Peripheral regulatory cells immunophenotyping in primary Sjögren's syndrome: a cross-sectional study

Abstract

Introduction: IL-10-producing B cells, Foxp3-expressing T cells (Tregs) and the IDO-expressing dendritic cells (pDC) are able to modulate inflammatory processes, to induce immunological tolerance and, in turn, to inhibit the pathogenesis of autoimmune disease. The aim of the study was to characterize and to enumerate peripheral IL-10--producing B cells, Tregs and pDCregs in primary Sjögren's Syndrome (pSS) patients in regard of their clinical and serologic activity.

Methods: Fifty pSS patients and 25 healthy individuals were included in the study. CD19⁺-expressing peripheral B lymphocytes were purified by positive selection. CD19⁺/CD24(hi)/CD38(hi)/IL-10-producing B cells, CD4⁺/CD25(hi)/Foxp3⁺ and CD8⁺/CD28⁺/Foxp3⁺ Tregs, as well as CCR6⁺/CD123⁺/IDO⁺ DCs, were quantitated by flow cytometry.

Results: Immature/transitional circulating IgA⁺ IL-10-producing B cells had higher levels in pSS patients versus control group, whereas CD19⁺/CD38(hi)/IgG⁺/IL-10⁺ cells had lower percentage versus control. Indeed CD19⁺/CD24(hi)/CD38(hi)/CD5⁺/IL-10⁺, CD19⁺/CD24(hi)/CD38(hi)/CD10⁺/IL-10⁺, CD19⁺/CD24(hi)/CD38(hi)/CD20⁺/IL-10⁺, CD19⁺/CD24(hi)/CD38(hi)/CD27⁻/IL-10⁺, and CD19⁺/CD24(hi)/CD38(hi)/CXCR7⁺/IL-10⁺ cells had higher frequency in clinical inactive pSS patients when compared with control group. Remarkably, only percentages of CD19⁺/CD24(hi)/CD38(hi)/CD10⁺/IL-10⁺ and CD19⁺/CD24(hi)/CD38(hi)/CD27⁻/IL-10⁺ subsets were increased in pSS serologic inactive versus control group (P < 0.05). The percentage of IDO-expressing pDC cells was higher in pSS patients regardless of their clinical or serologic activity. There were no statistically significant differences in the percentage of CD4⁺/CD25(hi)/Foxp3⁺ Tregs between patient groups versus controls. Nonetheless, a decrease in the frequency of CD8⁺/CD28⁻/Foxp3⁺ Tregs was found in inactive pSS patients versus controls (P < 0.05).

Conclusions: The findings of this exploratory study show that clinical inactive pSS patients have an increased frequency of IL-10--producing B cells and IDO-expressing pDC cells.

Figure 1

Clinical and laboratory data for patients with primary Sjögren's syndrome (pSS). Results are expressed as median, 10^th, 25^th, 75^th, and 90^th percentiles. *P < 0.05. SSDAI, Sjögren's syndrome disease activity index; ESSSDAI, European League Against Rheumatism Sjögren's syndrome disease activity index; Ig, immunobglobulin; ESR, erythrocyte sedimentation rate.

Figure 2

Percentage of circulating cells. (A) IL-10-producing B cells, (B) CCR6⁺/CD123^hi-, (C) CD8⁺/CD28^-- and (D) CD4⁺/CD25^hi-circulating cells. Results are expressed as mean. *P < 0.05, for comparison of pSS patients with control (CTL) group.

