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-DRB1 associations in individuals with single and multiple clinically relevant red blood cell antibodies.

BACKGROUND: A minority of red blood cell (RBC) alloantigen-exposed persons form antibodies. Responders are at high risk of developing additional antibodies upon subsequent transfusions. Several studies showed an association between particular HLA-DRB1 phenotypes and the development of specific RBC antibodies. This study evaluates the presence of HLA-DRB1 antigens in individuals with single or multiple RBC antibody specificities to explore whether the response against RBC antigens is associated with a summation of particular HLA-DRB1 susceptibility antigens. STUDY DESIGN AND METHODS: Frequencies of HLA-DRB1 alleles in individuals with antibodies against clinically relevant antigens were compared to a large population cohort to calculate odds ratios (ORs) for alloimmunization to different RBC antigens. RESULTS: The study cohort consisted of 941 individuals (female-to-male ratio, 3.8) possessing 1462 antibody specificities elicited by transfusion, pregnancy, transplantation, or a combination of these. Besides confirmation of known associations, new associations were identified for anti-E with DRB1*09 and for anti-S with DRB1*07 (ORs, 3.7 and 8.7, respectively). Multiple antibody formation was in a minority of cases associated with the presence of multiple DRB1 susceptibility genes. In multiple responders DRB1*15 was present in almost 40% of cases compared to approximately 25% in single-antibody responders and in the control population. CONCLUSION: This study suggests that HLA-DRB1 restriction plays an important role for a first RBC antibody response but multiple antibody formation seems less dependent on the presence of particular HLA restriction genes, while HLA-DRB1*15 may represent a susceptibility phenotype enhancing formation of multiple RBC antibody specificities.

Donor-specific HLA antibodies: evaluating the risk for graft loss in renal transplant recipients with isotype switch from complement fixing IgG1/IgG3 to noncomplement fixing IgG2/IgG4 anti-HLA alloantibodies.

Human leukocyte antigen alloantibodies have a multitude of damaging effects on the allograft, both complement (C') activation and Fc-independent ones. To date, the clinical significance of non-C' fixing (NCF) HLA donor-specific antibodies (DSA) is still unclear. In this study, we investigated whether renal transplant recipients with NCF-DSA subclasses (IgG2/IgG4, IgA1/IgA2) are at higher risk of graft loss compared to patients with exclusively C' fixing (IgG1/IgG3). Blood samples from 274 patients were analyzed for HLA IgG and IgA subclasses using a modified single-antigen bead assay. We identified 50 (18.2%) patients with circulating NCF antibodies either DSA (n=17) or against third-party HLA (n=33). NCF-DSAs were preferentially of IgG2/IgG4 isotype (11/17) and were mainly directed against HLA class II (13/17). NCF DSA were present as a mixture with strong C' fixing IgG1/IgG3. Graft survival was similar between patients with exclusively C' fixing antibodies and those with a mixture panel (log rang test P=0.162), and also among patients with different immunoglobulin isotype and subclasses (long-rank test, P=0.732). We conclude that expansion of DSA to NCF subclasses postrenal transplantation does not seem to be associated with worse graft survival as compared to the presence of exclusive C' fixing subclasses.

Clinical evaluation of the endothelial tie-2 crossmatch in ABO compatible and ABO incompatible renal transplants.

The necessity of detection of other than the classical major histocompatibility complex (MHC) and MHC class I-related chain A (MICA) directed antibodies prior to organ transplantation has already been repeatedly reported. A commercial flow cytometric endothelial crossmatch (CM) using isolated peripheral blood tie-2 positive cells provides a tool to detect non-MHC antibodies in addition to antibodies directed to MHC class I and II. The vast majority of circulating tie-2 positive cells expresses HLA-DR but not the A, B blood group antigens. Tie-2 cells are circulating surrogate endothelial cells. In this retrospective study we evaluated the endothelial CM in 51 renal transplantations, 30 with ABO compatible grafts and 21 with ABO incompatible grafts. Fifteen of the ABO compatible recipients (group A) developed unexplained rejection episodes (RE) while the remaining 15 had no RE (group B). Five cases of group A and none of group B had a positive tie-2 CM before transplantation (p=0.042). A positive tie-2 CM was also correlated with graft failure in ABO compatible transplants (p=0.02). No significant correlation was found between a positive pre-transplant tie-2 CM and RE in the ABO incompatible group. This study strongly suggest that a positive tie-2 CM may predict post-transplantation complications in ABO compatible grafts while negative reactions are not predictive. The test is not significantly correlated with RE in ABO incompatible grafts possibly due to applied desensitization.

