Luminex single antigen bead (SAB) kits from One Lambda (OL) and Lifecodes (LC) are widely used for HLA antibody detection but have substantial differences in design and assay protocol resulting in... Show moreLuminex single antigen bead (SAB) kits from One Lambda (OL) and Lifecodes (LC) are widely used for HLA antibody detection but have substantial differences in design and assay protocol resulting in different mean fluorescence intensity (MFI) values. Here, we present a non-linear modeling approach to accurately convert MFI values between two vendors and to establish user-independent MFI cutoffs when analyzing big datasets. HLA antibody data from a total of 47 EDTA-treated sera tested using both OL and LC SAB kits were analyzed. MFI comparisons were made for the common 84 HLA class I and 63 class II beads. In the exploration set (n = 24), a non-linear hyperbola model on raw MFI corrected by locus-specific highest self MFI subtraction yielded the highest correlation (class I r(2): 0.946, class II r(2): 0.898). Performance of the model was verified in an independent validation set (n = 12) (class I r(2): 0.952, class II r(2): 0.911). Furthermore, in an independent cohort of post-transplant serum samples (n = 11) using the vendor-specific MFI cutoffs dictated by the current model, we found 94% accuracy in bead-specific reactivity assignments by the two vendors. We recommend using the non-linear hyperbola modeling approach with self HLA correction and locus-specific analyzes to harmonize MFI values between two vendors in particular research datasets. As there are considerable variations between the two assays, using MFI conversion for individual patient samples is not recommended. Show less
Exposure of the adaptive immune system to a pathogen can result in the activation and expansion of T cells capable of recognizing not only the specific antigen but also different unrelated antigens... Show moreExposure of the adaptive immune system to a pathogen can result in the activation and expansion of T cells capable of recognizing not only the specific antigen but also different unrelated antigens, a process which is commonly referred to as heterologous immunity. While such cross-reactivity is favourable in amplifying protective immune responses to pathogens, induction of T cell-mediated heterologous immune responses to allo-antigens in the setting of solid organ transplantation can potentially lead to allograft rejection. In this review, we provide an overview of murine and human studies investigating the incidence and functional properties of virus-specific memory T cells cross-reacting with allo-antigens and discuss their potential relevance in the context of solid organ transplantation. Show less
Karahan, G.E.; Vaal, Y. de; Bakker, K.; Roelen, D.; Claas, F.H.J.; Heidt, S. 2021
Human leukocyte antigen (HLA) antibodies are induced by pregnancy, transfusion, or transplantation. Serum from transplant recipients is regularly screened for IgG HLA antibodies because of their... Show moreHuman leukocyte antigen (HLA) antibodies are induced by pregnancy, transfusion, or transplantation. Serum from transplant recipients is regularly screened for IgG HLA antibodies because of their clinical relevance for transplant outcome. While other isotypes of HLA antibodies, such as IgA may also contribute to the alloimmune response, validated detection assays for IgA HLA antibody detection are lacking. Therefore, we modified the commonly used luminex screening assay for IgG HLA antibody detection (IgG-LMX) into an IgA HLA antibody screening assay (IgA-LMX). Optimization and validation was performed with IgG, IgA1, and IgA2 isotype variants of HLA-specific human recombinant monoclonal antibodies (mAbs). Reactivity patterns of IgA1 and IgA2 isotype HLA-specific mAbs in IgA-LMX were identical to those of the IgG isotype. Cross-reactivity with IgG and IgM antibodies and nonspecific binding to the beads were excluded. Further assay validation showed the absence of IgA HLA antibodies in serum from individuals without alloantigen exposure (n = 18). When the IgA-LMX assay was applied to sera from 289 individuals with known alloantigen exposure through pregnancy (n = 91) or kidney transplantation (n = 198), IgA HLA antibodies were detected in 3.5% of individuals; eight patients on the kidney retransplant waitlist and two women immunized through pregnancy. The majority (90%) of IgA HLA antibodies were directed against HLA class II and were always present in conjunction with IgG HLA antibodies. Results of this study show that this validated IgA-LMX method can serve as a screening assay for IgA HLA antibodies and that the incidence of IgA HLA antibodies in alloantigen exposed individuals is low. Show less
