T lymphocyte cytokines distinguish responder from non-responder rheumatoid arthritis patients treated with anti-tumour necrosis factor alpha agents

J Bystrom1, T Taher1, M Al-Bogami1, S Kelly1, P Mangat2, A Jawad1, RA Mageed1. 1Queen Mary University of London, London, UK, 2University of London, Lonon, UK

Biologic anti-TNFα agents have revolutionized the treatment of patients with rheumatoid arthritis (RA). However ~30-40% of RA patients do not respond to treatment with these agents. Better knowledge of patients’ effector inflammatory pathways prior to treatment would guide knowledge-based choice of medication and reduce non-responsiveness to treatment. In this study, we assessed whether the measurement of cytokines in plasma and in culture supernatants of in vitro activated immune cells prior to treatment could predict the response of RA patients prior to treatment with biologic anti-TNFα agents.

Blood was drawn from 70 RA patients before the start of treatment with anti-TNFα and the level of cytokines in plasma and those produced by T lymphocytes and monocytes ex vivo was determined. The clinical response of patients to treatment with anti-TNFα agents was assessed after 12 weeks of treatment by determining changes in disease activity score 28 (DAS-28). Highly enriched T lymphocytes were stimulated with anti CD3/CD28 while monocytes were stimulated with lipopolysaccharide (LPS) for 48hr. The level of key pro-inflammatory and anti-inflammatory cytokines in plasma and culture supernatants was assessed using a multiplex technology (MSD technologies). In parallel, the cells were stimulated with PMA and ionomycin and surface markers and intracellular cytokines analysed by flow cytometry. The difference between responder and non-responder patients was assessed by the Mann-Whitney U test.

The study revealed that the level of GM-CSF was higher in the plasma of responders than non-responders to anti-TNFα agents before treatment (p=0.0257). Furthermore, activated ex vivo T lymphocytes from responder patients produced more GM-CSF that non-responder patients. These experiments also revealed that T lymphocytes produced significantly higher levels of GM-CSF in vitro than activated monocytes. In addition, T lymphocytes from responder patients produced significantly more IL-6 than non-responder patients (p=0.00554). In about 28% of responder patients T lymphocytes produced very high levels of GM-CSF (>2000pg/ml) and these patients also produced the highest levels of IL-6 (p= 0.00939). Assessment of TNFα production revealed that T lymphocytes produced higher level of the cytokine than monocytes. Furthermore, responder patients that produced the highest levels of GM-CSF and IL-6 also produced the highest levels of TNFα (p=0.0233). Intracellular staining for GM-CSF and TNFα revealed that only CD4+ and TNFα+ T lymphocytes produced GM-CSF.

These data indicate that a subset of CD4+ T lymphocytes are present at a higher frequency in the blood of RA patients that respond to biologic anti-TNFα agents compared with non-responder patients. T lymphocytes from these individuals produce high level of GM-CSF, TNFα and IL-6. Measurement of GM-CSF level in plasma could be used as a biomarker for predicting the response of a significant proportion of RA patients to treatment with biologic anti-TNFα agents.