SAT0021 CD28null T cells from myositis patients are cytotoxic to autologous muscle cells in vitro

Background T cells are implicated in the disease pathogenesis of dermatomyositis (DM) and polymyositis (PM) but their specificity and effector functions in the inflamed muscle remain unclear. We have previously demonstrated that CD28null T cells are the dominating T cell subset in affected muscle of patients with myositis1,2. These cells are apoptosis resistant3, pro-inflammatory2,4 and both CD4+CD28null and CD8+CD28null T cells from myositis patients contain perforin1, hence are potentially cytolytic. Objectives Here we investigated whether CD28null T cells are directly cytotoxic to autologous muscle cells in vitro Methods Autologous muscle cell - T cell co-cultures were performed in four patients with definite DM or PM and with >10% of CD4+CD28null T cells in peripheral blood. Biopsy specimens were obtained from tibialis anterior muscle. Muscle biopsies were enzymatically digested to obtain myoblasts and differentiated into myotubes. PBMC were isolated into CD4+CD28null, CD4+CD28+, CD8+CD28null and CD8+CD28+ T cell populations by flow cytometry. Before co-culturing, myotubes were labeled with calcein and T cell subsets were stimulated with mitogen PHA. The ratios used for T cells versus myotubes were mostly 5:1 and 30:1 (depending on cell yield). Co-culture supernatants were harvested after 24 hours, and calcein release, mirroring muscle cell lysis (myotoxicity), was measured. The results are expressed as percentage of maximal lysis with detergent Triton X-100, and were confirmed by morphological changes. Results We could demonstrate myotoxicity of CD4+CD28null T cells towards autologous muscle cells (PM1, 34.5%) and the effect was dose dependent (PM2, 43.4% (low ratio), 70% (high ratio); DM1, 15%, 25%; DM2, 2%, 9%). Surprisingly, the myotoxicity of CD8+ T cells was lower than that of CD4+ T cells. Still we could see a dose response also for CD8+CD28null T cells (PM1, 12.1% (low ratio), 39% (high ratio); PM2, 11.9%, 26.8%; DM1 0.7%, 5.1%; DM2 0.6%, 3.8%). Concanamycin A, an inhibitor of perforin-mediated cytotoxicity, reduced the CD28null myotoxicity up to 63%. The corresponding CD28+ T cell subsets demonstrated variable myotoxicity, which was also dependent on the cytokine output from these populations (preliminary data). Overall, myotoxicity was also reflected by morphological abnormalities in myotubes. Conclusions Herein we demonstrate for the first time that the muscle-dominating CD28null T cells harm autologous muscle cells from myositis patients. M