Chemotaxis of Vδ2 T cells to the joints contributes to the pathogenesis of rheumatoid arthritis

Abstract

Objectives: To explore the role of Vδ2 T cells in the pathogenesis of rheumatoid arthritis (RA).

Methods: Sixty-eight patients with RA, 21 patients with osteoarthritis and 21 healthy controls were enrolled in the study. All patients with RA fulfilled the 2010 American College of Rheumatology/European League Against Rheumatism criteria for RA. Peripheral Vδ2T population, chemokine receptor expression and proinflammatory cytokine secretion were quantified by flow cytometry. The infiltration of Vδ2 T cells within the synovium was examined by immunohistochemistry and flow cytometry. The effect of tumour necrosis factor (TNF)-α and interleukin (IL)-6 on Vδ2 T migration was determined by flow cytometry and transwell migration assay.

Results: Peripheral Vδ2T cells, but not Vδ1 T cells, were significantly lower in patients with RA, which was negatively correlated with disease activity gauged by Disease Activity Score in 28 joints. Vδ2 T cells from RA accumulated in the synovium and produced high levels of proinflammatory cytokines including interferon-γ and IL-17. Phenotypically, Vδ2 T cells from RA showed elevated chemotaxis potential and expressed high levels of chemokine receptors CCR5 and CXCR3, which was driven by increased serum TNF-α through nuclear factor kappa B signalling. In vivo, TNF-α neutralising therapy dramatically downregulated CCR5 and CXCR3 on Vδ2 T cells and repopulated the peripheral Vδ2 T cells in patients with RA.

Conclusions: High levels of TNF-α promoted CCR5 and CXCR3 expression in Vδ2 T cells from RA, which potentially infiltrated into the synovium and played crucial roles in the pathogenesis of RA. Targeting Vδ2 T cells might be a potential approach for RA.

Keywords: T cells; anti-TNF; rheumatoid arthritis.

Figure 1

Peripheral Vδ2 T cells were lower in patients with RA. Peripheral blood mononuclear cells obtained from patients with RA, patients with OA and HCs were stained with anti-CD3, anti-γδ TCR, anti-Vδ1 or anti-Vδ2 mAb followed by flow cytometry. The solid plots represent isotype controls, and the open plots represent indicated staining. The left panels show flow cytometry data of (A) γδ T cells, (B) Vδ1 T cells or (C) Vδ2 T cells. The right panels show bar graphs of the percentage of positively stained cells. Representative data of RA (n=30), HC (n=15) and OA (n=15) are shown. (D) The percentage of peripheral Vδ2 T cells in RA is negatively correlated with CRP, ESR and DAS28 (n=42). Results are expressed as mean±SEM. ns, no significance; **p<0.01 by one-way analysis of variance with Tukey-Kramer post-hoc test. Correlations are calculated using Spearman correlation analysis. Anti-CCP,  anti-cyclic citrullinated peptide; CRP, C reactive protein; DAS 28, Disease Activity Score in 28 joints; ESR, erythrocyte sedimentation rate; HC, healthy control; OA, osteoarthritis; RA, rheumatoid arthritis; RF, rheumatoid factor; TCR, T cell receptor.

Figure 2

Vδ2 T cells accumulated at the affected joints of RA and secreted high levels of IFN-γ and IL-17. (A,B) The percentage of Vδ2 T cells in (A) SF and (B) enzyme-digested fresh synovium analysed by flow cytometry. Representative data of OA (n=4) and RA (n=4) are shown. (C) Infiltrations of Vδ2 T cells in the knee joint synovium of RA and OA were examined by immunohistochemical staining. Representative data of OA (n=3) and RA (n=3) are shown. Scale bars represent 50 µm. (D–F) Flow cytometry analyses of the intracellular staining of (D) IFN-γ, (E) TNF-α and (F) IL-17 in Vδ2 T cells from RA and OA synovium were performed. Data are representative of three independent experiments. The right panels show bar graphs of the percentage or the average number of positively stained cells. Results are expressed as mean±SEM. *p<0.05 by Student’s t-test. FSC, forward scattering; IFN-γ; interferon-γ; IL-17, interleukin-17; OA, osteoarthritis; RA, rheumatoid arthritis; SF, synovial effusion; TNF-α, tumour necrosis factor-α.

