Jayesh M. Pandya

T Cell Infiltrates in the Muscles of Patients with Dermatomyositis and Polymyositis Are Dominated by CD28null T Cells

Dermatomyositis and polymyositis are disabling rheumatic diseases characterized by an appreciable number of T cells infiltrating muscle tissue. The precise phenotype, function and specificity of these cells remain elusive. In this study, we aimed to characterize T cells in muscle tissue and circulation and to investigate their association to clinical phenotype. Twenty-four patients with dermatomyositis and 42 with polymyositis were screened for frequency of CD4+CD28null and CD8+CD28null T cells in peripheral blood by flow cytometry. Presence of these cells in inflamed muscle tissue from 13 of these patients was analyzed by three-color immunofluorescence microscopy. Effector functions, proliferation and Ag specificity were analyzed by flow cytometry after in vitro stimulation. The clinical relevance of CD28null T cells was analyzed by multiple regression analyses including six separate and combined disease variables. We demonstrate that muscle-infiltrating T cells are predominantly CD4+CD28null and CD8+CD28null T cells in patients with dermatomyositis and polymyositis. Muscle-infiltrating CD28null T cells were found already at time of diagnosis. Disease activity correlated with the frequency of CD8+ T cells in the inflamed muscles of polymyositis patients. Circulating CD4+CD28null and CD8+CD28null T cells were significantly more frequent in human CMV (HCMV) seropositive individuals, responded to HCMV Ag stimulation, and correlated with disease duration. These cells also display a proinflammatory cytokine profile, contain perforin and lack the costimulatory molecule CD28. Our observations imply that CD28null T cells represent clinically important effector cells in dermatomyositis and polymyositis, and that HCMV might play a role in propagating disease in a subset of patients.

Expanded T cell receptor Vβ-restricted T cells from patients with sporadic inclusion body myositis are proinflammatory and cytotoxic CD28null T cells.

OBJECTIVE Sporadic inclusion body myositis (IBM) is characterized by T cell infiltrates in muscle tissue, but their functional role is unclear. Systemic signs of inflammation are lacking, and the absence of beneficial effects following immunosuppression has challenged the notion of a role for the immune system. This study was undertaken to investigate the phenotype and functionality of T cells, specifically a subset of proinflammatory, cytotoxic, and apoptosis-resistant T cells defined as CD28(null) T cells, in the pathogenesis of sporadic IBM. METHODS A cohort of 27 patients with sporadic IBM was analyzed for the frequency of circulating and muscle-infiltrating CD28(null) T cells. The T cell receptor (TCR) V(β) usage was determined using flow cytometry and immunohistochemistry. Anti-CD3-stimulated peripheral blood mononuclear cells were analyzed for intracellular interferon-γ and cytotoxic potential by flow cytometry. RESULTS We found striking accumulations of both CD8+CD28(null) and CD4+CD28(null) T cells, which represented the TCR V(β) -expanded T cells in sporadic IBM. Such CD28(null) T cells were abundant both in the inflamed muscle tissue and in the circulation. Although the specific TCR V(β) expansions varied between patients, both CD8+CD28(null) and CD4+CD28(null) T cells consistently displayed a highly proinflammatory and cytotoxic potential. CONCLUSION Our results suggest that CD28null T cell expansions represent the previously described expanded T cell subsets in sporadic IBM, and their proinflammatory capacity and presence in both muscle tissue and the circulation may imply a role of immune activation in sporadic IBM. In addition, CD4+CD28(null) T cells may exert cytotoxic effects directly on muscle fibers due to a cytotoxic potential similar to that in CD8+ T cells.

CD4+ and CD8+ CD28(null) T Cells Are Cytotoxic to Autologous Muscle Cells in Patients With Polymyositis.

Inflammatory T cell infiltrates in the skeletal muscle tissue of patients with polymyositis are dominated by CD28‐negative effector (CD28null) T cells of both the CD4 and CD8 lineage. These cells are potentially cytotoxic, and the aim of the present study was to develop a fully autologous cell culture system in which to investigate the functional contribution of such CD28null T cells to myotoxicity.

