HLA-DO polymorphism associated with resistance to type I diabetes detected with monoclonal antibodies, isoelectric point differences, and restriction fragment length polymorphism.

A new HLA-DQ-related genetic system with two alleles, 2B3 and TA10, defined serologically by MAbs and alloantisera, showed an almost perfect correlation with charge differences on DQ beta molecules, as well as with two polymorphic DNA fragments hybridizing with a DQ beta probe and various restriction enzymes on a panel of 14 DR4+ homozygous typing cells. It was therefore concluded that the serologically defined alleles 2B3 and TA10 are coded by the DQ beta gene and situated on the HLA-DQ beta chain. This 2B3/TA10 polymorphism is independent of HLA-D and segregates with HLA in families. The TA10 allele appears to be a new marker for resistance to type I diabetes, which is independent from the known resistance marker DR2, whereas no association was observed between this DQ beta polymorphism and rheumatoid arthritis.

The relationship between HLA class II polymorphisms and somatic deletions in testicular B cell lymphomas of Dutch patients.

Several risk factors including immune deficiencies, infections, and autoimmune diseases have been established for non-Hodgkin's lymphoma (NHL). For diffuse large B cell lymphoma (DLBCL), the most common type of lymphoma, no risk factors have been described, which may be due to the intrinsic heterogeneity of this disorder. Previously we reported that, in contrast to nodal DLBCLs, the majority of testicular DLBCLs manifested complete loss of HLA-DR and -DQ expression associated with homozygous deletions of the corresponding genes. To determine the correlation between HLA class II polymorphisms and these lymphomas, we applied DNA typing for HLA-DRB1 and HLA-DQB1 on 50 Dutch patients with testicular and 48 with nodal DLBCL and compared the frequencies with a cohort of healthy Dutch controls. Both the patients with nodal and those with testicular DLBCL manifested significantly higher frequencies of HLA-DRB1*15 than the controls (p < 0.018, odds ratio 2.09 and p < 0.013, odds ratio 2.12, respectively). Moreover, a positive association was seen with HLA-DRB1*12 (p = 0.043, odds ratio 4.17) in the patients with testicular DLBCL, and a negative association was seen with HLA-DRB1*07 (p = 0.022, odds ratio 0.13) in the patients with nodal DLBCL. Homozygous deletions of the HLA-DR/DQ region, evaluated by interphase fluorescence in situ hybridization were seen in 20 of 48 testicular tumors. No preferential loss or retention of a particular HLA-DR or -DQ allele was seen because all alleles were at least once retained or involved in a homozygous deletion.

Occasional memory cytotoxic T-cell responses of patients with human papillomavirus type 16-positive cervical lesions against a human leukocyte antigen-A *0201-restricted E7-encoded epitope.

Most cervical carcinoma (Cxca) cells constitutively express human papillomavirus type 16 (HPV16) E6 and E7 oncoproteins. These proteins are, therefore, attractive targets for T cell-based immunotherapy. Previously, we identified HVP16 E7-encoded CTL epitopes. In patients with cervical intraepithelial neoplasia or Cxca, little is known concerning T-cell activity against viruses in general and against HPV16 in particular. Here, we have screened the blood of 10 healthy donor controls and of 22 patients with HPV16+ cervical lesions for the presence of CTLs directed against HPV16 E7- and control influenza virus matrix-derived epitopes presented by HLA-A *0201. We detected influenza virus-specific CTLs in all donors and in the majority of patients, indicating that most patients have functioning T-cell responses despite their lesions or therapeutic interventions. Moreover, we show that patients with HPV16+ lesions occasionally have memory CTLs against a HPV16 E7-encoded epitope (sequence YMLD-LQPETT), providing evidence for natural CTL immunity against HPV16 in patients with cervical lesions. Combined, these findings raise possibilities for vaccination with HPV16 E7-encoded peptides to induce or augment CTL responses for treatment or prevention of Cxca.

