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Comprehensive and accurate human leukocyte antigen (HLA) typing within a short turnaround time is a crucial initial step for allocating deceased donor organs for transplantation. Erroneous HLA typing of deceased donors can be catastrophic and result in recipient death, failed transplant, and organ wastage due to inappropriately matched donors. The real-time polymerase chain reaction method is widely used as the sole method for HLA typing of deceased donors because of its simplified workflow. Herein, we have reported cases of four deceased donors showing discrepant HLA typing discovered using two independent methods concurrently. The HLA typing of these donors could have been erroneously reported if a single method had been used, which would have profound patient safety implications. In one case, the drop out of HLA-DR7 using a single method could have resulted in harmful organ allocation if the organ was transplanted after a virtual crossmatch to a sensitized candidate showing strong donor-specific HLA-DR7 antibodies. In conclusion, this case series suggests that concurrent dual typing is essential for accurate HLA typing of deceased donors. This strategy is vital because precise HLA typing is critical for accurate virtual crossmatching, which facilitates continuous distribution and broader geographic sharing of the deceased donor organ.

Human leukocyte antigens (HLA) have been reported to associate with the risk of aneurysmal subarachnoid hemorrhage (SAH) and poor outcome after SAH. Our aim was to identify HLA antigens that associate with the risk of fatal SAH in the Finnish population. Medical records of 600 cadaveric organ donors were reviewed to find organ donors that succumbed to SAH (n = 232) or brain trauma (n = 151). HLA antigen frequencies in these groups were compared with HLA frequencies in a reference population of 10,000 bone marrow donors. Chi-Square test with Bonferroni correction and multiplicative logistic regression models were used and false positive result probabilities (FPRP) were calculated. Alpha-level was 0.01. HLA-A3 associated with fatal SAH (p = 0.0014, OR 1.3 and 95%CI 1.1-1.6) and HLA-DR7 inversely associated with fatal SAH (p = 0.0040, OR 0.3 and 95%CI 0.2-0.6). HLA-A3 but not HLA-DR7 showed also a positive trend in donors with brain trauma. FPRP was below 0.5 for HLA-A3, but clearly above 0.5 for HLA-DR7. HLA-A3 seems to associate with fatal SAH in the Finnish population. Further studies are needed to reveal the pathobiologic mechanisms for how HLA-A3 associates with the risk of fatal SAH in Finns.

The production of human monoclonal antibodies was previously limited to very laborious and time-consuming processes involving EBV-transformation and/or hybridoma generation. Due to the development of molecular cloning techniques, it is now possible to produce human monoclonal antibody fragments quickly by panning phage display libraries against predefined antigenic specificities. Therefore, we tested this technology for producing human single chain Fv fragments (scFvs) against HLA-DR1 purified molecules immobilized on solid phase. Enrichment of DR1-specific phages was measured through five selection rounds of a synthetic library and revealed a 100-fold amplification. Soluble antibody fragments were then expressed and 7 out of 48 clones were found to secrete scFvs which specifically bind to DR1 molecules in ELISA. Further analysis revealed binding of the scFvs also to DR3 but not to DR5 or DR7 molecules correlating with the presence of particular polymorphic aminoacid residues in the DR beta chain. Western blot analysis indicated that the 7 scFvs react with the DR1 alpha/beta-dimer but not with free alpha- or beta- chains. This study shows that the innovative approach of phage display libraries can efficiently provide scFv fragments as useful reagents for the identification and dissection of HLA polymorphic epitopes.

To investigate the functional roles of DR alpha residues in T-cell recognition, 20 mutants of the DR alpha chain were constructed by site-directed mutagenesis. These DR alpha mutants were expressed with WT DR(beta 1*0701) on mouse L cells and used as APC for four DR7-restricted T-cell clones specific for rabies virus antigens. The results indicate that the DR alpha residues are differentially involved in recognition of rabies virus antigen by different T-cell clones. Mutations in the floor of the antigen-binding groove (positions 9, 11, 22, and 24), on the alpha-helix (47, 55, 65, 66, and 72), and surprisingly on the outer loop (15, 18, and 19), abrogated recognition by at least one T-cell clone. Most of these residues appear to be involved in either peptide or TCR contact, based on the DR1 crystal structure. The involvement in T-cell recognition of DR alpha residues located in the outer loop outside the binding groove suggests that these residues may directly contact TCR, or indirectly contribute to the conformation of peptide sitting in the groove.

