Abstract
The nature of activation signals is essential in determining T cell subset differentiation; however, the features that determine T cell subset preference acquired during intrathymic development remain elusive. Here we show that naive CD4+ T cells generated in the mouse thymic microenvironment lacking Scd1, encoding the enzyme catalyzing oleic acid (OA) production, exhibit enhanced regulatory T (Treg) cell differentiation and attenuated development of experimental autoimmune encephalomyelitis. Scd1 deletion in K14+ thymic epithelia recapitulated the enhanced Treg cell differentiation phenotype of Scd1-deficient mice. The dearth of OA permitted DOT1L to increase H3K79me2 levels at the Atp2a2 locus of thymocytes at the DN2–DN3 transition stage. Such epigenetic modification persisted in naive CD4+ T cells and facilitated Atp2a2 expression. Upon T cell receptor activation, ATP2A2 enhanced the activity of the calcium–NFAT1–Foxp3 axis to promote naive CD4+ T cells to differentiate into Treg cells. Therefore, OA availability is critical for preprogramming thymocytes with Treg cell differentiation propensities in the periphery.
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Data availability
The sequencing data generated and analyzed in this publication have been deposited in NCBI’s Gene Expression Omnibus. The Mus musculus reference genome (GRCm38/mm10 assembly; https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20/) was used for ChIP–seq and RNA-sequencing alignment. The genome-wide maps of chromatin accessibility in DN3 thymocytes and splenic naive CD4+ T cells are deposited under the accession number GSE209563. The mRNA-sequencing data for cortical and medullary thymic epithelial cells are available under accession GSE237819, while the H3K79me2 ChIP–seq analysis data on in vitro-developed DN3 cells are available under accession GSE237573. Source data are provided with this paper.
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Acknowledgements
We thank Xinhua Liao for providing K14cre mice, L. Qiu, J. Wu, X. Miao, Q. Chu, Zi Li, Y. Ma, K. Wang, Z. Weng, Zhigang Li, Y. Zhai, Y. Bu, S. Yan and F. Zhao from Institutional Center for Shared Technologies and Facilities of SINH, CAS for technical assistance. Ying Wang discloses support for the research of this work from the National Key R&D Program of China (2022YFA0807300), National Natural Science Foundation of China (32070872, 31771641) and the March of Dimes Birth Defects Foundation of China. Y.S. discloses support for the research of this work from the National Key R&D Program of China (2018YFA0107500), National Natural Science Foundation of China (81930085, 81530043, 31961133024, 81571612) and Scientific Innovation Project of the Chinese Academy of Sciences (XDA16020403). Liangyu Lin discloses support for the research of this work from the National Natural Science Foundation of China (32000626) and a Fellowship of China Postdoctoral Science Foundation (2020M671261). Liangyu Lin acknowledges the support of SANOFI Scholarship Program. Q.L. discloses support for the research of this work from the National Natural Science Foundation of China (82301571).
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Liangyu Lin and H.M. conceived the project, designed and performed experiments, analyzed the data, interpreted results and prepared the manuscript. Q.L., L.D., Y.X., F.Z., F.W., K.L., J.Y., X.J., X.W., Jiaqi Wang, J.Z., B.L. and H.X. helped to conduct experiments. Li Lin conducted bioinformatic analysis. Q.L. and Yu Wang assisted in constructing the manuscript. D.R.G., X.K., Y.Y. and D.F. advised on the project. Jinyong Wang provided OP9-DL1 cells. Ying Wang and Y.S. led the project and wrote the manuscript with the co-authors.
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Extended data
Extended Data Fig. 1 Properties of Treg cells of Scd1-/- mice.
(a) EAE was induced in Scd1-/- and Scd1+/- mice as described in Fig. 1a. The levels of Th17 and Treg cells in the central nervous system (CNS) were analyzed on day 17 post EAE induction (n = 7 mice for Scd1+/-+Iso, Scd1-/-+Iso, n = 5 mice for Scd1+/-+α-CD25, n = 6 for Scd1-/-+α-CD25). (b) The absolute number of CD4+Foxp3+ Treg cells and CD4+Foxp3− conventional T cells in various organs of Scd1-/- and Scd1+/- mice (n = 4 mice per group). (c) CD4+CD25+ T cells were isolated from the spleen of Scd1-/- mice and Scd1+/- littermate control mice, and co-cultured with activated CFSE-labeled CD4+ T cells (1 × 106) from wild type (WT) mice at indicated ratio. The proliferation of CD4+ T cells were determined by the dilution of CFSE. (d) The expression levels of CD25, CTLA-4, and GITR in CD4+Foxp3+ Treg cells were determined by flow cytometry. (e) Gating strategies (with the spleen as a representative example) for analyzing Helios expression in Treg cells. (f-g) The percentage and absolute number of Helios+ and Helios− Treg cells in the various tissues of Scd1-/- mice and Scd1+/- mice under steady status (n = 4 mice per group). Data are presented as Mean ± SEM. NS, not significant; P values were determined by unpaired two-tailed Student’s t-test.
