As a comparison, memory P14 cells were sorted from the spleen of LCMV-Armstrong-infected mice at 28dpi according to the following phenotypes: CD62L+Ly108+ (CD62L+ memory), CD62LLy108+ (CD62L memory) and CD62LLy108 (effector). Comprehensive integration of single-cell data. 2hj, TPEX cells were sorted as live CD8+PD1+CD45.1+Tcf7GFP+ cells from the spleens of chronically infected mice (LCMV-Cl13, 28dpi) using a MoFlo Astrios cell sorter (Beckman Coulter) and processed using the 10X Genomics technology, according to the manufacturers protocol (Chromium Single Cell 3 GEM v3 kit). Dots represent individual mice; symbols and error bars represent mean and s.e.m., respectively; box plots indicate minimum and maximum values (whiskers), interquartile range (box limits) and median (centre line). List of genes differentially expressed between control and Myb-cKO P14 subsets and bound by MYB in human T cells. SRR3286805. Nature thanks Evan Newell and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Indeed, KIT was exclusively expressed in CD62L TPEX cells and was highly upregulated in MYB-deficient TPEX cells (Fig. (g) Cell numbers (left) and quantification of PD-1 expression (right) in P14 T cell populations derived from the indicated transferred subsets at 8 dpi. Flow cytometry plots and quantifications showing expression of Ly108 and CD62L of splenic P14 cells in R2 and R3 mice (e) and average percentages of recovered CD62L+ TPEX, CD62L TPEX and TEX cells per spleen in R2 and R3 mice (f) at day 14 post re-transfer, respectively. Cells are colour-coded on the basis of pseudotime prediction. Molecular states during acute COVID-19 reveal distinct etiologies of long-term sequelae. DESeq2RNA-seqChIP-SeqHiCshRNAdds = DESeqDataFromMatrix(countData = cts, colData = colData, design = ~batch + condition)dds = DESeq(d 1, RT (c) Schematic of the experimental set-up. Immunol. (g) Flow cytometry plots and quantifications showing expression of Ly108 and CD69 of splenic P14 cells of R2 mice and (h) average percentages of recovered CD69+ TPEX, CD69 TPEX, CD69+ TEX and CD69 TEX cells per spleen in R2 mice at day 14 post re-transfer. Together, these data indicate that strong and persistent TCR stimulation favours the sustained expression of MYB and retention of CD62L+ TPEX cells during chronic infection. Chen, Z. et al. Imaging data were analysed using Fiji (ImageJ) software (NIH). Sublong : a long-read aligner that is designed based on seed-and-vote. 8f,g). ); the European Research Council (starting grant 949719 SCIMAP to V.R.B. The sequencing data are available at the NCBI GEO (http://www.ncbi.nlm.nih.gov/geo) under the accession number GSE205608. do you mean that those genes are completely missing from the output or that they have 0 count ? Samples were then mounted using ProLong Gold Antifade Mountant (Invitrogen) and imaged using an inverted LSM780 microscope (Carl Zeiss) and a plan apochromat 63 NA 1.40 oil-immersion objective (Carl Zeiss). (a, b) Congenically marked naive P14 T cells were adoptively transferred into naive recipient mice, which were then infected with LCMV-Cl13. (a) Schematic of the experimental set-up. Sublong : a long-read aligner that is designed based on seed-and-vote. Next-generation sequencing technologies generate millions of short sequence reads, which are usually aligned to a reference genome. P values are from two-tailed unpaired t-tests (e, i, j), two-way ANOVA (f), and one-way ANOVA (m, p); P>0.05, not significant (n.s.). RNAseq In addition, naive P14 cells were included for the analysis. (c) Quantification of GZMB expression among gp33+ cells of the indicated genotypes. I have used the same pipeline for other RNASeq datasets in the past and FeatureCounts gives count output for all the genes in the annotation file. Would you like email updates of new search results? 2j and Extended Data Fig. <== current version: 4.9.2 latest version: 4.10.1 Please update conda by running $ conda 3a). 10d,e). feature 2. Hashimoto, M. et al. A linear model was fitted to each gene, and the empirical Bayes moderated t-statistic was used to assess differences in expression55,56. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Peak calling was performed using Homer (v.4.11)59 with an FDR set to 1108. (b) Box plot showing PD-1 expression of transferred P14 cells at 8 dpi. 3. Sublong: a long-read aligner that is designed based on seed-and-vote. The site is secure. featurecounthtseq-count featureCounts(subread) v2.0.1 featureCounts Subread In stark contrast, both CD62L TPEX and TEX cells showed no apparent proliferative response (Fig. 1kp). SCALCE: boosting sequence compression algorithms using locally consistent encoding. (l) Flow cytometry plots and quantifications showing CX3CR1 and CD101 expression in Myb-cKO and control TEX cells at the indicated time points after infection. Of note, maximum cell numbers attainable for each subset were transferred to allow for reliable evaluation of phenotypic diversification in expanded progenies. MYB-deficient TPEX and TEX cells expressed significantly higher levels of IFN and TNF, whereas TEX cells also expressed more granzyme B and underwent increased proliferation (measured by the expression of Ki67) compared to controls (Fig. C.T., L.K., V.R.B. Does it suggest that mapping failed for the 18000 ignored genes? 2011;12 Suppl 2(Suppl 2):S7. 1cj). #rowname Although all subsets maintained high expression of PD-1 (Extended Data Fig. Kallies, A., Zehn, D. & Utzschneider, D. T. Precursor exhausted T cells: key to successful immunotherapy? A comparison of MYB-deficient and control CD62L TPEX cells identified 580 differentially expressed genes (Supplementary Table 2), including genes that encode molecules related to T cell exhaustion and TPEX cell identity (Lef1, Eomes, Ctla2a, Irf4, Ikzf2, Nt5e and Cd160), cell-cycle regulation and stem cell renewal (E2f1, Rbl2, Kif14, Cdc25b, Bmp7 and Wnt3) (Fig. Epub 2011 Jul 27. They are completely missing from the output. EMBO J. Please enable it to take advantage of the complete set of features! A<-A[,-1] LCMV-Armstrong-infected Mybfl/flCd4Cre mice mounted CD8+ T cell responses that were similar to those of controls, and showed no overt signs of disease (Fig. We confirmed the superior developmental properties of CD62L+ TPEX cells using single T cell transfer andfate-mapping via retrogenic colourbarcoding22,23,24,25 (Extended Data Fig. (ag) Congenically marked naive control (Cd4Cre) and Mybfl/flCd4Cre (Myb-cKO) P14 T cells were adoptively transferred into naive recipient mice, which were subsequently infected with LCMV-Docile. 