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Flow Cytometric Analysis of HIV-1 Antigen-Specific CD4+ T Cell Proliferation in HIV-Infected Long-Term Survivors S. Ghanekar*1, S. Stranford2, J. Ong1, J. Levy2, and V. C. Maino1.
1BD Biosci., San Jose, CA; and 2Univ. of California, San Francisco It has recently been demonstrated using a sensitive multiparametric flow cytometry based assay (Pitcher et al., Nature Medicine, 1999) that antigen specific CD4+ T cell cytokine responses to HIV-1 Gag proteins could be detected in all HIV-1 seropositive long term survivors. It was also demonstrated that these responses were depressed in subjects who had been on long term HAART therapy. In the present study our objective was to examine the effect of anti-retroviral therapy on the HIV-1 specific proliferation of CD4+ T cells in PBMC of long term HIV-positive healthy subjects.
We compared the proliferation responses of CD4+ T cells from subjects infected for >10 years who were on anti-retroviral thearpy (n=8) with those with no history of therapy (n=12). The frequencies of HIV-1 specific proliferating CD4+ T cells were measured using a flow cytometric assay, which allows the detection of incorporated BrdU after 48h in culture with HIV-1 (REMUNETM, gift of R. Moss, IRC, San Diego).
Memory CD4+ T cells from HIV-1 infected healthy subjects who were not on anti-retroviral therapy, exhibited higher frequencies (mean = 0.95%) of proliferating cells in response to in vitro stimulation with HIV-1 antigen. In contrast, the responses of CD4+ T cells obtained from HIV-positive individuals who were on anti-retroviral therapy exhibited significantly lower frequencies of HIV-1 specific proliferating CD4+ T cells (mean = 0.43%). These observations were also consistent when differences in absolute number of proliferating CD4+ cells in these groups were compared, suggesting a decline in the total frequency of HIV-specific memory CD4+ T cells in subjects on therapy (mean proliferating CD4+ T cells/ml blood = 2367 compared to mean HIV-1 specific CD4+ T cells/ml blood = 5871 in subjects without any anti-retroviral therapy). These data are consistent with the cytokine expression reported earlier in that suppression of HIV-1 viral load with therapy appears to be associated with loss of functional HIV-specific memory CD4+ T cells.
The observed loss of HIV-specific CD4+ T cells in the subjects on anti-retroviral therapy may contribute to the rapid rebound of HIV replication after termination of anti-retroviral therapy.
Key Words: flow cytomtery, HIV, proliferation
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