Activated LTB4 pathway in muscle tissue of patients with polymyositis or dermatomyositis

Objective To investigate the involvement of the leukotriene B4 (LTB4) pathway in polymyositis (PM) and dermatomyositis (DM) and the effect of immunosuppressive treatment on the LTB4 pathway. Methods 5-lipoxygenase (5-LO), 5-LO activating protein (FLAP) and LTB4 receptor-1 (BLT1) expression was analysed by immunohistochemistry in muscle tissue from patients with PM/DM before and after immunosuppressive treatment and from healthy individuals. In vivo LTB4 in thigh muscle was measured by microdialysis at rest and after acute exercise in another cohort of patients and healthy controls. Results The number of 5-LO-positive cells and BLT1-positive capillaries was higher in patients with PM/DM than in healthy individuals. The number of FLAP-expressing cells divided the patients into two groups (high/low expression). Treatment reduced the number of FLAP-positive cells in the group with initial high levels, however the expression remained high compared with healthy individuals. The number of BLT1-positive cells was also reduced while staining for 5-LO was unchanged. An inverse correlation was observed between the number of 5-LO or FLAP-positive cells in muscle tissue and muscle performance. LTB4 could be detected in dialysate of muscle tissue in vivo in both patients and healthy controls and was significantly increased after exercise in patients. Conclusion The LTB4 pathway is upregulated in muscle tissue from patients with PM/DM and this upregulation correlated negatively to muscle performance, suggesting a role for LTB4 in myositis muscle weakness. The immunosuppressive treatment was insufficient on the LTB4 pathway and, for patients with high expression of FLAP, FLAP inhibitors may be considered as possible therapy.

CD4+ and CD8+ CD28null T cells are Cytotoxic to Autologous Muscle Cells in Polymyositis Patients

Inflammatory T cell infiltrates in the skeletal muscle tissue of patients with polymyositis are dominated by CD28‐negative effector (CD28null) T cells of both the CD4 and CD8 lineage. These cells are potentially cytotoxic, and the aim of the present study was to develop a fully autologous cell culture system in which to investigate the functional contribution of such CD28null T cells to myotoxicity.

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

Background/purpose 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. The authors have previously demonstrated that CD28null T cells are the dominating T cell subset in affected muscle of patients with myositis. These cells are apoptosis resistant, pro-inflammatory and both CD4+CD28null and CD8+CD28null T cells from myositis patients contain perforin, hence are potentially cytolytic. Here, the authors investigated whether CD28null T cells are directly cytotoxic to autologous muscle cells in vitro. Method 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 h, 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. Result The authors 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 were lower than that of CD4+ T cells. Still, the authors could see a dose response also for CD8+CD28+ 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%). The corresponding CD28+ T cell subsets demonstrated variable myotoxicity and our preliminary data suggest that this is directly dependent on cytokine output from these populations. Overall, myotoxicity was also reflected by morphological abnormalities in myotubes. Conclusion Herein, the authors demonstrate for the first time that the muscle-dominating CD28null T cells harm autologous muscle cells from myositis patients. More experiments are needed to establish whether direct cytotoxicity or cytokine-mediated effects is the dominant pathway for myotoxicity. Irrespective of that outcome, theses data strengthen the notion that CD28null T cells may cause muscle fiber destruction and hence directly contribute to the chronic inflammation in myositis.

AB0023 Sex-based differences in association between circulating t cell subsets and disease activity in untreated early rheumatoid arthritis patients

