Expanded adaptive NKG2C+ NK cells exhibit potent ADCC and functional responses against HBV-infected hepatoma cell lines

Abstract

Background: Hepatitis B virus (HBV) infection remains a significant global health challenge, leading to chronic liver disease and hepatocellular carcinoma (HCC). Natural killer (NK) cells play an important role in the clearance of HBV-infected cells, but their efficacy is often compromised during chronic infection. Adaptive NK cells, characterised by NKG2C expression and enhanced functional responses, represent a promising therapeutic avenue for enhancing anti-HBV immunity and responses to HBV-driven cancers.

Methods: We applied an established protocol, involving K562-HLA-E expressing feeder cells and cytokines (IL-2), for the expansion of adaptive NK cells from cryopreserved T- and B cell depleted peripheral blood mononuclear cells (PBMCs) derived from donors with chronic HBV infection alone or with Human Immunodeficiency Virus (HIV) co-infection. We evaluated the adaptive profile of expanded NK cells, their antibody-dependent cellular cytotoxicity (ADCC) capacity and functional responses against hepatoma cell lines in the presence or absence of HBV infection.

Results: Expanded NK cells achieved >97% purity, with the NKG2C positive population exhibiting a mean 100-fold expansion. These cells demonstrated a predominantly adaptive phenotype with high surface expression of NKG2C and cytotoxic potential (Granzyme B). They maintained high levels of CD16 surface expression and upregulated CD2, essential for ADCC. Functionally, expanded adaptive NK cells showed enhanced ADCC capacity and functional responses to K562 targets, naive, HBV integrant-expressing, and de novo infected hepatoma cell lines. TGF-β preconditioning induced tissue-resident features (CD103, CD49a) in expanded adaptive NK cells, while preserving their adaptive phenotype and functionality, enhancing their potential for liver targeted immunotherapy. Further, expanded adaptive NK cells demonstrated minimal reactivity against autologous activated T cells, suggesting limited off-target effects.

Conclusions: Our study demonstrates the first successful expansion of adaptive NK cells with robust functional responses from donors with chronic viral infection. This approach creates opportunities for NK cell-based therapies alone or in combination with monoclonal antibodies contributing to HBV functional cure strategies and the treatment of HBV-driven cancers.

Keywords: Hepatitis B virus; adaptive immunity; antiviral therapy; immunotherapy; natural killer cells.

Figure 1.. Expanded NK cells display an adaptive phenotype.

(a) Expansion protocol; (b) Cell type frequency on day 0 depleted PBMC and day 11 expanded NK cells; (c) Fold expansion in number of total NK cells and NKG2C⁺ NK cells following the 11-day culture; (d) Relative expression of key adaptive markers shown on viSNE clustering and violin plots of sampled events to show frequencies of positive populations; (e) Heatmap of expression levels of depicted NK cell markers on day 0 and day 11. Significance determined by Wilcoxon paired test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 2.. Expanded adaptive NK cells exhibit enhanced ADCC capacity compared to pre-expansion.

(a) Representative flow plots of CD107a/IFNy co-expression on bulk NK cells on day 0 and day 11 post-expansion against K562 cells expressing HLA-E; (b) Relative expression of IFNy, CD107a and percentage killing on NK cells against K562 cells expressing HLA-E; (c) Representative flow plots of CD107a/IFNy co-expression on NK cells on day 0 and day 11 post-expansion against Raji cells in the presence of anti-CD20 antibody; (d) Relative expression of IFNy, CD107a and percentage killing of NK cells against Raji cells in the presence of anti-CD20 antibody. Significance determined by Wilcoxon paired test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 3.. Expanded aNK cells exhibit enhanced functional capacity against HBV-infected HepG2-NTCP cells compared to pre-expansion.

(a) Relative expression of CD107a and target cell killing of NK cells against PLC/PRF/5 cells of sampled events; (b) Relative expression of CD107a and target cell killing of NK cells against HepG2-NTCP cells of sampled events; (c) Normalised cell index of HepG2-NTCP cells in the absence or presence of expanded aNK cells; (d) Representative flow plots of CD107a/IFNy co-expression on day 11 expanded NK cells against uninfected HepG2-NTCP cells, HBV-infected HepG2-NTCP cells and HepG2.2.15 cells and relative expression of CD107a and target cell killing against uninfected HepG2-NTCP cells, HBV-infected HepG2-NTCP cells and HepG2.2.15 cells. Significance determined by Wilcoxon paired test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 4.. Pre-conditioning with TGF-β results in an expanded aNK cell population with retained adaptive features and adopted tissue-resident phenotype, while maintaining functional responses.

(a) Expansion timeline; (b) Cell type frequencies on day 0 depleted PBMC, day 11 IL-2 and day 11 TGF-β+IL-2 expanded aNK cells; (c) Histograms of expression of NKG2C, NKG2A, CD103, CD69, CD49a and CXCR3, summary graphs of expression for these phenotypic markers and heatmap of expression of other phenotypic markers; (d) Summary plot of relative CD107a expression against anti-CD20 coated Raji cells; (e) Summary plot of relative CD107a expression and estimated target cell killing against HepG2-NTCP cells. Significance determined by Friedman paired test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Significance on the heatmap in (c) is shown for comparisons between IL-2 vs day 0 and TGFβ+IL-2 vs day 0.

Figure 5.. Expanded aNK cells display minimal reactivity to activated CD4⁺ T cells.

(a) Assay design; (b) Representative flow plots of CD107a expression of day 0 NK cells and day 11 cryopreserved expanded aNK cells against autologous resting and activated CD4⁺ T cells; (c) Heatmap of CD107a expression on day 0 NK cells and day 11 cryopreserved expanded adaptive NK cells against autologous resting and activated CD4⁺ T cells from 5 donors; (d) Representative flow plots of NKG2C/CD107a co-expression of day 0 isolated ex vivo NK cells and day 11 cryopreserved expanded adaptive NK cells against autologous resting and activated CD4⁺ T cells; (e) Heatmap of expression of CD107a according to NKG2C expression of day 0 NK cells and day 11 cryopreserved expanded aNK cells against autologous resting and activated CD4⁺ T cells from 5 donors.