CD4+ CD25+ T cells with the phenotypic and functional characteristics of regulatory T cells are enriched in the synovial fluid of patients with rheumatoid arthritis

Abstract

CD4(+) CD25(+) regulatory T (T(reg)) cells play a critical role in the maintenance of peripheral tolerance and the prevention of autoimmunity. In the present study, we have explored the characteristics of CD4(+) CD25(+) T(reg) cells in patients with rheumatoid arthritis (RA). The frequency and phenotype of CD4(+) CD25(+) T cells in paired samples of synovial fluid (SF) and peripheral blood (PB) from patients with RA and PB from normal controls were analysed. An increased frequency of CD4+ cells T cells expressing CD25 was detected in SF compared to PB from patients with RA. No significant difference was observed in the numbers of CD4(+) CD25(+) T cells in PB from patients and controls. SF CD4(+) CD25(+) T cells expressed high levels of CTLA-4 (both surface and intracellular), GITR and OX40, as well as Foxp3 transcripts. Functionally, SF CD4(+) CD25(+) T cells were impaired in their proliferative responses and could suppress the proliferation of their CD4(+) CD25(-) counterparts. In conclusion, these data demonstrate that CD4(+) CD25(+) T(reg) cells, with the potential to regulate the function of effector T cells and antigen-presenting cells, accumulate in the synovium of patients with RA.

Fig. 1

Synovial fluid (SF) CD4⁺ T cells express increased levels of CD25. Representative FACS stainings of SF and peripheral blood mononuclear cells (PB) from one patient with rheumatoid arthritis (RA) is shown. Cells with light-scatter characteristics of lymphocytes were gated. Double stainings with anti-CD4 and anti-CD25 MoAbs are shown. Gates define the cells expressing CD25 at high levels (CD4⁺ CD25^high).

Fig. 2

Synovial fluid (SF) CD4⁺ CD25⁺ T cells from patients with rheumatoid arthritis (RA) express increased levels of markers associated with the regulatory T cell phenotype. CD4⁺ mononuclear cells with light scatter characteristics of lymphocytes were gated. The expression of markers was analysed on gated SF CD25⁺ and CD25^– cells and peripheral blood (PB) CD25⁺ cells from the corresponding patients as well as normal controls. Numbers indicate the percentages (mean ± s.d.) of positive cells. Number of samples analysed was six, except for (a) 10 and (b) nine. Representative stainings from one patient and one control are illustrated. *P < 0·05 when compared to SF CD4⁺ CD25^– cells and RA PB or normal PB CD4⁺ CD25⁺ cells.

Fig. 3

Typical pattern of CD25 expression on sorted CD4⁺ CD25^– and CD4⁺ CD25⁺ SF T cell fractions used for functional studies. Representative stainings of one patient sample are shown.

Fig. 4

(a) Synovial fluid (SF) CD4⁺ CD25⁺ T cells express high levels of Foxp3 trancripts. Relative expression of Foxp3 mRNA in CD4⁺ CD25⁺ and CD4⁺ CD25^– T cell populations in SF from three patients with rheumatoid arthritis was studied by real-time quantitative polymerase chain reaction (PCR). β-actin was used as a reference gene. Normalized Foxp3 mRNA abundance was derived from the ratio of Foxp3 mRNA to β-actin mRNA expression in each sample. (b) SF CD4⁺ CD25⁺ T cells are hyporesponsive. SF CD4⁺ CD25⁺ and CD4⁺ CD25^– cells (5–7·5 × 10⁴ cells/well) were stimulated with either anti-CD3 (1 µg/ml) + anti-CD28 (10 µg/ml) MoAbs or phytohaemagglutinin (PHA) (5 µg/ml) and the proliferative response was measured by tritiated thymidine incorporation after 4 days of culture. (c) SF CD4⁺ CD25⁺ T cells are able to suppress the proliferation of their SF CD4⁺ CD25^– counterparts. CD4⁺ CD25^– cells (5 × 10⁴ cells/well), CD4⁺ CD25⁺ cells (5 × 10⁴ cells/well) and co-cultures of both CD4⁺ CD25^– (5 × 10⁴ cells/well) and CD4⁺ CD25⁺ cells (2·5 × 10⁴ or 1·25 × 10⁴ cells/well) from two patients were stimulated with anti-CD3 (1 µg/ml) and anti-CD28 (10 µg/ml) MoAbs for 4 days.