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CRISPR screens unveil signal hubs for nutrient licensing of T cell immunity

Abstract

Nutrients are emerging regulators of adaptive immunity1. Selective nutrients interplay with immunological signals to activate mechanistic target of rapamycin complex 1 (mTORC1), a key driver of cell metabolism2,3,4, but how these environmental signals are integrated for immune regulation remains unclear. Here we use genome-wide CRISPR screening combined with protein–protein interaction networks to identify regulatory modules that mediate immune receptor- and nutrient-dependent signalling to mTORC1 in mouse regulatory T (Treg) cells. SEC31A is identified to promote mTORC1 activation by interacting with the GATOR2 component SEC13 to protect it from SKP1-dependent proteasomal degradation. Accordingly, loss of SEC31A impairs T cell priming and Treg suppressive function in mice. In addition, the SWI/SNF complex restricts expression of the amino acid sensor CASTOR1, thereby enhancing mTORC1 activation. Moreover, we reveal that the CCDC101-associated SAGA complex is a potent inhibitor of mTORC1, which limits the expression of glucose and amino acid transporters and maintains T cell quiescence in vivo. Specific deletion of Ccdc101 in mouse Treg cells results in uncontrolled inflammation but improved antitumour immunity. Collectively, our results establish epigenetic and post-translational mechanisms that underpin how nutrient transporters, sensors and transducers interplay with immune signals for three-tiered regulation of mTORC1 activity and identify their pivotal roles in licensing T cell immunity and immune tolerance.

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Fig. 1: Genome-wide CRISPR screening uncovers mTORC1 regulatory networks in Treg cells.
Fig. 2: SEC31A is crucial for nutrient and GATOR2-dependent mTORC1 activation and the abundance of SEC13.
Fig. 3: SEC31A protects SEC13 from SKP1-mediated proteasomal degradation.
Fig. 4: The SAGA complex suppresses nutrient transporter expression and mTORC1 activation.
Fig. 5: Steady state and tumour challenge phenotypes of Foxp3CreCcdc101fl/fl mice.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information. All microarray, ATAC-seq and scRNA-seq data described in the manuscript have been deposited in the NCBI Gene Expression Omnibus (GEO) database and are accessible through the GEO SuperSeries accession number 160598. Other resources: CRAPome database (https://reprint-apms.org/); Uniprot mouse database (https://www.uniprot.org/); STRING (v10) (https://string-db.org/); BioPlex (https://bioplex.hms.harvard.edu/). Source data are provided with this paper.

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Acknowledgements

We acknowledge M. Hendren and S. Rankin for animal colony management; J. Wen for help with phenotypic studies; C. Li and S. Zhou for technical and scientific insights; G. Neale and S. Olsen for assistance with sequencing; the St. Jude Immunology FACS core facility for cell sorting; and the St. Jude Communications and Science/Medical Content Outreach for artwork. This work was supported by ALSAC and by US National Institutes of Health grants R01AG053987 (to J.P.) and AI105887, AI131703, AI140761, AI150241, AI150514, CA250533 and CA253188 (to H.C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

Authors and Affiliations

Authors

Contributions

L.L. and J.W. conceived the project, designed and performed in vitro and in vivo experiments, analysed data and wrote the manuscript. S.A.L. performed tumour and scRNA-seq experiments. J.L.R. performed Seahorse experiments. H.S., I.R., Y.L. and Y.D. performed bioinformatic analyses. J.P.C. and S.M.P.-M. designed and generated the focused sgRNA library. H.W., B.X., M.N. and J.P. performed proteomics. C.G. performed imaging experiments. N.M.C., Y.W., H.H., W.S., A.K. and P.Z. helped with immunological experiments. G.F. performed LCMV infection experiments. J.S. helped with ATAC-seq sample preparation. Y.-D.W. and J.Y. analysed CRISPR–Cas9 screening data. P.V. provided histological analysis. H.C. helped to design experiments, co-wrote the manuscript, and provided overall direction.

Corresponding author

Correspondence to Hongbo Chi.

Ethics declarations

Competing interests

H.C. is a consultant for Kumquat Biosciences.

