BSHI and BTS UK guideline on the detection of alloantibodies in solid organ (and islet) transplantation.

Solid organ transplantation represents the best (and in many cases only) treatment option for patients with end-stage organ failure. The effectiveness and functioning life of these transplants has improved each decade due to surgical and clinical advances, and accurate histocompatibility assessment. Patient exposure to alloantigen from another individual is a common occurrence and takes place through pregnancies, blood transfusions or previous transplantation. Such exposure to alloantigen's can lead to the formation of circulating alloreactive antibodies which can be deleterious to solid organ transplant outcome. The purpose of these guidelines is to update to the previous BSHI/BTS guidelines 2016 on the relevance, assessment, and management of alloantibodies within solid organ transplantation.

Distinct HLA associations of LGI1 and CASPR2-antibody diseases.

The recent biochemical distinction between antibodies against leucine-rich, glioma-inactivated-1 (LGI1), contactin-associated protein-2 (CASPR2) and intracellular epitopes of voltage-gated potassium-channels (VGKCs) demands aetiological explanations. Given established associations between human leucocyte antigen (HLA) alleles and adverse drug reactions, and our clinical observation of frequent adverse drugs reactions in patients with LGI1 antibodies, we compared HLA alleles between healthy controls (n = 5553) and 111 Caucasian patients with VGKC-complex autoantibodies. In patients with LGI1 antibodies (n = 68), HLA-DRB1*07:01 was strongly represented [odds ratio = 27.6 (95% confidence interval 12.9-72.2), P = 4.1 × 10-26]. In contrast, patients with CASPR2 antibodies (n = 31) showed over-representation of HLA-DRB1*11:01 [odds ratio = 9.4 (95% confidence interval 4.6-19.3), P = 5.7 × 10-6]. Other allelic associations for patients with LGI1 antibodies reflected linkage, and significant haplotypic associations included HLA-DRB1*07:01-DQA1*02:01-DQB1*02:02, by comparison to DRB1*11:01-DQA1*05:01-DQB1*03:01 in CASPR2-antibody patients. Conditional analysis in LGI1-antibody patients resolved further independent class I and II associations. By comparison, patients with both LGI1 and CASPR2 antibodies (n = 3) carried yet another complement of HLA variants, and patients with intracellular VGKC antibodies (n = 9) lacked significant HLA associations. Within LGI1- or CASPR2-antibody patients, HLA associations did not correlate with clinical features. In silico predictions identified unique CASPR2- and LGI1-derived peptides potentially presented by the respective over-represented HLA molecules. These highly significant HLA associations dichotomize the underlying immunology in patients with LGI1 or CASPR2 antibodies, and inform T cell specificities and cellular interactions at disease initiation.10.1093/brain/awy109_video1awy109media15796480660001.

De novo donor-specific HLA antibodies after combined intestinal and vascularized composite allotransplantation - a retrospective study.

Combining vascularized composite allotransplantation (VCA) with intestinal transplantation to achieve primary abdominal closure has become a feasible procedure. Besides facilitating closure, the abdominal wall can be used to monitor intestinal rejection. As the inclusion of a VCA raises the possibility of an enhanced alloimmune response, we investigated the incidence and clinical effect of de novo donor-specific HLA antibodies (dnDSA) in a cohort of patients receiving an intestinal transplant with or without a VCA. The sequential clinical study includes 32 recipients of deceased donor intestinal and VCA transplants performed between 2008 and 2015; eight (25%) modified multivisceral transplants and 24 (75%) isolated small bowel transplants. A VCA was used in 18 (56.3%) cases. There were no episodes of intestinal rejection without VCA rejection. Fourteen patients (14 of 29; 48.3%) developed dnDSA. In the VCA group, fewer patients developed dnDSA; six of 16 (37.5%) VCA vs. eight of 13 (61.5%) non-VCA. There was no statistically significant difference in one- and 3-year overall graft survival stratified for the presence of dnDSA; P = 0.286. In the study, there is no evidence that the addition of a VCA increases the incidence of dnDSA formation compared to transplantation of the intestine alone.

A genome-wide association study for celiac disease identifies risk variants in the region harboring IL2 and IL21.

