Annu. a, Uniform manifold approximation and projection (UMAP) plot of 15,743 single exhausted T cells coloured according to cluster classification. Lahortiga, I. et al. As a consequence you will over-count, as typically you do not want to include reads in introns as expression signal. All data are representative of two independent experiments. eCollection 2022. 2g and Extended Data Fig. 07.Hisat2 Mapping Illumina paired-end sequencing was performed with 150 cycles on a Novaseq 6000. 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. Web Using DESeq2 with FeatureCounts is a much better-supported operation if your main interests are in gene-level DE. featureCounts (subread) sam bam . Pathway enrichment against the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways on the differentially expressed genes was performed using the kegga function also implemented in the limma package. ftp.sra.ebi.ac.uk/vol1/fastq/SRR12, subtype_MethyLevel, https://blog.csdn.net/weixin_43569478/article/details/108079250. Uncovering the molecular mechanisms of drought stress response has been one of hot research topics worldwide. 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. 10hj and Supplementary Table 2). 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. P values are from MannWhitney tests (hi); P>0.05, not significant (n.s.). Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Immunol. Note: in the display used, CFP emission appears on the diagonal between the BFP (x-axis) and T-Sapphire signal (y-axis) and is therefore indicated on both axes. After sorting, the whole content of each well was injected into separate C57BL/6 recipients. PubMed and S.J. Histone modification and transcription factors have been shown to function in the drought stress response featureCounts: a ultrafast and accurate read summarization program. Wu, T. et al. Cells were rested in fully supplemented RPMI medium (see above) at 37C for 10min, after which P14 cells were counted, and 5,000 P14 cells were injected intravenously into recipient mice before infection with LCMV. Google Scholar. Duplication of the MYB oncogene in T cell acute lymphoblastic leukemia. Immunol. Nat. g,h, Representative flow cytometry plots (g) and box plot (h) showing the fold change of frequencies of splenicpolyclonal PD1+CD8+ T cells in anti-PD-L1-treated versus PBS-treated mice. 20, 651668 (2020). 5l), and was further enhanced by the inhibition of PD-1 signalling in vivo (Extended Data Fig. (o) Flow cytometry plots and quantification showing the proportions of Ki67+ within the gp33+ compartment in Myb-cKO and control mice at day 8 post LCMV-Docile infection. (mn) Flow cytometry plots (m) and quantification (n) showing the frequencies of the PD-1+ cells among splenic CD8+ T cells. Google Scholar. Utzschneider, D. T. et al. Rev. 2kn and Extended Data Fig. Dots in graphs represent individual mice (h,j); horizontal lines and error bars of bar graphs indicate mean and s.e.m., respectively. Bookshelf #conda 8f,g). Did you finally find out why it is so? Developmental relationships of four exhausted CD8+ T cell subsets reveals underlying transcriptional and epigenetic landscape control mechanisms. To characterize the dynamics of Myb expression in chronic infection, we infected MybGFP reporter mice34 with LCMV-Docile (Fig. (c) Tracking of colour-barcoded single-cell-derived progenies at 8 dpi in the spleens of three representative recipient mice. Surface staining was performed for 30min at 4C in PBS supplemented with 2% FCS (FACS buffer) with the following antibodies: CD8a (53-6.7, BD), CD44 (IM7, BD), CD45.1 (A20, BD or Biolegend), CD45.2 (104, BD), CD90.1 (HIS52, Thermo Fisher Scientific) CX3CR1 (SA011F11, Biolegend), PD-1 (RMP1-30 or 29F.1A12, Biolegend), CD62L (MEL-14, Biolegend), TIM-3 (RMT3-23, Biolegend), CD101 (Moushi101, Thermo Fisher Scientific), Ly108 (eBio13G3-18D, BD), CD117 (KIT) (ACK2, Thermo Fisher Scientific), CD244 (2B4) (eBio244F4, Thermo Fisher Scientific), CD160 (eBioCNX46-3, eBioscience), TIGIT (GIGD7, Thermo Fisher Scientific) and KLRG1 (2F1, Biolegend). fh, Mixed bone marrow chimeric mice containing congenically marked Myb-cKO and Cd4Cre (control) T cells, infected with LCMV-Docile, were treated with anti-PD-L1 on days 33, 36, 39, 42 and 45 and analysed at 49dpi. RNA-seq 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 2d). featureCounts implements highly efficient chromosome hashing and feature blocking techniques. