LIMS1 risk genotype and T cell-mediated rejection in kidney transplant recipients.

BACKGROUND: This study aims to examine the association of LIM zinc finger domain containing 1 (LIMS1) genotype with allograft rejection in an independent kidney transplant cohort. METHODS: We genotyped 841 kidney transplant recipients for the LIMS1 rs893403 variant by Sanger sequencing followed by polymerase chain reaction confirmation of the deletion. Recipients who were homozygous for the LIMS1 rs893403 genotype GG were compared with the AA/AG genotypes. The primary outcome was T cell-mediated or antibody-mediated rejection (TCMR or ABMR, respectively) and secondary outcome was allograft loss. RESULTS: After a median follow-up of 11.4 years, the rate of TCMR was higher in recipients with the GG genotype (n = 200) compared with the AA/AG genotypes (n = 641) [25 (12.5%) versus 35 (5.5%); P = 0.001] while ABMR did not differ by genotype [18 (9.0%) versus 62 (9.7%)]. Recipients with the GG genotype had 2.4 times higher risk of TCMR than those who did not have this genotype [adjusted hazard ratio2.43 (95% confidence interval 1.44-4.12); P = 0.001]. A total of 189 (22.5%) recipients lost their allografts during follow-up. Kaplan-Meier estimates of 5-year (94.3% versus 94.4%; P = 0.99) and 10-year graft survival rates (86.9% versus 83.4%; P = 0.31) did not differ significantly in the GG versus AA/AG groups. CONCLUSIONS: Our study demonstrates that recipient LIMS1 risk genotype is associated with an increased risk of TCMR after kidney transplantation, confirming the role of the LIMS1 locus in allograft rejection. These findings may have clinical implications for the prediction and clinical management of kidney transplant rejection by pretransplant genetic testing of recipients and donors for LIMS1 risk genotype.

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR.

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 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.

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

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 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.

EFI region 8 Balkan external proficiency testing for HLA typing: Results of 15 years.

Istanbul Medical Faculty, Department of Medical Biology, as the first EFI-accredited HLA laboratory in Turkey since 1999 has been organizing both national and international quality control tests for HLA typing under the banner of the "Balkan external proficiency testing (BEPT)" encompassing countries from EFI regions 8. The first round of BEPT in 2004 was organized for low-resolution HLA-A,-B typing by molecular methods and 12 centres participated in the exercise. In 2005, low-resolution HLA-DR typing was added, and in 2007, low-resolution HLA-C and DQ typing were added to the exercise and 28 centres participated. In 2015, high-resolution (four digits or higher typing) HLA-A,-B,-C,-DR and -DQ typing added to the exercise and 40 centres participated. In the last 2 years, 2017 and 2018, the number of participating centres increased to 48 and 52, respectively. When the distribution of low-resolution typing methods applied in the exercises were investigated, it was found that 82% of the centres in the first round of BEPT in 2004 used PCR-SSP, whereas in the last round in 2018, 26% of the centres preferred SSP, while the rest used SSO (50%) or SSP and SSO (24%) methods. Methods for high-resolution typing were SBT (41%), NGS (18%), SSO (18%), SSP (6%), SBT + NGS (6%) and SBT + SSP (11%). In 15th trial for BEPT, nomenclature mistake rate was 12%, and erroneous result rate was 6% for HLA samples. The most common mistakes in the exercises were nomenclature mistakes. The EFI Standards Version 7.0 is stated "HLA type can be reported using a hyphen if homozygosity is not proven by family studies." In order to provide standard results among HLA laboratories and since accurate HLA typing leads to significant increase of graft and patient survival, quality control exercises should be performed in all HLA-based tests periodically.

Technical challenges and clinical relevance of single antigen bead C1q/C3d testing and IgG subclass analysis of human leukocyte antigen antibodies.

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 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.

Anti-HLA Class II Antibodies Are the Most Resistant to Desensitization in Crossmatch-positive Living-donor Kidney Transplantations: A Patient Series.