Additional information

Peer review information Nature thanks Jeffrey Rathmell and the other, anonymous, reviewers for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Two rounds of pooled CRISPR screening to identify novel regulators of nutrient and mTORC1 signalling.

Related to Fig. 1. (a) Flow cytometry analysis of IFNγ, IL-4, IL-17A or FOXP3 expression in cells cultured in TH0-, TH1-, TH2-, TH17- or induced Treg-polarizing condition (n = 3 samples each group). (b) Flow cytometry analysis of pS6 in TH1 and Treg cells with TCR stimulation for 0 or 1 h (n = 3 samples each group). (c) Induced Treg cells were stimulated with anti-CD3 and anti-CD28 in the presence or absence of amino acids (AA) or glucose for 3 h followed by flow cytometry analysis and quantification of pS6 level [based on mean fluorescence intensity (MFI)] (n = 3 samples each group). (d) Induced Treg cells were labelled with CellTrace Violet (CTV) and stimulated with anti-CD3 and anti-CD28 in the presence or absence of AA or glucose for 3 d, followed by flow cytometry analysis of CTV dilution (n = 3 samples each group). (e) Gating strategy used for sorting cells with the ≥ 10% highest (pS6hi) and ≤ 10% lowest (pS6lo) levels after stimulation with 0.25 or 4 μg–1 of anti-CD3 for 3 h (n = 2 samples each group). Mean ± s.e.m. (c). ***P < 0.001; one-way ANOVA (c). Data are representative of two (bd) or three (a) experiments.

Source data

Extended Data Fig. 2 Validation of individual candidate mTORC1 regulators.

Related to Fig. 1. (a) Flow cytometry analysis and quantification of pS6 level [based on mean fluorescence intensity (MFI)] in naïve or activated WT and Depdc5-deficient CD4+Foxp3 T cells (n = 4 samples each group). Naïve CD4+ T cells among freshly isolated splenocytes from WT and Cd4CreDepdc5fl/fl mice were gated (indicated as TCR 0 h), or naïve CD4+ T cells were sorted and stimulated with anti-CD3 and anti-CD28 overnight for flow cytometry analysis of pS6 level. (b) Quantification of relative pS6 level in induced Treg cells transduced with sgNTC, sgSec13, sgMios, sgSeh1l, or sgWdr24 (all Ametrine+) were stimulated with TCR for 3 h (n = 3 samples each group). (c) Diagram of dual-colour co-culture system to examine cell-intrinsic effects of deletion of a candidate gene on TCR-induced pS6, cell size and CD71 expression. Specifically, Cas9+ cells transduced with sgNTC (mCherry+ or GFP+; ‘spike’) were mixed with those transduced with those targeting a specific gene (Ametrine+), and stimulated with anti-CD3 for 3 h (for pS6) or with anti-CD3 and anti-CD28 for 20 h (for cell size and CD71). (d) Validation of dual-colour co-culture system by using two sgNTC-expressing vectors with different fluorophores. Cells transduced with sgNTC (GFP+; ‘spike’) were mixed with those transduced with sgNTC (Ametrine+), and stimulated with anti-CD3 for 3 h to examine pS6 (see phos-flow staining), or stimulated with anti-CD3 and anti-CD28 for 20 h (n = 3 samples each group) to measure cell size and CD71 expression (see surface staining). (e) Heat map summary of log2 (pS6hi/pS6lo) for individually validated candidate genes (63 positive and 21 negative regulators) including positive (Rheb, Rptor, Lamtor3, Rraga and Mtor) and negative (Cd5, Nprl3, Nprl2 and Tsc1) control genes (2 sgRNAs for each candidate). Specifically, Cas9-expressing CD4+ T cells transduced with sgRNA for target genes (Ametrine+) or non-targeting control sgRNA (sgNTC) (mCherry+; ‘spike’) were mixed and differentiated into induced Treg cells. These cells were then stimulated with anti-CD3 for 3 h (n = 3 samples each group). Relative pS6 level (normalized to ‘spike’) was analysed by flow cytometry. (f) Analysis of protein–protein interaction (PPI) networks of high-confidence regulators. Specifically, 286 positive and 60 negative high-confidence hits were integrated with the composite PPI databases that encompass STRING, BioPlex and InWeb_IM databases for the inference of functional modules. Red and blue circles represent genes the deletion of which represses and promotes mTORC1 activity, respectively. Mean ± s.e.m. (a, b). *P < 0.05; **P < 0.01; ***P < 0.001; one-way ANOVA (a, b). Data are representative of one (f) or three (d, e), or pooled from two (a) or three (b) experiments.

