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Nutrients are emerging regulators of adaptive immunity1. Selective nutrients interact with immune signals to activate the mechanistic target of rapamycin complex 1 (mTORC1), which is a key driver of cell metabolism2,3,4, but how these environmental signals are integrated for immune regulation remains unclear clear. Here, we use genome-wide CRISPR screens to bind protein-protein interaction networks to identify regulatory modules that mediate immune receptors and nutrient-dependent signal transduction to mTORC1 in mouse regulatory T (Treg) cells. SEC31A was identified as promoting mTORC1 activation by interacting with GATOR2 component SEC13 to protect it from SKP1-dependent proteasome degradation. Therefore, the loss of SEC31A will impair T cell activation and Treg suppression in mice. In addition, the SWI/SNF complex restricts the expression of the amino acid sensor CASTOR1, thereby enhancing the activation of mTORC1. In addition, we revealed that the SAGA complex related to CCDC101 is an effective inhibitor of mTORC1, which limits the expression of glucose and amino acid transporters and maintains the quiescence of T cells in the body. The specific deletion of Ccdc101 in mouse Treg cells can cause uncontrolled inflammation, but it can increase anti-tumor immunity. In summary, our results establish epigenetic and post-translational mechanisms that support how nutrient transporters, sensors, and sensors interact with immune signals to regulate mTORC1 activity in three layers, and determine that they are permitting T cells Key role in immunity and immune tolerance.

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The author declares that the data supporting the results of this study can be found in 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 Synthesis (GEO) database and can be accessed through 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/). This article provides source data.

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We thank M. Hendren and S. Rankin for animal community management; J. Wen for seeking help in phenotyping research; C. Li and S. Zhou for technical and scientific insights; G. Neale and S. Olsen for assisting in sequencing; for cell use Sorted St. Jude Immunology FACS core facilities; and St. Jude Communications and Science/Medical Content Outreach artwork. This work was supported by grants R01AG053987 (for JP) and AI105887, AI131703, AI140761, AI150241, AI150514, CA250533 and CA253188 (for HC) from ALSAC and the National Institutes of Health. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.

The contributions of these authors are the same: Ling Yunlong, Wei Jun

Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

Lingyun Long, Jun Wei, Seon Ah Lim, Jana L. Raynor, Hao Shi, Cliff Guy, Nicole M. Chapman, Guotong Fu, Yanyan Wang, Hongling Huang, Wei Su, Jordy Saravia, Isabel Risch, Yogesh Dhungana, Anil KC, Zhou Peipei & Chi Hongbo

Advanced Genome Engineering Center, St. Jude Children's Research Hospital, Memphis, Tennessee, U.S.

Jon P. Connelly & Shondra M. Pruett-Miller

Proteomics and Metabolomics Center, St. Jude Children's Research Hospital, Memphis, Tennessee, U.S.

Wang Hong, Xie Boer, Li Yuxin, Niu Mingming and Peng Junmin

Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee, U.S.

Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

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LL and JW conceived the project, designed and performed in vitro and in vivo experiments, analyzed the data and wrote the manuscript. SAL performed tumor and scRNA-seq experiments. JLR conducted a hippocampus experiment. HS, IR, YL, and YD were analyzed by bioinformatics. JPC and SMP-M. Designed and generated a key sgRNA library. HW, BX, MN and JP conducted proteomics. CG conducted imaging experiments. NMC, YW, HH, WS, AK, and PZ helped with immunological experiments. GF conducted an LCMV infection experiment. JS helps prepare ATAC-seq samples. Y.-DW and JY analyzed the CRISPR-Cas9 screening data. PV provides histological analysis. HC helped design the experiment, co-authored the manuscript, and provided general guidance.

HC is an advisor to Kumquat Biosciences.

