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. 2016 Jul:434:53-60.
doi: 10.1016/j.jim.2016.04.007. Epub 2016 Apr 16.

Assessment of the antiviral capacity of primary natural killer cells by optimized in vitro quantification of HIV-1 replication

Xuan He  1 , Camille R Simoneau  2 , Mitchell E Granoff  3 , Sebastian Lunemann  4 , Anne-Sophie Dugast  5 , Yiming Shao  6 , Marcus Altfeld  7 , Christian Körner  8
Affiliations

Assessment of the antiviral capacity of primary natural killer cells by optimized in vitro quantification of HIV-1 replication

Xuan He et al. J Immunol Methods. 2016 Jul.

Abstract

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.

Keywords: CD4(+) T cell; HIV-1; HIV-1 RNA; HIV-1 p24 ELISA; Natural killer cell.

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Figures

Figure 1
Figure 1. NK-cell-mediated inhibition of HIV-1 replication is dependent on the effector:target cell ratio between NK cells and CD4+ T cells
The figure display levels of HIV-1 replication (A–C) and NK-cell-mediated inhibition (D–F) measured as frequency of p24+ CD4+ T cells (p24 ICS), concentration of p24 Gag (p24 ELISA) and copy numbers of viral RNA (HIV RNA) in the culture supernatant. HIV-1-infected autologous CD4+ T cells from 13 healthy donors were either cultured alone or in the presence of increasing numbers of primary NK cells. Panels A–C show levels of HIV-1 replication after 14 days of co-culture. Panels D–F display NK-cell-mediated inhibition of HIV-1 replication as compared to CD4+ T cells alone. Wilcoxon matched-pairs signed rank test was used to identify differences between two groups.
Figure 2
Figure 2. Correlations between different methods for quantification of HIV-1 replication
Frequency of HIV-1 p24+ CD4+ T cells, p24 Gag concentration and HIV-1 RNA copy numbers in the supernatant of all individuals (n=13) and conditions were plotted against each other. Percentage of p24+ CD4+ T cells was assessed by flow cytometry, p24 Gag production was determined by ELISA and viral RNA was quantified by RT-qPCR respectively. Panel A displays absolute values of HIV-1 replication (52 data points); panel B shows log inhibition of HIV-1 replication as compared to CD4+ T cells alone (39 data points). Non-parametric spearman’s rank correlation was used to determine association between two methods.
Figure 3
Figure 3. Kinetics of NK-cell-mediated inhibition of HIV-1 replication
The figure displays the kinetics of NK cell-mediated viral inhibition stratified into three donor groups with different antiviral capacities (poor: bottom third (n=4), moderate: middle third (n=5), good: top third (n=4) at day 14 with NK:CD4+ T cell ratio 10:1). p24 Gag production (upper panel, A) and HIV-1 RNA copy numbers (lower panel, B) at different NK:CD4+ T cell ratios (0.1:1, 1:1 and 10:1) were measured longitudinally over the course of 14 days.
Figure 4
Figure 4. NK cell activation and effector/target cells numbers during NK/CD4+ T cell co-culture
Figure 4A displays the levels of NK cell degranulation (CD107a expression) following co-incubation with differentially stimulated CD4+ T cells at two effector:target cell ratios. CD4+ T cells were either stimulated with CD3/28 beads (squares) or PHA with (circles) or without (triangles) subsequent in vitro HIV-1 infection for 3 days and then co-incubated with enriched NK cells for 4 hours. Figure 4B shows the kinetics of NK cells and CD4+ T cells numbers during the co-culture. CD4+ T cell numbers following infection with HIV-1 (MOI: 0.01) in the presence (grey circles) or absence of NK cells (clear circles) are shown in the left panel of 4B, NK cell numbers on the right panel. The initial effector:target cell ratio was 10:1. Cell numbers were normalized to 5*104 and 5*105 per well respectively for better comparison. Median and IQR of 3 independent experiments (subjects) are displayed for both experiments. Statistical analysis was conducted using Friedman test and Dunn’s multiple comparison test.
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