HLA phenotyping of a leukemia patient of Caucasoid origin revealed the presence of the serological HLA-DR53 specificity. Comprehensive pedigree analysis demonstrated that the HLA-DR53 specificity segregated with the HLA-DR7, -DQ3 haplotype. High resolution PCR- SSP genotyping of the HLA class II genes revealed the presence of the HLA-DRB4*0101101 allele segregating together with the HLA-DRB1*0701, -DQA1*0201 and DQB1*03032 alleles. This finding is in contrast to known linkages in that thus far, the HLA-DR7, -DQ9 haplotype has only been described in association with the non-expressed HLA-DRB4*0103102N allele. The existence of this "novel" haplotype may be explained by a homologous recombinational event that occurred between the HLA-DR7, -DR53, -DQ2 and the HLA-DR7, -DQ9 haplotypes.

A Pvu II RFLP was mapped within the LST-1 gene, the human homologue of the mouse B144 sequence, establishing LST-1 as a new marker gene within the TNF region. We investigated the distribution of this Pvu II RFLP in 274 unrelated individuals, 132 additional HLA-DR7-positive individuals, 86 homozygous lymphoblastoid cell lines, and in four families. Seventeen of 274 individuals (6.2%) were heterozygous for the Pvu II restriction site (ADB1 = lack and ADB2 = presence of the Pvu II restriction site). In our study population the polymorphism has a much wider distribution than that previously reported in an analysis of selected haplotypes. Besides a strong association of ADB1 with HLA-B14, -DR7, we found a further association with HLA-B35. These results were also validated by family segregation studies and analyses of homozygous cell lines. In addition, five of 17 individuals carrying the ADB1 allele had HLA types other than B14 or B35, emphasizing that the presence of ADB1 is not limited to the HLA-B14, DR7 haplotype. LST-1 and its polymorphism may be used as an additional marker of the TNF region, where genes responsible for autoimmune diseases are suspected to be localized.

We have studied the relationship between HLA class II haplotypes and alleles, and the IgE antibody response to a highly purified allergen, Ole I, in allergic patients. Ole e I, is the major antigen from the pollen of olive tree that grows mainly in the Mediterranean. Genomic DNA typing was performed in 40 unrelated patients with seasonal allergic pollenosis who had specific IgE antibodies against Ole e I, detected by double-antibody radioimmunoassay. HLA-DRB and -DQB loci were analyzed by PCR-SSO and RFLP. Phenotypic frequencies were compared with those of 179 healthy unrelated individuals. Significant increases in the phenotypic frequencies of DR7 (pf = 67.5% vs 31.3% in the control population, p c = 0.0023) and DQ2 (pf = 90.0% vs 48.0%, p c = 0.0003) were found, indicating an association between DRB1∗0701/2, DQB1∗0201 alleles and the IgE antibody response to Ole e I. This first time that the HLA-DQ gene has been associated with a positive allergic response.

L cell fibroblasts transfected with HLA class II cDNA clones isolated from a cDNA library produced from a DR7 homozygous cell line were used as antigen-presenting cells (APC) for three HLA DR-restricted, diphtheria toxoid-specific T-cell clones in order to assess the antigen-presenting ability of the transfectants and to define the class II restriction of each clone. Class II-expressing transfectants are capable of presenting antigen to antigen-specific T-cell clones, although the transfectants are less efficient at antigen presentation than conventional APC. Paraformaldehyde fixation of transfectants prior to antigen pulsing abrogated antigen presentation, demonstrating that the transfectants require antigen processing. Antigen presentation by transfectants is completely inhibited by CD4-specific monoclonal antibodies (mAb) and one of four DR-specific mAb, whereas antigen presentation by conventional APC is only partially inhibited. Both the DR alpha:DR7 beta 1 and DR alpha:DR beta 4 (DR omega 53) molecules of the DR7 allotype serve as restriction elements for the diphtheria toxoid-specific T-cell clones. One clone is restricted by the DR7 beta 1 molecule, another clone by the DR beta 4(DR omega 53) molecule, and a third clone by a cross-reactive T cell epitope on DR7 beta 1 and DR beta 4(DR omega 53) molecules. The two DR beta 4(DR omega 53)-restricted clones react, however, differently with a panel of HLA-DR DR omega 53-positive human peripheral blood lymphocytes used as APC. Therefore the data presented here clearly document that the DR beta 4 (DR omega 53) chain may serve as restriction elements for DT-specific T-cell clones. They also provide the first evidence for functional cross-reactivity of the products of two different DR beta loci and in addition emphasize the high complexity of the supertypic HLA-DR omega 53 specificity.

