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LINE1 are spliced in non-canonical transcript variants to regulate T cell quiescence and exhaustion

Abstract

How gene expression is controlled to preserve human T cell quiescence is poorly understood. Here we show that non-canonical splicing variants containing long interspersed nuclear element 1 (LINE1) enforce naive CD4+ T cell quiescence. LINE1-containing transcripts are derived from CD4+ T cell-specific genes upregulated during T cell activation. In naive CD4+ T cells, LINE1-containing transcripts are regulated by the transcription factor IRF4 and kept at chromatin by nucleolin; these transcripts act in cis, hampering levels of histone 3 (H3) lysine 36 trimethyl (H3K36me3) and stalling gene expression. T cell activation induces LINE1-containing transcript downregulation by the splicing suppressor PTBP1 and promotes expression of the corresponding protein-coding genes by the elongating factor GTF2F1 through mTORC1. Dysfunctional T cells, exhausted in vitro or tumor-infiltrating lymphocytes (TILs), accumulate LINE1-containing transcripts at chromatin. Remarkably, depletion of LINE1-containing transcripts restores TIL effector function. Our study identifies a role for LINE1 elements in maintaining T cell quiescence and suggests that an abundance of LINE1-containing transcripts is critical for T cell effector function and exhaustion.

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Fig. 1: LINE1 elements are expressed and bound to chromatin in quiescent naive CD4+ T cells and downregulated by mTORC1 upon T cell activation.
Fig. 2: Intronic LINE1 elements are spliced in non-canonical transcript variants of cell-activation genes in naive CD4+ T cells.
Fig. 3: IRF4 regulates expression of LINE1-containing transcripts and canonical transcripts in CD4+ T cells.
Fig. 4: LINE1-containing transcripts are localized in cis and halt the expression of corresponding genes.
Fig. 5: LINE1-containing transcripts in complex with nucleolin control gene expression, hampering H3K36me3 deposition in quiescent CD4+ T cells.
Fig. 6: Upon T cell activation, LINE1-containing transcripts are suppressed by PTBP1, while GTF2F1 favors expression of canonical transcripts.
Fig. 7: IRF4 in cooperation with nucleolin cause re-accumulation of LINE1-containing transcripts in exhausted T cells.
Fig. 8: LINE1-containing transcripts modulate the dysfunctional phenotype of CD4+ and CD8+ TILs.

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Data availability

All data are available in the main text or in the Supplementary Information. Data that support the findings of this study have been deposited and are publicly available at ArrayExpress: E-MTAB-9572 and E-MTAB-10798 for ChIP–seq data, E-MTAB-9574 for short-read RNA-seq data and E-MTAB-10797 for long-read RNA-seq data. RNA-seq data from Bediaga et al.55 can be download from https://doi.org/10.1038/s41598-020-80165-9. RNA-seq data from Buratin et al.56 can be downloaded from https://doi.org/10.1182/bloodadvances.2020002337. RNA-seq data from mESCs from the ENCODE Project Consortium can be downloaded from https://doi.org/10.1038/nature11247. ChIP–seq data from the ENCODE Project Consortium can be downloaded from https://doi.org/10.1038/s41467-020-14743-w. All sequencing datasets are listed in Supplementary Table 12. Source data are provided with this paper.

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Acknowledgements

We are grateful to A. Lanzavecchia, V. Costanzo, P. Della Bona and C. Lanzuolo for discussions and critical revision of the manuscript. We acknowledge the scientific and technical assistance of the INGM Imaging Facility, in particular, C. Cordiglieri and A. Fasciani (Istituto Nazionale di Genetica Molecolare ‘Romeo ed Enrica Invernizzi’ (INGM), Milan, Italy). This work has been supported by the following grants: the Fondazione Cariplo (Bando Giovani, grant no. 2018-0321 to F.M.), the Fondazione AIRC under 5 per Mille 2018 (ID, AIRC 5x1000 programs: project 21147 ‘ISM’ and ID 21091 to S.A.), the Fondazione Regionale per la Ricerca Biomedica (FRRB CP2_12/2018) and the Fondazione Cariplo (grant no. 2019-3416 to B.B.).