Extended Data Fig. 2 Newly generated CD4+ T cells in Scd1-/- mice are prone to differentiate to Treg cells in WT recipient mice.
(a) Thymocytes were depleted of double positive and CD8 single positive cells by magnetic beads and then subjected to sort CD4+CD8−CD73−CD25− thymocytes by flow cytometry. The purity of sorted thymocytes was depicted. (b) The gating strategies (with the spleen as a representative example) for analyzing Treg cells in CD45.1+CD45.2+ WT mice receiving the co-transfer of CD4+CD8−CD73−CD25− thymocytes from CD45.1+WT and CD45.2+ Scd1-/- mice. (c) The percentage of Foxp3+ cells in CD4+ cells of CD45.1+WT and CD45.2+Scd1-/- origin were determined 6 weeks post thymocyte co-transfer (n = 6 mice per group). (d) The absolute number of WT CD45.1+CD4+Foxp3+ and Scd1-/- CD45.2+CD4+Foxp3+ cells in various organs of WT recipient mice were numerated (n = 6 mice per group). (e) The absolute number of CD4+ T cells derived from CD45.1+ and CD45.2+ donor cells were assessed (n = 6 mice per group). Data are presented as Mean ± SEM. NS, not significant; P values were determined by paired two-tailed Student’s t-test.
Extended Data Fig. 3 Functionality of CD4+ T Cells isolated from Scd1+/- and Scd1-/- mice.
(a) The percentage of CD44−CD62L+, CD44+CD62L−, and CD44+CD62L+ CD4+ T cells in the spleen of Scd1+/- (n = 7) and Scd1-/- mice (n = 5) at steady status. (b) CFSE-labeled splenic CD4+CD25− conventional T cells from Scd1+/- and Scd1-/- mice were subjected to anti-CD3/CD28 stimulation in vitro for 3 days and cell proliferation was detected by assessing CFSE dilution. (c-f) Splenic CD4+CD62L+T cells from Scd1-/- and Scd1+/- mice were cultured in Th1, Th2, or Th17 induction medium for 3 days (n = 3 for Th1 and Th17 differentiation, n = 4 for Th2 differentiation). The percentages of IFNγ+, IL-4+, or IL-17+ CD4+T cells were determined by flow cytometry. Data are presented as Mean ± SEM. NS, not significant; P values were determined by unpaired two-tailed Student’s t-test.
Extended Data Fig. 4 Treg cells are comparable in CD4creScd1loxP and Scd1loxP mice.
(a) mRNA levels of Scd1 in CD4+ T cells and hepatocytes isolated from CD4creScd1loxP mice and Scd1loxP littermate control mice (n = 5 mice for each group). (b) The percentages of stearic acid (18:0) and oleic acid (18:1n9) among total fatty acids in in vitro expanded CD4+ T cells isolated from CD4creScd1loxP mice and Scd1loxP mice (n = 3 for each group). (c) The CD4+ T cell reconstitution efficiency in Rag2-/- mice receiving co-transfer of CD45.1+WT CD4+ T cells and CD45.2+CD4creScd1loxP CD4+ T cells (n = 4 mice per group). (d) The percentage of Treg cells among CD4+ T cells in the spleen, auxiliary lymph node and central nervous system of CD4creScd1loxP mice and Scd1loxP mice on day 0 (steady, n = 3 mice per group), day 18 (peak, n = 5 for Scd1loxP mice, n = 3 for CD4creScd1loxP mice), and day 24 (recovery, n = 5 for Scd1loxP mice, n = 3 for CD4creScd1loxP mice) post EAE induction. Data are presented as Mean ± SEM. NS, not significant; P values were determined by unpaired two-tailed Student’s t-test.
Extended Data Fig. 5 The T cell reconstitution efficiency of WT and Scd1-/- bone marrow cells were comparable.
(a) Representative gating strategies in analyzing the thymocyte populations of the recipient mice as treated in Fig. 2a. The reconstitution efficiency of CD45.1+ and CD45.1+CD45.2+ cells within thymocytes at DN, DP, and SP stages were depicted. (b) The reconstitution efficiency of CD45.1+WT and CD45.1+CD45.2+Scd1-/- bone marrow cells transplantation (BMT) in different tissues of recipient mice as treated in Fig. 2a. (c) The percentages of donor CD4+ cells among CD45+ cells in the blood and different organs of recipient mice in Fig. 2a. (d) The thymic CD4−CD8− double negative (DN) subpopulation levels in Scd1+/- mice and Scd1-/- mice (n = 5 mice for each group). (e) The thymic DN, CD8SP, CD4SP, and DP levels in Scd1+/- mice and Scd1-/- mice (n = 6 mice for each group). Data are presented as Mean ± SEM. NS, not significant; P values were determined by unpaired two-tailed Student’s t-test.
