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. 2018 May 29;92(12):e00235-18.
doi: 10.1128/JVI.00235-18. Print 2018 Jun 15.

Interleukin-15-Stimulated Natural Killer Cells Clear HIV-1-Infected Cells following Latency Reversal Ex Vivo

Carolina Garrido  1   2 , Maria Abad-Fernandez  1   3 , Marina Tuyishime  4 , Justin J Pollara  4 , Guido Ferrari  4 , Natalia Soriano-Sarabia  1   2 , David M Margolis  5   2   3   6
Affiliations

Interleukin-15-Stimulated Natural Killer Cells Clear HIV-1-Infected Cells following Latency Reversal Ex Vivo

Carolina Garrido et al. J Virol. .

Erratum in

Abstract

Current efforts toward human immunodeficiency virus (HIV) eradication include approaches to augment immune recognition and elimination of persistently infected cells following latency reversal. Natural killer (NK) cells, the main effectors of the innate immune system, recognize and clear targets using different mechanisms than CD8+ T cells, offering an alternative or complementary approach for HIV clearance strategies. We assessed the impact of interleukin 15 (IL-15) treatment on NK cell function and the potential for stimulated NK cells to clear the HIV reservoir. We measured NK cell receptor expression, antibody-dependent cell-mediated cytotoxicity (ADCC), cytotoxicity, interferon gamma (IFN-γ) production, and antiviral activity in autologous HIV replication systems. All NK cell functions were uniformly improved by IL-15, and, more importantly, IL-15-treated NK cells were able to clear latently HIV-infected cells after exposure to vorinostat, a clinically relevant latency-reversing agent. We also demonstrate that NK cells from HIV-infected individuals aviremic on antiretroviral therapy can be efficiently stimulated with IL-15. Our work opens a promising line of investigation leading to future immunotherapies to clear persistent HIV infection using NK cells.IMPORTANCE In the search for an HIV cure, strategies to enhance immune function to allow recognition and clearance of HIV-infected cells following latency reversal are being evaluated. Natural killer (NK) cells possess characteristics that can be exploited for immunotherapy against persistent HIV infection. We demonstrate that NK cells from HIV-positive donors can be strongly stimulated with IL-15, improving their antiviral and cytotoxic potential and, more importantly, clearing HIV-infected cells after latency reversal with a clinically relevant drug. Our results encourage further investigation to design NK cell-based immunotherapies to achieve HIV eradication.

Keywords: ADCC; HIV; HIV eradication; IL-15; NK cell; SAHA; VOR; human immunodeficiency virus; immune function; immunotherapy; interleukins; kick and kill; latency reversal; natural killer cells; shock and kill; vorinostat.