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

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Related to Figure 1. (A) Flow cytometry analysis of IFNγ, IL-4, IL-17A or FOXP3 expression in cells cultured under TH0-, TH1-, TH2-, TH17- or induced Treg polarization conditions (n ​​= 3 per Group sampling). (b) Flow cytometry analysis of pS6 in TH1 and Treg cells 0 or 1 hour after TCR stimulation (n = 3 samples per group). (c) In the presence or absence of amino acids (AA) or glucose, stimulate the induced Treg cells with anti-CD3 and anti-CD28 for 3 hours, then perform flow cytometry analysis and quantification of pS6 levels [based on average fluorescence intensity ( MFI) )] (n = 3 samples per group). (d) The induced Treg cells were labeled with CellTrace Violet (CTV) and stimulated with anti-CD3 and anti-CD28 in the presence or absence of AA or glucose for 3 days, and then the CTV dilution was analyzed by flow cytometry (n = 3 samples) per group). (e) Gating strategy for sorting cells with ≥ 10% highest (pS6hi) and ≤ 10% lowest (pS6lo) levels after stimulation with 0.25 or 4 μg–1 anti-CD3 for 3 hours (n = 2 samples)). Mean ± sem (c). *** P <0.001; one-way analysis of variance (c). The data represents two (b–d) or three (a) experiments.

Related to Figure 1. (a) Flow cytometric analysis and quantification of pS6 levels [based on mean fluorescence intensity (MFI)] in naive or activated WT and CD4 Foxp3-T cells lacking Depdc5 (n = 4 samples per group). Naive CD4 T cells in freshly isolated splenocytes of WT and Cd4CreDepdc5fl/fl mice were gated (denoted as TCR 0 h), or naive CD4 T cells were sorted and stimulated with anti-CD3 and anti-CD28 overnight for pS6 Level flow cytometry analysis. (b) Quantification of relative pS6 levels in induced Treg cells transduced with sgNTC, sgSec13, sgMios, sgSeh1l or sgWdr24 (all amethyst), stimulated with TCR for 3 hours (n = 3 samples). (c) Two-color co-culture system diagram, used to examine the intrinsic effects of candidate gene deletion on TCR-induced pS6, cell size, and CD71 expression. Specifically, Cas9 cells transduced with sgNTC (mCherry or GFP; "spike") are mixed with those transduced with a specific gene (Ametrine) and stimulated with anti-CD3 for 3 hours (for pS6) or with anti-CD3 and Anti-CD28 for 20 hours (for cell size and CD71). (d) Verify the dual-color co-culture system by using two sgNTC expression vectors with different fluorophores. Cells transduced with sgNTC (GFP; "spike") are mixed with cells transduced with sgNTC (amethyst), stimulated with anti-CD3 for 3 hours to check pS6 (see phos-flow staining), or with anti-CD3 and anti- Stimulate-CD28 for 20 hours (n = 3 samples per group) to measure cell size and CD71 expression (see surface staining). (e) The log2 (pS6hi/pS6lo) heat map summary of individually verified 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 are used for target gene (Ametrine) or non-targeting control sgRNA (sgNTC) (mCherry;'spike') to mix and differentiate into inducible Treg cells. These cells were then stimulated with anti-CD3 for 3 hours (n = 3 samples per group). The relative pS6 level (normalized to "spike") was analyzed by flow cytometry. (F) Analyze the protein-protein interaction (PPI) network of high-confidence modifiers. Specifically, 286 positive and 60 negative high-confidence hits are integrated with a composite PPI database containing STRING, BioPlex and InWeb_IM databases to infer functional modules. The red and blue circles represent gene deletions, which inhibit and promote mTORC1 activity, respectively. Mean ± sem (a, b). *P <0.05; ** P <0.01; *** P <0.001; one-way analysis of variance (a, b). The data represents one (f) or three (d, e), or collected from two (a) or three (b) experiments.