In allogeneic transplantation, genetic disparities between patient and donor may lead to cellular and humoral immune responses mediated by both naive and memory alloreactive cells of the adaptive... Show moreIn allogeneic transplantation, genetic disparities between patient and donor may lead to cellular and humoral immune responses mediated by both naive and memory alloreactive cells of the adaptive immune system. This review will focus on alloreactive T and B cells with emphasis on the memory compartment, their role in relation to kidney rejection, andin vitroassays to detect these alloreactive cells. Finally, the potential additional value of utilizing donor-specific memory T and B cell assays supplementary to current routine pre-transplant risk assessment of kidney transplant recipients will be discussed. Show less
Kramer, C.S.M.; Franke-van Dijk, M.E.I.; Bakker, K.H.; Uyar-Mercankaya, M.; Karahan, G.E.; Roelen, D.L.; ... ; Heidt, S. 2020
In kidney transplantation, eplet mismatches between donor and recipient have been associated with de novo donor-specific antibody development. Eplets are theoretically defined configurations of... Show moreIn kidney transplantation, eplet mismatches between donor and recipient have been associated with de novo donor-specific antibody development. Eplets are theoretically defined configurations of polymorphic amino acids and require experimental verification to establish whether they can be bound by alloantibodies. Human HLA-specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we isolated single HLA-DR-specific memory B cells from peripheral blood of immunized individuals (n = 3) using HLA class II tetramers to generate recombinant human HLA-DR antigen-reactive mAbs (n = 5). Comparison of the amino acid composition of the reactive HLA alleles in relation to the antibody reactivity patterns led to identification of 3 configurations, 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognized, respectively, by HLA-DRB1*01:01, HLA-DRB1*04:01, and HLA-DRB1*07:01 antigen-reactive mAbs. The first 2 correspond to eplets 70QA and 31FYY and can now be considered antibody verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody verified. Generation and reactivity analysis of human HLA-DR mAbs allowed for identification of amino acid configurations corresponding to known eplets, whereas the other patterns may be used to redefine eplets with similar, but not identical predicted amino acid composition. Show less
Humoral alloimmunity mediated by anti-human leucocyte antigen (HLA) antibodies is a major challenge in kidney transplantation and impairs the longevity of the transplanted organ. The immunological... Show moreHumoral alloimmunity mediated by anti-human leucocyte antigen (HLA) antibodies is a major challenge in kidney transplantation and impairs the longevity of the transplanted organ. The immunological risk of an individual patient is currently mainly assessed by detection of HLA antibodies in the serum, which are produced by long-lived bone marrow-residing plasma cells. However, humoral alloimmunity is complex, and alloreactive memory B cells constitute an additional factor in the interplay of immune cells. These recirculating "silent" cells are responsible for the immunological recall response by differentiating into antibody-producing cells upon antigen re-encounter. Historically, due to the lack of appropriate and routinely applicable assays to determine the presence and HLA specificity of alloreactive memory B cells, their contribution to the humoral alloimmune response has clinically often been suspected but could not be determined. In this review, we give an overview of recent advances in techniques to detect alloreactive memory B cells and discuss their strengths and limitations. Furthermore, we summarize experiences with these techniques in alloimmunized individuals and transplant recipients, thereby emphasizing unmet needs to be addressed in future studies. Show less
Wehmeier, C.; Karahan, G.E.; Krop, J.; Vaal, Y. de; Langerak-Langerak, J.; Binet, I.; ... ; Swiss Transplant Cohort Study 2020
