Anti-West Nile virus activity of in vitro expanded human primary natural killer cells

Abstract

Background: Natural Killer (NK) cells are a crucial component of the host innate immune system with anti-viral and anti-cancer properties. However, the role of NK cells in West Nile virus (WNV) infection is controversial, with reported effects ranging from active suppression of virus to no effect at all. It was previously shown that K562-mb15-41BBL (K562D2) cells, which express IL-15 and 4-1BBL on the K562 cell surface, were able to expand and activate human primary NK cells of normal peripheral blood mononuclear cells (PBMC). The expanded NK cells were tested for their ability to inhibit WNV infection in vitro.

Results: Co-culture of PBMC with irradiated K562D2 cells expanded the NK cell number by 2-3 logs in 2-3 weeks, with more than 90% purity; upregulated NK cell surface activation receptors; downregulated inhibitory receptors; and boosted interferon gamma (IFN-gamma) production by approximately 33 fold. The expanded NK (D2NK) cell has strong natural killing activity against both K562 and Vero cells, and killed the WNV infected Vero cells through antibody-dependent cellular cytotoxicity (ADCC). The D2NK cell culture supernatants inhibited both WNV replication and WNV induced cytopathic effect (CPE) in Vero cells when added before or after infection. Anti-IFN-gamma neutralizing antibody blocked the NK supernatant-mediated anti-WNV effect, demonstrating a noncytolytic activity mediated through IFN-gamma.

Conclusions: Co-culture of PBMC with K562D2 stimulatory cells is an efficient technique to prepare large quantities of pure and active NK cells, and these expanded NK cells inhibited WNV infection of Vero cells through both cytolytic and noncytolytic activities, which may imply a potential role of NK cells in combating WNV infection.

Figure 1

In vitro expansion of PBMC enriched CD56+ cells. NK cells before and 2 weeks after expansion by co-culture with K562-mb15-41BBL (D2NK) cells were stained with anti-CD56-PE and anti-CD3-FITC antibodies as the markers for NK cells and T cells, and then analyzed by flow cytometry. The UL quadrant counts (NK cells) are 9.60% and 91.20% for PBMC-Day 0, D2NK-Day 14 respectively; The LR quadrant counts (T cells) are 53.22% and 6.60% for PBMC-Day 0, D2NK-Day 14 respectively. PBMC isolated from the Buffy coat were either cultured freshly, or after frozen storage in liquid nitrogen, with equivalent results. The data presented in this figure is typical of 12 expansions made from the PBMC of 5 different donors. Averaged over these 12 experiments, NK cells were 10.05% ± 4.42 and T cells 52.44% ± 6.71 CD3+ before expansion. After expansion, NK cells were 85.25% ± 12.53 and T cells 12.12% ± 5.52 CD3+.

Figure 2

Cytotoxicity of the D2NK cells. (a) Natural killing activities of NK92, NKL cell lines and in vitro expanded primary D2NK cells. The effector cells were NK92, NKL, D2NK cells, the target cells were K562 cells; the effector: target ratio were 10:1, and 1:1; reaction at 37°C for 4 hours. (b) Natural killing and ADCC of D2NK cells against normal and WNV infected Vero cells. The ADCC group was done with effector cell, target cells, and WNV specific hyper-immune ascetic fluid; the natural killing group was done with effector cells, target cells, but no antibody; the Ab control group was done with effector cell, target cells, and HIV-1 RT specific ascetic fluid as nonspecific antibody and ascetic fluid control; the target cells were Vero cells either uninfected or 48 hours post infection with 100 TCID50 units of WNV; the effector cells were in vitro expanded D2NK cells; the E:T ratio was 10:1; WNV hyper-immune acetic fluid and HIV-1 RT McAb in acetic fluid were used at 1:1000 dilutions for ADCC; All reactions were performed in triplicate. The SD is the standard deviation from 6 tests with NK cells expanded from 6 different donor PBMC.

Figure 3

Effects of in vitro expansion on expression of NK cell receptors. D2NK cells were stained with PE or FITC conjugated antibodies against (a) cell activating receptors, NKG2D, NKp30, NKp44, NKp46 NK, or (b) cell inhibitory receptors, CD158a, CD158b, NKB1, NKAT2 NK, and then analyzed by flow cytometry. CD94 can be either an activation or inhibitory KIR, according to which downstream receptor it activates. The data presented represent the combined results from two donors, one tested in singlicate, another tested in duplicate. The error bars represent standard deviations, according equal weight to all three assays.

Figure 4

IFN-γ production of the expanded NK cells. Supernatants from 6 × 10⁶ PBMC, D2NK cells cultured for 48 hours at a 6-well plate in 2 ml medium were tested with human IFN-γ EIA kit. The concentrations of IFN-γ from PBMC and D2NK are 154 ± 12 pg/ml, and 5052 ± 493 pg/ml respectively; the culture medium has no detectable human IFN-γ. The PBMC and D2NK from 3 different donors were tested in triplicate for each. The error bars represent SD.

Figure 5

NK cells and supernatants inhibited WNV replication in Vero cells. NK cells or culture supernatants were added to WNV-infected or control Vero cultures 2 hours post infection. Supernatants from testing and control Vero cell cultures were collected on day 10 post WNV infection, and WNV RNA was extracted with Qiagen viral extraction kit and then analyzed with TaqMan RT-PCR. The NK cell culture supernatant was from D2NK cells.

Figure 6

Anti-IFN-γ antibody neutralized NK cell supernatant inhibition of WNV replication in Vero cells. NK culture supernatants with and without antibodies to IFNs were added to WNV-infected or control Vero cultures 2 hours post infection. Supernatants from testing and control Vero cell cultures were collected 10 days post WNV infection, and WNV RNA was extracted with Qiagen viral extraction kit and then analyzed with TaqMan RT-PCR. The NK cell culture supernatant was from D2NK cells.

Figure 7

NK cell supernatant pretreatment made Vero cells resistant to WNV infection. Vero cell cultures were treated with NK cell culture supernatants or control supernatants overnight prior to infection with WNV. Supernatants from testing and control Vero cell cultures were collected 10 days post WNV infection, and WNV RNA was extracted with Qiagen viral extraction kit and then analyzed with TaqMan RT-PCR. The NK cell culture supernatant was from D2NK cells.