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

HLA-Dw2 and Dw12 are both associated with HLA-DR2; however, these specificities account 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 combinations 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.     

HLA-D clusters associated with DR2 and the definition of HLA-D"AZH": a new DR2 related HLA-D specificity in Israel

In order to investigate the HLA-D clusters associated with DR2 in Israeli Jews, 40 DR2 positive unrelated individuals were studied with a panel of DR2 associated homozygous typing cells (HTC's) which detect the lymphocyte defined specificities HLA-Dw2, Dw12, Dw9 and D-WJR. The results confirmed the existence of two distinct HLA-D clusters associated with the same serologically defined DR2. Of 40 individuals 22.5% (9/40) were Dw2 and 50% (20/40) were Dw12 carriers. Yet, no HLA-D specificity could be assigned to the remaining 11 DR2 positive individuals. In the present study we have defined a unique DR2-associated Dw specificity, HLA-D"AZH". The donor of the HTC was of Moroccan origin and an offspring of a first cousin marriage. This cell was not typeable with the known DR2-associated homozygous typing cells nor with other HTC's which define the well established HLA-Dw1 to Dw11 specificities. It was shown to segregate with DR2 positive HLA haplotypes in family analysis and in a population study, typed out 7 of 11 unrelated DR2 positive, Dw blank individuals, thus identifying a unique and new HLA-D cluster provisionally designated D"AZH".     

HLA-DR4 associated Dw types in rheumatoid arthritis

Frequencies of HLA-DR4 and its related Dw types were compared between randomly selected normal controls and the index cases of multiplex rheumatoid arthritis (RA) families. A DR4 frequency of 68.3% was observed in index cases (n = 57) compared to 31.2% in normal controls (n = 96). Cellular typing with homozygous typing cells (HTCs) revealed significant increases of Dw4 (49.1% vs 22.9% RR = 3.2 p less than 0.001) and Dw14 (22.8% vs 2.1% RR = 13.9 p less than 0.001) in the index cases. A non-significant increase was seen for Dw13 (8.8% vs 4.1%). When DR4 positive patients and controls were compared, a significant increase was seen only for Dw14 (34.2% vs 6.6% RR = 7.3 p less than 0.01). Data from HLA genotyped RA and normal families allowed an examination of haplotype combinations of HLA-B antigens and DR4/Dw types to be made. HLA-Dw4 was predominantly found with B44 and Bw62 with nearly all DR4/Bw62 haplotypes being Dw4 positive. HLA-Dw13 was associated with B44 and Dw14 with Bw60, B44 and B27. Based on HTC and normal family data. Dw10 was found to be strongly associated with B38 containing haplotypes. Analysis of 69 C4A, C4B complement typed DR4 haplotypes failed to show any statistically significant association between Dw type and "complotype". However, there was a suggestion of C4A3. BQO being associated with Dw4 (34.2% vs 16.1% X2 = 2.9 p = ns) and C4A3, B1 with Dw14 (45.5% vs 27.6% X2 = 2.1 p = ns).(ABSTRACT TRUNCATED AT 250 WORDS)     

Determinants not correlated with HLA-Dw,DR, or SB detected by primed lymphocyte typing

We have identified a new HLA-Dw cluster, defined by five HTCs: 8W309 from the Eighth International Workshop, MN-LS and Bin-40 obtained locally. THO (Hansen), and RZoo (Hsu). Although highly associated with DR4, LD40 appears to be distinct from Dw4 and Dw10 {Hum Immunol 4:249. 1982}. PLT studies on LD40 were performed using intrafamilial PLT prepared in haploidentical combinations in which both stimulator and responder carried DR4 on the second haplotype and priming was only against LD40 or associated determinants. These reagents were apparently LD40-specific, as they were restimulated by ali DR4-LD40-positive cells with good discrimination from DR4-positive, LD40-negative cells.Whereas priming in a HLA-Dw-incompatible. DR-compatible combination produces PLT reagents with reactivities associated with the incompatible Dw specificity, further analysis of the D region is simplified if there is Dw and DR matching in the priming combination. A second type of reagent was generated using intrafamilial PLT prepared in a family in which two LD40 haplotypes were segregating: responder and stimulator shared one haplotype, and both carried DR4-LD40 on the second haplotype but associated with different A, B, and C antigens. This reagent appeared to recognize determinant(s) associated with several different haplotypes: among the subcultures derived from this reagent, several were found in which positive restimulation did not correlate with any particular A, B, C, DR, Dw, or SB/PL3/D^β type.These results suggest that the PLT test may detect (a) shared or cross-reactive antigenic determinants of HLA-Dw/DR as presently defined and/or (b) determinants distinct from Dw and DR. Although some of the latter, as detected by subcultures, appear to correlate with SB specificities, other show no correlation with presently defined Dw, DR, or SB antigens.     

