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Glycolysis controls the induction of human regulatory T cells by modulating the expression of FOXP3 exon 2 splicing variants

Abstract

Human regulatory T cells (Treg cells) that develop from conventional T cells (Tconv cells) following suboptimal stimulation via the T cell antigen receptor (TCR) (induced Treg cells (iTreg cells)) express the transcription factor Foxp3, are suppressive, and display an active proliferative and metabolic state. Here we found that the induction and suppressive function of iTreg cells tightly depended on glycolysis, which controlled Foxp3 splicing variants containing exon 2 (Foxp3-E2) through the glycolytic enzyme enolase-1. The Foxp3-E2–related suppressive activity of iTreg cells was altered in human autoimmune diseases, including multiple sclerosis and type 1 diabetes, and was associated with impaired glycolysis and signaling via interleukin 2. This link between glycolysis and Foxp3-E2 variants via enolase-1 shows a previously unknown mechanism for controlling the induction and function of Treg cells in health and in autoimmunity.

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Figure 1: Generation of human iTreg cells from Tconv cells during weak stimulation of the TCR.
Figure 2: Generation of human iTreg cells from Tconv cells during weak stimulation of the TCR in the presence of inhibitors of either glycolysis or FAO.
Figure 3: Biochemical analysis of human iTreg cells.
Figure 4: Phenotypical characterization and metabolic programs of iTreg-CTR, iTreg-2DG and iTreg-Etx cells.
Figure 5: Glycolysis controls expression of the Foxp3-E2 variants, which are indispensable for the suppressive function of iTreg cells.
Figure 6: Recruitment of enolase-1 to FOXP3 regulatory regions controls Foxp3-E2 expression.
Figure 7: Impaired glycolysis, impaired IL-2–IL-2R–STAT5 signaling and altered Foxp3-E2 expression in iTreg cells from subjects with RRMS.
Figure 8: Impaired IL-2–IL-2R–STAT5 signaling and altered Foxp3-E2 expression in iTreg cells from subjects with T1D.

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Acknowledgements

We thank M.R. Montagna for technical support, and all members of the Laboratory of Immunology at Istituto di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche for assistance and support. Supported by the European Union IDEAS Programme European Research Council (“menTORingTregs” 310496 to G.M.), the Fondazione Italiana Sclerosi Multipla (2012/R/11 to G.M.), the Consiglio Nazionale delle Ricerche-Medicina Personalizzata (G.M.), the Ministero della Salute (GR-2010-2315414 to V.D.R.), the Fondo per gli Investimenti della Ricerca di Base (RBFR12I3UB_004 to V.D.R.), the Fondazione Italiana Sclerosi Multipla (2014/R/21 to V.D.R.), the Juvenile Diabetes Research Foundation (1-PNF-2015-115-S-B to M.G.), the US National Institutes of Health (AI109677 to A.L.C.), the PhD Program in Medicina Traslazionale dello Sviluppo e dell'Invecchiamento Attivo, Università degli Studi di Salerno (A.C.), the PhD Program in Medicina Molecolare e Biotecnologie Mediche (M.S.), the PhD Program in Biologia, Università degli Studi di Napoli “Federico II” (A.R.) and the “Fondazione Umberto Veronesi”, Milano (C.Z.).

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Contributions

V.D.R., M.G., A.P., A.L.C. and G.M. designed the study, interpreted data and wrote the manuscript; V.D.R., M.G., A.P., A.C., M.S., C.Z., A.R., S.D.S., C.P. and C.L.R. performed the experiments; V.D.R., M.G., A.P. and A.L.C. analyzed the data and interpreted results; V.D.R., M.G., A.P. and C.P. performed statistical analyses; and P.B.C., G.T.M., M.S., M.C.B., A.F. and E.M. obtained human samples from patients and were involved in discussions about data.

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Correspondence to Giuseppe Matarese.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Experimental procedure used for the generation of human iTreg cells from activated Tconv cells.

Human CD4+ T cells were isolated from PBMCs by negative selection with magnetic beads, and activated with anti-CD3+CD28 beads (0.1 bead/cells) for 36h. Analyses and flow-sorting were performed on the different three regions according to the expression of the CD25 molecule (CD25hi, CD25int and CD25lo).

Supplementary Figure 2 Transwell experiments and stability of iTreg cells’ suppressive properties.