Author information

Authors and Affiliations

Authors

Contributions

F.M. designed and performed experiments, analyzed and interpreted data and wrote and finalized the manuscript. S. Sinha designed and performed bioinformatic experiments, analyzed data and revised the manuscript. R.V. set up, performed and analyzed experiments. B.P. set up the strategy for LINE1-containing transcript selection, performed RNA-seq, ChIP–seq and Nanopore long-read sequencing analyses. V.R. supervised bioinformatic analyses and revised the manuscript. E.M.P. performed NGS sequencing. F.V.B. and M.G. performed experimental validations. M.C. supervised flow cytometry sorting and immunological assays. M.L.N. performed ChIP experiments. S.C. provided technical support for TIL isolation from tissues. S.N. supervised immunological assays of TILs. A.S.-B. and S. Siena provided tumor samples. D.P. provided buffy coats. G.M. provided blood samples from patients with kidney transplantation. G.V. provided tumor samples. O.T. and S.H. provided blood samples from patients with LAM. R.G. provided technical support for TIL isolation from tissues. G.S. conceived the protocol for RNA-seq library preparation. S.B. conceived the study of mTORC1 signaling, analyzed experiments and revised the manuscript. B.B. and S.A. conceived and conceptualized the study, designed experiments, analyzed and interpret data and wrote and finalized the manuscript.

Corresponding authors

Correspondence to Sergio Abrignani or Beatrice Bodega.

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Nature Genetics thanks Musa Mhlanga and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 LINE1 RNAs are enriched in open chromatin regions in naïve CD4+ T cells.

(a) Sorting of naïve and memory CD4+ and CD8+ T cells, TH1, TH2 and TH17 CD4+ T helper subsets from blood samples. (b) Confocal fluorescence microscopy images of Alu RNA FISH (red) of naïve and memory CD4+ and CD8+ T cells. Original magnification 63×. Scale bar 5 µm. (c) Quantification of Alu RNA FISH, at least 220 nuclei, four individuals. *** P < 1 x 10-15, One-way ANOVA, F = 47.5. (d) Alu expression by RT-qPCR in quiescent naïve and memory TH1, TH2 and TH17 CD4+ T cells and in quiescent naïve and memory CD8+ T cells, (n = 6 individuals for naïve CD4+: n = 5 for TH1 and CD8+ subsets, n = 4 for TH2 and TH17). For box-and-whisker plots, the central line, box and whiskers represent the median, interquartile range (IQR) from first to third quartiles, and 1.5 × IQR, respectively. (e) Confocal fluorescence microscopy images of HERV RNA FISH (red) performed on quiescent naïve and memory CD4+ and CD8+ T cells. Original magnification 63×. Scale bar 5 µm. (f) Quantification of HERV RNA FISH, at least 164 nuclei, three individuals. (g) HERV expression by RT-qPCR in quiescent naïve and memory TH1, TH2 and TH17 CD4+ T cells and in quiescent naïve and memory CD8+ T cells, (n = 10 individuals for naïve and TH1 CD4+, n = 6 TH17; n = 5 for TH2 and CD8+ subsets). (h) Expression of 18S and Xist, in cytosol, nucleoplasm and chromatin of naïve CD4+ T cells (n = 3 individuals). Data are represented as mean ± s.e.m. (i) Confocal fluorescence microscopy images of LINE1 RNA FISH (red) and immunofluorescent staining (gray) for H3K4me3 and H3K9me3 of naïve CD4+ T cells. Original magnification 63×. Scale bar 5 µm. (j) Pearson correlation of colocalization relative to (i), at least 103 nuclei, three individuals. H3K9me3 versus H3K4me3 colocalization: *** P < 1 x 10-15, Two-tailed Mann Whitney test. (k-l) Naïve CD4+ T cells were activated with TCR engagement and differentiated to (k) TH2 or (l) TH17. LINE1 expression was analyzed by RT-qPCR, (n = 4 individuals). For box-and-whisker plots, the central line, box and whiskers represent the median, interquartile range (IQR) from first to third quartiles, and 1.5 × IQR, respectively. (m) LINE1 expression by RT-qPCR in 72 hours activated naive CD4+ T treated with signaling pathway inhibitors (n = 4 individuals). Control versus rapamycin: * P = 0.02678 Two-tailed paired t test. For box-and-whisker plots, the central line, box and whiskers represent the median, interquartile range (IQR) from first to third quartiles, and 1.5 × IQR, respectively. (n) Phosphorylated S6 protein (pS6) levels by western blot in 72 hours activated naïve CD4+ T treated with rapamycin or CsA. (o) Confocal fluorescence microscopy images of LINE1 RNA FISH (red) of naive CD4+ T cells that were activated for 72 hours and then treated with rapamycin or CsA. Original magnification 63×. Scale bar 10 µm.