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Figures

FIG 1
FIG 1
IL-15 improves the antiviral activity of NK cells from ART-treated HIV-infected donors. (A) Viral replication measured as HIV gag p24 antigen in the supernatants of 7-day cultures with only infected CD4+ T cells (Targets alone) or in the presence of NK cells at different effector/target cell ratios. UT, untreated. The red asterisks indicate statistically significant differences compared to targets alone, and black asterisks indicate differences between untreated and IL-15-stimulated NK cells (n = 14). (B) Viral replication in viral inhibition assays performed with JR-CSF superinfection (n = 8) or autologous reservoir virus (n = 6). Wilcoxon matched-pairs signed-rank test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The error bars indicate standard error of the mean (SEM). (C) Representative flow cytometry plots of intracellular p24 in cells from one donor gated on the CD3+ population of the live fraction. (D) Proportion of live CD4+ T cells positive for intracellular p24 staining. Coculture of infected CD4 cells with IL-15-treated NK cells significantly reduced the proportion of live CD4+ T cells containing p24 antigen after 5 days in culture. The orange circles correspond to cells from HIV-negative donors (n = 2), and the purple squares correspond to cells from aviremic HIV-positive donors (n = 3). Mann-Whitney U test. (E) Interaction of an NK cell with an infected CD4+ T cell visualized with ImageStreamX.
FIG 2
FIG 2
IL-15 increases the cytotoxic function of NK cells. (A) (Left and middle) Degranulation of NK cells measured by CD107a expression after coculture with the cell line K562 or autologous HIV-superinfected CD4+ T cells (n = 21 and n = 8, respectively). (Right) IFN-γ production after coculture with K562 cells measured by intracellular staining (n = 17). Wilcoxon matched-pairs signed-rank test. (B) (Left and middle) Comparison between degranulation and IFN-γ production in NK cells from HIV-negative or HIV-positive donors. (Right) Fold change (IL-15/untreated) of CD107a expression and IFN-γ production in HIV-negative and HIV-positive donors. Mann-Whitney test. The orange circles correspond to cells from HIV-negative donors, and the purple squares correspond to cells from aviremic HIV-positive donors. The error bars indicate SEM.
FIG 3
FIG 3
NK cell-mediated cytotoxicity after treatment with different cytokines. The graphs show the proportions of NK cells degranulating or producing IFN-γ in the presence of K562 cells (red squares) or in the absence of target cells (green circles) after stimulation with different cytokines or combinations, as indicated in the table below. Degranulation after culture with the cell line K562 showed a target-specific increase in cytotoxicity after stimulation with any of the cytokines or the studied combinations (P = 0.03). However, IL-15 showed better stimulation than IL-12, IL-18, or IL-21 (P = 0.03), and no difference was found between IL-15 alone and IL-15 in addition to any of the other cytokines (P, NS). IFN-γ production upon culture with K562 cells is increased after stimulation with IL-15 or IL-12 alone or any of the cytokine combinations (P = 0.03). The IL-15 effect was improved when IL-12 or IL-12 and IL-18 were included. However, IFN-γ production in the absence of target cells (NK alone) also increased after IL-12 or IL-12 and IL-18 addition to IL-15, indicating nonspecific activation. Wilcoxon matched-pairs signed-rank test. The error bars indicate SEM.
FIG 4
FIG 4
ADCC improves after IL-15 stimulation of NK cells. NK cells were cultured with TV-1-infected CEM.NKR cells in the presence of HIV-negative or A300 (HIV-positive) plasma for 6 h, and specific target killing was recorded using different plasma dilutions. (A) Mean antibody-mediated specific killing with untreated NK cells or after overnight IL-15 stimulation using different plasma dilutions. Cytokine treatment improved ADCC activity in both HIV-negative (n = 6) and HIV-positive (n = 6) donors. Means with SEM are shown. (B) Antibody titers observed with untreated/treated NK cells (n = 12). (C) Areas under the curve at different plasma dilutions with untreated NK and IL-15-stimulated NK cells (n = 12). The orange circles correspond to cells from HIV-negative donors, and the purple squares correspond to cells from ART-treated aviremic HIV-positive donors. Wilcoxon matched-pairs signed-rank test.
FIG 5
FIG 5
Latency clearance assay. (A) Resting CD4+ T cells were incubated with PHA or VOR for 24 h, cocultured with or without autologous NK cells at an E:T ratio of 1:10 for another 24 h, and plated in 12 replicates in the presence of feeders for 19 days, refreshing the medium every 3 or 4 days. The supernatant was assayed for p24 ELISA to assess the presence of HIV replication. The graphs show the numbers of wells in which p24 was detected at day 19, with a different color for each donor. (Left) Reactivation with PHA (n = 6). (Right) Reactivation with 335 nM VOR (n = 8). Wilcoxon matched-pairs signed-rank test. (B) Latency clearance assays in which NK cells were depleted after the initial 24 h of coculture with VOR-reactivated CD4+ T cells (n = 5). IL-15-stimulated NK cells reduced the number of HIV+ wells during the first 24 h of coculture with VOR-reactivated CD4+ T cells. The error bars indicate SEM.
FIG 6
FIG 6
Expression of activating NK cell receptors before and after IL-15 exposure. Cytokine stimulation upregulated the expression of NKG2D and NKp30, while it decreased that of NKp46. The orange circles represent cells from healthy donors (n = 6), and the purple squares represent cells from HIV-infected donors (n = 6). Wilcoxon matched-pairs signed-rank test. *, P < 0.05.
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References

    1. Archin NM, Liberty AL, Kashuba AD, Choudhary SK, Kuruc JD, Crooks AM, Parker DC, Anderson EM, Kearney MF, Strain MC, Richman DD, Hudgens MG, Bosch RJ, Coffin JM, Eron JJ, Hazuda DJ, Margolis DM. 2012. Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy. Nature 487:482–485. doi:10.1038/nature11286. - DOI - PMC - PubMed
    1. Sogaard OS, Graversen ME, Leth S, Olesen R, Brinkmann CR, Nissen SK, Kjaer AS, Schleimann MH, Denton PW, Hey-Cunningham WJ, Koelsch KK, Pantaleo G, Krogsgaard K, Sommerfelt M, Fromentin R, Chomont N, Rasmussen TA, Ostergaard L, Tolstrup M. 2015. The depsipeptide romidepsin reverses HIV-1 latency in vivo. PLoS Pathog 11:e1005142. doi:10.1371/journal.ppat.1005142. - DOI - PMC - PubMed
    1. Rasmussen TA, Tolstrup M, Brinkmann CR, Olesen R, Erikstrup C, Solomon A, Winckelmann A, Palmer S, Dinarello C, Buzon M, Lichterfeld M, Lewin SR, Ostergaard L, Sogaard OS. 2014. Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial. Lancet HIV 1:e13–e21. doi:10.1016/S2352-3018(14)70014-1. - DOI - PubMed
    1. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, Mackey EW, Miller JD, Leslie AJ, DePierres C, Mncube Z, Duraiswamy J, Zhu B, Eichbaum Q, Altfeld M, Wherry EJ, Coovadia HM, Goulder PJ, Klenerman P, Ahmed R, Freeman GJ, Walker BD. 2006. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 443:350–354. doi:10.1038/nature05115. - DOI - PubMed
    1. Petrovas C, Casazza JP, Brenchley JM, Price DA, Gostick E, Adams WC, Precopio ML, Schacker T, Roederer M, Douek DC, Koup RA. 2006. PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection. J Exp Med 203:2281–2292. doi:10.1084/jem.20061496. - DOI - PMC - PubMed

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