Consensus guidelines on the testing and clinical management issues associated with HLA and non-HLA antibodies in transplantation.

BACKGROUND: The introduction of solid-phase immunoassay (SPI) technology for the detection and characterization of human leukocyte antigen (HLA) antibodies in transplantation while providing greater sensitivity than was obtainable by complement-dependent lymphocytotoxicity (CDC) assays has resulted in a new paradigm with respect to the interpretation of donor-specific antibodies (DSA). Although the SPI assay performed on the Luminex instrument (hereafter referred to as the Luminex assay), in particular, has permitted the detection of antibodies not detectable by CDC, the clinical significance of these antibodies is incompletely understood. Nevertheless, the detection of these antibodies has led to changes in the clinical management of sensitized patients. In addition, SPI testing raises technical issues that require resolution and careful consideration when interpreting antibody results. METHODS: With this background, The Transplantation Society convened a group of laboratory and clinical experts in the field of transplantation to prepare a consensus report and make recommendations on the use of this new technology based on both published evidence and expert opinion. Three working groups were formed to address (a) the technical issues with respect to the use of this technology, (b) the interpretation of pretransplantation antibody testing in the context of various clinical settings and organ transplant types (kidney, heart, lung, liver, pancreas, intestinal, and islet cells), and (c) the application of antibody testing in the posttransplantation setting. The three groups were established in November 2011 and convened for a "Consensus Conference on Antibodies in Transplantation" in Rome, Italy, in May 2012. The deliberations of the three groups meeting independently and then together are the bases for this report. RESULTS: A comprehensive list of recommendations was prepared by each group. A summary of the key recommendations follows. Technical Group: (a) SPI must be used for the detection of pretransplantation HLA antibodies in solid organ transplant recipients and, in particular, the use of the single-antigen bead assay to detect antibodies to HLA loci, such as Cw, DQA, DPA, and DPB, which are not readily detected by other methods. (b) The use of SPI for antibody detection should be supplemented with cell-based assays to examine the correlations between the two types of assays and to establish the likelihood of a positive crossmatch (XM). (c) There must be an awareness of the technical factors that can influence the results and their clinical interpretation when using the Luminex bead technology, such as variation in antigen density and the presence of denatured antigen on the beads. Pretransplantation Group: (a) Risk categories should be established based on the antibody and the XM results obtained. (b) DSA detected by CDC and a positive XM should be avoided due to their strong association with antibody-mediated rejection and graft loss. (c) A renal transplantation can be performed in the absence of a prospective XM if single-antigen bead screening for antibodies to all class I and II HLA loci is negative. This decision, however, needs to be taken in agreement with local clinical programs and the relevant regulatory bodies. (d) The presence of DSA HLA antibodies should be avoided in heart and lung transplantation and considered a risk factor for liver, intestinal, and islet cell transplantation. Posttransplantation Group: (a) High-risk patients (i.e., desensitized or DSA positive/XM negative) should be monitored by measurement of DSA and protocol biopsies in the first 3 months after transplantation. (b) Intermediate-risk patients (history of DSA but currently negative) should be monitored for DSA within the first month. If DSA is present, a biopsy should be performed. (c) Low-risk patients (nonsensitized first transplantation) should be screened for DSA at least once 3 to 12 months after transplantation. If DSA is detected, a biopsy should be performed. In all three categories, the recommendations for subsequent treatment are based on the biopsy results. CONCLUSIONS: A comprehensive list of recommendations is provided covering the technical and pretransplantation and posttransplantation monitoring of HLA antibodies in solid organ transplantation. The recommendations are intended to provide state-of-the-art guidance in the use and clinical application of recently developed methods for HLA antibody detection when used in conjunction with traditional methods.

