Vasilis Kosmoliaptsis

High density mapping of the MHC identifies a shared role for HLA-DRB1*01:03 in inflammatory bowel diseases and heterozygous advantage in ulcerative colitis

Genome-wide association studies of the related chronic inflammatory bowel diseases (IBD) known as Crohns disease and ulcerative colitis have shown strong evidence of association to the major histocompatibility complex (MHC). This region encodes a large number of immunological candidates, including the antigen-presenting classical human leukocyte antigen (HLA) molecules. Studies in IBD have indicated that multiple independent associations exist at HLA and non-HLA genes, but they have lacked the statistical power to define the architecture of association and causal alleles. To address this, we performed high-density SNP typing of the MHC in >32,000 individuals with IBD, implicating multiple HLA alleles, with a primary role for HLA-DRB1*01:03 in both Crohns disease and ulcerative colitis. Noteworthy differences were observed between these diseases, including a predominant role for class II HLA variants and heterozygous advantage observed in ulcerative colitis, suggesting an important role of the adaptive immune response in the colonic environment in the pathogenesis of IBD.

Detection of Immunoglobulin G Human Leukocyte Antigen-Specific Alloantibodies in Renal Transplant Patients Using Single-Antigen-Beads is Compromised by the Presence of Immunoglobulin M Human Leukocyte Antigen-Specific Alloantibodies

Background. Luminex-based single-antigen human leukocyte antigen (HLA) antibody detection beads (SAB) are a major advance for the characterization of HLA-specific antibodies but their clinical utility is limited unless the analysis is performed and interpreted optimally. Here, we identify problems encountered in routine monitoring of antibody levels that may give rise to misleading results, and describe how these can be overcome to provide more meaningful clinical information. Methods and Results. HLA class I specific antibody-binding levels were determined using SAB in the sera of 42 highly sensitized patients awaiting renal transplantation. Normalization of the results against the HLA class I specific monoclonal antibody W6/32 overcame the problems caused by variation in antigen density on SAB and also suggested the presence of alloantibodies directed against multiple HLA class I epitopes of a given specificity. Routine analysis using undiluted sera gave an incomplete assessment of antibody levels. On serum dilution, three patterns of antibody binding became apparent: most sera showed a sequential reduction in immunoglobulin G (IgG) binding levels but several sera displayed antibody binding which remained unchanged (suggesting antigen saturation) or increased IgG binding on serum dilution (suggesting inhibition of IgG binding using neat serum). The presence of immunoglobulin M (IgM) HLA-specific antibodies in sera correlated with inhibition of IgG antibody binding for the corresponding specificity and treatment of sera with dithiothreitol to eliminate IgM HLA-specific blocking antibodies restored maximum IgG antibody binding levels. Conclusion. When using SAB to monitor HLA-specific antibody binding levels, sera should be pretreated with dithiothreitol to eliminate blocking IgM HLA-specific antibodies that may mask clinically relevant allosensitisation.

HLA Class I Allelic Sequence and Conformation Regulate Leukocyte Ig-Like Receptor Binding

Leukocyte Ig-like receptors (LILRs) are a family of innate immune receptors predominantly expressed by myeloid cells that can alter the Ag presentation properties of macrophages and dendritic cells. Several LILRs bind HLA class I. Altered LILR recognition due to HLA allelic variation could be a contributing factor in disease. We comprehensively assessed LILR binding to >90 HLA class I alleles. The inhibitory receptors LILRB1 and LILRB2 varied in their level of binding to different HLA alleles, correlating in some cases with specific amino acid motifs. LILRB2 displayed the weakest binding to HLA-B*2705, an allele genetically associated with several autoimmune conditions and delayed progression of HIV infection. We also assessed the effect of HLA class I conformation on LILR binding. LILRB1 exclusively bound folded β2-microglobulin–associated class I, whereas LILRB2 bound both folded and free H chain forms. In contrast, the activating receptor LILRA1 and the soluble LILRA3 protein displayed a preference for binding to HLA-C free H chain. To our knowledge, this is the first study to identify the ligand of LILRA3. These findings support the hypothesis that LILR-mediated detection of unfolded versus folded MHC modulates immune responses during infection or inflammation.

