Homozygosity in any HLA locus is a risk factor for specific antibody production: the taboo concept 2.0.

Objective: In a cooperative study of the University Hospital Leipzig, University of Leipzig, and the Charité Berlin on kidney transplant patients, we analysed the occurrence of HLA-specific antibodies with respect to the HLA setup of the patients. We aimed at the definition of specific HLA antigens towards which the patients produced these antibodies. Methods: Patients were typed for the relevant HLA determinants using mainly the next-generation technology. Antibody screening was performed by the state-of-the-art multiplex-based technology using microspheres coupled with the respective HLA alleles of HLA class I and II determinants. Results: Patients homozygous for HLA-A*02, HLA-A*03, HLA-A*24, HLA-B*07, HLA-B*18, HLA-B*35, HLA-B*44, HLA-C*03, HLA-C*04, and HLA-C*07 in the class I group and HLA-DRB1*01, HLA-DRB1*03, HLA-DRB1*07, HLA-DRB1*15, HLA-DQA1*01, HLA-DQA1*05, HLA-DQB1*02, HLA-DQB1*03(7), HLA-DQB1*06, HLA-DPA1*01, and HLA-DPB1*04 in the class II group were found to have a significant higher antibody production compared to the heterozygous ones. In general, all HLA determinants are affected. Remarkably, HLA-A*24 homozygous patients can produce antibodies towards all HLA-A determinants, while HLA-B*18 homozygous ones make antibodies towards all HLA-B and selected HLA-A and C antigens, and are associated with an elevation of HLA-DRB1, parts of DQB1 and DPB1 alleles. Homozygosity for the HLA class II HLA-DRB1*01, and HLA-DRB1*15 seems to increase the risk for antibody responses against most of the HLA class I antigens (HLA-A, HLA-B, and HLA-C) in contrast to HLA-DQB1*03(7) where a lower risk towards few HLA-A and HLA-B alleles is found. The widely observed differential antibody response is therefore to be accounted to the patient's HLA type. Conclusion: Homozygous patients are at risk of producing HLA-specific antibodies hampering the outcome of transplantation. Including this information on the allocation procedure might reduce antibody-mediated immune reactivity and prevent graft loss in a patient at risk, increasing the life span of the transplanted organ.

A Short History of B-Cell HLA Epitopes.

Background: HLA epitopes are currently in the focus of transplantation immunogenetics. The main reason is the complexity of the HLA system with >38,000 alleles, the number of which increases steadily. These alleles are determined by the current state-of-the art typing methods like second- and third-generation sequencing. Screening for HLA antibodies is hampered by the lack of specific target beads with all possible alleles described. Summary: A way to circumvent the problem is to define HLA epitopes. The number of antibody-confirmed epitopes, on the other hand, was found to be 72 for HLA class I and 74 for HLA class II. Here, we elaborate on the current knowledge on these HLA epitopes. Absolute definitions of these structures are not yet available. Key Messages: Making use of eplets is a comparable way allowing statistical analyses. However, one should keep in mind that the results obtained are approximative or perhaps better associative. Continuous collaboration is needed for the full understanding of the HLA epitopes. The reactivity toward epitopes remains patient-specific.

Prognostic Role of Human Leukocyte Antigen Alleles and Cytokine Single-Nucleotide Polymorphisms in Patients with Chronic Myeloid Leukemia Treated with Tyrosine Kinase Inhibitor Drugs.

