Figure 10. CD73⁺ B-lymphocytes in DIDS treated mice are T1D suppressive.

NOD female mice were treated weekly with 50 mg/kg DIDS or vehicle from 8-16 weeks of age. (A) NOD-scid recipients were infused with 2×10⁶ purified splenic T-cells from vehicle-treated donors admixed with an equal number of purified total splenic B-lymphocytes from vehicle (VB) or total or CD73-depleted B-lymphocytes from DIDS-treated mice (DB). The NOD-scid recipients were then monitored for T1D development over 8 weeks. (B) 1.0×10⁵ NOD T-cells were labeled with Cell Proliferation Dye (CPD) eFluor670 and co-cultured for 4 days with 1.0×10⁵ CD73⁺ or CD73^- B-lymphocytes from pooled spleens of vehicle (n=6 biological replicates) or DIDS treated (n=6 biological replicates) mice under stimulation conditions consisting of soluble anti-CD40 (1 μg/mL), plate-bound anti-CD3ε (5 μg/mL) and soluble anti-CD28 (2 μg/mL) with 0 (baseline) or 10 μM AMP in the presence or absence of 100 μM APCP. Quantification of percent suppression from baseline. (C) 1×10⁵ sort-purified CD73⁺ or CD73^- B-lymphocytes from vehicle- or DIDS-treated NOD mice (n=6 biological replicates per group) were cultured for 3 days with 10 μg/mL LPS and culture supernatant IL-10 subsequently measured by ELISA. CD73-mediated in vitro suppression and IL-10 production data shown are each combined from two individual experiments. Incidence study p-value was calculated using Mantel-Cox analysis. Wilcoxon test was performed for suppression assay. Mann-Whitney analysis was performed for IL-10 production. All bar graphs show Mean±SEM.