High anti-HLA response in women exposed to intrauterine transfusions for severe alloimmune hemolytic disease is associated with mother-child HLA triplet mismatches, high anti-D titer, and new red blood cell antibody formation.

BACKGROUND: Women whose fetuses were treated with intrauterine transfusions (IUTs) for alloimmune hemolytic disease are high responders to red blood cell (RBC) antigens. We investigated the risk for HLA alloimmunization. STUDY DESIGN AND METHODS: Women and their children treated with IUT between 1987 and 2008 were included. Participants were HLA antigen typed and studied for the prevalence of HLA antibodies compared to age-matched parous nontransfused blood donors. Anti-D titer, the formation of new RBC antibodies after IUT, and the degree of fetomaternal HLA mismatches on HLA antibody formation and/or persistence were analyzed. RESULTS: A higher prevalence of HLA Class I antibodies was observed in these women compared to controls (41% vs. 23%). Both a higher anti-D titer (>8000) and formation of new RBC antibodies after IUT were associated with increased HLA immunization. HLA antibody formation was associated with the number of fetomaternal triplet epitope mismatches. Antigens within HLA-Bw4, HLA-B35/51/52/53/18/78-complex and A1/A9, were higher and mismatches within HLA-C were less immunogenic than expected. HLA antibodies against the IUT-treated fetus were more persistent than other antibodies. CONCLUSION: Women whose fetuses were treated with IUT had a high risk of developing and maintain fetal-specific HLA Class I antibodies. Factors associated with increased HLA immunization were a higher amount of fetomaternal HLA triplet mismatches, higher anti-D titer, and additional RBC antibody formation. We presume that the induction of HLA Class I antibodies is the result of increased fetomaternal hemorrhage during IUT, eliciting antibodies in women with an increased susceptibility to alloimmunization.

Stimulation of human EBV- and CMV-specific cytolytic effector function using allogeneic HLA molecules.

Viral infection is a major cause of morbidity and mortality, and there are few therapeutic options available to augment a virus-specific T cell response. Although allo-HLA cross-reactivity from virus-specific memory T cells is common, it is unclear whether priming with specific allogeneic cells could conversely elicit a viral peptide/self-HLA restricted cytotoxic T cell response in humans. First, we used the previously described allo-HLA-B*44:02 cross-reactivity of EBV peptide/HLA-B8 restricted T cells, to determine whether allogeneic HLA stimulation can elicit a cytolytic immune response against EBV. HLA-B8(+) HLA-B44(-) EBV-seropositive PBMCs were stimulated with either HLA-B*44:02(+) or HLA-B*44:03(+) mismatched irradiated PBMCs in a 7-10 d MLR. The allo-HLA stimulated responder cells were then evaluated for cytotoxicity using EBV peptide loaded autologous target cells and unloaded HLA-B8(+) EBV LCL target cells. PBMCs from EBV-seropositive donors gained EBV-specific cytolytic effector function following specific allo-HLA stimulation. Finally, we also elicited cytolytic CMV-specific responses using specific allogeneic cell stimulation, to confirm that this technique can be used to elicit viral peptide/self-HLA restricted responses even from nonpublic TCR responses. Allogeneic cell stimulation used as a cell therapy may be a potential tool to augment an antiviral T cell response in patients with EBV or CMV infection.

The functional polymorphism Ala258Ser in the innate receptor gene ficolin-2 in the donor predicts improved renal transplant outcome.

