Tumour necrosis factor RECEPTOR activated T cells of patients with systemic sclerosis are deficient in IL-10 expression but promote myofibroblast differentiation via IL-6 and TGF- 
Authors: T. Hugle, A. Krippner-Heidenreich, R. Simpson, M. Kraaij, S. O'Reilly, V. Bigley, M. Collin, L. Iversen, J. M. van Laar
Journal: Annals of the Rheumatic Diseases (2011)

<h3>Background and objectives</h3> Systemic sclerosis (SSc) is an auto-inflammatory multisystem disease leading to fibrosis of the skin and inner organs. T lymphocytes are key effector cells in the affected tissue. The role of lymphocytes in the transition from inflammation to fibrosis is unclear. Previous data suggested a possible pro-fibrotic effect of tumour necrosis factor–receptor (TNF-R) 2. <h3>Materials and methods</h3> Skin punch biopsies from affected skin of 12 SSc patients (six limited, six diffuse) were digested in dispase and collagenase and processed for multicolour flow cytometry (CD45, HLA-DR, auto-fluorescence, CD14, CD1a, C3, CD4, CD8, TNF-R1 and 2). α-Smooth muscle actin (α-SMA) positive myofibroblasts and CD3<sup>+</sup> lymphocytes were furthermore quantified in skin biopsies of 26 patients of a control SSc patient cohort by immunohistochemistry. For functional analyses, T lymphocytes were isolated from peripheral blood of 12 patients using CD3 selection and activation via ± CD3/CD28 beads. TNF-R were stimulated with selective CysTNF mutants at concentrations of 100 ng/ml. Real-time PCR and multiplex cytokine assay was performed for cytokine expression. Fibroblasts of SSc patients were incubated in conditioned (TNF-R stimulated) lymphocyte medium; α-SMA was detected by Western blot. <h3>Results</h3> Higher numbers of lymphocytes per CD45 cells (45.7% vs 26.6%), but less CD1a<sup>+</sup> (1.0% vs 4.1%) cells were detected in the dermis of patients with diffuse SSc compared to limited SSc. Lymphocyte infiltration correlated with skin thickening (modified Rodnan skin score, mRSS) of the patients (p=0.004) and myofibroblast infiltration (p&lt;0.05). TNF-R2 was significantly up-regulated on CD3, CD4 and CD8 subsets in patients with diffuse but not limited disease. TNF-R1 was not significantly upregulated on dermal lymphocytes. The mean intensity fluorescence of TNF-R2 but not 1 correlated with the mRSS (p=0.01). Activation of SSc lymphocytes selective TNF-R agonists showed that TNF-R1 stimulation resulted in a significantly higher interleukin (IL)-6 expression whereas TNF-R2 stimulation lead to a higher TGF-β expression, compared to healthy individuals. Conversely, CD3/CD28 activated lymphocytes of SSc patients expressed significantly less IL-10 upon TNF-R2 stimulation compared to lymphocytes from healthy individuals. When the authors incubated control fibroblasts in conditioned medium, medium from healthy TNF-activated lymphocytes <i>inhibited</i> α-SMA expression. In contrast, conditioned medium from TNF-activated SSc lymphocytes <i>increased</i> α-SMA expression. In dermis of a SSc patient who underwent lymphoablative treatment, the authors observed a reduced number of myofibroblasts and lymphocytes with a reversibility of TNF-R2 expression. <h3>Conclusion</h3> Lymphocyte infiltration and TNF-R expression play a role in SSc via enhanced IL-6, TGF-β and myofibroblast differentiation. Deficient IL-10 expression might impair the resolution of inflammation in SSc.