HLA antigens in patients with chronic inflammatory demyelinating polyneuropathy.

In a group of 52 patients with a chronic inflammatory demyelinating polyneuropathy (CIDP), no significant associations were found with any of the HLA-A, B, C, DR or DQ antigens. No HLA association was found between the presence or absence of anti-neural antibodies or between the group of patients improving or not improving after intravenous immunoglobulin. These findings mean that we could not confirm results from the literature that suggest that CIDP is associated with the HLA-A1,B8,DR3 haplotype or--as recently was suggested--with the HLA-Cw7 or DR2 antigen.

The effect of HLA matching on kidney graft survival in separate posttransplantation intervals.

The effect of matching for the HLA antigens has been well established as important in the prognosis of kidney grafts. By analyzing the effect of matching on first transplants from unrelated donors in specific intervals up to 3 years posttransplantation, we show that the effect of HLA-DR matching is strongest in the first 5 months following transplantation (relative risks of graft failure 1.31 and 1.77 for 1 and 2 HLA-DR mismatches, respectively, compared with no mismatches). For patients whose grafts remained functioning after 5 months, there was no significant further improvement in graft survival to 3 years (relative risks 1.16 and 0.98 for 1 and 2 HLA-DR mismatches, respectively, compared with no mismatches)--i.e., the gain in graft survival by matching for HLA-DR appears to be due to its influence in the first 5 months following transplantation. For HLA-B, the matching effect was evident both before and after 5 months (relative risks 1.11 and 1.27 for 1 and 2 HLA-B mismatches, respectively, compared with no mismatches and modelled as constant over the 3-year period), whereas no effect of HLA-A matching was evident in the period up to 3 years.

Prognostic indices to predict survival of first and second renal allografts.

In order to predict kidney graft survival, the influence of independent prognostic factors can be examined multivariately and the factors combined into a prognostic index. Data on 7121 patients receiving an unrelated first and 1033 patients receiving an unrelated second transplant from nonliving donors, between 1 January 1984 and 31 December 1987, were analyzed to ascertain the most important prognostic variables up to 4.5 years posttransplantation. Factors found to be significant for graft survival were donor and recipient age and sex, recipient blood group, whether the recipient was diabetic, cold ischemic period, number of HLA-B and - DR mismatches, highest percent panel-reactive antibody, transplant center, and--for second transplants--duration of first graft. A risk score for graft failure, based on the prognostic factors, was developed using these factors and five risk groups (from excellent to very poor prognosis) were identified. This index was tested on an independent data set and showed a good fit when compared with the observed Kaplan-Meier graft survival: patients allocated by the risk score into the "excellent prognosis" group had an observed one-year graft survival of 90.4%, compared with a predicted value of 90.3% for first transplants. Corresponding results for second transplants were 86.2% (observed) and 86.0% (predicted). For the "very poor" prognosis group, the results were 73.4% (observed) and 74.4% (predicted), for first transplants, and 60.9% (observed) and 60.1% (predicted), for second transplants. A prognostic index can therefore identify patients likely to have a high or low graft survival, leading to improved decision-making and aiding the choice of patient management once a recipient has been transplanted.

Factors contributing to long-term kidney graft survival in Eurotransplant.

We examined the graft survival of 12,883 first unrelated kidney grafts from nonliving donors, transplanted between 1 January 1971 and 31 December 1987 within 52 renal transplantation centers participating in the Eurotransplant organization. The 5-year graft survival increased from 38.8% for the period 1971-1975 to 66.0% for the period 1981-1987 for patients treated with cyclosporine, whereas the half-life increased by only 2 years, from 9.7 years to 11.6 years over the same period, based on grafts functioning at 1 year posttransplantation. Results per HLA locus showed considerable improvements within mismatch groups over the entire period. Large differences between mismatch groups for the early years were observed, but within the cyclosporine era only HLA-B showed a statistically significant difference in half-lives (13.2 versus 9.0 years, for 0 and 2 mismatches respectively, P = 0.013). When other prognostic factors were taken into account, it was revealed by means of an exponential model that number of HLA-B mismatches, donor and recipient age and sex, and recipient diagnosis of diabetes had significant effects on the long-term outcome of the grafts. Depending on the combination of these parameters, estimates of half-life varied from 4.9 to 14.5 years. These results show that matching for HLA-B is still of benefit in the longer term and that other prognostic factors play an important role in predicting the late outcome of renal allografts.

