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.     

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.     

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.     

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.     

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.     

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.     

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.     

False HLA—D Assignments May Be Caused by Cytotoxic Responder Lymphocytes

Lymphocytes from a healthy female repeatedly giving rise to MLC-typing response against HLA--D homozygous typing cells of three different specificities were investigated for cytotoxic capacity by the direct CML technique. Testing against a panel indicated the presence of circulating cytotoxic lymphocytes with specificity towards HLA--A2. When tested against selected HLA--D homozygous typing cells, the pattern of CML reactivity closely resembled the pattern of MLC-typing responses, i.e. typing responses were mostly restricted by the presence of HLA--A2 on the stimulator cells. This pattern was also found when time course studies of Cr--51 release were performed using experimental conditions identical to ordinary MLC typing, but involving chromium-51 labeled, irradiated homozygous typing cells as targets. These studies indicate that the presence of in vivo educated cytotoxic lymphocytes among the responder lymphocytes may in some instances mimic typing responses. Such lymphocytes are thought to lyse relevant stimulator lymphocytes prior to initiation of the proliferative response.     

The differential diagnosis of hairy cell leukemia with a panel of monoclonal antibodies

A panel of monoclonal antibodies reactive with normal human lymphoid cells and with hairy cells has been applied to the immunocytochemical analysis of hairy cell leukemia. Staining was performed by immunoenzymatic methods on frozen sections of bone marrow trephines and extramedullary tissues and on cell smears. Hairy cells reacted with antibodies against HLA-DR, leukocyte common antigen, B-cell antigens (antibodies To15 and B1) and with three anti-hairy cell monoclonal antibodies (S-HCL3, HC1, and HC2). Neoplastic cells in other B-cell lymphoproliferative disorders also expressed HLA-DR, leukocyte common, and B-cell antigens but were consistently negative for the antigen detected by monoclonal antibody S-HCL3. Furthermore, hairy cells differed from other neoplastic B-cells in that they were unreactive with monoclonal antibodies against C3b receptors, anti-Leu-1, Tü1, Tü33, and lacked a meshwork of dendritic reticulum cells. These findings establish a distinctive antigenic phenotype for hairy cell leukemia and indicate that it may be diagnosed reliably by immunoenzymatic labeling of tissue sections or cell smears.     

Hemizygous HLA mutants of lymphoblastoid cells: A new cell type for histocompatibility testing

HLA variants that have lost expression of multiple cis-linked alleles as determined serologically and enzymatically were analyzed for expression of HLA-D PLT-stimulating (PL) determinants using the primed lymphocyte test. All 19 variants that had lost HLA-DRw3 expression simultaneously had lost expression of the HLA-D region-associated PL specificity. PLT cells made by priming to a variant that was hemizygous for HLA genes resulted in priming to HLA-D PL determinants encoded for by just one haplotype, analogous to priming to an HLA homozygous cell.     

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.     

Use of a panel of monoclonal antibodies for the diagnosis of hairy cell leukaemia. An immunocytochemical study of 36 cases

The phenotype of 36 cases of hairy cell leukaemia has been investigated using a panel of monoclonal antibodies reactive with normal human lymphoid cells and with hairy cells. Staining was performed on frozen sections and/or cell smears by the recently developed APAAP immuno-alkaline phosphatase procedure. In about 90% of cases, neoplastic cells reacted strongly with antibodies against HLA-DR, leucocyte common antigen, B-cells (antibodies B1 and To15), hairy-associated antigens (antibodies KB-90, S-HCL3, HC2) and activated T-lymphocytes (antibodies anti-Tac and Tü69). The phenotype of 10% of cases was clearly different in that the neoplastic cells were negative or only weakly positive for one or more of the antigens recognized by HC2, anti-Tac and Tü69. Antibody HC1 reacted with tumour cells of only 50% of the hairy cell leukaemia cases investigated. Monoclonal antibody Ki-67 (which selectively detects proliferating cells) stained only a low percentage of cells in all but three of the cases studied. The neoplastic cells in all cases were unreactive with monoclonal antibodies anti-Leu1, Tü1, Tü33 and a meshwork of follicular dendritic cells was consistently absent from tissues infiltrated by hairy cells. The immunological phenotype associated with hairy cell leukaemia was not observed in any case of non-Hodgkin's lymphoma, suggesting that it represents a unique type of B-cell neoplasm.     

The Role in Primary MLC of the non HLA–D/DR Determinant PL3A

To test the influence in primary MLC of the newly PLT defined determinant PL3A (Termijtelen et al. 1980), 11 Dw3/DR3 homozygous individuals were tested in an MLC matrix. Two groups of mutually negative cells were detailed. The results appeared to correlate with the presence of PL3A. Cells from group 2 were stimulated by cells from group 1, but the reverse reactions were negative. A genetic model for this "one way" stimulation, observed in MLC as well as PLT, is discussed. We suggest that PL3A, which causes strong stimulation in PLT, also plays a role in primary MLC.     

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.     

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.     

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.     

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.