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| Status |
Public on Aug 23, 2020 |
| Title |
Innate, non-cytolytic CD8+ T cell-mediated suppression of HIV replication by MHC-independent inhibition of virus transcription |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
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.
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| 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.
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| Contributor(s) |
Zanoni M, Palesch D, Pinacchio C, Tharp GK, Paiardini M, Chahroudi A, Bosinger S, Kulpa D, Silvestri G |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Apr 26, 2019 |
| Last update date |
Aug 23, 2020 |
| Contact name |
Gregory K Tharp |
| E-mail(s) |
[email protected]
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| Phone |
404-727-7797
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| Organization name |
Yerkes National Primate Research Center
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| Department |
Developmental and Cognitive Neuroscience
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| Lab |
Genomics Core
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| Street address |
954 Gatewood Dr
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| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30329-4208 |
| Country |
USA |
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| Platforms (1) |
| GPL21290 |
Illumina HiSeq 3000 (Homo sapiens) |
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| Samples (32)
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| Relations |
| BioProject |
PRJNA539944 |
| SRA |
SRP193975 |
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