Sequence of a new HLA-DR4 allele with an unusual residue at position 88 that does not seem to affect T-cell allo recognition.

New HLA alleles can be identified by unorthodox patterns observed during low-resolution typing performed with sequence specific oligonucleotide probes (SSOP). One of the best examples is locus DRB1, where allelic subtypes are characterized by a combination of a limited number of residues located in three hypervariable regions of exon 2. HLA-DR oligotyping analysis of a female caucasoid bone marrow donor led to the identification of an individual that typed as DRB1*11, DRB3*02, DRB4*01, DQB1*0301-0302. This donor was, however, typed by serology as DR11 DR4, DR52, DR53, DQ7 DQ8. PCR-SSP typing for DR4 subtype revealed an amplification pattern typical for DRB1*0404. After sequencing the entire exon 2, a new DRB1 allele was identified: DRB1*04var that is identical to DRB1*0404, except for one nucleotide at codon 88 resulting in a Ser-->Arg exchange. This mutation had prevented amplification with the DR generic primers. Cellular typing by three HTCs-DRB1*0404/DW14 from the 9th Workshop showed that this DRB1*04var typed exactly like a DW14 cell. This suggests that residue 88 does not affect T cell recognition.

Apparent HLA DR triplet due to the coexpression of a DRB5-encoded molecule on a DR1 haplotype.

HLA DR1 molecules are coded by a single polymorphic DRB1 gene. We have observed rare DR1 cells in one Caucasoid family and three unrelated individuals that also reacted with some anti-DR2 sera. Since the second DR antigen was normally expressed, these cells appeared as triplets. Contrary to serology, the cells were not typed by HTCs defining Dw2, Dw12, and Dw21. Further investigations on these unusual DR1+2* haplotypes were conducted by DNA oligotyping and by sequencing of the DRB first-domain exon. The results showed that these DR1 haplotypes, besides their DRB1*0101 allele, carried also a DRB5*0101 allele.

Alloantisera can detect some of the variants of HLA DRw13 identified by oligotyping.

Oligotyping has revealed a considerable polymorphism of HLA DRw13; so far, 5 alleles coded by DRB1 have been identified. An even greater number of haplotypes are apparent when considering the association of DRw13 with alleles coded by DRB3 and DQB1. Serology can now define some of these variants which had formerly escaped detection. We have tested by serology and by oligoprobes 66 genotyped Caucasoid cells comprising the known DR and DQ alleles. Among the 28 DRw6 cells, 10 different haplotype patterns were selected, of which 8 concern DRw13 and 2 DRw14. Four variants of DRw13 were represented: DRB1 *1301, *1302, *1303 and *1305 associated with usual and with uncommon alleles belonging to DRB3 and to DQB1. We have centered our analysis on 19 sera of the XIth Workshop defining DRw6. Two sera (639, 826) were noteworthy since they reacted with certain subtypes of DRw13 and with DRB1 *1102, *1103 only, the other DR specificities remaining negative.

Selection of unrelated donors for bone marrow transplantation is improved by HLA class II genotyping with oligonucleotide hybridization.

As the demand for donors for bone marrow transplantation increases, the use of HLA-matched, genetically unrelated donors represents a promising strategy. It is well documented that the clinical outcome of bone marrow transplantation is directly dependent on optimal matching for HLA class I and class II specificities. Molecular studies have revealed the existence of a much larger number of HLA class II alleles than was anticipated, many of which cannot be recognized by routine serological typing. Currently this "hidden" polymorphism represents a major limitation to the generalized use of unrelated donors for bone marrow transplantation. It has recently become possible, however, to identify HLA allelic polymorphism directly at the DNA level by hybridization with sequence-specific oligonucleotide probes ("HLA oligotyping") after amplification of DNA by polymerase chain reaction. In this study, we have investigated whether donor-recipient pairs that are fully matched for HLA by serology are truly HLA-DR, -DQ, and -DP identical and to what extent class II differences influence the primary mixed lymphocyte culture. We show that HLA oligotyping, performed on 50 pairs of HLA class I and II serologically matched individuals, can indeed reveal phenotypically relevant allelic differences at either DRB or DQB loci in 56% of these pairs and can therefore improve HLA class II typing and the choice of bone marrow donors quite significantly. Oligotyping for DRB/DQB/DPB polymorphism also allows prediction of a positive mixed lymphocyte culture, as established in 31 donor/recipient combinations, and even detection of polymorphic differences that were not revealed by this test. This approach is well suited for accurate HLA typing of large pools of bone marrow donors and was successfully applied to select fully matched donors for bone marrow transplantation.

