Functional and molecular analysis of three distinct HLA-DR4 beta-chains responsible for the MLR between HLA-Dw4, Dw15, and DKT2

Differences in structure and function of HLA-class II molecules between HLA-DR4-related HLA-D specificities HLA-Dw4, Dw15, and DKT2 were investigated. Anti-HLA-DR framework monoclonal antibody HU-4 completely inhibited the one-way mixed lymphocyte reaction (MLR) between these specificities. HU-4 precipitated a monomorphic alpha-chain and a polymorphic beta-chain of human class II molecules from each B lymphoblastoid cell line (BLCL) homozygous for these three HLA-D specificities. We established IL 2-dependent T cell lines specific for streptococcal cell wall (SCW) antigen from peripheral blood lymphocytes (PBL) from four DR4-positive donors: an HLA-Dw4/DKT2 heterozygote, an HLA-Dw4/Dw12 heterozygote, an HLA-DKT2/D-blank heterozygote, and an HLA-Dw15/D-blank heterozygote. These T cell lines showed a proliferative response to SCW antigen in the presence of autologous or allogeneic antigen-presenting cells (APC) when T cell donors and APC donors shared at least one of the HLA-D specificities. This cooperation between the T cell line and APC was completely blocked by HU-4. We conclude from these data that the T cells could distinguish three distinct DR4 molecules expressed on APC of HLA-Dw4, Dw15, and DKT2 as restriction molecules at the presentation of SCW antigen.     

Two distinct class II molecules encoded by the genes within HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating and restriction molecules

By using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we investigated the difference in the HLA class II molecule between HLA-Dw2 and Dw12, both of which are typed as HLA-DR2 serologically. The anti-HLA-DR framework monoclonal antibody (MoAb) HU-4 precipitated an alpha-chain and two beta-chains of human class II molecules from both Dw2 and Dw12 homozygous B lymphoblastoid cell lines. It was demonstrated clearly that an alpha-chain (alpha 1) and one of the beta-chains (beta 1) showed no difference in mobility in the 2D-PAGE between Dw2 and Dw12, but that another beta chain (beta 2) of Dw2 was distinct from that of Dw12 in the 2D-PAGE profile. Thus, MoAb HU-4 precipitated alpha 1 beta 1 and alpha 1 beta 2 molecules from Dw2 and Dw12, and the alpha 1 beta 1 molecule appears to be an HLA-DR2 molecule. The alpha 1 beta 2 molecule, on the other hand, is a class II molecule distinct from those precipitated with anti-DR2, anti-DQw1 (DC1, MB1, MT1), or anti-FA MoAbs. MoAb HU-4 completely inhibited the mixed lymphocyte culture reaction (MLR) between Dw2 and Dw12, but anti-DR2 MoAb HU-30, which reacts only with the alpha 1 beta 1 molecule, did not show an inhibitory effect on the MLR between Dw2 and Dw12. The alpha 1 beta 2 molecule is therefore the molecule which elicits MLR between Dw2 and Dw12. An IL 2-dependent T cell line established from an HLA-Dw12/D blank heterozygous high responder to the streptococcal cell wall antigen (SCW) clearly distinguished the Dw2 specificity from Dw12 specificity expressed on the antigen-presenting cell (APC). Moreover, MoAb HU-4 markedly inhibited the cooperation between the T cell line and APC to respond to SCW. These observations indicate that the alpha 1 beta 2 molecule is recognized as a restriction molecule by the T cell line at the antigen presentation of SCW through APC MoAb HU-30 on the other hand partially inhibited the MLR between Dw2 or Dw12 homozygous cell as a stimulator cell and non DR2 cell as a responder cell. It markedly inhibited the proliferative response of the Dw12/D- heterozygous T cell line to SCW, presented by Dw2+ but Dw12- allogeneic APC, and the peripheral response of Dw2 or Dw12 homozygous peripheral blood lymphocytes to SCW. Thus, two distinct class II molecules encoded by the genes within the HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating molecules in the MLR and as restriction molecules in the antigen presentation by APC.     

