Fig 2: SLC5A12 Expression by CD4+ T Cells Is Regulated by Activating and Inflammatory Stimuli(A–C) Representative flow cytometry plots of SLC5A12 expression by CD4+ or CD8+ T cells from non-activated (n = 3; A) or anti-CD3 mAb-activated (n = 6; B) HC PBMCs. Quantification shown in (C).(D–F) Representative flow cytometry histograms (D and E) and quantification (F) of SLC5A12 expression by CD4+ T cells from non-activated HC (n = 4) and RA (n = 4; D and F), or anti-CD3 mAb-activated HC (n = 4) and RA (n = 5; E and F) PBMCs. CD4+ T cells from non-activated RA SFMCs (n = 8; E and F) were also analyzed. Briefly, PBMCs were cultured in RPMI medium supplemented with 5% RA or HC autologous blood serum (BS), or 5% RA synovial fluid (SF); SFMCs were cultured in RPMI medium supplemented with 5% autologous SF.(G) Representative flow cytometry histograms (left) and quantification (right) of SLC5A12 expression by CD4+ T cells from non-activated or anti-CD3 mAb-activated RA SFMCs. Briefly, cells were cultured in RPMI medium supplemented with 5% FBS (n = 3), 5% autologous BS (n = 8) or 5% autologous RA SF (n = 8). Activated RA PBMCs cultured in 5% BS RPMI (n = 5) were used as controls (H). MFI, mean fluorescent intensity.(H) Representative flow cytometry histograms (left) and quantification (right) of SLC5A12 expression by CD4+ T cells from RA SFMCs (n = 5) incubated with 3C7 mAb or control rat sera.(I and J) SLC5A12 mRNA (n = 5; I) and protein (representative western blots [left] and densitometric quantification [right; n = 3]; J) expression by CD4+ T cells isolated from HC PBMCs, then activated with anti-CD3 and anti-CD28 mAb in the presence of sodium lactate (10 mM) and/or SLC5A12 Ab, or left untreated. Lactate-untreated CD4+ T cells (CN, dotted line) set to 1.One-way ANOVA (C and F) or two-tailed Student’s t test (G–J). Data expressed as mean ± SEM. *p = 0.05; **p = 0.01; ***p = 0.001. See also Figures S1, S2, and S3.

Fig 3: Lactate Uptake by CD4+ T Cells Impacts Intracellular Utilization of Central Carbon Metabolic Pathways(A) Glucose and glutamine uptake rates for CD4+ T cells isolated from HC PBMCs, then activated with anti-CD3 and anti-CD28 mAbs for 24 h followed by further 48-h culture with lactate alone or in the presence of SLC5A12 Ab, or left untreated, in medium containing low glucose (5 mM) and 5% FBS (n = 3, each in duplicate).(B) NAD+ and NADH intracellular levels in CD4+ T cells (n = 2) treated with sodium lactate (10 mM) for the indicated time points after 72-h activation and shown as NAD+/NADH ratio. Lactate-untreated CD4+ T cells (CN, dotted line) set to 1.(C) Seahorse measurements of extracellular acidification (left) and oxygen consumption (right) rates (ECAR and OCR, respectively) by 12-h-activated CD4+ T cells (n = 3, technical replicates). 1 h prior to the experiment, cells were seeded in a 96-well microplate in XF Assay medium in the presence of 10 mM of glucose. Sodium lactate (10 mM) or PBS was injected during measurement. Data representative of n = 2 independent experiments.(D and E) 13C tracing of [U13C]-lactate into pyruvate and citrate. Activated CD4+ T cells were incubated for 48 h with [U13C]-lactate in the presence or absence of SLC5A12 Ab in medium containing low glucose (5 mM) and 5% FBS (n = 2, each in duplicate). Polar metabolites were extracted, analyzed by LC-MS and peak areas of mass isotopologues normalized to cell number are represented.(F and G) Acetyl-CoA (F) and citrate (G) intracellular levels in CD4+ T cells (n = 3) treated with sodium lactate (10 mM) for the indicated time points after 72-h activation. Lactate-untreated CD4+ T cells (CN, dotted line) set to 1.Two tailed Student’s t test. Data expressed as mean ± SEM. *p = 0.05; **p = 0.01; ***p = 0.001.