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.

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.

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.

CD4 and CD8 T cell responses to tumour-associated Epstein-Barr virus antigens in nasopharyngeal carcinoma patients.

Nasopharyngeal carcinoma (NPC), an Epstein-Barr virus (EBV)-associated tumour common in Southern Chinese populations, is a potentially important target for T cell-based immunotherapy. The tumour cells are HLA class I- and II-positive and express a limited subset of EBV latent proteins, namely the nuclear antigen EBNA1 and the latent membrane proteins LMP2 and (in some cases) LMP1. To ask whether the tumour develops in the presence of a potentially protective host response or in its absence, we set out to determine the prevailing levels of CD4+ and CD8+ T cell memory to these proteins in NPC patients at tumour diagnosis. We first screened healthy Chinese donors against Chinese strain EBNA1, LMP1 and LMP2 sequences in Elispot assays of interferon-gamma release and identified the immunodominant CD4+ and CD8+ epitope peptides presented by common Chinese HLA alleles. Then, comparing 60 patients with >70 healthy controls on peptide epitope mini-panels, we found that T cell memory to CD4 epitopes in all three proteins was unimpaired in the blood of patients at diagnosis. In most cases NPC patients also showed detectable responses to CD8 epitopes relevant to their HLA type, the one consistent exception being the absence in patients of a B*4001-restricted response to LMP2. We infer that NPC arises in patients whose prevailing levels of T cell memory to tumour-associated EBV proteins is largely intact; the therapeutic goal must therefore be to re-direct the existing memory repertoire more effectively against antigen-expressing tumour cells.

Transmission of class I/II multi-locus MHC haplotypes and multiple sclerosis susceptibility: accounting for linkage disequilibrium.

The human major histocompatibility complex (MHC) class II region is associated with genetic susceptibility to multiple sclerosis (MS). Roles for HLA class I loci have been supported in several case-control studies, but this methodology does not consider the known linkage disequilibrium (LD) between class I and II loci. In 1258 individuals from 294 MS families, we analysed class I and II interactions. Using transmission disequilibrium test and haplotype analyses, we found positive associations between MS and several HLA-DRB1*15-HLA-A haplotypes including HLA-DRB1*15-HLA-A*02 (P = 2.41 x 10(-5)) and -HLA-A*03 (P = 8.42 x 10(-6)) and several HLA-DRB1*15-HLA-B haplotypes including HLA-DRB1*15-HLA-B*07 (P = 2.23 x 10(-10)). HLA-DRB1*15 haplotypes divergent for reported HLA-A allelic associations were equally over-transmitted, illustrating no detectable effect of HLA-A or -B alleles in cis on susceptibility. HLA-A and -B alleles on haplotypes not bearing HLA-DRB1*15 were not over-transmitted. Similarly, general over-transmission of HLA-DRB1*15 haplotypes was independent of the HLA-B allele present. Furthermore, HLA-B*07 haplotypes from HLA-DRB1*X-HLA-B*X/HLA-DRB1*X-HLA-B*07 heterozygous parents were transmitted per random expectation giving no indication of HLA-B independence or trans complementation of HLA-DRB1*15 by HLA-DRB1*X-HLA-B*07 haplotypes. These results imply that many reports of class I allelic associations in MS are class II dependent, secondary to LD with class II loci. The lack of independent class I associations suggests that virus-related class I-antigen complexes are not T-cell targets in MS. The inability to replicate confirmed case-control associations highlights the importance of family-based analyses. The frequency of allelic associations not being replicated emphasizes the requirement for constructing multi-locus haplotypes in dissecting associations in regions of tight LD.

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 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.

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 pharmacogenetics of methotrexate in inflammatory bowel disease.

OBJECTIVES: Methotrexate (MTX) is an effective immunosuppressive treatment in inflammatory bowel disease (IBD) but its use is limited by unpredictable toxicity and efficacy. MTX metabolism is complex involving a number of enzymes. An individual's response to MTX may in part be genetically determined by functional genetic variation in genes encoding these enzymes. We report a pharmacogenetic evaluation of MTX therapy in IBD. METHODS: We studied 102 IBD patients treated with MTX, and 202 patients with Crohn's disease (CD), 205 patients with ulcerative colitis (UC) and 189 healthy volunteers served as controls to assess allele frequencies in the disease and healthy populations. All subjects were genotyped for four polymorphisms: G80A in the reduced folate carrier (RFC1) gene, G452T in the gamma-glutamyl hydrolase (GGH) gene and C677T and A1298C in the methylenetetrahydrofolate reductase (MTHFR) gene. Three non-conservative SNPs in the RFC1 and the MTHFR gene could not be detected in our patient cohort. Genotype-phenotype associations were evaluated with respect to efficacy and toxicity of MTX therapy. RESULTS: No significant differences in the allele frequencies between CD, UC and healthy controls were detected. Overall 21% of patients experienced MTX side effects. Patients homozygous for the MTHFR 1298C allele were more likely to experience one or more side effects compared to patients with the wild-type 1298AA genotype (21.0 vs. 6.3%, P < 0.05). None of the genotyped SNPs or haplotypes, either alone or in combination, was associated with short-term efficacy or sustained response. CONCLUSIONS: Side effects of MTX in IBD are associated with a SNP in the MTHFR gene but response cannot be predicted by any of the investigated SNPs.

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.

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.