Results of flow cytometric detection of gamma-deltaT cells in peripheral blood of patients with ankylosing spondylitis: a pilot study

Abstract

Previous studies have suggested that gamma-delta T cells play an important role in the pathogenesis of ankylosing spondylitis (AS). In this pilot study, the peripheral blood mononuclear cells (PBMCs) of patients with ankylosing spondylitis (AS) and healthy volunteers were stained and analyzed by flow cytometry to distinguish gamma-delta T cells and its subtypes, and then to report the distribution of gamma-delta T cells and iyts subtypes and their correlation with ankylosing spondylitis. A total of 17 patients with active AS and 10 age- and gender- matched healthy volunteers were enrolled in this study, and their peripheral blood were drawn to collect mononuclear cells (PBMCs). Flow cytometry was used to analyze gamma-delta T cell subpopulations by measuring the surface and intracellular expressions of phenotypic markers. Serum levels of inflammatory and bone turnover markers were measured, and their correlations with subpopulations of gamma-delta T cells were evaluated. In patients with AS, the Vdelta2 fractions within gamma-delta T cells and CD3+ T cells decreased significantly, in particular, the proportions of CD27+ Vdelta2 T cells, CD86+CD80+ Vdelta1 T cells, and IL17A-secreting and TNFalpha-secreting Vdelta1 T cells within the parental cells decreased significantly. gamma-delta T cells/PBMCs, Vdelta2 cells/gamma-delta T cells, and Vdelta2 cells/CD3+ T cells were negatively correlated with CRP, whereas Vdelta1 cells/CD3+ T cells were negatively correlated with ESR. Vdelta1 cells/gamma-delta T cells were positively correlated with CRP, gamma-deltaT cells/PBMCs were positively correlated with beta-CTx, CD69+CD25+ and IL-17A-secreting Vdelta1 cells were positively correlated with TP1NP, and CD69+CD25+ Vdelta1 and Vdelta2 cells were positively correlated with osteocalcin. Decreases in peripheral Vdelta2, CD27+ Vdelta2, CD86+CD80+ Vdelta1, and IL17A or TNFalpha-secreting Vdelta1 T cells are associated with AS. The correlations between gamma-delta T cell subpopulations and CRP and the CD69+CD25+ subpopulation with TP1NP or osteocalcin suggest that an imbalance in peripheral gamma-delta T cell subpopulations contributes to the pathogenesis of AS.

Fig. 1

Vδ1 and Vδ2 composition within total CD3+ T cells. The peripheral blood mononuclear cells (PBMCs) of patients with active ankylosing spondylitis (AS) and healthy controls were collected. Flow cytometry was used to distinguish between Vδ1 and Vδ2 cells based on the expression of surface (CD3/Vδ2/Vδ1/CD25/CD69/CD27/CD80/CD86) or intracellular (CD3/vδ2/vδ1/CD27) markers. Flow cytometry plots of total CD3+ cells from patients P3 and P14 are shown.

Fig. 2

Analysis of CD80 and CD86 expression in Vδ1 and Vδ2 subsets. The PBMCs of patients with active AS and healthy controls were collected. Flow cytometry was used to distinguish between Vδ1 and Vδ2 cells based on surface (CD3/Vδ2/Vδ1/CD25/CD69/CD27/CD80/CD86) or intracellular (CD3/vδ2/vδ1/CD27) marker expression. (A) Using flow cytometry, the Vδ1 and Vδ2 cells were isolated from the PBMCs of two patients (P1 and P10) and healthy controls (M11). (B) The Vδ1 and Vδ2 cells were further separated based on their expression of CD80 and CD86.

Fig. 3

Flow cytometry analysis of the fractions of IL17A-secreting and TNFα-secreting Vδ1 T cells within CD3⁺ T cells from P3 and P14. The PBMCs of patients with active AS and healthy controls were collected. Flow cytometry was used to distinguish between Vδ1 and Vδ2 cells based on surface (CD3/Vδ2/Vδ1/CD25/CD69/CD27/CD80/CD86) or intracellular (CD3/vδ2/vδ1/CD27) marker expression. Cytokine secretion was measured using antibodies against TNF-α/IFN-γ/IL-17A.

Fig. 4

Correlation analysis. (A–E) Spearman’s rank correlation was utilized to determine the relationship between cell subtype and inflammation or bone turnover marker expression. Each panel displays the coefficients r and corresponding P-values.