Vgamma9/Vdelta2 T lymphocytes in Italian patients with Behçet's disease: evidence for expansion, and tumour necrosis factor receptor II and interleukin-12 receptor beta1 expression in active disease

Abstract

Behçet's disease is a multisystem disease in which there is evidence of immunological dysregulation. It has been proposed that gamma/delta T cells are involved in its pathogenesis. The aim of the present study was to assess the capacity of gamma/delta T cells with phenotype Vgamma9/Vdelta2, from a group of Italian patients with Behçet's disease, to proliferate in the presence of various phosphoantigens and to express tumour necrosis factor (TNF) and IL-12 receptors. Twenty-five patients and 45 healthy individuals were studied. Vgamma9/Vdelta2 T cells were analyzed by fluorescence activated cell sorting, utilizing specific monoclonal antibodies. For the expansion of Vgamma9/Vdelta2 T cells, lymphocytes were cultured in the presence of various phosphoantigens. The expression of TNF receptor II and IL-12 receptor beta1 was evaluated with the simultaneous use of anti-TNF receptor II phycoerythrin-labelled (PE) or anti-IL-12 receptor beta1 PE and anti-Vdelta2 T-cell receptor fluorescein isothiocyanate. There was a certain hierarchy in the response of Vgamma9/Vdelta2 T cells toward the different phosphoantigens, with the highest expansion factor obtained with dimethylallyl pyrophosphate and the lowest with xylose 1P. The expansion factor was fivefold greater in patients with active disease than in those with inactive disease or in control individuals. TNF receptor II and IL-12 receptor beta1 expressions were increased in both patients and control individuals. The proportion of Vgamma9/Vdelta2 T cells bearing these receptors was raised in active disease when Vgamma9/Vdelta2 T cells were cultured in the presence of dimethylallyl pyrophosphate. These results indicate that Vgamma9/Vdelta2 T cell activation is correlated with disease progression and probably involved in the pathogenesis.

Figure 1

Expansion of Vγ9/Vδ2 T lymphocytes from patients with active or inactive Behçet's disease and healthy control individuals in response to various phosphoantigens. The Vγ9/Vδ2 expansion factor (EF) was then calculated by dividing the absolute number of Vγ9/Vδ2 T cells in specifically stimulated cultures by the absolute number of Vγ9/Vδ2 T cells cultured in the absence of any antigen. DMAPP, dimethylallyl pyrophosphate; IPP, isopentenyl pyrophosphate; RIB, ribose 1-P; TUBAg, Mycobacterium tuberculosis related phosphorylated components; XYL, xylose 1-P.

Figure 2

Cytofluorimetric analysis of Vγ9/Vδ2 T lymphocytes from a patient with active Behçet's disease (BD) and a healthy control individual in vitro, cultured with dimethylallyl pyrophosphate (DMAPP) or medium alone. The horizontal axis represents log10 fluorescence intensity of Vγ9/Vδ2 stained cells. Each analysis was repeated at least three times and was performed each time with cells from different donors. FITC, fluorescein isothiocyanate.

Figure 3

Expression of tumour necrosis factor receptor II (TNF-RII) and IL-12 receptor β₁ (IL-12Rβ1) on Vγ9/Vδ2 T lymphocytes from patients with active or inactive Behçet's disease (BD) and healthy control individuals. Results are expressed as percentage of Vγ9/Vδ2 T cells.

Figure 4

Tumour necrosis factor receptor II (TNF-RII) and IL-12 receptor β₁ (IL-12Rβ1) expression of Vγ9/Vδ2 T lymphocytes from a patient with active Behçet's disease (BD). The ordinate indicates the expression of phycoerythrin-labelled (PE) conjugated TNF-RII or IL-12Rβ1, and the abscissa indicates the expression of fluorescein isothiocyanate (FITC)-conjugated anti-Vγ9/Vδ2. Each analysis was repeated at least three times and was performed each time with cells from different patients. DMAPP, dimethylallyl pyrophosphate.