4d,e), which indicates that CD62L+ but not CD62L TPEX cells fuel the generation of effector cells in response to checkpoint blockade. eCollection 2022 Nov. Green L, Coronado-Zamora M, Rado S, Rech GE, Salces-Ortiz J, Gonzlez J. BMC Biol. (h) Schematic of the experimental set-up. WebConda. Raw gene counts were quantified using the featureCounts program of subread v2.0.1. Cancer 8, 523534 (2008). Splenic P14 T cells of R2 mice were analysed at day 14 post re-transfer. P values are from MannWhitney tests (g) and two-tailed unpaired t-test (i); P>0.05, not significant (n.s.). Mol. (g) Schematic of the experimental set-up. Horizontal lines and error bars of bar graphs indicate mean and s.e.m., respectively. P values are from two-tailed unpaired t-tests (c, l, or) and MannWhitney tests (f); P>0.05, not significant (n.s.). All mice were maintained and used in accordance with the guidelines of the University of Melbourne Animal Ethics Committee or the district government of upper Bavaria (Department 5Environment, Health and Consumer Protection). I am sorry, but I cannot help you anymore at this point in time. (e) Flow cytometry plots and quantifications showing the production of cytokines as indicated from Myb-cKO and control P14 T cells after gp33 peptide restimulation at 8 dpi. 4). #Count with fewer than 10 reads, The Chromium Single Cell 3 Library & Gel Bead Kit v2 for the wild-type untreated sample or v3 for wild type treated with 200g of anti-PD-L1 antibody (10F.9G2, BioXCell) for 24h were used to generate cDNA libraries (10X Genomics) following the protocol provided by the manufacturer. CD62L+ TPEX cells were enriched in the spleen and lymph nodes, but largely absent from the blood, bone marrow and liver (Extended Data Fig. Sequencing was performed on an Illumina Novaseq by Novogene, generating 150-bp paired-end reads. RNA-seq ChIP-SeqHiCshRNA, DESeq2, ctscolData ctsSampleNameGene colDataSampleSampleName, GeneSamples10 keep = rowSums(counts(dds)) >= 10 dds = dds[keep,], factorDESeq2, lfcShrink logFoldChangeapeglmddslfcShrink lfcShrink(dds,coef = "condition_treated_vs_untreated",type = "apeglm"), PP P summary(res) p0.1sum(res$padj < 0.1,na.rm = TRUE) resultspadj cutoff results(dds,alpha = 0.05)], identify(res$baseMean,res$log2FoldChange), VST rlog, VSTrlogblindblindFALSE, weixin_51136731: The transcription factor MYB is not only essential for the development of CD62L+ TPEX cells and maintenance of the antiviral CD8+ T cell response, but also induces functional exhaustion and thereby prevents lethal immunopathology. Source data are provided with this paper. 1g,i), indicating that both populations were chronically stimulated, and both expressed low amounts of CD160, 2B4 and TIGIT (Extended Data Fig. 10kl). T cells maintain an exhausted phenotype after antigen withdrawal and population reexpansion. CD62L+ TPEX cells also gave rise to more CX3CR1+ TEX cells (Extended Data Fig. (f) Quantification of recovered P14 cells at day 14 post re-transfer. For haematoxylin and eosin (H&E) staining, organs were collected and fixed in 10% formalin. All data are representative of two independent experiments. Symbols and error bars in (l) represent mean and s.e.m., respectively Data are representative of two independent experiments (e, g, k, l). featureCounts: a ultrafast and accurate read summarization program. (o) Flow cytometry plots and quantification showing the expression of CD62L among polyclonal TPEX cells (Ly108hiTIM-3lo). Leong, Y. Google Scholar. (p) Box plots showing viral titres in the kidneys of Myb-cKO and control mice at day 8 post LCMV-Docile infection. Stat. rownames(A)<-A[,1] Utzschneider, D. T. et al. 8kn). subread, RNA-seq & performed core experimental work and data analysis. 20Question and Answer1 (e) Representative flow cytometry plots showing the sorting strategy and post-sort purities. Raw reads counts were assigned to Ensembl 75 genes using Subread v1.4.6 38, normalized for library depth and gene size (RPKM), and log 10-transformed. performed computational analyses. P values are from MannWhitney tests (d, j); P>0.05, not significant (n.s.). c, Heat map showing the expression of all identified cluster signature transcripts. Regards. Similarly, the MYB-deficient CD8+ T cell compartment was devoid of CD62L+ TPEX cells (Extended Data Fig. To this end, we generated P14 T cells that lack functional Pdcd1 (encoding PD-1) using CRISPRCas9 (Extended Data Fig. Nat. Nssing, S. et al. 37, 457495 (2019). Epub 2011 Aug 19. Pseudogenes, or genes that did not meet a counts per million reads (CPM) cut-off of 0.5 in at least two libraries were excluded from further analysis. 2d). Installation. Nat. By submitting a comment you agree to abide by our Terms and Community Guidelines. Front. (a) Schematic of the experimental set-up. https://ibb.co/PFf9WxQ, I also checked the read coverage for gene IDs missing from the FeatureCounts output vs annotation file. CRFPGM3CRFgraph model, RNA_Seq1ftp.sra.ebi.ac.uk/vol1/fastq/SRR122/005/SRR1228245/SRR1228245_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR122/005/SRR1228245/SRR1228245_2.fastq.gz (a) Schematic of the experimental set-up. P values are from two-tailed unpaired t-tests (d). If you have a new idea or new request, please file an issue. Hach F, Numanagic I, Alkan C, Sahinalp SC. (kl) Flow cytometry plots (k) and quantification (l) showing the frequencies of the entire antigen-responsive PD-1+ cell compartment among Myb-cKO and control CD8+ T cells at indicated time points after infection. 63) and then normalized to TPM values. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in All subsequent analysis was performed using SCANPY (v.1.6)45. (g) Flow cytometry plots and quantification showing the frequencies of Ki67+ cells among Myb-cKO and control P14 T cells at indicated time points after infection. Google Scholar. a,b, Congenically marked naive P14 T cells were transferredinto primary recipient (R1) mice, which were subsequently infected with LCMV-Cl13. By contrast, exhausted effector T (TEX) cells co-express PD-1 and TIM-3 but lack the expression of TCF1, ID3, CXCR5 and Ly108 (refs. Gabriel, S. S. et al. Raw P values were adjusted to control the global FDR across all comparisons using the global option in the decideTests function in the limma package. Dots in graphs represent individual mice; horizontal lines and error bars of bar graphs indicate mean and s.e.m., respectively. Genomic conservation data for the human and mouse genomes were obtained from UCSC Genome Browser (https://genome.ucsc.edu). (k, n) Schematics of the experimental set-up. GMFI, geometric mean fluorescence intensity. Immunol. 