We tested 310,605 SNPs for association in 778 individuals with celiac disease and 1,422 controls. Outside the HLA region, the most significant finding (rs13119723; P = 2.0 x 10(-7)) was in the KIAA1109-TENR-IL2-IL21 linkage disequilibrium block. We independently confirmed association in two further collections (strongest association at rs6822844, 24 kb 5' of IL21; meta-analysis P = 1.3 x 10(-14), odds ratio = 0.63), suggesting that genetic variation in this region predisposes to celiac disease.

CD4-positive T-helper cell responses to the PASD1 protein in patients with diffuse large B-cell lymphoma.

BACKGROUND: Vaccine development targeting the novel immunogenic Per ARNT Sim Domain containing 1 (PASD1) cancer testis antigen represents an attractive therapeutic approach for the significant number of patients with diffuse large B-cell lymphoma who are refractory to conventional treatment. Since CD4-positive T helper cells have crucial roles in promoting and maintaining immune responses to tumor antigens, the presence of a CD4-positive T-helper immune response to the PASD1 antigen in patients with diffuse large B-cell lymphoma was investigated in the current study. DESIGN AND METHODS: Thirty-one patients with diffuse large B-cell lymphoma (25 with de novo, five with transformed and one with T-cell-rich B-cell lymphoma) were studied. Five immunogenic PASD1 peptides predicted to bind to several major histocompatibiliy complex, class II DR beta 1 alleles were identified using web-based algorithms. Peripheral blood mononuclear cells from patients were used to investigate the immunogenicity of these DR beta 1-restricted peptides in vitro using both gamma-interferon release enzyme-linked immunospot and cytolytic assays. RESULTS: Two of the five PASD1 peptides, PASD1(6) and PASD1(7), were shown to be immunogenic in 14 out of 32 patients studied in a gamma-interferon release assay. CD4-positive T-helper cell lines from two patients raised against PASD1 peptides were able to lyse cell lines derived from hematologic malignancies expressing endogenous PASD1 protein. CONCLUSIONS: This is the first report of a CD4-positive T-helper response to the PASD1 protein in patients with lymphoma. The immunogenic peptides described here represent valuable additional candidates for inclusion in a vaccine to treat patients with PASD1-positive diffuse large B-cell lymphoma whose disease is refractory to conventional therapies.

HLA class I alleles tag HLA-DRB1*1501 haplotypes for differential risk in multiple sclerosis susceptibility.

The major locus for multiple sclerosis (MS) susceptibility is located within the class II region of the Major Histocompatibility Complex (MHC). HLA-DRB1 alleles, constituting the strongest MS susceptibility factors, have been widely exploited in research including construction of transgenic animal models of MS. Many studies have concluded that HLA-DRB1*15 allele itself determines MS-associated susceptibility. If this were true, haplotypes bearing this allele would confer equal risk. If HLA-DRB1*15 bearing haplotypes differed for risk, roles for other loci in this region would be implied and further study of the fine structure of this locus would be compelling. We have tested the hypothesis comparing haplotypes stratified by HLA class I tagging. We show here that HLA-DRB1*15-bearing-haplotypes in 1970 individuals from 494 MS families are indeed heterogeneous. Some HLA-DRB1*15 haplotypes determine susceptibility while others do not. Three groups of class I tagged HLA-DRB1*15 haplotypes were not over-transmitted: (i) HLA-DRB1*15-HLA-B*08 (TR = 25, NT = 23, Odds Ratio = 1.09), (ii) -HLA-B*27 (TR = 18, NT = 17, Odds Ratio = 1.06), and (iii) rare HLA-DRB1*15 haplotypes (frequency <0.02). Rare haplotypes were significantly different from common haplotypes, and transmissions were remarkably similar to those for class-I-matched non-HLA-DRB1*15 haplotypes. These results unambiguously indicate that HLA-DRB1*15 is part of a susceptibility haplotype but cannot be the susceptibility allele itself, requiring either epistatic interactions, epigenetic modifications on some haplotypes, or nearby structural variation. These findings strongly imply that differences among HLA-DRB1*15 haplotypes will furnish the basis for MHC-associated susceptibility in MS and raise the possibility that the MHC haplotype is the fundamental unit of genetic control of immune response.

Cytolytic T-cell response to the PASD1 cancer testis antigen in patients with diffuse large B-cell lymphoma.