The impact of HLA-A matching on long-term survival of renal allografts.

An important contribution of HLA-A antigen matching in renal transplantation was reported initially, hut later publications showed a minor or absent role. We analyzed the contribution of HLA-A locus matching to graft survival in 17,672 first renal transplants from unrelated, nonliving donors. We show that an independent HLA-A matching effect still exists. Due to its relative weakness and late appearance, large numbers and longer follow-up periods are required. The HLA-A matching effect is a significant factor in first renal allograft survival up to 6 years after transplantation, with an increasing effect over time. This is in contrast to the strong, short-lived, effects of HLA-DR and -B matching, which can only be detected up to 6 months and 2 years after transplantation, respectively. A clear additive beneficial effect of HLA-A matching is shown in the group without B and DR mismatches. Therefore, prospective matching for the HLA-A antigens remains important for renal allograft survival.

Beneficial effect of HLA-DR matching on the survival of corneal allografts.

BACKGROUND: Although HLA typing and matching have been used for 3 decades, that practice has been poorly implemented in corneal transplantation, mainly because of inconclusive or contradictory analytical results. Consequently, we studied the immune response of corneal transplant recipients to HLA histoincompatibilities in a large homogeneous study. METHODS: All corneal transplantations performed by a single surgeon between 1976 and 1996 were studied. HLA-AB matching was used for recipient selection. All HLA typings were performed by a single experienced laboratory. Population genetic techniques were used to assess the validity of the HLA typings. Mono- and multivariate analyses were performed to identify the factors which significantly influence the survival of corneal allografts. Simulation studies were carried out to demonstrate the effects of mis-typed donor and recipient HLA-DR typings on analytical results. RESULTS: Retransplantation, degree of vascularization, HLA-AB and DR matching, endothelial cell count, graft size, recipient gender, and storage method were identified as significant factors by our monovariate analyses. A Cox proportional hazards survival analysis model identified degree of vascularization and HLA-AB and DR matching as significant prognostic factors when all immunological rejection episodes were used, P=0.000001. When only irreversible immunological rejection episodes were used, panel reactive antibodies, retransplantation, and number of rejection events were also identified, P=0.000001. Simulation studies showed that the effects of HLA-DR matching are abrogated by poor HLA-DR typings. CONCLUSIONS: Corneal allograft recipients have a normal alloimmune response to histoincompatibilities. Demonstration of that fact requires accurate HLA typings.

TA10 and 2B3, two new alleles in the HLA-DQ region recognized by monoclonal antibodies.

The monoclonal antibodies (MoAbs) A10/13 and IIB3 were studied in parallel with routine HLA-DR typings. The specificities TA10 and 2B3, recognized by A10/13 and IIB3 MoAb, respectively, showed clear segregation in families and never occurred together on the same haplotype. TA10 and 2B3 appeared to be in perfect Hardy-Weinberg equilibrium. The TA10 specificity is DQw3 related and is in linkage disequilibrium with DR5 (DRw11 and DRw12) and it is also weakly associated with DR4. The 2B3 specificity is DQw1 related and it is in linkage disequilibrium with DR2, DR4, DRw9, and DRw13. The 2B3 and TA10 specificities appear to be alleles of a polymorphic system, closely related, but different from HLA-DQ.

T-cell recognition of class II products that result from the combined presence of two different HLA haplotypes.

