Vγ9Vδ2 T cells recovered from eyes of patients with Behçet's disease recognize non-peptide prenyl pyrophosphate antigens

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Abstract

The phenotype and antigen-specificity of T cells expanded by mitogenic stimulation from intra-ocular fluid (IOF) samples of affected eyes of six Behçet's disease (BD) patients, and seven patients with other uveitis entities, were determined. High numbers of γδ T cells, predominantly Vγ9Vδ2 T cells, were only detected in the IOF-derived TCL of three BD patients. Whereas no TCL responded to heat shock protein (HSP) 65 kDa, reactivity to isopentyl pyrophosphate (IPP) and related non-peptide prenyl pyrophosphates (PPP) was restricted to the γδ T cell containing TCL. Upon IPP stimulation, these TCL secreted IFN-γ but no IL-4. By single-cell analysis of intracellular IFN-γ production and CD69 expression the IOF-derived IPP-specific T cells were identified as CD4CD8 γδ T cells. The data presented suggest the infiltration of PPP-specific Vγ9Vδ2 Th1-like cells into the eye of BD patients with uveitis.

Introduction

Behçet's disease (BD) is a refractory multisystemic inflammatory disorder characterized by recurrent ulceration of the oral and genital mucosa Serdaroglu, 1998, Sakane et al., 1999. The clinical spectrum of BD is diverse. Patients can suffer from lesions affecting the skin, blood vessels, joints, lungs, intestine, nervous system, and eyes. The disease has a chronic course with periodic exacerbations and progressive deterioration. The pathophysiological singularities of BD are vasculitis, hyperfunction of neutrophils and auto-immune responses. Recurrent ocular attacks can result in permanent loss of vision due to ischaemic damage to the retina and optic nerve as the main cause (Nussenblatt, 1997).

The etiopathogenesis of BD remains largely unknown. Infections with variety of microorganisms, like herpes simplex virus (HSV) and streptococci, have been implicated to induce BD in genetically predisposed (HLA-B51+) individuals Eglin et al., 1982, The Behçet's Disease Research Committee of Japan, 1998, Mizuki et al., 1997. Immunohistological studies have demonstrated a predominant T cell infiltration of inflamed tissues, including the eye, of BD patients Charteris et al., 1992, Gül et al., 1995, Inomata, 2000. Whereas the majority of the intra-lesional T cells are CD4+αβ+, enhanced percentages of γδ T cells in active lesions and peripheral blood of BD patients compared to disease controls have been reported Fortune et al., 1990, Suzuki et al., 1992, Hamzaoui et al., 1994, Hasan et al., 1996, Yamashita et al., 1997, Freysdottir et al., 1999. γδ T cells are considered to play a role in immunoregulation and the host defense towards various microorganisms (Kabelitz et al., 1999). In humans, γδ T cells account for ∼2–5% of peripheral blood T cells and mainly express the T cell receptor (TCR) variable (V) gene segments Vγ9Vδ2 (Kabelitz et al., 1999). The antigens recognized by γδ T cells include heat shock proteins (HSP) and the non-peptide antigens prenyl pyrophosphates (PPP) (De Libero et al., 1997). The PPP isopentenyl pyrophosphate (IPP) is a precursor for a variety of biological molecules, like cholesterol and vitamins. IPP and related PPP are essential metabolites for both prokaryotic and eukaryotic cells (De Libero et al., 1997). Given the wide variety of microorganisms implicated in the development of BD, it has been suggested that ubiquitous antigens of microbial origin may trigger cross-reactive autoimmune responses in BD patients. Several studies have shown cellular and humoral immune responses to mycobacterial 65-kDa HSP (HSP65) and the homologous human mitochondrial 60-kDa HSP in peripheral blood (PB) of BD patients, especially those with ocular involvement, suggesting a possible role of HSP in the etiopathogenesis of BD Pervin et al., 1993, Hasan et al., 1996, Kaneko et al., 1997, Direskeneli et al., 2000. Moreover, a role of HSP60/65-specific γδ T cells in the pathogenesis of BD has been postulated (Hasan et al., 1996).

Thus far, the antigen specificity of ocular infiltrating T cells in BD patients has not been analyzed in detail. In the present study, we report on the establishment and characterization of intra-ocular T cell lines (TCL) recovered from intra-ocular fluid (IOF) samples of six BD patients. As disease controls, four patients with Fuch's heterochromic cyclitis (FHC), two patients with panuveitis and one with pars planitis were included.

Section snippets

Clinical specimen collection and processing

Patients with uveitis presenting to the clinic were characterized on clinical grounds, with additional investigations to exclude systemic disease or, in case of extensive vasculitis (patients 1–3 and 6), herpesvirus-induced uveitis (Table 1). Patients 1–6 fulfilled the clinical criteria of BD as proposed by the International Study Group for Behçet's Disease (International Study Group for Behçet's Disease, 1990). Four FHC patients (patients 7–10), two patients with panuveitis (patient 11 and 12)

Generation of TCL from IOF samples of uveitis patients

Intra-ocular TCL were generated to determine the phenotype and functional characteristics of ocular infiltrating T cell in six BD and seven non-BD uveitis patients (Table 1). The IOF-derived TCL obtained consisted of >94% CD3+ cells and the ratio of CD4+ and CD8+ varied inter-individually (Table 2). In contrast to the non-BD patients, relatively high numbers of γδ T cells, predominantly CD3+CD4CD8 (Fig. 1A), were detected in the IOF-derived TCL of three out six BD patients (Table 2; patients

Discussion

The adult eye is an organ without constitutive lymphoid components. Therefore, T cells recovered from IOF samples of uveitis patients must have migrated into the eye following induction of disease. Information on antigen specificity and function of intra-ocular T cells may be relevant for our understanding of the role of T cells in the pathogenesis of BD. Due to limitation of IOF samples available and, especially in aqueous humor samples, the low numbers of T cells present these studies are

Acknowledgements

The authors thank M.E.M. Dings for technical assistance. rHSP65 was generously provided by R. van der Zee (Dept. Immunology, Veterinary faculty, Utrecht University, Utrecht, The Netherlands). Financial support was obtained from the “Stichting Wetenschappelijk Onderzoek Oogziekenhuis”, “Stichting HOF” and “Rotterdamse Vereniging Blindenbelangen” (GMGMV).

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