Dissection of HLA class II haplotypes in HLA-DR4 homozygous individuals

In order to identify better markers for HLA-DR4-associated autoimmune disorders, we have studied the complexity of the HLA class II region in DR4-positive cells at the DNA level and compared the DNA polymorphism with that defined by serology, mixed lymphocyte culture (MLC) reactivity, and protein chemistry. At the DNA level, HLA-DR4 can be characterized by a homogeneous pattern of bands hybridizing to HLA class II cDNA probes. Besides, subtypes can be defined within DR4 using HLA-DR , -DQ , and -DQ cDNA probes in Southern blot analysis. Three subtypes are found using the DR cDNA probe. One of these subtypes correlates with the cellularly defined Dw15 specificity, another with the serologically defined LB4 and LB14 specificities. None of the restriction fragment length polymorphism (RFLP) patterns coincide with the MLC-defined DR4 subtypes Dw4, DW10, Dw13, and Dw14 separately. Variation of two fragments hybridizing to the DQ cDNA probe obtained after either Pvu II or Taq I digestion yields three subtypes. Pvu II- and Eco RI-digested DR4 DNA give rise to three DQ detectable subtypes. Correlation between these subtypes, isoelectric point variation of DQ molecules, and the DQ-related allelic system TA10/2B3 are demonstrated. Some of the patterns obtained with DQ and DQ cDNA probes display heterozygosity in the DQ region, as demonstrated by family segregation. No correlation was observed between DQ and the cellularly defined Dw determinants. A new polymorphism has been obtained with the DQ probe, probably due to DX polymorphism. DR RFLP divides the LB 14 supertypic specificity into two new subtypes. A combination of the four different techniques applied to a panel of 16 DR4 homozygous cell lines reveals at least nine different haplotypes in DR4. These newly defined haplotypes may be of help in further studies concerning the relationship of micropolymorphism with several diseases.     

Immunochemistry of the HLA class II molecules isolated from a mouse cell transfected with DQ alpha and beta genes from a DR4 haplotype

Cells from a mouse B lymphoma were transfected by DQ alpha and DQ beta genes derived from a DR4 haplotype. Quantitatively, the resulting expression of human class II molecules was similar to that of human B lymphoblastoid cell lines. Qualitatively, the transformant class II molecules differed from normal class II molecules in their carbohydrate moiety. As for their antigenic specificity, they were shown to carry two determinants previously identified on DQ molecules controlled by DR4 haplotypes, i. e., DQw3 and DCHON. The transformant molecules did not carry a third DR4-associated specificity, DC5 (equivalent to TA10), and must possess a structure allelic to DC5. However, no corresponding alloantigenic specificity was detected by a screening of relevant alloantisera.     

Differential contribution of C4 and HLA-DQ genes to systemic lupus erythematosus susceptibility

The particular histocompatability antigen (HLA) gene(s) that may confer systemic lupus erythematosus (SLE) susceptibility remains unknown. In the present study, 58 unrelated patients and 69 controls have been analyzed for their class I and class II serologic antigens, class II (DR and DQ) DNA restriction fragment length polymorphism, their deduced DQA1 and B1 exon 2 nucleotide sequences and their corresponding amino acid residues. By using the etiologic fraction () as an almost absolute measure of the strongest linkage disequilibrium of an HLA marker to the putative SLE susceptibility locus, it has been found that the strength of association of the HLA marker may be quantified as follows: DQA1^*0501 (associated to DR3) or DQB1^*0201 (associated to DR3) > non Asp 57 DQ/Arg 52 DQ > DR3 > non Asp 57 DQ. Thus, molecular HLA DQ markers tend to be more accurate as susceptibility markers than the classical serologic markers (DR3). However, dominant or recessive non Asp 57 DQ susceptibility theories, as previously postulated for insulin-dependent diabetes mellitus, do not hold in our SLE nephritic population; indeed, three patients bear neither Arg 52 DQ nor Asp 57 DQ susceptibility factors. On the other hand, nonsusceptibility factors are included in our population in the A30B18CF130-DR3DQ2(Dw25) haplotype and not in A1B8CS01-DR3DQ2(Dw24); this distinctive association has also been recorded in type I diabetes mellitus and may reflect the existence of common pathogenic HLA-linked factors for both diseases only in the A30B18CF10DR3DQ2-(Dw 25) haplotype. Finally, the observed increase of deleted C4 genes (and not null C4 proteins) in nephritic patients shows that C4 genes are disease markers, but probably without a pathogenic role.     

