T-cell stimulatory capacity of in vitro–activated mouse pre-DCs.

Purified mouse blood pre-DC populations were tested for their ability to stimulate naive CD4⁺ T cells in an allostimulatory MLR. Freshly isolated pre-DC populations did not yield counts greater than the background obtained with T cells alone (fewer than 1000 counts per well), and only the results from in vitro–stimulated pre-DCs are shown. Pre-DCs were cultured overnight with their respective optimal stimuli: CD45RA⁻CD11c^int pre-DCs in GM-CSF and TNF-α and CD45RA^hiCD11c^lo/int pre-DCs in GM-CSF and CpG. In addition, the cells were cultured overnight with the optimal stimulus of the other population. After such preculture, the activated pre-DCs were washed, and live cells were counted and added to T cells for the MLR culture assay. The average cpm of triplicate values is shown for each time point, and the error bars represent the range of triplicate values. Data shown are from a single experiment that compared mouse pre-DC populations purified directly or after in vitro stimulation. Similar results were obtained in a second experiment. Activated CD45RA^hiCD11c^int pre-DCs from mouse spleen gave similar results, stimulating proliferation in allogeneic T cells though less effectively than conventional DCs.