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.

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.

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.

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.

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.

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.

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.

AIDS-protective HLA-B*27/B*57 and chimpanzee MHC class I molecules target analogous conserved areas of HIV-1/SIVcpz.

In the absence of treatment, most HIV-1-infected humans develop AIDS. However, a minority are long-term nonprogressors, and resistance is associated with the presence of particular HLA-B*27/B*57 molecules. In contrast, most HIV-1-infected chimpanzees do not contract AIDS. In comparison with humans, chimpanzees experienced an ancient selective sweep affecting the MHC class I repertoire. We have determined the peptide-binding properties of frequent chimpanzee MHC class I molecules, and show that, like HLA-B*27/B*57, they target similar conserved areas of HIV-1/SIV(cpz). In addition, many animals appear to possess multiple molecules targeting various conserved areas of the HIV-1/SIV(cpz) Gag protein, a quantitative aspect of the immune response that may further minimize the chance of viral escape. The functional characteristics of the contemporary chimpanzee MHC repertoire suggest that the selective sweep was caused by a lentiviral pandemic.

Allo-HLA reactivity of virus-specific memory T cells is common.

Graft-versus-host disease and graft rejection are major complications of allogeneic HLA-mismatched stem cell transplantation or organ transplantation that are caused by alloreactive T cells. Because a range of acute viral infections have been linked to initiating these complications, we hypothesized that the cross-reactive potential of virus-specific memory T cells to allogeneic (allo) HLA molecules may be able to mediate these complications. To analyze the allo-HLA reactivity, T cells specific for Epstein-Barr virus, cytomegalovirus, varicella zoster virus, and influenza virus were tested against a panel of HLA-typed target cells, and target cells transduced with single HLA molecules. Eighty percent of T-cell lines and 45% of virus-specific T-cell clones were shown to cross-react against allo-HLA molecules. The cross-reactivity of the CD8 and CD4 T-cell clones was directed primarily against HLA class I and II, respectively. However, a restricted number of CD8 T cells exhibited cross-reactivity to HLA class II. T-cell receptor (TCR) gene transfer confirmed that allo-HLA reactivity and virus specificity were mediated via the same TCR. These results demonstrate that a substantial proportion of virus-specific T cells exert allo-HLA reactivity, which may have important clinical implications in transplantation settings as well as adoptive transfer of third-party virus-specific T cells.

Phenotypic variance in childhood coeliac disease and the HLA-DQ/DR dose effect.

OBJECTIVE: Coeliac disease (CD) is associated with HLA-DQ2 and DQ8. The clinical picture is variable and certain human leucocyte antigen (HLA) DQ/DR combinations have a higher relative risk (RR) for CD than others. Moreover, the HLA-DQ gene-dose effect has an impact on the strength of the gluten-specific T-cell response and thus may correlate with clinical presentation and severity of CD. The aim of this study was to determine the correlation between HLA-DQ/DR-based genotypes and the variation in phenotypes of the disease. MATERIAL AND METHODS: A total of 113 non-related Caucasian children clinically diagnosed with CD during the period 1980-2003 with a known HLA type were included in the study. Patients were divided into four categories according to amount of disease expression predisposing to HLA-DQ2 or HLA-DQ8 molecules and the known RR of their HLA-DR/DQ type for CD: high (DR3DQ2 homozygous and DR3DQ2/DR7DQ2), substantial (DR3DQ2/DR5DQ7 and DR5DQ7/DR7DQ2), moderate (DR3DQ2-DR4DQ8 and DR3DQ2/DR*DQ*) and low (DR7DQ2/DR*DQ*, DR4DQ8- DR*DQ* and DR*DQ*- DR*DQ*). The clinical data and HLA genotypes of these patients were compared. RESULTS: The 113 children were diagnosed with CD at a mean age of 4.6 years and boys were significantly older than girls when diagnosed (p=0.01). RR for having CD was highest for the high HLA-risk group (RR 8.1). With the exception of a greater frequency of abdominal distension and fewer non-gastrointestinal symptoms in the substantial HLA-risk group, there were no significant differences in clinical characteristics or degree of severity of the small-bowel histological findings between the children in the different HLA-risk groups. CONCLUSION: No correlation was found between disease severity and a double HLA-DQ2 gene dose.

