Phenotype and functional changes of Vγ9/Vδ2 T lymphocytes in Behçet's disease and the effect of infliximab on Vγ9/Vδ2 T cell expansion, activation and cytotoxicity

Introduction Infliximab is a chimeric monoclonal antibody against tumor necrosis factor alpha (TNF-α) that has been introduced recently for Behçet's disease (BD) patients who were resistant to standard treatment. The aim of this study was to analyse the functional changes of Vγ9/Vδ2 T lymphocytes in both active and inactive disease and the effect of infliximab on Vγ9/Vδ2 T cell expansion, activation and cytotoxicity. Methods We investigated 1) cell expansion, 2) expression of TNFRII receptor, 3) perforin and gamma interferon (IFN) content, 4) release of granzyme A (GrA) and 5) phenotype changes, in vitro and in vivo, in Vγ9/Vδ2 T lymphocytes by means of fluorescence-activated cell sorter analysis of lymphocyte cultures from patients with active and inactive BD and healthy subjects. Results Cell expansion, expression of TNFRII, perforin and gamma IFN content and release of granzyme A were significantly higher in active patients. In vitro and ex vivo treatment with infliximab resulted in a significant reduction of all parameters together with changes in the phenotype of Vγ9/Vδ2 T cells. Conclusions All together these data indicate that infliximab is capable of interfering with Vγ9/Vδ2 T cell function in BD and although cell culture models cannot reliably predict all potential effects of the drug in vivo, our results present the possibility that this drug may find use in a range of immunological disorders, characterized by dysregulated cell-mediated immunity.


Introduction
Behçet's disease (BD) is a multisystemic inflammatory disorder characterized mainly by recurrent oral and genital apthous ulcerations and uveitis. The clinical spectrum of BD is wide, involving skin, blood vessels, joints, nervous system, lungs and intestines. The treatment of BD is based for most patients on the combination of corticosteroids and immunosuppressive agents. Despite the improvement obtained with this strategy, relapses and failure may occur, some patients being also refractory to all treatments. Infliximab a chimeric monoclonal anti-body against TNF-α that has been introduced for patients with Crohn's disease, rheumatoid arthritis, psoriasis, juvenile chronic arthritis and more recently for BD patients who were resistant to standard treatment [1,2].
The immunopathogenesis of BD remains unknown, but it is believed to be T cell-mediated [3][4][5]. Recently, attention has been focused on the role of γδ T cells and we have demonstrated that Vγ9/Vδ2 circulating lymphocytes are activated in patients with active disease and express increased levels of receptors for TNF-α and IL (interleukin)-12 [6]. Moreover, elevated levels of granzyme A both in the serum and Vγ9/Vδ2 cell supernatants of active BD patients are present, suggesting a role for this kind of lymphocytes in the pathogenesis and in the progression of the disease [7]. In this paper we analysed the functional changes of Vγ9/Vδ2 T lymphocytes in both active and inactive disease and of the effect of infliximab on Vγ9/Vδ2 cell expansion, activation and cytotoxicity.

Patients
Thirteen patients with BD (nine males and four females, mean age 42 ± 24 years), classified according to the International Study Group for Behçet's Disease [8] were studied. The activity of BD was assessed by collecting clinical symptoms defined according to the BDCAF score [9,10] that includes the presence of several manifestations of the disease, by the uveitis scoring system and by the visual activity measurement [11]. At the time of sampling, disease was active in six patients and inactive in seven. In five of the active patients blood for serum and lymphocyte studies was obtained before and after the anti-TNF-α (Infliximab) therapy. All patients were using colchicine (n = 13), and/or low dose corticosteroids (n = 8). Ten healthy volunteers (age range 21 to 47, mean 30 ± 8 years) were enrolled as controls. None of patients or controls were HIV, CMV, EBV infected. Human studies committee approval and individual informed consent from each patient were obtained.

Cell separation and in vitro expansion by Vγ9/Vδ2 T lymphocytes
Peripheral blood mononuclear cells (PBMC) were obtained from each individual by separating heparinized venous blood on Ficoll (Euroclone, Wetherby, Yorkshire, UK). The cells were washed in RPMI-1640 medium (Euroclone), and cultured in 24-well plates (Costar, Cambridge, MA, USA) at a concentration of 5 × 10 5 cells/ml in RPMI-1640 supplemented with 10% foetal calf serum (Euroclone) 20 mM Hepes (Euroclone), 2 mM L-glutamine (Euroclone) and 100 U/ml penicillin/streptomycin (Sigma, St Louis, MO, USA) at 37°C at 0,5% CO2. For the expansion of Vγ9/Vδ2 T cells, PBMCs were cultured for 10 days in medium alone or in the presence of 0,5 mM Dimethylallyl pyrophosphate (DMAPP, Sigma, St Louis). After 72 hours, cultures were supplemented with a 0,5 ml medium containing 40 U/ml recombinant human IL-2 (Genzyme, Cambridge, MA, USA). Every 72 h, 0.5 ml medium was replaced with a 0.5 ml fresh medium containing IL-2. After 10 days, cells were washed three times in medium, and expansion of Vγ9/Vδ2 T cells was assessed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) by using forward scatter/side scatter gating to select the lymphocyte population for analysis. The Vγ9/Vδ2 T cell expansion factor (EF) was then calculated as described above [6].
The number of Pf and TNF-RII molecules (MESF; molecular equivalents of soluble fluorochrome) was calculated by fluorescence-activated cell sorter analysis of cells stained with saturating amounts of PE labelled anti-TNF-RII and anti-Pf mAb of known PE/protein ratio and comparing the staining with a standard curve of microbeads labelled with defined numbers of PE molecules (Quantum Fluorescence Kit, Sigma). The analysis was done using Quickal Program for MESF Units for Windows.

