Loren Kell1, Samuel Taylor2, Kavina Shah2, Roel De Maeyer2, Debajit Sen2, Madhura Castelino2, Jo Cambridge2, David Isenberg2, Maria Leandro3, Arne Akbar2 and venkat Reddy2, 1University College London, Oxford, United Kingdom, 2University College London, London, United Kingdom, 3University College London/UCLH, London, United Kingdom
Background/Purpose: Activated T cells make a significant contribution to inflammation in systemic lupus erythematosus (SLE). We know that cellular metabolism regulates the activation of T cells. Evidence from patients with multiple sclerosis indicates that dimethyl fumarate (DMF), an electrophile, targets cellular metabolism to modulate CD4+T cell activation and function.
Here, we investigated whether DMF modulates T cell metabolism and function in samples from patients with SLE in a series of in vitro experiments.
Methods: All experiments were performed using isolated T cells from freshly drawn whole blood samples from patients with SLE. T cells were isolated using negative selection using Stem cell or Miltenyei magnetic bead separation kit. Isolated T cells were activated with anti-CD3 and IL-2 and incubated with either DMF at 25μM concentration or DMSO alone for three or seven days at 37°C and 5% CO2 before harvesting.
Results: In Seahorse experiments, after three days of incubation dimethyl fumarate (DMF) inhibited Figure A-i) the oxygen consumption rate (OCR) and A-ii) extra cellular acidification rate (ECAR) in isolated T cells when compared with samples incubated with vehicle, dimethyl sulfoxide (DMSO). Our results revealed that DMF significantly inhibited: 1) aerobic glycolysis and oxidative phosphorylation in activated T cells from patients with SLE (n=4) (Figure A- i and ii), in vitro; 2) T cell activation and proliferation as assessed by a reduction in the frequency of CD69 (n=4) and D) Ki67 (n=2) positivity, respectively (Figure B-E); 3) Collectively, these results suggest that DMF inhibits T cell activation and proliferation in samples from patients with SLE.
After 7 days of incubation, DMF significantly inhibited the expression of activating NK receptors CD158a and NKG2D on T cells whereas there was a trend toward enhancing the expression of inhibitory NK receptors NKG2A and CD158b (Figure F-I).
After 7 days of incubation, DMF significantly reduced intracellular expression of IFN-γ, TNF-α, IL-17 and secretion of pro-inflammatory cytokines IFN-γ and TNF-α in supernatants (Figure J – N).
Conclusion: Our preliminary data indicated that DMF modulates metabolic programming to inhibit activation, proliferation, and secretion of proinflammatory cytokines from T cells patients with SLE.
DMF inhibits glycolysis and effector functions of CD4+ T cells from patients with SLE. All experiments were performed using isolated T cells from freshly drawn whole blood samples from patients with SLE. T cells were isolated using negative selection using Stem cell or Miltenyei magnetic bead separation kit. Isolated T cells were activated with anti-CD3 and IL-2 and incubated with either DMF dissolved in 0.0025% DMSO to achieve 25μM concentration or DMSO alone for three or seven days at 37°C and 5% CO2 before harvesting. A-I) In Seahorse experiments, after three days of incubation dimethyl fumarate (DMF) inhibited the oxygen consumption rate (OCR) and A-ii) extra cellular acidification rate (ECAR) in isolated T cells when compared with samples incubated with vehicle, dimethyl sulfoxide (DMSO). These results revealed that DMF significantly inhibited aerobic glycolysis and oxidative phosphorylation in activated T cells from patients with SLE (n=4), in vitro.
B) Flow cytometry gating strategy to analyse for activated T cells CD3+CD69+ and proliferating T cells CD3+Ki67+. C) After three days of incubation, DMF significantly inhibited T cell activation and proliferation as assessed by a reduction in the frequency of CD69 (n=4) and D) Ki67 (n=2) positivity, respectively. E-i) After 7 days of incubation, the density of T cells at 10 X magnification in flat bottom wells without DMF was higher than E-ii) in the well in the presence of DMF. Collectively, these results suggest that DMF inhibits T cell activation and proliferation in samples from patients with SLE. After 7 days of incubation, DMF significantly inhibited the expression of activating NK receptors F) CD158a and G) NKG2D on T cells whereas DMF seemed to have a trend toward enhancing the expression of inhibitory NK receptors H) NKG2A and I) CD158b. After 7 days of incubation, DMF significantly reduced intracellular expression of J) IFN-γ, K) TNF-α, L) IL-17 and secretion of pro-inflammatory cytokines M) IFN-γ and N) TNF-α in supernatants. Thus our preliminary data indicated that DMF modulates metabolic programming to inhibit activation, proliferation and secretion of proinflammatory cytokines from T cells patients with SLE.
L. Kell: None; S. Taylor: None; K. Shah: None; R. De Maeyer: None; D. Sen: None; M. Castelino: None; J. Cambridge: None; D. Isenberg: None; M. Leandro: Roche Basel, 1; A. Akbar: None; v. Reddy: None.
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