Background RA CD8+ T cells (CD8s) present a pro-inflammatory, cytotoxic effector phenotype and may, therefore, play a major role in disease pathogenesis. The presence of such effector CD8s in the blood and synovial fluid suggests that they must adapt their metabolism to sustain their functional energetic demands. Thus, we hypothesised that modulation of CD8s-metabolism, by blocking essential glycolytic and citric acid cycle enzymes, might alter their phenotype and function. The ultimate goal was to explore the potential of this metabolic modulation as novel therapeutic opportunity in RA.
Materials and methods 35 RA patients and 16 age and gender matched healthy controls (HC) were recruited at the Heidelberg University Hospital. Blood CD8s were purified by negative magnetic separation selection and CFSE labelled. Anti-human CD28/CD3 stimulated CD8s were cultured in vitro for 3 days in medium containing [1,6-13C] glucose. After 24 hours, enzyme blockers were added in non-toxic concentrations. Proliferation, subset distribution, CD57, CD147, CD25, CD137, TIM-3 and PD-1 expression were assessed by FACS. Cytokines were quantified by cytometric bead array. Changes in lactate production were monitored by1H-NMR spectroscopy.
Results Upon inhibition of hexokinase II (3-Bromopzruvate, 3 BP), LDHA (FX11) and pyruvate dehydrogenase complex (CPI-613) both RA and HC CD8s significantly lost proliferative activity. We observed a loss of activity and cytotoxicity marker CD137, the effector function marker (CD25) and the exhaustion marker PD-1. The matrix metalloproteinase inductor and regulator of transmembrane monocarboxylate transport basigin (CD147) showed a strong downregulation. Interestingly, the lactate-dehydrogenase A (LDHA) blocker FX11, induced the strongest down-modulation of these surface molecules in RA CD8s. Metabolic interference also lead to changes in cytokine production. Concentrations of proinflammatory proteins IL-17 α, IL-6, IFN-γ, TNF-α, Perforin and VEGF were lower in the presence of 3 BP, FX11 and CPI-613. 1H-NMR spectroscopy showed diminished newly-synthesised lactate levels in cell supernatants.
Conclusions Metabolically altered CD8s presented a less proliferative, less pro-inflammatory and cytotoxic profile with lower de novo synthesis of lactate. Moreover, RA CD8s seemed to be more susceptible upon LDHA blockage than HC. This might indicate that CD8s metabolism, especially glycolytic pathways present a possible anti-inflammatory therapeutic target.
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