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
Jong, E. de; Quint, K.D.; Ghalbzouri, A. el; Verdijk, R.M.; Goeman, J.J.; Heidt, S.; ... ; Bavinck, J.N.B. 2023
Background: Solid organ-transplant recipients (SOTR) have an increased risk of cutaneous squamous-cell carcinoma (cSCC), metastasis and death from cSCC. In immunocompetent patients with mucosal SCC... Show moreBackground: Solid organ-transplant recipients (SOTR) have an increased risk of cutaneous squamous-cell carcinoma (cSCC), metastasis and death from cSCC. In immunocompetent patients with mucosal SCC, downregulation of HLA class I is associated with poor prognosis. Since the degree of HLA expression on tumor cells could play a role in immunogenicity and pathophysiology of cSCC metastasis, we hypothesized that decreased HLA expression is associated with an increased risk of metastasis.Methods: We compared HLA expression between primary metastasized cSCCs, their metastases, and nonmetastasized cSCCs from the same patients. Samples were stained for HLA-A, HLA-B/-C and quantified by calculating the difference in immunoreactivity score (IRS) of the primary cSCC compared with all nonmetastasized cSCCs. Results: The mean IRS score for HLA-B/C expression was 2.07 point higher in metastasized compared to nonmetastasized cSCCs (p = 0.065, 95 % CI -0.18-4.32). 83.3 % of the primary metastasized cSCCs had an IRS score of 4 or higher, compared to 42.9 % in non-metastasized cSCCs. Moderately to poorly differentiated cSCCs had more HLA class I expression compared to well-differentiated cSCCs. Conclusion: Contrary to immunocompetent patients, HLA-B/C expression tends to be upregulated in metastasized cSCC compared to non-metastasized cSCC in SOTR, suggesting that different tumor escape mechanisms play a role in SOTR compared to immunocompetent patients. Show less
Meng, T.; Bezstarosti, S.; Singh, U.; Yap, M.; Scott, L.; Petrosyan, N.; ... ; Heidt, S. 2022
Eplet 44KM is currently listed in the HLA Epitope Registry but does not adhere to the eplet definition of an amino acid configuration within a 3.5 angstrom radius. Eplet 44KM has been previously... Show moreEplet 44KM is currently listed in the HLA Epitope Registry but does not adhere to the eplet definition of an amino acid configuration within a 3.5 angstrom radius. Eplet 44KM has been previously redefined to the antibody-verified reactivity pattern 44K/150V/158V, based on reactivity analysis of monoclonal antibody VDK1D12. Since the three residues are always simultaneously present on common HLA alleles, methods to define which residue is crucial for antibody-induction and binding are limited. In this proof-of-concept study, we performed site-directed mutagenesis to narrow down the antibody-verified reactivity pattern 44K/150V/158V to a single amino acid and defined 44K as the eplet or functional epitope of mAb VDK1D12. Show less
Krop, J.; Zwan, A. van der; Ijsselsteijn, M.E.; Kapsenberg, H.; Luk, S.J.; Hendriks, S.H.; ... ; Eikmans, M. 2022
Although the immunological complexity of the maternal-fetal interface is well appreciated, the actual interaction of maternal immune cells and fetal trophoblasts is insufficiently understood. To... Show moreAlthough the immunological complexity of the maternal-fetal interface is well appreciated, the actual interaction of maternal immune cells and fetal trophoblasts is insufficiently understood. To comprehend the composition and spatial orientation of maternal immune cells and fetal extravillous trophoblasts, we applied imaging mass cytometry on decidua basalis of the three trimesters of healthy pregnancy. Within all trimesters, we observed considerably higher frequencies of myeloid cells in the decidua than is seen with single-cell suspension techniques. Moreover, they were the most pronounced cell type in the microenvironment of other decidual cells. In first trimester, HLA-DR- macrophages represented the most abundant myeloid subcluster and these cells were frequently observed in the vicinity of trophoblasts. At term, HLA-DR+ macrophage subclusters were abundantly present and frequently observed in the microenvironment of T cells. Taken together, our results highlight the dynamic role of myeloid cells at the human maternal-fetal interface throughout gestation. Show less