BACKGROUND: Innate immunity plays a role in controlling adaptive immune responses. METHODS: We investigated the clinical relevance of single nucleotide polymorphisms in 22 genes encoding innate, secreted, and signaling pattern recognition receptors in a total of 520 donor-recipient pairs of postmortem, human leukocyte antigen-DR-compatible kidney transplantations. Associations with rejection incidence were tested in an a priori randomized training set and validation set. RESULTS: Polymorphisms in TLR-3 (rs3775296) in the recipients and in ficolin-2 (rs7851696; Ala258Ser) and C1qR1 (rs7492) in the donors showed the strongest association with severe rejection. In multivariate analysis, presence of the ficolin-2 Ala258Ser variant in the donor predicted lower incidence of severe rejection (odds ratio=0.3; 95% confidence interval, 0.1-0.9; P=0.024) and of graft loss (hazard ratio=0.5; 95% confidence interval, 0.2-1.0; P=0.046) independently of clinical risk factors. Ficolin-2 messenger RNA expression was detected in pretransplantation biopsies from 69 donor grafts. Serum and tissue ficolin-2 levels were unaffected by genotype. Ficolin-2 protein, which bound to dying cells, was detected in donor kidneys in a passenger leukocyte-like pattern. Indeed, monocytes, monocyte-derived macrophages, and peripheral blood mononuclear cells expressed ficolin-2. Donor grafts with the ficolin-2 Ala258Ser variant contained significantly elevated expression of interleukin 6, having ascribed cytoprotective effects. It has been described that Ala258Ser leads to increased binding capacity of ficolin-2 to N-acetylglucosamine. CONCLUSIONS: Presence of the ficolin-2 Ala258Ser polymorphism in the donor independently predicts improved graft outcome. Based on mechanistic data, we propose that this functional polymorphism leads to more efficient handling of injured cells by phagocytozing cells, resulting in decreased intragraft exposure to danger signals and dampened alloimmune responses.

Detection of allo-HLA cross-reactivity by virus-specific memory T-cell clones using single HLA-transfected K562 cells.

The ability to directly measure virus-specific lymphocytes using fluorochrome-labeled tetrameric complexes has proven a great advancement for the transplantation field. Viral peptide/HLA tetrameric complexes allow the rapid generation of virus-specific clones using single cell sorting apparatus, permitting the determination of alloreactivity from a single TCR with known specificity. When combined with new target "detector" cells called single HLA antigen-transfected K562 cells (SALs), the human alloresponse can for the first time be examined specifically and reliably. Here we describe a method for detection of "heterologous immunity" from virus-specific memory T-cells using single HLA expressing cell lines as allogeneic targets.

Detection and clinical relevance of donor specific HLA antibodies: a matter of debate.

The introduction of new sensitive assays for the detection of HLA antibodies on basis of their binding to isolated HLA molecules has got an enormous impact on the decision-making process with respect to donor selection for sensitized patients. In the past, when only complement-dependent cytotoxicity was used as a tool to define HLA alloantibodies, the presence of donor specific antibodies (DSA) before transplantation was considered a contraindication for renal transplantation with that donor. The interpretation of the current DSA results is far more difficult and leads to a lot of discussions and controversy. The problems associated with the use of solid phase assays for clinical decision making and possible solutions are discussed.

Compatibility and kidney transplantation: the way to go.

Long lasting debates in the past questioned the relevance of any sort of compatibility in post mortal kidney transplantation. It is for no say that fully compatible transplants have the highest chances for a long patient and graft survival. In the present report the use of HLA-DR as a representative of the Major Histocompatibility Complex class II genes in the allocation of organs is discussed. The major arguments are the easiness to offer to patients a compatible graft in a relatively short waiting time, an increase in graft survival, the less sensitization during the transplantation period, and the lower waiting time for a retransplant. Even if the number of organ donors remains the same a lowering of the mean waiting time is expected because of the longer period of graft survival.

Vaccine-induced allo-HLA-reactive memory T cells in a kidney transplantation candidate.