Predicting Hla Class Ii Alloantigen Immunogenicity From the Number and Physiochemical Properties of Amino Acid Polymorphisms

Background. We have shown previously that human leukocyte antigen (HLA) class I immunogenicity can be predicted by the number, position, and physiochemical differences of polymorphic amino acids (AAs). We have now modeled the structural and physiochemical polymorphisms of HLA class II alloantigens and correlated these with humoral alloimmunity in sensitized patients awaiting kidney transplantation. Methods. Sera obtained from 30 patients with high levels of IgG HLA-specific antibodies were screened using single-antigen HLA antibody detection beads. A computer program was developed to determine the number of AA mismatches (after interlocus and intralocus subtraction) and their hydrophobicity and electrostatic mismatch score for each mismatched HLA-DR and -DQ specificity. Regression methods were used to compare these variables with the occurrence and magnitude of alloantibody responses. Results. HLA-specific antibody was detected against 879 (55%) of 1604 mismatched HLA specificities evaluated. There was a strong correlation between increasing number of AA mismatches and the occurrence (P<0.001, odds ratio 3.85 per AA) and magnitude of alloantibody responses (P<0.001); only 6% of alloantigens with 0 to 2 mismatched AA-induced alloantibody (median fluorescence intensity 37) compared with 82% of alloantigens with more than or equal to 20 mismatched AAs (median fluorescence intensity 9969). Hydrophobicity and electrostatic mismatch scores also correlated closely with alloantibody response (P<0.001), but neither variable had independent predictive value over the number of AA mismatches alone. Conclusion. Differences in the number of polymorphic AA mismatches and their physiochemical properties for a given recipient HLA type are strong predictors of class II alloantigen immunogenicity and alloantibody response before kidney transplantation.

Back to the future: application of contemporary technology to long-standing questions about the clinical relevance of human leukocyte antigen-specific alloantibodies in renal transplantation

Luminex technology allows the accurate identification of human leukocyte antigen (HLA) class I and class II-specific antibodies at levels below the threshold detectable by either conventional complement-dependent lymphocytotoxicity or flow cytometry. The technology enables the analysis of complex antibody profiles in sensitized patients and gives improved definition of acceptable and unacceptable HLA specificities to guide donor kidney allocation. This helps to facilitate virtual cross-matching and avoid inappropriate shipping of kidneys for incompatible patients in distant centers. Luminex allows the cause of a positive cross-match test to be determined in a clinically relevant time scale and, when used in conjunction with lymphocytotoxic and flow cytometric cross-matching, it provides an assessment of the level of immunological risk in patients being considered as potential recipients for a particular donor kidney. Information is now emerging to enable the full clinical potential of Luminex to be realized.

Predicting the immunogenicity of human leukocyte antigen class I alloantigens using structural epitope analysis determined by HLAMatchmaker.

Background. Human leukocyte antigen (HLA) matching strategies for kidney transplantation assign equal weighting to mismatches at a particular locus and take no account of variation in immunogenicity according to recipient HLA type. We examined the ability of intra- and interlocus analysis of amino-acid polymorphisms at continuous (triplet) and discontinuous positions (eplet) defined by the HLAMatchmaker program to predict alloantigen immunogenicity. Methods. Sera from highly sensitized patients were screened for HLA class-I alloantibodies and mismatched combinations were analyzed using HLAMatchmaker to determine the number of triplet or extended-triplet and eplet mismatches. Results. Logistic regression analysis revealed a strong correlation between the number of triplet or extended-triplet and eplet mismatches and both the presence and magnitude of alloantibody to mismatched HLA-A and -B specificities. The additional structural information provided by eplet analysis gave increased discrimination of mismatched-HLA specificities for alloantigens with greatest sequence disparity but this did not further improve the ability of triplet analysis to predict alloantigen immunogenicity. High antibody levels were observed for several mismatched-HLA combinations with zero triplet or eplet mismatches indicating that self triplets or eplets expressed in different conformations do not always predict nonimmunogenic epitopes. Conclusion. Analysis of recipient HLA type and mismatched-HLA alloantigens using the HLAMatchmaker algorithm allows prediction of immunogenic donor HLA types.

Preimplant Normothermic Liver Perfusion of a Suboptimal Liver Donated After Circulatory Death.

Livers retrieved after circulatory death are associated with an increased incidence of primary nonfunction, early allograft dysfunction, and biliary strictures. The authors report a case of preimplant normothermic perfusion of a suboptimal liver from a 57‐year‐old donor after circulatory death who had been hospitalized for 9 days; predonation alanine transaminase level was 63 IU/L, and the period from withdrawal of life‐supporting treatment to circulatory arrest was 150 minutes. After 5 hours of static cold storage, the liver was subject to normothermic machine perfusion with a plasma‐free red cell–based perfusate. Perfusate lactate level fell from 7.2 to 0.3 mmol/L within 74 minutes of ex situ perfusion, at which point perfusate alanine transaminase level was 1152 IU/L and urea concentration was 9.4 mmol/L. After 132 minutes, normothermic perfusion was stopped and implantation begun. After transplantation, the patient made an uneventful recovery and was discharged on day 8; liver biochemistry was normal by day 19 and has remained normal thereafter. Donor common bile duct excised at implantation showed preservation of peribiliary glands, and cholangiography 6 months posttransplantation showed no evidence of cholangiopathy. Preimplant ex situ normothermic perfusion of the liver appears to be a promising way to evaluate a marginal liver before transplantation and may modify the response to ischemia.

