CD40 activated human B cells can efficiently induce early T-cell activation. (A) Human B cells were enriched (EasySep, StemCell Technologies) from buffy coats of 4 healthy donors. Informed consent and approval of the institutional review board were previously obtained. A representative flow cytometry analysis of the 4 experiments is shown indicating high enrichment of B cells (> 90%) and efficient depletion of monocytes (< 0.2%). (B) Enriched B cells were activated over 7 days on a monolayer of NIH3T3 cells constitutively expressing the human CD40 ligand. Surface expression of CD80, CD86 and HLA-DR is significantly up-regulated after 7 days of coculture demonstrated by flow cytometry analysis (dark histograms). (C) A mixed lymphocyte reaction of CD40-activated B cells (CD40B) with allogeneic T cells (left column) was performed over 5 hours. Monensin was added after 1 hour. Corresponding cell free supernatants (SN) and culture media (ctrl) were used for T-cell cultures as controls. Subsequent analyses of CD40L (CD154) and CD107a expression on CD4⁺ T cells as early activation markers were performed by flow cytometry. Analog experiments were done with none-manipulated peripheral blood mononuclear cells (PBMC, right column). Supernatants from PBMC were taken from overnight cultures. Results are representatives of 4 experiments of each condition (CD40B versus PBMC). (D) Results of the coculture experiments are shown separately as means of the relative numbers of CD40L⁺ or CD107a⁺ T cells (n = 4 CD40B experiments; n = 4 PBMC experiments). SD is indicated for each column.