Patients with Wegener's granulomatosis demonstrate a relative deficiency and functional impairment of T-regulatory cells

Abstract

An increased proportion of CD4(+) CD25(+) T cells has been reported in Wegener's granulomatosis (WG) and may represent an accumulation of regulatory T cells (Treg). CD25 is also expressed on recently activated effector T cells. We have determined the relative proportion of these subsets in a large patient cohort. The fraction of Treg in peripheral blood mononuclear cells from patients and healthy controls was determined by assessment of Foxp3 expression on CD4(+) CD25(+) T cells. The functional activity of Treg was determined by their ability to suppress proliferation and cytokine production in response to proteinase-3. Although WG patients demonstrated an increased fraction of CD4(+) CD25(+) T cells, the percentage of Foxp3-positive cells was decreased. In addition, the percentage of Treg was inversely related to the rate of disease relapse. CD4(+) CD25(hi) T cells were able to suppress T-cell proliferation to proteinase-3 in healthy controls and anti-neutrophil cytoplasm antibody (ANCA)- negative patients (at time of sampling) but not in ANCA-positive patients. In patients with active disease, an increased proportion of CD4(+) Foxp3(+) cells was associated with a more rapid disease remission. Patients with WG demonstrate abnormalities in the number and function of Treg and this is most pronounced in those with most active disease. This information is of value in understanding the pathogenesis and potential treatment of this disease.

Figure 1

Flow cytometry analysis of markers of T-cell activation and regulation on peripheral blood mononuclear cells from patients with Wegener’s granulomatosis (PG1; n= 55) and age-matched healthy controls (HC, n= 33). Patients showed an increased percentage of CD4⁺ lymphocytes that were CD25^hi (a) (*P= 0·0006) and an increase in CD25 median fluorescent intensity (MFI) (b) (**P< 0·0001). There was no difference between patients and HC in the percentage of Foxp3⁺ CD4⁺ cells (c). There was a non-significant decrease in Foxp3 expression in the patient group (d) with a decreased proportion of Foxp3⁺ cells in the CD4⁺ CD25^hi fraction in the patient group (e) (***P< 0·0001) and therefore the increase in CD4⁺ CD25^hi in the patient group is likely to be the result of an increase in the frequency of activated and not regulatory CD4⁺ T cells.

Figure 2

Foxp3 expression on patients (PG2) with active disease and after 14 weeks of treatment. (a) The percentage of Foxp3⁺ CD4 T cells in patients with active disease (week 0) was higher in patients who subsequently entered remission by 14 weeks of treatment than those who had not entered remission by 14 weeks. The percentage of Foxp3⁺ CD4 T cells increased by week 14 of treatment compared with week 0 in both patients who had gone into remission by week 14 (b) and those who still had active disease at week 14 (c). Only patients who had gone into remission by week 14 showed an increase in the MFI for Foxp3 on Foxp3⁺ CD4 T cells by week 14 (d).

Figure 3

Magnetic bead depletion of CD25⁺ cells. Anti-CD25 coated magnetic beads were used to deplete CD25^hi cells that were virtually all confined to the CD4⁺ subset (a, c). (b, d) are gated on CD4⁺ lymphocytes and demonstrate that in this healthy control CD4⁺ CD25^hi cells are virtually all Foxp3⁺ (b, d; right upper quadrant). There are however some CD4⁺ Foxp3⁺ lymphocytes that express intermediate levels of CD25 (b, d; right lower quadrant). (f, g) In both healthy controls and patients, bead depletion led to a decrease in % of CD4⁺ cells that were CD25^hi or Foxp3⁺. The removed cells were highly enriched in Foxp3 expressing Treg. Bead depletion led to an increase in proliferation to anti-CD3/anti-CD28 beads, measured at day 3 (e). Add-back of the depleted CD25^hi cells led to suppression of this increased proliferation. (*P< 0·05).

Figure 4

The effect of CD25⁺ cell depletion on proliferative responses in T cells from PG3. Relative proliferation to the auto-antigen proteinase 3 (PR3) in the presence of CD25^hi cells was significantly increased in the anti-neutrophil cytoplasm antibody positive (ANCA⁺) group compared with either the currently ANCA⁻ group (*P= 0·036) or healthy controls (**P= 0·002) (a). There were no significant differences in proliferation to purified protein deriviative (PPD) between the three groups (b). Proliferation was measured at 11 days. Removal of CD25^hi led to an increase in proliferation in response to PR3 in both healthy controls (c) and currently ANCA⁻ patients (d) with this increase in proliferation being significantly greater in the ANCA⁻ patients than controls (P= 0·001). The ANCA⁺ patients showed a significant decrease in proliferation in response to PR3 (e). Both healthy controls and currently ANCA⁻ patients showed an increase in proliferation to PPD with removal of CD25^hi cells but there was no difference in the degree of this increase between the two groups.