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.     

Response of Primed LD Typing Cells to Homozygous Typing Cells

At least two different methods using cellular responses have been described for defining the determinants of the HLA-D region: typing with HLA-D homozygous cells and primed LD typing. Primed LD typing cells were generated in one-haplotype-different combinations and grouped on the basis of two or more cells appearing to define the same HLA-D-region-determined PL antigen. Such cells were restimulated with homozygous typing cells for several of the presently known HLA DW clusters. A very strong correlation was noted: PLT cells defining the antigen PL1 were restimulated with homozygous typing cells for DW3, those PLT cells defining the antigen PL2 were restimulated by homozygous typing cells for DW2, and those defining PL5 were restimulated by homozygous typing cells for DW1.     

Specificity of Human Lymphocytes Primed against Allogeneic Cells in vitro. II. Discrimination and Cross-Reactivity in Repeated Priming

When studying the specificity of human lymphocytes primed in vitro against HLA-D determinants on allogeneic cells, it was found that specific restimulation of the primed cells 10 days after the first priming did not influence their discriminatory power compared to cells primed only once. Likewise, priming to one HLA-D determinant and repeated priming against another HLA-D determinant did not change the discrimination for the first priming antigen. Neither was there any increased relative reactivity to the second priming determinant. On simultaneous priming against two HLA-D determinants carried by two different stimulating cells, a good discrimination for both antigens was obtained compared to third party cells, but if the stimulations were separated by 24 h or more, the second priming was mainly without effect. Antigeneic competition is thought to be the mechanism involved in this early restriction of specificity.     

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.     

A New Determinant, Defined by PLT, Coded for in the HLA Region and Apparently Independent of the HLA-D and DR Loci

A PLT cell was raised between a responder cell which carried the HLA-D and DR specificities Dw3, 8; DRw3, 8 and a stimulator cell which was most likely homozygous for HLA-Dw3, DRw3. The PLT cell appeared to recognize a determinant (PL3A) which was (1) different from the officially recognized HLA-D and DR determinants, (2) was associated with HLA-A1, B8, Dw3 and DRw3, and (3) segregated in three informative families with HLA. Another responder-stimulator combination could be selected to raise PLT cells which recognized the same determinant.     

Serological identification of five HLA-D associated (Ia-like) determinants.

Five antisera raised within HLA-A and -B compatible, HLA-D disparate combinations were reactive in a modified NIH microcytotoxicity test with B lymphocytes, but not with T lymphocytes from the immunizing donor, as well as with B lymphocytes from most or all donors sharing his immunizing HLA-D phenotype. Four antisera recognized structures closely associated with the HLA-D determinants Dw2, Dw3, Dw4 and LD 108. One serum had a broad reactivity pattern including Dw3, Dw6 and some unknown specificity(ies). In population and family studies, these B lymphocyte antigens behaved as if they were governed by one genetic locus in the B-D region of the HLA complex. We conclude that the antisera produced by this method recognize Ia-like antigens identical to or very closely associated with the HLA-D determinants.     

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.     

The Specificity of Secondary MLC Assayed by Titration of Primed Lymphocyte Populations

The specificity of responses in secondary MLC was studied by titration (100 x 10(3) to 5 x 10(3)) of responder primed lymphocytes. In all instances, significant proliferative responses of responder primed lymphocytes to the specific stimulator occurred using lower responding cell numbers. When tested against allogeneic stimulating donors, three patterns of responses were observed: no significant responses, responses only at higher (100 x 10(3)) responding cell densities, and responses at lower responding cell densities, similar to those with the priming donor. In instances where primed lymphocytes responded significantly to allogeneic stimulating cells at lower cell densities, the responses were considered positive and the stimulating cells positive for the PL determinant. In several instances, primed lymphocytes responded significantly to allogeneic stimulators negative for the specific HLA-D and/or HLA-DR specificities. On the other hand, in experiments where allogeneic stimulating donors shared either HLA-D or DR specificity with the priming donor, a significant response was always observed. Thus, the PL determinant was present on stimulating allogeneic cells negative for specific HLA-D and DR specificities. The present data suggest that an analysis of the specificity of primed populations could be profoundly affected by the responder cell density at which the assay is performed. Also, the data suggest that other MHC determinants, including non-HLA loci may be important in secondary MLC.     

