Detection of HLA-D Clusters and Segregation Studies Using Primed LD Typing

By testing a group of PLT cells over a panel of unrelated restimulating cells, the PLT's could be grouped into clusters according to their ability to discriminate antigen(s) in unrelated cells. The PLT clusters broadly correlated with the homozygous typing cell-defined HLA-D clusters represented on the panel. The PLTs grouped together clearly segregate with a particular HLA haplotype when tested in both unrelated families not possessing the sensitizing haplotype and in the family with the sensitizing haplotype. No influence of HLA SD antigens could be observed in PLT restimulation in the segregation studies.     

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.     

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.     

Typing an Unrelated Panel with PLT Cells: Association with DW Clusters

Primed LD typing (PLT) cells prepared in one Laboratory (Madison) were shipped in the frozen state and tested in Tübingen on a separate panel that had been typed with homozygous typing cells. Those PLT cells that had been grouped, on the basis of their reaction with test cells of the Madison panel, as defining an HLA PL antigen showed identical or nearly identical patterns of reactivity with the Tübingen panel. Clear association between certain PL antigens and DW clusters as defined with homozygous typing cells could be demonstrated. Of particular interest may be combinations of certain PLT reactions with D-locus-typed cells, where the primed cells do not react as expected from the target's HLA-D type.     

Quantitation of HLA-D Differences

The predictability of MLC non-reactivity by HTC typing was tested in a single checkerboard experiment which involved 39 unrelated individuals belonging to 12 different groups of HLA-D identical phenotypes. The strength of one-way MLC reactions in all possible responder-stimulator combinations (39 × 39) was quantitated by the Linear Clustering Analysis Program. Individuals who, by HTC typing, were identical for two HLA-D antigens gave 51% negative, 36% intermediate and 13% strong positive MLC responses. Identity for only one HLA-D antigen resulted in 3% negative, 40% intermediate and 57% strong MLC reactions. When no HLA-D antigen was shared, 88% of the reactions were strongly positive. HLA-D antigens behaved as equipotent stimulators in MLC between half-identical pairs. The only exception consisted of the higher frequency of weak responses displayed by the Dw5 positive individuals against stimulators differing by LD107. The same pattern was observed when LD107 homozygous cells were used as stimulators, suggesting that determinants of this specificity might be partially included in Dw5.     

A new homozygous typing cell with HLA-D"H" (DB6) specificity

A new homozygous typing cell (HTC), SK is described. Mixed leukocyte culture (MLC) showed that SK is homozygous for HLA-D"H" (DB6) defined by the HTC, Herluf, and that the HLA-D typing results obtained by Herluf and SK are highly significantly correlated (Kendall's R = 0.46, p = 2.5 X 10(-5)). Both Herluf and SK are also homozygous for a new class II determinant, DN-1, defined by the monoclonal anti-B-lymphocyte antibody, 9w925, developed by Aizawa. The corresponding DN-1 antigen was present in 2.2% of 136 random, unrelated Danes and in all of six unrelated HLA-D"H" positive but in none of 20 HLA-D"H" negative individuals. Thus, there is an absolute and highly significant (p = 4 X 10(-6)) association between the cellularly defined HLA-D"H" determinant and the serologically defined DN-1 antigen, which strongly suggests that HLA-D"H" can now be detected serologically. DN-1 may be identical to DRw12 which is, however, poorly defined. The HTC-donor SK was immunized by pregnancy, and her serum contains anti-HLA-B7, which can easily be absorbed, and anti-B-lymphocyte antibodies which reacted with cells from 87.7% of 536 unrelated Danes. The reaction pattern of this serum is negatively associated with DR3, w6, and w8. This serum may define a new broad, cross-reacting antigen belonging to the same group as DRw52 and DRw53 or DQw1-w3, but it is clearly different from each of these antigens.     