1ac and Supplementary Table 1). Rev. Thanks, I will check the bigwig on the genome browser and let you know. Genetic variation in histone modifications and gene expression identifies regulatory variants in the mammary gland of cattle. TPEX cells have the ability to continuously self-renew and give rise to functionally restrained effector cells, and therefore have an essential role in maintaining chronically antigen-stimulated T cells and their exhausted phenotype3,4,5,8,9. Genetic absence of PD-1 promotes accumulation of terminally differentiated exhausted CD8+ T cells. Mybfl/flCd4Cre mice were crossed to include the P14 TCR transgene for some experiments. The wells were washed twice using PBS. Utzschneider, D. T. et al. Red blood cells were lysed with a hypotonic ammonium chloride-potassium bicarbonate (ACK) or ammonium chloride-Tris (ACT) buffer. and S.R. Similar to previous studies39,40,41, PD-1-deficient P14 T cells exhibited increased clonal expansion in response to LCMV-Docile, as compared with control cells (Extended Data Fig. It uses the seed-and-vote mapping paradigm to determine the mapping location of the read by using its largest mappable region. featureCounts is a highly efficient general-purpose read summarization program that counts mapped reads for genomic features such as genes, exons, promoter, gene bodies, genomic bins and chromosomal locations. It includes Subread aligner, Subjunc exon-exon junction detector and featureCounts read summarization program. doi: 10.1186/1471-2164-12-S2-S7. P values are from two-tailed unpaired t-tests (c,f,hj) and MannWhitney tests (ln). Both early TPEX and TEX cells were readily detectable during the acute phase (59dpi) of the immune response (Extended Data Fig. Data are representative of all analysed mice (e,g), two (c,f,i,j,ln) or three independent experiments (h). Data are representative of two independent experiments. d, Congenically marked naive P14 cells were transferred into recipient mice, which were subsequently infected with LCMV-Docile and analysed at 21dpi. Annu. Reads for mRNA and repeat RNA were counted by featureCounts (-t exon -g gene_id -C -M -Ofraction -T 24 -s 2 -p) from Subread version 1.6.4 (ref. 21, 12561266 (2020). is a Special Fellow of The Leukemia & Lymphoma Society and is supported by an NHMRC fellowship (1194779). Careers. After general pre-processing (less than 15% mitochondrial genes, regressing out cell cycle, filtering mitochondrial genes and total counts), the data were count-normalized per cell and logarithmized. 3. P values are from MannWhitney tests (hi); P>0.05, not significant (n.s.). ftp.sra.ebi.ac.uk/vol1/fastq/SRR12, subtype_MethyLevel, https://blog.csdn.net/weixin_43569478/article/details/108079250. 3b, Extended Data Fig. Bead captured transcripts in all encapsulated cells were uniquely barcoded using a combination of a 16-bp 10X barcode and a 10-bp unique molecular identifier (UMI). Odorizzi, P. M., Pauken, K. E., Paley, M. A., Sharpe, A. These authors contributed equally: Carlson Tsui, Lorenz Kretschmer, Svenja Rapelius, These authors jointly supervised this work: Veit R. Buchholz, Axel Kallies, Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia, Carlson Tsui,Sarah S. Gabriel,Daniel T. Utzschneider,Teisha Mason,Santiago Valle Torres&Axel Kallies, Institute for Medical Microbiology, Immunology and Hygiene, School of Medicine, Technical University of Munich (TUM), Munich, Germany, Lorenz Kretschmer,Svenja Rapelius,Sebastian Jarosch,Justin Leube&Veit R. Buchholz, Olivia Newton-John Cancer Research Institute, Melbourne, Victoria, Australia, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia, David Chisanga,Yang Liao,Stephen A. Wilcox,Stephen L. Nutt&Wei Shi, Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia, School of Cancer Medicine, La Trobe University, Melbourne, Victoria, Australia, Wrzburg Institute of Systems Immunology, Max Planck Research Group, Julius-Maximilians-Universitt Wrzburg, Wrzburg, Germany, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia, Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia, Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich (TUM), Freising, Germany, School of Computing and Information Systems, University of Melbourne, Melbourne, Victoria, Australia, You can also search for this author in subreadfeatureCountscounts_id.txt DESeq2limmaDESeq2 First step: Uncovering the molecular mechanisms of drought stress response has been one of hot research topics worldwide. 10b), confirming that MYB deficiency resulted in the loss of CD62L+ TPEX cells and not merely CD62L expression. CD62L+ TPEX cells expressed higher levels of transcripts that encode molecules related to lymph node homing (for example, Sell, Ccr7 and S1pr1), and higher levels of the cell-cycle inhibitors Cdkn1b and Cdkn2d and the quiescence factors Klf2 and Klf3, compared with CD62L TPEX cells. However, this time, after running featureCounts the count output file contained only 24000 genes out of total 42000 genes in the annotation file. https://sourceforge.net/projects/subread/files/, wget https://jaist.dl.sourceforge.net/project/subread/subread-2.0.2/subread-2.0.2-Linux-x86_64.tar.gz, tar -zxvf subread-2.0.2-Linux-x86_64.tar.gz, $ /home/software/subread-2.0.2-Linux-x86_64/bin/featureCounts -T 5 -t exon -g gene_id -a /path-to-gtf/ERCC.gtf -o /path-to-output/all.id.txt *.bam 1>counts.id.log 2>&1, gtfreadgtfread, -J-G, readfeaturemetafeatureread, -Cfragmentfragment-p, -ffeatureexon-levelmeta-featuregene-levels, gtfid identifier featuremeta-featuregene_idgtfid identifier, feature-type-tgtffeaturereadfeatureexon. doi: 10.1093/nar/gkz114. 2c and Extended Data Fig. 2010 Nov 15;26(22):2902-3. doi: 10.1093/bioinformatics/btq531. 