Surface expression of CD28 is progressively down-modulated in a cell division–dependent manner in dividing CD8 but not CD4 T cells. (A) FCM histogram overlay of CD28 staining in total CD28⁺-purified splenocytes stimulated for 96 hours with immobilized anti-CD3 (solid line) or parallel nonstimulated control cultures (dashed line). Activation leads to the broadening of CD28 staining intensities, suggesting simultaneous maintenance, down-regulation, and moderate up-regulation of CD28 surface levels in different subpopulations of stimulated cells. The result is representative of 4 adult (5-7 years old) animals analyzed in 2 independent experiments. (B) Representative FCM dot plots of CD28 surface levels in different generations of dividing cells after 96 hours of anti-CD3 stimulation of CD28⁺-purified splenocytes, gated on CD4 and CD8 cells as indicated. There is an evident down-regulation of CD28 surface levels in CD8 cells, directly proportional to the number of divisions, whereas CD4 cells maintain or slightly up-regulate CD28 expression. (C) Relative CD28 staining intensity (relative fluorescence index, RFI) in different generations of dividing CD4 and CD8 cells, compared with the undivided population (RFI = 1). The data were obtained from 4 young adult animals (5-7 years old) and are representative of 2 independent experiments.