Human Ia-Like Antigens Associated with HLA-D

Antisera raised with HLA-A- and -B-compatible, HLA-D-disparate combinations were cytotoxic to B lymphocytes from the immunizing donor's HLA-D phenotype. Four antisera recognized structures closely associated with the HLA-D determinants Dw2, Dw3, Dw4, and LD 108. One antiserum had a broad reactivity pattern, including Dw3, Dw6, and some unknown specificity(ies). In population and family studies these B-lymphocyte antigens behaved as if they were governed by one genetic locus in the B-D region of the HLA complex. Furthermore, the antisera were cytotoxic to a minor concavalin-A-reactive T-cell subpopulation. The antisera had previously been shown to inhibit the stimulating cells in mixed lymphocyte culture and to be capable of inhibiting the Fc receptor in the EA rosette assay. We conclude that the antisera produced by this method recognize Ia-like antigens closely associated with the HLA-D determinants.     

In Vitro Sensitization of Human T Lymphocytes to Hapten (TNP)-Conjugated and Non-treated Autologous Cells is Restricted by Self-HLA-D

By co-culturing human T lymphocytes with TNP-treated autologous B lymphocytes and macrophages for 10 days in vitro, sensitization to TNP-treated autologous cells could be detected in a proliferative assay. By restimulation with different types of allogeneic cells and with cells from donors compatible or incompatible for the HLA-ABC or -D determinants, results were obtained suggesting that the TNP-specific response was restricted by the HLA-D but not the -ABC antigens of the autologous priming cells. Furthermore, our experiments demonstrate that T lymphocytes can also be primed against non-treated autologous cells in vitro and suggest that the HLA-D determinants may be involved also in this auto-sensitization.     

Human B-blast specific target determinants in CML: a family study

Human B blast specific target determinants, sclctivcly identified on PWM stimulated purified B lymphoblasts by in vitro generated CTLs, have previously been studied in the population and showed association to and inclusion of HLA-DR geneproducts. This report indicates that B blast specific target determinants are products of genes which in a codominant mcndclain way segregate with the HLA haplotypes in 4 selected families. Furthermore tests of families with HLA-B/D, DR and HLA-D, DR/GLO recombinations show that human B blast specific target determinants are coded from loci (locus) in the HLA-D region , between HLA-B and GLO.     

HLA Antigens in a Psoriatic Family: Comparative Studies with MLC, HTC, PLT and Serological HLA-DR Determinations

HLA genotypes were characterized in a large family of 48 individuals in three generations. In this family, carriers of the proband's disease-predisposing haplotype commonly expressed clinical signs of illness, manifested as psoriasis and/or arthritic lesions. From these data, and from other family studies, presented previously, we have concluded that cutaneous and/or joint lesions may be signs of disease in carriers of the predisposing HLA haplotype. We have investigated HLA-A, B, C and D/DR antigens in the family members. This gave us the opportunity to evaluate the validity of different assays for the determination of HLA-D alleles in a family material where the MLC tests formed the basis for a correct assignment of HLA-D determinants. The HTC method and the PLT assay were both afflicted with specific typing problems, partly due to the existence of cellular cross-reactions between HLA-D determinants, which seemed to be reminiscent of serological DR crossreactions.     

Selection of Haploid Spermatozoa and Its Application to HLA-D Typing

The expression of HLA-A, -B, and -D determinants on the surface of spermatozoa is haploid. Using cytotoxicity and appropriate HLA antisera and complement, we were able to select haploid spermatozoa. The surviving sperms behaved similarly to homozygous lymphoid cells when cultured in mixed sperm lymphocyte culture (SLC). This method is being applied to define new HLA-D specificities, to improve typing and matching for bone marrow transplantation and kidney transplantation and to study their association with various diseases.     

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.     