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. ); and the Deutsche Krebshilfe (DKH 70113918 to V.R.B). It uses the seed-and-vote mapping paradigm to determine the mapping location of the read by using its largest mappable region. Comparative analysis of algorithms for next-generation sequencing read alignment. The indicated subsets of P14 T cells were sorted at 28dpi and 310315103 cells were re-transferred to infection-matched secondary recipient (R2) mice. Dear all, I use Subread in Bash for mapping and FeatureCounts for count quantification for my RNASeq data. By contrast, the larger TPEX cell cluster showed low expression of Sell but was enriched for other TPEX-cell-associated transcripts, including Icos, Xcl1, Cxcl10, Cd28 and Eomes (CD62L TPEX cells; Fig. Immunol. CD62L+ TPEX cells were also found among endogenous gp33-specific and among polyclonal antigen-responsive PD-1+CD8+ T cells in LCMV-Docile-infected mice (Extended Data Fig. source activate python3 WebfeatureCountsubreadsubreadsubreadR featureCounts Feature: exon; Data are representative of at least two independent experiments (b,de,gh). 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. 2j and Extended Data Fig. I ran into the same problem. linux100101subread (rnaseq) root 12:08:22 ~ $ conda install -y subread Collecting package metadata (current_repodata.json): done Solving environment: done ==> WARNING: A newer version of conda exists. 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). Statistical analysis was performed in R (v.3.6.3). Hudson, W. H. et al. 7r,s). 3 and Extended Data Fig. Normalization and differential gene expression analysis was performed using DESeq2 (v.1.26.0). Quality control was performed with a High Sensitivity DNA Kit (Agilent 5067-4626) on a Bioanalyzer 2100, as recommended in the protocol. 2022(PPT & ppt In addition, naive P14 cells were included for the analysis. 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. Stat. (h) Schematic of the experimental set-up. 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. Consistent with the impaired expression of transcripts related to cell migration and lymph node homing (Ccr7, Cxcr5, S1pr1, Itgb1 and Itgb3), MYB-deficient antigen-specific CD8+ T cells were largely excluded from the lymph nodes (Extended Data Fig. Subread index: WebfeatureCounts 1. is a Senior Research Fellow of the NHMRC; D.T.U. 8). Nat. Immunity 54, 16981714 (2021). Red blood cells were lysed with a hypotonic ammonium chloride-potassium bicarbonate (ACK) or ammonium chloride-Tris (ACT) buffer. (ad) Single naive colour-barcoded P14 T cells were transferred to primary recipient mice, which were then infected with LCMV-Armstrong. Further information on research design is available in theNature Research Reporting Summary linked to this article. The indicated subsets of P14 T cells were sorted at 28 dpi and 330 x 103 cells were re-transferred to infection-matched secondary recipient mice (R2). 7lo), despite the viral titres being similar in both groups of mice (Extended Data Fig. Does it suggest that mapping failed for the 18000 ignored genes? 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. (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. Finally, the proportions of CD62L+ antigen-specific CD8+ T cells were 10-fold higher in LCMV-Docile versus LCMV-Armstrong infection (Extended Data Fig. Apparently the missing genes are all single-exon genes, represented in the gtf file by a single row, as "gene" only, no exons. eh, Survival curves of Myb-cKO and control mice and box plots showing the frequencies of gp33+CD8+ T cells at the indicated time points after infection with LCMV-Armstrong (e,f) or LCMV-Docile (g,h). CD45.1 or CD45.2 mice were obtained from the Australian Resources Centre or were purchased from Envigoat 68 weeks of age. We and others have shown that TPEX and TEX cells are controlled by specific transcriptional and metabolic networks that support their differentiation and maintenance13,14,15,16,17,18. PubMed Central -t # feature exon 1d), and about 30% of TPEX cells expressed CD62L, which gradually declined and stabilized at around 10% by three weeks after infection (Fig. For CD8+ T cell depletion, mice were injected intraperitoneally with 100g per mouse of anti-CD8 monoclonal antibody (YTS-169, BioXCell) on days 1, 3 and 5 of infection. Flow cytometry plots show the expression of PD-1, Ly108 and CD62L in splenic P14 T cells. LinuxCondaCondamacAapp Storecondacondacondacondaconda & Smyth, G. K. Testing significance relative to a fold-change threshold is a TREAT. Genome Biol.15, R29 (2014). 