Tyrosine kinase inhibitor (TKI) drugs have significantly improved chronic myeloid leukemia (CML) outcomes. Neopeptides from CML cells may induce specific immune responses, which are crucial for deep molecular (DMR) and treatment-free remission (TFR). In this study of Ethiopian patients with CML (n = 162), the HLA alleles and single-nucleotide polymorphisms of five cytokines revealed significant associations with clinical outcomes. Clinically unfavorable outcomes correlated with HLA alleles A*03:01/02, A*23:17:01, B*57:01/02/03, and HLA-DRB4*01:01 (p-value = 0.0347, p-value = 0.0285, p-value = 0.037, and p-value = 0.0127, respectively), while HLA-DRB4*01:03:01 was associated with favorable outcomes (p-value = 0.0058). After assigning values for the 'low', 'intermediate', and 'high' gene expression of the SNPs' respective cytokine genes, Kaplan-Meier estimates for relapse-free survival, adjusted for age, treatment duration, and relapse risk among patients after the administration of TKIs, indicated that a gene expression ratio above the overall median of TNF-α, IL-6, and the combination of TGF-ß1/IL-10, IFNγ, and IL-6/IL-10 TGF-ß1 was correlated with a higher likelihood of treatment failure ((RR: 3.01; 95% CI: 1.1-8.3; p-value = 0.0261) and (RR: 2.4; 95% CI: 1.1-5.2; p-value = 0.022), respectively). Multi-SNPs, surpassing single-SNPs, and HLA allele polymorphisms showed promise in predicting outcomes of patients with CML during TKI treatment, prompting further exploration into their potential utility.

Genetic Predisposition to SARS-CoV-2 Infection: Cytokine Polymorphism and Disease Transmission within Households.

We addressed the question of the influence of the molecular polymorphism of cytokines from different T helper subsets on the susceptibility to SARS-CoV-2 infection. From a cohort of 527 samples (collected from 26 May 2020 to 31 March 2022), we focused on individuals living in the same household (n = 58) with the SARS-CoV-2-infected person. We divided them into households with all individuals SARS-CoV-2 PCR positive (n = 29, households, 61 individuals), households with mixed PCR pattern (n = 24, 62) and negative households (n = 5, 15), respectively. TGF-ß1 and IL-6 were the only cytokines tested with a significant difference between the cohorts. We observed a shift toward Th2 and the regulatory Th17 and Treg subset regulation for households with all members infected compared to those without infection. These data indicate that the genetically determined balance between the cytokines acting on different T helper cell subsets may play a pivotal role in transmission of and susceptibility to SARS-CoV-2 infection. Contacts infected by their index persons were more likely to highly express TGF-ß1, indicating a reduced inflammatory response. Those not infected after contact had a polymorphism leading to a higher IL-6 expression. IL-6 acts in innate immunity, allergy and on the T helper cell differentiation, explaining the reduced susceptibility to SARS-CoV-2.

Individual Immune Response to SARS-CoV-2 Infection-The Role of Seasonal Coronaviruses and Human Leukocyte Antigen.

During the coronavirus pandemic, evidence is growing that the severity, susceptibility and host immune response to SARS-CoV-2 infection can be highly variable. Several influencing factors have been discussed. Here, we investigated the humoral immune response against SARS-CoV-2 spike, S1, S2, the RBD, nucleocapsid moieties and S1 of seasonal coronaviruses: hCoV-229E, hCoV-HKU1, hCoV-NL63 and hCoV-OC43, as well as MERS-CoV and SARS-CoV, in a cohort of 512 individuals. A bead-based multiplex assay allowed simultaneous testing for all the above antigens and the identification of different antibody patterns. Then, we correlated these patterns with 11 HLA loci. Regarding the seasonal coronaviruses, we found a moderate negative correlation between antibody levels against hCoV-229E, hCoV-HKU1 and hCoV-NL63 and the SARS-CoV-2 antigens. This could be an indication of the original immunological imprinting. High and low antibody response patterns were distinguishable, demonstrating the individuality of the humoral response towards the virus. An immunogenetical factor associated with a high antibody response (formation of ≥4 different antibodies) was the presence of HLA A*26:01, C*02:02 and DPB1*04:01 alleles, whereas the HLA alleles DRB3*01:01, DPB1*03:01 and DB1*10:01 were enriched in low responders. A better understanding of this variable immune response could enable more individualized protective measures.

Extended genomic HLA typing identifies previously unrecognized mismatches in living kidney transplantation.