Source data

Extended Data Fig. 3 SWI/SNF complex represses expression of nutrient sensor CASTOR1 to support mTORC1 activation.

Related to Fig. 1. (a) Quantification (normalized to ‘spike’) of relative pS6 level, cell size (FSC-A) and CD71 expression in induced Treg cells transduced with the indicated sgRNAs followed by stimulation with anti-CD3 for 3 h to measure pS6 level, or with anti-CD3 and anti-CD28 for 20 h to measure cell size (FSC-A) and CD71 expression by flow cytometry (n = 3 samples each group). (b) Imaging analysis and quantification of lysosome-associated mTOR [based on mean fluorescence intensity (MFI)] in sgNTC- or sgSmarcb1-transduced cells that were stimulated with anti-CD3 for 3 h, starved of amino acids (AA), and refed AA for 20 min (n > 230 cells per condition). Scale bars, 5 μm.  (c) Volcano plots of expression levels of transcripts, including Castor1, in sgNTC- or sgSmarcb1 (both Ametrine+)-transduced cells that were stimulated with TCR for 3 h (n = 4 samples each group). (d) Castor1 mRNA expression in sgNTC- or sgSmarcb1 (both Ametrine+)-transduced cells were stimulated with anti-CD3 for 3 h or with anti-CD3 and anti-CD28 for 20 h (n = 3 samples per group). (e) sgNTC- or sgSmarcb1 (both Ametrine+)-transduced cells were left unstimulated (indicated by 0 h) or stimulated with anti-CD3 for 3 h or anti-CD3 and anti-CD28 for 20 h. Immunoblot analysis and quantification of relative CASTOR1 expression (n = 3 samples each group). (f) Immunoblot analysis and quantification of relative pS6K1 and pS6 levels in cells transduced with empty vector or vector expressing Castor1, followed by stimulation with anti-CD3 and anti-CD28 for 2 d (n = 3 samples each group). Mean ± s.e.m. (a, b, df). **P < 0.01; ***P < 0.001; two-tailed unpaired Student’s t-test (f); one-way ANOVA (a, b, d, e). Data are representative of one (c) or two (b), or pooled from two (d, e) or three (a, f) experiments

Source data.

Extended Data Fig. 4 SEC31A is required for mTORC1 activation.

Related to Fig. 2. (a) Interaction of endogenous SEC13 with SEC31A in induced Treg cells as assessed by immunoprecipitation (IP)–immunoblot analysis. (b, c) sgNTC- or sgSec31a-transduced cells were starved of and refed amino acids (AA, b) or glucose (c) for 20 min, followed by immunoblot analysis of SEC31A, pS6K1, pS6 and β-actin. Bottom, quantification of relative pS6K1 and pS6 levels (n = 3 samples each group). (d) Cells transduced with the indicated sgRNAs (all Ametrine+) were mixed with sgNTC (mCherry+; ‘spike’)-transduced cells, and stimulated with anti-CD3 for 3 h to measure pS6 level or with anti-CD3 and anti-CD28 for 20 h to measure cell size (FSC-A) and CD71 expression by flow cytometry (normalized to ‘spike’) (n = 3 samples each group). (eg) sgNTC-, sgSec31a- or sgSec13 (all Ametrine+)-transduced cells were co-transduced with constitutively active RAGAQ66L (CA-RAGA)-expressing retrovirus (GFP+) or sgNprl2 (GFP+), followed by stimulated with TCR for 3 h to examine relative pS6 level by flow cytometry (n = 3 samples each group) (e), pS6K1 and pS6 levels by immunoblot analysis (n = 4 samples each group) (f), or lysosomal localization of mTOR [based on mean fluorescence intensity (MFI)] (n > 700 cells per condition). Scale bars, 5 µm (g). In f, two different exposures for pS6K1 were included to account for the differential intensities between mock and CA-RAGA or sgNprl2 conditions, and relative pS6K1 level was quantified from long exposure for mock and short exposure for CA-RAGA or sgNprl2 conditions (middle). Bottom, quantification of pS6 level. Mean ± s.e.m. (bg). NS, not significant; **P < 0.01; ***P < 0.001; one-way ANOVA (bg). Data are representative of two (eg) or four (a), or pooled from three (bd) experiments.