(a) Suppression of T cell proliferation (96 hours) in transwell by flow-sorted iTreg cells. Filled grey histograms correspond to proliferation in vitro of CD4+CFSE-labelled T cells stimulated with anti-CD3+CD28 mAbs; black lines in histograms correspond to proliferation of CD4+CFSE-labelled T cells co-cultured with flow-sorted iTreg cells either in transwell plate or in cell-to-cell contact. Representative experiment of two (left panels) and percent of proliferation of two independent experiments (right panel). Statistical analysis derives from two independent experiments in technical triplicates (n = 6; data are shown as mean ± s.e.m.) *P < 0.05; **P < 0.001 by paired two-tailed Student’s t-test. (b) Methylation of CpG island of FOXP3 CNS2 region evaluated in: freshly isolated Treg cells and Tconv cells, post-sorting CD25- and iTreg cells, iTreg cells upon 10 days of culture with anti-CD3+CD28 mAbs and IL-2 (50 IU/ml). Statistical analysis derives from two independent experiments in technical triplicates (n = 6; data are shown as mean ± s.e.m.) *P < 0.001; NS not significant by two-tailed Student’s t-test. (c) Suppression of T cell proliferation (96 hours) by flow-sorted iTreg cells after 10 days of culture with anti-CD3+CD28 and IL-2 (50 IU/ml). Filled grey histograms correspond to proliferation of CD4+CFSE-labelled T cells stimulated in vitro with anti-CD3+CD28 mAbs; black lines in histograms correspond to proliferation of CD4+CFSE-labelled T cells co-cultured with flow-sorted iTreg cells at different ratios (from 1:1 to 4:1). Representative experiment of three. (d) Representative dot plots showing expression of CD25, CD4 and Foxp3 and other Treg cell markers in flow-sorted iTreg cells after 10 days of culture with anti-CD3+CD28 mAbs and IL-2 (50 IU/ml). Percentages and MFI of positive cells are indicated. Representative experiment of three.

Supplementary Figure 3 Experimental procedure used for the generation of human iTreg cells from Tconv cells in the presence of metabolic inhibitors.

Human CD4+ T cells were isolated from PBMCs by negative selection with magnetic beads, and activated with anti-CD3+CD28 beads (0.1 bead/cells) for 36h in the presence or absence of 2DG or Etx. Flow-sorting was performed on each CD25hi cell population, defined as iTreg-CTR, iTreg-2DG and iTreg-Etx.

Supplementary Figure 4 Splicing variants of Foxp3 in iTreg-CTR, iTreg-2DG and iTreg-Etx cells at various doses of inhibitors.

(a) Representative flow cytometry plots showing levels of Foxp3 in iTreg-CTR, iTreg-2DG and iTreg-Etx cells stained with either Foxp3 Ab PCH101 (against all Foxp3 splicing variants) (upper panels) or Foxp3 mAb 150D/E4 (specific for the Foxp3 exon 2 form) (lower panels). Percentages and MFI of positive cells are indicated. Representative experiment of three. (b) Representative flow cytometry plots showing levels of Foxp3-all (upper panels) and Foxp3-E2 (lower panels) in iTreg cells generated in the presence of different doses of 2DG (250 μM, 500 μM, 1 mM and 2 mM). Representative experiment of three. (c) Representative flow cytometry plots showing levels of Foxp3-all (upper panels) and Foxp3-E2 (lower panels) in iTreg cells generated in the presence of different doses of Etx (50 μM, 100 μM, 200 μM and 400 μM). Representative experiment of three.

Supplementary Figure 5 Silencing of FOXP3 splicing variants and ENO1 mRNA in iTreg cells.

(a) Immunoblotting for Foxp3 (clone PCH101) showing specific silencing on Tconv cells stimulated for 36 hours with anti-CD3+CD28 mAbs in the presence of reported siRNA. One representative of three independent experiments. The graphs show relative densitometric quantitation of each Foxp3 band normalized on total Erk1/2 and shown as fold change over siRNA scrambled cells. Statistical analysis derives from three independent experiments in technical triplicates (n = 9; data are shown as individual data points and means) *P < 0.001; NS not significant by two-tailed Student’s t-test. (b) Western blotting analysis of cytoplasmic enolase-1 (48 kDa) levels in freshly isolated Tconv cells and iTreg-CTR cells. One representative experiment of two. Graph shows the absorbance values, shown as fold over Tconv cells, normalized on total Erk1/2. Statistical analysis derives from two independent experiments in technical triplicates (n = 6; data are shown as individual data points and means) *P < 0.01 by Wilcoxon. (c) RT-PCR for ENO1 showing specific silencing on iTreg-CTR and iTreg-2DG in the presence of reported siRNA. The graphs show ENO1 mRNA levels 18S-normalized and shown as fold over iTreg-CTR siRNA scrambled cells. Statistical analysis derives from two independent experiments in technical triplicates (n = 6; data are shown as mean ± s.e.m.) *P < 0.0001 by paired two-tailed Student’s t-test.

