Phenotypic and functional characterisation of CCR7+ and CCR7- CD4+ memory T cells homing to the joints in juvenile idiopathic arthritis

Abstract

The aim of the study was to characterise CCR7+ and CCR7- memory T cells infiltrating the inflamed joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the functional and anatomical heterogeneity of these cell subsets in relation to the expression of the inflammatory chemokine receptors CXCR3 and CCR5. Memory T cells freshly isolated from the peripheral blood and synovial fluid (SF) of 25 patients with JIA were tested for the expression of CCR7, CCR5, CXCR3 and interferon-gamma by flow cytometry. The chemotactic activity of CD4 SF memory T cells from eight patients with JIA to inflammatory (CXCL11 and CCL3) and homeostatic (CCL19, CCL21) chemokines was also evaluated. Paired serum and SF samples from 28 patients with JIA were tested for CCL21 concentrations. CCR7, CXCR3, CCR5 and CCL21 expression in synovial tissue from six patients with JIA was investigated by immunohistochemistry. Enrichment of CD4+, CCR7- memory T cells was demonstrated in SF in comparison with paired blood from patients with JIA. SF CD4+CCR7- memory T cells were enriched for CCR5+ and interferon-gamma+ cells, whereas CD4+CCR7+ memory T cells showed higher coexpression of CXCR3. Expression of CCL21 was detected in both SF and synovial membranes. SF CD4+ memory T cells displayed significant migration to both inflammatory and homeostatic chemokines. CCR7+ T cells were detected in the synovial tissue in either diffuse perivascular lymphocytic infiltrates or organised lymphoid aggregates. In synovial tissue, a large fraction of CCR7+ cells co-localised with CXCR3, especially inside lymphoid aggregates, whereas CCR5+ cells were enriched in the sublining of the superficial subintima. In conclusion, CCR7 may have a role in the synovial recruitment of memory T cells in JIA, irrespective of the pattern of lymphoid organisation. Moreover, discrete patterns of chemokine receptor expression are detected in the synovial tissue.

Figure 1

Expression of CXCR3 and interferon (IFN)-γ by (SF) CCR7⁺ and CCR7^- memory CD4⁺ cells from synovial fluid. IFN-γ expression was investigated by three-colour staining of freshly isolated SF CD45RO⁺ cells with CD4–fluorescein isothiocyanate (FITC), anti-CCR7–phycoerythrin (PE) and anti-CCR5–CyChrome monoclonal antibodies (mAbs) or CD4–TC (where TC stands for Tri-color), anti-CCR7–PE and anti-IFN-γ mAbs, respectively; CXCR3 expression was investigated by triple staining with CD4–TC, anti-CCR7–PE and anti-CXCR3–FITC, as described in the Methods section. Subsequently, cytofluorimetric analysis was performed by gating on the CD4⁺CCR7⁺ and CD4⁺CCR7^- lymphocyte subsets. Data are expressed as percentages of positive cells or/and mean fluorescence intensity. (a, b) Expression of IFN-γ (a) and CXCR3 (b) by SF CCR7⁺ and CCR7^- memory CD4⁺ cells from 10 patients with juvenile idiopathic arthritis (JIA). Boxes contain values falling between the 25th and 75th centiles; whiskers show lines that extend from the boxes represent the highest and lowest values for each subgroup. Differences between paired SF mononuclear cells were evaluated by the Wilcoxon rank test. (c, d) Dot plots show the cytofluorimetric analysis IFN-γ (c) and CXCR3 (d) expression by the gated CD4⁺CCR7⁺ (gate 1) and CD4⁺CCR7^- (gate 2) cell populations in three representative patients with JIA.

Figure 2

Expression of CD4, CD20, CD45RO, CCR7, CCR5 and CXCR3 in synovial tissue obtained from patients with juvenile idiopathic arthritis (JIA) after synoviectomy. (a, b) Presence of CCR7⁺ cells (red) in the sublining layer of a synovial tissue characterised by a diffuse lymphocytic infiltrate (scattered CD4⁺ cells, brown) from a 14-year-old girl with antinuclear antibody-positive (ANA⁺) oligoarticular JIA (no. 1, Table 3) (Magnification × 20). (c–n) Serial stainings with CD20, CD4, CCR7, CXCR3 and CCR5 monoclonal antibody in synovial tissue from a 12-year-old girl with ANA⁺ oligoarticular JIA (no. 4, Table 3). The distribution of cells positive for CCR7, CCR5 and CXCR3 is shown in two different areas containing T and B cell aggregates (magnification × 10). (o, p) Different expression of CCR7 (red) and CCR5 (brown) in synovial membrane infiltrate (patient no. 1, Table 3) (magnification × 4.5). CCR7⁺ cells are observed exclusively in the perivascular lymphocytic infiltrate of the deep sublining layer (open rectangle). Conversely, CCR5-positive cells are prevalently observed at the level of the lining layer and superficial subintima (*) and in the perivascular infiltrates of the sublining layer (**).

Figure 3

Chemotactic activity of CD4 memory T cells from the synovial fluid of eight patients with juvenile idiopathic arthritis to inflammatory (CXCL11 and CCL3) and homeostatic (CCL19, CCL21) chemokines. Results are expressed as the percentage of migrated cells in the total cell input (see also the Methods section).

Figure 4

Expression of CCL21 in synovial fluid and tissue. (a) CCL21 concentrations in sera from 15 age-matched healthy controls, paired sera (Sera) and synovial fluids (SF) from 28 patients with juvenile idiopathic arthritis (JIA). Lines represent median values. Boxes contain values falling between the 25th and 75th centiles; whiskers show lines that extend from the boxes represent the highest and lowest values for each subgroup. The heterogeneity test among the three subgroups was highly significant (Kruskal–Wallis analysis of variance test, P = 0.0045). At post hoc analysis, differences between paired sera and SF were evaluated by the Wilcoxon rank test. Difference between JIA sera and healthy controls were evaluated by the Mann–Whitney U-test. (b) Expression of CCL21 in perivascular aggregates and vascular endothelium in synovial tissue with diffuse lymphocytic infiltration from 10-year-old girl with persistent oligoarticular JIA. (c, d) Double staining with anti-CCR7 (red) and anti-CCL21 (brown) monoclonal antibodies at different magnifications (×10 and ×40, respectively) shows CCL21 expression by endothelial cells of vessels located in the sublining zone of the superficial subintima.