HLA-D clusters associated with DR2 and the definition of HLA-D"AZH": a new DR2 related HLA-D specificity in Israel

In order to investigate the HLA-D clusters associated with DR2 in Israeli Jews, 40 DR2 positive unrelated individuals were studied with a panel of DR2 associated homozygous typing cells (HTC's) which detect the lymphocyte defined specificities HLA-Dw2, Dw12, Dw9 and D-WJR. The results confirmed the existence of two distinct HLA-D clusters associated with the same serologically defined DR2. Of 40 individuals 22.5% (9/40) were Dw2 and 50% (20/40) were Dw12 carriers. Yet, no HLA-D specificity could be assigned to the remaining 11 DR2 positive individuals. In the present study we have defined a unique DR2-associated Dw specificity, HLA-D"AZH". The donor of the HTC was of Moroccan origin and an offspring of a first cousin marriage. This cell was not typeable with the known DR2-associated homozygous typing cells nor with other HTC's which define the well established HLA-Dw1 to Dw11 specificities. It was shown to segregate with DR2 positive HLA haplotypes in family analysis and in a population study, typed out 7 of 11 unrelated DR2 positive, Dw blank individuals, thus identifying a unique and new HLA-D cluster provisionally designated D"AZH".     

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.     

Comparison with monoclonal anti-Ia antibodies of antigenic determinants inducing lymphocyte proliferation and immune interferon production in primary and secondary murine mixed lymphocyte reactions

The nature of Ia antigens inducing lymphocyte proliferation and immune interferon (IFN-γ) production in primary and secondary murine mixed lymphocyte cultures was studied by means of monoclonal anti-Ia antibodies. In primary mixed lymphocyte cultures, both lymphocyte proliferation and IFN-γ production were found to be controlled by Ia determinants coded for by the I-A subregion. E alloantigen-recognizing antibodies displayed consistent stimulation of both lymphocyte functions, instead of inhibition, suggesting a regulatory mechanism of the E molecule on lymphocyte activation. In secondary mixed lymphocyte cultures fewer Ia determinants seemed to be involved in lymphocyte proliferation, since only antibodies directed against specificities Ia.17 and Ia.19 were found capable of blocking lymphocyte proliferation. In contrast, IFN-γ production, although significantly decreased by antibodies against specificities Ia.17 and Ia.19, was not completely abrogated, even by a mixture of all anti-Ia antibodies. The data indicate that (a) in primary mixed lymphocyte reaction lymphocyte proliferation and IFN-γ production are triggered by the same Ia specificities coded by the I-A subregion and (b) in secondary mixed lymphocyte reaction, whereas lymphocyte proliferation is restricted to fewer determinants, IFN-γ production shows a lower threshold of activation, being induced also by determinants ‘silent’ in primary mixed lymphocyte reaction.     

Insulin-dependent diabetes--associated HLA-D region encoded determinants

We have studied the relative frequency of Dw specificities (defined with homozygous typing cells or primed LD (lymphocyte defined) typing reagents) associated with DR4 and DR2 in the normal and insulin-dependent diabetic population. Our findings demonstrate that there is a highly significantly increased frequency of Dw4 in DR4 positive diabetics as compared with normals and a significantly decreased frequency of Dw2 and Dw12 in the few DR2 positive insulin-dependent diabetics that we have found. In addition, we have used PLT reagents to define a new LD specificity, LD-MN2, that is associated with DR2 and is found significantly more frequently in DR2+ IDD patients than in DR2+ normals. These results suggest that determinants of import in the association between HLA-D and IDD may be more closely related to Dw than to DR.     

Cells homozygous for two HLA-DR7-associated HLA-D specificities express highly similar DR molecules but different DQ molecules

The Ia molecules expressed by cells homozygous for two distinct HLA-DR7-associated HLA-D specificities, Dw7S and Dw11L, were compared. The complete Ia phenotypes of these cells are DR7, DRw53, DQw2, Dw7S, DPw4 and DR7, DRw53, DQw3, Dw11L, DPw4, respectively. Immunofluorescence analysis revealed that three DQ-specific monoclonal antibodies (Leu-10, 33.1, and HK-19), which detect polymorphic DQ determinants that do not correspond to known serologic specificities, are nonreactive with DR7, Dw7S cells but are reactive with DR7, Dw11L cells. The DR molecules isolated from Dw7S and Dw11L cells are very similar and comigrate when analyzed together by two-dimensional gel electrophoresis. In contrast, the DQ molecules isolated from these cells are structurally distinct: the DQ beta chains of DQw2-bearing molecules from Dw7S cells are very basic, while those of DQw3-bearing DQ molecules from Dw11L cells are more acidic. The finding that two DR7, D-different cells express indistinguishable DR molecules and structurally distinct DQ molecules documents a unique pattern of Ia molecular organization which is different from those previously described for the DR2-, DR4-, or DRw8-associated HLA-D 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.     

