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.     

Detection of HLA-D Clusters Using Primed LD Typing

Using a set of 17 primed LD typing (PLT) cells tested on a panel of 35 unrelated cells, we showed that certain groups of PLT cells tended to detect similar unrelated cells. The PLT cells were grouped into seven clusters and these tended to correlate with the seven HLA-D specificities represented on the panel, as determined by HTC testing. These data suggest that the antigens that cause restimulation in PLT are similar to those HLA-D antigens detected by the homozygous typing cell (HTC) test or, alternatively but more unlikely, that the two typing methods are detecting genes in close linkage disequilibrium with the HLA-D region.     

Typing for HLA-D Determinants. Comparison of Typing Results using Homozygous Stimulating Cells and Primed Cultures

Typing for HLA-D determinants, using primed lymphocyte typing (PLT), has been compared to the results obtained using conventional homozygous stimulating cell primary MLC tests. The HLA-Dwl, -Dw2 and -Dw3 specificities were studied. Preliminary results indicate that these two methods essentially type for the HLA-D determinants and that reproducible results can be obtained employing the PLT technique after 24 hours of incubation in the secondary cultures.     

Primed LD Typing: Reagent Preparation and Definition of the HLA-D-Region Antigens

Primed LD typing cells were prepared against single HLA haplotypes within families and used to type a random panel of 48 individuals. PLT cells could be grouped on the basis of highly correlated responses with the panel test cells; the 21 different PLT cells could be used to define 6 different PL antigens. One of these, PL 3, was split by the responses of two of the 21 PLT cells which measured an antigen called PL 3.1.     

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.     

Confounding factors in HLA-DW 2 typing of human leucocytes.

Three healthy HLA-B7 homozygous subjects were found with similar but not identical HLA-D antigens; one was DW 2 homozygous according to independent typing results. This could be an expression of "long" and "short" HLA-D antigens or be due to differences in weak antigens outside the HLA-D region. Two further healthy HLA-B7 homozygous subjects were studied; one was apparently heterozygous for DW 2, the other apparently carried no DW 2 antigen. Both could discriminate between different DW 2 homozygous test cells. Two such test cells--one from a patient with multiple sclerosis (MS) and the other from a man with two children with MS--gave variable and absurb reactions with cells from the two subjects in question. It is tentatively suggested that genes exist which, when present in both moities in a mixed leucocyte reaction (MLR), can impair the MLR and give false "typing" reactions. This might be more common among patients with MS and perhaps also some other diseases (certain arthritides, e.g. rheumatoid arthritis) than among healthy subjects and can complicate or make impossible the interpretation of HLA-D typing data. It could also explain the previously-described impaired MLR between cells from patients with these diseases.     

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 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.     

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.     

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%.     

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.     

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.     

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 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.     

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.     

Relation Between HLA-DW and the B-Lymphocyte Specificities

Homozygous DW typing cells were tested for six B-lymphocyte specificities. All four of the second locus specificities of B lymphocytes were strongly associated with the DW specificities. DW1 typing cells were B group 6, DW2 were B4, DW3 were B5, and LD107 were B3. The first B-cell locus antigens 1 and 2 tended to be uniform within the DW groups. From an analysis of the typing responses of a panel of cells to the homozygous typing cells, it has become apparent that the first B-locus specificity present on the homozygous typing cells also plays a role in determining whether a typing response is obtained or not. Thus, the DW3 typing cells were themselves B2 and B5, and cells having B2-B5 were most frequently nonreactive to DW3 in mixed lymphocyte culture. Homozygous typing cells therefore mainly detect the second B-cell locus antigens and, to a lesser degree, the first locus specificities. Stated another way, homozygous typing cells do not define a single specificity, but rather the presence of two B-lymphocyte specificities, even though their responses often reflect matching of only the second B-locus specificity.     

Lymphoblastoid Cell Lines of Homozygous Typing Cells Used for Sensitization in PLT

Lymphoblastoid cell lines of homozygous typing cells were used as the sensitizing cells in MLC to prepare PLT cells. Results obtained using such cells against a panel of restimulating cells were compared to those obtained using regular PLT cells in which priming had been accomplished with normal peripheral blood lymphocytes. It appears that lymphoblastoid cell lines can be used for this purpose; the advantages of such an approach are given.     

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.