Gene products of the HLA-D region in normal and malignant tissues of nonlymphoid origin

Indirect immunofluorescence staining with monoclonal antibodies has shown a differential distribution of HLA-DR, DQ, and DP antigens in normal tissues of nonlymphoid origin. The distribution of HLA-DP antigens is similar to that of HLA-DR antigens, while that of HLA-DQ antigens is more restricted. Malignant transformation of cells of nonlymphoid origin may be associated with the appearance of the gene products of the HLA-D region. HLA-DR antigens appear more frequently than the other two types of HLA class II antigens and HLA-DP antigens more frequently than HLA-DQ antigens. Differential expression of the gene products of the HLA-D region was also found in autologous metastases removed from different anatomic sites from patients with melanoma. The HLA class II phenotype of surgically removed malignant lesions did not correlate with the degree of differentiation of tumor cells and/or with the expression and/or cellular distribution of HLA class I antigens. Furthermore, in melanoma lesions, no relationship was found between the HLA class II phenotype and the expression of 3 membrane bound and 1 cytoplasmic melanoma associated antigen recognized by monoclonal antibodies. The functional significance and the practical implications of the differential expression of the gene products of the HLA-D region by tumor cells are discussed.     

Joint Report of the B Lymphocyte Specificities Workshop of the France-one Region by FRANCE-ONE with the collaboration of

The analysis of 182 selected anti-B cell sera on 102 cells allowed us to identifysseveral clusters of sera. They showed no correlation with HLA-A, B or C specificities but many associations with the HLA-D determinants. In 10 families, most of these sera segregated with HLA, and five recombinants showed a linkage with the BD or D end part of the MHC. The panel distribution and the family analysis indicated that at least one (the Ly-Li system) and probably two segregant series could exist close to the HLA-D locus.     

LB-Q1 and LB-Q2: two determinants defined in the primed lymphocyte test and independent of HLA-D/DR, MB/LB-E, or SB

The primed lymphocyte test (PLT) was used to prepare reagents between HLA-D/DR identical individuals. Two sets of primed lymphocytes were obtained which recognized the new determinants referred to as LB-Q1 and LB-Q2, respectively. LB-Q2 is significantly associated with HLA-A1, -B8, and -D/DR3. Nevertheless, LB-Q1 and LB-Q2 seem to be distinct from any of the established HLA antigens. No association with any of the alleles of the SB system was observed. A recombination between HLA-D/DR and HLA-B suggested that the locus encoding LB-Q2 is situated on the telomeric side of HLA-D/DR.     

Xenogeneic recognition of soluble and cell surface HLA class II antigens by proliferative murine T cells

In order to characterize the murine anti-human xenogeneic mixed lymphocyte reactions (MLR), we studied T cell proliferative responses against various human lymphoid cells by immunization of mice either with cellular or purified HLA-DR antigens. Data presented here indicated that small amounts of soluble HLA-DR antigen were able to prime mice, and that the xenogeneic MLR depends on the expression of HLA class II antigens on the stimulating cells. Experiments using a mutant cell line clearly showed that HLA-DP molecules were also sufficient in eliciting a primary or a secondary xenogeneic MLR while no secondary proliferative response was obtained with cells expressing only HLA class I molecules. Using a large panel of human cells with various haplotypes, our results also showed that (a) nonpolymorphic determinants of HLA class II antigens trigger dominantly the murine T cells and (b) the xenogeneic response required I-E and L3T4 accessory molecules and was not inhibited with anti I-A and monomorphic anti-HLA class II antigen monoclonal antibodies. Altogether these results suggest that HLA class II antigens act as nominal antigens in triggering a murine anti-human proliferative response.     

HLA-D region products are expressed in endothelial cells

The mixed lymphocyte endothelial cell culture was studied by the primed lymphocyte typing (PLT) technique. By comparing the HLA-D/DR specificity of the secondary response when using either peripheral blood mononuclear cells (PBM) or endothelial cells from umbilical cords for priming or restimulation of lymphocytes, it was found that PBM from newborn would induce a clear-cut specificity for HLA-D/DR when used for priming a well a for restimulation. No HLA-D/DR specificity was seen, however, when endothelial cells were used for restimulation of lymphocytes primed to HLA-D/DR on PBM. On the other hand, lymphocytes primed to endothelial cells showed significant, albeit not very strong specificity for HLA-D/DR when restimulated with PBM. Our experiments suggest that HLA-D region products are present on endotheial cells, and thus confirm and extend serological studies using anti DR antisera.