Supplementary Figure 6 Characterization of CD4+Foxp3-E2+ T cells from PBMCs of human healthy donors.

(a) Percentage of Foxp3-all+ cells (black column) and Foxp3-E2+ cells (grey column) in freshly isolated PBMCs of human healthy donors (n = 19), gated on CD4+ cells, (cumulative data are shown as mean ± s.e.m.) *P < 0.0001 by paired two-tailed Student’s t-test. (b) Representative dot plots showing expression of Treg cell-specific markers in freshly isolated PBMCs of human healthy donors, gated on CD4+Foxp3-all cells (upper panel) and CD4+Foxp3-E2 cells (lower panel). MFI and percent of expression of reported markers are indicated. Representative experiment of three. (c) Percentage of expression of Treg cell-specific markers in freshly isolated PBMCs of human healthy donors, gated on CD4+Foxp3-all cells and CD4+Foxp3-E2 cells. Statistical analysis derives from three independent experiments, data are shown as individual data points and means (n = 7). **P < 0.005; *P < 0.05; NS not significant by paired two-tailed Student’s t-test. (d) Percentage of ki67+ and p-S6+ cells gated on CD4+Foxp3-all cells and CD4+Foxp3-E2 cells in freshly isolated PBMCs of human healthy donors. One representative plot out of four (left panel). MFI and percentage of expression of reported markers are indicated. Statistical analysis derives from cumulative data of four independent experiments, data are shown as individual points and means (n = 21 for ki67, n = 11 for p-S6) (right panel) **P < 0.005 by paired two-tailed Student’s t-test.

Supplementary Figure 7 Selective impairment of glycolysis and Foxp3-E2 expression in iTreg cells from autoimmune subjects.

(a) Cumulative data of Tconv cells proliferation after anti-CD3+CD28 mAbs stimulation, in RRMS (n = 25) and healthy (n = 42) subjects, measured as (3H) thymidine incorporation. Data are shown as mean ± s.e.m.; NS not significant by Wilcoxon. (b) Basal glycolysis (after glucose addition), maximal glycolysis (after oligomycin addition) and glycolytic capacity (calculated as the difference of oligomycin-induced ECAR and 2DG-induced ECAR) of Tconv cells from naive-to-treatment RRMS (n = 6) and healthy subjects (n = 14). Cumulative data are expressed as mean ± s.e.m. *P < 0.001 by Wilcoxon. (c) Western blot analysis of Foxp3-E2 protein after 24 and 36 hours of in vitro stimulation with anti-CD3+CD28 mAbs of Tconv cells from RRMS and healthy subjects. Graphs show the relative densitometric quantitation of Foxp3-E2 bands normalized to total Erk1/2, shown as fold change over Tconv cells from healthy donor, 24 hour after in vitro stimulation with anti-CD3+CD28 mAbs. One representative of three independent experiments. Statistical analysis derives from three independent experiments performed on three healthy and three RRMS subjects, in technical triplicates (n = 9; data are shown as individual data points and means) *P < 0.001 by Wilcoxon. (d) Western blot analysis of Foxp3-E2 protein after 24 and 36 hours of in vitro stimulation with anti-CD3+CD28 mAbs of Tconv cells from T1D and healthy subjects. Graphs show the relative densitometric quantitation of Foxp3-E2 bands normalized to total Erk1/2, shown as fold change over Tconv cells from healthy donor, 24 hour after in vitro stimulation with anti-CD3+CD28 mAbs. One representative of three independent experiments. Statistical analysis derives from three independent experiments performed on two healthy and three T1D subjects in technical triplicates (n = 6 for healthy and n = 9 for T1D; data are shown as individual data points and means) **P < 0.005; NS not significant by Wilcoxon.

Supplementary Figure 8 Model of the glycolytic control of Foxp3-E2 splicing variants in human iTreg cells in health and autoimmune diseases.

Tconv cells from healthy subjects engage glycolysis during in vitro weak-TCR stimulation, which leads to generation of fully suppressive iTreg cells representing the highly proliferative and glycolytic fraction of Tconv cells (CD25hi, ki67+, p-S6+, Foxp3-E2+) (upper panel). On the contrary, in subjects with autoimmunity, Tconv cells show an impaired glycolysis during TCR stimulation, which accounts for an altered Foxp3-E2 induction, reduced generation and function of iTreg cells and loss of immune tolerance (lower panel).

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De Rosa, V., Galgani, M., Porcellini, A. et al. Glycolysis controls the induction of human regulatory T cells by modulating the expression of FOXP3 exon 2 splicing variants. Nat Immunol 16, 1174–1184 (2015). https://doi.org/10.1038/ni.3269

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