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".     

Mixed Lymphocyte Culture Search for HLA-D Homozygous Typing Cells in the Hungarian Inbred Population of Ivád

Six functionally HLA-D homozygous typing cells were identified by a restricted investigation into the Hungarian inbred population of Ivád. These putative HLA-D homozygous typing cells were then tested against a highly selected Scandinavian population sample of 60 individuals previously typed by histocompatibility reference reagents. The different HLA-D specificities could thus be identified: one closely matching HLA-Dw5, another resembling the Oslo LDoH specificity, while the last seems to be unique. Only one of the typing cells thus ascertained were HLA-B homozygous and were selected on the basis of the Ivád family structure and not on the basis of serological HLA typing.     

Ninth International Histocompatibility pre-workshop testing of Dw6 HTCs. Two subtypes of Dw6

In the scope of the cellular part of the 9th International Histocompatibility Workshop, the offered homozygous typing cells (HTCs) of several specificities have been screened in a pre-Workshop. In Leiden and Seattle all HTCs typing for "HLA-Dw6" have been tested. This implied that in both laboratories mixed lymphocyte culture (MLC) matrices were performed between the Dw6 HTCs and that all HTCs were tested as stimulator cells against a panel of heterozygous responder cells. The results clearly demonstrated that "HLA-Dw6" as defined by the different participating laboratories can be split into two major groups, Dw6.a and Dw6.b. This observation confirms and extends previous reports. None of the 9th Workshop B-cell sera could discriminate between the two presently described subgroups of HLA-Dw6.     

Alloreactive T cells: Expression of HLA-D antigens, stimulation of autologous MLR, and possible immunoregulatory function

Primed in MLC with allogeneic stimulators T cells acquire the capacity of expressing HLA-D and DR antigens and of stimulating the MLC response of autologous lymphocytes When primed T cells from HTCs are used as stimulators, a bimodal distribution of response with clear-cut “typing responses” and no significant “back stimulation” is observed This pattern may be due to the expansion during priming of a population of HLA-D restricted suppressors since irradiated primed T cells inhibit the MLC responsiveness of HLA-D “compatible” lymphocytes. The development and size of such a population is not dependent, however, on the strength of the antigenic stimulus used for priming since no differences were seen between the pattern of reactions induced by T cells primed against HLA-D identical or HLA-D different cells Primed OKT4⁺ and OKT8⁺ T cells share the capacity of expressing Ia antigens and of inducing “HLA-D restricted suppression.” We suspected that a similar phenomena accounted for the behavior of two HLA-D heterozygous cells as if they were HTCs Although no suppression was found, the fact that these cells typed for their “silent” antigen when tested as responders, yet failed to express it when tested as stimulation, supports the theory that different genes control the MLC-responding and stimulating capacities.     

Primed lymphocyte typing predicts MLC reactivity between unrelated individuals

The present study was carried out to evaluate the ability of primed lymphocyte typing (PLT) to predict mixed lymphocyte culture (MLC) reactivity between unrelated individuals. Eleven PLT cells generated against independent familial haplotypes, and one PLT cell generated against a homozygous typing cell (HTC), were tested for their response to cells of a random panel of 53 unrelated individuals. From Chi-square analysis of the response patterns of these 12 cells, nine "PLT specificities" could be defined. Most panel members (81.2%) types for 1 or 2 of these specificities; equal numbers (9.4%) types for 3 or none, respectively. Four of these 9 specificities were shown to be significantly correlated with HLA-Dw antigens defined by HTCs. Typing for these nine PLT specificities was found to be predictive of subsequent primary MLC reactivity between panel members: a) pairs of panel members sharing PLT specificities produced three-fold lower MLC results on the average than pairs of panel members disparate for PLT specificities (p less than 0.0001), and b) in MLC combinations, an increase in number of foreign PLT specificities presented by the stimulator cell was paralleled by a statistically significant increase in MLC reactivity. To the extent that low MLC reactivity is correlated with improved graft survival, the PLT method could have significant value in selecting histocompatible donors for organ transplantation.     