Background.HLA-specific memory B cells may contribute to the serum HLA antibody pool upon antigen reexposure. The aim of this pilot study was to investigate the presence of concurrent donor... Show moreBackground.HLA-specific memory B cells may contribute to the serum HLA antibody pool upon antigen reexposure. The aim of this pilot study was to investigate the presence of concurrent donor-specific memory B cell-derived HLA antibodies (DSA-M) in renal allograft recipients with pretransplant donor-specific HLA antibodies (DSA) and its association with occurrence of antibody-mediated rejection (AMR) using a recently developed method.Methods.Twenty patients with Luminex single antigen bead (SAB) assay-defined DSA but negative complement-dependent cytotoxicity crossmatches were enrolled. Plasma samples and peripheral blood mononuclear cells were collected at 3 timepoints (pretransplant, mo 6, mo 12). We analyzed IgG-purified and concentrated culture supernatants from polyclonally activated peripheral blood mononuclear cells using SAB assays and compared HLA antibody profiles with same day plasma results.Results.Plasma SAB analysis revealed 35 DSA in 20 patients pretransplant. DSA-M were detected in 9 of 20 (45%) patients and for 10 of 35 specificities (29%). While median mean fluorescence intensity values of DSA with concurrent DSA-M (5877) were higher than those of DSA without DSA-M (1476), 3 of 6 patients with AMR and low mean fluorescence intensity DSA (<3000) had DSA-M. Overall, pretransplant DSA/DSA-M-pos allograft recipients showed a higher incidence of biopsy-proven (sub)clinical AMR (P = 0.032) and a higher extent (g >= 1 + ptc >= 1) of microvascular inflammation (67% vs 9%, P = 0.02). In 17 patients (28 DSA) with posttransplant analyses, persisting DSA posttransplant had more often DSA-M (6/12; 50%) than nonpersisting DSA (2/16; 13%).Conclusions.Assessment of DSA-M might be a novel tool to supplement serum HLA antibody analysis for pretransplant risk stratification in patients with DSA. Show less
Luminex single antigen bead assays revolutionized human leukocyte antigen (HLA) antibody detection owing to their superior sensitivity compared to conventional methods. Nevertheless, the advent of... Show moreLuminex single antigen bead assays revolutionized human leukocyte antigen (HLA) antibody detection owing to their superior sensitivity compared to conventional methods. Nevertheless, the advent of higher sensitivity came at the expense of difficulty in clinical decision-making, since not all luminex detectable antibodies are clinically relevant. Therefore, new tools such as C1q/C3d assays and IgG subclass analysis emerged with the aim to discriminate the inert antibodies from the deleterious ones. Here, we provide an overview on the technical challenges related to these different HLA antibody detection systems and briefly refer to the recent literature regarding the clinical relevance of these assays, mainly in the field of kidney transplantation. Show less
The aim of this thesis was to develop tools to detect and quantify HLA-specific memory B cells in peripheral blood of HLA-immunized individuals and to assess the applicability of the newly... Show moreThe aim of this thesis was to develop tools to detect and quantify HLA-specific memory B cells in peripheral blood of HLA-immunized individuals and to assess the applicability of the newly developed assays in the setting of clinical transplantation. Transplant patients who have already made a defense response to the foreign HLA on donor organ have an increased risk of developing antibody-mediated rejection, which may adversely affect the survival of the graft. In diagnostic HLA laboratories, serum of the patients is tested for the presence of HLA-specific antibodies before and after transplantation. Serum HLA antibodies are produced by plasma cells located in the bone marrow however, memory B cells can also play a role in antibody production against the donor organ. So far, the role of these cells has been neglected in the diagnostics. The research conducted in my PhD thesis enabled us to develop methods to detect HLA-specific memory B cells. By this means, patients who may potentially harbor HLA-specific memory B cells such as repeat transplant candidates, women receiving transplants from their partner or child and patients undergoing desensitization treatments may benefit from the assays that are described in this PhD thesis. Show less