Figure 3

CCR5 and CXCR3 upregulation promoted Vδ2 T cell chemotaxis in RA. (A) Transwell migration assay: freshly isolated peripheral blood mononuclear cells from HC, OA and RA were loaded in the upper chamber, and SFs of OA, RA or medium were loaded in the lower chamber in the transwell invasion model. (B) The percentage and MFI of indicated chemokine receptors on Vδ1/Vδ2 T cells of RA. (C) Comparison of the proportion and MFI of indicated chemokine receptors in Vδ2 T cells from RA, HC and OA. (D) Vδ2 T cell migration assay with RA serum in the presence or absence of neutralising antibodies against CCR5, CXCR3 and CXCR6. (E) The concentration of known ligands of CCR5 and CXCR3 in SF of RA (n=22) and OA (n=10), and serum of RA (n=12) and OA (n=7). Data were pooled from three independent experiments (A,D) or five independent experiments (B,C). Results are expressed as mean±SEM. *p<0.05, **p<0.01 by one-way analysis of variance with Tukey-Kramer post-hoc test (A,C,D,E) and Student’s t-test (B). HC, healthy control; MFI, mean fluorescence intensity; OA, osteoarthritis; RA, rheumatoid arthritis; SF, synovial effusion.

Figure 4

TNF-α augmented the expression of CCR5 and CXCR3 on Vδ2 T cells. Flow cytometry analysis of (A) CCR5 and CXCR3 expression on Vδ2 T cells at indicated time points in the presence of HC, OA or RA serum; or (B) RA serum in combination with neutralising antibodies against TNF-α or IL-17 for 3 days; or (C) with medium in the presence or absence of TNF-α for indicated days. Data were pooled from three independent experiments. Results are expressed as mean±SEM. ns, no significance; *p<0.05, **p<0.01 by two-way ANOVA (A,C) or one-way ANOVA (B). ANOVA, analysis of variance; HC, healthy control; IL-17, interleukin-17; MFI, mean fluorescence intensity; OA, osteoarthritis; RA, rheumatoid arthritis; TNF-α, tumour necrosis factor-α.

Figure 5

NF-κB signalling pathway was involved in the expression of CCR5 and CXCR3 on Vδ2 T cells. (A) Vδ2 T cells were treated with TNF-α (100 ng/mL) for indicated time. The cells were permeabilised and stained with antibodies against NF-κB p65 (pS529), JNK1/2 (pT183/pY185) or p38 MAPK (pT180/pY182). The data represent one of three independent experiments. The right panels show bar graphs of MFI of Vδ2 T cells stimulated with TNF-α in 15 min. (B) Flow cytometric analysis of chemokine receptor expressions on TNF-α-stimulated HC Vδ2 T cells pretreated with QNZ (5 µM) or dimethyl sulfoxide (DMSO) for 1 hour. The solid plots represent isotype controls, and the open plots represent indicated staining. Results are expressed as mean±SEM. *p<0.05, **p<0.01 by paired t-test (A) and one-way analysis of variance with Tukey-Kramer post-hoc test (B). HC, healthy control; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MFI, mean fluorescence intensity; NF-κB, nuclear factor kappa B; TNF-α, tumour necrosis factor-α.

Figure 6

TNF-α antagonist therapy repopulated peripheral Vδ2 T cells and downregulated CCR5 and CXCR3 expressions in patients with RA. Treatment-naïve patients with RA (n=12) were treated with etanercept in combination with methotrexate for 3 months. Flow cytometry was performed for the analysis of (A) the percentage of peripheral Vδ2 T cells and the expressions of (B) CCR5 and (C) CXCR3 on Vδ2 T cells before and after treatment. The solid plots represent isotype control, and the open plots represent Vδ2 T cells staining. *p<0.05, **p<0.01 by paired t-test. RA, rheumatoid arthritis; TNF-α, tumour necrosis factor-α.