Extended Data Fig. 6 DN3 and DN4 cells of Scd1 deficient origin generated CD4+ T cells with intrinsic Treg propensities upon injection into the thymus of WT mice.
(a) Gating strategies (with the spleen as a representative sample) used to analyze the Treg cell proportion among donor-derived CD4+ T cells. (b-c) Thymocyte population from CD45.2+Scd1-/- mice and CD45.1+WT mice at the DN1, DN2, DN3, or DN4 stage were mixed at a 1:1 ratio and co-transferred into irradiated (3 Gy) CD45.1+CD45.2+ WT recipient mice via intrathymic injection (n = 5 mice for DN1, DN2 transfer; n = 6 mice for DN3, DN4 transfer). Four weeks post transfer, the recipient mice received WT and Scd1-/- DN thymocytes were euthanized to numerate the absolute number of CD4+ T cells (b) and CD4+Foxp3+ Treg cells (c) in various organs. Data are presented as Mean ± SEM. NS, not significant; P values were determined by paired (b, c) two-tailed Student’s t-test.
Extended Data Fig. 7 The influence of SCD1 inhibitor on in vitro T cell development.
(a) The progression of in vitro T cell development in the OP9-DL1 and HSC co-culture system. The effect of SCD1 inhibitor at a concentration of 40 nM was tested. The expression of CD44 and CD25 was used to monitor T cell developmental stages. (b) The proliferation of OP9-DL1 cells treated with SCD1 inhibitor at indicated doses was measured by CCK8 (n = 5 for each group). (c) Apoptosis in OP9-DL1 cells treated with SCD1 inhibitor at indicated doses was determined by Propidium Iodide/Annexin V staining (n = 4 for each group). (d) Schematic representation of different treatment protocols used in the in vitro T cell developmental system. HSCs with indicated background cultured on OP9-DL1 cells with different treatments were matured into DN3 stages and then co-transferred to Rag2-/- mice. (e) Representative gating strategies (with the spleen as a representative example) for analyzing T cells isolated from Rag2-/- mice receiving DN3 co-transferred as described in Extended Data Fig. 7d. (f) DN3 derived Treg cells in different tissues of Rag2-/- recipient mice were analyzed by flow cytometry (n = 7 mice). (g) The levels of CD4+Foxp3+ Treg cells in the blood, liver, and lungs of mice in Fig. 3e. (h-i) The percentage and absolute number of Helios−RORγt+ and Helios+ RORγt− cells in the colon of mice with treatment as described in Fig. 3e (n = 5 mice for WT-ND, WT-OA; n = 4 mice for KO-DN, KO-OA). Data are presented as Mean ± SEM. NS, not significant; P values were determined by unpaired two-tailed Student’s t-test (b, c, g, h, i) or paired two-tailed Student’s t-test (f).
Extended Data Fig. 8 The impact of Scd1 deficiency on the health status and thymic epithelial cells.
(a) Weight of body, spleen, and MLNs of K14creScd1loxP and Scd1loxP mice (n = 5 mice for each group). (b-c) The whole image and H&E staining of various organs of K14creScd1loxP and Scd1loxP mice. (d) Gating strategies used for identifying cortical thymic epithelial cells (cTEC) and medullar thymic epithelial cells (mTEC) of Scd1+/- and Scd1-/- mice, with subsequent calculation of their absolute numbers (n = 3 mice for each group). (e-f) cTEC and mTEC of Scd1+/- and Scd1-/- mice were isolated and subjected to mRNA sequencing analysis. The expression levels of critical regulators governing thymocyte migration, development, and selection in thymic epithelial cells were shown (e). The differentially expressed genes in cTEC or mTEC of Scd1+/- and Scd1-/- mice were shown in a volcano plot (f). (g) The percentage and absolute number of Helios−RORγt+Foxp3+ and Helios+ RORγt−Foxp3+ cells in the colon of K14creScdloxP (n = 5) and Scd1loxP (n = 7) mice. (h-i) Average MALDI-TOF mass spectra of oleic acid in Fig. 4a. Data are presented as Mean ± SEM. NS, not significant; P values were determined by unpaired two-tailed Student’s t-test.
Extended Data Fig. 9 Evaluation on OT-II T cells developed in K14creScd1loxP mice.
