Natural Killer Cell Characteristics in Patients With Chronic Hepatitis B Virus (HBV) Infection Are Associated With HBV Surface Antigen Clearance After Combination Treatment With Pegylated Interferon Alfa-2a and Adefovir

MAJOR ARTICLE Natural Killer Cell Characteristics in Patients With Chronic Hepatitis B Virus (HBV) Infection Are Associated With HBV Surface Antigen Clearance After Combination Treatment With Pegylated Interferon Alfa-2a and Adefovir Femke Stelma,1,2 Annikki de Niet,1,2 Marjan J. Tempelmans Plat-Sinnige,2 Louis Jansen,1,2 R. Bart Takkenberg,1 Hendrik W. Reesink,1,2 Neeltje A. Kootstra,2 and Ester M. M. van Leeuwen2 Department of Gastroenterology and Hepatology, and 2Experimental Immunology, Academic Medical Center, Amsterdam, The Netherlands Background. The role of natural killer (NK) cells in the process of hepatitis B virus (HBV) surface antigen (HBsAg) clearance and whether their phenotype is related to treatment outcome in patients with chronic hepatitis B are currently unknown. Methods. Patients with chronic hepatitis B (HBV DNA load, >17 000 IU/mL) were treated with pegylated interferon alfa-2a and adefovir for 48 weeks. NK cell phenotype and function were analyzed in 7 responders (defined as individuals with HBsAg clearance by week 72; 3 HBV e antigen [HBeAg]-positive and 4 HBeAg-negative), 7 matched nonresponders, and 7 healthy controls. Subsequently, 34 baseline samples from HBeAg-positive patients with chronic hepatitis B were analyzed. Results. During treatment, the percentage and absolute number of CD56bright NK cells increased significantly, whereas the percentage and absolute number of CD56dim NK cells decreased. At baseline, responders had a significantly lower expression of chemokine receptor CX3CR1 on CD56bright NK cells and inhibitory receptor NKG2A on CD56dim NK cells, compared with nonresponders. In addition, responders had higher CD56bright TRAIL expression and interferon γ production at end of treatment. These baseline differences were not found in HBeAg-positive patients who had HBeAg seroconversion without HBsAg clearance. Conclusions. Combination therapy significantly influences NK cell phenotype and function. Differences between patients with chronic hepatitis B with HBsAg clearance and nonresponders suggest that NK cells play a role in the clearance of HBsAg during interferon-based combination therapy. Keywords. Hepatitis B; combination therapy; interferon alfa; NK cells; innate immunity; TRAIL. Hepatitis B virus (HBV) infection, with approximately 240 million chronic cases worldwide, is a global health problem [1]. Patients with chronic hepatitis B (CHB) Received 9 January 2015; accepted 11 March 2015; electronically published 19 March 2015. Presented in part: Annual Meeting of the Dutch Society for Immunology, 17–19 December 2014, Kaatsheuvel, The Netherlands. Abstract 79. Correspondence: Hendrik W. Reesink, MD, PhD, Academic Medical Center, Department Gastroenterology and Hepatology, Rm G4-215, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands (). The Journal of Infectious Diseases® 2015;212:1042–51 © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: . DOI: 10.1093/infdis/jiv180 1042 • JID 2015:212 (1 October) • Stelma et al are at increased risk of developing serious hepatic complications, such as cirrhosis and hepatocellular carcinoma [2]. Current treatment options for CHB include pegylated interferon alfa-2a or nucleos(t)ide analogue (NUC) treatment. While NUCs can potently block viral replication, pegylated interferon alfa-2a exerts its action via direct antiviral activity and immunomodulatory effects. The immunomodulatory effects of pegylated interferon alfa-2a have been investigated in innate and adaptive effector cells. Pegylated interferon alfa-2a therapy has been shown to cause expansion and activation of CD56bright natural killer (NK) cells [3]. T cells, on the other hand, remain present at low frequency in the peripheral blood, owing to the suppressive effects on bone marrow function 1 interferon alfa-2a/NUC combination therapy in patients with CHB and related these changes to treatment outcome. MATERIALS AND METHODS Patient Samples In a previously described investigator-initiated study (clinical trials registration ISRCTN 77073364) [14], patients with CHB and a high HBV DNA load (>17 000 IU/mL) received combination therapy of pegylated interferon alfa-a2 and adefovir for 48 weeks. All patients gave informed consent, and the study was approved by the Ethical Review Board of the Academic Medical Center Amsterdam. Samples were obtained at baseline, during treatment (day 3, week 1, week 24, and week 48), and during follow-up (week 72). Peripheral blood mononuclear cells (PBMCs) were isolated using standard density-gradient centrifugation and cryopreserved for later analysis. From this cohort, 14 patients with CHB were selected (Supplementary Figure 1A). Half of this group consisted of 7 responders with HBsAg clearance at week 72, defined according to European Association for the Study of the Liver guidelines [20] as having persistently undetectable HBsAg and a negative result of HBV DNA testing, with or without development of antibody to HBsAg (anti-HBs; for HBeAg-positive patients, this also included HBeAg seroconversion). The other half comprised 7 nonresponders (matched on the basis of genotype, ethnicity, and HBeAg status at baseline) with no HBsAg clearance and, for HBeAg positive patients, no HBeAg clearance (Table 1). All 7 nonresponders had to be retreated with NUC within 2 years after the end of treatment Table 1. Baseline Characteristics of Patients With Chronic Hepatitis B Treated With Combination Therapy of Pegylated Interferon Alfa-2a With Adefovir Case Sex Age, y HBeAg Status HBV DNA Level, Log10 IU/mL HBsAg Level, Log10 IU/mL ALT Level, U/L Genotype Interferon Treatment Naive T72 Response 1 M 37 Negative 4.05 2.41 36 D No HBsAg clearancea 2 M 30 Negative 5.92 3.63 79 D Yes No response 3 4 M M 46 31 Positive Positive 8.72 8.65 4.91 4.75 81 1256 A A No Yes Seroconversionb No response 5 M 39 Positive 6.85 3.18 327 A Yes Seroconversion 6 7 M M 42 46 Positive Negative 7.28 4.89 4.63 3.22 106 52 A E No Yes No response Seroconversion 8 M 30 Negative 7.72 4.04 214 E Yes No response 9 10 F M 69 27 Negative Negative 3.63 5.37 2.54 3.44 50 49 C C No Yes HBsAg clearance No response 11 M 24 Negative 5.48 1.62 38 A Yes Seroconversion 12 13 M M 27 48 Negative Positive 7.51 8.84 3.62 5.00 91 80 A A Yes Yes No response Seroconversion 14 M 44 Positive 9.81 5.27 105 A No No response Abbreviations: ALT, alanine aminotransferase; HBeAg, hepatitis B virus e antigen; HBsAg, hepatitis B virus surface antigen; HBV, hepatitis B virus; T72, 72 weeks after the start of treatment. a Persistently undetectable HBsAg, combined with negative HBV DNA test results or a HBV DNA level of <20 IU/mL. b HBsAg clearance with subsequent development of antibodies to HBsAg. NK Cell Characteristics in Chronic Hepatitis B • JID (...truncated)