Source data

Extended Data Fig. 2 Human CD4+ T cells express evolutionarily old LINE1 elements, whereas mESCs express evolutionarily young LINE1.

(a) Heatmap of TEs expression at class, family and subfamily level in chromatin and nucleoplasm RNA-seq of naïve CD4+ T cells, four individuals are plotted. Z-score was computed on the log2 transformed normalized read count using DESeq2. (b) Scatter plot of LINE1 subfamilies expressions in nucleoplasm (x-axis) and chromatin (y-axis) RNA-seq of naïve CD4+ T cells. Subfamilies are color coded based on evolutive origin: mammalian-specific (L1M, orange), primate-specific (L1P, blue), human-specific (L1Hs, green), HAL (yellow). (c) Heatmap of TEs expression at class, superfamily and subfamily level in mESCs RNA-seq, two biological replicates are plotted. Z-score was computed on the log2 transformed normalized read count using DESeq2 (d) Pie-chart representing distribution of LINE1 chimeric and not chimeric reads in human CD4+ T cells. (e) Pie chart representing genomic distribution of LINE1 reads among intergenic regions, protein coding, lncRNAs, pseudogenes and noncoding RNAs transcriptional units in human CD4+ T cells. (f) Pie-chart representing distribution of LINE1 chimeric and not chimeric reads in mESCs. (g) Pie chart representing genomic distribution of LINE1 reads among intergenic regions, protein coding, lncRNAs, pseudogenes and noncoding RNAs transcriptional units in mESCs.

Extended Data Fig. 3 Validation of LINE1-containing transcripts by long reads sequencing and by RT-PCR.

(a) Examples of long reads covering the LINE1-containing transcripts reconstructed by de novo transcriptome assembly. (b) Scheme of canonical transcripts and LINE1-containing transcripts; the LINE1 exon is represented in red. In the middle, schemes of the PCR primers designed to verify the presence of the LINE1-containing transcripts and canonical transcripts are reported. Below, agarose gels representing RT-PCR results of LINE1-containing transcripts. PCRs have been repeated on three individuals obtaining same results.

Source data

Extended Data Fig. 4 Characterization of LINE1 elements spliced in novel exons of non-canonical splicing variants.

(a) Length distribution of the LINE1 elements spliced in the LINE1-containing transcripts. (b) Distribution and enrichment of the expressed LINE1 regions in respect to a full length LINE1 sequence (6kb). Primers used for LINE1 RT-qPCR, probes for LINE1 RNA FISH and antisense oligonucleotide (ASOs) for LINE1 knock down experiments are shown. Right, percentage of the expressed LINE1 elements complementary to ORF1, ORF2, 5′UTR and 3′UTR regions of the full length LINE1. (c) Bar plot showing the percentage of the most enriched LINE1 subfamilies in the LINE1-containing transcripts. (d) LINE1 distribution among introns, exons, promoters, 5′UTR and 3′UTR of the LINE1 containing protein coding genes. (e) Consensus motifs of the donor and acceptor splicing sites of the LINE1 exons. (f) Scheme of ARPC2.L1 transcript. The novel exon containing LINE1 element is zoomed. Long reads (blue) and short reads (green) of chromatin RNA-seq that support the novel exon reconstruction are shown.

Extended Data Fig. 5 Upon T cell activation, LINE1-containing transcripts are downregulated while canonical transcripts are upregulated.