Source data

Extended Data Fig. 5 SEC31A–SEC13 axis promotes mTORC1 activation and cell proliferation in vivo.

Related to Fig. 2. (a) Cells transduced with sgNTC or sgSec31a (both Ametrine+) were labelled with CellTrace Violet (CTV) and transferred into Rag1–/– mice. Flow cytometry analysis of CTV dilution, and quantification of percentage of proliferated (CTVlo) cells at 7 d after transfer (n = 5 samples each group). (b) WT, Rptor- and Sec31a-null Treg cells (all CD45.1+Ametrine+) were mixed with conventional CD4+ T cells (Tconv; CD45.2+) at a 1:4 ratio and transferred into Rag1–/– mice. Quantification of the accumulation of conventional T cells in the spleen at 7 d after transfer (n = 5 samples each group). (c) Naïve CD4+ T cells were stimulated with anti-CD3 and anti-CD28 for 0, 24, 48 or 72 h followed by immunoblot analysis of the indicated protein expression, and quantification of pS6K1, SEC13, SEC31A, and TSC2 (n = 3 samples each group). (d) sgNTC-, sgSec31a- and sgSec13-transduced cells were sorted and lysed with CHAPS buffer for immunoprecipitation (IP) with an antibody against WDR24. The immunoprecipitated proteins were analysed by immunoblot for WDR24, WDR59, MIOS, SEH1L, SEC13, SEC31A, SEC23A and β-Actin. Mean ± s.e.m. (ac). **P < 0.01; ***P < 0.001; two-tailed unpaired Student’s t-test (a); one-way ANOVA (b). Data are representative of one (a, b) or two (c, d) experiments.

Source data

Extended Data Fig. 6 SEC31A protects SEC13 from proteasomal degradation to sustain mTORC1 activation.

Related to Fig. 3. (a) Sec13 mRNA expression in sgNTC- or sgSec31a-transduced induced Treg cells (n = 3 samples each group). (b) sgNTC- or sgSec31a-transduced induced Treg cells were sorted and treated with cycloheximide (CHX) for the indicated times. Total protein extracts of cells transduced with sgNTC (5 μg) or sgSec31a (12.5 µg; more protein was loaded to equalize basal SEC13 amount between these cells) were used for immunoblot analysis and quantification of relative SEC13 abundance (n = 4 samples each group). (c) Naïve CD4+ T cells were stimulated with anti-CD3 and anti-CD28 for 0, 24, or 48 h and then treated with DMSO or MG132 at 48–72 h of stimulation, followed by immunoblot analysis and quantification of SEC13 and SEC31A expression (n = 3 samples each group). (d) HEK293T cells were transfected with HA-tagged SEC13 and 6× His-tagged WT-, K48R- or K63R-ubiquitin (Ub) and treated with MG132 for 6 h. Ni-nitrilotriacetic acid (Ni-NTA) bead-based pull-down and immunoblot analysis of HA-SEC13. Bottom, expression of indicated proteins in whole cell lysates (WCL). (e) sgNTC- or sgSec31a-transduced HEK293T cells were transfected with HA-tagged SEC13 and 6× His-tagged WT ubiquitin (His-tagged Ub), and treated with MG132 for 6 h. Left, Ni-NTA bead-based pull-down of His-tagged Ub-labelled proteins followed by immunoblot analysis for HA-SEC13. Right, immunoblot analysis of WCL for expression of endogenous SEC31A or HA-SEC13, His-tagged Ub, and β-Actin. Mean ± s.e.m. (ac). NS, not significant; **P < 0.01; ***P < 0.001; two-tailed unpaired Student’s t-test (a); two-way ANOVA (b); one-way ANOVA (c). Data are representative of two (d, e) or three (a), or pooled from two (b) or three (c) experiments.