The identification of immunogenic cancer testis antigens (CTAs) as immunotherapeutic targets represents one approach to improve treatment options for diffuse large B-cell lymphoma (DLBCL). We previously identified PASD1 [PAS (Per ARNT Sim) domain containing 1 (PASD1)], a DLBCL-associated CTA that was expressed in a range of hematopoietic malignancies. The aim of the present study was to investigate the presence of a cytotoxic T-cell (CTL) response to PASD1 in DLBCL patients. A significant gamma-interferon (IFN) release was detected in 21/29 HLA-A*0201-positive DLBCL patients (18 de novo DLBCL, two transformed DLBCL and one T-cell rich B-cell lymphoma) following short-term culture of their peripheral blood mononuclear cells stimulated with five HLA-A*0201-restricted PASD1 peptides. No significant responses were detected in 21 HLA-A*0201-negative DLBCL patients (12 de novo DLBCL, seven transformed DLBCL, two T-cell rich B-cell lymphoma) or six normal subjects. CTL cell lines were able to lyse PASD1-positive tumour cells in a major histocompatibility complex-Class I dependent manner. The presence of a gamma-IFN response correlated with PASD1 protein expression in the tumour cells in 12/15 cases studied. This is the first report of a CTL response to a CTA in DLBCL. Our results provide additional valuable evidence supporting PASD1 as a potential immunotherapeutic target for the treatment of DLBCL and other malignancies.

Contribution of histological, serological, and genetic factors to the clinical heterogeneity of adult-onset coeliac disease.

OBJECTIVE: Although the factors predisposing to coeliac disease (CD) are largely understood, it remains unclear what determines the clinical heterogeneity of the disease. The aim of this study was to explore the contribution of histological, serological, and genetic factors to disease presentation. MATERIAL AND METHODS: The study was designed as a retrospective chart review of 384 unrelated Caucasian patients diagnosed with CD after the age of 16 at a single UK centre. RESULTS: We found that 8.8% of IgA-competent CD patients were endomysial antibody (EMA)-negative. Compared with the EMA-positive group, EMA-negative CD patients had a lower prevalence of iron deficiency (52.0% versus 72.6%, p=0.03) and Marsh IIIb-c lesions (66.7% versus 85.3%, p=0.03). Histological severity at diagnosis correlated with anaemia (p<0.01), folate deficiency (p<0.01), and iron deficiency (p=0.05), but no other laboratory or clinical features. Compared with human leucocyte antigen (HLA)-DQ2.5-positive patients, those carrying HLA-DQ2.2 were similar in terms of all the characteristics we considered, whereas those carrying HLA-DQ8 had a lower frequency of EMA positivity (62.5% versus 92.6%, p<0.01). The proportion of EMA-positive patients increased with frequency of the HLA-DQB1*0201 allele (76.7% versus 92.3% versus 96.4% for 0 versus 1 versus 2 alleles, p<0.01); no other evidence of a gene-dose effect of HLA-DQB1*0201 was observed. CONCLUSIONS: Histological severity at diagnosis of CD is associated with anaemia and some micronutrient deficiencies, but no other clinical features. The proportion of EMA-positive patients is higher amongst those carrying HLA-DQ2 than in those carrying HLA-DQ8, and is highest in HLA-DQ2 homozygotes. We found no correlation between frequency of the HLA-DQ alleles encoding HLA-DQ2.5 and CD severity.

CD4 T-helper responses to the anaplastic lymphoma kinase (ALK) protein in patients with ALK-positive anaplastic large-cell lymphoma.

We have previously shown both humoral and CTL responses to anaplastic lymphoma kinase (ALK) in patients with ALK-positive anaplastic large-cell lymphoma (ALCL). However, because CD4(+) T-helper (Th) cells also play a vital role in developing and maintaining tumor immunity, we investigated the presence of a CD4(+) Th response in ALK-positive ALCL. Using an IFN-gamma ELISPOT assay, we identified two ALK-derived DRB1-restricted 24-mer promiscuous peptides, ALK1(278-301) and ALK2(233-256), as being immunogenic in six ALK-positive ALCL patients but not in two ALK-negative ALCL patients or five normal subjects. A significant interleukin-4 response to the ALK peptides was detected in only one ALK-positive patient. CD4(+) Th cell lines lysed ALK-positive ALCL cell lines in a MHC class II-restricted manner. This first report of a CD4(+) Th response to ALK provides valuable information for developing future immunotherapeutic options for ALK-positive ALCL patients who fail to respond well to conventional therapies.

