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Background:  Long-term replication kinetics of HIV in primary CD4⁺ T-lymphocytes have not been studied in vitro because activated, proliferating CD4⁺ T-cells eventually undergo activation-induced cell death. We have developed a novel culture system that allows activated CD4⁺ T-cells to become resting memory cells and permits their maintenance for many months. Using this system, we have asked whether latently infected cells are formed in vitro.

Methods:  Anti-CD3-stimulated primary human CD4⁺ T cells were grown for 3 weeks and then co-cultured on an attached tumor line, H80, to rescue them from activation-induced cell death. Cells were phenotyped by analyzing cell-activation markers and cell-cycle status by flow cytometry. HIV-infected primary CD4 T-cells were co-cultured on H80 cells for 2 to 4 months and phenotyped. Viral synthesis was measured by p24 EIA, intracellular p24-staining, in-situ RNA hybridization. Prostratin was used to stimulate any latent HIV from quiescent cells.

Results:  The H80 cell line could rescue activated, proliferating CD4⁺ T-cells from activation-induced cell death and keep them alive for more than 5 months. During co-culture, 99% of CD4 cells remained in G₀/G₁ phase of cell cycle, did not express CD25 and HLADR on their surface, and a minimal level of cellular DNA synthesis was observed. A secreted soluble factor(s) present in H80-conditioned media could rescue these cells from activation-induced cell death, and that could not be blocked by specific kinase inhibitors for PI3’K/Akt, MEK/ERK, and Jak/STAT pathways. Similarly, HIV-infected activated primary CD4⁺ T cells when co-cultured on H80 became quiescent, but about 8 to 10% of the cells continued to produce virus chronically as observed in viral assays. Interestingly, when these cells were treated with 1- to 5-mM prostratin, the percentage of HIV-p24⁺ cells increased by some 2-fold in the absence of cell proliferation, suggesting that latently infected cells were also generated in this culture system.

Conclusions:  These results demonstrate a novel culture system in which activated, proliferating primary CD4⁺ T cells can be rescued from activation-induced cell death and maintained viably for many months in a non-cycling state. Using this system, we demonstrate the formation of latently infected CD4 T cells in vitro. This system provides the opportunity to study the mechanism of latent HIV infection in primary CD4 T cells in vitro at the molecular level, as well as the biology of chronically HIV-producing primary CD4 T cells that are frequently observed in vivo.

Keywords: A novel culture system; Long-survival of primary CD4 T-cells ; Latent HIV