Polymorphisms within the HLA-DRw6 haplotype. III. DQ alpha and DQ beta polymorphism associated with HLA-D

We have studied HLA-DQ encoded antigens from HLA-DRw6 homozygous cells and analyzed the DQ region at the DNA level. HLA-DQ molecules were isolated from EBV transformed B-cell lines and analyzed for DQ alpha and DQ beta polymorphism. From the same set of cells, DNA was isolated and analyzed for RFLP. Polymorphism could be detected by both techniques, i.e., on the protein level and on the DNA. The variation in pI of the DQ alpha and beta chains correlated with the polymorphism as detected by HTC typing, as did the variation in molecular weight of the bands hybridizing to DQ specific cDNA probes; identical patterns were detected for cells of one HLA-D specificity and different patterns for different HLA-D types. Additionally, DQ reactive PLT reagents were raised against DRw6 positive cells. Panel studies revealed that these DQ reactive proliferative T cells can discriminate between the polymorphic DQ antigens on cells with different HLA-D specificities.     

Allogeneic Responses In Vitro Induced by Fetomaternal Alloimmunization*

ABSTRACT: The present study was undertaken in order to determine what type(s) of pregnancy-induced allogeneic reaction could alter MLC (mixed lymphocyte culture) reactivity in routine HLA-D typing of lymphocytes in multi-parous women (MW) possessing antibodies against paternal HLA-DR antigens. Unresponsiveness to homozygous typing cells (HTC) representing a paternal and probably fetal HLA-DR determinant was frequently observed. Kinetics experiments ruled out an early secondary proliferative response to HTC representing the paternal HLA-D determinant, which would be missed in a classical long-term mixed lymphocyte culture. Direct cytotoxicity against paternal or panel target cells was not always associated with inhibition of proliferative response to the same stimulator cell. Specific anti-HLA-DR blocking activity (antibodies?) in the supernates of restimulation reactions of lymphocytes from MW could be responsible for this inhibitory effect. Moreover, the study points to the existence of suppressor cells in the immunized MW acting independently of specific restimulation. The in vitro suppression appeared to be selective, restricted to cells sharing HLA-D linked structures with the suppressor cells, and suggests that auto-regulator mechanisms could be induced in pregnancy in order to modulate antibody production.     

Population Data on Two New HLA-D Determinants, El and RE

Homozygous typing cells (HTC) for two new HLA-D determinants, EI and RE, defined by family studies, are described. The HLA-D typing experiments among more than 300 unrelated individuals showed phenotype frequencies of 0.045 for EI and 0.098 for RE. Since the tested population was also typed for its HLA-A and -B alleles, linkage disequilibrium parameters could be calculated: HLA-D type EI was statistically significantly associated with HLA-B13 and Bw17, HLA-D type RE with HLA-Bw40. These data support the working hypothesis that both EI and RE are new alleles of the HLA-D series.     

Population studies of HLA-linked SB antigens and their relative importance in primary MLC typing. Analysis of HLA-D homozygous typing cells and normal heterozygous populations

SB phenotyping was undertaken on 96 HLA-D homozygous typing cells (HTCs) and 129 normal unselected heterozygous donors in the German population, using Interleukin-2-propagated primed lymphocyte typing (PLT) reagents. The results showed that the SB antigens in the normal population behave as a system of alleles at a single locus in Hardy-Weinberg equilibrium (p approximately equal to 0.20). Estimated gene frequencies in the German population appeared to be significantly different (p less than 0.002) from the North American Caucasian population: the principal differences were increased frequencies of the specificities SB1 and SB4, and decreased frequencies of blanks. Of HLA heterozygous donors 41% typed for two distinct SB specificities; 57% typed for one; and 2% were blank. In the HTC group, 20% typed for two specificities; 68% typed for one; and 12% were blank. Thus, a significant proportion of HLA-D homozygous test cells were, nonetheless, heterozygous for HLA-linked SB antigens. Performance of checkerboard mixed leukocyte cultures (MLCs) between 16 SB typed HLA-Dw3 HTCs, however, did not indicate that the observed mutual or one-way responses were influenced in any simple way by SB antigens; neither heterozygosity nor assumed homozygosity for SB antigens appeared to influence the frequency of MLC typing responses of HLA-Dw3-positive donors on these HTCs. These results add further confirmation of the genetic and functional independence of the SB gene product(s) and the HLA-D/DR gene product(s).     

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".     

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.     

Cellology of HLA I. The Apparent Homogeneity of HLA-D

We have demonstrated that there is a similarity in the distribution of restimulation responses of primed lymphocyte typing (PLT) cells raised between related and unrelated individuals, thus leading to the suggestion that HLA-D is a homogeneous determinant. It is proposed that within HLA a hitherto unrecognized heterogeneous structure may exist that is closely associated with HLA-D and which may give rise to intermediate reactions in primary mixed lymphocyte reactions and PLT.     

