic condition, H) for 2 hours
Project description:We have reported that intra-tumoral treatment with 1V270, a phospholipid-conjugated TLR7 agonist,induces local expansion an systemic dispersion of oligoclonal tumor-specific T cells by TCR repertoire analysis using next generation RNAseq methodology. Here, we examined whether systemic 1V270 therapy also induced oligoclonal expansion of tumor-specific T cells. Two groups of BALB/c mice (n=4/group) were i.p. treated with 1V270,a phospholipid-conjugated TLR7 agonist. One cohort of mice was i.v. injected with 4T1-GLF cells (2×104) on day 0. Another cohort did not receive i.v. tumor injection (no tumor-exposed mice). 4T1 cells were orthotopically inoculated on day 21. To examine clonal specificity of tumor-specific T cells, CD8+ cells were isolated from the spleens and the tumor infiltrating lymphocytes of secondarily challenged tumors after initial 1V270 therapy. The TCR repertoires were assessed by next generation RNA sequencing of both TCRαand TCR β genes.
Project description:In this work, we investigated the frequency and oligoclonal expansion of effector CD28-CD57+ memory cells in CD8+ T cell compartments and the T-cell Receptor (TCR) Vβ repertoire in acquired aplastic anemia (AA) to provide additional evidence to the immune hypothesis in AA pathophysiology. Using flow cytometry, deep sequencing, and computational methods, we examined the blood of 32 AA patients and 29 appropriate controls for Vβ usage; eight of these patients showing CD8+CD57+ expansion and three healthy subjects were subjected to TCR deep sequencing to confirm the clonality. We showed that CD8+CD57+ cells were frequently expanded with oligoclonal characteristics in AA, and patients and healthy donors shared CD8+ clonotypes by complementarity-determining region 3 sequences analysis. Overall design: For VDJ combination and CDR3 sequence profiling, DNA was isolated from FACS-sorted CD4+ and CD8+ T cells from nine SAA patients with CD8+CD57+ cell expansion and four healthy subjects (mean, 2.4 μg of DNA; range, 1.2 – 3.4 μg). DNA was also isolated from beads-sorted (Miltenyi Biotec Inc., San Diego, CA) CD8+CD57+ cells from two of the eight SAA patients with enough cells for further analysis (mean, 1.6 μg of DNA; range, 1.0 – 2.3 μg). TCR repertoire sequencing was performed with Illumina HiSeq 2000 sequencer (Illumina, Inc., San Diego, CA).
Project description:The methylation profile of human cytotoxic CD4 T cells was compared to their naive counterpart, to central memory Th1 cells, and to naive and cytotoxic CD8 T cells in order to elucidate the epigenetic basis of cytotoxic differentiation in CD4 T cells. Overall design: Total genomic DNA obtained from ex-vivo isolated blood lymphocytes of 5 CMV-seropositive healthy adults. For each subject, five populations were sorted: naive CD4, naive CD8, central memory CD4 Th1 cells, cytotoxic CD28- CD4 T cells and cytotoxic CD27- CD8 T cells.
Project description:The transcriptional profile of human cytotoxic CD4 T cells was compared to their naive counterpart, to central memory Th1 cells, and to naive and cytotoxic CD8 T cells in order to elucidate the epigenetic basis of cytotoxic differentiation in CD4 T cells. Overall design: Total RNA obtained from ex-vivo isolated blood lymphocytes of 5 CMV-seropositive healthy adults. For each subject, five populations were sorted: naive CD4, naive CD8, central memory CD4 Th1 cells, cytotoxic CD28- CD4 T cells and cytotoxic CD27- CD8 T cells.
Project description:SH-SY5Y cells were transfected with an siRNA against T-UC.300A (final concentration 50nM) or negative control siRNA (Dharmacon Negative Control #1, final concentration 50 nM) using the transfection reagent Lipofectamine (Invitrogen). Media was changed after 24hrs. RNA was extracted 120hrs after transfection. Gene expression microarray analysis was carried out using Roche NimbleGens 4x72K Homo Sapiens gene expression array.