Martin Barnardo

T cells in peripheral blood after gluten challenge in coeliac disease

Background: Current understanding of T cell epitopes in coeliac disease (CD) largely derives from intestinal T cell clones in vitro. T cell clones allow identification of gluten peptides that stimulate T cells but do not quantify their contribution to the overall gluten specific T cell response in individuals with CD when exposed to gluten in vivo. Aims: To determine the contribution of a putative dominant T cell epitope to the overall gliadin T cell response in HLA-DQ2 CD in vivo. Patients: HLA-DQ2+ individuals with CD and healthy controls. Methods: Subjects consumed 20 g of gluten daily for three days. Interferon γ (IFN-γ) ELISPOT was performed using peripheral blood mononuclear cells (PBMC) to enumerate and characterise peptide and gliadin specific T cells before and after gluten challenge. Results: In 50/59 CD subjects, irrespective of homo- or heterozygosity for HLA-DQ2, IFN-γ ELISPOT responses for an optimal concentration of A-gliadin 57–73 Q-E65 were between 10 and 1500 per million PBMC, equivalent to a median 51% of the response for a “near optimal” concentration of deamidated gliadin. Whole deamidated gliadin and gliadin epitope specific T cells induced in peripheral blood expressed an intestinal homing integrin (α4β7) and were HLA-DQ2 restricted. Peripheral blood T cells specific for A-gliadin 57–73 Q-E65 are rare in untreated CD but can be predictably induced two weeks after gluten exclusion. Conclusion: In vivo gluten challenge is a simple safe method that allows relevant T cells to be analysed and quantified in peripheral blood by ELISPOT, and should permit comprehensive high throughput mapping of gluten T cell epitopes in large numbers of individuals with CD.

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.

HLA-DQA1-HLA-DRB1 variants confer susceptibility to pancreatitis induced by thiopurine immunosuppressants

Pancreatitis occurs in approximately 4% of patients treated with the thiopurines azathioprine or mercaptopurine. Its development is unpredictable and almost always leads to drug withdrawal. We identified patients with inflammatory bowel disease (IBD) who had developed pancreatitis within 3 months of starting these drugs from 168 sites around the world. After detailed case adjudication, we performed a genome-wide association study on 172 cases and 2,035 controls with IBD. We identified strong evidence of association within the class II HLA region, with the most significant association identified at rs2647087 (odds ratio 2.59, 95% confidence interval 2.07–3.26, P = 2 × 10−16). We replicated these findings in an independent set of 78 cases and 472 controls with IBD matched for drug exposure. Fine mapping of the HLA region identified association with the HLA-DQA1*02:01–HLA-DRB1*07:01 haplotype. Patients heterozygous at rs2647087 have a 9% risk of developing pancreatitis after administration of a thiopurine, whereas homozygotes have a 17% risk.

Mother-to-child transmission of HIV infection and CTL escape through HLA-A2-SLYNTVATL epitope sequence variation

Cytotoxic T lymphocytes (CTL) play a central role in containment of HIV infection. Evasion of the immune response by CTL escape is associated with progression to disease. It is therefore hypothesised that transmitted viruses encode escape mutations within epitopes that are required for successful control of viraemia. In order to test this hypothesis, escape through the dominant HLA-A2-restricted CTL epitope SLYNTVATL (p17 Gag residues 77-85 SL9) in the setting of mother-to-child-transmission (MTCT) was investigated. Initial data from two families in which the HIV-infected mother expressed HLA-A*0201 and had transmitted the virus to other family members were consistent with this hypothesis. In addition, analysis of the gag sequence phylogeny in one family demonstrated that CTL escape variants can be successfully transmitted both horizontally and vertically. To test the hypothesis further, a larger cohort of transmitting mothers (n=8) and non-transmitters (n=14) were studied. Variation within the SL9 epitope was associated with expression of HLA-A2 (P=0.04) but overall no clear link between variation from the SL9 consensus sequence and MTCT was established. However, the high level of background diversity within p17 Gag served to obscure any possible association between escape and MTCT. In conclusion, these studies highlighted the obstacles to demonstrating CTL escape arising at this particular epitope. Alternative strategies likely to be more definitive are discussed.

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-γ 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.

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.

A correlation between HLA-C matching and donor antirecipient CTL precursor frequency in bone marrow transplantation

A newly developed, reliable, DNA-based method for typing for alleles of the HLA-C locus has been applied in the context of unrelated, volunteer donors for bone marrow transplantation. Some donors matched for HLA-A, -B, -DR, and -DQ have been found to generate in vitro high frequencies of CTL reactive with the recipients cells. Here we demonstrate that there is a highly significant correlation of the frequencies of CTL precursors and incompatibility at the HLA-C locus. These data indicate that HLA-C locus incompatibility should be avoided in unrelated donor bone marrow transplantation.

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 γ‐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 γ‐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.

Detection of HLA-specific IgG antibodies using single recombinant HLA alleles - The monoLISA assay

BACKGROUND Because of the presence of confounding antigens, the assignment of HLA antibody specificity is difficult in highly sensitized patients, and the definition of an acceptable HLA mismatch requires a significant workload per patient. We describe a new ELISA method, monoLISA, for detection of immunoglobulin (Ig)G HLA antibody using single recombinant HLA class I monomers bound to microtiter plates. METHODS HLA-A2 and -B8 monomers were synthesized and used as screening targets for 85 sera from renal patients. The sera contained various IgG and IgM HLA-specific antibodies, including anti-A2 and anti-B8,defined in a conventional complement-dependent cytotoxicity test (CDC). Investigations were performed to determine possible effects on antibody binding of differential monomer peptide presentation as well as lack of glycosylation. RESULTS A good correlation was found between CDC-defined specificities and the reactivity observed with HLA monomers. MonoLISA attained means of 100% sensitivity and 92.5% specificity compared with CDC. Neither the presence of different peptides, nor the absence of glycosylation of the monomer affected the ability of monoLISA to detect antibody. CONCLUSION This study demonstrates that the mono-LISA method for HLA antibody detection is valid. Because this has the potential to reduce the work involved in screening sensitized patients awaiting transplantation for HLA antibodies, resources aimed at increasing the number of constructed monomers would be well targeted.