High resolution HLA haplotyping by imputation for a British population bioresource.

This study aimed to establish the occurrence and frequency of HLA alleles and haplotypes for a healthy British Caucasian population bioresource from Oxfordshire. We present the results of imputation from HLA SNP genotyping data using SNP2HLA for 5553 individuals from Oxford Biobank, defining one- and two-field alleles together with amino acid polymorphisms. We show that this achieves a high level of accuracy with validation using sequence-specific primer amplification PCR. We define six- and eight-locus HLA haplotypes for this population by Bayesian methods implemented using PHASE. We determine patterns of linkage disequilibrium and recombination for these individuals involving classical HLA loci and show how analysis within a haplotype block structure may be more tractable for imputed data. Our findings contribute to knowledge of HLA diversity in healthy populations and further validate future large-scale use of HLA imputation as an informative approach in population bioresources.

Sp17 Protein Expression and Major Histocompatibility Class I and II Epitope Presentation in Diffuse Large B Cell Lymphoma Patients.

Improved therapies are urgently needed for patients with diffuse large B cell lymphoma (DLBCL). Success using immune checkpoint inhibitors and chimeric antigen receptor T cell technology has fuelled demand for validated cancer epitopes. Immunogenic cancer testis antigens (CTAs), with their widespread expression in many tumours but highly restricted normal tissue distribution, represent attractive immunotherapeutic targets that may improve treatment options for DLBCL and other malignancies. Sperm protein 17 (Sp17), a CTA reported to be immunogenic in ovarian cancer and myeloma patients, is expressed in DLBCL. The aim of the present study was to investigate Sp17 epitope presentation via the presence of a cytotoxic T cell (CTL) and a CD4 T-helper (Th) response in DLBCL patients. A significant γ-interferon CTL response was detected in peripheral blood mononuclear cells of 13/31 DLBCL patients following short-term cell stimulation with two novel HLA-A⁎0201 peptides and one previously reported HLA-A⁎0101-restricted nine-mer Sp17 peptide. No significant responses were detected in the HLA-A⁎0201-negative DLBCL patients or four healthy subjects. A novel immunogenic 20-mer CD4 Th Sp17 peptide was detected in 8/17 DLBCL patients. This is the first report of a CTL and a CD4 Th response to Sp17 in DLBCL and supports Sp17 as a potential immunotherapeutic target for DLBCL.

Replication of the association of HLA-B7 with Alzheimer's disease: a role for homozygosity?

BACKGROUND: There are reasons to expect an association with Alzheimer's disease (AD) within the HLA region. The HLA-B & C genes have, however, been relatively understudied. A geographically specific association with HLA-B7 & HLA-Cw*0702 had been suggested by our previous, small study. METHODS: We studied the HLA-B & C alleles in 196 cases of 'definite' or 'probable' AD and 199 elderly controls of the OPTIMA cohort, the largest full study of these alleles in AD to date. RESULTS: We replicated the association of HLA-B7 with AD (overall, adjusted odds ratio = 2.3, 95% confidence interval = 1.4-3.7, p = 0.001), but not the previously suggested interaction with the epsilon4 allele of apolipoprotein E. Results for HLA-Cw*0702, which is in tight linkage disequilibrium with HLA-B7, were consistent with those for the latter. Homozygotes of both alleles appeared to be at particularly high risk of AD. CONCLUSION: HLA-B7 and HLA-Cw*0702 are associated with AD in the Oxford population. Because of the contradictions between cohorts in our previous study, we suggest that these results may be geographically specific. This might be because of differences between populations in the structure of linkage disequilibrium or in interactions with environmental, genetic or epigenetic factors. A much larger study will be needed to clarify the role of homozygosity of HLA alleles in AD risk.

The association between HLA DR, DQ antigens, and vulval lichen sclerosus in the UK: HLA DRB112 and its associated DRB112/DQB10301/04/09/010 haplotype confers susceptibility to vulval lichen sclerosus, and HLA DRB10301/04 and its associated DRB10301/04/DQB10201/02/03 haplotype protects from vulval lichen sclerosus.

