Lymphocyte Proliferation Assays May Underestimate Antigen Responsiveness in Human Immunodeficiency Virus Infection

The objective of this study was to examine the relationships among lymphocyte proliferation, interferon-γ (IFN-γ) production, and apoptosis in peripheral blood mononuclear cells (PBMC) of HIV-1-infected patients and controls. PBMC were prepared from 19 HIV-1-infected patients and 16 healthy controls. Using tetanus toxoid (TT) as a recall antigen, we assessed lymphocyte proliferation using [3H]thymidine incorporation after 2, 4, 6, and 7 daysʹ culture and IFN-γ production in 48-h culture supernatants by ELISA. Apoptosis was measured using TdT-mediated dUTP nick-end labeling. Median stimulation indices (SIs) in HIV-1-infected patients were 2.8 and 3.7 as opposed to 24.9 and 25.1 in healthy controls after 6 and 7 daysʹ culture, respectively (P < 0.001). Among the controls, peak proliferation was seen after 7 days in culture whereas in patients, SIs peaked at 4 days and fell progressively by days 6 and 7. At 2 and 4 days of stimulation with tetanus, patientsʹ T cells showed increased apoptosis (19 and 25%) vs 12 and 15% apoptosis seen in controlsʹ cells, P < 0.05. Interferon-γ in 48-h supernatants of TT-stimulated PBMC was comparable among patients and controls. Whereas in our system, 6 and 7 day assays of lymphocyte proliferation provide increasing responses to TT among healthy controls, these durations of culture may underestimate antigen responsiveness in HIV-1 infection. Cell death due to apoptosis may account for this phenomenon. Whether shorter term or longer term assays of lymphocyte responsiveness more accurately reflect in vivo immune competence is unknown. Nonetheless, shorter duration assays may provide a more realistic estimate of the frequency of antigen-reactive cells in persons with HIV-1 infection.