Introduction: Antibody mediated rejection (ABMR) is the most common cause of long-term allograft loss in kidney transplantation (KT). Therefore, a low human leukocyte antigen (HLA) mismatch (MM) load is favorable for KT outcomes. Hitherto, serological or low-resolution molecular HLA typing have been adapted in parallel. Here, we aimed to identify previously missed HLA mismatches and corresponding antibodies by high resolution HLA genotyping in a living-donor KT cohort. Methods: 103 donor/recipient pairs transplanted at the University of Leipzig Medical Center between 1998 and 2018 were re-typed using next generation sequencing (NGS) of the HLA loci -A, -B, -C, -DRB1, -DRB345, -DQA1, -DQB1, -DPA1, and -DPB1. Based on these data, we compiled HLA MM counts for each pair and comparatively evaluated genomic HLA-typing with pre-transplant obtained serological/low-resolution HLA (=one-field) typing results. NGS HLA typing (=two-field) data was further used for reclassification of de novo HLA antibodies as "donor-specific". Results: By two-field HLA re-typing, we were able to identify additional MM in 64.1% (n=66) of cases for HLA loci -A, -B, -C, -DRB1 and -DQB1 that were not observed by one-field HLA typing. In patients with biopsy proven ABMR, two-field calculated MM count was significantly higher than by one-field HLA typing. For additional typed HLA loci -DRB345, -DQA1, -DPA1, and -DPB1 we observed 2, 26, 3, and 23 MM, respectively. In total, 37.3% (69/185) of de novo donor specific antibodies (DSA) formation was directed against these loci (DRB345 ➔ n=33, DQA1 ➔ n=33, DPA1 ➔ n=1, DPB1 ➔ n=10). Conclusion: Our results indicate that two-field HLA typing is feasible and provides significantly more sensitive HLA MM recognition in living-donor KT. Furthermore, accurate HLA typing plays an important role in graft management as it can improve discrimination between donor and non-donor HLA directed cellular and humoral alloreactivity in the long range. The inclusion of additional HLA loci against which antibodies can be readily detected, HLA-DRB345, -DQA1, -DQB1, -DPA1, and -DPB1, will allow a more precise virtual crossmatch and better prediction of potential DSA. Furthermore, in living KT, two-field HLA typing could contribute to the selection of the immunologically most suitable donors.

Outcome differences in HPV-driven head and neck squamous cell carcinoma attributable to altered human leukocyte antigen frequencies.

Background: Effective immune surveillance requires a functioning immune system and natural killer (NK) and T cells for adequate innate and antigen-specific immune responses critically depending on human leukocyte antigens (HLAs) and haplotypes representing advantageous combinations of HLA antigens. Recently, we reported a link between altered frequencies of HLA alleles and haplotypes and developing head and neck squamous cell carcinoma (HNSCC). Whereas the majority of HNSCCs seem to be related to classical risk factors alcohol and tobacco, a subset of HNSCC and especially oropharyngeal squamous cell carcinoma (OPSCC) were etiologically linked to human papillomavirus (HPV) recently. Here, we demonstrate in HPV-driven (p16-positive high risk-HPV DNA-positive) HNSCC a deviating distribution of HLA antigens and haplotypes and their relevance to outcome. Methods: Leukocyte DNA of n = 94 HPV-driven HNSCC patients (n = 57 OPSCC, n = 37 outside oropharynx) underwent HLA SSO typing, allowing allele, antigen (allele group), and haplo-typing. Besides comparing these frequencies with those of German blood donors, we analyzed their impact on outcome using Kaplan-Meier plots and Cox proportional hazard regression. Results: Antigen and haplotype frequencies demonstrate enrichment of rare antigens and haplotypes. The HLA score for unselected HNSCC patients was not predictive for outcome here. However, together with alcohol consumption, tobacco smoking, T category, and extranodal extension of locoregional metastases and treatment applied, eight HLA traits allow for predicting progression-free and tumor-specific survival. Conclusion: Patients can be categorized into low, intermediate-low, intermediate-high, and high risk groups. Using a new PFS risk score for HPV-driven HNSCC may allow to improve prognostication.

Immunogenetic Predisposition to SARS-CoV-2 Infection.