Figure 3

Percentage of immunoglobulin (Ig) expression on IL-10-producing B peripheral-cell subtype in patients with primary Sjögren's syndrome (pSS). CD19⁺ blood B cells were obtained by positive selection with microbeads. (A) Representative unstained and permeabilized sample of peripheral blood mononuclear cells (autofluorescence control). (B) phycoerythrin (PE)-labeled-anti rat-IL-10 IgG isotype control. (C-E) IgG₁-fluorescein isothiocyanate (FITC)/IgG₁-PE/CD45-PeCy5 mouse IgG₁, k isotype controls (BD Tritest™, BD Biosciences). (F) Representative contour plot of CD19⁺ B cells. An electronic gate was made for CD38^hi cells. (G) From the gate F CD19⁺⁄CD38^hi/IgM⁺ cells were determined. (H) From the latter CD19⁺⁄CD38^hi⁄IgM⁺⁄IL-10⁺ cells were defined. A total of 50,000 to 100,000 events were recorded for each sample before any gate setting, and were analyzed with the CellQuestPro software (BD Biosciences). Bar graphs show percentage of (I) CD19⁺⁄CD38^hi⁄IgM⁺⁄IL-10⁺ (J) CD19⁺⁄CD38^hi⁄IgG⁺⁄IL-10⁺, and (K) CD19⁺⁄CD38^hi⁄IgA⁺⁄IL-10⁺ cells. (I-K) Results are expressed as median, 10^th, 25^th, 75^th, and 90^th percentiles. *P < 0.05. APC, allophycocyanine.

Figure 4

Percentage of immature/transitional IL-10-producing B peripheral-cell subtype in patients with primary Sjögren's syndrome (pSS). CD19⁺ blood B cells were obtained by positive selection with microbeads. (A) Representative contour plot of CD19⁺ B cells. An electronic gate was made for CD38^hi cells. (B) From the gate A CD19⁺⁄CD38^hi⁄CD24^hi cells were determined. (C) From the gate B CD19⁺⁄CD38^hi⁄CD24^hi⁄CD20⁺ were defined. (D) From the latter CD19⁺⁄CD38^hi⁄CD24^hi⁄CD20⁺⁄IL-10⁺ cells were determined. Bar graphs show percentage of (E) CD19⁺⁄CD38^hi⁄CD24^hi⁄CD5⁺⁄IL-10⁺, (F) CD19⁺⁄CD38^hi⁄CD24^hi⁄CD10⁺⁄IL-10⁺, (G) CD19⁺⁄CD38^hi⁄CD24^hi⁄CD20⁺⁄IL-10⁺, (H) CD19⁺⁄CD38^hi⁄CD24^hi⁄CD27^-⁄IL-10⁺, (I) CD19⁺⁄CD38^hi⁄CD24^hi⁄CXCR4⁺⁄IL-10⁺-, and (J) CD19⁺⁄CD38^hi⁄CD24^hi⁄CXCR7⁺⁄IL-10⁺-producing B peripheral cells. A total of 50,000 to 100,000 events were recorded for each sample before any gate setting and were analyzed with the CellQuestPro software (BD Biosciences). (E-J) Results are expressed as median, 10^th, 25^th, 75^th, and 90^th percentiles. *P < 0.05.

Figure 5

Percentage of indoleamine 2,3-dioxygenase (IDO)- and Foxp3-expressing peripheral blood cells in patients with primary Sjögren's syndrome (pSS). (A,B) Control of fluorescein isothiocyanate (FITC)-labeled-rabbit anti-sheep specificity staining. (C) An electronic gate was made for CCR6⁺ cells. (D) From the gate C CCR6⁺⁄CD123^hi cells were determined. (E) From the latter CCR6⁺⁄CD123^hi⁄IDO⁺ cells were defined. (F) An electronic gate was made for CD4⁺ cells. (G) From the gate F CD4⁺⁄CD25^hi were determined and an electronic gate was made for double positive cells. (H) From the latter CD4⁺⁄CD25^hi⁄Foxp3⁺ cells were defined. Bar graphs show percentage of (I) CCR6⁺⁄CD123^hi⁄IDO⁺ cells, (J) CD4⁺⁄CD25^hi⁄Foxp3⁺ cells, (K) CD8⁺⁄CD28^-⁄Foxp3⁺ cells. A total of 100,000 to 250,000 events were recorded for each sample before any gate setting, and were analyzed with the CellQuestPro software (BD Biosciences). (I-K) Results are expressed as median, 10^th, 25^th, 75^th, and 90^th percentiles. *P < 0.05.