FACS-sorted and CFSE-labeled na?ve B cells (1105, CD19+IgD+CD27?) were stimulated with IgM then co-cultured with the DCs in the presence of 20 units/ml IL-2, 50 nM CpG and 100 ng/ml agonisticCD40 mAb (clone 12E12)

FACS-sorted and CFSE-labeled na?ve B cells (1105, CD19+IgD+CD27?) were stimulated with IgM then co-cultured with the DCs in the presence of 20 units/ml IL-2, 50 nM CpG and 100 ng/ml agonisticCD40 mAb (clone 12E12). assessed. (F) Staining of IFNDCs and TNFDCs with LOX-1 (8B4) mAb (upper left). B cells were co-cultured with LOX-1-treated IFNDCs, as in (E). On day 6, cells proliferation and PB differentiation were assessed. (lower left). On day12, culture supernatants were analyzed to measure the amount of Igs by ELISA (right). (G) CFSE-labeled 5105 PBMCs were cultured for 7 days in plates coated with 2g/ml LOX-1 or control IgG. PB differentiation was assessed (left). On day 12, the amounts of Igsin the supernatants were assessed by ELISA. Error bars indicate SD of triplicate assays from two impartial experiments.Physique S2. LOX-1-treated DCs promote na?ve B cell differentiation into Ig-secreting PBs. (A) IL-4DCs (5103/well) were incubated overnight in plates coated with LOX-1 (8B4) or control IgG. FACS-sorted and CFSE-labeled na?ve B cells (1105, CD19+IgD+CD27?) were stimulated with IgM then co-cultured with the DCs 1alpha, 24, 25-Trihydroxy VD2 in the presence of 20 units/ml IL-2, 50 nM CpG and 100 ng/ml agonistic CD40 mAb (clone 12E12). On day 6, B cells were stained for HLA-DR. (B) Na?ve B cell culture in (A) were performed in the absence or presence of DCs. On day 6, B cells were stained and assessed for PB differentiation. Two impartial experiments using cells from different donors showed similar results. (C) Culture supernatants of the DC-B 1alpha, 24, 25-Trihydroxy VD2 cell co-culture in (A) were harvested on day 12 and the amounts of Igs were measured by ELISA. Physique S3. LOX-1 mAb does not induce 7 integrin, CCR6, or CCR9 expression on na?ve B cells co-cultured with DCs. IL-4DCs (5103/well) were incubated overnight in plates coated 1alpha, 24, 25-Trihydroxy VD2 with 2g/mlLOX-1 or control IgG. FACS-sorted and CFSE-labeled na?ve B cells (1105, CD19+IgD+CD27?) were stimulated with IgM then co-cultured with the DCs in the presence of 20 units/ml IL-2, 50 nM CpG and 100 ng/ml agonisticCD40 mAb (clone 12E12). On day 6, cells were stained withCD19 andCD38 along with indicated antibodies. CD19+CD38+ live cells were gated to assess the surface expression levels of 7 integrin, CCR6, and CCR9. Physique S4. LOX-1 (8B4) mAb can induce DCs to secrete APRIL and BAFF and further promotes Ig-secreting B cell responses. (A) 1105 IL-4DCs were cultured 72h in plates coated with the indicated mAbs (2g/ml). The amounts of APRIL and BAFF in the supernatants were measured by ELISA. Each dot represents data generated with cells from different healthy donors. (B) IL-4DCs (5103/well) were incubated overnight in plates coated with 2 g/ml LOX-1 (8B4), DCIR (9E8),Dectin-1 (15E2),DC-SIGN (24G3), or control IgG. FACS-sorted and CFSE-labeled na?ve B cells (1105, CD19+IgD+CD27?) were stimulated with IgM then co-cultured with the DCs in the presence of 20 units/ml IL-2, 50 nM CpG, and 100 ng/ml agonisticCD40 mAb (clone 12E12). Culture supernatants were harvested on day 12 and the amounts of Igs were measured by ELISA. Error bars indicate SD of triplicate assays. Two impartial experiments using cells from different healthy donors 1alpha, 24, 25-Trihydroxy VD2 showed comparable results. (C) 1 105 IL-4DCs were cultured 72h in plates coated with the indicated mAbs (2g/ml). The amounts of APRIL and BAFF in the supernatants were measured by ELISA. Each dot 1alpha, 24, 25-Trihydroxy VD2 represents data generated with cells from different healthy donors. Physique S5. ox-LDL can Sirt2 activate B cells. Purified CD19+B cells (1105/well) were cultured in the presence or absence of 30g/ml ox-LDL for 12 days. 20 units/ml IL-2 was added into the culture. Culture supernatants were analyzed to measure the amount of Igs by ELISA. Two impartial experiments using cells from different donors were performed. Each experiment was performed with a triplicate assay. Error bars indicate SD. Physique S6. LOX-1 mAb binds to rhesus macaque LOX-1 and also binds to the surface of CD11c+ and CD14+ cells, but not CD3+.