(a) Expression of the canonical transcripts and of three random set of control genes that i) do not retain genomic LINE1 elements (control genes no LINE1) or ii) retain LINE1 elements but do not generate LINE1-containing transcripts (control genes with LINE1) in RNA-seq datasets of quiescent and activated naïve CD4+ T cells (n = 3 individuals). *** P < 0.001, Two-tailed Wilcoxon signed rank test for paired samples, corrected accordingly to Bonferroni for multiple testing, was performed to compare canonical transcripts with control genes in naïve (P values from left to right: 1.89 x 10-12, 6.42 x 10-13, 1.91 x 10-12, 1.27 x 10-13, 1.04 x 10-12, 3.13 x 10-10) or activated T cells (P values from left to right: 3.06 x 10-07, 3.20 x 10-08, 1.15 x 10-06, 6.41 x 10-11, 1.55 x 10-07, 4.98 x 10-05). For box-and-whisker plots, the central line, box and whiskers represent the median, interquartile range (IQR) from first to third quartiles, and 1.5 × IQR, respectively. (b) Expression of LINE1-containing transcripts and canonical transcripts by RT-qPCR in quiescent and 16 hours activated naïve CD4+ T cells (n = 3 individuals). Data are represented as mean ± s.e.m. LINE1-containing transcripts in naïve versus activated CD4+ T cells: ** P = 0.0096, F = 9.647, Two-way ANOVA; canonical transcripts in naïve versus activated CD4+ T cells: *** P = 0.00045, F = 22.85, Two-way ANOVA. (c) IRF4 Transcription Factors (TFs) binding motif research was performed on promoter regions of the LINE1-containing genes, selecting TFs upregulated in CD4+ naïve T cells. (d) IRF4 expression by RT-qPCR in quiescent and 16 hours activated CD4+ T cells (n = 3 individuals). Data are represented as mean ± s.e.m. naïve versus activated CD4+ T cells: * P = 0.03975, Two-tailed paired t test. (e) IRF4 expression by RT-qPCR and (f) IRF4 protein by FACS analysis in quiescent naïve CD4+ T cells treated with IRF4 or control (scr) ASOs. Data are represented as mean ± s.e.m (n = 3 individuals). scr versus IRF4 ASO groups: ** P = 0.0042, Two-tailed paired t test.

Extended Data Fig. 6 LINE1-containing transcripts keep the expression of the corresponding canonical transcripts paused.

(a) Scheme of RAB22A.L1 knock down experiments in quiescent naïve CD4+ T cells. (b) Expression of RAB22A.L1 and of ARPC2, IFNGR2, EED, HIRA, ASH2L, RAB22A canonical transcripts in quiescent naïve CD4+ T cells treated with RAB22A.L1 or control (scr) ASOs (n = 6 individuals). RAB22A.L1 in scr versus RAB22A.L1 ASO groups: ** P = 0.00798, Two-tailed paired t test; RAB22A in scr versus RAB22A.L1 ASO groups: ** P = 0.0041, Two-tailed paired t test. (c) Design of the L1MC5a element deletion in ARPC2 gene with CRISPR-Cas9 RNP complexes: the intronic L1MC5a element was deleted using sgRNAs complementary to the non-repetitive regions flanking the repeat. (d) Expression of ARPC2.L1 and of ARPC2, IFNGR2, EED, HIRA, ASH2L, RAB22A canonical transcripts in quiescent naïve CD4+ T cells nucleofected with control Cas9 RNPs – or Cas9-RNPs with sgRNAs targeting L1MC5a in ARPC2 (see Supplementary Table 6) (n = 5 individuals). Data are represented as mean ± s.e.m. ARPC2.L1 in control versus L1MC5a Cas9 RNPs groups * P = 0.0201, Two-tailed paired t test; ARPC2 in control versus L1MC5a Cas9 RNPs groups * P = 0.0310, Two-tailed paired t test. (e) ARPC2 protein by FACS analysis in quiescent naïve CD4+ T cells nucleofected with control Cas9 RNPs – or Cas9-RNPs with sgRNAs targeting L1MC5a in ARPC2 gene and then activated (n = 3 individuals). Data are represented as mean ± s.e.m. ARPC2 in control versus groups L1MC5a Cas9 RNPs * P = 0.0273, Two-tailed paired t test. (f-g) Sanger sequencing of the PCR amplification products performed to assess L1MC5 and L1MC5a deletion from IFNGR2 and ARPC2. PCRs have been performed on four individuals in (f) and on five individuals in (g).

Source data

Extended Data Fig. 7 LINE1-containing transcripts are bound by Nucleolin and regulate H3K36me3 levels in naïve CD4+ T cells.

