Occurrence of paternal and maternal HLA-B/D recombinants in a single family

Two B/D recombinant offspring in one family were identified by HLA-A,B,C,D, and DR typing with the Eighth International Workshop sera and cells, intrafamily MLC and PLT test.

Restimulation of cells primed against the four parental haplotypes showed good discrimination between family members who shared the D alleles and those who did not. When the B/D recombinant haplotypes were primed, accelerated proliferative response was observed only with cells sharing the HLA-D region. Cells that shared with the priming cells the HLA-A to B interval but differed for the D region did not restimulate.

These results demonstrated the role of HLA-D disparity in evoking secondary proliferative response and showed that the determinant(s) mapping between HLA-A and B did not restimulate.     

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.     

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.     

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-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.     

Specificity of Human Lymphocytes Primed against Allogeneic Cells in vitro. I Optimalization of Culture Conditions for Detection of HLA—D Specificity

The specificity of lymphocytes primed against allogeneic cells in vitro has been examined in order to find the optimal conditions for detection in the restimulating response of HLA--D specificity, as defined by homozygous cell typing. It was found that reducing the number of stimulating cells to less than the number of responding cells (ratio R/S = 4 : 1) in the priming culture, and increasing the number of stimulating cells at restimulation gave better discrimination. Priming with HLA--D heterozygous stimulating cells, where the responding and stimulating cells shared the other HLA--D determinant, rendered the primed cells somewhat more discriminatory than priming with HLA--D homozygous cells. The optimal length of the secondary culture was found to be about 38 h.     

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.     

Inhibition of human mixed lymphocyte culture stimulation by anti-HLA-D-associated antisera: studies with primed responding cells.

The inhibitory effects of HLA-D-associated antisera on stimulating lymphocytes when cultured together with allogeneic primed responding lymphocytes were investigated. The responding lymphocytes were primed for either the HLA-Dw2 or Dw3 determinants. The presence of HLA-D-associated (Ia-like) antibodies during the culture period specifically inhibited the stimulatory capability of lymphocytes possessing the HLA-D determinants with which the antisera were apparently reacting, as long as the stimulating cells carried the determinant for which the responding cells were primed. HLA-D-associated antisera of other specificities caused no decrease in the stimulatory capability. These antisera, therefore, apparently contain antibodies which are reactive with determinants closely associated or identical to the HLA-D determinants and may be human analogues to the mouse Ia antigens.     

Alloreactive T cells: Expression of HLA-D antigens, stimulation of autologous MLR, and possible immunoregulatory function

Primed in MLC with allogeneic stimulators T cells acquire the capacity of expressing HLA-D and DR antigens and of stimulating the MLC response of autologous lymphocytes When primed T cells from HTCs are used as stimulators, a bimodal distribution of response with clear-cut “typing responses” and no significant “back stimulation” is observed This pattern may be due to the expansion during priming of a population of HLA-D restricted suppressors since irradiated primed T cells inhibit the MLC responsiveness of HLA-D “compatible” lymphocytes. The development and size of such a population is not dependent, however, on the strength of the antigenic stimulus used for priming since no differences were seen between the pattern of reactions induced by T cells primed against HLA-D identical or HLA-D different cells Primed OKT4⁺ and OKT8⁺ T cells share the capacity of expressing Ia antigens and of inducing “HLA-D restricted suppression.” We suspected that a similar phenomena accounted for the behavior of two HLA-D heterozygous cells as if they were HTCs Although no suppression was found, the fact that these cells typed for their “silent” antigen when tested as responders, yet failed to express it when tested as stimulation, supports the theory that different genes control the MLC-responding and stimulating capacities.     

Priming potency of the products of individual regions of the H-2 complex and their role in the restimulation in secondary MLC.

Genetic analysis of the determinants causing an early proliferation in MLC after priming with allogeneic cells in vivo was performed. The active determinants were localized in the I-A subregion of the H-2 complex by using appropriate recombinant mouse strains. Upon priming with antigens coded by the whole H-2 complex, the lymphocytes gave a strong MLC response only to the priming stimulator cells and cells sharing the I-A subregion determinants of the latter. The response to third party stimulator cells and cells not sharing I-A subregion determinants was low. Upon priming with various H-2 region products, only lymphocytes primed against I-A subregion products gave an early proliferative MLC response to the priming stimulator cells, whereas lymphocytes primed against other H-2 region products gave none.     

Delayed hypersensitivity in the mouse induced by hapten-carrier complexes.

Delayed hypersensitivity (DH) in the mouse was studied with complexes of dinitrophenyl (DHP) as hapten and bovine serum albumin (BSA), mouse immunoglobulin (MIg) and polyvinylpyrrolidone (PVP) as carrier. Priming with BSA induced strong DH against this carrier, but DN of decreasing strength against complexes with increasing DNP:carrier ratio. Priming with DNP-BSA complexes never resulted in a DH against BSA or DNP 3-minusBSA. Injections of DNP 16-minusBSA and DNP 28-minusBSA induced positive DH which increased with the hapten:carrier ratio of the eliciting antigen. The complexes with an isologous carrier DNP 48-minusMIg or DNP 90-minusMIg induced positive reactions against both complexes but not against the weakly substituted DNP 11-minusMIg. The latter only primed for itself. The importance of the DNP groups as determinant in these DH reactions is stressed by the cross-reactions between DNP-BSA and DNP-MIg complexes and by the induction by DNP 16-minusPVP of positive DH against DNP 28-minusBSA. Cyclophosphamide (Cy) treatment before priming with complexes induced enhanced DH against complexes with sufficient hapten:carrier ratio. Priming with carrier under Cy treatment induced no DH against complexes. All these results indicate that carrier determinants are not involved in the DH against complexes. After priming wtih complexes with a low hapten:carrier ratio the DH is directed against new antigenic determinants (NAD) groups. After priming with complexes with high ratios DH is directed against DNP groups. With adoptive local transfer of spleen cells of primed animals and pretreatment of these cells with anti-thymocyte serum or anti-plasma cell serum and complement it was possible to demonstrate that the T cell was responsible for the DH reactions. The involvement of different determinant groups on the hapten-carrier complexes in immune reactions is discussed.     

