Induction of apoptosis by cross-linking antibodies bound to human B-lymphoma cells: expression of Annexin V binding sites on the antibody cap.

unlabelledThere are many reports that cross-linking antibodies (Abs) bound to the surface of B-lymphoma cells can induce apoptosis and/or cell death, especially with anti-CD20 Abs. This study was intended to extend our understanding of these effects. To determine if CD20 is a unique target in this respect, or whether Abs to other antigens would have similar effects, six Abs were tested, with and without cross-linking with a secondary Ab, on three target cell lines. We utilized assays that distinguish between apoptotic, dead, and viable cells. Two assays were used: Annexin V plus propidium iodide, and JC-1 plus SYTOX green (Molecular Probes, Eugene, OR). Most of the Abs tested induced a low level of apoptosis and cell death in Ramos cells, but not in the other two cell lines (Raji and RL). In general, the level of toxicity was correlated with the level of antigen expression, with Abs to high-density antigens having the strongest effects. However, since the majority of Ramos cells continued to multiply, it is questionable whether toxicity at this level can provide a significant clinical benefit. Unexpectedly, there was also a population of cells that stained weakly with Annexin V. These cells were distinct from classical apoptotic cells, and appeared to belong to the viable cell population. In these cells, Annexin V stained the region of the Ab cap, in contrast to the ringed staining of classical apoptotic cells. IN

conclusion1) Low-level induction of apoptosis was not unique for anti-CD20 Abs, but occurred similarly with other Abs, and 2) results of Annexin V staining experiments may need to be reevaluated. Further studies are required to explain why Annexin V binding sites are exposed in the region of an Ab cap.