Anti-fibrotic characteristics of Vγ9+ γδ T cells in systemic sclerosis.

OBJECTIVES: γδ T cells of the Vγ9Vδ2 subtype secrete anti-fibrotic cytokines upon isopentenyl pyrophosphate (IPP) stimulation. In this study, we sought to compare IPP and Zoledronate, an up-regulator of IPP, effects on proliferation and cytokine secretion of Vγ9+ T cells from systemic sclerosis (SSc) patients and healthy controls (HCs). We also examined the effect of IPP-triggered peripheral blood mononuclear cells (PBMC) on fibroblast procolla- gen secretion. METHODS: PBMC from SSc patients and HCs were stimulated by increasing concentrations of Zoledronate, with or without IPP, and Vγ9+ T cell percentages were calculated using FACScan analysis. Subsequently, PBMC were cultured with IPP or toxic shock syndrome toxin-1 (TSST-1), and contents of the anti-fibrotic cytokines tumour necrosis factor (TNF)-α and interferon (IFN)-γ were measured by ELISA kits. Finally, supernatants of IPP-triggered Vγ9+ T cells from SSc patients were added to fibroblast cultures, and relative intensities of procollagen α1 chains were determined by densinometry. RESULTS: Higher concentrations of Zoledronate were required for maximal proliferation of Vγ9+ T cells in 9 SSc patients compared to 9 HCs, irrespective of exogenous IPP. When compared to stimulation by TSST-1, a non-Vγ9+ selective reagent, secretion of the anti-fibrotic cytokines TNF-α and IFN-γ in response to IPP was relatively diminished in SSc but not in HCs. Reduction of procollagen secretion by fibroblasts cultured with supernatants of IPP-stimulated PBMC was observed only in some SSc patients. CONCLUSIONS: Activated Vγ9+ T cells could act as anti-fibrotic mediators in SSc, although decreased responsiveness to IPP may play a role in the pathological fibrosis of this disease.

Cellular interactions of synovial fluid γδ T cells in juvenile idiopathic arthritis.

The pathogenesis of juvenile idiopathic arthritis (JIA) is thought to involve multiple components of the cellular immune system, including subsets of γδ T cells. In this study, we conducted experiments to define the functional roles of one of the major synovial fluid (SF) T cell subsets, Vγ9(+)Vδ2(+) (Vγ9(+)) T cells, in JIA. We found that as opposed to CD4(+) T cells, equally high percentages (∼35%) of Vγ9(+) T cells in SF and peripheral blood (PB) produced TNF-α and IFN-γ. Furthermore, stimulation with isopentenyl pyrophosphate (IPP), a metabolite in the mevalonate pathway, which is a specific potent Ag for Vγ9Jγ1.2(+) T cells, similarly amplified cytokine secretion by SF and PB Vγ9(+) T cells. Significantly, the SF subset expressed higher levels of CD69 in situ, suggesting their recent activation. Furthermore, 24-h coculturing with SF-derived fibroblasts enhanced CD69 on the SF > PB Vγ9(+) T cells, a phenomenon strongly augmented by zoledronate, a farnesyl pyrophosphate synthase inhibitor that increases endogenous intracellular IPP. Importantly, although Vγ9(+) T cell proliferation in response to IPP was significantly lower in SF than PBMC cultures, it could be enhanced by depleting SF CD4(+)CD25(+)FOXP3(+) cells (regulatory T cells). Furthermore, coculture with the Vγ9(+) T cells in medium containing zoledronate or IPP strongly increased SF-derived fibroblasts' apoptosis. The findings that IPP-responsive proinflammatory synovial Vγ9(+) T cells for which proliferation is partly controlled by regulatory T cells can recognize and become activated by SF fibroblasts and then induce their apoptosis suggest their crucial role in the pathogenesis and control of synovial inflammation.

GammadeltaT cells in juvenile idiopathic arthritis: higher percentages of synovial Vdelta1+ and Vgamma9+ T cell subsets are associated with milder disease.