Reshuffling of ancient peptide binding motifs between HLA-DRB multigene family members: old wine served in new skins.

In most primate species, the class II region of the Major Histocompatibility Complex (MHC) displays diversity with regard to gene copy number and combination of DRB genes present per region configuration. Some of these loci exhibit extremely high levels of allelic variability, whereas others display only moderate levels of polymorphism. To understand the evolutionary history of the various HLA-DR region genes, a large number of full-length sequences of rhesus macaques, chimpanzees and humans were determined. The exon-intron organisation of the DRA gene, displaying only low levels of polymorphism, appears to have been highly conserved during primate evolution. The physical length of various DRB genes/alleles, however, fluctuates significantly in primates due to the presence of indels (insertions/deletions), mainly mapping to intron 1. Phylogenetic evidence supports the notion that the generation of new DRB genes is a dynamic and steadily ongoing process. Indeed, most of the primate DRB alleles investigated represent relatively young entities, possessing species-unique sequences. This seems to contradict the current view that the highly similar peptide binding motifs of many HLA-, Patr- and Mamu-DR molecules, encoded by exon 2 of the DRB gene, represent old entities, which predate primate speciation. As no evidence was found for convergent evolution, the combination of these two observations indicates that ancient peptide binding motifs are frequently reshuffled among duplicated members of the HLA-DRB multigene family.

Selecting highly affine and well-expressed TCRs for gene therapy of melanoma.

A recent phase 1 trial has demonstrated that the generation of tumor-reactive T lymphocytes by transfer of specific T-cell receptor (TCR) genes into autologous lymphocytes is feasible. However, compared with results obtained by infusion of tumor-infiltrating lymphocytes, the response rate observed in this first TCR gene therapy trial is low. One strategy that is likely to enhance the success rate of TCR gene therapy is the use of tumor-reactive TCRs with a higher capacity for tumor cell recognition. We therefore sought to develop standardized procedures for the selection of well-expressed, high-affinity, and safe human TCRs. Here we show that TCR surface expression can be improved by modification of TCR alpha and beta sequences and that such improvement has a marked effect on the in vivo function of TCR gene-modified T cells. From a panel of human, melanoma-reactive TCRs we subsequently selected the TCR with the highest affinity. Furthermore, a generally applicable assay was used to assess the lack of alloreactivity of this TCR against a large series of common human leukocyte antigen alleles. The procedures described in this study should be of general value for the selection of well- and stably expressed, high-affinity, and safe human TCRs for subsequent clinical testing.

Simpler and equitable allocation of kidneys from postmortem donors primarily based on full HLA-DR compatibility.

BACKGROUND: The introduction of human leukocyte antigen (HLA)-matching in nonliving kidney transplantation has resulted into a better graft outcome, but also in an increase of waiting time, especially for patients with rare HLA phenotypes. We addressed the question of the differential influence of HLA-DR-matching versus HLA-A,B in clinical kidney transplantation. METHODS: We used Kaplan-Meier product limit method to estimate survival rates, and Cox proportional hazard regression for the estimation of relative risks (Hazard-ratios) for different variables. RESULTS: A single center study (n=456 transplants, performed between 1985 and 1999) showed that full HLA-DR compatibility leads to a lower incidence of biopsy confirmed acute rejections in the first 180 posttransplantation days. These results were substantiated using the Eurotransplant database (n=39,205 transplants performed between 1985 and 2005) where graft survival in the full HLA-DR compatible group was significantly better than in the incompatible. An additional positive effect of HLA-A,B matching was only found in the full HLA-DR compatible group. In both studies, the introduction of a single HLA-DR incompatibility eliminates the HLA-A,B matching effect. CONCLUSIONS: We propose to allocate postmortem kidneys only to patients with full HLA-DR compatibility, and use HLA-A,B compatibility as an additional selection criterion. All patients, irrespective of their ethnic origin, will profit since the polymorphism of HLA-DR is by far lower than that of HLA-A,B. Excessive kidney travel and cold ischemia time will be significantly reduced.