To analyze DR2 haplotypes as recognized by alloreactive T cells, lymphocytes from a DR7; DQw2 homozygous donor were cocultured with irradiated lymphocytes that were DRw15, DR7; DQw6, DQw2 heterozygous. In this report, we focus on two HLA-DQ-specific T-cell clones obtained from this priming. These two clones (c3518 and c3523) responded to the positive control (original stimulator) and five of 66 panel donors. Three of these donors typed DRw15, DR7; DQw6, DQw2, as did the positive control. One stimulatory donor typed DRw15, DR7; DQw6, DQw9 and one stimulatory donor typed DRw14, DR7; DQw5, DQw2. Oligonucleotide typing revealed that recognition by the clones depended on the simultaneous presence of the DQB1*0602 gene on one haplotype and DRB1*0701 or DQA*0201 on the other. The hypothesis that c3518 and c3523 recognize an HLA class II product that results from the combination of two different HLA haplotypes was further confirmed in family studies. In three families, it was shown that the DRw15, DR7; DQw6, (DQw2 or DQw9)-positive individuals were recognized, whereas the cells carrying either DRw15; DQw6, DR7; DQw2, or DR7; DQw9 were nonstimulatory. Our results can be explained in two ways: (a) the T cells recognize a class II dimer that results from trans-complementation of DQA1*0101 and DQB1*0602, and (2) the T cells recognize a DR7-derived peptide that is presented by DQw6.

Human leukocyte antigen-DQ and DR polymorphisms predict rheumatoid arthritis outcome better than DR alone.

Conflicting data have been published on the value of the shared epitope (SE) hypothesis in predicting disease outcome in rheumatoid arthritis (RA). Recently we have proposed an alternative hypothesis, referred to as the RA protection (RAP) model. In this model, the HLA-DQ loci carry predisposition while HLA-DRB1 alleles encoding the motif DERAA provide protection against severe RA. In the present study, we have compared the respective values of the models in predicting both remission and erosions in early RA patients. We made use of an early arthritis clinic in which 158 RA patients and 138 patients with undifferentiated arthritis were enrolled. Patients were typed for HLA-DQ and -DR using high resolution DNA typing methods. Homozygosity for predisposing HLA-DQ alleles was associated with no remission and high erosion score. The presence of DERAA-bearing DRB1 alleles was negatively associated with erosions in otherwise predisposed individuals and increased the chance of being in remission. We found that the RAP model was significantly better than the SE model in predicting remission rate and erosion scores at one and two years in early RA patients. We conclude that HLA polymorphism does not only affect RA susceptibility, but also protects against severe disease at early stage.

HLA and elephantiasis in lymphatic filariasis.

Lymphatic filariasis presents a spectrum of manifestations with infection-free asymptomatics at one end and elephantiasis at the other. In order to determine if any HLA antigens are associated with the development of elephantiasis, we compared the HLA frequencies in 55 elephantiasis patients with those in 40 controls consisting of individuals older than 45 years of age without any signs of elephantiasis. The only significant difference in class I antigen frequencies was observed for B27, which was present in 11% of the patients and absent in the controls. More differences were observed in HLA class II antigen frequencies. Both DR3 and the 2B3 epitope (on DQ6, DQ8, and DQ9 molecules) were significantly decreased in patients with elephantiasis whereas the DQ5 frequency was significantly higher in patients than in controls. Analysis of specific antibody isotype profiles revealed that DQ5-positive individuals had increased levels of antifilarial IgG3, an isotype known to be involved in tissue damage. These data suggest that HLA class II genes may control the course of Brugian filariasis by influencing the T-cell-dependent antibody repertoire.

Increasing complexity of HLA-DR2 as detected by serology and oligonucleotide typing.