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. The opposing roles of the transcription factor E2A and its antagonist Id3 that orchestrate and enforce the naive fate of T cells. P values are from two-tailed unpaired t-tests (d, fh) and MannWhitney tests (a, c, e, i); P>0.05, not significant (n.s.). Data are representative of two independent experiments (e, f, i, j) and all analysed mice (m, p). GFOLD Generalized fold change for a, Schematic of the experimental set-up. Dots in graph represent individual mice; box plots indicate range, interquartile and median. ce, Congenically marked naive P14 T cells were transferred into CD4-depleted R1 mice, which were subsequently infected with LCMV-Cl13. Author summary Water deficiency has become one of the greatest concerns limiting sustainable crop production. 08.sam to bamreads b, Numbers of recovered P14 T cells (left), percentages of KLRG1+ (middle) and splenic viral loads (right). Does it suggest PCR amplification-based duplicated reads? Chen, Z. et al. (q) Box plots showing the expression of PD-1 (left) and TIM-3 (right) among gp33+ CD8+ T cells of control and Myb-cKO mice at day 8 post LCMV-Docile infection. Transcriptional regulatory network for the establishment of CD8+ T cell exhaustion, TOX transcriptionally and epigenetically programs CD8+ T cell exhaustion, Ectopic Tcf1 expression instills a stem-like program in exhausted CD8+ T cells to enhance viral and tumor immunity, BATF regulates progenitor to cytolytic effector CD8+ T cell transition during chronic viral infection, Antigen-driven EGR2 expression is required for exhausted CD8+ T cell stability and maintenance, Early precursor T cells establish and propagate T cell exhaustion in chronic infection, PTPN2 regulates the generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity, Extended Data Fig. (ei) Colour-barcoded naive P14 T cells were transferred into primary recipient mice (R1), which were subsequently infected with LCMV-Cl13. Similar results were obtained from adoptively transferred MYB-deficient and control P14 T cells (Extended Data Fig. #rowname For further analysis in this study, datasets GSM5135522 and GSM5135523 (ref. 7 MYB is required to limit CD8, Extended Data Fig. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. (ab) Flow cytometry plots showing (a) splenic antigen-specific (gp33+) CD8+ cells and (b) expression of CD62L and KLRG1 among antigen-specific cells in Myb-cKO and control mice at indicated time points post LCMV-Armstrong infection. (ij) Flow cytometry plots (i) and quantification (j) showing kinetics of splenic polyclonal PD-1+ TPEX cells among Myb-cKO and control CD8+ T cells after infection. conda activate python3 Recombinant adeno-associated virus tools for enhanced microglial transduction in mice are reported. Subread aligner can be used to align both gDNA-seq and RNA-seq reads. do you mean that those genes are completely missing from the output or that they have 0 count ? I also ran HTSeq-counts and it also did not provide the count data for all the genes. Nat. -RA: a parallel sparse index for genomic read alignment. (a, b) Congenically marked naive P14 T cells were adoptively transferred into naive recipient mice, which were then infected with LCMV-Cl13. Use of this site constitutes acceptance of our User Agreement and Privacy Ouzhan Klekci M, Hon WK, Shah R, Scott Vitter J, Xu B. BMC Genomics. 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: 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. If you have a new idea or new request, please file an issue. How is read coverage for missing genes per exon (for eg take one and check)? Q32798724 Relating to the data in Fig. For infection of Tcra/ mice, a dosage of 2103 PFU was used. S.S.G. 2022 Dec 8;20(1):275. doi: 10.1186/s12915-022-01479-w. Prowse-Wilkins CP, Lopdell TJ, Xiang R, Vander Jagt CJ, Littlejohn MD, Chamberlain AJ, Goddard ME. 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. Street, K. et al. Please also post featurecounts summary, if possible. 39, 19951998 (2007). $ cat dir_6.txt Author summary Water deficiency has become one of the greatest concerns limiting sustainable crop production. 