(a) Schematic representation of the experimental design to assess the differentiation potential of OT-II cells developed in K14creScd1loxP or Scd1loxP mice. (b) Gating strategies (with the spleen as a representative example) for identifying transferred OT-II T cells in the recipient mice (c) OT-II T cells developed in K14creScd1loxP or Scd1loxP mice were labeled with CFSE and co-cultured with splenocytes from CD45.1+WT mice in the presence of OVA323-339 peptide. The CFSE dilution in OT-II T cells was measured 96 h post-stimulation. (d-g) CD45.2+CD4+CD62L+TCRVβ5.1+ cells were isolated from the spleen of Scd1loxP or K14creScd1loxP mice and intravenously transferred to CD45.1+ WT recipient mice at a dose of 1 × 107 cells per mouse. To test Treg cell generation, recipient mice were supplied with 2 mg/mL OVA in drinking water for 7 consecutive days and examined for the percentage and number of Foxp3+ OT-II T cells in the spleen, MLNs and colon (n = 5 mice for each group) (d). To test Th1 and Th17 cell induction, recipient mice were s.c. injected with OVA emulsified in CFA and examined for the percentage and number of IL-17- and IFNγ-secreting OT-II T cells in the draining lymph nodes (DLNs) and lungs (n = 7 mice for each group) (e-f). To test Th2 cell induction, recipient mice were i.p. injected with OVA absorbed to alum adjuvant and examined for the percentage and number of IL-4-secreting OT-II T cells in the spleen and liver (n = 4 mice for each group) (g). The percentage and absolute number of distinct OT-II cell subsets were determined. (h-k) OVA323-339 peptide was supplied to the co-culture of CD45.2+CD4+CD62L+TCRVβ5.1+ cells and CD45.1+WT splenocytes to activate OT-II T cells in Th1 (n = 4 biologically independent samples), Th2 (n = 4 biologically independent samples), Th17 (n = 3 biologically independent samples), or Treg (n = 3 biologically independent samples) cell induction medium. Ninety-six hours later, the expression of IFNγ, IL-4, IL-17, or Foxp3 was determined by flow cytometry. Data are presented as Mean ± SEM. NS, not significant; P values were determined by unpaired two-tailed Student’s t-test.
Extended Data Fig. 10 Scd1 deficiency promotes ATP2A2 expression through modulating DOT1L activities.
(a) Heat map display of ATAC-seq data from naïve CD4+ T and DN3 cells. (b) Venn diagram of overlapped differential ATAC peaks related genes. (c) Atp2a2 mRNA levels in naïve CD4+ T cells (n = 4 per group). (d) SCD1 inhibitor A939572 (SCD1i, 40 nM) was supplied to the in vitro T cell developmental system. DN3 thymocytes were harvested for ATP2A2 analysis (repeated twice). (e-f) WT naïve T cells were cultured in the Treg induction medium with or without SCD1i. The ATP2A2 protein (e) and the Treg (f) levels were determined (repeated twice). (g) The statistical analysis of western blot as described in Fig. 6c from 3 independent experiments. (h) Scramble siRNA (NC) or Atp2a2-targeting siRNA was transfected to CD4+CD62L+ T cells. The knockdown efficiency was determined 24 h after transfection (repeated twice). (i) SOCE in Scd1-/-CD4+CD62L+ T cells transfected with Atp2a2 -targeting siRNA or NC was detected. (j-k) WT CD4+CD62L+ T cells were transfected with adenovirus particles to overexpress ATP2A2 (n = 3 for each group) (j). T cells were subsequently cultured in the Treg induction medium for another 3 days to evaluate Treg levels (k). (l) The heat map showing the H3K79me2 levels at the genome of DN3 developed with or without SCD1i. (m) The H3K79me2 levels at the genetic locus of Atp2a2. (n-o) The statistical analysis of Fig. 7f (n) and 7h (o) obtained from 4 independent experiments. (p) SCD1i and DOT1L inhibitor EPZ005676 were added to the in vitro T cell development system. The resultant DN3 thymocytes were examined for ATP2A2 expression (repeated twice). (q) The levels of oleic acid and stearic acid in the nucleus of thymocytes from Scd1-/- or Scd1+/- mice (n = 6 for each group). (r) Palmitoleic acid (n = 4) or Palmitic acid (n = 3) was diluted to the indicated concentrations with 10% DMSO and supplied to an enzymatic reaction catalyzed by DOT1L. Data are presented as Mean ± SEM. NS, not significant; P values were determined by unpaired two-tailed Student’s t-test (c, f, k, q) or paired two-tailed Student’s t-test (n, o).
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Lin, L., Hu, M., Li, Q. et al. Oleic acid availability impacts thymocyte preprogramming and subsequent peripheral Treg cell differentiation. Nat Immunol 25, 54–65 (2024). https://doi.org/10.1038/s41590-023-01672-1
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DOI: https://doi.org/10.1038/s41590-023-01672-1
