CD4 mAb induced apoptosis of peripheral T cells: multiparameter subpopulation analysis by flow cytometry using Attractors (TM)

Studies describing the induction of apoptosis for CD4 mAbs do not delineate between epitope-dependent and Fc-driven epitope cross-linking induced cell death. Keliximab and clenoliximab are two CD4 mAbs that differ only in the ir heavy chain isotypes, being an IgG1 and a modified IgG4, respectively. T hese antibodies suppress CD4 T cell responses in vitro and in vivo and have been in human clinical trials for the treatment of RA and asthma. Here we compared the apoptotic activity of these mAbs to differentiate between the contributions of epitope-dependent vs. Fc-driven epitope cross-linking indu ced cell death in vitro as a link to differential CD4 cell depletion in viv o. We developed a simple flow cytometry procedure that measures apoptosis w ithin intact and compromised subpopulations of PBMCs within a few hours of culture. Attractors (TM) software was used to quantitate the percentage of apoptotic CD4 T cells, which generate reactive oxygen species (ROS), expres s external phosphatidyl serine (PS) and cleaved fluorescein diacetate (FDA) , within the intact and compromised lymphocyte populations. Treatment of fr eshly isolated PBMCs with keliximab resulted in the appearance of character istic apoptotic condensed CD4 T cells that contained reactive oxygen specie s. were annexin V positive and had intact esterase activity. Apoptosis was evident within 3 h and continued throughout the 72-h culture period. In con trast, clenoliximab alone did not induce apoptosis. The use of multiparamet er flow cytometry and Attractors (TM) to analyze subpopulations based on sc atter properties and biochemical processes during apoptosis provides a sens itive assay in which to quantitate and characterize the induction of cell d eath. Depletion of CD4 T cells in vivo by keliximab may reflect, in part, a ntibody-mediated apoptosis of these cells that is dependent on Fc gamma rec eptors. (C) 2001 Elsevier Science B.V. All rights reserved.