T and B cell responses to chimeric proteins containing heterologous T helper epitopes inserted at different positions.

The ability of a T helper (Th) epitope to induce help for B cells recognizing different determinants within a multideterminant antigen was investigated. Chimeric fusion proteins, containing inserts of single or multiple copies of the Th epitope ovalbumin 323-339 (ova) at two different positions, were compared with respect to their ability to induce specific antibody production and ova-specific T cell activation. The antibody responses against B cell determinants at the amino and carboxy terminus, respectively was differently influenced by the molecular positioning of the inserted Th determinant. All ova-containing fusion proteins induced antibody production against the B cell determinant at the amino terminal end irrespective of the positioning of ova. In addition, multiple copies of ova in any position led to increased levels of antibody production against this epitope. In contrast, T cell help for antibody production against the determinant at the carboxy terminus was more effective after insertion of multiple copies of ova in a distal than in an adjacent position. Furthermore a fusion protein, containing four copies of ova effectively elicited T cell help for high levels of antibody production against both examined B cell determinants, showing that activated Th cells recognizing a single epitope could simultaneously provide help for distinct sets of B cells specific for widely separated epitopes within a protein. Immunodominant T cell recognition of ova in all chimeric peptides, independently of its position, was demonstrated by lymph node cell (LNC) proliferation of primed BALB/c mice. The level of ova-specific T cell proliferation was similar, irrespective of which chimeric peptide that had been used for priming, and thus did not reveal any differences in T cell priming efficiencies related to the number of ova copies in the fusion proteins. However, when the peptides were presented to a ova-specific T cell line by A20 B lymphoma cells, a close correlation between IL-2 production by the clonal T cells and the number of ova epitopes in the chimeric peptides was observed. Thus, increased cytokine production by ova-specific T cells may be important for the increased level of in vivo antibody production observed in response to multiple copies of ova in the chimeric antigens.     

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.     

PRIMING POTENCY OF THE PRODUCTS OF INDIVIDUAL REGIONS OF THE H-2 COMPLEX AND THEIR ROLE IN THE RESTIMULATION IN SECONDARY MLC

Genetic analysis of the determinants causing an early proliferation in MLC after priming with allogeneic cells in vivo was performed. The active determinants were localized in the I-A subregion of the H-2 complex by using appropriate recombinant mouse strains. Upon priming with antigens coded by the whole H-2 complex, the lymphocytes gave a strong MLC response only to the priming stimulator cells and cells sharing the I-A subregion determinants of the latter. The response to third party stimulator cells and cells not sharing I-A subregion determinants was low. Upon priming with various H-2 region products, only lymphocytes primed against I-A subregion products gave an early proliferative MLC response to the priming stimulator cells, whereas lymphocytes primed against other H-2 region products gave none.     

Stimulation in primary MLR caused by a PLT defined non-HLA-D/DR determinant, EP1

The influence in primary mixed lymphocyte culture reaction (MLR) of a primed lymphocyte typing (PLT) defined non-HLA-D/DR determinant, EP1, was investigated. In primary MLR between HLA-D/DR compatible lymphocytes, the response of the lymphocytes from 14 EP1-negative HLA-D/DR heterozygous individuals towards two EP1-positive homozygous typing cells (HTCs) was on an average approximately 35% higher than the response towards two EP1-negative HTCs ( P < 0.01). The strength of the MLR between lymphocytes from 25 EP1-negative and 10 EP1-positive individuals matched for two HLA-D/DR antigens was investigated. The average responses of EP1-negative lymphocytes against EP1-positive lymphocytes were approximately 40% higher than the average responses against EP1-negative lymphocytes ( P < 0.01). These data indicate that the PLT defined determinant EP1 causes stimulation in primary MLR.     

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.     

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.     

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.     

Mixed lymphocyte reactivity of human lymphocytes primed in vitro. I. Secondary response to allogenic lymphocytes.

In order to study the mixed lymphocyte culture reactivity of human lymphocytes primed in vitro, a nucleopore culture chamber technique allowing human lymphocytes to be cultured for a period of at least two weeks has been developed. During the primary culture period in nucleopore chambers, human lymphocytes were sensitized against mitomycin-treated allogenic stimulating cells. It was shown that the stimulated lymphocytes underwent a blastogenic reaction and the results suggest a reversion to the state of small, resting, primed lymphocytes. In vitro primed lymphocytes displayed allogenic memory. This was characteristic of a secondary response, which is shown by the following: 1) acceleration, the peak of thymidine incorporation occurring on day 4,2) specificity, the accelerated response was observed only when the primed lymphocytes were confronted with the cell used for priming. Contact with a third party cell did not produce this kind of activation. 3) Amplitude; the peak DNA synthesis response was greater than that of unprimed lymphocytes cultivated for the same length of time.