Human Ia-like DRw lymphocyte antigens stimulating activity in primary mixed lymphocyte reaction.

The intensity of the primary mixed lymphocyte reaction (MLR) seems to be influenced by at least two distinct determinants of the HLA-D region: the HLA-Dw stimulating products and the human Ia-like B lymphocyte DR (D-related) antigens. In three families, the primary MLR is significantly higher in case of HLA haploidentity, when stimulating and responding cells differ with regard to both Dw and DRw determinants, than when only Dw products are different. Thus, an additional effect of DR antigens during primary MLR is observed. The role of DRw antigens in primary MLR and the results obtained by a primed lymphocyte test which can discriminate between Dw3 and DRw3, indicate that DRw and Dw products could be distinct determinants.     

Analysis of HLA-DRB and -DQB gene RFLPs in DR7 homozygous cell lines: associations with Dw11, Dw17 and DB1

The DR7-associated Dw specificities, Dw11, Dw17 and DB1 were investigated with regard to DRB- and DQB-gene polymorphism, as revealed by RFLP analysis using the restriction enzyme TaqI. In the 22 DR7 homozygous cell lines investigated, each of these Dw specificities was found to correlate to one specific RFLP defined DR-DQ haplotype. In addition, a clear linkage disequilibrium to a specific HLA-B locus allele for each Dw specificity was noted, indicating that the Dw subtypes of DR7 often are associated with a conserved HLA-B-DR-DQ haplotype. Only one genetically homozygous cell line, PLH, deviated from these correlations. This cell line, notably derived from an individual with a deletion of the 21-hydroxylase B-gene (21-OHB), caries the HLA haplotype Bw47, DR7, DQw2, DB1, but displayed a DRB RFLP otherwise found in association with Dw17.     

COMPLEXITY OF THE BOVINE MHC CLASS-II SPECIFICITY DW3 AS DEFINED BY ALLOANTISERA

Alloantisera related to the bovine major histocompatibility complex (MHC) class-II specificity Dw3 were investigated by cross-absorption experiments and by application of the monoclonal antibody-specific immobilization of lymphocyte antigen assay (MAILA). The absorption study revealed antibodies specific for an antigenic determinant shared by all Ds03 (Dw3)-positive animals, and several other antibody populations recognizing the locally defined specificites Ds10, Ds11 and Ds15, that are closely associated with Ds03. The results of the MAILA-assay indicate that the Ds03 specificity is probably encoded by DQ, whereas specificities Ds10 and Ds11 are more closely associated with DR molecules. The data presented here provide the first evidence that bovine DR and DQ specificities can be identified separately by serological methods using alloimmune antisera.     

Functional analysis of MHC class II-restricted T cells derived from a Caucasian with a DR4, Dw15, DQw8 haplotype

Rabies virus-specific CD4+ T lymphocyte clones were isolated from a Caucasian male vaccine recipient (DR4/7, DQw2/w8; DPw4) and studied for their major histocompatibility complex restricting elements. None of the rabies-specific T-cell clones could be induced to proliferate to antigen by either lymphoblastoid cells or DR-transfected L cells expressing DR4 molecules of the Dw subtypes commonly found on Caucasian individuals (Dw4, Dw10, Dw13, Dw14). The HLA-Dw subtype of the rabies vaccine recipient was determined by conventional mixed lymphocyte culture, and the results revealed that this individual had a DR4 (Dw15), DR7 (Dw7) phenotype. The presence of the DR4, Dw15 antigen was confirmed by nucleotide sequencing of the DR4B1 gene corresponding to the DRB1*0405 allele. Significant antigen-induced T-cell proliferative responses were obtained with two DR4, Dw15, DQw4 homozygous lymphoblastoid cell lines of Japanese origin (HAS-15 and KT-3) and with a L-cell transfectant expressing the DR4, Dw15 molecule. The existence of the DR4, Dw15 antigen in the Japanese has been reported to be associated with the DQw4 specificity. However, the presence of DQw8 (previously designated DQw3.2) and the absence of DQw4 in the lymphoblastoid cells of the Caucasian rabies vaccine was confirmed with monoclonal antibodies IVD12 (anti-DQw7 + DQw8 + DQw9) and HU46 (anti-DQw4) and by the reactivity of a DQw8-restricted antigen-specific T-cell clone. These studies indicate, contrary to previous findings, that the DR4, Dw15 molecule may be present in Caucasian (non-Japanese) individuals in association with DQw8.     