Ten-year experience of selective omission of the pretransplant crossmatch test in deceased donor kidney transplantation.

Background. A pretransplant lymphocyte crossmatch (XM) test is usually considered mandatory but may delay deceased donor renal transplantation. We report on the safety and clinical efficacy of omitting the XM when it is predicted to be negative based on sensitization history and human leukocyte antigen-specific antibody screening. Methods. From 1998 to 2008, 606 deceased donor kidney transplants were performed at our center and the prospective donor-recipient XM omitted in 257 (42%). In all cases, a negative XM was confirmed retrospectively. Four hundred fourteen (68%) kidneys were donated after brain death (DBD) and 192 (32%) after cardiac death (DCD). The effect of this policy on cold ischemia time (CIT), delayed graft function (DGF), and transplant survival was assessed. Results. Mean CIT was 16.7 hr with a prospective XM and 14.3 hr when it was omitted (P<0.001). The beneficial effect of omitting the XM on DGF was only apparent in recipients of DBD kidneys, where the DGF rate was 28% with a prospective XM and 18% without a prospective XM (P=0.03). The corresponding DGF rate in recipients of DCD kidneys was 52% with a prospective XM and 54% without a prospective XM. Logistic regression analysis, after adjustment for variables that influenced DGF, showed that the odds on suffering DGF were lower when the pretransplant XM test was omitted (P=0.04). Neither acute rejection rate nor long-term graft survival was influenced by omission of the XM. Conclusion. Rigorous recording of potential allosensitizing events and comprehensive antibody screening allows the XM to be safely omitted in selected patients and this helps limit CIT and may reduce DGF.

Improved Luminex-based human leukocyte antigen-specific antibody screening using dithiothreitol-treated sera

Solid-phase binding assays using purified human leukocyte antigen (HLA) allow the accurate identification and characterization of HLA-specific antibodies in organ transplant patients, but sera may contain factors that block the detection of clinically relevant alloantibodies. The effect of treating patient sera with dithiothreitol (DTT) on alloantibody detection was examined. In 49 sera submitted for routine HLA-specific antibody monitoring, DTT made little difference to immunoglobulin G HLA-specific antibody detection using LABScreen HLA class I and class II mixed beads. However, in sera submitted for antibody identification using single antigen beads (SAB), DTT markedly increased (>twofold increased median fluorescence intensity) antibody binding to HLA class I and/or class II specificities in 14 of 76 (18%) patient sera. In a cohort of highly sensitized patients, treatment of sera with DTT enhanced antibody-binding levels in 14 of 15 (93%) sera. This study highlights the need to consider routine testing of sera with and without DTT for analysis of HLA-specific antibodies by SAB.

High-resolution, three-dimensional modeling of human leukocyte antigen class I structure and surface electrostatic potential reveals the molecular basis for alloantibody binding epitopes

The potential of human leukocyte antigens (HLA) to stimulate humoral alloimmunity depends on the orientation, accessibility and physiochemical properties of polymorphic amino acids. We have generated high-resolution structural and physiochemical models of all common HLA class I alleles and analyzed the impact of amino acid polymorphisms on surface electrostatic potential. Atomic resolution three-dimensional structural models of HLA class I molecules were generated using the MODELLER computer algorithm. The molecular surface electrostatic potential was calculated using the DelPhi program. To confirm that electrostatic surface topography reflects known HLA B cell epitopes, we examined Bw4 and Bw6 and ascertained the impact of amino acid polymorphisms on their tertiary and physiochemical composition. The HLA protein structures generated performed well when subjected to stereochemical and energy-based testing for structural integrity. The electrostatic pattern and conformation of Bw4 and Bw6 epitopes are maintained among HLA molecules even when expressed in a different structural context. Importantly, variation in epitope amino acid composition does not always translate into a different electrostatic motif, providing an explanation for serologic cross-reactivity. Mutations of critical amino acids that abrogate antibody binding also induce distinct changes in epitope electrostatic properties. In conclusion, high-resolution structural modeling provides a physiochemical explanation for serologic patterns of antibody binding and provides novel insights into HLA immunogenicity.