477 Non-Replicating HIV-1 Virions Induce Increased Expression of Activation Markers in Primary T-cells in a Strain-dependent Manner G. Holm*1,2, D. Gabuzda1,2 1Dana-Farber Cancer Inst, Boston, MA and 2Harvard Med Sch, Boston, MA
Background: During HIV-1 infection, both infected and uninfected CD4 and CD8 T-cells undergo apoptosis, contributing to T-cell depletion. The mechanism for apoptosis of bystander T-cells is controversial. Some groups propose that HIV-1 Env or other stimuli sensitizes T-cells to undergo activation-induced cell death while others suggest that Env directly induces pro-apoptotic signaling. We previously demonstrated that the ability of HIV-1 to induce bystander apoptosis in T-cells is strain-dependent and is enhanced by increased affinity of the envelope glycoproteins for CD4 and/or increased fusogenicity. We investigated the ability of HIV-1 viruses that differ in these properties to alter the expression of activation markers in primary T-cells.
Methods: PBMC from a normal uninfected donor were PHA-stimulated for 3 days. T-cells were negatively selected by magnetic cell sorting and then incubated with UV-inactivated ELI-1 or ELI-6 for 3 or 5 days. ELI-1 is a primary CXCR4-tropic HIV-1 virus that was serially passaged to derive ELI-6, which contains point mutations in the Env that increase CD4 affinity and fusogenicity. On days 3 and 5, cultures were stained with anti-CD4 or anti-CD8, and anti-CD25, anti-CD38, anti-CD95, anti-CD95L, or anti-HLA-DR. Primary T-cell cultures were incubated with or without anti-Fas and/or ELI-6 for 3 or 5 days and stained with 7AAD, either anti-CD4 or anti-CD8, and TUNEL.
Results: Incubation of primary T-cells with UV-inactivated ELI-1 or ELI-6 virions increased the expression levels of T-cell activation markers CD25, CD38, CD95, CD95L, and HLA-DR on day 3 and/or day 5 as indicated by the percentage of positive cells and/or mean fluorescence intensity. This effect was observed in both CD4+ and CD8+ T-cells and was strain dependent; cells incubated with ELI-6 exhibited higher expression levels of activation markers than cells incubated with ELI-1. Non-replicating ELI-6 virions induced significantly higher levels of apoptosis in primary T-cells than ELI-1 or control supernatant. However, apoptosis was not inhibited by anti-Fas, implicating a Fas-independent pathway of activation-induced cell death.
Conclusions: Non-replicating HIV-1 virions increase the expression of activation markers on primary CD4 and CD8 T-cells in a strain-dependent manner. HIV-1 variants with increased affinity for CD4 and/or increased fusogenicity may increase apoptosis of bystander T-cells through a Fas-independent pathway of activation-induced cell death.