Background: In HLA-incompatible kidney transplantation, the efficacy of desensitization in terms of anti-HLA antibody kinetics is not well characterized. We present an overview of the course of anti-HLA antibodies throughout plasma exchange (PE) desensitization in a series of crossmatch-positive patients. Methods: All consecutive candidates in the Dutch HLA-incompatible kidney transplantation program between November 2012 and January 2022 were included. The eligibility criteria were a positive crossmatch with a living kidney donor and no options for compatible transplantation. Desensitization consisted of 5-10 PE with low-dose IVIg. Results: A total of 16 patient-donor pairs were included. Patients had median virtual panel-reactive antibody of 99.58%. Cumulative donor-specific anti-HLA antibody (cumDSA) mean fluorescence intensity (MFI) was 31 399 median, and immunodominant DSA (iDSA) MFI was 18 677 for class I and 21 893 for class II. Median anti-HLA antibody MFI response to desensitization was worse in class II as compared with class I (P < 0.001), particularly for HLA-DQ. Class I cumDSA MFI decreased 68% after 4 PE versus 53% in class II. The decrease between the fifth and the 10th PE sessions was modest with 21% in class I versus 9% in class II. Antibody-mediated rejection occurred in 85% of patients, with the iDSA directed to the same mismatched HLA as before desensitization, except for 3 patients, of whom 2 had vigorous rebound of antibodies to repeated mismatches (RMMs). Rebound was highest (86%) in RMM-DSA with prior grafts removed (transplantectomy n = 7), lower (39%) in non-RMM-DSA (n = 30), and lowest (11%) for RMM-DSA with in situ grafts (n = 5; P = 0.018 for RMM-DSA transplantectomy versus RMM-DSA graft in situ). With a median follow-up of 59 mo, 1 patient had died resulting in a death-censored graft survival of 73%. Conclusions: Patients with class II DSA, and particularly those directed against HLA-DQ locus, were difficult to desensitize.

Memory B-cell derived donor-specific antibodies do not predict outcome in sensitized kidney transplant recipients: a retrospective single-center study.

Background: Repeated exposure to sensitizing events can activate HLA-specific memory B cells, leading to the production of donor-specific memory B cell antibodies (DSAm) that pose a risk for antibody-mediated rejection (ABMR) in kidney transplant recipients (KTRs). This single-center retrospective study aimed to identify DSAm and assess their association with outcomes in a cohort of KTRs with pretransplant serum donor-specific antibodies (DSA). Methods: We polyclonally activated pretransplant peripheral blood mononuclear cells (PBMCs) from 60 KTRs in vitro, isolated and quantified IgG from the culture supernatant using ELISA, and analyzed the HLA antibodies of eluates with single antigen bead (SAB) assays, comparing them to the donor HLA typing for potential DSAm. Biopsies from 41 KTRs were evaluated for rejection based on BANFF 2019 criteria. Results: At transplantation, a total of 37 DSAm were detected in 26 of 60 patients (43%), of which 13 (35%) were found to be undetectable in serum. No significant association was found between pretransplant DSAm and ABMR (P=0.53). Similar results were observed in a Kaplan-Meier analysis for ABMR within the first year posttransplant (P=0.29). Additionally, MFI levels of DSAm showed no significant association with ABMR (P=0.28). Conclusion: This study suggests no significant association between DSAm and biopsy-proven clinical ABMR. Further prospective research is needed to determine whether assessing DSAm could enhance existing immunological risk assessment methods for monitoring KTRs, particularly in non-sensitized KTRs.

FGF-23 associated with the progression of coronary artery calcification in hemodialysis patients.

BACKGROUND: Disordered mineral metabolism is implicated in the pathogenesis of vascular calcification in hemodialysis (HD) patients. Fibroblast growth factor 23 (FGF-23) is the main regulator of phosphate metabolism. In this prospective study, we aimed to investigate the association of serum FGF-23 with progression of coronary artery calcification in HD patients. METHODS: Seventy-four HD patients (36 male/38 female, mean age: 52 ± 14 years) were included. Serum FGF-23 levels were measured by ELISA. Coronary artery calcification score (CACS) was measured twice with one year interval. Patients were grouped as progressive (PG) (36 patients-48%) and non-progressive (NPG). RESULTS: Age, serum phosphorus, baseline and first year CACS were found to be significantly higher in the PG compared to NPG group. Serum FGF-23 levels were significantly higher in PG [155 (80-468) vs 147 (82-234), p = 0.04]. Patients were divided into two groups according to baseline CACS (low group, CACS ≤ 30; high group, CACS > 30). Serum FGF-23 levels were significantly correlated with the progression of CACS (ΔCACS) in the low baseline CACS group (r = 0.51, p = 0.006), but this association was not found in high baseline CACS group (r = 0.11, p = 0.44). In logistic regression analysis for predicting the PG patients; serum FGF-23, phosphorus levels and baseline CACS were retained as significant factors in the model. CONCLUSIONS: Serum FGF-23 was found to be related to progression of CACS independent of serum phosphorus levels. FGF-23 may play a major role in the progression of vascular calcification especially at the early stages of calcification process in HD patients.

A modeling approach for mean fluorescence intensity value harmonization and cutoff prediction for luminex single antigen bead assays of two different vendors.

