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

Abstract

BackgroundNatural 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.ResultsCo-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-γ) production by ~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-γ neutralizing antibody blocked the NK supernatant-mediated anti-WNV effect, demonstrating a noncytolytic activity mediated through IFN-γ.ConclusionsCo-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.

Allogenic Natural Killer Cell Immunotherapy Combined with Irreversible Electroporation for Stage IV Hepatocellular Carcinoma: Survival Outcome

“…It is dedicated for the expansion and activation of NK cells in peripheral blood or umbilical cord blood mononuclear cells in vitro, the preparation of NK cells with higher quantity, purity and activity, namely HANK cells [15]. The final cell count and quality control inspection were performed at day 9 of culture, and the qualified indicators included proportion of living cells ≥ 90%, proportion of CD56+CD3-cells ≥ 85% (detection by flowcytometry was shown previously [15]), endotoxin content ≤ 1 EU/ml, cell viability ≥ 80% (K562 cells were used as target cells, cytotoxicity assay was shown previously [15]), Bacteria, fungi and mycoplasma culture negative. 80 ml peripheral blood from allogenic donors was drawn 7 days before IRE and the immunotherapy was given 3 days after IRE.…”

Allogenic natural killer cell immunotherapy of sizeable ovarian cancer: A case report

“…The human high activity NK cell in vitro preparation kit was used (Hank Bioengineering Co. Ltd, Shenzhen, China) that contained chimeric active cellular factors on K562 cell membranes (3), plasma treatment fluid, lymphocyte culture fluid additives, serum-free medium additives and cell infusion additives. This kit is intended for expanding and activating NK cells in peripheral blood mononuclear cells in vitro to prepare NK cells of higher quantity, purity and activity, namely highly activated NK (HANK) cells (4 (3), and bacteria-, fungi-and mycoplasma-negative culture. After a 14-day culture, the HANK cells were divided into three parts and infused intravenously on days 15-17.…”

The Immunopathogenesis of Neurotropic Flavivirus Infection

“…Indeed, to date they have been regarded as redundant in flavivirus infection, since upregulated MHC expression on infected cells reduces NK cell recognition. However, in vitro, under artificially-stimulated conditions, NK cells are cytotoxic for WNV-infected cells (M. Zhang et al, 2010). The lack of neutrophil infiltration is perhaps not surprising as there is little or no CXCL1 expression in the brain during infection.…”