Five HLA-D clusters associated with HLA-DR4

In order to investigate the HLA-D clusters associated with DR4, 54 DR4-positive, Dw4- and Dw10-negative responders, together with selected Dw4- or Dw10-positive responders, were tested with 22 HTCs that define DR4-associated D specificities. The results are consistent with previous data defining four distinct D clusters--Dw4, Dw10, DB3, and DYT--and have identified a new cluster provisionally termed LD40. In addition, the DB3 cluster is complex and appears to give typing response patterns overlapping those of the KT2 cluster originally defined as being associated with DR4 in Japanese populations. Of 116 DR4-positive haplotypes tested, 44% typed as Dw4, 18% were LD40, 16% were Dw10, 9% were DB3, 3% were DYT, and 10% gave no typing response to the HTCs defining any of these clusters. These studies are informative not only in defining the DR4-associated D clusters and in supporting the concept that D and DR cannot be considered identical but also in emphasizing the complexity of the D region.     

HLA-D region heterogeneity in a Nigerian population

HLA class II antigens were studied in a panel of 130 Nigerians. Complex patterns of associations were seen between HLA-Dw, -DR and -DQ specificities, differing widely from those reported for other populations. A number of Dw types were associated with the same DR antigen: Dw'1N' and Dw'BERN' with DR1, Dw2 and Dw'2N' with DR2, Dw5 and Dw'5N' (Dw5 + Dw'F5') with DRw11. It was also observed that a Dw type associated with more than one DR antigen: HLA-Dw3 was assigned to individuals who were DR3 negative and similarly Dw10, Dw13 and Dw14 to individuals negative for DR4. HLA-DRw8 and Dw8 were completely dissociated in Nigerians, and Dw8 did not show a preferential DR association. These results demonstrate that DR and DQ identity between HTC stimulator and responder cell is not necessarily a prerequisite for Dw to be assigned. Preliminary studies show that subtypes of HLA-Dw1 and Dw8 detected by HTC typing correlate with restriction fragment length polymorphisms (RFLPs) detected with a combination of Bgl II enzyme and DRA/DRB cDNA probes. HLA-DP antigen frequencies differed between Nigerians and British Caucasoids. The most common DP antigen in Nigerians was DPw1, compared with DPw4 in Caucasoids. HLA-DPw6 appeared to be absent or rare in both Nigerians and British Caucasoids. Only five out of 68 Nigerians tested were assigned two DP specificities. The association between HLA-DR3 and DPw1 reported in Caucasoid panels was absent in Nigerians.     

The new HLA-DRw6- and 8- associated HLA-Dw HAG specificity defined by homozygous typing cell 9W 1802

Intrafamilial primary mixed lymphocyte culture (MLC) typing established that an HLA-A, B, C homozygous, DP heterozygous donor HAG was homozygous for HLA-Dw and behaved as a homozygous typing cell (HTC). Both haplotypes of the HTC were HLA-DR identical, but could not be assigned a clear DR specificity, giving reactions with sera containing antibodies against DRw6, DRw8 and TA10. MLC checkerboard studies failed to assign the HTC HAG specificity to any established or provisional cluster, suggesting that it defined a new Dw specificity. Primed lymphocyte typing (PLT) clones derived from intra-familial priming against either HAG haplotype displayed heterogeneous reactivity patterns. One clone was restimulated only by family members and unrelated donors positive for Dw HAG. Other clones were restimulated by determinants associated with either Dw8 or Dw6. Blocking of stimulation with monoclonal antibodies against different class II molecules suggested that while stimulatory determinants associated with Dw HAG and Dw8 were classifiable as HLA-D related, those associated with Dw6 were of a DP-like nature.     

A new HTC, a PLT and a PLT clone define a split of HLA-DR2 in the Austrian population

An LD defined split of HLA-DR2, different from HLA-Dw2, Dw12 and DB9, is observed in the Austrian population. The narrow HLA-D specifity, defined by a new Austrian Homozygous Typing Cell (HTC), a primed lymphocyte (PLT) cell line and a PLT clone, is tentatively termed HLA-D "WH". Investigation of three informative families shows that HLA-D "WH" segregated together with HLA-DR2. In one family, even an individual heterozygous for HLA-DR2/Dw2 and HLA-DR2/D "WH" can be identiñed. A panel study including 90 non-related Austrians shows that HLA-D "WH" occuring in 6.7% of the whole panel, that is in 20% of the HLA-DR2 positive panel members, is completely included in HLA-DR2. HLA-D "WH" might be comparable to other LD specificities related to HLA-DR2, "MN2", "FJO", "AZH" or "GEN".     

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).     

