Assessment of the antiviral capacity of primary natural killer cells by optimized in vitro quantification of HIV-1 replication.
Despite a growing number of studies investigating the impact of natural killer (NK) cells on HIV-1 pathogenesis, the exact mechanism by which NK cells recognize HIV-1-infected cells and exert immunological pressure on HIV-1 remains unknown. Previously several groups including ours have introduced autologous HIV-1-infected CD4(+) T cells as suitable target cells to study NK-cell function in response to HIV-1 infection in vitro. Here, we re-evaluated and optimized a standardized in vitro assay that allows assessing the antiviral capacity of NK cells. This includes the implementation of HIV-1 RNA copy numbers as readout for NK-cell-mediated inhibition of HIV-1 replication and the investigation of inter-assay variation in comparison to previous methods, such as HIV-1 p24 Gag production and frequency of p24(+) CD4(+) T cells. Furthermore, we investigated the possibility to hasten the duration of the assay and provide concepts for downstream applications. Autologous CD4(+) T cells and NK cells were obtained from peripheral blood of HIV-negative healthy individuals and were separately enriched through negative selection. CD4(+) T cells were infected with the HIV-1 strain JR-CSF at an MOI of 0.01. Infected CD4(+) T cells were then co-cultured with primary NK cells at various effector:target ratios for up to 14days. Supernatants obtained from media exchanged at days 4, 7, 11 and 14 were used for quantification of HIV-1 p24 Gag and HIV-1 RNA copy numbers. In addition, frequency of infected CD4(+) T cells was determined by flow cytometric detection of intracellular p24 Gag. The assay displayed minimal inter-assay variation when utilizing viral RNA quantification or p24 Gag concentration for the assessment of viral replication. Viral RNA quantification was more rigorous to display magnitude and kinetics of NK-cell-mediated inhibition of HIV-1 replication, longitudinally and between tested individuals. The results of this study demonstrate that NK-cell-mediated inhibition of HIV-1 replication can be reliably quantified in vitro, and that viral RNA quantification is comparable to p24 Gag quantification via ELISA, providing a robust measurement for NK-cell-mediated inhibition of viral replication. Overall, the described assay provides an optimized tool to study the antiviral capacity of NK cells against HIV-1 and an additional experimental tool to investigate the molecular determinants of NK-cell recognition of virus-infected cells.
Ragon Institute of MGH, MIT and Harvard, 400 Technology Square, Cambridge, MA 02139, USA; State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, 155 Changbai Road, Changping District, Beijing 102206, People's Republic of China. Electronic address: hexuan09@yahoo.com.
Xuan
X
He
Ragon Institute of MGH, MIT and Harvard, 400 Technology Square, Cambridge, MA 02139, USA. Electronic address: csimoneau@mgh.harvard.edu.
Camille R
CR
Simoneau
Ragon Institute of MGH, MIT and Harvard, 400 Technology Square, Cambridge, MA 02139, USA. Electronic address: mitchell.granoff@gmail.com.
Mitchell E
ME
Granoff
Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Martinistraße 52, 20251 Hamburg, Germany. Electronic address: sebastian.lunemann@hpi.uni-hamburg.de.
Sebastian
S
Lunemann
Ragon Institute of MGH, MIT and Harvard, 400 Technology Square, Cambridge, MA 02139, USA. Electronic address: adugast@merrimack.com.
Anne-Sophie
AS
Dugast
State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, 155 Changbai Road, Changping District, Beijing 102206, People's Republic of China. Electronic address: yshao@bjmu.edu.cn.
Yiming
Y
Shao
Ragon Institute of MGH, MIT and Harvard, 400 Technology Square, Cambridge, MA 02139, USA; Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Martinistraße 52, 20251 Hamburg, Germany. Electronic address: marcus.altfeld@hpi.uni-hamburg.de.
Marcus
M
Altfeld
Ragon Institute of MGH, MIT and Harvard, 400 Technology Square, Cambridge, MA 02139, USA; Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Martinistraße 52, 20251 Hamburg, Germany. Electronic address: christian.koerner@hpi.uni-hamburg.de.
Christian
C
Körner
D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D002478:Cells, Cultured; D016655:HIV Core Protein p24 / Q000032:analysis; D015658:HIV Infections / Q000276:immunology; D015497:HIV-1 / Q000276:immunology* / Q000502:physiology; D064368:Healthy Volunteers; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology* / Q000821:virology; D012367:RNA, Viral / Q000032:analysis; D014779:Virus Replication
D016655:HIV Core Protein p24; D012367:RNA, Viral
CD4(+) T cell; HIV-1; HIV-1 RNA; HIV-1 p24 ELISA; Natural killer cell