Generation of discriminatory PL against HLA-D antigens not detected by serology

A family was studied in which the father and the mother were both homozygous for the HLA-DR antigens. All children responded to the parents in MLC, even though each had a copy of the parental DR antigen, implying that they recognized a difference. When the father or the mother was sensitized in PLT against any child, all siblings could restimulate equally well. However, by sensitizing a family member with one copy of the DR phenotype against another family member with the alternate form of the phenotype, discriminatory PL could be produced. This may help define the subtypic specificities of PL antigens.     

Heterogeneity of HLA-D(DR)4 detected by primary and secondary mixed-lymphocyte-culture reactions and by individual HLA-DR4 antisera

One of our panel families (Sb), in which the paternal haplotypes Dw4, DR4 (a) and Dwblank, DR4 (b) segregate, was tested in primary mixed lymphocyte culture (1.MLC) and in the primed lymphocyte test (PLT). In the 1.MLC, cells which carry the a haplotype strongly stimulate b-haplotype cells, and vice versa. For the PLT, lymphocytes of two family-members were primed against the a haplotype and two against the b haplotype. A strong positive restimulation (RR greater than or equal to 60%) occurred only with cells bearing the original stimulating haplotype. The PLs were tested later against families St and Sm, which possess DR4 haplotypes, and against a panel of 73 unrelated persons. The results show heterogeneity of D(DR)4, suggesting at least three different subgroups: D(DR)4a, present on DR4 cells which strongly restimulate the anti-a PLs; D(DR)4b, on DR4 cells which strongly restimulate the anti-b PLs, and D(DR)4c, on the DR4 cells, which do not restimulate any of the PLs tested here. It seems also possible to differentiate between these subgroups with conventional DR-serology, as the 8W sera 903 and 981 react only with a-haplotype cells of family Sb, and ths 8W sera 872 and 1045 react only with b-haplotype cells.     

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.     

Cloned cellular reagents to define antigens encoded between HLA-DR and glyoxylase

Primed lymphocyte typing reagents have been used to define antigens encoded by genes of a locus (loci) mapping between HLA-DR and glyoxalase I. This locus, which we shall refer to as the third locus of the HLA-D region, has been variously referred to as D beta, PL beta, PL3, and SB. Generating discriminatory primed lymphocyte typing reagents which can be used to define these antigens, however, has been extremely difficult. Donors of responding and stimulating cells for the priming combinations have usually been matched not only for the DR, D, and MB/MT antigens but also for the HLA-A, -B, and -C antigens. Even under these very restricted conditions, not all bulk primed lymphocyte typing reagents that are generated are discriminatory enough to be useful for antigen definition. We have derived "clones" from bulk priming combinations in which stimulator and responder differed for known antigens of this third locus. Even though the bulk reagents that were prepared did not provide discriminatory results, approximately 7-12% of the clones derived from the bulk priming combination proved to be highly discriminatory. We have been able to obtain these results with regard to all three antigens of the third locus so far evaluated. The very ease of screening clones and deriving discriminatory reagents, as compared with screening responder-stimulator combinations, allows the ready derivation of cellular reagents that define the antigens of this third locus.     

Restimulation properties of cloned alloactivated lymphocytes: Detection of a novel type of HLA-D/DR region determinant and allelic subtypes for HLA-Dw3 using cloned reagents

Lymphocytes alloactivated by Dw3 homozygous typing cells were cloned by the method of limiting dilution and cultured for prolonged periods using T-cell growth factor and irradiated pooled leukocytes (as feeder cells). Restimulation specificity of two clones functioning as primed lymphocyte typing reagents was investigated in panel and family studies. Cells from one of the clones (12-2) were always specifically stimulated by HLA-Dw3 antigens shared between the original priming cells and the stimulating panel cells. In an informative family KOH, however, cells from this clone seemed to detect a split in the Dw3 cluster. Cells from the other clone (12-8) failed to respond to Dw3 antigens as expressed by the original priming cells or by panel stimulating cells; rather, specificity of restimulation seemed to be associated with the expression of Dw4 antigens. Family segregation analysis did not support this conclusion however, since stimulating products segregated with one of the three Dw3-bearing haplotypes and with none of three Dw4-bearing haplotypes. This suggested that 12-8 cells may be responsive to antigens different from those detected by homozygous typing cells.     

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.     

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-Dw"SHY": a new lymphocyte defined specificity associated with HLA-DRw10

HLA-DRw10 is a relatively new class II serologically-defined alloantigen first described in the 8th International Histocompatibility Workshop. To date the HLA-Dw related to the DRw10 specificity has not been detected. We have studied a Jewish family ("SHY") of Yemenite origin, in which the parents are first cousins who share one HLA haplotype: A29,Cw2,B7,BfF,DRw10,GL02. Two of the offspring in this family are homozygous for this haplotype. MLC family study confirmed that each of the two individuals was homozygous for HLA-Dw. No currently defined HLA-Dw specificity could be assigned to "SHY" using a selected panel of Caucasoid and local HTcs. HLA-Dw"SHY" was shown to segregate with DRw10 positive/Dw blank haplotypes in two families and in 64% (7/11) of DRw10 positive unrelated positive/Dw blank haplotypes in two families and in 64% (7/11) of DRw10 positive unrelated individuals. HTC"SHY" thus expresses the first HLA-Dw specificity associated with DRw10.     

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.     

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.     

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.     

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.     

