Prevention of apoptosis prevents b cell death and loss in vitro

Cellular immunologists all know that normal B lymphocytes die in vitro in a few days even under the most favorable culture conditions. Our laboratory has demonstrated that small dense resting B cells behave in vitro as if an apoptosis program is already underway, progressing to apoptosis much faster than in vivo lifespan estimates would predict. The simplest way to reconcile these data would be to postulate that B cell apoptosis rates can be regulated to very low levels in favorable environments. We have now shown that the combination of S ug/ml dextran sulfate CDS), 0.1 ug/ml cycloheximide (CHX), 100 ng/ml PMA and 20 U/ml IL-4 can hold the proportion of apoptotic B cells to the levels seen in fresh cells for at least 64 hr. Loss of viability by propidium iodide uptake and loss of recoverable cells also ceases under these conditions. B cells that survive a 64 hr culture retain normal responsiveness to LPS measured by acridine orange cell cycle analysis. Strikingly, when apoptosis is prevented by IL-4, PMA, CHX and DS during the response to LPS, the percent of cells jncycle rises to about 90% regardless of their preincubation history. So apoptosis accounts for virtually all cell death or loss in short term cultures, and also limits cell cycle entry in polyclonally activated cells.