Layer guided-acoustic plate mode biosensors for monitoring MHC-peptide interactions.

The transduction signals from the immobilisation of a class I heavy chain, HLA-A2, on a layer guided acoustic plate mode device, followed by binding of beta(2)-microglobulin and subsequent selective binding of a target peptide are reported.

Progress made towards the development of a CMV peptide vaccine.

Cytomegalovirus disease still remains a major cause of morbidity and mortality in hematopoietic stem cell transplantation recipients. The cell-mediated immune response is essential in the maintenance of latency and the resolution of primary infections. The identification of immunodominant cytomegalovirus antigens has enabled researchers to determine the best candidate antigens to be included in a cytomegalovirus vaccine. Such a vaccine would have to stimulate both a cell-mediated and humoral immune response. Recent advances have enabled the rapid identification of minimal cytotoxic epitopes required to trigger such responses. Epitope mapping to date has mainly focused on the pp65 antigen but other antigens such as IE1 are starting to be mapped. A human leukocyte antigen allele hierarchy is starting to emerge that is dependent on the alleles present in an individual; this is relevant when considering what peptides should be included in a vaccine. This review looks at the current methods available for epitope mapping and the progress that has been made to date.

CD4(+) bias in T cells cloned from a CML patient with active graft versus leukemia effect.

BACKGROUND: The ability to generate a GvL response by infusion of donor leukocytes (DL) in patients following relapse after BMT is now well documented and has been demonstrated to be particularly effective in patients with CML. METHODS: We generated T-cell lines from a patient who was undergoing an active GvL response following withdrawal of immunosuppression for cytogenetic relapse of CML. Cryopreserved pre-transplant leukemic cells were used as stimulators, to generate T-cell lines and oligoclonal lines from the lymphocytes. In total 38 sub-lines were generated from different bulk cultures. The lines were tested for their proliferative and cytotoxic capability to patient pre-transplant leukemic cells, PHA-transformed lymphoblasts, allogeneic CML cells, and autologous and allogeneic B-LCL. RESULTS: Four of the cloned lines tested recognized the patient's pre-transplant leukemic cells. Specifically, two were both cytotoxic and proliferative in response to patient leukemic cells and two were cytotoxic only. Six clonal lines recognized PHA blasts only and were proliferative; one was specific for PHA blasts and CML cells. The sub-lines were phenotyped for cell-surface markers and all were CD4(+) CD8(-) CD 16/56(-). The proliferative response of the leukemia-specific clonal lines could be blocked with anti-MHC Class II MAbs. DISCUSSION: These data suggest that CD4(+) cells play a crucial role in mediating the GvL effect in CML patients. Our observations can be used to delineate strategies for enhancing and investigating the GvL effect in CML.

HLA tetramers and anti-CMV immune responses: from epitope to immunotherapy.

BACKGROUND: Identification of HLA class I-restricted CMV epitopes, and the subsequent synthesis of HLA class I-peptide tetrameric complexes, have provided investigators with an important tool for visualising and quantifying the precise in vivo CTL response to CMV reactivation following stem cell transplantation. In conjunction with PCR-monitoring of the viral load, the magnitude and dynamic of the host's specific CD8(+) T cell response to viral replication can be studied. METHODS: CMV peptide epitopes can be identified be searching the CMV-pp65 antigen for HLA class I allele binding motifs, by testing their binding affinity and ability to generate CTLs, and by screening for CTL responses in as many individuals as possible to assess their general applicability for monitoring large number of patients. HLA tetramers are synthesized by refolding recombinant class I heavy chains and beta(2)m with CMV-pp65(495-503) peptide. After biotinylation and tetramerisation to PE-conjugated streptavidin, they are used to stain CD8(+) T cells taken from patients at different time points after SCT. RESULTS: The T-cell mediated immune response is mainly directed against epitopes derived from the CMV tegument protein pp65. CMV-specific CTL's confer protection against CMV reactivation above a threshold level of 10(7) to 2 x 10(7)/L. CMV reactivation is required to stimulate CTL responses. Transfer of CMV immunity from seropositive donors is associated with better outcome and steroids suppress the Ag-specific immune response. DISCUSSION: Initial studies with CMV-specific HLA class I tetramers have helped to define the nature of anti-CMV T cell response in SCT patients and to determine a threshold CTL level required for controlling CMV reactivation. Monitoring patients with HLA-tetramers should therefore allow clinicians to predict and assess the risk of reactivation and to balance the risks and benefits of early anti-viral treatment, thereby avoiding the hazards of anti-viral prophylaxis. HLA-tetramers can also be used to isolate antigen-specific cells for further in vitro expansion and transfer to patients for antiviral immunotherapy. The threshold level determined from patient monitoring can be used as a guide for estimating an effective target cell dose.

Direct evidence that leukemic cells present HLA-associated immunogenic peptides derived from the BCR-ABL b3a2 fusion protein.

