Quantification of HLA class II-specific memory B cells in HLA-sensitized individuals Gonca E. Karahan a , Yvonne J.H. de Vaal a , Dave L. Roelen a , Rico Buchli b , Frans H.J. Claas a , Sebastiaan Heidt a,⇑ a Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands b R&D, Pure Protein LLC, Oklahoma City, OK, USA article info Article history: Received 12 September 2014 Accepted 14 January 2015 Available online 27 January 2015 Keywords: HLA antibodies Luminex Pregnancy-immunized women Kidney transplantation Chronic rejection abstract For the quantification of HLA-specific memory B cells from peripheral blood of sensitized individuals, a limited number of methods are available. However, none of these are capable of detecting memory B cells directed at HLA class II molecules. Since the majority of antibodies that occur after transplantation appear to be specific for HLA class II, our aim was to develop an assay to detect and quantify HLA class II-specific memory B cells from peripheral blood. By using biotinylated soluble HLA class II molecules as detection agent, we were able to develop an HLA class II-specific memory B cell ELISPOT assay. The assay was validated using B cell-derived hybridomas that produce human monoclonal antibodies directed at specific HLA class II molecules. In pregnancy- immunized females, we found memory B cell frequencies ranging from 25 to 756 spots per 10 6 B cells specific for the immunizing paternal HLA class II molecules, whereas in non-immunized males no signif- icant spot formation was detected. Here, we present a novel ELISPOT assay for quantifying HLA class II-specific memory B cells from peripheral blood. This technique provides a unique tool for monitoring the HLA class II-specific memory B cell pool in sensitized transplant recipients. Ó 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. 1. Introduction Pre-existing as well as de novo produced HLA antibodies repre- sent an important risk factor for adverse transplantation outcome [1]. In the last decade, major advances in HLA antibody detection have enabled a clear definition of antibody specificities in the serum of transplant recipients, and thereby improved the predic- tion of immunologically low and high-risk patients [2–4]. How- ever, none of the currently available methods to detect serum HLA antibodies provide information on the presence or absence of HLA-specific memory B cells. Since serum antibodies are mainly produced by bone marrow residing plasma cells, serum antibody levels may not be represen- tative for the size of the peripheral memory B cell pool [5,6]. Upon a re-encounter with antigen, memory B cells can rapidly differen- tiate into antibody secreting cells to drive anamnestic responses [7]. Therefore, while serum antibody levels may be undetectable prior to transplant, accelerated antibody mediated rejection can occur in case memory B cells directed at donor HLA antigens are present [8]. Furthermore, apart from antibody production, memory B cells are potent cytokine producing cells [9] and can also function as antigen presenting cells [10], potentially driving the rejection process by activating alloantigen-specific T cells. Determining the level of HLA-specific memory B cells pre-transplant would benefit the risk assessment for early humoral rejection, whereas monitor- ing these cells post-transplant may provide a better understanding on how B cells may affect transplant outcomes in other ways than by antibody production [11]. As a result of the technical challenge of detecting and enumer- ating the relatively low numbers of HLA-specific memory B cells in peripheral blood, only a few studies exist aiming at quantification of HLA-specific memory B cells in sensitized individuals [12–18]. http://dx.doi.org/10.1016/j.humimm.2015.01.014 0198-8859/Ó 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. Abbreviations: ELISPOT, enzyme-linked immunosorbent spot; B-LCL, B-lympho- blastoid cell line; DSA, donor-specific antibody; FCS, fetal calf serum; HLA, human leukocyte antigen; IMDM, Iscove’s modified Dulbecco’s medium; ITS, insulin- transferrin-sodium selenite; mAb, monoclonal antibody; MFI, mean fluorescence intensity; ODN, oligodeoxynucleotides; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; SFU, spot forming unit; TLR, toll-like receptor. ⇑ Corresponding author at: Leiden University Medical Center, Department of Immunohaematology and Blood Transfusion, Albinusdreef 2, 2300 RC Leiden, The Netherlands. Fax: +31 71 5265267. E-mail address: S.Heidt@lumc.nl (S. Heidt). Human Immunology 76 (2015) 129–136 Contents lists available at ScienceDirect www.ashi-hla.org journal homepage: www.elsevier.com/locate/humimm