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 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.     

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.     

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.     

Lack of shared lymphocyte-stimulating determinants between H-2I and HLA-D/DR using mouse primed lymphocyte typing cells

A number of anti-H-2 alloantisera containing antibody reactive with I region gene products (Ia) of the major histocompatibility complex cross-react with determinants expressed by human peripheral blood B lymphocytes. Such data have led to the conclusion that Ia and DR antigens share cross-reacting determinants. We have attempted to generate mouse primed T lymphocyte populations specific for defined I region gene product determinants which concomitantly recognize DR determinants on human peripheral blood leukocytes in primed lymphocyte typing (PLT) analysis. Mouse PLT cells were generated in primary MLC using strain combinations identical to those in which positive mouse/human cross-reacting antisera have been obtained. The resulting PLT cells exhibited strong, yet specific, secondary MLC responses against mouse cells expressing the Ia determinants used as the primary stimulus. In contrast, when examined on panels of human peripheral blood leukocytes, no reactivity was detected. This lack of cross-reactivity suggests that mouse T cells primed toward Ia determinants do not regularly recognize cross-reacting determinants of DR or D-associated antigens expressed on human PBLs. Consequently, mPLT cells are not a useful reagent in defining HLA-D region polymorphism.     

Insulin-dependent diabetes--associated HLA-D region encoded determinants

We have studied the relative frequency of Dw specificities (defined with homozygous typing cells or primed LD (lymphocyte defined) typing reagents) associated with DR4 and DR2 in the normal and insulin-dependent diabetic population. Our findings demonstrate that there is a highly significantly increased frequency of Dw4 in DR4 positive diabetics as compared with normals and a significantly decreased frequency of Dw2 and Dw12 in the few DR2 positive insulin-dependent diabetics that we have found. In addition, we have used PLT reagents to define a new LD specificity, LD-MN2, that is associated with DR2 and is found significantly more frequently in DR2+ IDD patients than in DR2+ normals. These results suggest that determinants of import in the association between HLA-D and IDD may be more closely related to Dw than to DR.     

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-DR, D recombination in a kidney transplant recipient

Immunogenetic studies of a consanguineous family revealed discordance in the inheritance pattern of the HLA-D and HLA-DR antigens in one offspring. The findings suggest a recombination between the HLA-D and HLA-DR loci in one of the paternal chromosomes. Results on segregation of B-cell alloantigens. MLC reactivity, and glyoxalase isoenzyme determination map the DR gene between the HLA-B and D loci.     

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.     

Typing for human alloantigens with the primed lymphocyte typing (PLT) technique with notes on the interpretation of PLT data

A typing system for HLA-D/DR-associated PLT-defined determinants, which have been called “DP” antigens, is reported. Some of the results concerning a data interpretation system, the reproducibility of PLT results, and the correlations between the results of HLA-D, -DR, and DP typing are presented. Also, a “new” human alloantigen, EP1, not belonging to the series of DP antigens, is defined with PLT.     

Detection of HLA-D region products by primed lymphocyte typing (PLT)

In vitro priming experiments were pormed with lymphocytes from members of two different families carrying Dw-,DR2 or Dw2,DR2 haplotypes. It was demonstrated that lymphocytes could be primed to allogeneic HLA-D determinants without detectable priming to the associated HLA-DR determinant, even when the priming cell was also HLA-DR incompatible to the responding cell. It was further shown that the unknown HLA-D determinants of the two families (e.g., Dw-) were different, one of them showing cross-reactivity to Dw2. Priming to MT1 determinants or to Lewis antigens could not be detected.     

HLA-D clusters associated with DR4 in the Japanese population

A study of HLA-D clusters associated with DR4 was performed in the Japanese population. These clusters consist of DYT, DKT2, DB3, and Dw4. Forty-two Japanese typed as DR4 were investigated, and it was found that 17 (40.5%) were DYT. 7 (16.7%) DKT2. 7 (16.7%) DB3, and 4 (9.5%) Dw4.     

Is predisposition to pemphigus vulgaris in Jewish patients mediated by HLA-Dw10 and DR4?

Twenty-one Israeli Jewish pemphigus vulgaris (PV) patients were studied for the HLA-D lymphocyte defined determinants and the serologically defined antigens of the HLA-A, B, and DR series. HLA-D typing revealed that Dw10 is significantly associated with PV: 86% of patients vs 18% of controls carried Dw10, and DR4 was present in 86% of patients as compared to 38% in the controls. The most striking observation was that all Dw10 positive patients were also positive for DR4, and no other patient carried DR4 alone. The relative risk for a Dw10-DR4 carrier to develop PV was estimated at 31.9, higher than that observed for Dw10 alone (RR 26.7) or DR4 alone (RR 9.6). Probably HLA-Dw10 predisposes for pemphigus vulgaris.     

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.     