Solid organ transplantation is the treatment of choice for various end-stage diseases, but requires the continuous need for immunosuppression to prevent allograft rejection. This comes with serious... Show moreSolid organ transplantation is the treatment of choice for various end-stage diseases, but requires the continuous need for immunosuppression to prevent allograft rejection. This comes with serious side effects including increased infection rates and development of malignancies. Thus, there is a clinical need to promote transplantation tolerance to prevent organ rejection with minimal or no immunosuppressive treatment. Polyclonal regulatory T-cells (Tregs) are a potential tool to induce transplantation tolerance, but lack specificity and therefore require administration of high doses. Redirecting Tregs towards mismatched donor HLA molecules by modifying these cells with chimeric antigen receptors (CAR) would render Tregs far more effective at preventing allograft rejection. Several studies on HLA-A2 specific CAR Tregs have demonstrated that these cells are highly antigen-specific and show a superior homing capacity to HLA-A2+ allografts compared to polyclonal Tregs. HLA-A2 CAR Tregs have been shown to prolong survival of HLA-A2+ allografts in several pre-clinical humanized mouse models. Although promising, concerns about safety and stability need to be addressed. In this review the current research, obstacles of CAR Treg therapy, and its potential future in solid organ transplantation will be discussed. Show less
MHC class I molecules play an important role in adaptive immune responses against intracellular pathogens. These molecules are highly polymorphic, and many allotypes have been characterized. In a... Show moreMHC class I molecules play an important role in adaptive immune responses against intracellular pathogens. These molecules are highly polymorphic, and many allotypes have been characterized. In a transplantation setting, a mismatch between MHC allotypes may initiate an alloimmune response. Rhesus macaques (Macaca mulatta, Mamu) are valuable as a preclinical model species in transplantation research as well as to evaluate the safety and efficacy of vaccine candidates. In both lines of research, the availability of nonhuman primate MHC-reactive mAbs may enable in vitro monitoring and detection of presence of particular Mamu molecules. In this study, we screened a collection of thoroughly characterized HLA class I–specific human mAbs for cross-reactivity with rhesus macaque MHC class I allotypes. Two mAbs, OK4F9 and OK4F10, recognize an epitope that is defined by isoleucine (I) at amino acid position 142 that is present on the Indian rhesus macaque Mamu-B*008:01 allotype, which is an allotype known to be associated with elite control of SIV replication. The reactive pattern of a third mAb, MUS4H4, is more complex and includes an epitope shared on Mamu-A2*05:01 and -B*001:01–encoded Ags. This is the first description, to our knowledge, of human HLA-reactive mAbs that can recognize Mamu allotypes, and these can be useful tools for in vitro monitoring the presence of the relevant allelic products. Moreover, OK4F9 and OK4F10 can be powerful mAbs for application in SIV-related research. Show less
Over the past decade, high HLA epitope mismatch scores have been associated with inferior transplant outcomes using several tools, of which HLAMatchmaker is most well-known. This software uses... Show moreOver the past decade, high HLA epitope mismatch scores have been associated with inferior transplant outcomes using several tools, of which HLAMatchmaker is most well-known. This software uses theoretically defined polymorphic amino acid configurations, called eplets, for HLA compatibility analysis. Although consideration of eplet mismatch loads has potential for immunological risk stratification of transplant patients, the use of eplet matching in organ allocation algorithms is hindered by lacking knowledge of the immunogenicity of individual eplets, and the possibility that single mismatched amino acids, rather than complete eplets, are responsible for HLA antibody induction.There are several approaches to define eplet immunogenicity, such as antibody verification of individual eplets, and data-driven approaches using large datasets that correlate specific eplet mismatches to donor specific antibody formation or inferior transplant outcomes. Data-driven approaches can also be used to define whether single amino acid mismatches may be more informative than eplet mismatches for predicting HLA antibody induction.When using epitope knowledge for the assignment of unacceptable antigens, it important to realize that alleles sharing an eplet to which antibodies have formed are not automatically all unacceptable since multiple contact sites determine the binding strength and thus biological function and pathogenicity of an antibody, which may differ between reactive alleles.While the future looks bright for using HLA epitopes in clinical decision making, major steps need to be taken to make this a clinical reality.(c) 2021 The Author(s). Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/). Show less