OBJECTIVE: To analyze γδT cell subsets in peripheral blood (PB) and synovial fluid (SF) of patients with juvenile idiopathic arthritis (JIA), and to correlate γδT cell subsets with clinical characteristics. METHODS: γδT cell subsets as percentages of CD3+ T cells in samples of PB (n = 25) and SF (n = 93) were analyzed by flow cytometry in 93 JIA patients. The percentage of Vγ9+ γδT cells after 10 days of in vitro expansion with either interleukin 2 (IL-2) or isopentenyl pyrophosphate (IPP) plus IL-2 was determined. RESULTS: Both Vδ1+ and Vγ9+ γδT cell subsets were detected in SF of all patients, but only the percentage of Vδ1+ cells was higher in SF compared to PB (p < 0.01). The distribution of γδT cell subsets was similar in different JIA subgroups, whereas antinuclear antibody (ANA)-positive patients had a higher percentage of SF Vδ1+ T cells than ANA-negative patients (p < 0.01). The percentage of SF Vδ1+ T cells was inversely associated with age at onset, recurrence of synovitis, and erythrocyte sedimentation rate; and that of SF Vγ9+ T cells was inversely correlated with age at onset and was higher in patients who recovered from disease (n = 15). IPP-induced expansion of SF Vγ9+ T cells correlated with disease remission, whereas the expansion of SF Vγ9+ T cells in media with IL-2 alone was significantly greater in patients with uveitis. CONCLUSION: The percentage of Vδ1+ and Vγ9+ γδT cells among the SF T cells and their ability to respond to IPP or IL-2 correlated with specific outcomes of JIA, suggesting their role in the immunopathogenesis of this disease.

Vgamma9+ gammadelta T cells in systemic sclerosis patients are numerically and functionally preserved and induce fibroblast apoptosis.

Vdelta1 expressing gammadelta T cells are oligoclonally expanded in systemic sclerosis (SSc) (scleroderma) and thought to play an immunopathogenic role, whereas that of Vgamma9+ gammadelta T cells is unclear. In studies of 16 patients and 16 healthy controls (HCs) we found that whereas the percent of Vdelta1+ gammadelta T cells was significantly elevated among the peripheral blood T cells in patients without radiographic evidence of interstitial lung disease (n=7), Vgamma9+ T cells were equally and persistently represented irrespective of pulmonary disease or cyclophosphamide treatment, at levels similar to healthy controls. Furthermore, ex vivo triggering of patient Vgamma9+ T cells with isopentenyl pyrophosphate plus interleukin-2-induced dose-dependent expansion, secretion of tumor necrosis factor alpha, and contact-dependent apoptosis of co-cultured fibroblasts similarly to Vgamma9+ T cells of controls. Fully functional Vgamma9+ T cells persisting in the peripheral blood of patients with progressive systemic sclerosis could potentially play an immunopathogenic role in vivo by secreting cytokines and inducing death of fibroblasts in a contact-dependent manner when activated by specific antigens.

Altered self-erythrocyte recognition and destruction in an inbred line of tilapia (Oreochromis aureus).

Carboxyfluorescein diacetate (cFDA)-stained autologous and syngeneic tilapia (Oreochromis aureus) erythrocytes are recognized by effector peripheral blood leukocytes and lysed after a short culture period of 4 h. The hemolysis level was evaluated by measuring the fluorescence of the released cFDA. The degree of lysis of stained target erythrocytes of 60 individuals revealed a trimodal distribution statistically stratified into three groups of low (LR), intermediate (IR), and high (HR) responders. Depletion of the majority of phagocytes from leukocytes lowered the lysis level of HR to that of LR. A highly significant increase of LR cytotoxicity was obtained after the addition of conditioned medium from HR but only in the presence of phagocytes. Genetic analysis of offspring from four crosses (IR x HR, IR x LR, HR x LR, and LR x LR) revealed a quantitative trait locus (QTL) segregating for the level of response linked to markers UNH207 and UNH231 on linkage group 6 of tilapia. Based on segregation analysis of 58 gynogenetic BIU-1 offspring, the distances from the centromere were estimated as 21.5, 11.5, and 9.0 cM for UNH207, UNH231, and the QTL, respectively. It is suggested that 1) self-target recognition and destruction requires both cFDA-altered self-erythrocyte membrane and membrane structures normally present in autologous, syngeneic, and xenogeneic targets; 2) natural cytotoxic cells and/or macrophages are involved in erythrocyte lysis; and 3) the lysis level is codominantly inherited by a QTL segregating on tilapia linkage group 6.