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 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 r2 : 0.946, class II r2 : 0.898). Performance of the model was verified in an independent validation set (n = 12) (class I r2 : 0.952, class II r2 : 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.

Heterologous Immunity of Virus-Specific T Cells Leading to Alloreactivity: Possible Implications for Solid Organ Transplantation.

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, 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.

Comparison of different luminex single antigen bead kits for memory B cell-derived HLA antibody detection.

Detection of HLA-specific memory B cells can provide additional information on sensitization of alloantigen-exposed individuals and refine immunological risk assessment. We have recently developed an assay enabling profiling of memory B cell-derived HLA antibodies using luminex single antigen bead (SAB) assay. Here, we compared the performance of the SAB kits from two vendors for memory B cell-derived HLA antibody detection. IgG was isolated from culture supernatants of polyclonally activated B cells from alloantigen-exposed (n = 7) or nonexposed (n = 5) individuals, using our previously established method. Eluates containing isolated IgG from culture supernatants were tested for the presence of HLA antibodies using luminex SAB analysis from both One Lambda and Lifecodes (Immucor). In contrast to Lifecodes, high mean fluorescence intensity (MFI) signals were found for negative control beads in One Lambda (median MFI for class I:1730 and for class II:728), accompanied by high MFI values for self HLA-coated beads, especially for HLA-C. Despite high background in the One Lambda assays, 91% concordance for HLA class I and 85% concordance for HLA class II were found between the specificities detected using SAB kits from the two vendors. Our results show that HLA-specific memory B cells can be profiled using kits from both vendors. However, when analyzing One Lambda results one should be aware of the restrictions related to nonspecific binding particularly in HLA-C-coated beads, and pay attention to self HLA-coated beads in order to accurately identify the reactivities leading to the definition of the actual HLA antibody specificities.

Low incidence of IgA isotype of HLA antibodies in alloantigen exposed individuals.

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 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.

Association of HLA phenotypes of end-stage renal disease patients preparing for first transplantation with anti-HLA antibody status.

Patients with pre-transplantation high levels of panel reactive antibody (PRA) have an increased risk of graft failure, and renal transplantation in sensitized patients remains a highly significant challenge worldwide. The influence of anti-human leukocyte antigen (HLA) antibodies on the development of rejection episodes depends on patient-specific clinical factors and differs from patient to patient. The HLA typing of the recipient might influence the development of anti-HLA antibodies. Some HLA antigens appear to be more immunogenic than others. The aim of this study is to demonstrate the distribution of HLA phenotypes in PRA-positive and PRA-negative end-stage renal disease (ESRD) patients on the basis of having sensitizing events or not. Our study included 642 (mean age: 41.54; female/male: 310/332) ESRD patients preparing for the first transplantation and who are on the cadaveric kidney transplantation waiting list of Istanbul Medical Faculty in 2008-2009. Class I HLA-A,B typing was performed by complement-dependent cytotoxicity (CDC) method, whereas class II HLA-DRB1 typing was performed by low-resolution polymerase chain reaction (PCR)-sequence-specific primer (SSP). All serum samples were screened for the presence of IgG type of anti-HLA class I- and II-specific antibodies by enzyme-linked-immunosorbent assay (ELISA). PRA-negative group consisted of 558 (86.9%) and PRA-positive group included 84 (13.1%) patients. We have found statistically significant frequency of HLA-A3 (p=0.018), HLA-A66 (p=0.04), and HLA-B18 (p=0.006) antigens in PRA-positive patients and DRB1*07 (p=0.02) having the highest frequency in patients with sensitizing event history but no anti-HLA development suggesting that DRB1*07 might be associated with low risk of anti-HLA antibody formation.

Pre-existing Alloreactive T and B Cells and Their Possible Relevance for Pre-transplant Risk Estimation in Kidney Transplant Recipients.

In allogeneic transplantation, genetic disparities between patient and donor may lead to cellular and humoral immune responses mediated by both naïve 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, and in vitro assays 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.

Donor-specific B Cell Memory in Alloimmunized Kidney Transplant Recipients: First Clinical Application of a Novel Method.

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-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-Mpos 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.

The genetic architecture of membranous nephropathy and its potential to improve non-invasive diagnosis.

