Background/Purpose:  Studies by our team and others have shown that CD8 T cells (CD8s) in RA have a pro-inflammatory, cytotoxic effector phenotype and may, therefore, play a major role in RA. The presence of such effector CD8s in the blood and synovial fluid suggests that they have to adapt their metabolism to sustain their functional energetic demands in both oxygenated and in oxygen-deprived environments. Thus we focused on the characterization of the metabolic processes by ¹³C NMR isotopomer analysis (to simultaneously monitor the glycolytic and citric acid cycle fluxes) in CD8s from RA, PsA and SpA patients comparing to healthy controls (HC). We also assessed whether metabolic changes accompanied alterations in the production of cytokines and cytotoxic molecules. The ultimate goal was to explore the potential of CD8s metabolic processes as new diagnostic tools to distinguish RA from PsA and SpA.

Methods:  50 RA, 17 PsA and 20 SpA patients (all fulfilling the ACR criteria), and 21 age and gender matched HC where recruited at the Heidelberg University Hospital. Blood CD8s were purified by negative selection magnetic separation. CSFE-labeled CD8s were cultured in vitro for 3 days in the presence of anti-human CD28/CD3, in medium containing [U-¹³C]glucose. Proliferation, subset distribution and Caspase 3 and PD-1 expression were assessed by FACS. Cytokines and cytotoxic molecules were quantified by cytometric bead arrays. Changes in metabolic enzymes were quantified by western blot. Changes in lactate and acetate production were assessed by ¹H-NMR.

Results:  At rest, in HC, SpA and PsA patients levels of [U-¹³C]lactate, derived from the [U-¹³C]glucose in the media, were quite low, denoting a basal metabolism not dominated by aerobic glycolysis and consistent with low biosynthetic activity. In contrast, in RA patient’s unstimulated CD8s the [U-¹³C]lactate levels were significantly higher, compatible with higher energetic and biosynthetic/proliferative demands even at rest. Upon in vitro activation levels of [U-¹³C]lactate in the medium increased significantly, particularly in RA and PsA patients, and were consistent with an activation of aerobic glycolysis to cope with the greater biosynthetic demands for phenotypic changes, i.e. the transition from an naïve (CD45RA⁺CCR7⁺) to an effector phenotype (CD45RA⁺CCR7^–), higher proliferation, and production of pro-inflammatory cytokines (TNF-α; IFN-γ IL-6) and cytotoxic molecules (Perforin; Granzyme B). This profile of arthritic CD8s was confirmed by a significantly higher expression of the enzymes pyruvate kinase M2, hexokinase 2 and lactate dehydrogenase, and reduced glutamate dehydrogenase expression. Receiver operating curves based on the levels of IL-6, TNF-α, Granzyme B, Perforin and [U-¹³C]lactate could distinguish RA patients from PsA and SpA patients with high sensitivity and specificity (>70%).

Conclusion:  CD8s from chronic arthritis patients present a Warburg-like metabolic profile, which may allow them to adapt to the environment of the inflamed synovium, and exert their pro-inflammatory and cytotoxic functions. More importantly, this altered CD8s metabolic profile provided a differential diagnosis for RA (including seronegative) from PsA and SpA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/specific-metabolic-and-functional-changes-in-peripheral-cd8-t-cells-specifically-distinguish-ra-from-psa-and-spa/