Serological and oligonucleotide typing was performed on a number of HLA-DR2-positive cells from different ethnic origin, including DR2 haplotypes with various DQ associations. Exons 2 of DRB1 and DRB5 of DR2-positive individuals were locus-specific amplified and hybridized with a number of different oligonucleotides capable of discriminating between the various Dw2, Dw12, Dw21, and Dw22 associated sequences. The linkage of DRB with DQA1 and DQB1 in these haplotypes was analyzed. Among the DR2- positive cells we could define 10 different DR DQ haplotypes by serology and 13 by oligonucleotide typing. The DR2.ES specificity is a serological DRw15 variant which could not be discriminated by oligonucleotide typing from a DRw15 DQw5 haplotype. The DR2.JA variant represents a unique DRB1*1602 DRB5*0101 haplotype. The DR1+2s haplotype consists of a DRB1 DQ region from a Dw1 and a DRB5 gene from a Dw2 haplotype. Its short DR2 serum pattern can be explained by the absence of a DR2 DRB1 gene product. DRB5*0101 sequences were found in association with DRB1*1501, *1502, *1602, and *0101 alleles. Since the DRB5 gene is capable of such different associations it is comparable to the DRB3 and DRB4 genes. This may have implications for the definition of the broad DR2 specificity which is predominantly encoded by the DRB5 gene product. New DR2 haplotypes included the following DQ combinations: DQw2-positive DQA1/B1*0301/0201 and DQw6-positive DQA1/B1*0102/0601 and *0102/0603 haplotypes.

Validation of haplotype frequency estimation methods.

In order to investigate the performance of haplotype frequency estimation methods using unrelated individuals, we compared the results of three estimation methods with those from the haplotypes deduced from family pedigrees. To that end we used the HLA phenotypes of the parents of 1040 families as data for the estimation methods and the full pedigree information as data for the deductive method. We evaluated the results of the following estimation methods: the method using two by two tables described by Mattiuz et al., the maximum likelihood method described by Yasuda and Tsuji and a crude method that uses the information on homozygosity in the phenotypes. All estimation methods generate reliable haplotype frequencies for the more frequent haplotypes, but are unreliable for the less frequent haplotypes. The maximum likelihood estimation method shows the best overall correlation with the results of the deductive method.

Unambiguous typing for HLA-DQ TA10 and 2B3 specificities using specific oligonucleotide probes.

Oligonucleotide probes specific for the serologically defined TA10 and 2B3 specificities were selected based on a comparison of the available HLA-DQ beta sequences. Panel and family segregation studies confirm a complete correlation between the reactivities of the selected probes and the TA10/IIB3 antibodies. The Glu residue at position 45 of the HLA-DQ beta chain is specific for the TA10 determinants, and a DQ beta Gly-Val-Tyr sequence is found at position 45-47 for all 2B3-positive DQ beta chains.

Biotinylated DRB sequence-specific oligonucleotides. Comparison to serologic HLA-DR typing of organ donors in eurotransplant.

A novel HLA-DR typing method was applied using PCR-amplified fragments and biotin-labeled oligonucleotides (PCR-biotin-SSO). The PCR-biotin-SSO method can be used efficiently to perform HLA-DR typing for a large number of individuals when time is not the limiting factor. The reliability of HLA typing of cadaveric organ donors is of vital importance for organ exchange organizations such as ET. Due to lack of time, these typings are usually performed by the complement-dependent cytotoxicity. The individual donor center typings are immediately reported to ET, where the recipient selection procedure is started. DNA isolated from donor spleen material, sent to the ETRL for retyping purposes, was subjected to PCR-biotin-SSO typing. The results were compared with the serological HLA-DR typings as reported to ET. The analysis of 1052 donor samples for the broad DR1-DR10 antigens revealed a concordance rate of over 90% between the donor center and the ETRL. The majority of the discrepancies involved specificities of the HLA-DR5, DR6, and DR8 cross-reacting group, with DR6 as the predominant discordant specificity. The results indicate (a) that PCR-biotin-SSO is a reliable technique for DNA-based HLA-DR typing and (b) that HLA-DR serology is still a useful technique when time is limited, such as for cadaveric donor typing.