Correlations between HLA-D/DR associated Primed Lymphocyte Typing (PLT) defined DP-antigens, HLA-D and HLA-DR antigens

A panel of 79 individuals were typed for HLA-D/DR associated Primed Lymphocyte Typing (PLT) defined "DP"-antigens, HLA-D and HLA-DR antigens. Typing for DP-antigens was carried out with local PLT-cells. HLA-D and-DR typing was performed with all homozygous typing cells and all DR-antisera included in the 8th International Histocompatibility Workshop. Assignments of DP-, HLA-D-and HLA-DR-antigens were done independently and the correlations between DP/D/DR1–8 were analyzed. The panel included random unrelated individuals, and individuals previously found to have one or no identifiable HLA-D antigen (B). In the random group, 80% of the individuals were assigned to possess the same antigen with the 3 techniques, while this was only the case in 46% of B-group individuals. The overall correlation coefficients, r, for the antigens HLA-Dw/-DR/DP1–8 were 0.95 (DP/D), 0.94 (DP/DR), and 0.89 (D/DR). There is a remarkably strong correlation between HLA-D and-DR typing results concerning D/DR1–8, in particular in random individuals. It is possible to select PLT-cells that give typing results which are almost identical to those of HLA-D and-DR typing. When discrepant results were seen, HLA-DR was in general "broader" than DP which in turn was broader than HLA-D, indicating that it may be possible to split HLA-DR/DP1–8 into more "narrow" specificities.     

HLA-D typing in Austria. A panel study using 26 newly defined homozygous typing cells

A panel of 120 HLA-A, -B, -C and -DR typed Austrians has been typed for HLA-D by the use of 26 Homozygous Typing Cells (HTC). The new Austrian HTC, partly defined by the 9th International Histocompatibility Workshop (9WS), partly by a checkerboard experiment with internationally well defined reference HTC, type for HLA-Dw1 to -Dw7 and an obviously new, so far unknown HLA-DR2 related HLA-D determinant. Associations of HLA-DR and HLA-D antigens in Austria and their frequencies are determined. Antigen frequencies in Austria are compared to frequencies in other Caucasoid populations.     

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.     

Definition of DW8.2 by primary and secondary mixed lymphocyte cultures

Using HLA-DW8 homozygous typing cells (HTC) of different ethnic origin it is possible to identify three subgroups of the DW8/DRW8 product (Mickelson et al., 1983). To further characterize the DW8.2 subgroup defined by HTCs of Amerindian origin we have now generated bulk PLTs within members of one extended Amerindian family and within selected HTCs of Caucasian, Oriental, and Amerindian origin. A panel of 61 DRW8 positive and negative donors of the three ethnic groups was used to test 15 different PLTs. Our results demonstrate that it is possible to generate DW8.1, 8.2, or 8.3 sensitized lymphocytes which distinguish in secondary cultures between each of the three subgroups of the DW8/DRW8 products. Of 40 DRW8 cells tested, 100% Caucasians typed as DW8.1, 100% Amerindians were 8.2; 75% Orientals were DW8.3; 8.3% were DW8.2, and 16.6% could not be classified within any of these subgroups. DRW8 individuals of mixed ethnic origin typed as either DW8.1 or DW8.2 and one DRW8 homozygous donor behaved as heterozygous 8.1/8.2. These results confirm the subdivision of the DW8/DRW8 product and explain the poor correlation and unexpected responses reported in MLC with DW8 HTCs and DRW8 donors of different ethnic origin.     

HLA-DR genes and antigens in the Danish population.

Five hundred unrelated Danes, including 202 healthy individuals, 35 cadaveric kidney donors, and 263 patients in terminal uraemia were typed for the HLA-DR antigens DR1—w8.
The HLA-DR gene frequencies of healthy Danes are in good agreement with frequencies estimated during The Eighth International Histocompatibility Workshop of the Scandinavian population, but differ to some extent from other groups of European Caucasians. This indicates geographical variation in the distribution of DR genes within Europe.
Very close associations were found between the HLA-DR antigens and their corresponding HLA-D specificities, except for Dw4 and Dw6, which were included in DR4 and DRw6, respectively.
Linkage disequilibrium was calculated between alleles of two loci ( HLA-A,-DR; -B-DR and -C,-DR ) using the population data. Furthermore, linkage disequilibrium involving HLA-DR alleles was assessed for multiple loci by using haplotype data from 32 HLA-A,-B,-C,-D,-DR, and Bf typed Danish families.
The haplotype data showed that HLA-DR4 is strongly associated with HLA-B15 only in haplotypes together with HLA-Dw4. This indicates genetic heterogeneity of HLA—DR4 which, however, does not appear serologically in this study.     