Related to Figure 1. (a) Quantification of relative pS6 level, cell size (FSC-A) and CD71 expression (normalized to "spike") in induced Treg cells transduced with the specified sgRNA, and then stimulated with anti-CD3 for 3 h to measure pS6 level, or Measure cell size (FSC-A) and CD71 expression by flow cytometry using anti-CD3 and anti-CD28 for 20 hours (n = 3 samples per group). (b) Imaging analysis and quantification of lysosomal-associated mTOR in sgNTC or sgSmarcb1 transduced cells [based on mean fluorescence intensity (MFI)], these cells were stimulated by anti-CD3 for 3 hours, lacking amino acids (AA), and re-added AA 20 Minutes (n> 230 cells per condition). Scale bar, 5 μm. (c) Volcano plot of transcript expression levels including Castor1 in cells transduced with sgNTC or sgSmarcb1 (both ametrine) stimulated with TCR for 3 hours (n = 4 samples per group). (D) Castor1 mRNA expression in cells transduced with sgNTC- or sgSmarcb1 (both amethyst) was stimulated with anti-CD3 for 3 hours or stimulated with anti-CD3 and anti-CD28 for 20 hours (n=3 samples per group). (E) sgNTC- or sgSmarcb1 (both ametrine)-transduced cells were unstimulated (represented by 0 hours) or stimulated with anti-CD3 for 3 hours or with anti-CD3 and anti-CD28 for 20 hours. Western blot analysis and quantification of relative CASTOR1 expression (n = 3 samples per group). (f) Western blot analysis and quantification of the relative pS6K1 and pS6 levels in cells transduced with an empty vector or a vector expressing Castor1, and then stimulated with anti-CD3 and anti-CD28 for 2 days (n = 3 samples per group). Mean ± sem (a, b, d–f). ** P <0.01; *** P <0.001; two-tailed unpaired Student's t-test (f); one-way analysis of variance (a, b, d, e). Data represents one (c) or two (b), or summarized from two (d, e) or three (a, f) experiments

Related to Figure 2. (A) The interaction of endogenous SEC13 and SEC31A in induced Treg cells, as assessed by immunoprecipitation (IP)-immunoblotting analysis. (b, c) Cells transduced by sgNTC or sgSec31a were starved and supplemented with amino acids (AA, b) or glucose (c) for 20 minutes, and then western blot analysis was performed on SEC31A, pS6K1, pS6 and β-actin. Bottom, quantification of relative pS6K1 and pS6 levels (n = 3 samples per group). (d) Cells transduced with designated sgRNA (all Ametrine) are mixed with cells transduced with sgNTC (mCherry;'spike') and stimulated with anti-CD3 for 3 hours to measure pS6 levels or with anti-CD3 and anti-CD28 for 20 hours Cell size (FSC-A) and CD71 expression (normalized to "spike") were measured by flow cytometry (n = 3 samples per group). (eg) sgNTC-, sgSec31a- or sgSec13 (all Ametrine)-transduced cells with constitutively active RAGAQ66L (CA-RAGA)-expressing retrovirus (GFP) or sgNprl2 (GFP) co-transduced and then stimulated with TCR3 After hours, the relative pS6 level was detected by flow cytometry (n = 3 samples per group) (e), and the pS6K1 and pS6 levels were detected by Western blot analysis (n = 4 samples per group) (f), or the dissolution of mTOR Enzymatic localization [based on mean fluorescence intensity (MFI)] (n> 700 cells per condition). Scale bar, 5 µm (g). In f, two different exposures of pS6K1 are included to explain the difference in intensity between the simulated and CA-RAGA or sgNprl2 conditions, and the relative pS6K1 level is from the simulated long-term exposure and the short-term exposure under CA-RAGA or sgNprl2 Quantification (middle). Bottom, pS6 level quantification. Mean ± sem (bg). NS, not significant; ** P <0.01; *** P <0.001; one-way analysis of variance (bg). The data represents two (eg) or four (a), or aggregated from three (bd) experiments.

