Introduction Innate/adaptive immunity interplay is crucial to control hepatitis B virus (HBV) replication. In infancy-acquired chronic hepatitis B, low viral load is associated with high numbers of natural killer cells (NKC), high expression of their activation markers/receptors (CD69, CD107a, CD161 and NKG2D) and possibly of their inhibitory receptor NKG2A, but there is no information on NKC functional subsets and their interaction with adaptive immunity.

Aim To investigate NKC functional subsets ex-vivo and after exposure to K562 cells in relation to HBV-specific Th1 immune response and viral load.

Method 30 infancy-acquired chronic hepatitis B children (median age 13 y, 14 boys) divided into: group A (HBeAg+/HBsAg+/normal ALT; n=8), group B (HBeAg+/HBsAg+/elevated ALT; n=8), group C (HBeAg-/HBsAg+; n=9) and group D (HBeAg-/HBsAg-; n=5). NKC were obtained by negative magnetic bead isolation from PBMC and after exposure to K562 cells (post-K562). CD107a degranulation, IFN-g intracellular staining and NKC receptor expression (NKG2A/2D) were assessed concomitantly by flow cytometry. HBV-specific immune response was tested by IFN-g intracellular staining after PBMC incubation with HBV core antigen (HBcAg) and HBV DNA viral load by real-time PCR.

Results Frequency of NKC producing IFNg only (CD107a-IFNg+), polyfunctional NKC (CD107a+IFNg+) and NKC expressing NKG2A or NKG2D only (CD107a-NKG2A/D+) was higher in group D than in groups A–C, both ex-vivo ((%CD107a-IFNg+: 24.5vs7.3, 11.3, 16.3, p=0.03) (%CD107a+IFNg+: 25.5vs14.6, 18.6, 20.2, p=0.04) (%CD107a-NKG2A+: 10.9vs3.1, 4.5, 4.6, p=0.02) and (% CD107a-NKG2D+: 27.3 vs 16.7, 20.2, 22.6, p=0.05)) and post-K562 ((%CD107a-IFNg+: 31.1 vs 13.2, 16.4, 19.6, p=0.04) (%CD107a+IFNg+: 39.5 vs 20.3, 24.1, 31.1, p=0.05) (%CD107a-NKG2A+: 12.3 vs 3.7, 4.1, 5.6, p=0.03) and (%CD107a-NKG2D+: 35.3 vs 20.1, 22.6, 23.4, p=0.05)). % NKG2D+CD107a+ NKC was higher in group D than in groups A-C ex-vivo (32.4 vs 17.1, 20.1, 22.4, p=0.03) and post-K562 (39.7 vs 17.9, 21.7, 26.2, p=0.04), while NKG2A+/CD107a+ NKC number was similar in all groups. % HBcAg-specific IFN-g producing cells was higher in group D than groups A–C (CD4+/IFN-g+: 7.2±1.2 vs 2.3±0.3, 2.7±0.5, 3.1±0.9, p=0.04). Polyfunctional NKC CD107a+/IFN-g+ number correlated with that of HBcAg-specific IFN-g producing cells (r=0.5, p=0.04) and negatively with HBV DNA viral load (r=−0.42, p=0.05).

Conclusion High numbers of NKC producing IFN-g, polyfunctional, and with high NKG2D expression are associated with low HBV DNA replication. The strong correlations between polyfunctional NKC and HBV-specific T-helper 1 cells and HBV DNA viral load indicate a joint action between innate and adaptive immunity in controlling HBV infection.