The BCR-ABL oncogene is central in the pathogenesis of chronic myeloid leukemia (CML). Here, tandem nanospray mass spectrometry was used to demonstrate cell surface HLA-associated expression of the BCR-ABL peptide KQSSKALQR on class I-negative CML cells transfected with HLA-A*0301, and on primary CML cells from HLA-A3-positive patients. These patients mounted a cytotoxic T-lymphocyte response to KQSSKALQR that also killed autologous CML cells, and tetramer staining demonstrated the presence of circulating KQSSKALQR-specific T cells. The findings are the first demonstration that CML cells express HLA-associated leukemia-specific immunogenic peptides and provide a sound basis for immunization studies against BCR-ABL.

Cytomegalovirus-specific cellular immune responses and viremia in recipients of allogeneic stem cell transplants.

The immune suppression inherent in allogeneic stem cell transplantation (SCT) offers a favorable environment for infection by opportunistic agents, such as human cytomegalovirus (CMV). Despite the application of potent antiviral prophylaxis, patients remain at risk for CMV infection until adequate immunity is restored. CMV-specific CD8(+) T cell counts were monitored, using HLA-A2 tetrameric complexes, to establish the level of immune response to the viral phosphoprotein UL83 in patients after allogeneic SCT. Correlating this with viral replication and clinical status shows that the level of tetramer-positive T cells provides an assessment of CMV immune reconstitution after stem cell transplantation. Most patients with seropositive donors did reconstitute long-term CMV immunity, unless prolonged immunosuppression to control graft-versus-host disease was induced. Together with polymerase chain reaction testing, this technique provides measurable parameters that can be a guide to therapeutic decision making and can form the basis of CMV immunotherapy.

A high resolution HLA class I and class II matching method for bone marrow donor selection.

HLA matching in bone marrow transplantation has an important role in determining successful outcome. However HLA typing of both potential related and unrelated donors can be both time-consuming and laborious, and does not always resolve accurately the true level of histocompatibility. We have utilised a method, reference strand mediated conformation analysis (RSCA), which is technically simple and allows high resolution matching for all HLA loci, for the typing of 48 patients and their potential 120 donors. The results indicate that RSCA can detect many mismatches that are not routinely identified by conventional HLA typing methods. In addition, RSCA can be applied for the simultaneous analysis of multiple potential BM donor samples in order to quickly identify the best match for each patient.

Family study of the major histocompatibility complex in HLA DR3 negative patients with systemic lupus erythematosus.

Susceptibility to systemic lupus erythematosus (SLE) is known to be governed by genes in the HLA region of the 6th chromosome. From previous studies it has not been possible to distinguish between the effects of null genes for the complement component C4 and HLA-DR3, because of the marked linkage disequilibrium between DR3 and a null allele of C4A (C4A QO) in caucasoid populations. We report here an immunogenetic study of 44 cases of SLE, selected because they were DR3 negative. Eighteen of the 30 Caucasoid cases (60%) had extended HLA haplotypes with a C4 null allele, compared with 22 of 60 (37%) of a control panel of 60 DR3 negative normal Caucasoid subjects. This difference is significant (chi 2 = 4.41; 0.05 greater than P greater than 0.01). Of 14 non-caucasoid patients analysed, 10 had a C4 null allele. It is concluded that the null alleles of the C4 A and B genes are themselves directly responsible for conferring susceptibility to SLE.

Multiple sclerosis in north-east Scotland. An association with HLA-DQw1.

This study reports the frequencies of HLA antigens and the polymorphic variants of C4, C2, and Bf for 200 patients with multiple sclerosis (MS) living in the Grampian region of Scotland, an area of high disease prevalence. A group of 128 normal subjects from the same region were typed for comparison. Although the frequencies of HLA-B7 and DR2 in the patient group (43.3% and 49.4%, respectively) were found to be similar to those reported for other Northern European HLA studies on patients with MS, high frequencies of these antigens were also observed in the group of normal Grampian subjects (38.3% and 40.6%) the differences were not statistically significant. However, a significant association was found between the recently defined Class II HLA antigen, DQw1, and MS (P less than 0.006) when compared with controls. There were no significant differences in frequency of the polymorphisms of C4, C2, and Bf when the group of patients with MS was compared with the control group of normal subjects. The patients were subdivided according to disease severity, remittent versus progressive clinical course, age of onset of the disease and initial symptoms. The frequencies of the HLA and complement polymorphisms (C4, C2, and Bf) were analysed in these subdivisions. DQw1 was found with similar frequency in severe and benign disease (78% and 80%, respectively) but DR2 was most frequent in the group of patients with remittent disease (54%). There were no significant differences in frequency of the polymorphisms of C4, C2, and Bf between the above subgroups of patients and overall no significant HLA associations were found with age of onset of disease or initial symptoms. The findings suggest that in an area of high prevalence of MS, the disease is more closely associated with DQw1 than DR2. Furthermore, there was no evidence to support the hypothesis that the HLA region complement gene polymorphisms show significant association with a putative HLA-linked MS susceptibility gene.

HLA genetic determinants in familial MS. A study from the Grampian region of Scotland.

