Objectives and design:The expression of the accessory molecule CD28 was compared in various populations of T and natural killer (NK) cells from HIV-1-negative and HIV-1-positive individuals and correlated with activation using mitogensin vitro.Methods:Multiparameter flow cytometric analysis using combinations of CD3 CD28 and other markers was performed together with absolute cell counting in peripheral blood. Blast transformation and proliferative responses were also quantitated using the Cytoronabsoluteafter stimulation with phytohaemagglutinin (PHA) and anti-CD3. CD28− cells were also purified to confirm the observations.Results:In HIV-1-negative individuals >90% of CD3+ T cells were CD28+ and responded to stimulation, while CD3− CD16+ CD57+ NK-like cells were CD28-and failed to respond. In HIV-1-positive individuals the expression of CD28 was greatly reduced and the proportion of CD3+CD28− T cells expanded. CD8 lymphocytosis was caused entirely by the accumulation of CD28− T cells and many of these expressed activation markers human lymphocyte antigen-DR, CD38 and CD45RO on their membrane and molecules such as TIA-1 and perform, associated with cytolytic function, in their cytoplasm. The strong positive correlation (r = 0.66) between the lack of CD28 expression and the poor proliferation from HIV-1-positive individuals was confirmed by demonstrating that only CD28+ cells transformed into lymphoblasts and proliferated. Although the CD28− including CD3+ T cells transiently expressed CD25 (interleukin-2Rct), they did not undergo blastogenesis or activation measured by bromodeoxyuridine uptake and died after 3–4 days in culture. These observations were confirmed in costimulation experiments with anti-CD2 and anti-CD28.Conclusion:In HIV-1 infection activated CD3+CD28− T cells accumulate but are unresponsive to mitogens and anti-CD28. These cells appear to represent terminally differentiated effector cells which fail to respond to further stimuli because of the absence of a CD28 second signal.