In vitro effect of Infliximab on Vγ9/Vδ2 cultures
In order to examine the effects on Vγ9/Vδ2 expansion, TNF-RII expression, perforin and IFN-γ content, and supernatant levels of GrA, Infliximab (Remicade; Centocor Inc., Malvern, PA, USA; Schering Plough SpA, Segrate (Mi), Italy) was added in the medium at a final concentration of 10, 50 (for 3 days) and 100 μg/ml (for 3 and 10 days).

Effect of Infliximab therapy
Five patients with active disease were treated with Infliximab, 5 mg/Kg, by a two hour infusion, at weeks 0, 2, 4 and the patients observed for a further two hours without adverse effects. Sampling, for Vγ9/Vδ2 studies, were performed before the start of therapy and at the time of the second infusion.

Statistics
All values are expressed as mean ± SD. We performed analysis of significance in Prism (GraphPad, La Jolla, CA, USA) by the two-tailed t test analysis and by two-way ANOVA.

Discussion
The immunopathogenesis of BD is unknown. Various micro-organisms such as streptococci and herpes simplex virus have been implicated in the pathogenesis in genetically susceptible HLA-B51+ individuals [12,13]. There is evidence of immunological dysregulation, including neutrophil hyperfunction [14,15], autoimmune manifestations [16] and several phenotypic and functional lymphocyte abnormalities, most of the immunological studies suggesting a central role for T cells in the pathogenesis of this disease [17]. Cytotoxic T cells are considered to play a role in the development of disease. Recent studies [18,19] and our own [6] point also for a role of activated Vγ9/Vδ2 T lymphocytes in the progression and probably in the pathogenesis of the disease.
The treatment of BD comprises mainly systemic corticosteroids for most manifestations of BD. Supplemental therapy with other immunomodulatory agents is often necessary to control serious manifestations such as uveitis and meningoencephalitis and to reduce the incidence of long-term steroid toxicity. Drugs usually used in BD such as glucocorticoids, pentoxifylline and cyclosporine have been demonstrated to modulate peripheral blood gamma delta T lymphocytes [20][21][22].
The role of TNF-blocking agents on gamma delta T lymphocyte functions has not yet been investigated. In BD, increased serum levels of TNF-α and soluble TNF-RII has been observed during the active stage of disease suggesting a role for TNF-α in the pathogenesis [23][24][25]. In addition, TNF-α, that has been reported also to be pro-   duced by γδ T cells, might stimulate the TNF receptor bearing γδ T cells, in an autocrine or paracrine manner or both, to proliferate [26,27].
In this study we investigated the functional changes of Vγ9/Vδ2 T lymphocytes in both active and inactive Behçet's disease and the effect of Infliximab on Vγ9/Vδ2 T cell expansion, activation and cytotoxicity.
Infliximab is a high affinity monoclonal anti TNF-α antibody that has been introduced for Crohn's disease and rheumatoid arthritis treatment in patients who are resistant to standard therapy. Our previous studies demonstrated a complete remission of all disease manifestations in BD patients with ocular involvement and cerebral vasculitis [1,2].
Infliximab interferes with Vγ9/Vδ2 T cell functions. In particular, in vitro and in vivo studies demonstrated that this drug was able to suppress the Vγ9/Vδ2 T cell expansion and activation (TNF-RII expression and IFN-γ production) induced by DMAPP. Infliximab interferes also with the potential cytotoxic activity of these cells that we evaluated through the expression of the cytoplasmic granule-associated molecules perforin and the GrA release in the medium of cultures.
In this paper we did not study the phenotype of circulating Vγ9/Vδ2 T lymphocytes being our cytofluorimetric analysis not sensitive enough to measure membrane antigens in a relatively low number of cells. Although these preliminary observations have to be properly defined, after phosphoantigen stimulation, however, we observed frequently that the Vγ9/Vδ2 subpopulation was mainly composed by effector cells in active patients and by memory cells in inactive patients and controls. After in vitro exposure to Infliximab, there was a lack of effector cells, suggesting that this drug might block the lineage pattern of differentiation (naive → memory → effector → terminally differentiated cells), as a consequence of expansion and activation inhibition.

Conclusions
Our observations of disease-specific changes in Vγ9/Vδ2 T cell functions are consistent with the hypothesis that these cells play a role in the pathogenesis of BD. Despite the relatively low number of patients enrolled, data collected in this study point also to a critical role in the regulation of cellular activation and function of Vγ9/Vδ2 T cells by Infliximab and lead to encourage the possibility that this drug may find widespread use in the treatment of BD.