HLA-A, B, C, D, DR Antigens and Primed Lymphocyte Typing (PLT) Defined DP-antigens in Juvenile Chronic Arthritis

A total of 48 patients with juvenile chronic arthritis (JCA) were typed for HLA-A, -B, -C, -D and -DR antigens and 36 patients were also typed for HLA-D/DR associated "DP"-antigens with the primed lymphocyte typing technique. In the total group of patients, we found increased frequencies of HLA-B27, HLA-Dw/DP5 and HLA-Dw/DP8, and decreased frequencies of HLA-Dw/-DR/DP2. The increased frequencies of HLA-Dw/DP8 and the decreased frequencies of HLA-Dw/-DR/DP2 were primarily found among patients with persistent pauciarticular arthritis. The frequencies of HLA-Dw/-DR/DP4 were increased in patients with polyarticular arthritis. The frequencies of HLA-B27 and -Dw/DP5 were increased in both pauciarticular and polyarticular arthritis.
The results indicate (i) that genetic factors controlled by HLA confer susceptibility and/or resistance to JCA, and (ii) that the clinical subdivision of JCA into pauciarticular and polyarticular JCA can be supported by the presence of different genetic markers (HLA-antigens) in the two groups of JCA-patients.
If these data can be confirmed HLA-D, -DR or DP typing may be of value in the prognostic evaluation of patients with pauciarticular onset JCA.     

Stimulation in primary MLR caused by a PLT defined non-HLA-D/DR determinant, EP1

The influence in primary mixed lymphocyte culture reaction (MLR) of a primed lymphocyte typing (PLT) defined non-HLA-D/DR determinant, EP1, was investigated. In primary MLR between HLA-D/DR compatible lymphocytes, the response of the lymphocytes from 14 EP1-negative HLA-D/DR heterozygous individuals towards two EP1-positive homozygous typing cells (HTCs) was on an average approximately 35% higher than the response towards two EP1-negative HTCs ( P < 0.01). The strength of the MLR between lymphocytes from 25 EP1-negative and 10 EP1-positive individuals matched for two HLA-D/DR antigens was investigated. The average responses of EP1-negative lymphocytes against EP1-positive lymphocytes were approximately 40% higher than the average responses against EP1-negative lymphocytes ( P < 0.01). These data indicate that the PLT defined determinant EP1 causes stimulation in primary MLR.     

A reliable and efficient high resolution typing method for HLA-C using sequence-based typing

Abstract: Serological typing of HLA-C has been poor and almost half of its alleles are serologically undetectable blanks in most populations. Therefore, DNA typing techniques have been used to identify and type for the HLA-C gene. Sequence-based typing (SBT) has proven a major typing strategy for highly polymorphic HLA genes. The technique enables direct identification of all sequence motifs without the need to continuously adjust primers. Here we describe a reliable solid-phase SBT strategy for HLA-C which can be used to distinguish all currently known HLA-C alleles without prior knowledge gained by low resolution typing. Exons 2 and 3 were amplified and sequenced and if necessary sequences of exons 1 and 5 were determined. A total of 257 individuals were typed for HLA-C using this protocol and 30 of the 42 known HLA-C alleles were detected. All heterozygous combinations found in this study were unambiguously discriminated.
One hundred and forty-four individuals from the Dutch population were typed randomly. In this group Cw*0701 and *0702 were the most frequently detected alleles. Of the serological Cw blank alleles Cw*1203 was found to have the highest frequency (16%). From the total group 212 individuals were typed serologically and 106 were retyped with 97 selected antisera to further compare serological and molecular defined phenorypes. Discrepancies between serological typing and SBT are mainly attributable to the serologically Cw blank alleles Cw*12–18.
The high resolution SBT protocol described will be a valuable tool for the identification of HLA-C alleles and the determination of the role of HLA-C in marrow and organ transplantation.     