Multiple Ia-like molecules characterize HLA-DR2-associated haplotypes which differ in HLA-D

Two-dimensional gel electrophoresis of immunoprecipitated human Ia-like molecules was used to investigate the structural relationship between HLA-D and HLA-DR2. Eight different lymphoblastoid lines derived from HLA-DR2-associated homozygous typing cells, representing five distinct HLA-D clusters, were compared. Two or three distinct beta chain molecules from DR-like loci were identified in the DR2 homozygous cell lines studied. Furthermore, one of these molecules was present in all lines tested, while the others were highly variable. The electrophoretic mobility of these variable DR-like molecules correlated very well with HLA-D typing studies, suggesting that the HLA-DR specificity and the HLA-D specificity on these DR2 cells may be present on separate, but related, molecules.     

Genetic and molecular analyses of lymphocyte-defined HLA-D region specificities

Determinants encoded in the HLA-D region have been studied with both cellular (PLT) and molecular (SDS-IEF) methods. When the PLT response against a lymphoblastoid cell line was analyzed by limiting dilution culture and determination of the reactivity of individual cultures against a panel of loss mutants of the initial stimulating LCL, a large fraction of the cultures showed the same pattern, apparently recognizing a determinant associated with DR. In two-dimensional gel analysis of several DR4-positive HLA-D region homozygous cells, the IEF pattern of the DR beta chain correlated with the Dw specificity expressed by the cell. These two pieces of evidence suggest that, although many determinants may contribute to reactivity in mixed leucocyte culture or PLT, an immunodominant determinant associated with the DR beta chain may be the most important single factor in the assignment of Dw specificity.     

HLA-D/DR Restriction of Macrophage-dependent Antigen Activation of Immune T Lymphocytes: Cross-reacting Allogeneic HLA-D/DR May Partly Substitute for Self HLA-D/DR

Optimal proliferative response of T lymphocytes to purified protein derivative of tuberculin (PPD) in vitro requires that antigen be presented by autologous macrophages or allogeneic macrophages sharing HLA-D/DR determinants with the T cell donor. In some cases, however, T cells may respond to a limited extent to PPD in association with macrophages expressing different HLA-D/DR determinants. In this paper experiments are presented where various combinations of T cells and HLA-D/DR disparate macrophages were stimulated with PPD. We often found a stronger PPD-specific response in HLA-D/DR-incompatible combinations in which the macrophages carried HLA-DR antigens known from serological studies to be cross-reactive with those of the T cell donor than in combinations in which this was not the case. Thus, the cross-reactions detected by serology may sometimes be reflected on a functional level in T lymphocyte/macrophage cooperation.     

HLA-D/DR Restriction of Langerhans Cell-Dependent Antigen Activation of T Lymphocytes

In previous studies we have shown that the response of T cells to antigen presented by epidermal Langerhans cells (LC) is restricted by products of the HLA-D region. An optimal antigen-specific response required that the LC used for antigen presentation shared both, or at least one, of the D/DR determinants of the T-cell donor. These studies were, however, disturbed by a strong allogeneic response induced by the foreign D/DR determinants of the LC. We report here that by separating the antigen-specific T cells from those that are alloreactive, a clearer picture of the D/DR restriction phenomenon may be obtained. Furthermore, the present studies demonstrate that the same D/DR determinants function as restriction elements on peripheral blood monocytes and LC.     

Cell mediated cytotoxicity against HLA-D region products expressed in monocytes and B lymphocytes. III. Inhibition by monoclonal antibodies and study of an HLA-B/DR recombinant

Attempts to further define the antigens recognized by HLA-D region specific cytotoxic lymphocytes were undertaken using monocytes and transformed B cell lines as target cells. Monoclonal antibody against framework determinants of HLA-DR antigens partially blocked cell mediated lysis, suggesting that at least a portion of the D-region specific cytotoxic cells recognized the HLA-DR determinants. The study of a family with an HLA-B/DR recombinant showed that the determinants recognized by allogeneic anti-HLA-D-region cytotoxic lymphocytes are encoded outside of HLA-B. In addition, cytotoxic lymphocytes specific for the HLA-D region could be generated with cells identical throughout the interval from HLA-A to B and disparate only to the left of HLA-B.     

Influence of HLA-D/DR antigen disparity in CTL generation in vitro

This report describes the influence of HLA-D/DR antigen disparity upon the level of cytotoxicity in allogeneic in vitro cultures. Allogeneic cultures, between unrelated HLA-D/-DR full house donors, tested in CML gave three different levels of cytotoxicity, termed weak, intermediate and strong cytotoxicity. HLA-D/-DR compatibility predicts weak cytotoxicity and two HLA-B antigen incompatibility predicts strong cytotoxicity. On the contrary, HLA-A antigens have no major influence upon the strength of cytotoxicity. Accepting that the MLC/CML reaction is an in vitro parallel to the in vivo transplantation of allogeneic tissue, the observations are in accordance with the results of HLA-D/-DR matching for graft survival in human renal transplantation.