Related to Figure 2. (a) Cells transduced with sgNTC or sgSec31a (both Ametrine) were labeled with CellTrace Violet (CTV) and transferred to Rag1–/– mice. Flow cytometry analysis of CTV dilution and quantification of the percentage of proliferating (CTVlo) cells on day 7 after transfer (n = 5 samples per group). (b) WT, Rptor- and Sec31a-null Treg cells (all CD45.1 amethyst) were mixed with regular CD4 T cells (Tconv; CD45.2) at a ratio of 1:4 and transferred to Rag1–/– mice Medium. Quantify the accumulation of conventional T cells in the spleen on day 7 after transfer (n = 5 samples per group). (c) Stimulate naive CD4 T cells with anti-CD3 and anti-CD28 for 0, 24, 48, or 72 hours, then perform immunoblotting analysis on the expression of the specified protein, and analyze pS6K1, SEC13, SEC31A and TSC2 (n = 3 per group sample). (d) sgNTC-, sgSec31a- and sgSec13-transduced cells were sorted and lysed with CHAPS buffer for immunoprecipitation (IP) and anti-WDR24 antibodies. The immunoprecipitated proteins were analyzed by immunoblotting of WDR24, WDR59, MIOS, SEH1L, SEC13, SEC31A, SEC23A and β-actin. Mean ± sem (ac). ** P <0.01; *** P <0.001; two-tailed unpaired Student's t-test (a); one-way analysis of variance (b). The data represents one (a, b) or two (c, d) experiments.

Related to Figure 3. (a) Sec13 mRNA expression in Treg cells induced by sgNTC or sgSec31a transduction (n = 3 samples per group). (B) sgNTC- or sgSec31a-transduced induced Treg cells are sorted and treated with cycloheximide (CHX) for the specified time. Cell total protein extracts using sgNTC (5 μg) or sgSec31a (12.5 μg; load more protein to balance the basal SEC13 amount between these cells) were used for western blot analysis and quantification of relative SEC13 abundance (n = (4 samples). (c) The initial CD4 T cells were stimulated with anti-CD3 and anti-CD28 for 0, 24, or 48 hours, and then treated with DMSO or MG132 at 48-72 hours of stimulation, and then subjected to Western blot analysis and SEC13 and SEC31A Quantitative expression (n = 3 samples per group). (D) HEK293T cells were transfected with HA-labeled SEC13 and 6×His-labeled WT-, K48R- or K63R-ubiquitin (Ub) and treated with MG132 for 6 hours. The pull-down and western blot analysis of HA-SEC13 based on Ni-nitrilotriacetic acid (Ni-NTA) beads. At the bottom, the expression of the specified protein in the whole cell lysate (WCL). (E) sgNTC- or sgSec31a-transduced HEK293T cells were transfected with HA-labeled SEC13 and 6×His-labeled WT ubiquitin (His-labeled Ub), and treated with MG132 for 6 hours. On the left, pull-down His-labeled Ub-labeled protein based on Ni-NTA beads, and then perform Western blot analysis on HA-SEC13. Right image, Western blot analysis of WCL for expression of endogenous SEC31A or HA-SEC13, His-labeled Ub and β-actin. Mean ± sem (ac). NS, not significant; ** P <0.01; *** P <0.001; two-tailed unpaired Student's t-test (a); two-way analysis of variance (b); one-way analysis of variance (c). Data represents two (d, e) or three (a), or aggregates from two (b) or three (c) experiments.