BACKGROUND: Allo-human leukocyte antigen (HLA) reactivity by naturally acquired viral-specific memory T cells is common. However, the effect of successful vaccination on the alloreactive memory T-cell repertoire is unclear. We hypothesized that vaccination could specifically induce allo-HLA-reactive memory T cells. METHODS: A varicella-zoster virus (VZV) immediate early 62 (IE62)-specific CD8 memory T-cell clone was single cell sorted from a VZV seronegative renal transplant candidate after response to live attenuated varicella vaccination. To analyze the allo-HLA reactivity, the VZV IE62-specific T-cell clone was tested against HLA-typed target cells and target cells transfected with HLA molecules, in both cytokine production and cytotoxicity assays. RESULTS: The varicella vaccine-induced VZV IE62-specific T-cell clone specifically produced interferon-γ when stimulated with HLA-B*55:01-expressing Epstein-Barr virus-transformed B cells and HLA-B*55:01-transfected K562 cells (single HLA antigen expressing cell line [SALs]) only. The clone also demonstrated specific cytolytic effector function against HLA-B*55:01 SALs and phytohemagglutinin blasts. Cytotoxicity assays using proximal tubular epithelial cell and human umbilical vein endothelial cell targets confirmed the kidney tissue specificity of the allo-HLA-B*55:01 reactivity, and the relevance of the cross-reactivity to clinical kidney transplantation. The results also suggest that molecular mimicry, and not bystander proliferation, is the mechanism underlying vaccine-induced alloreactivity. CONCLUSIONS: Varicella vaccination generated a de novo alloreactive kidney cell-specific cytolytic effector memory T cell in a patient awaiting renal transplantation. Vaccination-induced alloreactivity may have important clinical implications, especially for vaccine timing and recipient monitoring.

Tissue specificity of cross-reactive allogeneic responses by EBV EBNA3A-specific memory T cells.

BACKGROUND: The crossreactivity of Epstein-Barr virus (EBV Epstein-Barr virus nuclear antigen 3A [EBNA3A])-specific CD8 T cells against allogeneic human leukocyte antigen (HLA)-B*44:02 has been shown to be dependent on presentation of self-peptide EEYLQAFTY by the target antigen. In this study, we report that allogeneic HLA-B*44:02 proximal tubular epithelial cells (PTECs) and human umbilical vein endothelial cells (HUVECs) are poor targets for EBV EBNA3A-specific T cells. METHODS: The EEY peptide was exogenously loaded onto HLA-B*44:02 and HLA-B*44:03-expressing PTECs and HUVECs. EEY-peptide-loaded, and unloaded, PTECs and HUVECs were then incubated with serial dilutions of our EBNA3A T-cell clone, in a cytotoxicity assay. RESULTS: Although HLA-B*44:02-expressing PTECs were specifically lysed in proportion to the effector/target ratio by the EBNA3A T-cell clone, without peptide loading, lysis was greatly increased by exogenous EEY peptide loading (15% vs. 75%; P<0.0001). HLA-B*44:02-expressing HUVECs were only lysed when loaded with exogenous EEY peptide (0% vs. 64%; P<0.0001). Lack of HLA expression and lack of ABCD3 gene expression were excluded as a cause for these results. PTECs and HUVECs were specifically targeted by another alloreactive T-cell clone without exogenous peptide loading, suggesting that the lack of recognition of HLA-B*44:02 epithelial and endothelial cells by the EBV EBNA3A T-cell clone was due to lack of EEYLQAFTY peptide presentation. CONCLUSIONS: Tissue-specific (peptide dependent) alloreactivity may have important implications for transplantation monitoring and rejection.

IgM antibodies towards pre-endothelial cells: strong indication for an association with accelerated rejection. A case report.

A 27-year-old woman developed a graft loss due to an accelerated humoral rejection after receiving a blood group identical, human leucocyte antigens (HLA) haploidentical living-related kidney, despite the fact that she did not refer any sensitization event before transplantation. The complement-dependent cytotoxicity and flow cytometry crossmatches were negative for T and B cells. Retrospectively, IgM antibodies against donor precursor endothelial Tie-2(+) cells were detected using a commercially available assay and the pre-transplant serum sample. This case illustrates the necessity of detection of other than the classical HLA-directed antibodies prior organ grafting.

Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes.

Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.