Decreased numbers of blood dendritic cells and defective function of regulatory T cells in antineutrophil cytoplasmic antibody-associated vasculitis

Abstract

Background: Dendritic cells (DC) and regulatory cells (Treg) play pivotal roles in controlling both normal and autoimmune adaptive immune responses. DC are the main antigen-presenting cells to T cells, and they also control Treg functions. In this study, we examined the frequency and phenotype of DC subsets, and the frequency and function of Treg from patients with ANCA-associated vasculitis (AAV).

Methodology/principal findings: Blood samples from 19 untreated patients with AAV during flares and before any immunosuppressive treatment were analyzed, along with 15 AAV patients in remission and 18 age-matched healthy controls. DC and Treg numbers, and phenotypes were assessed by flow cytometry, and in vitro suppressive function of Treg was determined by co-culture assay. When compared to healthy volunteers, absolute numbers of conventional and plasmacytoid DC were decreased in AAV patients. During the acute phase this decrease was significantly more pronounced and was associated with an increased DC expression of CD62L. Absolute numbers of Treg (CD4(+)CD25(high)CD127(low/-) Tcells) were moderately decreased in patients. FOXP3 and CD39 were expressed at similar levels on Treg from patients as compared to controls. The suppressive function of Treg from AAV patients was dramatically decreased as compared to controls, and this defect was more pronounced during flares than remission. This Treg functional deficiency occurred in the absence of obvious Th17 deviation.

Conclusion: In conclusion, these data show that AAV flares are associated with both a decrease number and altered phenotype of circulating DC and point to a role for Treg functional deficiency in the pathogenesis of AAV.

Figure 1. Characterization and count of dendritic cells using flow cytometry.

A, Gating strategy for identification of DC subsets, and expression of CD86, CD62L and CCR7 (blue line) vs. isotype control (red line) on DC subset. B, Quantification of DC, myeloid DC and plasmacytoid DC using flow cytometry is shown for healthy controls (HC ; n = 18), remission phase (RP ; n = 15) and acute phase (AP ; n = 19) AAV patients. Data are presented as boxplots with whiskers from minimum to maximum. C, Left: the histogram shows an exemple of CD62 staining on pDC from a control subject (empty histogram) and an AAV patient in acute phase (grey histogram) as compared to isotype control (dotted line); Right: CD62L expression on pDC and mDC is shown as MFI of CDL62 – MFI of isotype control. Data are presented as boxplots with whiskers from minimum to maximum.

Figure 2. Characterization of Treg using flow cytometry in controls and patients.

A, Expression of CD25, CD127 and FOX-P3 in CD4⁺ T cells. Representative dot plots for healthy controls (HC), AAV patients in remission phase (AAV-RP) and AAV patients in acute phase (AAV-AP) showing CD25 vs. CD127 and CD25 vs. FOXP3 expression in gated CD4⁺CD3⁺ T cells are shown. The gate use to identify Treg in fresh blood is shown in each CD25 vs. CD127 dot plot. B. Representative histograms showing expression of FOX-P3 (bold line) vs. isotype control (dotted line) and CD39 (inset) in CD4⁺CD25^highCD127 ^low cells as gated in A.

Figure 3. Frequency, number and phenotype of Treg in patients vs. controls.

Histograms represent: A, Proportion of CD4⁺CD25^highCD127^low/− T cells within CD4⁺CD3⁺ T cells (as gated in figure 2A) for HC (mean, 6.8%, n = 18), remission, (mean, 6.9%, n = 15), and acute (mean, 6.7%, n = 19) AAV patients. No significant difference was observed for percentage of Treg cells between the 3 groups. B, Absolute numbers of CD4⁺CD25^highCD127^low/− T cells in whole blood for HC (mean, 60.3 cells/µL), remission (mean, 47.5 cells/µL), and acute (mean, 40.1cells/µL) AAV patients. C, MFI of FOX-P3 in gated Treg and D, Mean percentage of CD39⁺ cells within Treg. Data are presented as boxplots with whiskers from minimum to maximum.

Figure 4. Suppressive function of circulating T regulatory cells.

A, Gating strategy for Treg sorting and typical results after sorting. B, Proliferative responses of Treg, effector responder T cells and Treg:Tresp co-cultures (ratio1/1) upon CD3+CD28 stimulation. Histograms are expressed as mean value +/− SD of cpm for healthy controls (HC ; n = 18), remission patients (RP ; n = 15), and active patients (PA ; n = 19). C, The percentage of suppression of responding cells was determined as 1−(proliferation of co-culture/proliferation of responder population alone)×100. Data are presented as boxplots with whiskers from minimum to maximum. D, A suppressive assay mixing Treg and effector T cells from and AP patient and a healthy volunteer was performed. Black bars represent suppression with HC Treg, and white bars with patient Treg. The results of one experiment out of three with similar results are shown.

Figure 5. Concentration of IL-17 in co-culture supernatants.

IL-17 was quantified in supernatant using a multiplex fluorescent bead immunoassay: the levels of IL-17 produced by Treg, responder T cells and co-culture were not statistically different between the 3 groups.