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: 7q), which suggests that the increased function and proliferation of effector cells was not due to impaired expression of inhibitory receptors. ac, Congenically marked Mybfl/flCd4Cre (Myb-cKO) and Cd4Cre (control) P14 T cells were adoptively transferred into naive recipient mice, which were then infected with LCMV-Docile. Representative histogram and normalized quantification show GMFI of Myb-GFP expression in CD8+ T cells stimulated with plate-bound anti-CD3 at the indicated concentrations. 41, e108 (2013). McLane, L. M., Abdel-Hakeem, M. S. & Wherry, E. J. CD8 T cell exhaustion during chronic viral infection and cancer. Chen, Z. et al. AoB Plants. featureCounts: a software program developed for counting reads to genomic features such as genes, exons, promoters and genomic bins. Accessibility (c) Schematic of the experimental set-up. ADS government site. featureCountscountscpmtpm setwd("~/Desktop")# countdata<-read.table(" APP IT RNAseq 1d and Extended Data Fig. Splenic P14 T cells of R2 mice were analysed at day 28 post re-transfer. Data are representative of two independent experiments (b, c, e, h, i, k) and all analysed mice (d, f, j, l). The process of counting reads is called read summarization. Natl Acad. 10kl). (d) Box plots show frequencies and numbers of CD62L+ TPEX cells among control and PD-1-deficient P14 cells. (c) Flow cytometry plots and frequencies of TPEX (Ly108hiTIM-3lo) and TEX (Ly108loTIM-3hi) cells. -T # , Geneidensemble 12d). WebConda. (k) Representative flow cytometry plots depict Ly108 and Myb-GFP expression among antigen-specific (gp33+) CD8+ T cells. Sequencing was performed on an Illumina Novaseq by Novogene, generating 150-bp paired-end reads. Biol. 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. Subread package: high-performance read alignment, quantification and mutation discovery. MeSH 11 MYB directly regulates target gene expression, and CD62L, Extended Data Fig. USA 102, 1554515550 (2005). Startfeature, Did you check the chromosome names in reference vs GTF? (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. (j) Volcano plot highlighting genes differentially expressed (FDR<0.15) between control and Myb-cKO TEX cells with genes of interested annotated. 5k), and found that the expression of Myb was highest in CD62L+ TPEX cells (Fig. (k) Survival curve of CD8-depleted Myb-cKO mice post LCMV-Docile infection. 10c and Supplementary Table 2). We present featureCounts, a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments. 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. featureCounts -p -T 8 -G ricegen.fa -t exon -g gene_id -a riceann.gtf -F GTF -M -O --fraction -o STRcounts.txt Q1.bam Q2.bam Q3.bam V1.bam V2.bam V3.bam, HTSeq-counts: 4, 11841191 (2009). Splenic P14 T cells of R2 mice were analysed 8 days after infection with LCMV-Armstrong. Hi, Selected lists of genes were then further explored using enrichment analyses against existing RNA-seq datasets13,20. Tsui, C., Kretschmer, L., Rapelius, S. et al. Adult 8-12 weeks Mybfl/flCd4Cre (Myb-cKO) and littermate Mybfl/fl control (Ctrl) mice were euthanized, and T cell populations were analysed in the thymus, spleen and lymph nodes. Data are representative of two independent experiments (d). CD62L+ and CD62L TPEX cells expressed high levels of PD-1, the activation marker CD44, the exhaustion-associated transcription factor TOX and the co-stimulatory molecule ICOS (Extended Data Fig. (d) Numbers of splenic gp33+CD8+ T cells in Myb-cKO and control mice at indicated time points post LCMV-Armstrong infection. featureCounts an efficient general-purpose read quantifier. McCarthy, D. J. Can you also post the snapshot of the gtf file? conda install -c bioconda subread --yes Command 1, eaai8593 (2016). 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). Nature 571, 211218 (2019). Epigenetic scarring of exhausted T cells hinders memory differentiation upon eliminating chronic antigenic stimulation. (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. 209, 24852499 (2012). Subjunc aligner was specified designed for the detection 50,52). Thus, exhausted CD8+ T cells constitute a dynamic network of phenotypically and functionally distinct populations that ultimately depend on the functionality of TPEX cells. Nssing, S. et al. 8kn). WebfeatureCounts: a software program developed for counting reads to genomic features such as genes, exons, promoters and genomic bins. Data show all analysed mice (h, i). 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. Web Using DESeq2 with FeatureCounts is a much better-supported operation if your main interests are in gene-level DE. featureCounts (subread) sam bam . 