THU0050 CXCL13 IS A KEY DRIVER FOR MIGRATION AND DIFFERENTIATION OF REGULATORY B CELLS

Abstract

Background:Regulatory B cells in human still need to be characterized. Given the absence of a phenotypical definition of these cells, a functional definition based on their ability to secrete IL-10 is often used (corresponding to B10+ cells). Chemokine receptors (CKR) profiles are useful to characterize some populations of T cells but have never been explored among B10+cells. Moreover, very little is known about B10+cell migration. Chemokines (CK) have also been implicated in the differentiation of naïve T cells towards regulatory T cells.Objectives:Therefore, the aims of our study were to first characterize the profile of CKR on B10+cells, and second to investigate CK implicated in their migration and differentiation, this, both in control (CTL) and in patients with rheumatoid arthritis (RA).Methods:B cells were isolated with Rosette Sep Human B cells enrichment followed by Ficoll separation. B cells were then activated 24 hours with CpG and CD40L to generate B10+cells. IL-10 secretion from B cells was assessed by FACs and ELISA. We compared the expression of several CKR between B10+and IL-10negB cells (B10neg) from CTL and RA patients by flow cytometry. For migration assay, B10+and B10negcells were sorted by FACSaria. Their ability to migrate, in response to ligand of CKR found differentially expressed in the first part (CCL21, CCL22, CXCL11, CXCL12 or CXCL13) or synovial fluid (SF) from RA patients, were evaluated by migration assay in 5μM Transwell chambers and expressed as fold increase compare to basal migration towards control media.Results:B10+cells expressed a different profile of CKR compared to B10negboth in CTL and RA patients and these profiles differed between B10+cells of CTL and RA patients. However, no CKR profile could phenotypically define B10+cells. Of note, CXCR5 was under-expressed on B10+cell surface compared to B10negin CTL (75% [IQR 72.9-81.4] positive cells among B10+vs 99.2% [98.4-99.4] positive cells among B10neg, p=0.006, n=10) and also in RA patients (78.3% [70.8-82.3] vs 98.2% [96.9-99.54, p=0.008, n=8). Nevertheless, mRNA expression of CXCR5 was higher among B10+versus B10negcells in CTL and RA patients. As CpG-stimulated cells over-expressed CXCL13, ligand of CXCR5, we hypothesized that the binding of its ligand induced the internalisation of CXCR5. Indeed, among all CK tested, only CXCL13, attracted significantly more B10+than B10negfrom CTL (9.1[5.6-14.6] fold increase migration of B10+vs 5.2 [3.1-7.5] fold increase migration of B10neg, p<0.0001, n=21). This was also true in RA patients (10.9 [3.6-29.9] fold increase migration of B10+vs 4.8[2.1-7.7] fold increase migration of B10neg, p=0.009, n=12). SF from RA patients induced a significant migration of B10+cells in CTL (7.3-fold increase [4.1-21.7], p=0.004, n=9) and RA patients (5.7-fold increase [2.3-7.9], p=0.008, n=10). This migration was correlated with the levels of CXCL13 in these SF, in CTL (r=0.7, p=0.05, n=9) but not in RA patients (n=10). Lastly, CXCL13 was also found to increase IL-10 secretion in B cells stimulated with CpG in CTL (1.5-fold increase [1.3-1.5], p=0.0002, n=13) and in RA patients (1.2-fold increase [1.1-1.3], p=0.005, n=12).Conclusion:We showed that CXCL13 is a key driver for migration and differentiation of B10+ cells in CTL and in RA patients. However, the migration of B10+cells in RA patients was not correlated with the level of CXCL13 in SF from RA patients, suggesting the implication of other CK in the migration of B10+cells in RA.Disclosure of Interests:None declared