(a-b) RIP assays in naïve CD4+ T cells with anti NCL antibody (n = 3 individuals). Data are represented as mean % of input ± s.e.m. (c) Co-IP and western blots of NCL and KAP1 in CD4+ T cells. Co-IP has been repeated on two individuals obtaining similar results. (d) NCL expression by RT-qPCR and (e) NCL western blot in quiescent naïve CD4+ T cells treated with NCL or control (scr) ASOs. Data are represented as mean ± s.e.m (n = 4 individuals). NCL expression in scr versus NCL ASO groups: * P = 0.0482 Two-tailed paired t test. (f) Expression of GAPDH and MALAT1 in cytoplasm, nucleoplasm and chromatin of naïve CD4+ T cells treated with NCL or scr ASOs (n = 3 individuals). Data are represented as mean ± s.e.m. (g) LINE1 expression by RT-qPCR in quiescent naïve CD4+ T cells treated with LINE1 or scr ASOs (n = 8 individuals). LINE1 ASOs were designed to target the ORF2 LINE1 region included in the LINE1-containing transcripts (see Extended Data Fig. 4b). Data are represented as mean ± s.e.m. Scr versus LINE1 ASO groups: *** P = 3 x 10-6, Two-tailed paired t test. (h) Confocal fluorescence microscopy images of LINE1 RNA FISH (red) of quiescent naïve CD4+ T cells treated with LINE1 or control (scr) ASOs. LINE1 ASOs target the ORF2 LINE1 region of LINE1-containing transcripts. Original magnification 63×. Scale bar 5 µm. (i) Quantification of (h), at least 832 nuclei, two individuals. scr versus LINE1 ASO groups: *** P < 1 x 10-15, Two-tailed Mann Whitney test. (j) Confocal fluorescence microscopy images of LINE1 RNA FISH (red) and immunofluorescent staining (gray) for H3K36me3 or H3K4me3 of naïve CD4+ T cells treated with LINE1 or control (scr) ASOs. Original magnification 63×. Scale bar 5 µm. (k) Quantification of (j), at least 267 nuclei, two individuals. H3K36me3 in scr versus LINE1 ASO groups: *** P < 1 x 10-15, Two-tailed Mann-Whitney test. (l) Confocal fluorescence microscopy images of LINE1 RNA FISH (red) and immunofluorescent staining (gray) for H3K36me3 of quiescent naïve CD4+ T cells treated with NCL ASOs or control (scr) ASOs. Original magnification 63×. Scale bar 5 µm. (m) Quantification of (l), at least 259 nuclei, three individuals. H3K36me3 in scr versus LINE1 ASO groups: *** P < 1 x 10-15, Two-tailed Mann Whitney test.

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Extended Data Fig. 8 Controls for ChIP-seq and for PTBP1 and GTF2F1 knock down.

(a-b) Representative H3K36me3 ChIP-seq tracks for (a) ERGIC2 LINE1 containing gene and (b) FUCA2 control gene in quiescent and 16 hours activated naïve CD4+ T cells and naïve CD4+ T cells treated with LINE1 ASOs. (c-d) ChIP assays for H3K36me3 at LINE1 containing genes and HECW1 (control) in (c) quiescent naïve CD4+ T cells treated with LINE1 or control (scr) ASOs and in (d) quiescent and 16 hours activated naïve CD4+ T cells (n = 3 individuals). Data are represented as mean % of input ± s.e.m. H3K36me3 ChIP in scr versus LINE1 ASO groups: *** P = 6.2 x 10-7, F = 90.24, Two-way ANOVA. H3K36me3 ChIP in naïve CD4+ versus activated CD4+: *** P = 3.6 x 10-4, F = 40.45 Two-way ANOVA. (e) PTBP1 expression by RT-qPCR and (f) PTBP1 protein by FACS analysis in quiescent naïve CD4+ T cells treated with PTBP1 or scr ASOs and then activated for 16 hours. Data are represented as mean ± s.e.m (n = 3 individuals). PTBP1 expression in scr versus PTBP1 ASO groups: ** P = 0.0014 Two-tailed paired t test. (g) GTF2F1 expression by RT-qPCR and (h) GTF2F1 protein by western blot in quiescent naïve CD4+ T cells treated with GTF2F1 or scr ASOs and then activated for 16 hours. Data are represented as mean ± s.e.m (n = 3 individuals). GTF2F1 expression in scr versus GTF2F1 ASO groups: ** P = 0.0031, Two-tailed paired t test.

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Extended Data Fig. 9 IRF4 and Nucleolin are overexpressed in in vitro exhausted CD4+ and CD8+ T cells while GTF2F1 is downregulated.