HLA-DRw11, DQw7 has four cellular subtypes revealed by homozygous typing cells and undetected by restriction fragment length polymorphism.

HLA-DRw11 is in strong linkage disequilibrium with DQw7. We have investigated the heterogeneity of DRw11, DQw7 by cellular typing and by HLA cDNA probes. The results obtained by local and other homozygous typing cells in four informative Caucasoid families and 20 genotyped individuals demonstrate the existence of at least four different cellular subtypes. The frequency of DRw11, DQw7 is 25.7% with the following distribution of subtypes: Dw5 (17.3%), JAC (4.2%), JVM (2.8%), TIS (0.7%), and blank (0.7%). JVM (10W 9039) has been previously described; TIS (10W 9042) and JAC are postulated new specificities from our laboratory. None of these subtypes typed for DRw11, DQw1 cells. The cellular heterogeneity contrasts with the absence of polymorphism observed by serology and by restriction fragment length polymorphism after hybridization with DRB, DQA, and DQB probes. Variation in amino acids of the DRB1 chain has been reported for at least three variants: Dw5, JVM, and TIS (more recently).

Diversity among DRw13 DQw7 haplotypes as revealed by serology Dw typing and restriction fragment length polymorphism.

HLA-DRw13 is in linkage disequilibrium with DQw6; an unusual association, DRw13 DQw7, is found in 2% of our Caucasoid population. Investigation of genotyped individuals and of families by two allosera and by Dw typing revealed two subtypes: one recognized by homozygous typing cell HAG and by two allosera, the other subtype remained unreactive with both reagents. Analysis of DNA fragments with DNA probes indicated that the HAG-negative subset had DNA fragments in common with DRw6 while the HAG-positive subset shared DNA fragments with DR5. However, all DRw13 DQw7 cells are Dw24 as seen by hybridization with DRB probe.

DNA typing of DRw6 subtypes: correlation with DRB1 and DRB3 allelic sequences by hybridization with oligonucleotide probes.

Among MHC class II antigens, the DRw6/Dw6 complex represents a special situation where typing on a routine basis is often troublesome, mainly because monospecific alloantisera are rare and individual subtypes numerous. We demonstrate here that the use of oligonucleotide DNA typing permits an analysis of the polymorphism within DRw6 haplotypes and provides a molecular basis for correlations with functional data. Synthetic oligonucleotide probes, most of them locus- and allele-specific, were derived from the DNA sequences of three alleles of locus DRB1 and three alleles of locus DRB3. These probes allow the positive identification of distinct DRw6 subtypes. As analyzed on a panel of 26 well-defined DRw6 cell lines, oligotyping allows a direct and absolute correlation with the DRw13 serologic specificity and with the cellularly defined Dw9,Dw16,Dw18, and Dw19 specificities. Correlations of the polymorphism at the DRB1 locus with the polymorphism at the DRB3 locus (DRw52 alleles) allow us to identify preferential allelic associations such as DRw13-Dw18-DRw52a/52b, DRw13-Dw19-DRw52c, and DRw13/Dw19 haplotype, the Dw19 cellular reactivity might involve, at least DRw14-Dw9-DRw52b. In view of the absolute segregation of the DRw52c allele with the DRw13/Dw19 haplotype, the Dw19 cellular reactivity might involve, at least in part, epitopes on the DRw52c allele. The identification of DRw6 subtypes, as well as of other HLA class II subspecificities, by oligotyping can now complement and possibly replace serologic and cellular typing. It represents a particularly useful contribution to the optimization of class II matching in the case of bone marrow transplantation with unrelated donors.

Both HLA-DR and HLA-DQ determinants contribute to HLA-Dw typing.