Role of the ABO, Secretor, and Lewis determinants in the primed lymphocyte test

The antigenic determinants of the combined ABO, Lewis, and Secretor genes have been detected on the surface of lymphocytes by the lymphocytotoxicity test. We have studied the role of these determinants in the primed lymphocyte test (PLT), and the data demonstrate that Lewis incompatibility causes proliferative responses in PLT. On the other hand, no effects of ABO and Secretor incompatibilities were observed in this test. The frequency of the alloantigen-reactive cells (ARC) responding to Lewis and HLA-DR antigens in PLT was estimated by the limiting dilution analysis. The frequency of ARC to allogeneic Lewis-negative donors, who are positive for the sensitizing HLA-DR antigens ranged between 1:58 and 1:97. The incidence of ARC to Lewis-positive allogeneic donors who did not carry the sensitizing HLA-DR specificity was 1:94 to 1:142. These results demonstrate the presence of lymphocyte clones that are able to respond to antigens of the Lewis system. This study suggests that non-HLA antigens belonging to the Lewis system can cause stimulation of lymphocytes in the PLT test.     

Rapid matching for transplantation: PHA primed lymphocytes

Human lymphocytes, 10 days after treatment with phytohemagglutinin P (PHA-P), show PLT (secondary in vitro restimulation) reaction patterns which correspond to their cellular (HLA-D) type. Lymphocytes with shared cellular type stimulate PHA primed cells to a lesser degree than the majority of lymphocytes with unshared HLA-D type, which stimulate a strong response. Using a standard 3H thymidine incorporation technique, it is possible to detect responses in as early as 12 hr total incubation time, although usually a 22-48 hr total incubation time is normally required. This method is simple, gives results that correspond to the primary mixed leukocyte response, and thus may be useful as a cross-matching technique for cadaveric renal transplantation.     

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).     

A new HLA-DP (SB) specificity (Cp63) defined in Japanese and Caucasian cell populations by cloned PLT cells

Cp61, Cp62, and Cp63 are HLA-DP (SB) specificities detected by PLT testing using cloned PLT cells derived from Japanese cell donors. It was confirmed, using Japanese and Caucasian cell panels, that Cp61 is identical to DPw5 and Cp62 is identical to DPw4. Cp63, however, was found not to correlate with any known HLA-DP specificities. The gene frequency of Cp63 was found to be 3.6% in Japanese (N = 42) and 1.9% in Caucasian (N = 52) cell panels of limited size. No triplet assignment of DPw specificity including Cp63 was found in the Japanese and Caucasian cell populations. The relationship between the Cp63 and the HLA antigens demonstrated no significant correlation with HLA-A,B,C,DR, or MT antigens in this study. Linkage between Cp63 and HLA was confirmed from family segregation studies. This is the first report of a new DP specificity termed Cp63.     

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.     

Homozygous typing cell-defined HLA-Dw specificities correlate better than serologically defined HLA-DR specificities with restriction elements for influenza virus-specific proliferative human T lymphocyte clones

Human T lymphocyte clones with specific proliferative response to influenza A virus were derived by limiting dilution from peripheral blood lymphocytes (PBL) after in vitro stimulation with autologous irradiated, virus-infected PBL. Four OKT3+4+8- T lymphocyte clones (TLC) that showed HLA-restricted antigen-specific proliferative responses were used for a detailed analysis of the restriction elements for antigen presentation. None of the clones showed alloreactivity and all required the presence on the antigen-presenting cell of HLA class II antigens of one or other haplotype of the donor. Restriction elements for two clones were correlated with Dw1 rather than DR1, and for two others with Dw6 rather than DRw6. These latter clones showed differential recognition of HLA-Dw6 subtypes as defined tentatively by homozygous typing cells, without relationship to putative serological "splits" of DRw6. One of the Dw6-restricted clones was specific for a Dw6.1 (now Dw18) "subtype," confirmed by family segregation analysis, the other for a broad Dw6 (Dw18 and Dw19) specificity. Studies with a panel of monoclonal antibodies against monomorphic determinants of HLA class II antigens revealed heterogeneous patterns of blocking activity, distinguishing between clones of different restriction specificity. Inhibition patterns were partly as predictable from the known activity of the monoclonal antibody in alloantigeneic PLT systems. These results provide evidence that certain structures that function as restriction elements for antigen presentation also carry alloantigeneic determinants.     

Monoclonal and xenoantibodies specific for HLA-DR inhibit primary responses to HLA-D but fail to inhibit secondary proliferative (PLT) responses to allogeneic cells

Monoclonal antibodies (DA-2 and CA-2) and xenoantisera (rabbit anti-human, p23,30) specific for HLA-DR framework determinants were added to primary and secondary mixed lymphocyte cultures. Although such antisera were shown to inhibit primary MLC, primed lymphocytes were much less sensitive to the blocking effects of these antibodies. In the studies shown here, the concentration of antibody required to inhibit primary MLC reactions was 0.1-1.5 micrograms/ml).     

Evidence for a new HLA-region determinant detected by human T-lymphocyte clones (TLCs)

Human peripheral blood T-lymphocytes were stimulated by allogeneic cells in primary MLC and subsequently cloned by limiting dilution in the presence of lymphocyte conditioned medium (LyCM). Following expansion, clones were tested for specific proliferation against a panel of 32 stimulator cells including cells from the family of the original stimulator (FLAM). Two clones, TLC 14-14 and TLC 14-86, responded to FLAM and to a cell homozygous for Dw5 (JPSU), but not to other unrelated panel members, reactivity segregated with the haplotype containing Dw1 in FLAM's family. In separate experiments, TLC 14-14 was restimulated by an antigen encoded by the maternal “c” haplotype in JPSU's family. This antigen may be a new determinant on the same molecule as Dw1 and 5 or, more likely, encoded by a new gene associated with these specificities.