(a) Scheme of in vitro exhaustion of CD4+ T cells. (b) On the left, cell count of effector and exhausted CD4+ T cells (n = 5 individuals). Data are represented as mean ± s.e.m, *** P < 2.7 x 10-7, F = 57.22 Two-way ANOVA. In the middle, PD-1 positive cells (n = 4 individuals). Data are represented as mean ± s.e.m, *** P = 0.00004, F = 40.3 Two-way ANOVA. On the right, IFN-γ positive cells (n = 4 individuals). Data are represented as mean ± s.e.m. IFN-γ: * P = 0.0319, One-tailed paired t test. (c) Scheme of in vitro exhaustion of CD8+ T cells. (d) On the left, cell count of effector and exhausted CD8+ T cells. Data are mean and ± s.e.m, N = 4 individuals. *** P = 0.0003, F = 26.05 Two-way ANOVA. In the middle, PD-1 positive cells. Data are mean and ± s.e.m, N = 4 individuals. *** P = 0.00003, F = 58.1, Two-way ANOVA. On the right, IFN-γ, GrzB and PerfA positive cells. Data are mean and ± s.e.m, N = 4 individuals. IFN-γ: * P = 0.0108, Two-tailed Paired t test; GrzB * P = 0.0243, Two-tailed Paired t test. (e) IRF4, NCL, GTF2F1 and PTBP1 by western blot performed in nuclear extracts of effector and exhausted CD4+ and CD8+ T cells. Data are represented as mean ± s.e.m (n = 2 individuals). (f) RIP assays for NCL, PTBP1 and GTF2F1 in effector and exhausted CD8+ T cells (n = 3 individuals). Data are represented as mean % of input ± s.e.m. NCL RIP in effector versus exhausted: * P = 0.0488, F = 5.54, Two-way ANOVA. (g-h) LINE1-containing transcripts and canonical transcripts expression by RT-qPCR in exhausted CD8+ T cells treated with (g) IRF4 or control (scr) ASOs or with (h) NCL or scr ASOs (n = 3 individuals). Data are represented as mean ± s.e.m. LINE1-containing transcripts in scr versus IRF4 ASO groups: *** P = 0.0004, F = 50.6. Two-way ANOVA; canonical transcripts in scr versus IRF4 ASO groups: ** P = 0.0024, F = 25.3. Two-way ANOVA. Data are represented as mean ± s.e.m. Canonical transcripts in scr versus NCL ASO groups: ** P = 0.0026, F = 24.5. Two-way ANOVA.

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Extended Data Fig. 10 LINE1-containing transcripts regulate effector functions of exhausted CD4+ and CD8+ T cells.

(a) Sorting of tumor-infiltrating memory CD4+ and CD8+ T cells. (b-c) LINE1 expression by RT-qPCR in memory CD4+ (a) and CD8+ (b) TILs treated with LINE1 or control (scr) ASOs (n = 2 individuals). Data are represented as mean ± s.e.m. (d) Scheme of the immunological assays performed on in vitro exhausted CD4+ and CD8+ T cells treated with LINE1 or scr ASOs to evaluate their effector properties (g-l). (e-f) LINE1 expression by RT-qPCR in exhausted (e) CD4+ and (f) CD8+ T cells (n = 4 individuals). Data are represented as mean ± s.e.m. scr versus LINE1 ASO exhausted CD4+: ** P = 0.0083, Two-tailed paired t test; scr versus LINE1 ASO exhausted CD8+: ** P = 0.0073, Two-tailed paired t test. (g) IFN-γ+ or GrzB+ exhausted CD4+ T cells (n = 4 individuals). Data are represented as mean ± s.e.m. IFN-γ in scr versus LINE1 ASO groups: * P = 0.0351 One-tailed paired t test. (h) IFN-γ+, GrzB+ or PerfA+ exhausted CD8+ T cells (n = 4 individuals). Data are represented as mean ± s.e.m. IFN-γ in scr versus LINE1 ASO groups: ** P = 0.0016, Two-tailed paired t test; GrzB in scr versus LINE1 ASO groups: ** P = 0.0039, PerfA in scr versus LINE1 ASO: *** P = 0.0002, Two-tailed paired t test. (i-j) Percentage of dead heterologous monocytes co-cultured for 12 hours with exhausted (i) CD4+ or (j) CD8+ T cells. Percentage of monocytes self-lysis is indicated (w/o T cells, in blue). n = 3 individuals for CD4+ and n = 4 individuals for CD8+, data for each individual are shown separately, monocytes self-lysis is represented as mean ± s.e.m. CD4+ scr versus LINE1 ASO: ** P = 0.0092, Two-tailed paired t test; CD8+ scr versus LINE1 ASO: ** P = 0.0084, Two-tailed paired t test. (k-l) Proliferation assay with cell trace in exhausted (j) CD4+ or (k) CD8+ T cells. (m) Scheme of how LINE1-containing transcripts control gene expression in T cell quiescence and exhaustion.

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Marasca, F., Sinha, S., Vadalà, R. et al. LINE1 are spliced in non-canonical transcript variants to regulate T cell quiescence and exhaustion. Nat Genet 54, 180–193 (2022). https://doi.org/10.1038/s41588-021-00989-7

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