Related to Figure 3. (a) HA-labeled WT or designated SEC13 lysine mutant constructs were respectively transfected into HEK293T cells. Western blot analysis of HA and Hsp90. (B) HA-labeled WT or K260R mutant SEC13 was transfected into HEK293T cells together with His-Ub only K48, and then subjected to MG132 treatment and anti-HA immunoprecipitation (IP). Western blot analysis of HA, His-Ub and β-actin. WCL, whole cell lysate. (c) Use sgNTC or sgSec31a retrovirus (Ametrine) together with WT or K260R mutant SEC13 expressing retrovirus (GFP) to transduce Cas9-expressing CD4 T cells. Stimulate ametrine GFPlo cells with TCR (see gate on flow cytometer diagram, top) for 0 or 3 hours. Western blot analysis and quantification of relative SEC13 and pS6 levels (n = 4 samples per group). (d) Volcano map of proteins, including SKP1, interacting with HA-SEC13 in induced Treg cells, as identified by mass spectrometry (n = 3 samples per group). (e) Inducible Treg cells transduced with HA-labeled SEC13 or retrovirus expressing an empty vector were lysed with CHAPS buffer, and then subjected to anti-HA immunoprecipitation (IP) and immunoblotting analysis of HA and SKP1. (F) The induced Treg cells were lysed with CHAPS buffer, then the endogenous SKP1 was immunoprecipitated and the SKP1 and SEC13 were subjected to immunoblotting analysis. (g) The interaction between endogenous SKP1 and SEC13 in sgNTC or sgSec31a transduced cells. (h) Stimulate naive CD4 T cells with anti-CD3 and anti-CD28 for 0, 24, 48 or 72 hours. Western blot analysis of SKP1, SEC13 and β-actin anti-SKP1 immunoprecipitants and WCL (n = 2 samples per group). (i) Western blot analysis and relative expression quantification of the specified protein in cells transduced with sgNTC or sgSkp1 (both amethyst) (n = 3 samples per group). (j) The designated sgRNA-transduced cells were labeled with CellTrace Violet (CTV) and stimulated with anti-CD3 and anti-CD28 for 72 hours, and then subjected to flow cytometry analysis and quantitative CTV dilution (n = 3 samples per group). (k ) System diagram of SMARTA T cell transfer and LCMV infection. In short, SMARTA-Cas9 CD4 T cells (CD45.1) use sgRNA to transduce the candidate gene (CD45.1 amethyst) and mix with sgNTC (CD45.1 mCherry;'spike')-transduced cells in 1 : 1 ratio, and adoptively transferred to naive (unchallenged; CD45. (l) Quantification of the relative proportion (normalized to "spike") of donor-derived (CD45.1) T cells in the spleen of uninfected mice 7 days after transfer (n = 6 mice per group). (m) Cells transduced with sgNTC (Ametrine), sgSec31a (Ametrine) or sgSec31a/Skp1 (GFP and Ametrine) were classified and stimulated with anti-CD3 and anti-CD28 for 20 hours (n = 6-7 samples per group), followed by It measures the extracellular acidification rate (ECAR). Oligo, oligomycin; FCCP, fluorocarbonyl cyanophenylhydrazone; rot, rotenone. Mean ± sem (c, i, j, l, m). ** P <0.01; *** P <0.001; one-way analysis of variance (c, i, j, l, m, right); two-way analysis of variance (m, left). The data represents one (d, j, l, m) or two (a, b, eh), or pooled from two (c) or three (i) experiments.

Related to Figure 4. (A) Relative pS6 level, cell size (FSC-A) and CD71 expression quantification (normalized to "spike") in inducible Treg cells transduced with Ccdc101 or Taf6l sgRNA, and then measured with anti-CD3 for 3 hours pS6 level (left) or 20 hours with anti-CD3 and anti-CD28 to measure cell size (FSC-A; middle) and CD71 (right) expression (n = 3 samples per group) by flow cytometry. (b) Western blot analysis and quantification of relative pS6K1 and pS6 expression in sgNTC or sgCcdc101-transduced cells lacking amino acids (AA) for 20 minutes (n = 4 samples per group). Mean ± sem (a, b). *P <0.05; ** P <0.01; *** P <0.001; one-way analysis of variance (a, b). The data represents three (a) or pooled from three (b) experiments.

Related to Figure 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 per group) or 20 hours (n = 4 samples per group)). (B) Slc2a1, Slc16a10 or Slc43a1 mRNA expression in cells transduced with sgNTC or sgCcdc101 under steady state (n = 3 samples per group). (c) Western blot analysis and quantification of relative GLUT1 expression in sgNTC or sgCcdc101 transduced cells stimulated with anti-CD3 and anti-CD28 for 0 or 20 hours (n = 3 samples per group). (d) Flow cytometry analysis and quantitative analysis of 2-NBDG uptake in sgNTC or sgCcdc101 (amethyst) transduced cells, stimulated with anti-CD3 and anti-CD28 for 20 hours (n = 4 samples per group). (e, f) Quantification of relative pS6 levels in cells transduced with designated sgRNAs stimulated with TCR for 3 hours (n = 3 samples per group). (g) Principal Component Analysis (PCA) of ATAC-seq of cells transduced with sgNTC (n = 4 samples) or sgCcdc101 (amethyst) (n = 3 samples) and stimulated with anti-CD3 and anti-CD28 for 20 hours . (h) Motif enrichment analysis of ATAC-seq of sgNTC and sgCcdc101 transduced cells (n = 4 samples per group). (i) Footprint analysis of Sp3 binding in ATAC-seq. (j) The accessibility of the Sp3 locus in sgNTC and sgCcdc101 transduced cells, as identified by ATAC-seq. The peaks highlighted in the red box represent different accessible areas. (k) Western blot analysis of SP3 expression in sgNTC or sgCcdc101 (both amethyst) transduced cells. Mean ± sem (b–f). *** P <0.001; two-tailed unpaired Student's t-test (b, d); one-way analysis of variance (c, e, f). Data represents one (a, g–j), two (b, e, f, k) or data collected from two (c, d) experiments.