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Extended Data Fig. 7 SEC31A protects SEC13 from SKP1-mediated proteasomal degradation and supports T cell functional fitness.

Related to Fig. 3. (a) HA-tagged WT or the indicated lysine mutant constructs of SEC13 were transfected into HEK293T cells individually. Immunoblot analysis of HA and Hsp90. (b) HA-tagged WT or K260R mutant SEC13 was transfected into HEK293T cells together with the K48-only His-Ub followed by MG132 treatment and anti-HA immunoprecipitation (IP). Immunoblot analysis for HA, His-Ub and β-actin. WCL, whole cell lysate. (c) Cas9-expressing CD4+ T cells were transduced with sgNTC or sgSec31a retrovirus (Ametrine+) together with WT or K260R mutant SEC13-expressing retrovirus (GFP+). Ametrine+GFPlo cells (see gating on flow cytometry plot, top) were stimulated with TCR for 0 or 3 h. Immunoblot analysis and quantification of relative SEC13 and pS6 levels (n = 4 samples each group). (d) Volcano plot of proteins, including SKP1, that interact with HA-SEC13 in induced Treg cells as identified by mass spectrometry (n = 3 samples each group). (e) Induced Treg cells transduced with HA-tagged-SEC13- or empty vector-expressing retrovirus were lysed with CHAPS buffer followed by anti-HA immunoprecipitation (IP) and immunoblot analysis of HA and SKP1. (f) Induced Treg cells were lysed with CHAPS buffer followed by immunoprecipitation of endogenous SKP1 and immunoblot analysis of SKP1 and SEC13. (g) Interaction of endogenous SKP1 with SEC13 in sgNTC- or sgSec31a-transduced cells. (h) Naïve CD4+ T cells were stimulated with anti-CD3 and anti-CD28 for 0, 24, 48 or 72 h. Immunoblot analysis of anti-SKP1 immunoprecipitants and WCL for SKP1, SEC13, and β-Actin (n = 2 samples per group). (i) Immunoblot analysis and quantification of relative expression of indicated proteins in sgNTC- or sgSkp1 (both Ametrine+)-transduced cells (n = 3 samples per group). (j) The indicated sgRNA-transduced cells were labelled with CellTrace Violet (CTV), and stimulated with anti-CD3 and anti-CD28 for 72 h, followed by flow cytometry analysis and quantification of CTV dilution (n = 3 samples each group). (k) Diagram of SMARTA T cell transfer and LCMV infection system. In brief, SMARTA–Cas9 CD4+ T cells (CD45.1+) transduced with sgRNA for candidate genes (CD45.1+Ametrine+) and mixed with sgNTC (CD45.1+mCherry+; ‘spike’)-transduced cells at a 1:1 ratio, and adoptively transferred into naïve (unchallenged; CD45.2+) mice that were left uninfected (see l) or challenged with LCMV infection (see Fig. 3f). (l) Quantification of the relative proportion (normalized to ‘spike’) of donor-derived (CD45.1+) T cells in the spleen of uninfected mice at 7 d after transfer (n = 6 mice per group). (m) Cells transduced with sgNTC (Ametrine+), sgSec31a (Ametrine+) or sgSec31a/Skp1 (GFP+ and Ametrine+) were sorted and stimulated with anti-CD3 and anti-CD28 for 20 h (n = 6-7 samples per group), followed by the measurement of extracellular acidification rate (ECAR). Oligo, oligomycin; FCCP, fluoro-carbonyl cyanide phenylhydrazone; Rot, rotenone. Mean ± s.e.m. (c, i, j, l, m). **P < 0.01; ***P < 0.001; one-way ANOVA (c, i, j, l, m, right); two-way ANOVA (m, left). Data are representative of one (d, j, l, m) or two (a, b, eh), or pooled from two (c) or three (i) experiments.

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Extended Data Fig. 8 The SAGA complex suppresses mTORC1 activation.

