635. In HIV-Infected Patients Killing of Latently HIV-Infected CD4 T Cells by Autologous CD8 T Cells Is Modulated by Nef

BACKGROUND: The PBMC of HIV-infected patients contain HIV-specific CD8 T cells and their potential targets, CD4 T cells latently infected by HIV. The role of HIV-specific CD8 T cells in the course of HIV infection and the way they affect the virus that resides in the latent reservoir, the resting memory CD4 T cells, is unknown. The association between HIV Nef protein and the cellular ASK1 protein protects the HIV-infected CD4 T cells from killing by CD8 T cells. METHODS: CD8 and autologous CD4 T cells procured from PBMC of acute, chronic untreated, treated and AIDS patients were isolated by magnetic beads and co-incubated. Resting memory CD4 T cells (CD25(−), CD69(−) and HLA-DR(−)) were isolated from activated CD4 T cells using a two-step bead depletion purification procedure. Formation of CD8-CD4 T-cell conjugates was observed by fluorescence microscopy and in situ PCR of HIV LTR DNA. Both conjugation and apoptosis were observed and quantified by imaging flow cytometry (ImageStream) using anti-human activated caspase 3 antibody and TUNEL assay. Formation of immunological synapse was observed by using anti-Perforin, anti γ-tubulin, and anti-LCK antibodies. RESULTS: Following co-incubation we observed that CD8 T cells conjugate with and induce apoptosis of autologous CD4 T cells. In patients with acute infection or AIDS the conjugation activity and apoptosis were much higher compared with chronic HIV-infected patients. In patients on anti-retroviral therapy (ART) low grade conjugation of CD4 T cells was observed by fluorescence microscopy (2.3 ± 0.3%), by in situ PCR of HIV DNA (3 ± 0.6%) and by ImageStream analysis (2.5 ± 0.5%). After co-incubation with autologous CD8 T cells 2.1 ± 0.4% of the CD4 T cells procured from patients on ART were undergoing apoptosis. Resting memory CD4 T cells were conjugated (1.9 ± 0.3%) and killed (2.2 ± 0.3%) by autologous CD8 T cells. Delivering a peptide that interferes with the Nef-ASK1 interaction, into the CD4 T cells, resulted in twofold enhancement of their apoptosis by the autologous CD8 T cells (from 2.1 ± 0.5% to 4.0 ± 0.4%), with no effect on conjugation. CONCLUSION: CD8 T cells conjugate with and kill HIV-infected CD4 T cells throughout the course of HIV infection. We suggest that Nef inhibition may result in the elimination of the latent reservoir CD4 T cells by CD8 T cells. [Image: see text] DISCLOSURES: All authors: No reported disclosures.