Matching strategies based on HLA eplets instead of HLA antigens in solid organ transplantation may not only increase the donor pool for highly sensitized patients, but also decrease the incidence... Show moreMatching strategies based on HLA eplets instead of HLA antigens in solid organ transplantation may not only increase the donor pool for highly sensitized patients, but also decrease the incidence of de novo donor-specific antibody formation. However, since not all eplets are equally capable of inducing an immune response, antibody verification is needed to confirm their ability to be bound by antibodies, such that only clinically relevant eplets are considered. The HLA Epitope Registry has documented all theoretically defined HLA eplets along with their antibody verification status and has been the foundation for many clinical studies investigating eplet mismatch in transplantation. The verification methods for eplets in the Registry range from polyclonal sera from multi- and uni-parous women to murine and human monoclonal antibodies (mAbs), and antibodies purified by adsorption and elution from sera of HLA immunized individuals. The classification of antibody verification based on different methods for validation is problematic, since not all approaches represent the same level of evidence. In this study, we introduce a classification system to evaluate the level of evidence for the antibody-verified status of all eplets in the HLA Epitope Registry. We demonstrate that for a considerable number of eplets, the antibody-verified status is solely based on polyclonal serum reactivity of multiparous women or on reactivity of murine mAbs. Furthermore, we noted that a substantial proportion of patient sera analyses and human mAb data presented in the HLA Epitope Registry Database has never been published in a peer-reviewed journal. Therefore, we tested several unpublished human HLA-specific mAbs by luminex single antigen beads assay to analyze their HLA reactivity for eplet antibody verification. Although the majority of analyzed mAbs indeed verified their assigned eplets, this was not the case for a number of eplets. This comprehensive overview of evidence for antibody verification of eplets in the HLA Epitope Registry is instrumental for future investigations towards eplet immunogenicity and clinical studies considering antibody-verified eplet mismatch in transplantation and warrants further standardization of antibody verification using high quality data. Show less
Bezstarosti, S.; Kramer, C.S.M.; Franke-van Dijk, M.E.I.; Vergunst, M.; Bakker, K.H.; Uyar-Mercankaya, M.; ... ; Heidt, S. 2022
HLA-DQ donor-specific antibodies (DSA) are the most prevalent type of DSA after renal transplantation and have been associated with eplet mismatches between donor and recipient HLA. Eplets are... Show moreHLA-DQ donor-specific antibodies (DSA) are the most prevalent type of DSA after renal transplantation and have been associated with eplet mismatches between donor and recipient HLA. Eplets are theoretically defined configurations of surface exposed amino acids on HLA molecules that require verification to confirm that they can be recognized by alloantibodies and are therefore clinically relevant. In this study, we isolated HLA-DQ specific memory B cells from immunized individuals by using biotinylated HLA-DQ monomers to generate 15 recombinant human HLA-DQ specific monoclonal antibodies (mAb) with six distinct specificities. Single antigen bead reactivity patterns were analyzed with HLA-EMMA to identify amino acids that were uniquely shared by the reactive HLA alleles to define functional epitopes which were mapped to known eplets. The HLA-DQB1*03:01-specific mAb LB_DQB0301_A and the HLA-DQB1*03-specific mAb LB_DQB0303_C supported the antibody-verification of eplets 45EV and 55PP respectively, while mAbs LB_DQB0402_A and LB_DQB0602_B verified eplet 55R on HLA-DQB1*04/05/06. For three mAbs, multiple uniquely shared amino acid configurations were identified, warranting further studies to define the inducing functional epitope and corresponding eplet. Our unique set of HLA-DQ specific mAbs will be further expanded and will facilitate the in-depth analysis of HLA-DQ epitopes, which is relevant for further studies of HLA-DQ alloantibody pathogenicity in transplantation. Show less
Krop, J.; Keur, C. van der; Kapsenberg, J.M.; Hollander, F. den; Hoorn, M.L.P. van der; Heidt, S.; ... ; Eikmans, M. 2022
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