Herein, we included 527 individuals from two Hospitals, Chemnitz and University-Hospital Leipzig. In total, 199 were negative for PCR and 328 were positive upon first admission. We used next generation sequencing for HLA-A, B, C, DRB1, DRB345, DQA1, DQB1, DPA1, and DPB1, and in some cases, HLA-E, F, G, and H. Furthermore, we molecularly defined 22 blood group systems comprising 26 genes and 5 platelet antigen genes. We observed a significant enrichment of homozygosity for DQA/DQB in the positive group. Within the negative subjects, HLA-B*57:01, HLA-B*55:01, DRB1*13:01, and DRB1*01:01 were enriched, and in the positive group, homozygosity for DQA/DQB, DRB1*09:01, and DRB1*15:01 was observed. DQA1*01:01, DQA1*02:01, and DQA1*01:03 were enriched in the negative group. HLA-DQB1*06:02 was enriched in the positive group, and HLA-DQB1*05:01 and HLA-DQB1*06:03 were enriched in the negative group. For the blood group systems MNS, RH, LE, FY, JK, YT, DO, and KN, enrichment was seen in both groups, depending on the antigen under observation. Homozygosity for D-positive RHD alleles, as well as the phenotypes M-N+ of the MNS blood group system and Yk(a-) of the KN system, were enriched in the positive group. All of these significances disappeared upon correction. Subjects who carried homozygous HPA-1a were more frequent in the negative group, contrasting with the finding that HPA-1ab was enriched in the positive group.

Hidden Patterns of Anti-HLA Class I Alloreactivity Revealed Through Machine Learning.

Detection of alloreactive anti-HLA antibodies is a frequent and mandatory test before and after organ transplantation to determine the antigenic targets of the antibodies. Nowadays, this test involves the measurement of fluorescent signals generated through antibody-antigen reactions on multi-beads flow cytometers. In this study, in a cohort of 1,066 patients from one country, anti-HLA class I responses were analyzed on a panel of 98 different antigens. Knowing that the immune system responds typically to "shared" antigenic targets, we studied the clustering patterns of antibody responses against HLA class I antigens without any a priori hypothesis, applying two unsupervised machine learning approaches. At first, the principal component analysis (PCA) projections of intra-locus specific responses showed that anti-HLA-A and anti-HLA-C were the most distantly projected responses in the population with the anti-HLA-B responses to be projected between them. When PCA was applied on the responses against antigens belonging to a single locus, some already known groupings were confirmed while several new cross-reactive patterns of alloreactivity were detected. Anti-HLA-A responses projected through PCA suggested that three cross-reactive groups accounted for about 70% of the variance observed in the population, while anti-HLA-B responses were mainly characterized by a distinction between previously described Bw4 and Bw6 cross-reactive groups followed by several yet undocumented or poorly described ones. Furthermore, anti-HLA-C responses could be explained by two major cross-reactive groups completely overlapping with previously described C1 and C2 allelic groups. A second feature-based analysis of all antigenic specificities, projected as a dendrogram, generated a robust measure of allelic antigenic distances depicting bead-array defined cross reactive groups. Finally, amino acid combinations explaining major population specific cross-reactive groups were described. The interpretation of the results was based on the current knowledge of the antigenic targets of the antibodies as they have been characterized either experimentally or computationally and appear at the HLA epitope registry.

Patterns of 1,748 Unique Human Alloimmune Responses Seen by Simple Machine Learning Algorithms.

Allele specific antibody response against the polymorphic system of HLA is the allogeneic response marker determining the immunological risk for graft acceptance before and after organ transplantation and therefore routinely studied during the patient's workup. Experimentally, bead bound antigen- antibody reactions are detected using a special multicolor flow cytometer (Luminex). Routinely for each sample, antibody responses against 96 different HLA antigen groups are measured simultaneously and a 96-dimensional immune response vector is created. Under a common experimental protocol, using unsupervised clustering algorithms, we analyzed these immune intensity vectors of anti HLA class II responses from a dataset of 1,748 patients before or after renal transplantation residing in a single country. Each patient contributes only one serum sample in the analysis. A population view of linear correlations of hierarchically ordered fluorescence intensities reveals patterns in human immune responses with striking similarities with the previously described CREGs but also brings new information on the antigenic properties of class II HLA molecules. The same analysis affirms that "public" anti-DP antigenic responses are not correlated to anti DR and anti DQ responses which tend to cluster together. Principal Component Analysis (PCA) projections also demonstrate ordering patterns clearly differentiating anti DP responses from anti DR and DQ on several orthogonal planes. We conclude that a computer vision of human alloresponse by use of several dimensionality reduction algorithms rediscovers proven patterns of immune reactivity without any a priori assumption and might prove helpful for a more accurate definition of public immunogenic antigenic structures of HLA molecules. Furthermore, the use of Eigen decomposition on the Immune Response generates new hypotheses that may guide the design of more effective patient monitoring tests.