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. table(duplicated(lnc$id)) Each cell staining reaction was preceded by a 10-min incubation with purified anti-mouse CD16/32 Ab (FcgRII/III block; 2.4G2) and (fixable) viability dye (Thermo Fisher Scientific). featureCountssubreadsubjuncsubread featureCounts 1.BAM/ SAM bambam Immunol. RNA-seq reads were aligned to the mouse reference genome GRCm38/mm10 using the Subread aligner (Rsubread v.2.2.6)50. 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. Next-generation sequencing technologies generate millions of short sequence reads, which are usually aligned to a reference genome. WebfeatureCounts 1. Nature 560, 494498 (2018). 204, 23082315 (2020). 10, RNA extraction from sorted P14 T cells was performed using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturers instructions. (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. (dj) Congenically marked naive P14 T cells were transferred into primary recipient mice (R1), which were then infected with LCMV-Cl13. (f) Spleen size (left) and haematoxylin and eosin staining of sections showing infiltration of immune cells (arrows) in livers (middle) and lungs (right) in Myb-cKO and control mice at 8 dpi. Transforming growth factor--regulated mTOR activity preserves cellular metabolism to maintain long-term T cell responses in chronic infection. (h) Flow cytometry plots showing the frequencies of TPEX cells among gp33+ cells at 49 dpi. Careers. FeatureCounts: featureCounts -p -T 8 -G ricegen.fa -t exon -g LCMV-derived Db/gp33-41 tetramers were obtained from the NIH Tetramer Facility; tetramer staining was performed for 3060 min at 4C in FACS buffer. 21, 12561266 (2020). Sublong : a long-read aligner that is designed based on seed-and-vote. FDM; FineSplice Enhanced splice junction detection and estimation from RNA-Seq data. This observation concurred with a premature termination of cell-cycle activity in MYB-deficient TPEX and TEX cells and a marked contraction of the entire antigen-specific compartment (Fig. (e) Schematic of the experimental set-up. P14 transgenic Tcf7GFP mice on a CD45.1 background were generated in the laboratory of D.Z. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Cancer 8, 523534 (2008). I cannot understand why is it so? Unfortunately not. Littermate Mybfl/fl mice were used as controls. (f) Flow cytometry plots and quantification showing the frequencies of TPEX cells among Myb-cKO and control P14 T cells at the indicated time points after infection. Imaging data were analysed using Fiji (ImageJ) software (NIH). To this end, we generated P14 T cells that lack functional Pdcd1 (encoding PD-1) using CRISPRCas9 (Extended Data Fig. The library was sequenced with 20,000 reads per cell. 5k). Nat. The sequencing data shown in Extended Data Fig. By contrast, LCMV-Docile-infected Mybfl/flCd4Cre but not control mice exhibited signs of severe immunopathology and most became moribund within 10dpi (Fig. Statistical methods to predetermine sample size were not used. (cj) Naive congenically marked (CD45.1+) Id3-GFP P14 cells were transferred to naive recipients (Ly.5.2), which were then infected with LCMV-Docile. (a) Schematic of the experimental set-up. sampleIDread. To directly test the role of CD62L+ TPEX cells incheckpoint blockade, we performed adoptive transfer experiments in the context of therapeutic PD-1 inhibition (Fig. Installation. Availability and implementation: 1e). Wolf, F. A., Angerer, P. & Theis, F. J. SCANPY: large-scale single-cell gene expression data analysis. Federal government websites often end in .gov or .mil. Transcription factor IRF4 promotes CD8+ T cell exhaustion and limits the development of memory-like T cells during chronic infection. New filters are being implemented. The sequencing data are available at the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo) under the accession number GSE168282 (ref. Mansour, M. R. et al. PubMed Overall, these data suggest a one-way developmental trajectory that originates from CD62L+ TPEX cells. (b) Gene set enrichment analysis showing loss of CD62L+ TPEX transcriptional signature in Myb-cKO TPEX cells compared to control TPEX cells. ); the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; SFB-TRR 338/1 2021452881907, SFB 1054210592381 to V.R.B. ); the European Research Council (starting grant 949719 SCIMAP to V.R.B. Dear all, Overlap between differentially expressed genes from the RNA-seq data (mouse) and ChIPseq data (human) was performed by first transforming the human genes associated with each annotated peak to their corresponding mouse homologues using information available in the Ensembl database through the biomaRt Bioconductor package60. 