A study of the HLA-Dw determinants and their relationship to DR and DQ antigens in three South African population groups: South African Caucasoids, South African Negroes, and Cape coloureds

The HLA-Dw specificities of a group of 177 unrelated randomly selected healthy individuals consisting of 67 South African Negroes (Xhosa), 57 Cape Coloureds, and 53 South African Caucasoids were determined. HLA-Dw specificities were determined in a mixed lymphocyte culture test using HTCs. Antigen and gene frequencies as well as the association between HLA-DR, DQ, and Dw were established in three populations. HLA-Dw gene frequencies in the South African Caucasoids agreed with these frequencies in other Caucasoid groups. The HLA-Dw1 frequency was decreased in the Cape Coloureds and South African Negroes compared to the Caucasoids. The gene frequency of Dw3 was low in the South African Negroes in spite of the fact that DR3 is a common DR antigen in this group. A high frequency of Dw 'blank' was observed in the South African Negroes and Cape Coloureds, suggesting the existence of undefined HLA-Dw specificities in these populations. Data concerning the HLA-Dw, DR, and DQ relationships showed that once a certain Dw specificity was associated with a particular DR and DQ antigen, this association remained a fixed entity in the different population groups.     

HLA-DP incompatibilities induce significant proliferation in primary mixed lymphocyte cultures in HLA-A, -B, -DR and -DQ compatible individuals: Implications for allogeneic bone marrow transplantation

The major part of the proliferative response in primary mixed lymphocyte cultures (MLC) is caused by HLA-DRB1 incompatibilities. In DRB1-matched pairs the proliferation induced by HLA-DRB3, -DQ and -DP mismatches may be unmasked. In most previous studies the influence of HLA-DP incompatibilities in primary MLC has been investigated in homozygous typing cells representing only a few Dw specificities. We were interested in determining the stimulatory capacity of isolated HLA-DP mismatches, ascertained by RFLP analysis, in primary MLC in HLA-A, -B, -DR and -DQ compatible, unrelated heterozygous individuals of many different Dw specificities. Thirty-eight MLCs performed with cells from related pairs and 67 with cells from unrelated pairs were evaluated. All but nine of the MLCs were analyzed in both directions, giving a total of 201 investigated reactions. The relative responses (RR) in the three MLCs performed between DP incompatible, related pairs were all positive (RR greater than or equal to 8%). Eighty of 82 MLCs performed with cells from DP incompatible, unrelated individuals were positive, whereas 37 of 46 MLCs between DP compatible, unrelated pairs were negative (RR less than 8%) (p less than 10(-10)). The magnitude of the RR was influenced by the number of DP mismatches. Thus, the mean RR was approximately twice as high in MLCs in which responder and stimulator cells differed by two DP antigens (mean RR 60.5%) compared with reactions with only one DP mismatch (mean RR 35.4%) (p less than 10(-3)). RFLP-defined HLA-DP incompatibilities predict a positive primary MLC in HLA-A, -B, -DR and -DQ matched individuals with a high degree of accuracy (98%).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

New Dw8 associated class II specificities defined by cytotoxic T lymphocyte clones

Two Leu2(-), Leu3(+), Leu4(+) human cytotoxic T lymphocyte (CTL) clones, BE-11 and AF-3, were generated against Epstein-Barr virus (EBV)-transformed cell line GI (Dw8/DRw8/DQWa homozygous). Blocking experiments with various monoclonal antibodies (MoAbs) revealed that the former recognized the DR molecule and the latter recognized the DQ molecule, respectively. Panel studies showed that CTL clone BE-11 lysed not only DRw8-positive cells but also DR1-positive ones. CTL clone AF-3 exhibited cytotoxicity against only Dw8/DRw8/DQWa typed cells. Until now, such specificities have not been defined serologically or biochemically. These results demonstrated that the previously unknown DR and DQ specificities could be defined by CTL clones, suggesting that CTL clones might be especially valuable tools for investigating the structural polymorphism of HLA antigens.     

Ninth International Histocompatibility pre-workshop testing of Dw6 HTCs. Two subtypes of Dw6

In the scope of the cellular part of the 9th International Histocompatibility Workshop, the offered homozygous typing cells (HTCs) of several specificities have been screened in a pre-Workshop. In Leiden and Seattle all HTCs typing for "HLA-Dw6" have been tested. This implied that in both laboratories mixed lymphocyte culture (MLC) matrices were performed between the Dw6 HTCs and that all HTCs were tested as stimulator cells against a panel of heterozygous responder cells. The results clearly demonstrated that "HLA-Dw6" as defined by the different participating laboratories can be split into two major groups, Dw6.a and Dw6.b. This observation confirms and extends previous reports. None of the 9th Workshop B-cell sera could discriminate between the two presently described subgroups of HLA-Dw6.     

