An Easy and Sensitive Method to Profile the Antibody Specificities of HLA-Specific Memory B Cells

Background Pre-transplant immunological risk assessment is currently based on donor-specific HLA antibodies in serum. Despite being an excellent source for antibodies produced by bone marrow-residing plasma cells, serum analysis does not provide information on the memory B cell compartment. While B cell culture supernatants can be used to detect memory B cell derived HLA antibodies, low IgG concentrations can preclude detectability of HLA antibodies in luminex single antigen bead (SAB) assays. Method Culture supernatants of polyclonally activated B cells from alloantigen exposed (n=13) or non-exposed (n=10) individuals were either concentrated 10-fold, or IgG was isolated by using a protein G affinity purification method to increase the IgG concentration. These processed culture supernatants, as well as paired serum samples were tested for the presence of HLA antibodies using luminex SAB analysis. Results In immunized individuals, 64% were found to have HLA-specific B cell memory in concentrated supernatants, whereas 82% showed HLA-specific B cell memory when IgG isolated supernatants were used for HLA antibody detection. IgG isolated supernatants showed higher MFI values compared to concentrated supernatants without increased background. In some individuals, HLA-specific B cell memory was detected in the absence of accompanying serum antibody specificities. Conclusions We developed a novel, highly sensitive method to assess the HLA-specific memory B cell compartment using luminex SAB technology. This assay, allows direct comparison to the serum compartment and may therefore provide a more complete picture of the humoral alloimmune response in patients with a history of alloantigen exposure. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Corresponding author: Gonca E. Karahan, Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2-2333 ZA Leiden, the Netherlands. e-mail: g.e.karahan@lumc.nl Funding: National Reference Center for Histocompatibility Testing, the Netherlands and the Dutch Transplant Society Astellas Transplantation Research Award 2017. CW was funded by the Swiss National Science Foundation. Conflict of interest: The authors declare no conflicts of interest. Authorship GEK: designed the study, performed experiments, analysed data, wrote manuscript JK/YdV/JLL: performed experiments CW: performed experiments, edited manuscript NML/FJB: provided study samples ItB/MEJR: provided study samples, edited manuscript CvK: designed study, edited manuscript FHJC: interpreted results, edited manuscript SH: designed the study, interpreted results, wrote the manuscript Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.

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