Related to Fig. 4. (a) Quantification (normalized to ‘spike’) of relative pS6 level, cell size (FSC-A) and CD71 expression in induced Treg cells transduced with sgRNA for Ccdc101 or Taf6l, followed by stimulation with anti-CD3 for 3 h to measure pS6 level (left) or with anti-CD3 and anti-CD28 for 20 h to measure cell size (FSC-A; middle) and CD71 (right) expression by flow cytometry (n = 3 samples each group). (b) Immunoblot analysis and quantification of relative pS6K1 and pS6 expression in sgNTC- or sgCcdc101-transduced cells that were starved of and refed amino acids (AA) for 20 min (n = 4 samples each group). Mean ± s.e.m. (a, b). *P < 0.05; **P < 0.01; ***P < 0.001; one-way ANOVA (a, b). Data are representative of three (a), or pooled from three (b) experiments.

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Extended Data Fig. 9 The SAGA complex represses the expression of nutrient transporters and mTORC1 activation.

Related to Fig. 4. (a) Heat map of differentially expressed genes in Ccdc101-null Treg cells stimulated with anti-CD3 and anti-CD28 for 0 (n = 3 samples each group) or 20 h (n = 4 samples each group). (b) Slc2a1, Slc16a10 or Slc43a1 mRNA expression in sgNTC- or sgCcdc101-transduced cells at steady state (n = 3 samples each group). (c) Immunoblot analysis and quantification of relative GLUT1 expression in sgNTC- or sgCcdc101-transduced cells that were stimulated with anti-CD3 and anti-CD28 for 0 or 20 h (n = 3 samples each group). (d) Flow cytometry analysis and quantification of 2-NBDG uptake in sgNTC- or sgCcdc101 (both Ametrine+)-transduced cells were stimulated with anti-CD3 and anti-CD28for 20 h (n = 4 samples each group). (e, f) Quantification of relative pS6 level in cells transduced with the indicated sgRNAs that were stimulated with TCR for 3 h (n = 3 samples each group). (g) Principal component analysis (PCA) of ATAC-seq for cells transduced with sgNTC (n = 4 samples) or sgCcdc101 (both Ametrine+) (n = 3 samples) and stimulated with anti-CD3 and anti-CD28 for 20 h. (h) Motif enrichment analysis of ATAC-seq of sgNTC- and sgCcdc101-transduced cells (n = 4 samples each group). (i) Footprinting analysis of Sp3 binding in ATAC-seq. (j) Accessibility of the Sp3 locus in sgNTC- and sgCcdc101-transduced cells as identified by ATAC-seq. Highlighted peaks in the red box indicate differential accessible regions. (k) Immunoblot analysis of SP3 expression in sgNTC- or sgCcdc101 (both Ametrine+)-transduced cells. Mean ± s.e.m. (bf). ***P < 0.001; two-tailed unpaired Student’s t-test (b, d); one-way ANOVA (c, e, f). Data are representative of one (a, gj), two (b, e, f, k), or pooled from two (c, d) experiments.

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Extended Data Fig. 10 The SAGA complex prevents mTORC1 hyperactivation to enforce immune homeostasis in vivo.

Related to Fig. 4. (a, b) Induced Treg cells transduced with the indicated sgRNAs were stimulated with anti-CD3 and anti-CD28 for 20 h (n = 3 samples each group). Flow cytometry analysis and quantification of staining with active caspase-3 (a) and fixable viability dye (FVD, b) (n = 3 per group). (c) Cells transduced with the indicated sgRNAs were stimulated with anti-CD3 and anti-CD28 for 20 h (n = 5–7 samples per group), followed by the measurement of extracellular acidification rate (ECAR). Oligo, oligomycin; FCCP, fluoro-carbonyl cyanide phenylhydrazone; Rot, rotenone. (d) Immunoblot analysis and quantification of CCDC101 expression in naïve CD4+ T cells from WT and Cd4CreCcdc101fl/fl mice (n = 3 mice per group). (e) Quantification of CD71 expression on naïve or activated WT and Ccdc101-deficient CD4+ T cells. Naïve CD4+ T cells among freshly isolated splenocytes from WT and Cd4CreCcdc101fl/fl mice (n = 4 mice per group) were gated (indicated as 0 h), or naïve CD4+ T cells were stimulated with anti-CD3 and anti-CD28 for 20 h. (f) Flow cytometry analysis and quantification of numbers of total, double-negative (DN), double-positive (DP), CD4 single-positive (CD4SP), and CD8 single-positive (CD8SP) thymocytes from WT and Cd4CreCcdc101fl/fl mice (n = 4 mice each group). (g) Flow cytometry analysis and quantification of proportions and numbers of splenic CD4+ and CD8+ T cells from WT and Cd4CreCcdc101fl/fl mice (n = 4 mice each group). (h) Flow cytometry analysis and normalized ratio of CD122+ versus CD122 cells among CD44hi populations (gated on splenic CD8+ T cells) from indicated mice (n = 4 mice each group). Mean ± s.e.m. (ah). NS, not significant; **P < 0.01; ***P < 0.001; two-tailed unpaired Student’s t-test (dh); one-way ANOVA (a, b, c, right); two-way ANOVA (c, left). Data are representative of one (c) or two (a, b), or pooled from three (dh) experiments.