Comparison of typing results by serology and polymerase chain reaction with sequence-specific primers for HLA-Cw in 650 individuals

HLA-Cw typing by standard serological techniques is associated with a high frequency of blanks, and reliable typing reagents for several of the Cw specificities are scarce. We evaluated the PCR-SSP technique for Cw typing in 370 kidney transplant patients and 280 healthy blood donors. Serological typing of all individuals was performed in our laboratory from 1995 to 1997 using commercially available tissue-typing trays. Comparison between serological and PCR-SSP typing revealed a discrepancy rate of 33.6% ( n = 94) in blood donors and 32.4% ( n =120) in kidney recipients. Incorrect antigen assignments occurred only rarely (3.6% of the blood donors and 3.2% of the kidney recipients). The vast majority of discrepancies were due to antigens that were not detected serologically. In 26 individuals no Cw antigen was detected by serological typing, whereas PCR-SSP showed 1 allele in 13 and 2 alleles in the other 13 cases. Another 269 individuals were typed serologically with one blank (presumably homozygous). Of these, only 108 were confirmed to be homozygous, whereas an additional Cw allele was found in the remaining 161 cases using the SSP technique. Most of the "missed" specificities (86.5%) were those for which serological reagents were not available (HLA-Cw*12-*17). The most commonly "missed" specificity was HLA-Cw*1203, which occurred in 13.9% of the healthy blood donors. These results indicate that serological HLA-Cw typing is insufficient for examining the clinical importance of HLA-Cw matching in transplantation. Future studies based on molecular typing should allow the proper investigation of HLA-Cw matching in kidney and bone marrow transplantation.     

Role of the mixed lymphocyte culture (MLC) reaction in marrow donor selection: Matching for transplants from related haploidentical donors

Abstract: The utility of the MLC assay as a test of HLA-D region matching and predictor of acute graft-versus-host disease (GvHD) was evaluated in 157 patients receiving marrow grafts from HLA-A, B identical related haploidentical donors. All donors and recipients were tested by HLA-DR serology, by Dw phenotyping with homozygous typing cells (HTC) and by standard MLC. Ninety-nine of the donor-recipient pairs were mismatched for a serologically defined HLA-DR antigen while 109 pairs were mismatched for the HLA-D region by HTC typing. Donor anti-recipient relative responses (RR) in MLC, corresponding to the GvHD vector in marrow transplantation, ranged from -4% to 100%, with a median of 25%. A comparison of reactivity in MLC with presence or absence of matching by Dw phenotyping, however, showed a significant overlap in the distribution of RRs from HLA-Dw matched versus Dw mismatched pairs, suggesting that the MLC was not a reliable predictor of HLA-Dw matching. Using an optimally-defined cutoff of 3% RR, the MLC was correlated with risk of developing clinically significant grades II–IV acute GvHD (p = 0.03) but not with risk of developing severe grades III-IV GvHD (p = 0.18). In contrast, matching by Dw phenotype was a significant predictor of GvHD, with Dw-compatible transplant recipients less likely to develop either grades II-IV (p = 0.004) or III-IV (p = 0.036) GvHD than Dw-incompatible transplant recipients. Overall, these results underscore the difficulty in using the MLC to measure HLA-D region compatibility and predict the risk of severe graft-versus-host disease among patients receiving related haploidentical marrow grafts. HLA-D (HTC) typing results correlate primarily with DRB compatibility, and with the advent of DRB1 allele matching by sequence-specific oligonucleotide probes (SSOP) or by direct sequencing, the precision in donor matching achievable with these methods is far greater than with either HLA-D typing or direct MLC testing.     

