Cell density plays a critical role in ex vivo expansion of T cells for adoptive immunotherapy.

The successful ex vivo expansion of a large numbers of T cells is a prerequisite for adoptive immunotherapy. In this study, we found that cell density had important effects on the process of expansion of T cells in vitro. Resting T cells were activated to expand at high cell density but failed to be activated at low cell density. Activated T cells (ATCs) expanded rapidly at high cell density but underwent apoptosis at low cell density. Our studies indicated that low-cell-density related ATC death is mediated by oxidative stress. Antioxidants N-acetylcysteine, catalase, and albumin suppressed elevated reactive oxygen species (ROS) levels in low-density cultures and protected ATCs from apoptosis. The viability of ATCs at low density was preserved by conditioned medium from high-density cultures of ATCs in which the autocrine survival factor was identified as catalase. We also found that costimulatory signal CD28 increases T cell activation at lower cell density, paralleled by an increase in catalase secretion. Our findings highlight the importance of cell density in T cell activation, proliferation, survival and apoptosis and support the importance of maintaining T cells at high density for their successful expansion in vitro.