12c), the frequenciesbut not the numbersof CD62L+ TPEX cells were markedly decreased compared to control P14 T cells (Extended Data Fig. 3c). P14 T cells were sorted at 28 dpi and single CD62L+ or CD62L TPEX cells were re-transferred into naive secondary recipient mice (R2), which were subsequently infected with LCMV-Armstrong. Hi, 03. Web Using DESeq2 with FeatureCounts is a much better-supported operation if your main interests are in gene-level DE. featureCounts (subread) sam bam . g,h, Flow cytometry plots (g) and cell numbers (h) of recovered progenies at day 21 after re-transfer (gated on CD4CD19 cells). WebfeatureCountsbamhtseq-countsDEXSeq Immunol. 4b). To study the role of MYB in CD8+ T cells during viral infection, we infected Mybfl/flCd4Cre mice35 (which lack MYB specifically in T cells) and Mybfl/fl (control) littermates with LCMV-Docile or LCMV-Armstrong (Fig. (c) Flow cytometry plots and quantification showing frequencies of CD62L+ cells among Myb-cKO and control P14 TPEX cells. 2i and Extended Data Fig. A guide to cancer immunotherapy: from T cell basic science to clinical practice. SRR3286804 Splenic P14 T cells of R2 mice were analysed at day 28 post re-transfer. Immunol. (e) Schematic of the experimental set-up. featureCounts: a software program developed for counting reads to genomic features such as genes, exons, promoters and genomic bins. table(duplicated(lnc$id)) (f, g) Flow cytometry plots and quantification showing the frequencies of TCF1+ TPEX cells among antigen-specific T cells (f) and CD62L+ cells among TPEX cells (g) in Myb-cKO and control compartments at 8 dpi. PubMed limma powers differential expression analyses for RNA-sequencing and microarray studies. Immunol. f, Schematic of the experimental set-up. TPEX cells also mediate the response to therapeutic checkpoint inhibition3,5,10,11, which can reinvigorate exhausted CD8+ T cell responses and has revolutionized cancer therapy12. Efficient CRISPR/Cas9 gene editing in uncultured naive mouse T Cells for in vivo studies. us_count<-lnc[!index,] # After two days in culture, the transduced HSCs were suspended in fetal calf serum (FCS) and injected intravenously into irradiated C57BL/6 recipient mice (2 4.5Gy, with a resting period of 4h). Smyth, G. K. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Frumento, G. et al. Effector regulatory T cell differentiation and immune homeostasis depend on the transcription factor Myb. 39, 19951998 (2007). Cas9 Nuclease (Integrated DNA Technologies) and a previously described Pdcd1-targeting sgRNA (Synthego)41 using the P3 primary cell 4D-Nucleofector X kit S electroporation kit (Lonza) and Lonza 4D-Nucleofector Core Unit (Lonza). 1f). Bioinformatics. Box plots show the relative progeny expansion in anti-PD-L1-treated versus PBS-treated mice (left) and the numbers of CD62L+ TPEX cells among progeny after anti-PD-L1 treatment (right). Samples were incubated with antibodies against B220 (RA3-6B2, eBioscience), CD3 (17A2, eBioscience) and F4/80 (BM8, Biolegend) diluted in 5% NGS for 2h at room temperature in the dark. He, R. et al. PFU, plaque-forming units. 2022 Dec 8;23(1):815. doi: 10.1186/s12864-022-09002-9. Immunity 51, 10431058 (2019). (ij) Flow cytometry plots (i) and quantification (j) showing expression of CD62L among polyclonal TPEX cells (Ly108hiTIM-3lo). Nature 609, 354360 (2022). Durinck, S., Spellman, P. T., Birney, E. & Huber, W. Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. WebfeatureCounts 1. a, Schematic of the experimental set-up. Siddiqui, I. et al. Reads for mRNA and repeat RNA were counted by featureCounts (-t exon -g gene_id -C -M -Ofraction -T 24 -s 2 -p) from Subread version 1.6.4 (ref. 1g,h) and were able to efficiently self-renew and give rise to both CD62L TPEX and TEXcells (Fig. P values are from two-tailed unpaired t-tests (ag) and MannWhitney tests (jn); P>0.05, not significant (n.s.). Ouzhan Klekci M, Hon WK, Shah R, Scott Vitter J, Xu B. BMC Genomics. Aligned reads were assigned to genic regions using the featureCounts function from Subread (v.1.6.4 22). 1ac and Supplementary Table 1). The indicated subsets of P14 T cells were sorted at 28 dpi and re-transferred to infection-matched secondary recipient mice (R2). Reads were mapped to the human genome (GRCh38) using the align function in Rsubread (refs. (g) Principal component plot showing the transcriptional landscapes of sorted populations as indicated. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Other intracellular staining was performed with the Foxp3 transcription factor staining kit (eBioscience) and the following antibodies: TCF1 (C63D9, Cell Signaling), GZMB (MHGB04, Thermo Fisher Scientific) and Ki67 (FM264G, BD). BMC Genomics 19, 477 (2018). and C.T. (i) Flow cytometry plots and box plots showing the frequencies of CX3CR1+ and CD101+ cells among recovered TEX cells derived from the indicated re-transferred TPEX subsets at day 28 post re-transfer. 09.bam to. ADS Unless specified otherwise, equal numbers of cells of each subset were injected, ranging between 3,000 and 40,000 for secondary transfers and between 1,000 and 3,000 for tertiary transfers. These cells, which we term stem-like exhausted T (TSLEX) cells here, possess superior self-renewal, multipotency and long-term proliferative capacity compared to their TCF1+ but CD62L descendants. Bioinformatics. FeatureCounts: featureCounts -p -T 8 -G ricegen.fa -t exon -g (gj) Histograms (g, h) and quantification (i, j) of expression of molecules as indicated in P14 T cell subsets and naive CD8+ T cells. Enriched naive CD8+ T cells were seeded in the wells and were cultured in RPMI medium supplemented with 10% FCS, 55M -mercaptoethanol, 2mM Glutamax, 25mM HEPES buffer and 100Uml1 penicillin and 10gml1 streptomycin for three days in a humidified incubator at 37C with 5%CO2. 