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".     

HLA-DR2 and DR4 further defined by two new HLA-D specificities (HTC) derived from Israeli Jewish donors: comparative study in Caucasian, Korean, Eskimo and Israeli populations

Two newly-identified HLA-D antigens were characterized by testing selected homozygous typing cells (HTC) against responder panels derived from Caucasian. Korean, Alaskan Eskimo and Israeli Jewish populations. The first specificity, defined by typing cell "AZH", is associated with DR2 haplotypes and is detected primarily in Israelis. The second specificity, defined by HTC "TAS", is associated with DR4 haplotypes and is detected with relatively high frequency in Koreans and Alaskan Eskimos. The data indicate that HTC-AZH and -TAS can be used to identify previously undefined splits or variants of lymphocyte-defined (LD) determinants associated with DR2 and DR4 haplotypes. Further, the study demonstrates the utility of comparative population analysis in identifying and characterizing alleles encoded by the HLA-D region and provides additional evidence of heterogeneity within the family of serologically-defined HLA-DR haplotypes.     

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.     

The MHC in human bone marrow allotransplantation

In this chapter, we have considered the theoretical and practical background of bone marrow transplantation. The immune response and its regulation by genes within the major histocompatibility complex, particularly of the I region of the mouse and of the HLA-D/DR region in man, is of central importance in both graft acceptance (rejection) and graft-versus-host disease. Methods which are available for typing alleles at the HLA-A, -C, -B, -DR and complotype (BF, C2, C4A, C4B) loci, have been considered in detail. The extent to which recombination affects specific alleles on haplotypes within families is discussed, as is the occurrence of linkage disequilibrium and extended haplotypes in populations of unrelated individuals. Because the HLA-DR and complotype region in man is thought to be critical for the success of bone marrow transplantation, methods for typing of HLA-D by both the HTC and PLT approaches have been examined. Although HLA-D/DR assignments are easily made in normal subjects, they are ambiguous in about 50 per cent of candidates for bone marrow transplantation, including, particularly, patients with aplastic anaemia, leukaemia, and severe combined immunodeficiency. In this setting, it is particularly important to obtain additional information by modification of HLA-D typing procedures and through complotype and GLO allele determinations in all family members. Finally, we can hope that there will be an increased possibility of using non-family donors through methods for removing cytotoxic T cells from donor marrow and through the identification, in the general population, of individuals who are genotypically similar or identical to the recipient. In this regard, the recognition that some 30 per cent of chromosome 6 in caucasians (50 per cent of individuals) bear extended haplotypes, which include a relatively fixed set of alleles particularly in the HLA-B, -DR, complotype and GLO regions, offers considerable promise.     

Generation of HLA-D specific primed lymphocyte typing (PLT) cells and cross-reactions of PLT-cells primed with homozygous typing cells

An approach for the selection of HLA-D specific primed lymphocyte typing (PLT) cells is described. The responder cells were primed with homozygous typing cells. Reproducible extra reactions were found and were analyzed in relation to HLA-D antigens defined by homozygous in cells (HTC's). The secondary response of 105 different PLT-cell combinations generated by 29 different primary responders against 19 different homozygous typing cells of the specificifies Dw1 to Dw8 and the local specificity "H" were tested in secondary PLT toward 17 different homozygous typing cells and 10 heterozygous cells. Cross-reactions were defined as reactions equal to or higher than the lowest HLA-D specific reaction observed. The entire experimental design and data analysis gave rise to a conservative definition of cross-reactivity. Two main groups of cross-reacting HLA-D determinants seem to exist: (i) Dwl, 3, 4, 7, and the local specificity "H", and (ii) Dw2, 5, 6, 8, and "H". The primary pairwise cross reactions were in group (i): Dw1-3, Dw1-"H", Dw3-4, Dw3-7, Dw7-"H", and in group (ii): Dw2-6, Dw2-8, Dw5-8, and Dw5-"H". The existence of such cross-reactions is likely to interfere with the results of PLT-typing and should be taken into account when attempts are made to develop HLA-D specific PLT-cells.