11g), progeny that were derived from CD62L+ TPEX cells expanded more efficiently (Fig. It can be used to count both RNA-seq and genomic WebfeatureCountsubreadsubreadsubreadR featureCounts Feature: exon; subreadfeatureCountscounts_id.txt DESeq2limmaDESeq2 First step: The Subread software package is a tool kit for processing next-gen sequencing data. Splenic P14 TPEX cells were analysed at day 8 post-infection. Subjunc aligner was specified designed for the detection Subsequent data analysis was performed using Seurat R package (v.3.2)43. Dots in graphs represent individual mice; horizontal lines and error bars of bar graphs indicate mean and s.e.m., respectively. 1kp). Results: We present featureCounts, a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments. 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. Nat. Splenic P14 T cells were analysed at day 8 post LCMV-Armstrong infection. (j) RNA velocity analysis showing developmental trajectories of TPEX cells, together with the expression of Tcf7 (left) and Sell (right). C.T., L.K., V.R.B. performed computational analyses. We next performed RNA-seq of control and MYB-deficient P14 TEX cells and TPEX cells sorted for differential expression of CD62L (Fig. 3gl). PanGen mPDAC MS proteomics ISSN 1476-4687 (online) Immunity 52, 825841.e8 (2020). PubMed Central SRR3286805. 1ac and Supplementary Table 1). 10b), confirming that MYB deficiency resulted in the loss of CD62L+ TPEX cells and not merely CD62L expression. (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. Raw gene counts were quantified using the featureCounts program of subread v2.0.1. and J.L. Although the frequencies of TPEX and TEX cells were unaffected by the loss of PD-1 (Extended Data Fig. FeatureCounts: featureCounts -p -T 8 -G ricegen.fa -t exon -g 10d,e). Correspondence to 10 MYB regulates the expression of multiple genes that are critical to exhausted T cell function and maintenance. Alfei, F. et al. (gj) Histograms (g, h) and quantification (i, j) of expression of molecules as indicated in P14 T cell subsets and naive CD8+ T cells. Flow cytometry was performed using a Fortessa or Cytoflex LX (Beckman Coulter) and sort purification was performed on a BD FACSAria Fusion or MoFlo Astrios (Beckman Coulter). 9ag). Indeed, KIT was exclusively expressed in CD62L TPEX cells and was highly upregulated in MYB-deficient TPEX cells (Fig. The site is secure. 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. FDM; FineSplice Enhanced splice junction detection and estimation from RNA-Seq data. (f) Schematic of the experimental set-up. featurecounthtseq-count featureCounts(subread) v2.0.1 featureCounts Subread Biotechnol. K. Kanev, W.K., D.T.U., S.N., T.M., S.V.T., S.A.W., I.A.P., S.J. Length Subjunc aligner was specified designed for the detection PubMed Moreover, they show that MYB is a transcriptional orchestrator of two fundamental aspects of exhausted T cell responses: the downregulation of effector function and the long-term preservation of self-renewal capacity. Bioinformatics 31, 166169 (2015). DCANomogram, 1.1:1 2.VIPC. (h) Size of single-T-cell-derived progenies and frequencies of CD62L+, KLRG1+ and (i) PD-1+ cells therein. (i) Confocal images of CD3 and B220 expression in frozen spleen sections showing the distribution of B and T cells in the spleens of Myb-cKO and control mice at day 8 post LCMV-Docile infection. Relating to the dataset in Extended Data Fig. Gautam, S. et al. Installation. Raw gene counts were quantified using the featureCounts program of subread v2.0.1. It is considerably faster than existing methods (by an order of magnitude for They are completely missing from the output. The 2,000 most variable genes were included for the anchoring process and used for downstream analysis to calculate principal components of log-normalized and scaled expression data. (gl) Congenically marked naive P14 T cells were transferred into primary recipient mice (R1), which were then infected with LCMV-Cl13. featureCounts: a software program developed for counting reads to genomic features such as genes, exons, promoters and genomic bins. (s) Quantification showing the absolute numbers of splenic CD8+, gp33+, CD62L+ TPEX, CD62L TPEX and TEX cells in Myb-cKO and control mice at day 8 post LCMV-Docile infection. Immunol. SRR3286803 (a) Schematic of the experimental set-up for the naive P14 single-cell transfer. Early emergence of T central memory precursors programs clonal dominance during chronic viral infection. Recombinant adeno-associated virus tools for enhanced microglial transduction in mice are reported. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. 20, 337349 (2019). Front. (p) Naive MybGFP and wild-type (non-reporter, control) CD8+ T cells were stimulated and cultured in vitro using plate-bound anti-CD3. 03. 2012 Dec 1;28(23):3051-7. doi: 10.1093/bioinformatics/bts593. 2011;12 Suppl 2(Suppl 2):S7. For primary population transfer experiments, 2,00010,000 naive P14 T cells were injected into naive congenically marked primary recipients. 10h,j). Proc. Buchholz, V. R. et al. It works with either single or paired-end reads and provides a wide range of options appropriate for different sequencing applications. There were no major differences in cytokine production between the two TPEX subsets, but IFN+ cells were enriched among CD62L+ TPEX cells (Extended Data Fig. The TCF1Bcl6 axis counteracts type I interferon to repress exhaustion and maintain T cell stemness. 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. A<-A[,-1] MYB orchestrates T cell exhaustion and response to checkpoint inhibition. He, R. et al. Mol. Google Scholar. C.T., L.K. Epub 2011 Aug 19. (b) P14 T cell frequencies and numbers of indicated genotypes. (j) Image and box plot showing the size and weights of spleens in untreated and CD8+ T-cell-depleted Myb-cKO mice at day 8 post LCMV-Docile infection. Subramanian, A. et al. Right, quantification showing the geometric mean fluorescence intensity (GMFI) of MybGFP among CD62L+ TPEX, CD62L TPEX and TEX cells as fold change over naive CD8+ T cells. Data are representative of two independent experiments. Dots in graph represent individual mice; box plots indicate minimum and maximum values (whiskers), interquartile range (box limits) and median (centre line). -g # meta-featuregene_id 7j,k), indicating that MYB-deficient CD8+ T cells mediated the fatal immunopathology in chronic LCMV infection. Immunity 51, 10431058 (2019). (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. Im, S. J. et al. Fixed samples were embedded in paraffin and sectioned at 10m, mounted on SuperFrostPlus Adhesion glass and stained using H&E. MYB also mediates functional exhaustion during chronic infection by restricting the initial expansion and effector function of antigen-responsive CD8+ effector T cells. (hj) Congenically marked naive Tcf7-GFP P14 T cells were adoptively transferred into naive mice, which were then infected with LCMV-Cl13. Bioinformatics. Data are representative of at least two independent experiments. b, Normalized gene expression of Tcf7, Sell, Gzmb and Cx3cr1 projected onto the UMAP. Rev. 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. Ritchie, M. E. et al. WebConda. ; APP2001719 to I.A.P. Results: We present featureCounts, a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments. Kallies, A., Zehn, D. & Utzschneider, D. T. Precursor exhausted T cells: key to successful immunotherapy? (m, n) Quantification of IFN expression (m), and granzyme B (GZMB) expression in TPEX and TEX cells (n) in Myb-cKO and control mice at day 8 post LCMV-Docile infection. WebSubread is a read aligner. All subsequent analysis was performed using SCANPY (v.1.6)45. (kp) Congenically marked naive Nur77-GFP reporter P14 T cells were transferred into naive (km) or CD4-T-cell-depleted (np) recipient mice, which were subsequently infected with LCMV-Cl13. 02. Furthermore, the superior proliferative and developmental potential of TSLEX cells makes them prime targets of adoptive T cell transfer and chimeric antigen receptor (CAR) T cell therapies. 9 MYB limits proliferation and cytotoxicity and sustains the long-term self-renewal of exhausted CD8. The wells were washed twice using PBS. The two remaining clusters expressed intermediate levels of both TPEX and TEX cell marker genes (cluster 3) or cell-cycle-related genes such as Mki67, Ccnb2 and E2f1 (cluster 4) (Fig. miR-150 regulates memory CD8 T cell differentiation via c-Myb. 