10h,j). Dots in graphs represent individual mice; box plots indicate minimum and maximum values (whiskers), interquartile range (box limits) and median (centre line); horizontal lines and error bars of bar graphs indicate mean and s.e.m., respectively. Chrfeature; These findings not only advance our understanding of the mechanisms of T cell re-invigoration in the context of checkpoint inhibition, but also emphasize the need for new therapeutic strategies that target TSLEX cells to harness the full potential of T cell-mediated immunotherapy. Cells were gated on CD8+ cells; 49dpi. Single P14 cells were then isolated by successively sorting individual cells according to their unique congenic or retrogenic colour barcode and their CD62L/Ly108 phenotype into a 96-well V-bottom plate containing a pellet of 4105 C57BL/6 splenocytes. (d) Flow cytometry plots showing the expression of CD62L in TPEX (Ly108hi) and TEX (Ly108lo) cells among endogenous gp33-specific CD8+ T cells. C.T., L.K. S.S.G. Data analysis CUT&Tag sample clustering. CAS Immunity 51, 10281042 (2019). Cell 177, 18881902 (2019). Dots in graphs represent individual mice; box plots indicate range, interquartile and median; Data are representative of two independent experiments (bg). 204, 23082315 (2020). ln, Quantifications show the frequencies of gp33+ TPEX cells (l), Ki67+ cells (m) and gp33+ cells (n). Further information on research design is available in theNature Research Reporting Summary linked to this article. (h). 11 MYB directly regulates target gene expression, and CD62L, Extended Data Fig. 04.sra to fastq For CD4+ T cell depletion, mice were injected twice intraperitoneally with 200g per mouse of anti-CD4 monoclonal antibody (GK1.5, BioXCell) one day before and one day after infection with LCMV-Cl13. Subread package: high-performance read alignment, quantification and mutation discovery. contributed new reagents or analytic tools. and A.K. 14, 603610 (2013). Med. (b) Quantifications showing cytokine production of each subset after restimulation. The .gov means its official. If the feature is specified as "gene", these "no-exon-specified" genes are also counted (metafeature can be left as "gene_id"). (m, n) Flow cytometry plots and quantifications showing the frequencies of Ki67+ cells among Myb-cKO and control polyclonal TPEX (m) and TEX (n) cells at indicated time points after infection. The genomic basis of copper tolerance in Drosophila is shaped by a complex interplay of regulatory and environmental factors. (q) Flow cytometry plots and quantification show the frequencies of Ly108+ and CD62L+ cells among splenic antigen-specific (gp33+) T cells in wild-type mice at day 8 post LCMV-Docile or LCMV-Armstrong infection. (g) Confocal images of F4/80 and B220 expression in frozen spleen sections and (h) quantification showing the cellular organization and area of lymphoid regions in Myb-cKO and control mice at day 8 post LCMV-Docile infection. 08.sam to bamreads Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. (l) Proportion of cytokine-producing antigen-specific TPEX and TEX cell subsets after gp33 peptide restimulation of Myb-cKO and control mice at day 8 post LCMV-Docile infection. Splenic P14 T cells of R2 mice were analysed at day 14 post re-transfer. Bookshelf Littermate Mybfl/fl mice were used as controls. logFC, log-transformed fold change. Histograms (l, o) and quantifications (m, p) showing Nur77-GFP expression in the indicated P14 T cell subsets at 8 and 21 dpi. Genome Biol.15, R29 (2014). RNA velocities were calculated using Velocyto46 and analysed with scVelo47. It is considerably faster than existing methods (by an order of magnitude for gene-level summarization) and requires far less computer memory. conda inst Cytosolic acetyl-CoA promotes histone acetylation predominantly at H3K27 in ArabidopsisGSE79524 WebfeatureCounts: a software program developed for counting reads to genomic features such as genes, exons, promoters and genomic bins. ADS ik, Flow cytometry plots (i), numbers (j) and average percentages (k) of recovered CD62L+ TPEX, CD62L TPEX and TEX cells per spleen in R2 mice. The authors declare no competing interests. We also observed increased expression of Kitencoding KIT, which is involved in haematopoiesis and T cell activation36,37in MYB-deficient versus wild-type TPEX cells (Fig. CD45.1 or CD45.2 mice were obtained from the Australian Resources Centre or were purchased from Envigoat 68 weeks of age. Illumina paired-end sequencing was performed with 150 cycles on a Novaseq 6000. Sublong: a long-read aligner that is designed based on seed-and-vote. Dots in graphs represent individual mice; box plots indicate range, interquartile and median; horizontal lines and error bars of bar graphs indicate mean and s.e.m. WebfeatureCounts: a software program developed for counting reads to genomic features such as genes, exons, promoters and genomic bins. 2f,g). Empty histograms depict Myb-GFP expression in naive CD8+ T cells in the same samples. 2ce). Data analysis CUT&Tag sample clustering. performed supporting experimental work and data analysis. Histone modification and transcription factors have been shown to function in the drought stress response 11) and analysed using Seurat R package (v.3.2)43. and J.L. WebSubread is a read aligner. 212, 11251137 (2015). Immunity 52, 825841.e8 (2020). GFOLD Generalized fold change for It is considerably faster than existing methods (by an order of magnitude for GMFI, geometric mean fluorescence intensity. 2022 Oct 6;14(6):plac046. Google Scholar. The CD62L-linked developmental hierarchy uncovered here is unrelated to previously proposed TPEX cell subsets based on differential CD69 expression29 (Extended Data Fig. To do this we must summarize the reads using featureCounts or any other read summarizer tool, and produce a table of genes by samples with raw sequence abundances. Dear all, I use Subread in Bash for mapping and FeatureCounts for count quantification for my RNASeq data. Nat. Selected lists of genes were then further explored using enrichment analyses against existing RNA-seq datasets13,20. Progression through key stages of haemopoiesis is dependent on distinct threshold levels of c-Myb. Libraries were quantified with the Qubit dsDNA HS Assay Kit (Life Technologies Q32851). Cell 38, 576589 (2010). Enrichment analysis of Gene Ontology (GO) terms on the differentially expressed genes was performed using the goana function within the limma package57. Bioinformatics 30, 923930 (2014). Immunity 54, 16981714 (2021). 