Fig 1.
Kinetics of proliferation and FOXP3 expression in human Treg subsets post activation.
(A) FACS isolated Tregs and naïve CD4+ T conventional cells were cultured in Treg expansion conditions (red) and iTreg generation conditions (blue), respectively. (B) Cells were stained with Cell Trace Violet and their dilution was compared at day 3 post activation in representative histograms and proliferation index that indicates mean frequency of cell division (n = 9 from 6 individual experiments). (C) Line plot data shows population doubling during in vitro culture of 5 different donors from 2 independent experiments. Plotted number and s.d. bars indicate mean population doublings and s.d., respectively. (D) FOXP3 expression by flow analysis over time. Data, mean ± s.d. of at least 5 independent donors for each time points from 6 individual experiments, are expressed as the MFI ratio of FOXP3. The MFI ratio of FOXP3 were calculated as MFI of FOXP3 divided by MFI of isotype control on live CD4+ cells.
Fig 2.
Metabolic traits of in vitro activated human tTregs and iTregs.
Real-time measurements of (A) extra cellular acidification rates (ECAR) and (B) oxygen consumption rates (OCR) of activated tTregs (red) and iTregs (blue) were assessed in response to glucose, oligomycin, and 2-deoxy-D-glucose (2DG) (glycolysis stress test, A) and oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and rotenone/ antimycin (mito-cell stress test, B) at day 3 and 7 post activation, respectively. Representative real-time data are shown from 5 individuals for day 3 and 4 individuals for day 7 (mean ±s.d. of n = 3 samples per group). (C) Metabolic profiling data of day3 (left side) and day 7 (right side). Glycolysis and glycolytic capacity from glycolysis stress test and basal OCR, MAX OCR and OCR/ECAR ratio from mito-cell stress test, are plotted with mean line and s.d. bar, respectively. Statistical analysis shown only on significant different data (*P<0.05, **P<0.01 ***P<0.001, One way ANOVA followed by Tukey’s multiple comparison).
Fig 3.
The timing of 2DG treatment has distinct effects on the proliferation of Treg subsets.
tTregs (red) and iTregs (blue) were cultured with 0.5mM 2-deoxy-D-glucose (2DG) from onset to day 3 post activation (n = 9). (A) 2DG effect on survival of human Treg subsets was analyzed by annexin-7AAD staining. Impact of Inhibiting glycolysis on (B) cell biomass by forward scatter, and (C) proliferation by Cell Trace Violet dilution assay were assessed. Data are shown as non-treated control (Cont) and treated Tregs (tTreg 2DG or iTreg 2DG) as solid and dot lines in representative histograms, respectively, with isotype control (filled gray). In bar graphs, solid (Cont, non-treated) and checked (2DG, treated) boxes indicate the mean ±s.d. (D) Line plot data show mean fold expansion rate (± s.d.) treated with (dot) or without (solid) 0.5mM 2DG from day 3 post activation (n = 5). Representative data is shown from at least 3 independent experiments. Statistical analyses were performed by Student’s t-test or One way ANOVA followed by Tukey’s multiple comparison (*** P<0.001 is shown only on significant different data in multiple comparison).
Fig 4.
Critical requirement of glucose metabolism in Tregs for optimal expression of functional molecules during initial activation.
0.5mM 2-deoxy-D-glucose (2DG, used to inhibit glycolysis, was added from the onset to day3 (A, B and D), and from 3 to 7 days post activation (C) in tTreg (red) and iTreg (blue). Flow analyses for FOXP3, CD25, CTLA-4 and ICOS were performed gated on live CD4+ cells. Data are shown as non-treated control and 2DG treated as solid and dot lines in representative histograms with isotype control staining (filled gray). Solid (non-treated, Cont) and checked (2DG treated, 2DG) boxes with bar graph indicates the mean fluorescence intensity (MFI) ±s.d.. (B) Quantitative PCR analysis of all FOXP3 mRNA were performed with post sorted naïve T conventional cells (Naïve) and 3 days cultured products (tTreg and iTreg±2DG). Representative data is shown from at least 3 independent experiments. Statistical analyses were performed by Student’s t-test or One way ANOVA following Tukey’s multiple comparison (*P<0.05, *** P<0.001 is shown only on significant different data in multiple comparison).
Fig 5.
2DG treatment at day 3 did not affect suppressive capability of Treg subsets.
Suppression assays were performed with 7 days cultured tTreg (red) and iTreg (blue) after they were rested in 50IU/ml IL-2 medium for an additional 2 days prior to assay. Autologous CD4 enriched T responders were stimulated by CD3/CD28 microbeads at 1:5 bead to cell ratio for 84hrs in absence or presence of suppressors indicated effector to suppressor ratio. Representative histogram gated on live CD4+ responder show the CFSE dilution profiles with stimulation control (open black) and unstimulated control (filled grey). % inhibition of CD4+ T cell proliferation was calculated as %proliferating (stimulated-sample)/ %proliferating cells (stimulated—unstimulated). Representative data is shown from 3 independent experiments of total 6 individual donors with experimental duplicates.
Fig 6.
Effect of 0.5mM 2DG on T conventional cells.
Th0 cells (green) culturing performed with 0.5mM of 2-deoxy-D-glucose from onset to day 3 post activation (A and B) or from day 3 to day 7 post activation (C and D). Cell proliferation (A) and survival (B) status were analyzed at day3 post activation by cell tracer dilution assay and Annexin-7AAD, respectively. (C) Line plot data show mean fold expansion rate (± s.d.) treated with (dot) or without (solid) 2DG (n = 6). Dots plot show the individual plots of fold expansion rate at day 7 post activation. (D) Representative plots show % IFN gamma producing cells in live Th0 cells treated with or without 2DG. Solid (non-treated, Cont) and checked (2DG treated, 2DG) boxes with bar graph indicates the mean fluorescence intensity (MFI) ±s.d.. Representative data is shown from at least 3 independent experiments of total 6 individual donors. Statistical analyses were performed by Student’s t-test.