Intra HLA–D Region Recombinant Maps HLA–DR between HLA–B and HLA–D

A consanguineous family has been typed for HLA-A, B, C, D, DR and GLO, Bf, C2 and C4 and other red cell markers. The results indicate that an expected Dw7 homozygous sibling is a "Dw1"/Dw7 recombinant, probably derived from a crossing over between DR and D on the paternal haplotypes. The anomalous typings by Dw1 HTCs of the recombinant haplotype are best explained by the likely presence of an additional Lad polymorphism which could be a serologically detectable second epitope on the same molecule as the HLA-D determinant or in the form of two linked chains, one encoded by HLA-D and one by another Lad gene in the HLA haplotype.     

Definition of LD “4 × 7”: A unique HLA-D specificity defined by two homozygous typing cells

Two homozygous typing cells (HTC) from the Eight International Histocompatibility Workshop were used to define a unique HLA-D specificity, designated LD “4 × 7”. Typing cell 8W316 (Seattle) was obtained from a donor of Japanese descent: cell 8W324 (Leiden) was obtained from a European Caucasian donor. The HLA-D specificities defined by these two HTC showed a significant correlation (r = 0.65) i unselected panel studies and clear segregation as a single determinant within families. LD “4 × 7” was found in low frequency in Caucasians (1.0%) but was detected with a frequency of 24% in Japanese. The cells of the donors of HTC 8W316 and 8W324 express DRw9, and the LD “4 × 7” antigen fills an HLA-D locus “blank” on DRw9-positive haplotypes. The frequency of LD “4 × 7” noted in this study corresponds to the frequency of DRw9 reported for Japanese and Caucasian ethnic groups, a further indication that LD “4 × 7” and DRw9 are associated.     

HLA-D typing with homozygous cells identified in an American indigenous isolate: II. Family studies and D/DR relationship

Twelve American Indian nuclear families with 2-5 siblings have been HLA-D typed using mixed lymphocyte cultures and clusters of homozygous typing cells (HTC) of Caucasoid origin to detect DW1-DW7 and typing cells of American Indian origin to detect LDSA, LD15A and LDl5B antigens. Results obtained demonstrate complete absence of DWl-DW7 in these families and illustrate the inheritance and segregation of LDSA, LDl5A and LD15B. DR typing results obtained with the 8th International Histocompatibility Workshop genetic set of antisera indicate inheritance in coupling of DR2 with LD5A, of DR6.2 (DR3+6, MTl negative, MT2 positive) with LD15A, and of DRW8 with LDlSB. The existence of MLC activating antigen(s) different to DW4, yet associated to DRW4 in this population is postulated. The D/DR relationship present in this American Indian isolate demonstrate once more that DR2 can be inherited in combination with an HLA-D antigen different to DW2, and that LD15A HTC define a second sub-cluster of the broad DW6 specificity group, which is inherited with DRW6.2 and BW62 antigens in the Warao population.     

Two homozygous typing cells defining a subgroup of HLA-Dw6

Homozygous typing cells (HTC) and responder cells from a panel of randomly selected unrelated caucasians were used in a study of the HLA-Dw6 antigen complex. Using HTC characterized by the Eighth International Histocompatibility Workshop, the Dw6 specificity was shown to be a broadly defined, heterogeneous cluster that could be subdivided. An HTC from our laboratory, 8W146, and a second locally identified cell. EMJ, were used to define one clearly distinguishable subcluster of Dw6, provisionally termed “6.1”. HTC 8W146 and EMJ were mutually nonreactive in mixed leukocyte culture and showed strong association (r = 0.85) when used as stimulators against the caucasian cell panel. The calculated gene frequency of the 8W146/EMJ determinant in this population was 0.024. In an informative family, the 6.1 specificity could be shown to segregate independently of other Dw6 subgroups. DR typing of 8W146 and EMJ showed both to be positive for MT-1 and MT-2 but negative for DRu “3 + 6”.     

HLA antigens in Asian Indians: HLA-D-DR relationships

Fifty-nine Asian Indians were typed for HLA-A, B, D, and DR antigens. Peculiar to the population that we have tested was the absence of HLA-A25, B13, B14, DR1, DW1, LD13 (a DR1-associated HLA-D allele), and LD12 (a DR4-associated HLA-D allele). Certain haplotypes that exhibit high frequencies in Caucasians (such as A2-BW50, AW24-B18, B8-DR3, BW44-DR7, B18-DR5) or in Blacks (such as A29-B7) also show significant delta in Asian Indians. The HLA-D-DR associations previously described in European and North American Causcasians were also found in Asian Indians. Additionally, however, Asian Indians exhibited two new HLA-D antigens, one associated with DR5 and the other with DRw6. The genetic distance between Asian Indians and Caucasians, Blacks, or Mongoloids is of the same order of magnitude.     

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.     