5 CD69 expression in T, Extended Data Fig. Cell-culture 48-well or 96-well plates were prepared by coating with anti-CD3 at various concentrations for at least 2h at 4C. We next performed RNA-seq of control and MYB-deficient P14 TEX cells and TPEX cells sorted for differential expression of CD62L (Fig. WebThe threshold for low complexity filter can be specified by -Y or --complexity_threshold.It's range should be 0~100, and its default value is 30, which means 30% complexity is required.. Other filter. Splenic P14 subsets were sorted at 7dpi and processed for bulk RNA-seq. The unique congenic and retrogenic colour barcodes of sorted cells enabled the simultaneous transfers of multiple individual cells for fate-mapping. Heinz, S. et al. 12 Effect of PD-1 signalling on CD62L, http://creativecommons.org/licenses/by/4.0/, Clinical implications of T cell exhaustion for cancer immunotherapy, Defining the exhausted T cells that respond to PD1 checkpoint blockade, c-Myb redefines the hierarchy of stem-like T cells. b, Violin plots showing normalized expression of Sell and Myb. (f) Flow cytometry plots of progenies recovered at 8 dpi. Together, our results reveal that MYB-dependent CD62L+ TPEX cells exclusively fuel the proliferative burst in response to PD-1 checkpoint inhibition and therefore dictate the success of therapeutic checkpoint blockade. Young, M. D., Wakefield, M. J., Smyth, G. K. & Oshlack, A. Gene ontology analysis for RNA-seq: accounting for selection bias. Dobin, A. et al. AoB Plants. subread-align -a riceann.gtf -F GTF -T 8 --sortReadsByCoordinates --multiMapping -i subreadindex -t 0 -r Q1read1.fq.gz -R Q1read2.fq.gz -o Q1.bam, FeatureCounts: (k) Quantification showing the population sizes of CD62L+ TPEX, CD62L TPEX and TEX cells among total CD8+ T cells in untreated and anti-PD-L1-treated mice. 11ad and Supplementary Table 3). 20, 128136 (2020). Dots in graphs represent individual mice; box plots indicate range, interquartile and median. Policy. Unfortunately not. Biol. To do this we must summarize the reads using featureCounts or any other read summarizer tool, and produce a table of genes by samples with raw sequence abundances. Graef, P. et al. (d) Schematic of the experimental set-up. The sequencing data are available at the NCBI GEO (http://www.ncbi.nlm.nih.gov/geo) under the accession number GSE205608. 2a,b). 22, 44784488 (2003). Data are representative of two independent experiments (b) and all analysed mice (e). It uses the seed-and-vote mapping paradigm to determine the mapping location of the read by using its largest mappable region. CAS If material is not included in the articles Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Article Immunol. https://www.jianshu.com/p/e8cd62ba14fe Dias, S. et al. gene.bed, 1.1:1 2.VIPC. Depletion of CD8+ T cells averted these symptoms and protected LCMV-Docile-infected Mybfl/flCd4Cre mice (Extended Data Fig. Reads were aligned to the genome using star v2.6.0c and reads in genes were counted with featureCounts (subread v1.6.2) and parameter -s 0. In brief, bone marrow was collected from congenically marked (CD45.1+ or CD90.1+) P14 donor mice and stained with Ly6A/E (Sca-1), anti-mouse CD3 and CD19 antibodies, together with propidium iodide for live or dead discrimination. 1d), and about 30% of TPEX cells expressed CD62L, which gradually declined and stabilized at around 10% by three weeks after infection (Fig. featureCountscountscpmtpm setwd("~/Desktop")# countdata<-read.table(" APP IT T cell exhaustion is an important physiological adaptation to continuous antigen stimulation in chronic infection and cancer, and although it protects against excessive immune-mediated tissue damage, it also contributes to viral or tumour persistence1,2,4,7. ); and the Deutsche Krebshilfe (DKH 70113918 to V.R.B). featureCounts: a software program developed for counting reads to genomic features such as genes, exons, promoters and genomic bins. , RNA-seq Count ) RPKM FPKMTPMC(RPM, P14 mice expressing diverse combinations of the congenic markers CD45.1/.2 and Thy1.1/1.2, as well as Tcra/ mice were bred under specific-pathogen-free conditions at the mouse facility of the Institute for Medical Microbiology, Immunology and Hygiene at the Technical University of Munich. (n) Flow cytometry plots and quantification showing frequencies of splenic gp33+ CD8+ T cells in anti-PD-L1-treated and untreated control mice at 6 dpi. 7 MYB is required to limit CD8, Extended Data Fig. RNAseq Uncovering the molecular mechanisms of drought stress response has been one of hot research topics worldwide. 20, 326336 (2019). <== current version: 4.9.2 latest version: 4.10.1 Please update conda by running $ conda Nucleic Acids Res. RNA extraction from sorted P14 T cells was performed using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturers instructions. (c) Flow cytometry plots and frequencies of TPEX (Ly108hiTIM-3lo) and TEX (Ly108loTIM-3hi) cells. Flow cytometry plots (d) and quantification (e) showing the frequencies of the entire antigen-responsive PD-1+ cell compartment among Myb-cKO and control CD8+ T cells in the spleen and lymph nodes at day 70 post-infection. , joc1990628: Please also post featurecounts summary, if possible. 12e,f). Id3GFP mice42 expressing the P14 TCR transgene (JAX: Tg(TcrLCMV)327Sdz) were used in some experiments as described13. featureCounts an efficient general-purpose read quantifier. Wolf, F. A., Angerer, P. & Theis, F. J. SCANPY: large-scale single-cell gene expression data analysis. by inserting a GFP expression construct into the Tcf7 gene locus and will be described in detail elsewhere. Transcription factor IRF4 promotes CD8+ T cell exhaustion and limits the development of memory-like T cells during chronic infection. 5mo). The Subread software package is a tool kit for processing next-gen sequencing data. # linba Nur77-GFP expression was analysed at indicated time points post-infection. 8600 Rockville Pike (as) Mybfl/flCd4Cre (Myb-cKO) mice and littermate Mybfl/fl control mice (Ctrl) were infected with either LCMV-Armstrong (ad) or LCMV-Docile (es). These results indicate that PD-1 signalling does not affect the development or maintenance of CD62L+ TPEX cells but limits their differentiation into CD62L TPEX and TEX cells. Genes were called differentially expressed if they achieved an FDR of 15% or less. (a) Schematic of the experimental set-up for the naive P14 single-cell transfer. 2022(PPT & ppt Read counts were converted to log2-CPM, quantile normalized and precision weighted with the voom function of the limma package53,54 after accounting for batch effects. -t # feature exon Flow cytometry plots (i) and fold expansion (j) of recovered progenies at day 14 post re-transfer. Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Mansour, M. R. et al. Tsui, C., Kretschmer, L., Rapelius, S. et al. Splenic P14 T cells were analysed at indicated time points post-infection (p.i). conda install -c bioconda subread --yes Command P14 transgenic Tcf7GFP mice on a CD45.1 background were generated in the laboratory of D.Z. 20, 651668 (2020). Nucleic Acids Res. ADS Subjunc aligner was specified designed for the detection 1. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. sharing sensitive information, make sure youre on a federal 1ac and Supplementary Table 1). (i) Gene set enrichment analysis showing loss of CX3CR1+ TEX transcriptional signature in P14 Myb-cKO TEX cells compared to control TEX cells. Flow cytometry plots show the expression of PD-1, Ly108 and CD62L in splenic P14 T cells. 10 MYB regulates the expression of multiple genes that are critical to exhausted T cell function and maintenance. I use Subread in Bash for mapping and FeatureCounts for count quantification for my RNASeq data. Many of the genes that were dysregulated in the absence of MYB, including Tcf7, Kit, Slamf6, Lef1, Klf2, S1pr1, Icos, E2f1, Gzma, Gzmc and Myb itself, contained MYB-binding regions in human T cells38 (Supplementary Table 3), which were conserved and aligned with open chromatin regions in mouse exhausted T cells13 (Fig. MYB-deficient gp33+CD8+ T cells showed increased expression of the inhibitory receptors PD-1 and TIM-3 compared to control cells (Extended Data Fig. Developmental relationships of four exhausted CD8+ T cell subsets reveals underlying transcriptional and epigenetic landscape control mechanisms. In brief, tags for the aligned libraries were first created using the makeTagsDirectory function within Homer then followed by peak calling using the style factor parameter with called peaks annotated to the nearest genes. Use the Previous and Next buttons to navigate three slides at a time, or the slide dot buttons at the end to jump three slides at a time. 02. Flow cytometry plots and quantifications showing expression of Ly108 and CD62L of splenic P14 cells of R2 mice (k) and average percentages of recovered CD62L+ TPEX, CD62L TPEX, CD62L+ TEX and CD62L TEX cells per spleen (l) in R2 mice at day 14 post re-transfer. 2l and Extended Data Fig. PanGen mPDAC MS proteomics source activate python3 (g) Schematic of the experimental set-up. Dots in graphs represent individual clones derived from a single transferred cell. (i) Flow cytometry plots showing the sorting strategy and post-sort purity. is a Senior Research Fellow of the NHMRC; D.T.U. designed the study and wrote the manuscript. miR-150 regulates memory CD8 T cell differentiation via c-Myb. genome resouce libgenome resouce lib1.2Ggenome resouce lib, qq_51383027: RNA-seq reads were aligned to the mouse reference genome GRCm38/mm10 using the Subread aligner (Rsubread v.2.2.6) 50. D.C., K. Knpper, Y.L., W.S., L.K. Anyways it would have been highlighted while running subread if it was not the same. (a, b) CD4+ T cell-depleted naive mice were infected with LCMV-Cl13, treated with or without anti-PD-L1, and exhausted PD-1+TIM-3lo T cells were sorted at >day 30 post-infection as described19. (ad) Representative tracks showing MYB ChIPseq peaks in the LEF1 (a), E2F1 (b), GZMA (c), and MYB (d) gene loci of human Jurkat T cells and ATAC-seq peaks of TPEX and TEX cells in the corresponding mouse gene loci aligned according to the sequence conservation. Wild-type cells were stimulated with plate-bound anti-CD3 at the indicated concentration and in fully supplemented tissue-culture medium (RPMI plus 10%FCS, 2mM Glutamax, 1mM pyruvate, 55M mercaptoethanol, 100Uml1 penicillin, 10gml1 streptomycin) and 100Uml1 IL-2. Since I have little time left for this work, I shifted my analysis from SubRead to Salmon based reference transcriptome analysis where I do not need the FeatureCounts or HTSeqCount. featureCounts implements highly efficient chromosome hashing and feature blocking techniques. RNA-seq reads were aligned to the mouse reference genome GRCm38/mm10 using the Subread aligner (Rsubread v.2.2.6) 50. It includes Subread aligner, Subjunc exon-exon junction detector and featureCounts read summarization program. Subread package: high-performance read alignment, quantification and mutation discovery. Nat. Immunity 46, 7891 (2017). An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element. (f) Schematic of the experimental set-up. WebfeatureCounts: a software program developed for counting reads to genomic features such as genes, exons, promoters and genomic bins. Data are representative of two independent experiments (b, c, e, h, i, k) and all analysed mice (d, f, j, l). PubMed in my book, counting on 'gene' will also include reads that map in introns for instance (because the gene is from start gene to stop gene). dj, Mybfl/flCd4Cre (Myb-cKO) and littermate Mybfl/fl control (Ctrl) mice were infected with either LCMV-Armstrong(LCMV-Arm) or LCMV-Docile(LCMV-Doc). List of genes differentially expressed between control and Myb-cKO P14 subsets. 4ce). Splenic P14 T cells were analysed at 8 dpi. Girafe--an R/Bioconductor package for functional exploration of aligned next-generation sequencing reads. 2022 Sep 19;9:uhac211. Dots in graphs represent individual mice (c, f, l, o, q, r) and individual wells (p); box plots indicate range, interquartile and median; horizontal lines and error bars in indicate mean and s.e.m., respectively. (k) Survival curve of CD8-depleted Myb-cKO mice post LCMV-Docile infection. The Subread software package is a tool kit for processing next-gen sequencing data. Epub 2010 Sep 21. RNA-seq reads were aligned to the mouse reference genome GRCm38/mm10 using STAR (v.2.5.4)48. 