Fourteen multiplex MS families, 9 single-case MS families and 11 normal families from the Grampian region of North-East Scotland were studied. The prevalence rate of MS for individuals in multiplex families was calculated at 809/100,000; 4.5 times the prevalence rate for the general population in this region. The distribution of shared haplotypes in 12 affected and 19 unaffected sib-pair comparisons did not differ significantly from that expected by chance. Furthermore there was no evidence that homozygosity of a particular HLA gene was required for increased susceptibility to the disease. HLA-B7, C4A3, C4B1, BfS, HLA-DR2, HLA-DQw1 was the commonest haplotype accounting for 18.9% and 24.2% of parental haplotypes from multiplex and single-case families, respectively, compared with 2.3% of parental haplotypes from control families (p less than 0.05 and p less than 0.01, respectively). No significant differences were observed in the frequencies of complement gene polymorphisms (Factor B and C4). The data suggests that a MS susceptibility gene exists, in the HLA complex, and is in closest linkage disequilibrium with the HLA-D region; although other factors, environmental and/or independent genetic loci, may have an important influence.

HLA-typing in oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a disease of the mouth and oropharynx characterised by progressive deposition of collagen leading to severe limitation of movement of the jaw in advanced cases. It is almost completely confined to inhabitants of, or migrants from India who chew 'betel nut'. The histopathological and clinical features suggest that autoimmune mechanisms may be involved. Because all chronic autoimmune diseases show disturbance in the frequencies of HLA antigens, we have HLA typed 50 OSF patients and a similar number of healthy subjects of the same ethnic origin. Raised frequencies of A10 and DR3 were observed. The results support the concept that OSF is a chronic autoimmune disease, initiated by constituents of betel nut, and occurring in genetically susceptible individuals. Genes situated in the HLA region are important determinants of genetic susceptibility in OSF.

C4 and HLA haplotypes associated with partial inhibition of anti-Rg and anti-Ch.

Rg and Ch typing was performed, by serum inhibition, on 145 families that had been typed for HLA/C4/BF/C2 with a view to assessing partial inhibition (p.i.) of anti-Rg/Ch and its haplotype associations. Rg p.i. was found predominantly with the C4A*3A*2,B*QO homoduplicated C4 haplotype and BFF. The original type of Ch p.i. (Nordhagen et al., 1980) was closely associated with the allotype C4B 2, which also occasionally exhibited complete inhibition (c.i.), but this Ch p.i. was also found with the C4A*1,B*QO haplotype (Rittner et al., 1984a). The second type of Ch p.i. (Giles, 1984) was closely associated with the C4B 1 allotype most frequently in the haplotype C4A*6,B*1 but also with C4A*3,B*1. Both types of Ch p.i. are usually found with BF S. The present data indicate that the determinants of Rg and Ch are not directly related to any particular C4 allotype or extended haplotype. Further examples of C4A 1 with Ch and C4B 5 without Ch determinants have been detected and theoretical considerations are discussed as to how they might have arisen from unequal crossovers in homologous regions that result in hybrid protein molecules.

Family study of the major histocompatibility complex in patients with systemic lupus erythematosus: importance of null alleles of C4A and C4B in determining disease susceptibility.

The families of 29 patients with systemic lupus erythematosus and 42 normal subjects were studied to determine the inheritance of the HLA-A, B, C, and DR antigens and also the complement polymorphisms for C2, C4A, C4B, and Bf, which are encoded in the same region of the sixth chromosome. Null (silent) alleles for C4A, C4B, or C2 were found in 24 of the 29 (83%) patients compared with 18 of the 42 (43%) normal controls. HLA-DR3 was present in 20 (69%) of the patients and seven out of 39 (18%) of the normal controls. There was strong linkage disequilibrium between DR3 and the null alleles for C4A and C4B. The data did not permit the relative contributions of DR3 and null factors of C4A and C4B as genetic risk factors to be distinguished. The known association of systemic lupus erythematosus with uncommon inherited and acquired deficiencies of complement components suggests, however, that the presence of null alleles for C4A and C4B, as well as C2, found in most of the patients, is relevant to their genetic susceptibility to this disease.

Histocompatibility antigens in coal miners with pneumoconiosis.

Twenty-five histocompatibility antigens have been measured in 100 coal miners with pneumoconiosis attending a pneumoconiosis medical panel and the results compared with a panel of 200 normal volunteers not exposed to dust. Chest radiographs were read independently by three readers according to the ILO U/C classification. On a combined score, 40 men were thought to have simple pneumoconiosis and 60 men complicated pneumoconiosis. The number of antigens tested and associations between antigens caused difficulties in assessing the statistical significance of differences in prevalence of antigens between groups of men. Using stringent criteria for statistical significance, no significant differences were found in antigen prevalences between miners and controls, or miners with simple or complicated pneumoconiosis. When a less stringent statistical approach was applied, three antigens appeared to have abnormal prevalences in these 100 miners by comparison with the normal volunteers. More detailed examination of these antigen prevalences in relation to radiographic category of pneumoconiosis did not provide any supportive evidence that these slight associations were of statistical or clinical significance. Reports on histocompatibility antigens in miners with pneumoconiosis are reviewed briefly and the results compared. There is no good evidence that any of the histocompatibility antigens so far tested are associated with a clinically important altered risk of simple or complicated pneumoconiosis when dust is inhaled.