More screening criteria were listed in Supplementary Fig. 1. At the end of this process 26 individuals from the cohort recruited were defined as authentic non-responders based on producing

BMS-777607 mouse anti-HBs levels of less than 10 mIU/ml after having received a total of six doses of vaccine administered over two consecutive rounds of vaccination schedule. DNA samples from 20 of these non-responders were available for use in this study. For comparative purpose, after considering almost the same criteria for screening non-responders, a group of vaccine responders were identified on the basis of having produced anti-HBs levels equal to or more than 100 mIU/ml after having received the standard 3 doses of vaccine. Finally 45 responders were randomly selected and there are no significant differences between the responders and non-responders in age (age range 25–60 for responders vs. age range 30–59 for non-responders, P = 0.0512) and gender (23F/22M for responders vs. 7F/13M for non-responders, P = 0.2291). The detailed demographic data of the Y-27632 research buy 20 non-responders and 45 responders is shown in Supplementary Table 1. Since no peripheral blood mononuclear cells (PBMC) were available from the non-responders and responders, 29 healthy adults who had physical examination in Peking University Third Hospital without evidence of prior HBV

infection were also enrolled for further experiments. This study was approved by the Ethics Committee of the Peking University Health Science Center and all subjects provided signed informed consent. Six TfH associated molecules CXCR5,

ICOS, CXCL13, IL-21, BCL6 and CD40L were selected for SNP analysis. Altogether 24 SNPs within these genes were chosen for the analysis (Supplementary Table 2), according to the following 2 criteria: first, the minor allele frequency (MAF) obtained from NCBI SNP database (http://www.ncbi.nlm.nih.gov/SNP/) or the SNP browser software 4.0 (Applied Biosystems) should be higher than 10% in the ethnic Han Chinese population. Second, there should be published evidence showing that the Megestrol Acetate SNP is associated with some disease. Genomic DNA extracted as previously described was dissolved in sterile double distilled water and stored at −20 °C [4]. SNP genotyping was undertaken by Bioyong Technology using Sequenom MassARRAY technology (Bioyong Technology Co., Beijing, China). Peripheral Blood Mononuclear Cells were isolated using Histopaque-1077 (Sigma, 10771) according to the manufacturer’s instructions and stored at −80 °C. For flow cytometry assays, recovered cells were incubated for 30 min with a cocktail of antibodies that included eFluor450 conjugated anti-CD3 mAb (eBioscience, 48-0038), PE-Cy7 conjugated anti-CD4 mAb (BD, 557852), APC conjugated anti-CD19 mAb (BD, 555415) and PE conjugated anti-CXCR5 mAb (eBioscience, 12-9185). Following incubation the cells were washed with PBS and fixed with 2% paraformaldehyde.