Activation of B lymphocytes with anti-CD40.

<p>(A) B lymphocytes sorted from tumor bearing C57Black/6 mice were treated <i>ex vivo</i> with 100 ng/ml anti-CD40 and analyzed by intracellular staining against the indicated phospho-proteins and flow cytometry; cells were stimulated for 30 min prior to harvesting. Results are the average of median fluorescence intensity of 3 independent experiments (indicated); (B) analysis of the expression of activation markers in peripheral lymph node cell suspensions determined by flow cytometry after 14 to 16 hours treatment. A representative experiment of 3 independent ones is shown (each experiment with at least 3 mice per group), and the average and standard deviation of the frequencies of the double-positive populations is shown in each dot-plot. Differences between control and treated cells were tested by t-test, * indicates p<0.05. (C) and (D) CellTrace labeled T lymphocytes from tumor bearing donors were incubated with sorted B lymphocytes from tumor bearing donors treated or not with 100 ng/ml anti-CD40 for 16 hours, in a 1:1 ratio. Phytohemagglutinin, (PHA) (1:100), or 5 μg/ml HPV16 E6 and E7 peptides (pep), were added to the cultures. Four days later, cells were harvested labeled with antibodies for CD4, CD8, CD154 and CD137 and analyzed by flow cytometry. The ratios of the percentage of CellTrace dim cells from cultures with anti-CD40 treated and control B lymphocytes (C) and expression ratio of CD154 or CD137, or both (DP) in cultures with treated and control B lymphocytes (D) are displayed. Ratios of three independent experiments, each one in biological triplicates (cells from 3 different mice, and our cultures were also plated in triplicates). The dashed lines indicate ratio value 1, where activation was the same in the two different conditions. The black solid short line, in C, indicates the proliferation ratio of T cells in neat cultures treated with PHA and compared to untreated T cells.