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Extended Data Fig. 11 Treg-specific deletion of Ccdc101 disrupts immune homeostasis and boosts antitumour response.

Related to Fig. 5. (a) Quantification of relative pS6 level in splenic CD4+FOXP3+ cells from WT and Foxp3CreCcdc101fl/fl (around 8 weeks old) mice (n = 4 mice per group). (b) Quantification of relative FOXP3 expression (gated on splenic FOXP3+CD4+ T cells) from WT and Foxp3CreCcdc101fl/fl mice (n = 4 mice per group). (c) Quantification of percentages of effector/memory (CD44hiCD62Llo) subsets in splenic CD4+FOXP3 and CD8+ T cells from WT and Foxp3CreCcdc101fl/fl (around 8 weeks old) mice (n = 4 mice each group). (d) Representative flow cytometry analysis of IL-2+ or IFNγ+ population of splenic CD4+Foxp3 and CD8+ T cells from WT and Foxp3CreCcdc101fl/fl (around 8 weeks old) mice. (eg) WT and Foxp3CreCcdc101fl/fl mice were inoculated with MC38 colon adenocarcinoma cells. Treg cells (CD45+CD4+YFP+), non-Treg immune cells (CD45+YFPCD11b) and myeloid cells (CD45+CD11b+) were isolated and sorted from tumours, and mixed at a 1:2:1 ratio for scRNA-seq analysis (2 biological replicates, pooled from 3-4 mice each, per group) at 19 d after tumour inoculation. Dot plot showing the differentially expressed marker genes for 4 subclusters of CD8+ T cells in the MC38 tumours (e; see Methods for details). UMAP embeddings of CD8+ T cells grouped by genotype (f, left) and indicated subclusters (f, right). Frequencies of the indicated subclusters were quantified for each genotype (g). Teff-like, effector-like CD8+ T cells; Tex-like, exhaustion-like CD8+ T cells; Tcm-like, central memory-like CD8+ T cells; Tem-like, effector/memory-like CD8+ T cells. (h) Flow cytometry analysis and quantification of the percentages of CD44hiCD62Llo intratumoral CD8+ T cells from WT and Foxp3CreCcdc101fl/fl mice (n ≥ 5 per group). (i) Flow cytometry analysis and quantification of IFNγ+ and TNF+ cells among intratumoral CD8+ T cells from WT and Foxp3CreCcdc101fl/fl mice (n ≥ 5 per group). (j) Violin plots of scRNA-seq data depicting Icos, Tnfrsf18, Ctla4 and Ifng expression in intratumoral Treg cells. (k) Flow cytometry analysis and quantification of ICOS, GITR and CTLA-4 for intratumoral Treg cells from WT and Foxp3CreCcdc101fl/fl mice (n ≥ 5 per group). (l) Flow cytometry analysis and quantification of IFNγ expression in intratumoral Treg cells from WT and Foxp3CreCcdc101fl/fl mice (n ≥ 5 per group). Mean ± s.e.m. (ac, h, i, k, l). *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed unpaired Student’s t-test (ac, h, i, k, l); two-sided Wilcoxon rank sum test in j. Data are representative of one (el) or two (d), or pooled from two (ac) experiments.

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Extended Data Fig. 12 Schematic of applying CRISPR screening and integrative analyses to dissect nutrient and mTORC1 signalling in primary T cells.