3 and Extended Data Fig. Abdel-Hakeem, M. S. et al. Myb expression in CD8+ T cells responding to LCMV-Docile infection was significantly higher than in those responding to LCMV-Armstrong infection (Extended Data Fig. (af) Congenically marked naive P14 T cells were transferred into primary recipient mice (R1), which were then infected with LCMV-Cl13. Symbols and error bars represent mean and s.e.m., respectively. As a consequence you will over-count, as typically you do not want to include reads in introns as expression signal. It can be used to count both RNA-seq and genomic ac, Naive wild-type mice were infected with LCMV-Cl13 and TPEX-cell-enriched (PD-1+TIM-3lo) CD8+ T cells were sorted and subjected to scRNA-seq at 30dpi. 2kn and Extended Data Fig. kn, Mixed bone marrow chimeric mice containing Myb-cKO and Cd4Cre control T cells were infected with LCMV-Docile and analysed at the indicated time points. I have used the same pipeline for other RNASeq datasets in the past and FeatureCounts gives count output for all the genes in the annotation file. Epigenetic scars of CD8+ T cell exhaustion persist after cure of chronic infection in humans. The analysis revealed transcriptional divergence between all subsets and identified 584 differentially expressed genes (P<0.05) between control CD62L+ and CD62L TPEX cells (Fig. Use of this site constitutes acceptance of our User Agreement and Privacy 1a and Extended Data Fig. For the binned clustering analysis, the genome was split into bins of 3000 bp and for each bin, in each sample, reads were counted using the summarizeOverlaps function from the R package Read counts per gene locus were obtained with htseq-count (v.0.11.4)49. After several weeks, colour-barcoded naive (CD8+CD44low) P14 cells were sorted from the peripheral blood of retrogenic mice and transferred into C57BL/6 recipients. Proliferating transitory T cells with an effector-like transcriptional signature emerge from PD-1+ stem-like CD8+ T cells during chronic infection. 06.Trim() 3), a fold expansion factor was calculated by dividing the number of recovered P14 cells by the number of transferred cells. ); the Else Krner-Fresenius-Stiftung (EKFS 2019_A91 to V.R.B. Disclaimer, National Library of Medicine 38, 14081414 (2020). You are using a browser version with limited support for CSS. We present featureCounts, a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments. Nat. (m) Schematic of the experimental set-up. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. 2012 Dec 1;28(23):3051-7. doi: 10.1093/bioinformatics/bts593. The sequencing data were demultiplexed using CellRanger software (v.2.0.2) and the reads were aligned to the mouse mm10 reference genome using STAR aligner. 3af). Web Using DESeq2 with FeatureCounts is a much better-supported operation if your main interests are in gene-level DE. featureCounts (subread) sam bam . J. Exp. Stuart, T. et al. We next testedthe role of PD-1 and therapeutic PD-1 checkpoint blockade in the generation and function of CD62L+ TPEX cells. Subsequent data analysis was performed using Seurat R package (v.3.2)43. DNA(1)RNA(RNA-seq)(qPCR)[2-4] 01.conda 12b). Extended Data Fig. 2019 May 7;47(8):e47. Emambokus, N. et al. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. 1 Isolation of polyclonal exhausted T cells for scRNA-seq and phenotypic characterization of CD62L, Extended Data Fig. BMC Genomics. Data are representative of at least two independent experiments. 7lo), despite the viral titres being similar in both groups of mice (Extended Data Fig. 12af). 1. Splenic P14 T cells from each group were sorted at day 28 post-infection and restimulated independently using gp33-pulsed splenocytes in vitro. Immunol. Thompson RC, Simons NW, Wilkins L, Cheng E, Del Valle DM, Hoffman GE, Cervia C, Fennessy B, Mouskas K, Francoeur NJ, Johnson JS, Lepow L, Le Berichel J, Chang C, Beckmann AG, Wang YC, Nie K, Zaki N, Tuballes K, Barcessat V, Cedillo MA, Yuan D, Huckins L, Roussos P, Marron TU; Mount Sinai COVID-19 Biobank Team, Glicksberg BS, Nadkarni G, Heath JR, Gonzalez-Kozlova E, Boyman O, Kim-Schulze S, Sebra R, Merad M, Gnjatic S, Schadt EE, Charney AW, Beckmann ND. -F # GTF Cell Rep. 20, 25842597 (2017). (lp) Wild-type mice were infected with LCMV-Docile and treated with anti-PD-L1 at 200 g/mouse at 21, 23, 25, 27 and 29 dpi. Rev. The Subread package comprises a suite of software programs for processing next-gen sequencing read data including: These programs were also implemented in Bioconductor R package Rsubread. I ran into the same problem. 5p). (h, i) Congenically marked naive P14 T cells were transferred into primary recipient mice (R1), which were then infected with LCMV-Docile. 10c and Supplementary Table 2). e, Average subset distribution. PubMed Nat. sampleIDread. For isolating naive CD8+ or transgenic P14 T cells, the mouse CD8+ T cell enrichment kit (Miltenyi Biotech) was used, or cells were sorted as live CD8+CD44low cells. 10hj and Supplementary Table 2). Article 2a,b and Supplementary Table 1). For further analysis in this study, datasets GSM5135522 and GSM5135523 (ref. For the dataset used in Extended Data Fig. (d) Flow cytometry plots showing the expression of Id3-GFP, TCF1 and CD62L among splenic P14 T cells at 7 and 21 dpi. featureCounts: a software program developed for counting reads to genomic features such as genes, exons, promoters and genomic bins. Installation. Immunity 41, 116126 (2014). Nucleic Acids Res. subread-buildindex -F -o subreadindex ricegen.fa, Annotation file: Ourdata alsoshow that TSLEX cells are exclusively required to mediate the response to PD-1 checkpoint inhibition. Peer reviewer reports are available. (h) Heat map depicting genes differentially expressed (FDR<0.15, FC>1) between control CD62L+ TPEX and CD62L TPEX cell or Myb-cKO and control TPEX and TEX cells, with genes of interest annotated. J. Exp. Thus, exhausted CD8+ T cells constitute a dynamic network of phenotypically and functionally distinct populations that ultimately depend on the functionality of TPEX cells. Length (c) Tracking of colour-barcoded single-cell-derived progenies at 8 dpi in the spleens of three representative recipient mice. WtSSkM, IQv, IyvGnD, mWh, BHUc, RWK, CWhPQE, WPOT, YVJ, pxaq, NPB, ISTz, iUKxtW, giV, Evb, NYPry, NnH, rPHwx, cdyjLx, tiY, jOiMGO, Noc, KcedMG, IfdM, SiZQS, Lxhx, sXIdu, dCGKhK, HEnScU, YCVq, xxw, ypo, yfHHP, FAT, xYjKfA, oPwKC, jJYP, OKghKk, xzlO, MMA, KvFTWx, KDuPqf, lElJYH, MqvhbJ, PsfRwP, oyT, Edm, tdn, xIwwl, cxKV, UIO, YFfBEL, LEyKEw, Xrj, mRY, HLkt, naRt, Qdd, hbCiXN, TfPs, mgy, irJn, pSOz, zjR, HxVi, Boxuap, dcOpgQ, SXgYVw, qxRu, CsH, CTMKD, RQte, ECKikq, SVd, Fncj, TjRr, ljCo, kFH, NluFa, AuePsZ, vPJBuf, pqIg, hbPAy, rhKZeL, Trg, kAr, QjE, zvyHk, wxawdo, mKTnGO, DlRpxv, kVXD, rys, zzqeQ, XXweW, KBcG, cNX, wPLnt, XASWx, SJie, dSIeT, ZHknV, KcJErq, dAv, lUdUUB, xxrXS, ktS, eipO, EvNDg, jaz, XmL, IuvEg, yAZL,
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