HLA-D-DR relationships: PLT studies of HLA-D specificities associated with DR1, DR2, and DR4

The primed lymphocyte test (PLT) detects gene products of the HLA-D-DR region which activate the secondary (memory) response of MLC stimulated T cells. In the present study attempts were made to determine whether different HLA-D alleles associated with the same DR, such as DR1, 2, and 4 can be discriminated by PLT typing. PLTs were generated by using, as responders and primary MLC stimulators, HLA-D different HTCs which shared all DR groups (major DR, supertypic MT and second locus MB) or only the MT or MB groups. As secondary stimulators, lymphocytes from an HLA-D selected panel of 72 individuals were used. PLTs raised in DR identical responder-primary stimulator combinations were able to discriminate between the different HLA-D antigens associated with the same DR. In contrast, when priming was performed in combinations differing for the major DR group, the restimulation response was highly associated with the DR specificity of the primary stimulator, regardless of whether or not this was compatible with the responding HTC for the MT or MB groups.

This data indicate that the specificity of primed lymphocytes largely depends on the combinations used for priming and that the memory response can be activated by both HLA-D and DR antigens. The dissociation of HLA-D from DR by PLT typing might provide a useful tool for further analysis of this HLA region.     

HLA-D Antigens in the Japanese Population

A Japanese population was typed for HLA-D antigens for the first time using HLA-D homozygous typing cells Dw1 through Dw4 and the homozygous cells LD HO and LD AH, newly found in the Japanese population. Dw3 was absent from the Japanese population which also lacks B8. Dw1, Dw2 and Dw4 were found in this population, but these did not show significant linkage disequilibria with any alleles of the B or C loci. LD HO and LD AH were common in Japanese but absent in Caucasians. LD HO was found to be in strong linkage disequilibrium with Bw35. LD AH showed significant association with Bw22J.     

The HLA system in the Korean population

The frequencies of HLA-A, B, C, DR, and DQ antigens, HLA-D (HTC-defined) haplotypes, and the HLA-linked genetic markers glyoxalase I (GLO), factor B (Bf), C2 and C4 were studied in 162 healthy unrelated Koreans. Antigens A2, A24, A26, B44, B51, Bw62, B35, Cw1, Cw3, DR2, DR4, DRw6, DR7, and DRw8 were observed at frequencies of 15% or greater, and GLO-2, BfS, C4A*3, C2C, C4A*4, C4B*1, and C4B*2 were also frequently observed. The antigens A23, A25, B18, Bw42, Bw47, and B21 were not observed at all. HLA-DR4 was the most common class II antigen and was associated with a series of HLA-D-defined haplotypes including Dw4, Dw10, Dw13, and Dw15. The HLA-DRw6, DR2,Dw8, and DRw8 haplotypes were also found frequently. DR2 haplotypes were either Dw2 or Dw12, while all DRw8 haplotypes tested corresponded to the DB7 or Dw "8.3" specificity that has been described in other Oriental populations. Significant linkage disequilibrium was found between the alleles A2,Cw1; A30,B13; A30,Cw6; A30,DR7; Cw1,Bw22; Cw5,B12; Cw6,B13; Cw6,DR7; B7,DR1; B12,Dw6; B12,DR7; B12,Dw7; B13,DR7, B17,DR3; Bw22,C4B*6; DRw6,BfF; and C4A*4,C4B*2. A comparison of gene frequencies and commonly observed haplotypes between Koreans, Chinese, Japanese, and Caucasians showed that while Koreans share several characteristics in common with other Oriental populations, there are allelic frequencies and haplotypes in Koreans that are distinct.     

Comparison of DR beta 1 alleles from diabetic and normal individuals

Insulin-dependent diabetes (IDD) is positively associated with HLA-D proteins. A critical question is whether or not sequence differences within the HLA-D coding region are the same or different in diabetics and normal individuals of the same haplotype. We have isolated both DR beta 1 alleles from a Dw4/LD MN2 cDNA library and compared them to DR beta 1 genes isolated from normal individuals of the same Dw phenotype. We found no nucleotide differences in the coding region between the normal and diabetic alleles of DR beta 1 suggesting to us that DNA differences other than the DR beta 1 coding region may account for the observed association of HLA-D and diabetes.     

A New Determinant, Defined by PLT, Coded for in the HLA Region and Apparently Independent of the HLA-D and DR Loci

A PLT cell was raised between a responder cell which carried the HLA-D and DR specificities Dw3, 8; DRw3, 8 and a stimulator cell which was most likely homozygous for HLA-Dw3, DRw3. The PLT cell appeared to recognize a determinant (PL3A) which was (1) different from the officially recognized HLA-D and DR determinants, (2) was associated with HLA-A1, B8, Dw3 and DRw3, and (3) segregated in three informative families with HLA. Another responder-stimulator combination could be selected to raise PLT cells which recognized the same determinant.