GSE130368
Innate, non-cytolytic CD8+ T cell-mediated suppression of HIV replication by MHC-independent inhibition of virus transcription
MHC-I-restricted, virus-specific cytotoxic CD8+ T cells control HIV/SIV replication via the recognition and killing of productively infected CD4+ T cells. Several studies in SIV-infected macaques suggest that CD8+ T cells may also decrease virus production by suppressing viral transcription. RNA sequencing is used to identify candidate cellular pathways that are involved in the virus-silencing mediated by these CD8+ T cells. Overall design: Peripheral blood mononuclear cells (PBMCs) from 8 donors were obtained using Ficoll-Hypaque density gradient centrifugation. CD4+ and CD8+ T cells were purified by negative selection (Miltenyi Biotec) and stimulated with human CD3/CD28/CD2 antibody-coated beads (Miltenyi Biotec) at 1:1 bead-to-cell ratio, in the presence of 50 IU/ml of rhIL-2 (Miltenyi Biotec) for three days. A replication-competent and an Envelope (Env)-defective NL4-3 reporter virus expressing eGFP under control of the HIV- 1 LTR were kindly provided by F Kirchhoff58 (Ulm University Medical Center, Ulm, Germany). Envelope (Env)-defective NL4-3_eGFP was then complemented in trans with a dual-tropic Env (pSVIII-92HT593.1) obtained through the NIH-NIH AIDS Reagent Program from Dr. Beatrice Hahn (cat# 3077). A variant of the (Env)-defective NL4-3 IRES-eGFP reporter virus encoding the short-lived D2eGFP version of the protein was generated as previously described32 by inserting the protein-destabilizing mouse ornithine decarboxylase (MODC) domain at the Emory Custom Cloning Core Division using standard cloning techniques.  Viral stocks were produced by single or co-transfection of Lenti-X 293T cells with the respective HIV-1 constructs, using FuGene6 (Promega) in OptiMEM (Thermo Fisher). For the dual-color co-culture assays, target CD4+ T cells were labelled with CellTrace Violet dye (Vio) prior to infection. Briefly, TCR-activated CD4+ T cells were labelled with 5 µM of CellTrace Violet dye (Vio) (Thermo Fisher) per 10x106 cells and were subsequently infected by spinoculation (2h, 37°C, 2,500 rpm) using 100 ?l of virus stock/0.6x106 cells in flat-bottom 96-well plates. Autologous TCR-activated CD8+ T cells were labelled with 5 µM of CellTrace Red dye (Red) (Thermo Fisher) per 10x106 cells and added to the Vio+CD4+ T cells at 1:1 and 5:1 effector-to-target cell (E:T) ratios in the presence of 50 IU/ml of rhIL-2 after infection in 48-well plates.