Figure 1

Robust expansion of “donor” B cells to be used as stimulators using ultraCD40L (n = 8). Peripheral blood was obtained from healthy volunteers and PBMC were isolated using Ficoll centrifugation. Either PBMC or CD19⁺ B cells purified using Miltenyi microbeads were cultured for 14 days in B cell culture media containing 20% ultraCD40L and 40ng/mL IL-4. Cyclosporine at 400ng/mL concentration was also included with PBMC cultures to prevent the growth of T cells. Cells were counted and flow cytometric analysis were performed on indicated days. (A) Absolute number of cells in the culture (mean ± SD); (B) Percentage of CD19⁺ B cells in the cultures where the starting cells were total PBMC; (C) A representative flow cytometry analysis showing the gating strategy for the classic B cell marker (CD19) and activation markers (CD80, CD86, and HLA-DR) in CD19⁺ gated cells. (D) The percentages CD19⁺ cells that expressed the activation markers on indicated days. Please note the robust expansion of B cells displaying high expression of HLA-DR, CD80 and CD86, hallmark of effective antigen presenting cells. (E) The expanded B cells that were to be used as stimulator cells were irradiated at indicated centi-Grey and cultured in 15% Human AB serum supplemented RPMI-1640 medium (15% HAB Medium). No viable B cells could be observed beyond 1 week of the culture, indicating that no B cell contamination was to be expected in the final expanded Treg product (of Fig. 2).