HLA-D "BG" in Israel

An homozygous typing cell for a local HLA-D determinant"BG", defined by family study, is described. HLA-D "BG" recognizes a specificity identical to the Japanese HLA-DHO (Dw12). Two families and 102 randomly selected healthy Israeli individuals were typed for the HLA-D "BG" determinant. HLA-D "BG" showed segregation as a single determinant within families. The gene frequency for this allele was 0.045, compared to the frequency of 0.148 for DHO in Japanese. In the Israeli population "BG" is in strong association with HLA-Bw52 (joint occurrence = 7.80%) and DR2 (joint occurrence = 8.80%), resembling the data reported foar DHO (Dw12) in Japan. The frequency of this allele may partially account for the low frequency of HLA-Dw2 in the Israeli population.     

Typing for HLA-D/DR Associated DP-Antigens with the Primed Lymphocyte Typing (PLT) Technique

A total of 74 healthy unrelated random individuals and 36 patients with juvenile rheumatoid arthritis (JRA) were typed for HLA-D antigens with the homozygous typing cell technique and typed for HLA-D/DR associated DP-antigens with the primed lymphocyte typing (PLT) technique. All patients and some of the controls were also HLA-DR typed with a limited battery of anti-DR sera. Selected PLT-cells, specific for the HLA-D/DR antigens D/DRw1-8 and the local specificity D"H" were used. The results of the PLT-experiments were evaluated with the Normalized Median Response (NMR) method and the further procedure of DP-antigen assignment was analyzed. The DP-antigen assignments could be done solely according the NMR-values in approximately two thirds of the individuals. In the remaining individuals, further interpretation of the experimental data had to be done for the assignment of DP-antigens. The correlation coefficients were estimated between the HLA-D assignments and (i) the individual PLT-cell NMR-values with a fixed cut-off for positive reactions and (ii) the DP-antigen assignments. These coefficients were 0.79 and 0.92, respectively. The correlations between HLA-D, -DR and DP-antigen assignments of the specificities HLA-D, -DR and DP1, 2, 3, 4, 7 and 8 were analyzed in 42 controls and 36 JRA patients. The total correlation coefficients were: (i) HLA-D/DR: r = 0.78; HLA-DR/DP: 0.77; and HLA-D/DP: 0.96. The DP-antigen assignments correlated significantly better with HLA-D than with the HLA-DR antigen assignments, which does not agree with other studies. The DP-antigen frequencies among the controls were calculated and the estimated sum of gene frequency corresponding to definable DP-antigens was 0.94 indicating that about 12% of random individuals possess as yet undefined DP-antigens.     

The Influence of Matching for SB on MLC Typing Is Significant but Marginal

To investigate the role of SB in MLC typing responses, reactions of lymphocytes from 23 DW3-positive, HLA-D-heterozygous individuals against 9 Dw3 homozygous typing cells (HTCs) were evaluated. Significantly more clear typing reactions were observed in those combinations that were matched for SB as compared with those that were mismatched. Nevertheless, MLC responses towards HTCs that were HLA-D/DR- and SB-compatible could be very strong. An additional analysis of the influence of HLA-B and the newly defined determinants LB-Q1 and LB-Q2 demonstrated that in combinations that were matched for these markers as well, stabilized relative responses could still be over 100%.     

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.     

Identification of four SB antigens by cloned cells: Population studies of Norwegians

By priming in vitro with allogeneic HLA-DR compatible and also HLA-A,B mostly compatible lymphoid cells, PLT cells resulted in recognizing a group of non D/DR allelic antigens provisionally named K, L, M and N. To improve discrimination these bulk primed typing reagents were cloned and expanded. By typing of previously SB typed lymphoblastoid B cell lines (LCL) the provisional specificities could be identified as SB1, 4, 3 and 2, respectively. Typing of 186 unrelated Norwegians gave the following gene frequences: SB1: 0.05, SB2: 0.16, SB3: 0.13, SB4: 0.42 and SB blank: 0.24. No triplets were found, the calculated gene frequencies fit with Hardy-Weinberg equilibrium, and typing of a B-DR recombinant family confirmed that the SB locus is situated centromeric to B. Associations between SB and A, B, DR antigens in the same material were generally weak, the most significant associations found were between SB1-DR3 and SB4-DR2.