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. Histone modification and transcription factors have been shown to function in the drought stress response Fold expansion of recovered progenies was then normalized to distinct input numbers. Trajectories were predicted using the Slingshot 1.4.0 package44, using the function slingshot with default settings and starting with the CD62L+ TPEX cell cluster. (d) Flow cytometry plots and quantification showing the expression of granzyme B (GZMB) in Myb-cKO and control TEX P14 cells at 8 dpi. 7ei). (de) Mixed bone marrow chimeric mice containing congenically marked Myb-cKO and control CD8+ T cells were infected with LCMV-Docile. 8hj). GMFI, geometric mean fluorescence intensity. (c) Quantification of central memory (TCM), effector memory (TEM), CX3CR1+ and KLRG1+ cells among gp33+ CD8+ cells in Myb-cKO and control mice at indicated time points post LCMV-Armstrong infection. These characteristics were maintained even after repetitive adoptive transfers (Extended Data Fig. featureCountssubreadsubjuncsubread featureCounts 1.BAM/ SAM bambam Regards. Please enable it to take advantage of the complete set of features! contributed new reagents or analytic tools. We next sorted MYB-deficient and control P14 TPEX cells from LCMV-Docile-infected mice and performed transcriptional profiling by RNA-seq (Extended Data Fig. For intracellular cytokine staining, splenocytes were ex vivo restimulated with gp33-41 (gp33) peptide (5mM) for 5h in the presence of brefeldin A (Sigma) for the last 4.5h, fixed and permeabilized using the Cytofix/Cytoperm (BD) or transcription factor staining kit (eBioscience) and stained with anti-IFN (XMG1.2, Thermo Fisher Scientific), TNF (MP6-XT22, Thermo Fisher Scientific). Data are representative of two independent experiments (c, l, o, p) and all analysed mice (f, q, r). 4d,e). This table will then be used to perform statistical analysis and find differentially expressed genes. P values are from two-tailed unpaired t-tests (c,f,hj) and MannWhitney tests (ln). For LCMV-Armstrong infection, frozen stocks were diluted in PBS and 2105 PFU were injected intraperitoneally. WebfeatureCounts: a software program developed for counting reads to genomic features such as genes, exons, promoters and genomic bins. 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. Annu. 1ac and Supplementary Table 1). PubMed 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. Thank you for visiting nature.com. Subread package: high-performance read alignment, quantification and mutation discovery. c, Volcano plot highlighting genes that are differentially expressed (false discovery rate (FDR)<0.15) between Myb-cKO TPEX and control CD62L TPEX cells, with genes of interest annotated. PFU, plaque-forming units. Both early TPEX and TEX cells were readily detectable during the acute phase (59dpi) of the immune response (Extended Data Fig. Flow cytometric analysis revealed a lack of CX3CR1+ cells and an increase in terminally exhausted CD101+ cells among MYB-deficient TEX cells compared to controls (Extended Data Fig. a,b, Congenically marked naive P14 T cells were transferredinto primary recipient (R1) mice, which were subsequently infected with LCMV-Cl13. e, UMAP plot showing two predicted developmental trajectories generated using Slingshot analysis. The smaller of these clusters was characterized by high expression of transcripts that are typically associated with naive or central memory T cells, including Sell (encoding CD62L), Ccr7, S1pr1, Lef1, Satb1 and Bach2 (referred to as CD62L+ TPEX cells; Fig. 3a). 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. Nature thanks Evan Newell and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 10f,g). Molecular states during acute COVID-19 reveal distinct etiologies of long-term sequelae. 2f,g, the indicated subsets of exhausted P14 cells (CD62L+ TPEX, CD62L TPEX, TEX) were sorted from the spleen of chronically infected mice (LCMV-Cl13) at 28dpi. 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). Peer reviewer reports are available. doi: 10.1186/1471-2164-12-S2-S7. RNA extraction from sorted P14 T cells was performed using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturers instructions. Dias, S. et al. In the meantime, to ensure continued support, we are displaying the site without styles 43, e47 (2015). 2e,f and Extended Data Fig. Results: We present featureCounts, a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments. 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