Related to Figure 4. (A, b) Inducible Treg cells transduced with the designated sgRNA were stimulated with anti-CD3 and anti-CD28 for 20 hours (n = 3 samples per group). Flow cytometry analysis and quantification of staining active caspase-3 (a) and fixable vitality dyes (FVD, b) (n = 3 per group). (c) Cells transduced with the designated sgRNA were stimulated with anti-CD3 and anti-CD28 for 20 hours (n = 5-7 samples per group), and then the extracellular acidification rate (ECAR) was measured. Oligo, oligomycin; FCCP, fluorocarbonyl cyanophenylhydrazone; rot, rotenone. (d) Western blot analysis and quantification of CCDC101 expression in naive CD4 T cells from WT and Cd4CreCcdc101fl/fl mice (n = 3 mice per group). (e) Quantification of CD71 expression on initial or activated WT and Ccdc101-deficient CD4 T cells. Naive CD4 T cells in freshly isolated splenocytes from WT and Cd4CreCcdc101fl/fl mice (n = 4 mice per group) were gated (indicated as 0 hours), or naive CD4 T cells were treated with anti-CD3 and anti- CD28 stimulation for 20 hours. (f) Flow cytometry analysis and quantification of total, double negative (DN), double positive (DP), CD4 single positive (CD4SP) and CD8 single positive (CD8SP) thymus from WT and Cd4CreCcdc101fl/fl Number of cells in mice (n = 4 mice per group). (g) Flow cytometry analysis and quantification of the proportion and number of CD4 and CD8 T cells in the spleen of WT and Cd4CreCcdc101fl/fl mice (n = 4 mice per group). (h) Flow cytometry analysis and normalized ratio of CD122 to CD122- cells in the CD44hi population (gated on splenic CD8 T cells) from designated mice (n = 4 mice per group). Mean ± sem (ah). NS, not significant; ** P <0.01; *** P <0.001; two-tailed unpaired Student's t-test (d–h); one-way analysis of variance (a, b, c, right); two-way analysis of variance ( c, left). The data represents one (c) or two (a, b), or summarized from three (d–h) experiments.