Lichen sclerosus (LS) is considered to have an immunogenetic background. Several small studies, using serological typing, have reported that HLA-DR11, DR12, and DQ7 were increased in LS, with DR17 less frequent. This study aimed to validate and detect new HLA-DR and DQ associations with LS in females and its characteristic clinical parameters. The cases, 187 female LS patients, and 354 healthy controls were all UK North Europeans. PCR-sequence specific primers method was applied to genotype the HLA-DR, DQ polymorphisms that correspond to 17 serologically defined DR and seven DQ antigens. Statistical analysis was performed with two-tailed Fisher's exact test with Bonferroni adjustment (p value after Bonferrroni adjustment, Pc). We found increased frequency of DRB1*12 (DR12) (11.2%vs 2.5%, pc < 0.01) and the haplotype DRB1*12/DQB1*0301/04/09/010 (11.2%vs 2.5%, p < 0.001, pc < 0.05), and a lower frequency of DRB1*0301/04 (DR17) (11.8%vs 25.8%, pc < 0.01) and the haplotype DRB1*03/DQB1*02DRB1*0301/DQB1*0201/02/03 (11.2%vs 24.6%, pc < 0.0001) in patients compared with controls. HLA DR and DQ antigens were not associated with time of onset of disease, site of involvement, structural changes of genitals, and response to treatment with potent topical steroids. In conclusion, HLA-DR and DQ antigens or their haplotypes appear to be involved in both susceptibility to and protection from LS.

Complete mapping of a novel HLA A*6801-restricted HIV-1 Tat epitope directly with a rapid modified enzyme-linked immunospot assay.

We identified a novel HLA A*6801-restricted HIV-1 Tat-derived cytotoxic T lymphocyte (CTL) epitope using an adapted enzyme-linked immunospot assay that allows the rapid ex vivo identification of CTL epitopes together with their associated HLA Class I restriction elements. The optimal 11 amino acid residue Tat epitope efficiently stabilized the refolding of monomeric peptide-HLA A6801 complexes in vitro and fluorochrome-labelled, tetrameric peptide-HLA A6801 complexes stained CD8 T cells specific for this epitope directly ex vivo.

Is there MHC Class II restriction of the response to MHC Class I in transplant patients?

BACKGROUND: In this study, we evaluated distinct HLA-DRB1 alleles to determine class II restriction of the production of HLA-A2-specific antibodies in renal transplant patients. METHODS: Data from 217 renal transplant patients who received an HLA-A2-mismatched renal graft were analyzed with regard to HLA-A2 humoral responsiveness. High-resolution DNA typing of class II HLA-DR alleles was performed by polymerase chain reaction-sequence-specific primer. Patients who had one of the following eight HLA-DRB1 alleles were included in the study: -*0101, -*0301, -*0401, -*0701, -*1101, -*1301, -*1401, and -*1501. Serum samples were screened posttransplantation with the standard complement-dependent cytotoxicity procedure. In addition, recombinant HLA-A2 monomers (the "MonoLISA" assay) were used as a target for the detection of HLA-A2 group-specific antibodies. The following HLA-A2 amino acid positions (termed "epitopes") that are responsible for the induction of an antibody response were defined: 74H, 65-66GK, 62G, 114H, 142-145TTKH, and 107W-127K. The definition of the "HLA-DR permittors" of anti-HLA-A2 response was based on a "class II restriction table" designed for this purpose. Prediction of immunogenic and/or nonimmunogenic HLA-A2 peptides was based on an MHC database. RESULTS: The HLA-DRB1-*0101 and -*1401 alleles had a trend toward a positive correlation with the production of HLA class I-specific antibodies against the HLA-A2 shared (public) epitopes 65-66GK and -62G, respectively. Only the DRB1-*1501 allele had higher trend toward a positive correlation with the production of antibodies against the HLA-A2 private (74H) epitope. In 42 patients with the HLA-DRB1-*1501 allele, 11 (26%) patients produced HLA-specific antibodies against the HLA-A2 group of epitope(s). Moreover, in these patients, spreading of the alloreactivity against "other" HLA antigens was detected. Many of these other HLA antigens did not belong to HLA-A2 group but had newly defined shared epitopes with this group. Furthermore, the epitope prediction, based on an MHC database, revealed differences in the ligation strength (score) to the HLA allele (class I and II) for a specific HLA-A2 peptide in the 42 patients (responders and nonresponders). CONCLUSIONS: The data presented in this paper suggest that the HLA class II allele and the type of the bound allopeptide may influence the humoral and cellular response. The immunogenicity of these allopeptides could be predicted with an MHC database (high-scored peptide=activating peptide and low-scored peptide=suppressor peptide). In the future, production of synthetic peptide analogues, on the basis of these predictions, could be used for induction of T-cell anergy and/or tolerance. In the short term, algorithms, on the basis of our approach, could be tested for influence on graft survival and allosensitization in current high-quality data sets.