J. D. Bignon, M. L. Cheneau, P. Herry, F. Bonneville, A. Cesbron, J. Y. Muller. Strong linkage disequilibrium of HLA DPw11 with the HLA B44-DR7-DQw2 extended haplotype. Tissue Antigens 1992: 39: 35-37.

Class II molecules were isolated from consanguineous HTCs (DR1-DRw8) by sequential immunoprecipitation with the monoclonal antibodies 7.3.19.1 (anti-DRw52-like), B8.11.2 (anti-DR backbone), and 7.5.10.1 (anti-HLA class II backbone). Depending upon the DR-serotype of the cell line used, two or three class II antigen families, distinct in molecular weight, could be isolated (see Hum Immunol 9:221, 1984). Immunoprecipitated class II molecules were treated with NaNase and then analyzed on 1D-IEF gels. Each HLA class II antigen family contained two alpha chains conserved in pI. Furthermore, the various haplotypes show distinct electrophoretic beta chain patterns. The number of beta chain charge configurations detected varies from 2 to 5, depending upon the antigen family or haplotype studied. Some of these chains have a pI which is specific for a given class II serotype whereas other beta chain pIs are invariant and shared among more antigen families or haplotypes.

Association between HLA-DR7 and Burkitt's lymphoma previously reported has been confirmed by a second study. Analysis of additional data from a second study of 33 Ghanaian patients with African Burkitt's lymphoma and 54 Ghanaian controls matched for age and ethnic origin showed that 39.4% of cases, but only 14.8% of controls, had HLA-DR7 (p less than 0.01). The relative risk of 3.7 is similar to that observed in the earlier study (3.3). Combining the earlier and present studies, analysis of clinical data from 94 patients with Burkitt's lymphoma and 116 controls shows the relative risk of Burkitt's lymphoma among individuals with HLA-DR7 was 3.4 (p less than 0.001). There was an increased relative risk of the disease associated with HLA-DR7 in: patients under 10 years of age; and patients with advanced stages of disease (Stage III or IV). However, comparison of relative risks by sequential analysis of 2 X 2 tables showed that these differences by age and stage were not statistically significant.

The class II antigens from a homozygous DR7 human B cell line were isolated by immunoprecipitation with human alloantisera or monoclonal antibodies. Three class II light chains and three class II heavy chains were identified. The heavy and light chain subunits of the DR7 antigen were distinct from those of the MB2 antigen as assessed by two-dimensional gel electrophoresis. A third light chain in addition to the DR7 and MB2 subunits was precipitated by some alloantisera containing MT3 antibodies. The monoclonal antibodies examined immunoprecipitated one or more of the three class II antigens. The MB2 antigen appeared to be the same as the major antigen recognized by L227 and the DS7 antigen precipitated by SG171.

A monoclonal antibody, PL2, has been produced that reacts with a new supertypic determinant expressed on the peripheral blood B lymphocytes and B-leukemic cells (B-CLL) from all individuals who are HLA-DR7 and some individuals who are HLA-DR5 positive. The genetic linkage of the PL2 determinant to the HLA region was demonstrated by family segregation studies. When cultured Epstein-Barr virus (EBV) transformed B cell lines were examined, PL2 was again found to be expressed on all cell lines homozygous for HLA-DR7 and the DRw11(5) subtype of HLA-DR5 positive cells, while one DRw12(5) cell line was negative, suggesting PL2 may distinguish between these DR5 subtypes. In addition, using the panel of EBV-transformed B-cell lines, PL2 was also found to be weakly expressed on HLA-DRw14(w6), -DRw13(w6), -DR3, and -DR2 positive cells but was completely absent from HLA-DR1 and -DR4 positive cells, and is probably absent also from DRw8- and DRw10-positive cells. From titration analysis and quantitative absorption studies the PL2 determinant was found to be expressed at quantitatively different levels in the following order: DR7 greater than DRw11, DRw14 greater than DRw13 greater than DR3 greater than DR2. The molecules carrying the PL2 determinant on DR7 cells have been characterized biochemically to be a subpopulation of HLA class II molecules recognized by the DR specific monoclonal antibody, L243. Furthermore, by two-dimensional gel analysis, PL2 immunoprecipitated only two of three beta chains associated with the DR-apha chain, which are the same two chains that carry the DR7 allodeterminants.(ABSTRACT TRUNCATED AT 250 WORDS)