HLA-DPB1*03 as Risk Allele and HLA-DPB1*04 as Protective Allele for Both Early- and Adult-Onset Multiple Sclerosis in a Hellenic Cohort.

Background: Human Leucocyte Antigens (HLA) represent the genetic loci most strongly linked to Multiple Sclerosis (MS). Apart from HLA-DR and HLA-DQ, HLA-DP alleles have been previously studied regarding their role in MS pathogenesis, but to a much lesser extent. Our objective was to investigate the risk/resistance influence of HLA-DPB1 alleles in Hellenic patients with early- and adult-onset MS (EOMS/AOMS), and possible associations with the HLA-DRB1*15:01 risk allele. Methods: One hundred MS-patients (28 EOMS, 72 AOMS) fulfilling the McDonald-2010 criteria were enrolled. HLA genotyping was performed with standard low-resolution Sequence-Specific Oligonucleotide techniques. Demographics, clinical and laboratory data were statistically processed using well-defined parametric and nonparametric methods and the SPSSv22.0 software. Results: No significant HLA-DPB1 differences were found between EOMS and AOMS patients for 23 distinct HLA-DPB1 and 12 HLA-DRB1 alleles. The HLA-DPB1*03 allele frequency was found to be significantly increased, and the HLA-DPB1*02 allele frequency significantly decreased, in AOMS patients compared to controls. The HLA-DPB1*04 allele was to be found significantly decreased in AOMS and EOMS patients compared to controls. Conclusions: Our study supports the previously reported risk susceptibility role of the HLA-DPB1*03 allele in AOMS among Caucasians. Additionally, we report for the first time a protective role of the HLA-DPB1*04 allele among Hellenic patients with both EOMS and AOMS.

HLA Class II Allele Analyses Implicate Common Genetic Components in Type 1 and Non-Insulin-Treated Type 2 Diabetes.

CONTEXT: Common genetic susceptibility may underlie the frequently observed co-occurrence of type 1 and type 2 diabetes in families. Given the role of HLA class II genes in the pathophysiology of type 1 diabetes, the aim of the present study was to test the association of high density imputed human leukocyte antigen (HLA) genotypes with type 2 diabetes. OBJECTIVES AND DESIGN: Three cohorts (Ntotal = 10 413) from Leipzig, Germany were included in this study: LIFE-Adult (N = 4649), LIFE-Heart (N = 4815) and the Sorbs (N = 949) cohort. Detailed metabolic phenotyping and genome-wide single nucleotide polymorphism (SNP) data were available for all subjects. Using 1000 Genome imputation data, HLA genotypes were imputed on 4-digit level and association tests for type 2 diabetes, and related metabolic traits were conducted. RESULTS: In a meta-analysis including all 3 cohorts, the absence of HLA-DRB5 was associated with increased risk of type 2 diabetes (P = 0.001). In contrast, HLA-DQB*06:02 and HLA-DQA*01:02 had a protective effect on type 2 diabetes (P = 0.005 and 0.003, respectively). Both alleles are part of the well-established type 1 diabetes protective haplotype DRB1*15:01~DQA1*01:02~DQB1*06:02, which was also associated with reduced risk of type 2 diabetes (OR 0.84; P = 0.005). On the contrary, the DRB1*07:01~DQA1*02:01~DQB1*03:03 was identified as a risk haplotype in non-insulin-treated diabetes (OR 1.37; P = 0.002). CONCLUSIONS: Genetic variation in the HLA class II locus exerts risk and protective effects on non-insulin-treated type 2 diabetes. Our data suggest that the genetic architecture of type 1 diabetes and type 2 diabetes might share common components on the HLA class II locus.