Background Genetic association studies strongly support the role of CD4+ T cells in promoting RA pathology. In a cohort of untreated early RA patients, we recently demonstrated that the balance of helper T cell subsets in blood of ueRA patients is skewed towards Th2 cells relative to healthy controls.1 RA has been shown to be a sexually dimorphic condition with current data suggesting that prevalence, disease course and treatment outcome varies between men and women. Objectives It is not known if sex-based disparities in immunological factors contribute to the disease process in rheumatoid arthritis (RA). Hence, we examined whether circulating T cell subset proportions and their association with disease activity differed in male and female patients with untreated early rheumatoid arthritis. Methods Proportions of T cell subsets were analysed in peripheral blood from 70 ueRA DMARD and prednisolone naïve patients with untreated early Rheumatoid arthritis (50 females and 20 males) and in 31 healthy age-matched controls. Broad analysis of helper and regulatory CD4+ T cell subsets was done using flow cytometry. Disease activity in patients was assessed using DAS28, CDAI, swollen joint counts, tender joint counts, CRP and ESR. Results Multivariate factor analyses showed that male and female untreated early rheumatoid arthritis patients display distinct profiles of association between disease activity and circulating T cell subset proportions. In male, but not female, ueRA patients Th2 cells showed a positive association with disease activity and correlated significantly with DAS28-ESR, CDAI and tender joint counts. Likewise, proportions of non-regulatory CTLA-4+ T cells associated positively with disease activity in male patients only, and correlated with DAS28-ESR. In contrast, there was a negative relation between Th1Th17 subset proportions and disease activity in males only. Proportions of Th1 and Th17 cells showed no relation to disease activity in either males or females. There were no significant differences in proportions of T cell subsets between the sexes in patients with untreated early rheumatoid arthritis. Conclusions In conclusion, our findings show sex-based differences in the association between T cell subsets and disease activity in ueRA patients, and that Th2 helper T cells may have a stronger role in the regulation of disease activity in male patients. Reference [1] Pandya JM, et al. Circulating T helper and T regulatory subsets in untreated early rheumatoid arthritis and healthy control subjects. J Leukoc Biol2016;100(4):823–833. Disclosure of Interest J. Aldridge: None declared, J. Pandya: None declared, L. Meurs: None declared, A. Kerstin: None declared, I. Nordström: None declared, E. Theander Employee of: Janssen Cilag, A.-C. Lundell: None declared, A. Rudin Consultant for: AstraZeneca

A3.11 Immunosuppressive Effects of Glucocorticoids and Regulatory T Cells on CD28null T Cells in Vitro

Background and Objectives CD28null T cells are terminally differentiated T cells lacking CD28 co-receptor. These cells display properties of proinflammatory killer cells and are suggested to be resistant to apoptosis in vivo. Frequencies of CD28null T cells are increased in various chronic, inflammatory diseases. CD28null T cells dominate both in the affected muscle and peripheral blood of patients with idiopathic inflammatory myopathies (myositis), suggesting a role these cells in disease mechanism and muscle pathology. Recently, it was found in our lab that after conventional glucocorticoid treatment, the relative number of regulatory T cells (Tregs) was unchanged or decreased, while the CD28null T cell proportion was mainly increased in muscle tissue of myositis patients. This lead to our working hypothesis that CD28null T cells are resistant to immunosuppression mediated by glucocorticoids in the setting of myositis. Such resistance could also be against Tregs mediated immunosuppression due to distinct phenotype of CD28null T cells. The aim of this study was to evaluate the immunosuppressive effects of glucocorticoids and Tregs on CD28null T cells in an in vitro system. Method Peripheral blood mononuclear cells (PBMCs) were obtained from 3 healthy individuals using Ficoll separation. CD3+CD4+ CD25++(high) cells were sorted as Tregs by flow cytometry. For glucocorticoid or Tregs mediated T cell suppression assays, PBMCs were stimulated with plate bound α-CD3 antibody in presence of 4uM glucocorticoid (methyl prednisolone sodium succinate) or optimal proportion of Tregs. Up-regulation of the early activation marker CD69 was measured by flow cytometry. Suppression was estimated based on % reduction in CD69 mean fluorescent intensity compared to stimulated control cells. Results CD4+CD28null T cells (median % suppression: 40.1%) displayed lower sensitivity towards glucocorticoid-mediated suppression compared to CD28+ counterparts (median: 54.7%), seen in all individuals tested. Similarly, CD4+CD28null T cells (median: 17.5%) were less sensitive towards Tregs mediated suppression compared to CD28+ counterparts (median: 34.4%) in all individuals. No clear trend could be observed in CD8 compartment so far. Conclusions Although, more individuals need to be tested, the above in vitro data support our in vivo findings that CD28null T cells are relatively resistant to glucocorticoid and Tregs mediated immunosuppression. Lower sensitivity of CD28null T cells towards glucocorticoid and Tregs mediated suppression support their treatment resistance nature in myositis and a role in chronic inflammation and autoimmunity.