ARTHRITIS & RHEUMATOLOGY Vol. 68, No. 2, February 2016, pp 494–504 DOI 10.1002/art.39437 V C 2016, American College of Rheumatology Breg Cells Are Numerically Decreased and Functionally Impaired in Patients With Systemic Sclerosis Athanasios Mavropoulos, Theodora Simopoulou, Areti Varna, Christos Liaskos, Christina G. Katsiari, Dimitrios P. Bogdanos, and Lazaros I. Sakkas Objective. Breg cells, a regulatory cell subset that produces interleukin-10 (IL-10), play a significant role in suppressing autoimmune responses and preventing auto- immunity. This study was undertaken to examine the num- ber and function of Breg cells in patients with systemic sclerosis (SSc), a disease with many autoantibodies. Methods. Forty-five patients with SSc (12 with early SSc, 33 with established disease including 16 with SSc-associated pulmonary fibrosis [PF]), 12 healthy control subjects, and 10 patients with rheumatoid arth- ritis (RA)–associated PF were studied. The phenotypes of immature/transitional Breg cells (CD191CD24 high CD38 high ) and memory Breg cells (CD191CD271 CD24 high ) were evaluated by flow cytometry. The func- tion of Breg cells was assessed by measuring the pro- duction of IL-10 after B cell activation. In addition, activation of p38 MAPK and STAT-3 was measured fol- lowing stimulation of the cells with B cell receptor (BCR) and Toll-like receptor 9 (TLR-9). Results. Percentages of memory Breg cells were decreased in patients with early SSc (mean 6 SEM 1.85 6 0.38%), those with established SSc (1.6 6 0.88%), those with SSc-associated PF (1.52 6 0.17%), and those with RA- associated PF (1.58 6 0.26%), compared to healthy controls (6.3 6 0.49%; each P < 0.001). Percentages of transitional Breg cells were also decreased. Expression of IL-10 by Breg cells after stimulation with TLR-9 was impaired in patients with SSc, particularly those with SSc-associated PF. Activa- tion of STAT-3 and p38 MAPK was impaired in naive and memory B cells from patients with SSc after stimulation with BCR and TLR-9. Expression of the stimulatory CD19 receptor was increased in B cells and also increased, to a lesser extent, in Breg cells from patients with SSc com- pared to healthy controls. Percentages of memory B cells were decreased in patients with SSc, particularly in those with SSc-associated PF. Conclusion. This is the first study to demonstrate that Breg cells are phenotypically and functionally im- paired in patients with SSc. Furthermore, in SSc, B cells exhibit impaired p38 MAPK and STAT-3 activation upon stimulation with BCR and TLR-9. The findings of decreased numbers of Breg cells along with increased expression of CD19 support the idea of B cell autoaggres- sion acting as an immunopathogenic mediator in SSc. Systemic sclerosis (SSc) is a chronic disease char- acterized by microvasculopathy (Raynaud’s phenome- non and fibrointima proliferation), production of auto- antibodies, and excessive collagen deposition in the skin and internal organs. The pathogenesis of SSc is incom- pletely understood, but immune cells are likely to be involved. There is evidence to suggest that T cells are implicated in the pathogenesis of the disease (1). Although frequencies of B cell infiltrates in SSc lesions vary (2–4), emerging evidence implicates B cells in the pathogenesis of SSc. B cells from patients with SSc overexpress CD19, a stimulatory B cell receptor (BCR), and similar overex- pression of CD19 in transgenic mice leads to spontane- ous autoantibody production (5). In contrast, CD19 Presented in part at EULAR 2015, the 16th Annual European Congress of Rheumatology, Rome, Italy, June 2015. Supported by the Special Fund for Research Grants (ELKE), University of Thessaly, Greece. Athanasios Mavropoulos, PhD(UK), Theodora Simopoulou, MD, PhD, Areti Varna, MD, Christos Liaskos, MD, PhD, Christina G. Katsiari, MD, PhD, Dimitrios P. Bogdanos, MD, PhD(UK), Lazaros I. Sakkas, MD, DM, PhD(UK), FRCP(UK): University of Thessaly, Larissa, Greece. Dr. Katsiari has received speaking fees from Bristol-Myers Squibb and Actelion (less than $10,000 each). Dr. Sakkas has received speaking fees and/or honoraria from Bristol-Myers Squibb, Novartis Hellas, Janssen-Cilag, MSD, Pfizer Hellas, and Actelion (less than $10,000 each). Address correspondence to Lazaros I. Sakkas, MD, DM, PhD(UK), FRCP(UK), University of Thessaly, Department of Rheu- matology, Faculty of Medicine, Biopolis, Larissa 41110, Greece. E-mail: lsakkas@med.uth.gr. Submitted for publication January 27, 2015; accepted in revised form September 10, 2015. 494