The long and winding road towards epitope matching in clinical transplantation.

Recent data suggest that HLA epitope matching is beneficial for the prevention of de novo donor specific antibody (DSA) formation after transplantation. In this review, different approaches to predict the immunogenicity of an HLA mismatch will be discussed. The parameters used in these models are often called epitopes but the actual antibody epitope is far more complex. Exact knowledge of the antibody epitope is crucial if epitope matching is also used as a tool to select compatible donors for (highly) sensitized patients. Evidence is provided that it is not always possible to give an exact definition of an antibody epitope. We conclude that HLA "epitope" matching is superior over HLA antigen matching with respect to the prevention of de novo DSA formation and will enhance the prediction of acceptable HLA mismatches for sensitized patients. However, epitope matching at our current level of knowledge will not solve all histocompatibility problems as unexpected antibody reactivity still may occur.

Fibroblast-adapted human CMV vaccines elicit predominantly conventional CD8 T cell responses in humans.

Cytomegalovirus (CMV)-based vaccines have shown remarkable efficacy in the rhesus macaque model of acquired immune deficiency syndrome, enabling 50% of vaccinated monkeys to clear a subsequent virulent simian immunodeficiency virus challenge. The protective vaccine elicited unconventional CD8 T cell responses that were entirely restricted by MHC II or the nonclassical MHC I molecule, MHC-E. These unconventional responses were only elicited by a fibroblast-adapted rhesus CMV vector with limited tissue tropism; a repaired vector with normal tropism elicited conventional responses. Testing whether these unusual protective CD8 T responses could be elicited in humans requires vaccinating human subjects with a fibroblast-adapted mutant of human CMV (HCMV). In this study, we describe the CD8 T cell responses of human subjects vaccinated with two fibroblast-adapted HCMV vaccines. Most responses were identified as conventional classically MHC I restricted, and we found no evidence for MHC II or HLA-E restriction. These results indicate that fibroblast adaptation alone is unlikely to explain the unconventional responses observed in macaques.

Association of high HLA-E expression during acute cellular rejection and numbers of HLA class I leader peptide mismatches with reduced renal allograft survival.

Non-classical Human Leukocyte Antigen (HLA)-E preferentially presents leader peptides derived from classical HLA-class I molecules. HLA-E can trigger opposed immune responses by interacting with inhibitory NKG2A or by activating NKG2C receptors on NK and T-cells. We studied the impact of HLA-E on renal allograft survival during acute cellular rejection. HLA-E expression was up-regulated in acute cellular rejection (ACR) biopsies (n=12) compared to biopsies from 13 renal allografts with no rejection-signs. HLA-E up-regulation was correlated with numbers of HLA-class I leader peptide mismatches (p=0.04). CD8+ and CD56+ infiltrating cells correlated with HLA-E expression (p<0.0001 and p=0.0009, respectively). Activating NKG2C receptor dominated on effector cells in biopsies and peripheral blood during ACR potentially allowing HLA-E-mediated immune activation. Moreover, HLA-E expression correlated with deterioration in renal allograft function (p<0.008) and reduced allograft survival (p=0.002). Our findings provide evidence that during renal allograft rejection HLA-E along with high numbers of mismatched HLA-class I leader peptides might represent additional targets for immune-activating responses.

The HLA-DRB1*15 phenotype is associated with multiple red blood cell and HLA antibody responsiveness.