Membranous Nephropathy (MN) is a rare autoimmune cause of kidney failure. Here we report a genome-wide association study (GWAS) for primary MN in 3,782 cases and 9,038 controls of East Asian and European ancestries. We discover two previously unreported loci, NFKB1 (rs230540, OR = 1.25, P = 3.4 × 10-12) and IRF4 (rs9405192, OR = 1.29, P = 1.4 × 10-14), fine-map the PLA2R1 locus (rs17831251, OR = 2.25, P = 4.7 × 10-103) and report ancestry-specific effects of three classical HLA alleles: DRB1*1501 in East Asians (OR = 3.81, P = 2.0 × 10-49), DQA1*0501 in Europeans (OR = 2.88, P = 5.7 × 10-93), and DRB1*0301 in both ethnicities (OR = 3.50, P = 9.2 × 10-23 and OR = 3.39, P = 5.2 × 10-82, respectively). GWAS loci explain 32% of disease risk in East Asians and 25% in Europeans, and correctly re-classify 20-37% of the cases in validation cohorts that are antibody-negative by the serum anti-PLA2R ELISA diagnostic test. Our findings highlight an unusual genetic architecture of MN, with four loci and their interactions accounting for nearly one-third of the disease risk.

Impact of HLA on the underlying primary diseases in Turkish patients with end-stage renal disease.

The number of patients with end stage renal disease (ESRD) is increasing faster than the number of renal transplantations performed per year worldwide. Of the primary diseases leading to ESRD, diabetic nephropathy is the leading cause. The purpose of the present study is to investigate the association of HLA with the primary diseases leading to ESRD in Turkish patients. A total of 3230 individuals comprising 587 ESRD patients and 2643 healthy controls were enrolled into the study. Class I HLA-A, -B typing was performed by CDC method, while class II HLA-DRB1 typing was performed by low resolution PCR-SSP. We found a significant negative association between almost all A locus antigens and primary disease groups classified as chronic glomerulonephritis and hypertensive nephrosclerosis (p < 0.05). HLA-B58 and HLA-DRB1*03 significantly correlated with amyloidosis and diabetic nephropathy, respectively. Determination of HLAs as risk factors for primary diseases leading to ESRD might be beneficial in preventing progression to ESRD and recurrence of the primary disease post-transplantation.

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

BACKGROUND: Pretransplant 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. Although 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. METHODS: Culture supernatants of polyclonally activated B cells from alloantigen exposed (n = 13) or nonexposed (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 mean fluorescence intensity values compared with 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.

Recombinant human monoclonal HLA antibodies of different IgG subclasses recognising the same epitope: Excellent tools to study differential effects of donor-specific antibodies.

In the field of transplantation, the humoural immune response against mismatched HLA antigens of the donor is associated with inferior graft survival, but not in every patient. Donor-specific HLA antibodies (DSA) of different immunoglobulin G (IgG) subclasses may have differential effects on the transplanted organ. Recombinant technology allows for the generation of IgG subclasses of a human monoclonal antibody (mAb), while retaining its epitope specificity. In order to enable studies on the biological function of IgG subclass HLA antibodies, we used recombinant technology to generate recombinant human HLA mAbs from established heterohybridomas. We generated all four IgG subclasses of a human HLA class I and class II mAb and showed that the different subclasses had a comparable affinity, normal human Fc glycosylation, and retained HLA epitope specificity. For both mAbs, the IgG1 and IgG3 isotypes were capable of binding complement component 3d (C3d) and efficient in complement-dependent cell lysis against their specific targets, while the IgG2 and IgG4 subclasses were not able to induce cytotoxicity. Considering the fact that the antibody-binding site and properties remained unaffected, these IgG subclass HLA mAbs are excellent tools to study the function of individual IgG subclass HLA class I and class II-specific antibodies in a controlled fashion.

No Evidence for Cross-reactivity of Virus-specific Antibodies With HLA Alloantigens.

BACKGROUND: Antibodies directed against HLA can develop through pregnancy, blood transfusions, or organ transplants. Anecdotal evidence suggests that virus-specific antibodies may have the capacity to cross-react with HLA, a phenomenon called heterologous immunity, which is well described for T-cell alloreactivity. METHODS: To determine whether antibody cross-reactivity between viral antigens and HLA is common, we tested 51 virus-specific human monoclonal antibodies (mAbs) specific for human immunodeficiency virus, varicella zoster virus, cytomegalovirus, and parvovirus, for reactivity against HLA class I and class II in single-antigen bead assays. In addition, we tested the reactivity of 41 HLA-specific human mAbs against common viral antigens of cytomegalovirus, varicella zoster virus, human immunodeficiency virus, Epstein-Barr virus, and BK polyomavirus. RESULTS: No cross-reactivity of any of the virus-specific mAbs with either HLA class I or class II molecules, as well as no cross-reactivity of any of the HLA-specific mAbs with any of the viral antigens was observed. CONCLUSIONS: These findings indicate that the frequency of cross-reactivity on the antibody level between viral antigens and HLA, if present at all, is low. The emergence of HLA antibodies upon viral infection or vaccination is therefore probably due to bystander activation of dormant HLA-specific memory B cells.