A Memory B Cell Crossmatch Assay for Quantification of Donor-Specific Memory B Cells in the Peripheral Blood of HLA-Immunized Individuals

Abstract

Humoral responses against mismatched donor HLA are routinely measured as serum HLA antibodies, which are mainly produced by bone marrow-residing plasma cells. Individuals with a history of alloimmunization but lacking serum antibodies may harbor circulating dormant memory B cells, which may rapidly become plasma cells on antigen reencounter. Currently available methods to detect HLA-specific memory B cells are scarce and insufficient in quantifying the complete donor-specific memory B cell response due to their dependence on synthetic HLA molecules. We present a highly sensitive and specific tool for quantifying donor-specific memory B cells in peripheral blood of individuals using cell lysates covering the complete HLA class I and class II repertoire of an individual. Using this enzyme-linked immunospot (ELISpot) assay, we found a median frequency of 31 HLA class I and 89 HLA class II-specific memory B cells per million IgG-producing cells directed at paternal HLA in peripheral blood samples from women (n = 22) with a history of pregnancy, using cell lysates from spouses. The donor-specific memory B cell ELISpot can be used in HLA diagnostic laboratories as a cross-match assay to quantify donor-specific memory B cells in patients with a history of sensitizing events.

HLA‐specific memory B‐cell detection in kidney transplantation: Insights and future challenges

“…These cocktails are based on ligation of either the BCR, Toll‐like receptors and/or costimulatory molecules, and are often supplemented with a combination of B‐cell cytokines and growth factors (Heidt et al., 2008; Karahan, Eikmans, Anholts, Claas, & Heidt, 2014). Among the described cocktails, stimulation with Toll‐like receptor 7/8 agonist (Resiquimod‐R848) and interleukin 2 has been shown to preferentially stimulate memory B cells among peripheral blood mononuclear cells without causing isotype switching in the naïve B‐cell population (Karahan et al., 2017; Pinna, Corti, Jarrossay, Sallusto, & Lanzavecchia, 2009) and is, therefore, increasingly applied. The second approach of HLA‐specific memory B cells analysis is based on profiling of memory B‐cell‐derived HLA antibodies in culture supernatant produced upon such polyclonal stimulation by using luminex SAB assays (Bernasconi et al., 2002; Han, Rogers, Lavingia, & Stastny, 2009; Karahan et al., 2019) (Figure 1, middle row).…”

“…Importantly, since ELISpot assays rely on the capacity of activated B cells to produce antibodies, viability of the stimulated cells at the end of the pre‐culture phase plays a major role for the quality and accuracy of the results. Hence, in contrast to supernatant analysis favouring longer culture periods up to 10–12 days to achieve accumulation of IgG in the culture supernatants (Karahan et al., 2019), cells to be used for ELISpot assays are generally cultured for 6–7 days in order to obtain an optimum of antibody‐producing viable cells (Heidt et al., 2012; Karahan et al., 2014, 2017). Following culturing, in most protocols activated B cells are transferred to anti‐IgG‐coated ELISpot plates and incubated overnight.…”

“…Following culturing, in most protocols activated B cells are transferred to anti‐IgG‐coated ELISpot plates and incubated overnight. In a second phase (visualization), antibody fingerprints can be visualized as a single spot representative of one HLA antibody‐producing cell using either synthetic HLA molecules (monomeric or multimeric), or donor lysate‐derived HLA molecules serving as detection matrix (Heidt et al., 2012; Karahan et al., 2017; Karahan, de Vaal, et al., 2015; Lucia et al., 2015). In order to enable detection of two different HLA specificities in the same plate well, a fluorescence‐based technique using HLA dextramers labelled by distinct fluorochromes was recently described (Luque et al., 2019).…”

“…Since no HLA antibody production is expected to occur against the recipient's own HLA, autologous controls using self HLA for detection serve as negative controls. For reporting of the frequency of HLA‐specific memory B cells, usually expressed as a ratio of HLA‐specific memory B cells per number of IgG‐producing cells, both controls need to be taken into account (Karahan et al., 2017; Lucia et al., 2015).…”

Refinement of humoral immune monitoring in kidney transplantation: the role of “hidden” alloreactive memory B cells

“…They assessed 22 healthy women with a history of pregnancy with cell lysates of the respective husbands and 10 males without any prior immunizing events. As a result, 50% of women with previous pregnancy harbored mBC directed to paternal HLA antigens . With the aim to better characterize the role of donor‐specific mBC in human transplantation, our group recently reported a novel HLA‐specific B‐cell ELISPOT assay approach to quantify HLA‐specific mBC from peripheral blood .…”