Related to Figure 5. (A) Quantification of relative pS6 levels in spleen CD4 FOXP3 cells from WT and Foxp3CreCcdc101fl/fl (approximately 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 the percentage of effector/memory (CD44hiCD62Llo) subpopulations in spleen CD4 FOXP3- and CD8 T cells in WT and Foxp3CreCcdc101fl/fl (approximately 8 weeks old) mice (n = 4 mice per group). (d) Representative flow cytometry analysis of IL-2 or IFNγ populations of splenic CD4 Foxp3- and CD8 T cells from WT and Foxp3CreCcdc101fl/fl (approximately 8 weeks old) mice. (eg) WT and Foxp3CreCcdc101fl/fl mice were inoculated with MC38 colon adenocarcinoma cells. Isolate and sort Treg cells (CD45 CD4 YFP), non-Treg immune cells (CD45 YFP–CD11b–) and bone marrow cells (CD45 CD11b) from tumors, and mix them at a ratio of 1:2:1 for scRNA-seq analysis (2 biological replicates, 3-4 mice in each group combined) 19 days after tumor inoculation. The dot plot shows the differentially expressed marker genes of 4 CD8 T cell subsets in MC38 tumors (e; see methods for details). The UMAP embeddings of CD8 T cells are grouped by genotype (f, left) and indicated subgroups (f, right). Quantify the frequency of the specified subgroup for each genotype (g). Teff-like, effector-like CD8 T cells; Tex-like, depletion-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 percentage of CD8 T cells in CD44hiCD62Llo tumors from WT and Foxp3CreCcdc101fl/fl mice (n ≥ 5 per group). (i) Flow cytometry analysis and quantification of IFNγ and TNF cells in intratumoral CD8 T cells from WT and Foxp3CreCcdc101fl/fl mice (n ≥ 5 in each group). (j) Violin plot of scRNA-seq data, depicting the expression of Icos, Tnfrsf18, Ctla4, and Ifng in Treg cells within the tumor. (k) Flow cytometry analysis and quantification of ICOS, GITR and CTLA-4 of intratumoral Treg cells in WT and Foxp3CreCcdc101fl/fl mice (n ≥ 5 in each group). (l) Flow cytometry analysis and quantification of IFNγ expression in intratumoral Treg cells from WT and Foxp3CreCcdc101fl/fl mice (n ≥ 5 in each group). Mean ± sem (a–c, 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. The data represents one (el) or two (d), or pooled from two (ac) experiments.

Related to Figure 5. Through two rounds of genome-wide and focused CRISPR screening, we identified 346 high-confidence mTORC1 signaling factors, including many new activators and inhibitors, as well as known modulators that have not yet been discovered (identified in other systems). Study in the generation of T cells. It is worth noting that, using the analysis of the protein-protein interaction (PPI) network and unbiased functional and proteomic methods, we have further established the epigenetic and post-translational mechanisms of the three-layer regulatory module that supports nutritional signals. Nutrient transporter composition (e.g. by influencing the expression of GLUT1 and other transporters through the SAGA complex), sensors (e.g. through the epigenetic regulation of CASTOR1 expression by the SWI/SNF complex) and sensors (e.g., shaping the stability of the GATOR2 complex through SEC31A ; Regulate SEC13 ubiquitination at lysine 260), they transmit immune and nutritional mTORC1 signaling to properly regulate T cell activity in vivo and in vitro.

This file contains Supplementary Figure 1.

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

Supplementary Table 2. This file contains sgRNA sequences from the CRISPR sgRNA library used for the second round of focused CRISPR screening. Sort by target gene symbol.

Supplementary Table 3. This file contains the gene levels analyzed in pipeline 1 (Tab a) and pipeline 2 (Tab b), or the analysis results of sgRNA levels analyzed in pipeline 1 (Tab c) against the second round of key CRISPR library screening data) and pipeline 2 ( Table d). A comparison of two stimulation conditions (0.25 and 4 μg ml-1 anti-CD3) (pS6high and pS6low) is shown. Sort by target gene symbol.

Supplementary Table 4. This file contains the sgRNA sequence used for validation in this study. Sort by target gene symbol.

Supplementary Table 5 reg cells after deletion of Smarcb1. This file contains 2,023 differentially expressed (|log2 FC|> 0.55, FDR <0.05) genes, Smarcb1-null and control at 3 hours [sgSmarcb1-3 h and sgNTC-3 h (columns E and F)] or 20 hours [ sgSmarcb1 -20 hours vs. sgNTC-20 hours (columns G and H)] After TCR stimulation, analyze by microarray analysis.

Supplementary Table 6 reg cells after deletion of Ccdc101. This file contains 1,120 differentially expressed (|log2 FC|> 0.55, FDR <0.05) genes, Ccdc101-null and control at 0 h [sgCcdc101-0 h vs sgNTC-0 h (columns E and F)] or 20 h Cells [sgCcdc101-20 h and sgNTC-20 h (columns G and H)] were analyzed by microarray analysis after TCR stimulation.

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

Long, L., Wei, J., Lim, SA, etc. CRISPR screening reveals the signal center of T cell immunonutrition permission. Natural (2021). https://doi.org/10.1038/s41586-021-04109-7

DOI: https://doi.org/10.1038/s41586-021-04109-7

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