Irregular polymerase chain reaction-sequence-specific oligonucleotide hybridization patterns reveal seven new HLA-DRB1 alleles related to DR2, DR3, DR6, DR8, and DR11. Implications for sequence-specific priming.

In the past 3 years we have typed over 7000 individuals for HLA-DRB using a nonradioactive PCR-SSO method. The use of locally developed computer programs simplified data input and the interpretation of the DRB PCR-SSO readings. In this way we detected a number of samples with unexpected hybridization patterns. DRB1 exon 2 segments of these samples were amplified, cloned, and sequenced and appeared to identify seven new DRB alleles: DRB1*0304, a DRB1*0301 variant, was observed in three unrelated Caucasoid individuals; DRB1*1606, which is very similar to *1603; DRB1*1113 is a *1101 variant with some *1401 sequences; DRB1*1310 is *1301-like; DRB1*1311 is similar to *1305 and *1307; DRB1*1416 is a *1401 sequence with a HV3 derived from *1301; DRB1*0808 was found in an Ethiopian individual. Next, we studied the effectiveness of PCR-SSP typing of the newly defined DRB1 alleles. Only two variants were distinguished as odd by PCR-SSP and two were typed as regular specificities. Three alleles were not amplified by the primer sets used. As compared to PCR-SSO, the PCR-SSP typing method using currently available typing kits clearly has limitations as far as the recognition of new and variant alleles is concerned. The products of some of these new alleles may be distinguished using conventional serology.

The HLA dictionary 2001: a summary of HLA-A, -B, -C, -DRB1/3/4/5, -DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR, and -DQ antigens.

This report presents the serologic equivalents of 123 HLA-A, 272 HLA-B, and 155 HLA-DRB1 alleles. The equivalents cover over 64 percent of the presently identified HLA-A, -B, and -DRB1 alleles. The dictionary is an update of the one published in 1999 (Schreuder GMTh, Hurley CK, Marsh SGE, Lau M, Maiers M, Kollman C, Noreen H. The HLA dictionary 1999: a summary of HLA-A, -B, -C, -DRB1/3/4/5, -DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR and -DQ antigens. Tissue Antigens 54:407, 1999) and also includes equivalents for HLA-C, DRB3, DRB4, DRB5, and DQB1 alleles. The data summarize information obtained by the WHO Nomenclature Committee for Factors of the HLA System, the International Cell Exchange (UCLA), the National Marrow Donor Program (NMDP), and individual laboratories. In addition, a listing is provided of alleles which are expressed as antigens with serologic reaction patterns that differ from the well-established HLA specificities. The equivalents provided will be useful in guiding searches for unrelated hematopoietic stem cell donors in which patients and/or potential donors are typed by either serology or DNA-based methods. These equivalents will also serve typing and matching procedures for organ transplant programs where HLA typings from donors and from recipients on waiting lists represent mixtures of serologic and molecular typings. The tables with HLA equivalents and a questionnaire for submission of serologic reaction patterns for poorly identified allelic products will also be available on the WMDA web page: www.worldmarrow.org.

Cell membrane polymorphisms coded for in the HLA-D/DR region. I. Relation between D and DR.

The relation of HLA-D and -DR determinants was studied in Dutch Caucasoids. The recognition of subgroups of DR4, DR5, and DR7, and the specificities LB12 and LB13 are described. Phenotype and gene frequencies and a Hardy--Weinberg analysis of DR and local (LB) B-cell groups are given. Excellent correlation between D and DR typing was obtained when HTCs were studied by selected B-cell antisera. When the same sera were used to type a panel of D typed cells, the correlation was decreased (with the exception of DR3 and Dw3). In the case of discrepancies the DR specificity, but not the corresponding D specificity, always could be found and not the other way around. The data fit best the assumption that HLA-D and -DR are carried by the same molecule, although they might be different determinants on this molecule. A number of possible explanations for the observed discrepancies has been given.