We have used antigen-specific human T-cell clones to study the relationship between MHC and antigen recognition specificities expressed by T cells. Tetanus toxoid (TT)-specific T-lymphocyte clones were derived from a immunized HLA-DR2,7 heterozygous donor by limiting dilution from peripheral blood mononuclear cells (PBM) restimulated with TT in vitro. Clones were screened for MHC-restricted antigen recognition against antigen-presenting cells (APC) from a panel of HLA-typed donors, using an in vitro T-cell proliferation assay. Several distinct patterns of antigen recognition were identified. In addition to T cells that recognized TT in association with donor class II MHC antigens, we found clones that simultaneously expressed self-restricted antigen recognition and alloreactivity, and clones with specificity for antigen in the context of MHC antigens not expressed by the T-cell donor. This was confirmed in inhibition studies using well-characterized monoclonal antibodies against class II MHC antigens to block specific proliferative responses. We propose a possible structure for the determinant recognized by two of the clones. These results suggest that the T-cell antigen receptor undergoes random or antigen-dependent changes in vitro, and that this may be a mechanism for somatic diversification of the T-cell repertoire.

Three of four parental haplotypes of a kindred from the Old Order Amish religious isolate carried HLA-DR7 and specificities of the HLA-Dw7 “cluster”. Intrafamily primary and secondary mixed lymphocyte responses clearly distinguished the three Dw7-related allelic specificities. Two of the specificities fall within the Dw11 crossreacting group, designated here as Dw11 “short” (Dw11S) and Dw11 “long” (Dw11L), while the third is more closely related to Dw7. Reaction patterns in this family illustrate the complexity of antigen recognition in primary and secondary mixed lymphoyte responses and the important role played by the responder cell in generating discriminatory primed lymphocyte typing reagents.

The Ia molecules expressed by cells homozygous for two distinct HLA-DR7-associated HLA-D specificities, Dw7S and Dw11L, were compared. The complete Ia phenotypes of these cells are DR7, DRw53, DQw2, Dw7S, DPw4 and DR7, DRw53, DQw3, Dw11L, DPw4, respectively. Immunofluorescence analysis revealed that three DQ-specific monoclonal antibodies (Leu-10, 33.1, and HK-19), which detect polymorphic DQ determinants that do not correspond to known serologic specificities, are nonreactive with DR7, Dw7S cells but are reactive with DR7, Dw11L cells. The DR molecules isolated from Dw7S and Dw11L cells are very similar and comigrate when analyzed together by two-dimensional gel electrophoresis. In contrast, the DQ molecules isolated from these cells are structurally distinct: the DQ beta chains of DQw2-bearing molecules from Dw7S cells are very basic, while those of DQw3-bearing DQ molecules from Dw11L cells are more acidic. The finding that two DR7, D-different cells express indistinguishable DR molecules and structurally distinct DQ molecules documents a unique pattern of Ia molecular organization which is different from those previously described for the DR2-, DR4-, or DRw8-associated HLA-D specificities.

The HLA-DRw53 specificity has not until now been shown to demonstrate polymorphism. We have studied 33 DRw53 haplotypes, comprising 19 DR4, 10 DR7, and 4 DR9 haplotypes, from 6 homozygous typing cells, 11 families, and 8 random individuals. All the subjects studied were South African blacks or of mixed ancestry (Cape Coloureds), with the exception of four homozygous typing cells from whites. The DNA was digested with TaqI and, after Southern blotting, was hybridized with a full-length DRB cDNA probe. Fragments correlations with DR4 (5.5 kb), DR7 (4.0 kb), and DR9 (4.1 kb) were observed. Two fragments of 14.5 and 2.8 kb correlated with DRw53. In addition, two pairs of fragments demonstrated a diallelic pattern, which is likely to correlate with a polymorphism of the DRB4 (DRw53) gene, since one or other of the two patterns was observed in all cells carrying the DRw53 specificity. The first allelic pattern, called DRw53a, was characterized by the presence of 7.5- and 2.6-kb fragments, while the second pattern, called DRw53b, had 5.8- and 2.7-kb fragments. DRW53a occurred in 10 of the 19 DR4 haplotypes and 7 of the 10 DR7 haplotypes. All three DR9, DRw2 haplotypes were also associated with DRw53a. These findings may have important implications for disease associations and the use of unrelated donors for organ transplantation.