The respective contribution of HLA-DR and HLA-DQ gene products in the induction of allogeneic proliferative responses in primary mixed lymphocyte reaction and, therefore, in HLA-Dw typing, is still unclear or controversial. This is in part due to a strong linkage disequilibrium between HLA-DR and -DQ genes. We used DR- or DQ-restricted influenza-specific T-cell clones to define DR and DQ products on a large panel of allogeneic antigen presenting cells. With this functional screening assay, we identified two haplotypes with unusual DR/DQ associations. Cells of these haplotypes were then used as responder cells in mixed lymphocyte culture and stimulated by homozygous typing cells displaying DR or DQ incompatibilities. Our results indicate that DR or DQ incompatibilities alone can give rise, in both cases, to strong T-cell proliferation in a mixed lymphocyte reaction. This was further verified by blocking experiments of secondary mixed lymphocyte reactions by HLA-specific monoclonal antibodies. Anti-DQ, but not anti-DR, antibodies inhibited DQ-incompatible responses. Conversely, anti-DR, but not anti-DQ, antibodies could block DR-incompatible mixed lymphocyte reactions. Together, the results suggest that both HLA-DR and DQ gene products can be involved in HLA-Dw typing. Finally, in dual DR- and DQ-incompatible mixed lymphocyte reaction combinations, HLA-DR molecules seem to have an immunodominant effect, because the response is mostly inhibited by anti-DR antibodies. Immunodominance of HLA-DR allodeterminants may, at least in part, explain some of the controversial conclusions reported by others concerning the role of HLA-DQ molecules in HLA-Dw typing.

Cellular and serological subtypes of HLA-DR2.

HLA-DR2 and its subtypes were investigated in informative families and unrelated Caucasoid individuals. Serological and cellular typing indicate that DR2 can be divided into DR2 long and DR2 short (sh), each of which can be further subdivided either by HLA-D typing or primed lymphocyte reagents. DR2 long includes Dw2, Dw12 and a new subtype temporarily called D-GEN. The cellular equivalent of DR2sh was recognized by mixed lymphocyte culture (MLC) practised in a family (family FJO); the typing cell individualized was termed FJO. The reactivity of this cell is compared with similar DR2sh varieties described in other laboratories: MN2 (Bach, Minneapolis), AZH (Brautbar, Jerusalem), DB9 (Layrisse, Caracas). The distinction between FJO, MN2 and AZH is not clear, suggesting the existence of shared epitopes; DB9 cells gave typing responses only in family FJO but typed no one else in our panel. Primed cellular reagents FJO anti Dw2 and Dw2 anti FJO when tested against DR2 subtypes support the distinction between DR2 long and DR2sh. The use of these subgroups of DR2 in functional studies, the biochemical analysis of their class II molecules, the pattern of their DNA recombinant fragments and the correlation established with some diseases will be discussed, since they further contribute to the cleavage between DR2 subtypes.

A division of HLA-DQw1 associated with DR1, DR1x-DQw1, DR2 short, DRw10, and DRw14. I. Definition by alloantiserum LY 1327.

We have identified an alloantiserum, LY 1327, directed against part of DQw1-positive cells. This split of DQw1 includes DR1, DR1x, DR2sh, DRw10, and DRw14; the other DQ-associated specificities, -DR2 long and DRw13, are unreactive. Segregation was ascertained in 11 informative Caucasoid families and in 33 genotyped individuals. DR1x refers to a specificity typing as DR blank DQw1, detected by certain anti-DR1 sera and recognized cellularly by HTC DwBON DR blank DQw1 (A. Cambon, Toulouse). Biochemical analysis by two-dimensional gel electrophoresis and DNA analysis by restriction fragment length polymorphism will be discussed since they support the existence of this division of DQw1.

Biochemistry of HLA-DRw6: evidence for seven distinct haplotypes.

