The same conclusion was true for the MFI value of CXCR5. However, no significant difference was observed when similar analysis was carried out on rs676925 (Supplementary Fig. 2). These results suggested that rs3922 might be involved in non-responsiveness to HBV vaccination through affecting the level of CXCR5 expression. Targetscan (http://www.targetscan.org/) prediction suggested that the rs3922 SNP is located in a potential microRNA binding site for miR-558 when the A allele is present, but not the G allele. To investigate whether allelic change in rs3922 can result in

miR-558 regulated differences in the expression of CXCR5, luciferase vectors pGL3-3922A-luc and pGL3-3922G-luc differing only in the allelic version of the potential miRNA binding site were constructed (Fig. 3A). These TSA HDAC luciferase vectors were independently co-transfected into HEK293T cells together with either miR-558 expressing or U6 control plasmids. Strikingly, cells co-transfected with pGL3-3922A-luc produced

significantly lower luciferase activity than those co-transfected with pGL3-3922G-luc irrespective of whether the co-transfection was with the U6 control plasmid or that expressing miR-558 (Fig. 3B). Similarly, when only the luciferase reporter vector alone was transfected into cells, the lowest relative level of luciferase activity was recorded from pGL3-3922A-luc and the difference between the level of luciferase Thymidine kinase expressed by the pGL3-3922A-luc and that by the pGL3-3922G-luc was statistically significant (Fig. 3C). The standard HDAC phosphorylation HBV vaccination regime provides protection from HBV infection in most vaccinees, leaving only 5–10% of recipients defined as non-responders. A variety of factors, including gene polymorphisms, have been found to cause inadequate antibody production and hence limit the efficacy of the HBV vaccine [4] and [24]. Following

the recognition that TfH cells play an important role in antibody responses, this study focused on the genes encoding 6 molecules associated with TfH cells (CXCR5, CXCL13, ICOS, CD40L, IL-21 and BCL6), to evaluate possible associations of polymorphisms in them with immune responses made to HBV vaccination. This SNP based association analysis clearly showed that polymorphisms in CXCR5 and CXCL13 were associated with non-responsiveness to the HBV vaccine. CXCR5 and CXCL13 appear to be inter-related not only in terms of anatomical location, but also in terms of the functioning of TfH cells [25]. These two molecules are expressed both by TfH cells and B cells [26] and [27]. The encounter between a CD4+ helper T cell and a cognate B cell is essential for TfH cells to offer help in the production of antibody by B cells and it has been suggested that proper interplay between CXCR5 and CXCL13 is the impetus for TfH cells and B cells to migrate to B cell follicles [28].