Background. There is an unmet need for noninvasive tools for diagnosis of rejection after kidney transplantation. The aim of this study was to determine the discriminative value of a combined... Show moreBackground. There is an unmet need for noninvasive tools for diagnosis of rejection after kidney transplantation. The aim of this study was to determine the discriminative value of a combined cellular and molecular biomarker platform in urine for the detection of rejection. Methods. First, microRNA (miR) molecules were screened in transplant biopsies and urine sediments of patients with acute rejection and patients without rejection and stable graft function. Second, the expression of 15 selected miRs was quantified in an independent set of 115 urine sediments of patients with rejection and 55 urine sediments of patients without histological signs of rejection on protocol biopsy. Additionally, CXCL-9 and CXCL-10 protein levels were quantified in the urine supernatant. Results. Levels of miR-155-5p (5.7-fold), miR-126-3p (4.2-fold), miR-21-5p (3.7-fold), miR-25-3p (2.5-fold), and miR-615-3p (0.4-fold) were significantly different between rejection and no-rejection urine sediments. CXCL-9 and CXCL-10 levels were significantly elevated in urine from recipients with rejection. In a multivariable model (sensitivity: 89.1%, specificity: 75.6%, area under the curve: 0.94, P < 0.001), miR-155-5p, miR-615-3p, and CXCL-9 levels were independent predictors of rejection. Stratified 10-fold cross validation of the model resulted in an area under the curve of 0.92. Conclusions. A combined urinary microRNA and chemokine profile discriminates kidney transplant rejection from stable graft conditions. Show less
Highly sensitized kidney patients accrue on the transplant waiting list due to their broad immunization against non-self Human Leucocyte Antigens (HLA). Although challenging, the best option for... Show moreHighly sensitized kidney patients accrue on the transplant waiting list due to their broad immunization against non-self Human Leucocyte Antigens (HLA). Although challenging, the best option for highly sensitized patients is transplantation with a crossmatch negative donor without any additional therapeutic intervention. The Eurotransplant Acceptable Mismatch (AM) program was initiated more than 30 years ago with the intention to increase the chance for highly sensitized patients to be transplanted with such a compatible donor. The AM program allows for enhanced transplantation to this difficult to transplant patient group by allocating deceased donor kidneys on the basis of a match with the recipient's own HLA antigens in combination with predefined acceptable antigens. Acceptable antigens are those HLA antigens towards which the patients has never formed antibodies, as determined by extensive laboratory testing. By using this extended HLA phenotype for allocation and giving priority whenever a compatible donor organ becomes available, organ offers are made for roughly 80% of patients in this program. Up till now, more than 1700 highly sensitized patients have been transplanted through the AM program. Recent studies have shown that the concept of acceptable mismatches being truly immunologically acceptable holds true for both rejection rates and long-term graft survival. Patients that were transplanted through the AM program had a similar rejection incidence and long-term graft survival rates identical to non-sensitized patients transplanted through regular allocation. However, a subset of patients included in the AM program does not receive an organ offer within a reasonable time frame. As these are often patients with a rare HLA phenotype in comparison to the Eurotransplant donor population, extension of the donor pool for these specific patients through further European collaboration would significantly increase their chances of being transplanted. For those patients that will not benefit from such strategy, desensitization is the ultimate solution. Show less
van't Hof, L.J.; Schotvanger, N.; Haasnoot, G.W.; Keur, C. van der; Roelen, D.L.; Lashley, L.E.E.L.O.; ... ; Hoorn, M.L.P. van der 2021
IntroductionIn pregnancy, the mother and fetus differ in HLA antigens, and yet the maternal immune system generally tolerates the fetus. KIR receptors expressed by maternal uterine NK cells at the... Show moreIntroductionIn pregnancy, the mother and fetus differ in HLA antigens, and yet the maternal immune system generally tolerates the fetus. KIR receptors expressed by maternal uterine NK cells at the maternal-fetal interface directly interact with HLA-C on extravillous trophoblast cells for optimal placental development. In this study, we aimed to determine whether there is a preferential selection for HLA compatibility and specific KIR/HLA-C combinations in uncomplicated and preeclamptic naturally conceived pregnancies compared to what would be expected by chance.MethodsGenotyping for maternal and fetal HLA-A, -B, -C, -DR, and -DQ, and maternal KIR was performed for 451 uncomplicated pregnancies and 77 pregnancies complicated with preeclampsia. The number of HLA antigen (mis)matches between mother and fetus was calculated and compared to expected values obtained