BACKGROUND: Once a patient has produced a red blood cell (RBC) antibody, there is an increased risk of additional antibody formation after subsequent RBC exposure. Recently, we observed that HLA-DRB1*15 was overrepresented in 379 multiple RBC antibody responders compared to controls or 562 patients with a single RBC antibody (odds ratio [OR], 1.7; 95% confidence interval [CI], 1.3-2.3). In this study we evaluated whether the HLA-DRB1*15 represents a responder phenotype against HLA and/or RBC antigens. STUDY DESIGN AND METHODS: HLA-DRB1*15 frequencies in single and multiple antibody responders were compared between three groups of individuals: 1) those with HLA antibodies, 2) those with RBC antibodies, and 3) those with both RBC and HLA antibodies. RESULTS: A total of 3959 immunized patients (female-to-male ratio, 2.3) had been HLA-DRB1 typed. Among the 3275 individuals with HLA antibodies, the frequency of the DRB1*15 phenotype differed significantly from 19.7% in patients with a panel reactivity (PRA) of not more than 20% to 26.9% in patients with PRA of more than 80% (OR, 1.5; 95% CI, 1.2-1.9). This association between DRB1*15 and multiresponsiveness was mainly due to pregnancy-induced HLA immunization. In the 257 individuals with RBC and HLA antibodies, the frequency of DRB1*15 was 4.2 times (95% CI, 1.1-16) higher in those with multiple RBC antibodies and HLA-PRA of more than 50% compared to only single RBC responders with PRA of less than 20%. CONCLUSION: The HLA-DRB1*15 phenotype is associated with broad RBC and HLA immunization.

Factors associated with persistence of red blood cell antibodies in woman after pregnancies complicated by fetal alloimmune haemolytic disease treated with intrauterine transfusions.

Red blood cell (RBC) antibodies can persist for decades or decrease quickly to undetectable levels. Antibody persistence has not been systematically studied. Women whose children are treated with intrauterine transfusions (IUT) for haemolytic disease of the fetus (HDFN) often produce additional antibodies, which can be evoked by the intrauterine transfusion or by fetomaternal haemorrhage during the procedure. Factors associated with persistence of both the antibodies responsible for HDFN and additional antibodies were studied in 260 women whose children were treated with IUT between 1988 and 2008. They possessed 499 (205 anti-D and 294 non-D) antibodies after the last IUT. After a median follow-up of 8·7 years, all 260 antibodies primarily responsible for HDFN had persisted. Additional antibodies directed against antigens of the children persisted in 70·6%, and in 32·3% if they were not child-specific (P < 0·001). Antibodies induced by irradiated IUT persisted in only 7·1%. Multivariate analyses showed that non-HDFN antibody persistence was dependent on the antibody titre and specificity. In conclusion, persistence of antibodies mainly depends on antibody strength and specificity. Difference between fetal or non-fetal immunogens suggests maintenance of antigenic stimulation possibly by long-term fetomaternal chimerism.

HLA monomers as a tool to monitor indirect allorecognition.

BACKGROUND: Recognition of donor antigens can occur through two separate pathways: the direct pathway (non-self HLA on donor cells) and the indirect pathway (self-restricted presentation of donor derived peptides on recipient cells). Indirect allorecognition is important in the development of humoral rejection; therefore, there is an increasing interest in the monitoring of indirect alloreactive T-cells. We have used an in vitro model to determine the optimal requirements for indirect presentation and assessed the risk for semidirect presentation in this system. METHODS: HLA-typed monocyte-derived dendritic cells (moDCs) were incubated with cellular fragments or necrotic cells and incubated with either indirect or direct alloreactive T-cell clones. T-cell reactivity was measured through proliferation or cytokine secretion. HLA-typed moDC, monocytes, or PBMCs were incubated with HLA class I monomers, in combination with either direct/indirect T-cell clones. RESULTS: Although both were efficiently taken up, alloreactivity was limited to the semi-direct pathway, as measured by allospecific CD4 (indirect) and CD8 T-cell clones (direct) when cells were used. In contrast, HLA-A2 monomers were not only efficiently taken up but also processed and presented by HLA-typed moDC, monocytes, and PBMCs. Activation was shown by a dose-dependent induction of IFN-γ production and proliferation by the CD4 T-cell clone. Antigen presentation was most efficient when the monomers were cultured for longer periods (24-48 hr) in the presence of the T-cells. Using this method, no reactivity was observed by the CD8 T-cell clone, confirming no semidirect alloreactivity. CONCLUSION: We have developed a system that could be used to monitor indirect alloreactive T-cells.