Abnormalities of CD4 T cell subpopulations in ANCA-associated vasculitis

Abstract

In patients with ANCA-associated vasculitis (AAV), CD25 expression is increased on circulating T cells. Although in animal experiments the role of CD4(+) CD25(+) T-regulatory-cells (T(reg)) in protection against autoimmunity is well established, the role of these cells in AAV is unknown. To investigate the hypothesis that an increased expression of CD25 on T cells is related to persistent T cell activation and not to disturbances in T(reg) cells in AAV (34 patients, six of them after renal transplantation), we investigated CD25 expression in different subpopulations of CD4(+) cells and FOXP3 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR). In addition, T cell proliferation and cytokine secretion after stimulation with anti-CD3 and anti-CD28 and intracellular cytokine production after stimulation with phorbol myristate acetate (PMA)-ionomycin was determined. Controls were non-vasculitic renal transplant patients (n = 9) and healthy controls (HC) (n = 13). In AAV the total number of lymphocytes, CD4(+) lymphocytes and the percentage of naive T cells are lower than in HC and RTX. An increased percentage of CD25(+) cells was found in AAV and AAV/RTX, irrespective of disease activity, but not in HC or RTX. This was confined to the naive (CD4(+) CD45RB(high)) population only. FOXP3 mRNA expression in CD4(+) T cells did not differ between AAV patients and healthy controls. In vitro T cell proliferation was enhanced in AAV patients compared to HC (P < 0.01). PBMC of AAV patients produced significantly less interleukin (IL)-10 and interferon (IFN)-gamma after anti-CD3/CD28 stimulation. The percentage of IL-10 and IL-12, but not IFN-gamma, IL-4 or tumour necrosis factor (TNF)-alpha-producing cells was significantly higher in patients compared to HC. These findings were confined to the memory population of CD4(+) cells. We conclude that AAV patients are lymphopenic and have low numbers of CD4(+) T cells, which seem to be in a persistent state of activation.

Fig. 1

Differences in CD25 expression in CD45RB subsets of CD4⁺ T cells in healthy controls and AAV patients. (a) Peripheral blood of healthy controls (HC) and vasculitis patients (AAV) were stained for CD4, CD45RB and CD25 using directly conjugated antibodies. CD45RB subsets were gated and analysed within those gates for the expression of CD25. The results of a representative experiment (n = 33) are depicted. (b) Similar staining as in (a) with peripheral blood obtained from a PR-3-ANCA positive patient with infectious endocarditis, but without evidence for vasculitis. M1 represents the region for positive cells.

Fig. 2

Immunohistochemical analysis of CD4⁺ and CD25⁺ cells in renal lesions of AAV and RTX patients. Cryostat sections were stained either with monoclonal antibodies directed against CD4 (upper panels) or CD25 (lower panels). In RTX patients both CD4⁺ and CD25⁺ T cells were easily detectable in the lesions. In contrast, in lesions of AAV patients CD4 was expressed abundantly, while the expression of CD25 in the inflammatory lesions of these patients was weak. Original magnification 100×.

Fig. 3

FOXP3 expression in CD4⁺ T cells. CD4⁺ T cells were isolated from 24 AAV patients (filled circles) and 10 healthy controls (open circles). FOXP3 expression was determined using LightCycler RT-PCR. The results are expressed as quotient of FOXP3 and G6PDH. There were no significant differences between AAV patients and HC.

Fig. 4

Proliferation of anti-CD3 and IL-2 stimulated CD4⁺ CD45RB subsets of AAV patients and HC. PBMC were labelled with CSFE and subsequently stimulated for 5 days with anti-CD3 in the absence (upper graph) or presence (lower graph) of IL-2 (50 U/ml). The cells were stained with anti-CD4 and anti-CD45RB to assess the different CD45RB subsets. Proliferation was measured in each of these subsets by gating. Ten patients in the AAV group and eight individuals in the HC group were tested. n.s.: not significant

Fig. 5

Proliferation of PBMC and purified CD4⁺ cells from AAV patients (n = 9) and HC (n = 9). PBMC (filled circles) and purified CD4⁺ (open circles) were labelled with CSFE and stimulated with either 0·5 µg of cross-linked anti-CD3 (upper graph) or with the combination of cross-linked anti-CD3 (0·1 µg) and anti-CD28 (1 µg) (lower graph). Proliferation was assessed after 5 days by means of FACS analysis. The bold line shows mean proliferation in each group.

Fig. 6

Cytokine production in PBMC (filled circles) and purified CD4⁺ cells (open circles) of AAV patients and HC (n = 8 for both). PBMC or purified CD4⁺ cells were stimulated for 5 days using plate-bound anti-CD3 and anti-CD28 (1 µg for both). Hereafter the supernatants were collected and assessed for IFN-γ (upper graph) and IL-10 (lower graph) production by ELISA. n.s.: not significant. Mean cytokine production is represented by the bold line.

Fig. 7

(a) Percentage of IL-10-producing cells. PBMC were isolated from 27 AAV patients (filled circles) and 10 healthy controls (open circles). After 4 h stimulation with PMA/ionomycin cells were harvested and fixed. IL-10 production was analysed by intracellular staining and subsequent FACS analysis. CD4⁺ T cells from AAV patients produced significantly more IL-10 compared to HC (P < 0·01). This was due mainly to an increase in IL-10-producing cells in the memory subpopulation (CD45RO⁺, P < 0·05). (b) Percentage of IL-12-producing cells. PBMC were isolated from 27 AAV patients (filled circles) and 10 healthy controls (open circles). After 4 h stimulation with PMA/ionomycin, cells were harvested and fixed. IL-12 production was analysed by intracellular staining and subsequent FACS analysis. CD4⁺ T cells from AAV patients produced significantly more IL-12 compared to HC (P < 0·01). This was due mainly to an increase in IL-12-producing cells in the memory subpopulation (CD45RO⁺, P < 0·05).