The DRw6 specificity, which has a frequency of 11% in the Caucasian population, cannot be positively defined, since no monospecific allo-antiserum is available. This particular status among DR specificities led us to study the DRw6 haplotypes at the molecular level. We performed 2D-PAGE analysis of HLA-DR molecules in 44 different DRw6 haplotypes. The data were obtained from six homozygous typing cells, eight families informative for the segregation of the DRw6 haplotype, and 15 unrelated donors. Five unique beta-chain electrophoretic patterns were detected, indicating the existence of five structurally distinct DRw6 beta-chains. Each haplotype expresses one or two beta-chains. The different combinations of the DR beta-chains present in a single haplotype allow to characterize seven unique DRw6 haplotypes. In contrast to what has been previously found for DR2 and DR4, there is no DR beta-chain common to all the DRw6 cells. Correlation of the biochemical data with the recent serologic (DRw13 vs DRw14) and cellular (Dw9, Dw18, Dw19) splits of the DRw6 specificity will be discussed.

Despite lack of monospecific anti-DRw13 sera, a corresponding class II determinant can be defined by a DRw13 restricted anti-influenza virus T cell clone.

The study of a T3+ T4+ T8- human T cell clone COTC2 with both specific proliferative response and cytolytic activity for influenza A virus infected cells reveals that: the restricting element of this clone is strongly associated with DRw13 molecule(s) as seen by the study of a large panel of antigen presenting cells (APC) and by the observation that monoclonal antibodies (MoAb) specific for DR molecules inhibit its proliferative activity while anti-DQ MoAb do not. These results indicate that there exists a DRw13 associated determinant that can be defined at the functional level by COTC2 recognition despite the absence of monospecific anti-DRw13 serum. In contrast to the results found by other groups, the restriction of this DRw13 restricted clone follows the DRw13 serological definition irrespective of the DW type of the APC. These results indicate that the polymorphism of HLA class II molecules can be further defined at the functional level by monoclonal populations of T cells in conjunction with molecular definition.

A study of HLA-DR2 associated HLA-Dw/LD specificities.

HLA-Dw2 and Dw12 are both associated with HLA-DR2; however, these specificities accounts for only 86% (161/188) of the DR2+ haplotypes in our North American Caucasian panel. In an attempt to identify new DR2 associated antigenic clusters, we have generated four primed lymphocyte (LD) typing (PLT) reagents in haploidentical familial combination against DR2+ Dw blank haplotypes. These reagents were positively restimulated by 11 of 16 DR2+ Dw blank cells tested, with good discrimination from Dw2 and Dw12+ cells, thus identifying a new antigenic cluster provisionally termed LD-MN2. We have compared the LD-MN2 specificity with the specificity LD-5a defined by two DR2+ HTCs, BAS and REM, (Layrisse, Caracas) which have been included in the pre-1984 Workshop Cluster DB9. Although none of our DR2+ cells gave typing responses to these two HTCs defining LD-5a, PLT studies did indicate an interrelationship between these specificities and with the specificity tb24 defined with the HTC, FJO (Betuel). The LD-5a HTCs, four LD-5a heterozygous cells, and two additional HTCs (WJR-Hansen, Seattle and FJO/tb24--Betuel, Lyon) significantly restimulated the anti-MN2 PLT reagents, though usually not as strongly as the MN2+ cells. MN2+ cells primed against the LD-5a HTCs were restimulated by only the LD-5a+ cells. Dw2+ cells primed against FJO were restimulated by some, but not all MN2+ cells. These results suggest that MN2, tb24, and LD-5a share some determinants, not shared with most cells which type as Dw2 and Dw12, though differing by other stimulatory determinants. These studies emphasize the necessity of studying new antigenic clusters by both PLT and HTC methodologies as well as testing different ethnic groups.

A possible new HLA-DR allele.

In routine screening of anti-HLA DR reagents, serum 901 was obtained from a woman of negroid origin ten days after delivery of a second child. The spouse was also of black origin. This serum contained polyspecific HLA A and B antibodies. After platelet absorption it reacted with the B cells of 10 out of 119 European Caucasoid panel donors (8.4%) typed for DR1 to DRW10 and for MT1, MT2. Each of these ten donors had only one recognized DR antigen, the other was "blank." Serum 901 gave negative reactions with the homozygous typing cells (HTC) DW1 to DW8. In 18 normal informative Caucasoid families, serum 901 recognized one HLA haplotype in one (or both) parents and segregated with this haplotype in one or more than one child. In one family in which both parents reacted with serum 901, two children were homozygous for this marker and reacted as HTCs. One of these HTCs typed as DW9 and was found to be identical to a DW9.HTC (8W207).