C4d deposition in early renal allograft protocol biopsies.

BACKGROUND: Deposition of the complement protein C4d in renal allograft biopsies obtained during graft dysfunction and rejection has been proposed to be a sensitive marker of antibody-mediated acute rejection. To determine the diagnostic specificity of C4d deposition, it is important to study biopsies from allografts with no evidence of dysfunction. In this study, we examined C4d deposition in protocol biopsies obtained irrespective of clinical status. METHODS: Immunohistochemistry for C4d was performed on routine protocol biopsies preimplantation and on day 7 posttransplantation from 48 unselected renal allografts. Serum samples obtained up to 1 month after transplantation were assayed for donor-reactive antibodies (DRA). Results were correlated with histopathology and clinical outcome measures. RESULTS: Diffuse C4d deposition was detected in the peritubular capillaries of 6 of 48 (13%) biopsies. C4d deposition was present in 5 of 15 (33%) biopsies that showed acute rejection (Banff 97, category 4) but only in 1 of 33 (3%) biopsies with no rejection (P=0.003, 97% specificity). Posttransplant DRAs were detected in 21 of 48 (44%) patients. All five recipients with C4d deposition and rejection had posttransplant DRA; the recipient whose biopsy showed C4d positivity, but not rejection, did not have detectable DRA. C4d deposition was not treated with plasmapheresis or intravenous immunoglobulin and was not associated with poor posttransplant graft outcome at 1-year follow-up. CONCLUSIONS: Our results show that in early posttransplant protocol biopsies, C4d is a specific marker for the presence of humoral rejection, as indicated by its association with DRA and acute histologic rejection.

Role of anti-vimentin antibodies in renal transplantation.

BACKGROUND: The role of non-HLA antibodies in rejection is not clear. We investigate whether antibodies to vimentin are made after renal transplantation and if production is associated with interstitial fibrosis and tubular atrophy (IFTA). METHODS: In this retrospective study, sera from 70 recipients of renal allografts (40 controls, 30 IFTA) were studied. The biopsy diagnosis of interstitial fibrosis and tubular atrophy (IFTA) was based on random, cause-indicating biopsies. Sera were collected pretransplant and at 3 monthly intervals up to 5 years posttransplant or diagnosis of IFTA and assayed by ELISA for IgM and IgG anti-vimentin antibodies (AVA) and HLA antibodies. RESULTS: Mean titers of IgM AVA were higher at every year after transplantation compared with pretransplant for both IFTA and controls groups (P<0.001). There was no difference in the mean level of IgM AVA achieved by IFTA and control groups. The mean pretransplant levels of IgG AVA in the IFTA and control group were 18.2±11.7 and 11.0±8.1, respectively (P=0.001). There was a significant increase between the pretransplant mean levels of IgG AVA and the levels at years 1 to 4 in the IFTA group (years 1-3, P<0.0001, year 4 P=0.003) but not in the controls. There was no significant difference between the numbers of IFTA or control patients achieving a positive value (mean+2SD of pretransplant antibody titers) of IgM AVA (50% versus 37.5%, respectively) or IgG AVA (26.6% versus 12.5%, respectively). There was no association between production of HLA and AVA antibodies. CONCLUSION: Posttransplant production of IgM AVA is not associated with IFTA. The production of IgG AVA by a minority of IFTA patients suggests that in some individuals, IgG AVA may be involved in the pathology of IFTA.