FLOW CYTOMETRIC ANALYSIS OF THE STIMULATORY RESPONSE OF T-CELL SUBSETS FROM NORMAL AND HIV-1(-VITRO() INDIVIDUALS TO VARIOUS MITOGENIC STIMULI IN)

A novel technique is described which allows the study of the responses of T cell subpopulations stimulated in bulk cultures without interfer ing with cell-cell interactions. The number and phenotype of lymphobla sts developing following stimulation with phytohaemagglutinin (PHA), a nti-CD3, staphylococcal protein A (SPA) and pokeweed mitogen (PWM) was determined in HIV-1(-) and HIV-1(+) patients using a new five-paramet er how cytometric method. We found that normal T cells responded faste r to PHA than to any of the other mitogens tested. The peak of the PHA response occurred on day 3, followed by anti-CD3 and SPA on day 4 and PWM mitogen on day 5. Although PHA and anti-CD3 stimulated up to 95% and 80% of lymphocytes, respectively, SPA and PWM stimulated only 40% and 30% of cells, respectively. A defective T cell response was observ ed in lymphocytes cultured from asymptomatic HIV-1(+) patients compare d with negative controls. This loss of response was related to a selec tive mortality of T cells following mitogenic stimulation, referred to as activation-associated lymphocyte death (AALD). The results showed that stronger mitogens (PHA and anti-CD3) induced AALD in a larger pro portion (50-60%) of T cells than weaker mitogens such as SPA and PWM ( 30-40%), and that AALD affected different lymphocyte subsets to differ ent extents. AALD occurred more frequently in total CD8(+) and CD45RO( +) T cells compared with CD4(+) and CD45RA(+) T cells, but memory CD4( +) T cells were the population most severely affected in samples from HIV-1(+) donors.