Effects of cyclosporin A on in vitro lymphocyte response to autologous or allogeneic stimulation: influence of HLA phenotypes

In this study we investigated whether the interindividual variability of lymphocyte sensitivity to cyclosporin A (CsA) could be controlled by the HLA region. The models used were the in vitro primary and secondary autologous (AMLR) and allogeneic mixed lymphocyte (MLR) cultures of cells from 32 healthy subjects from our HLA reference panel. Our results show that CsA inhibited primary allogeneic MLR to a much greater extent than primary AMLR (-81 +/- 2% vs -38 +/- 8%, P less than 0.001). The same pattern was observed when cells harvested from CsA-treated primary cultures were rechallenged in secondary cultures with the original sensitizing stimulator cells (-40 +/- 6% vs -17 +/- 9%, P less than 0.05). No differences were observed in primary autologous and allogeneic cultures among responders of different HLA phenotypes. In contrast, the secondary responses did vary according to the HLA types: in secondary AMLR, CsA-priming did not lower, or even enhance, the proliferative responses of DR5+ and/or DR2+ lymphocytes (+7 +/- 13%), whereas it significantly lowered the responses of DR2-5- cells (-46 +/- 8%). In secondary MLR, lymphocytes proliferation was lowered by CsA-priming in all but DRW11(5)+ subjects (-45 +/- 7% vs +2 +/- 23%, P less than 0.05). It is concluded that the individual HLA phenotype influences the pattern of lymphocyte sensitivity to CsA.     

Dw subtypes of serologically defined DR-DQ specificities restrict recognition of cytomegalovirus

We have investigated the relationship between serologically defined (Ia) and T lymphocyte-defined (LD/Dw) determinants in restricted recognition of cytomegalovirus (CMV) by human T lymphocytes. T lymphocytes isolated from CMV seropositive individuals expressing DQw3/DR4/Dw4 antigens were "sensitized" to CMV in vitro; CMV-specific blasts were isolated and tested for their ability to recognize CMV presented by cells expressing different DR4-associated Dw antigens (i.e., Dw4, Dw10, Dw13, Dw14, and Dw15). Similar studies were also performed using T lymphocytes from individuals expressing DQw1/DR2/Dw2 specificities and antigen presenting cells (APC) expressing the DR2-associated Dw/LD subtypes, Dw2, Dw12, and LD-MN2. CMV-specific T cell blasts were used as responding cells in order to reduce nonspecific background alloresponses which occur with allogeneic APC. In all cases it was found that the determinants involved in restricted recognition of CMV were subtypic to the DR-associated Ia specificities. To distinguish whether Dw specificities associated with DQ or with DR molecules, or both, were involved in these responses, we used anti-DR (L243) and an anti-DQwl (S3/4) monoclonal antibodies (MoAb) to block CMV-specific responses. Both MoAb significantly blocked responses, suggesting that determinants associated with both DR and DQ molecules are involved in restricted recognition of CMV by T cells.     

Discrete PLT clones detecting HLA-D and new HLA-non-D encoded cell surface determinants

Clones of alloreactive T-lymphocytes were isolated from 6-day-old mixed leukocyte cultures (MLCs) by limiting dilution in the presence of filler cells (either pooled irradiated peripheral blood mononuclear cells (PBMC_X) or autologous PBMC_X) followed by expansion of clonal progeny with Intcrlcukin 2 (IL 2). Using allogeneic IL 2, produced by stimulating pooled PBMC with phytohaemagglutinin (PHA), in conjunction with pooled filler cells for the limiting dilution cloning, over half the clones obtained had primed lymphocyte typing (PLT) function. The majority of these discriminated priming cell HLA-D type with fine specificity. However, clones which were restimulated by cells of specificities other than the priming HLA—D type were also found, and, moreover, some ("wild" clones) were not restimulated by the original priming cells. Antigens responsible for restimulating these clones included hitherto undetected HLA-encoded products (different from HLA-A, B, C, D, DR, MT, MB or SB). Such "wild" clones were also obtained after cloning procedures in which autologous filler cells were used. Autologous IL 2, depleted of mitogenic PHA, and used in conjunction with autologous filler cells, also failed to prevent generation of such clones. These results indicate that although the PLT-functional lymphocyte population alloactivated by a single HLA—DR/Dw specificity expressed by HLA—D homozygous stimulating cells consists of a majority of clones exclusively recognising specific priming-type HLA-DR/Dw products, it also contains clones recognising different HLA-DR/Dw specificities, as well as clones recognising lymphocyte stimulating determinants hitherto undetected by serological or cellular methods.     

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.     

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.