191-OR: Targeting IL-8/CXCR1-CXCR2 to Unleash Regulatory B Cells in Type 1 Diabetes

Introduction and Objective: The IL-8/CXCR1-CXCR2 axis has been demonstrated to be impaired in T1D, thus we hypothesize that targeting IL-8/CXCR1-CXCR2 axis may contribute to the expansion and differentiation of regulatory B cells. Methods: We profiled by FACS analysis murine and human Bregs (CD19+IL-10+ cells) isolated from patients with T1D as compared to healthy controls (HC) for IL-8/CXCR1-CXCR2 expression under resting/stimulating conditions. We tested the effect of murine Bregs expanded by LPS/rIL-2 in the presence of different titrations of Reparixin (1nM, 10nM and 50nM) in a co-culture assay with anti-CD3/anti-CD28 stimulated T cells or with diabetogenic T-cells (BDC2.5 stimulated CD4+ T cells), during a proliferation assay. Results: Our data showed that IL-8 and CXCR2 were found significantly higher on Bregs and also on non-Bregs (CD19+IL-10- cells) in patients with T1D as compared to HC (p<0.05). While under stimulating conditions (anti-CD40L/LPS), CXCR1 and CXCR2 were found significantly higher on Bregs from patients with T1D as compared to HC (p<0.05). The murine data in NOD on Bregs generated under stimulating conditions (LPS/IL-2) revealed an upregulation of CXCL1 and CXCR1 during the time course of T1D as NOD hyperglycemic showed the highest expression while those naturally protected from T1D showed the lowest expression (p<0.05). We next studied the effect of targeting IL-8/CXCR1-CXCR2 during murine Breg generation. A significant suppression of CD4+/CD8+ T cell effector proliferation in all conditions where Bregs were added as compared to T cells alone (p<0.001). Particularly, a reduced T-cell response toward islet autoantigens with Bregs expanded in the presence of Reparixin 10nM showed the most potent effect as compared to untreated (p<0.001). Lastly, we confirmed the immunoregulatory capacities of Bregs generated using Reparixin through an inhibition of IFN-γ production by diabetogenic T-cells (p<0.05). Conclusion: Altogether, targeting IL-8/CXCR1-CXCR2 axis may help to establish a novel tailored Breg-based immunotherapy in T1D. Disclosure S. Khalefa: None. M. Ben Nasr: None. V. Usuelli: None. P. Yerra: None. E. Assi: None. A. Petrazzuolo: None. M. Zocchi: None. F. D'Addio: Advisory Panel; Sanofi. C. Loretelli: None. I. Pastore: Advisory Panel; Sanofi. Board Member; Novo Nordisk. L. Montefusco: None. A. Rossi: Consultant; Roche Diabetes Care, Ascensia Diabetes Care, Ypsomed AG. M. Lunati: None. G. Zuccotti: None. M. Allegretti: Employee; Dompé. P. Fiorina: Consultant; Novo Nordisk, AstraZeneca. Board Member; Boehringer-Ingelheim. Funding The study is part of the activities planned in the co? financed project "Ladari.un as new Juvenile Diabetes Inhibitory Agent (LJDIA)" presented by Dompe under the Call "Fondo crescita sostenibile - Proposal n. 1410 bando MISE DM 02/08/2019 and consecutive DD 02/10/2019" ("MISE Grant").