by randomization of the HLA haplotype, inherited from the father, over the existing maternal haplotype of the fetuses. A similar methodology was executed for analysis of the KIR/HLA-C data (n=309).ResultsIn uncomplicated pregnancies, the degree of maternal-fetal HLA matching was not different than expected-by-chance values. In preeclamptic pregnancies, the degree of maternal-fetal HLA matching was different in observed compared to expected-by-chance values (p=0.012). More specifically, the degree of maternal-fetal matching of HLA-C was higher in the actual preeclamptic pregnancies than was expected-by-chance (p=0.007). Preeclamptic pregnancies showed an overall tendency towards higher maternal-fetal HLA compatibility, for total HLA matches (p=0.021), HLA class I (p=0.038) and HLA-C (p=0.025) compared to uncomplicated pregnancies.ConclusionThe data suggest that there is no preferential selection of maternal-fetal HLA compatibility in uncomplicated pregnancies. In contrast, increased total HLA, HLA class I and, especially, HLA-C compatibility is associated with preeclampsia, suggestive for a role of HLA mismatches in immune regulation leading to uncomplicated pregnancy. Show less
MHC class I molecules play an important role in adaptive immune responses against intracellular pathogens. These molecules are highly polymorphic, and many allotypes have been characterized. In a... Show moreMHC class I molecules play an important role in adaptive immune responses against intracellular pathogens. These molecules are highly polymorphic, and many allotypes have been characterized. In a transplantation setting, a mismatch between MHC allotypes may initiate an alloimmune response. Rhesus macaques (Macaca mulatta, Mamu) are valuable as a preclinical model species in transplantation research as well as to evaluate the safety and efficacy of vaccine candidates. In both lines of research, the availability of nonhuman primate MHC-reactive mAbs may enable in vitro monitoring and detection of presence of particular Mamu molecules. In this study, we screened a collection of thoroughly characterized HLA class I-specific human mAbs for crossreactivity with rhesus macaque MHC class I allotypes. Two mAbs, OK4F9 and OK4F10, recognize an epitope that is defined by isoleucine (I) at amino acid position 142 that is present on the Indian rhesus macaque Mamu-B*008:01 allotype, which is an allotype known to be associated with elite control of SIV replication. The reactive pattern of a third mAb, MUS4H4, is more complex and includes an epitope shared on Mamu-A2*05:01 and -B*001:01-encoded Ags. This is the first description, to our knowledge, of human HLA-reactive mAbs that can recognize Mamu allotypes, and these can be useful tools for in vitro monitoring the presence of the relevant allelic products. Moreover, OK4F9 and OK4F10 can be powerful mAbs for application in SIV-related research. Show less
Johnsen, G.M.; Fjeldstad, H.E.S.; Drabbels, J.J.M.; Haasnoot, G.W.; Eikmans, M.; Storvold, G.L.; ... ; Staff, A.C. 2021
HLA-G, a non-classical HLA molecule expressed by extravillous trophoblasts, plays a role in the maternal immune tolerance towards fetal cells. HLA-G expression is regulated by genetic polymorphisms... Show moreHLA-G, a non-classical HLA molecule expressed by extravillous trophoblasts, plays a role in the maternal immune tolerance towards fetal cells. HLA-G expression is regulated by genetic polymorphisms in the 3' untranslated region (3'UTR). Low levels of HLA-G in the maternal circulation and placental tissue are linked to preeclampsia.Our objective was to investigate whether variants of the 3'UTR of the HLA-G gene in mother and fetus are associated with acute atherosis, a pregnancy specific arterial lesion of the decidua basalis that is prevalent in preeclampsia.Paired maternal and fetal DNA samples from 83 normotensive and 83 preeclamptic pregnancies were analyzed. We sequenced the part of the HLA-G 3'UTR containing a 14-bp insertion/deletion region and seven single nucleotide polymorphisms (SNPs). Associations with acute atherosis were tested by logistic regression.The frequency of heterozygosity for the 14-bp polymorphism (Ins/Del) and the +3142 SNP (C/G) variant in the fetus are associated with acute atherosis in preeclampsia (66.7 % vs. 39.6 %, p = 0.039, and 69.0 % vs. 43.4 %, p = 0.024). Furthermore, the fetal UTR-3 haplotype, which encompasses the 14-bp deletion and the +3142G variant, is associated with acute atherosis in preeclampsia (15 % vs. 3.8 %, p = 0.016).In conclusion, HLA-G polymorphisms in the fetus are associated with acute atherosis. We hypothesize that these polymorphisms lead to altered HLA-G expression in the decidua basalis, affecting local feto-maternal immune tolerance and development of acute atherosis. Show less