Related to Fig. 5. With two rounds of genome-wide and focused CRISPR screenings, we identify 346 high-confidence mTORC1 signalling factors, including many novel activators and inhibitors, as well as known regulators (identified in other systems) that have not been studied in primary T cells. Notably, using analysis of protein–protein interaction (PPI) networks and unbiased functional and proteomic approaches, we further establish the epigenetic and post-translational mechanisms underpinning the three-tier regulatory modules of nutrient signalling, composed of nutrient transporters (e.g. via affecting expression of GLUT1 and other transporters by SAGA complex), sensors (e.g. via epigenetic regulation of CASTOR1 expression by SWI/SNF complex) and transducers (e.g. via shaping GATOR2 complex stability by SEC31A; regulating SEC13 ubiquitination at lysine 260), which transmit immunological and nutrient cues to mTORC1 signalling for proper regulation of T cell activity in vivo and in vitro.

Supplementary information

Supplementary Figure 1

This file contains Supplementary Fig. 1.

Reporting Summary

41586_2021_4109_MOESM3_ESM.xlsx

Supplementary Table 1 Data of the first-round genome-wide CRISPR screening via the Brie library. This file contains the output of the analysis of the first-round genome-wide Brie library CRISPR screening data at the gene level analysed by pipeline 1 (Tab a) and pipeline 2 (Tab b), or at the sgRNA level analysed by pipeline 1 (Tab c) and pipeline 2 (Tab d). The comparison (pS6high versus pS6low) for two stimulation conditions (0.25 and 4 μg ml−1 anti-CD3) is shown. Ranked by target gene symbol.

41586_2021_4109_MOESM4_ESM.xlsx

Supplementary Table 2 List of sgRNA sequence information in the second-round focused CRISPR library. This file contains the sgRNA sequences in the CRISPR sgRNA library used in the second-round focused CRISPR screening. Ranked by target gene symbol.

41586_2021_4109_MOESM5_ESM.xlsx

Supplementary Table 3 Data of the second-round focused CRISPR library screening. This file contains the output of the analysis of the second-round focused CRISPR library screening data at the gene level analysed by pipeline 1 (Tab a) and pipeline 2 (Tab b), or at the sgRNA level analysed by pipeline 1 (Tab c) and pipeline 2 (Tab d). The comparison (pS6high versus pS6low) for two stimulation conditions (0.25 and 4 μg ml−1 anti-CD3) is shown. Ranked by target gene symbol.

41586_2021_4109_MOESM6_ESM.xlsx

Supplementary Table 4 List of sgRNA sequence information. This file contains the sgRNA sequences used for validation in this study. Ranked by target gene symbol.

41586_2021_4109_MOESM7_ESM.xlsx

Supplementary Table 5 Differentially expressed gene list in induced Treg cells upon Smarcb1 deletion. This file contains 2,023 differentially expressed (|log2 FC| > 0.55, FDR < 0.05) genes in Smarcb1-null versus control at 3 h [sgSmarcb1-3 h versus sgNTC-3 h (columns E and F)] or 20 h [sgSmarcb1-20 h versus sgNTC-20 h (columns G and H)] after TCR stimulation as profiled by microarray analysis.

41586_2021_4109_MOESM8_ESM.xlsx

Supplementary Table 6 Differentially expressed gene list in induced Treg cells upon Ccdc101 deletion. This file contains 1,120 differentially expressed (|log2 FC| > 0.55, FDR < 0.05) genes of in Ccdc101-null versus control cells at 0 h [sgCcdc101-0 h versus sgNTC-0 h (columns E and F)] or 20 h [sgCcdc101-20 h versus sgNTC-20 h (columns G and H)] after TCR stimulation as profiled by microarray analysis.

41586_2021_4109_MOESM9_ESM.xlsx

Supplementary Table 7 Primers used to generate SEC13 mutants. This file contains the forward and reverse primer sequences used to generate the various single or double mutants of SEC13.

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Long, L., Wei, J., Lim, S.A. et al. CRISPR screens unveil signal hubs for nutrient licensing of T cell immunity. Nature 600, 308–313 (2021). https://doi.org/10.1038/s41586-021-04109-7

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