A possible association between basic amino acids of position 13 of DRB1 chains and autoimmune hepatitis

Fifty-one patients with autoimmune hepatitis have been studied for HLA association by conventional serology and also by modified polymerase chain reaction-restriction fragment lenght polymorphism (PCR-RFLP) genotyping.HLA-DR4 was significantly associated with autoimmmune hepatitis (46 of 51 patients, 90.2%). DNA typing of the DRB1 gene for 43 DR4-positive patients by using the PCR-RFLP technique revealed that of 43 patients, 33 had DRB1 ^* 0405 (Dw15), five had DRB1 ^* 0406 (DwKT2), four had DRB1 ^* 0403 (Dw13a), two had DRB1 ^* 0401 (Dw4), two of 43 had DRB1 ^* 0407 (Dw13b) and one had DRB1 ^* 0408 (Dw14b). Thus, there was no significant difference in Dw frequencies between DR4-positive patients and DR4-positive healthy subjects. These findings suggest that the DR4-specific sequence (Val 11 and His 13 at amino acid positions 11 and 13, respectively), but not particular Dw-associated DR4 sequence, in the first domain of the DRB1 chain contributes to susceptibility to autoimmune hepatitis among Japanese. Interestingly, all five of the DR4-negative patients had the DR2 specificity (DRB1 ¹⁵⁰² or 1601). Taken together, these results imply that the basic amino acids at position 13, which is present only on the DR2 and DR4 B1 molecules (Arg on DR2 and His on DR4), are most important for determining the predisposition to autoimmune hepatitis. Address correspondence and offprint requests to: M. Ota.     

A novel HLA class II molecule

Molecular evidence has been obtained for a novel monomorphic HLA class II molecule distinct from HLA-DP/DQ/DR using a panel of lymphoblastoid cells which include HLA-loss mutants. The expression of this molecule was investigated using monomorphic affinity-purified mouse monoclonal antibodies (mAbs), including one of the IgG_2a subclass designated EDUA. This antibody reacts strongly in a cell-binding radioimmunoassay with HLA-DR and -DQ loss mutants derived from a lymphoblastoid parental cell. The EDU-1 mAb also reacted with a local panel of homozygous Epstein-Barr virus-transformed cell lines. The reactive molecules were further detected on allostimulated T-cell clones and various leukemic cells including those of myeloid origin which lack surface expression of HLA-DQ molecules. Thus the class II molecule described in this study corresponds to a monomorphic HLA class II determinant expressed on a variety of cells of different origin and HLA phenotypes. Moreover, this antigen structure is distinct from that of HLA-DP/DQ/DR as shown by direct immunoprecipitation, serial immunodepletion experiments, and two-dimensional gel electrophoresis. The molecule could be specified by new class II genes between DP and DQ. An alternative explanation for the genetic basis of the novel molecule is the existence of isotypic associations between alpha and beta chains of various class II molecules (DP, DX, DZ, and DO) but not DR and DQ as the mutant cells tested lack the latter genes.     

Functional definition of HLA-DR and -DQ epitopes specific for DR2-short,DwFJO haplotype

T-lymphocyte clones specific for the influenza A/Texas virus were obtained by limiting dilution of activated T cells from an HLA A2/3, B7/39, Cw -/-, DR2-short/2 short, DQw1/w1, DwFJO/FJO donor. Among the proliferating clones studied, and irrespective of their antigenic specificities, most of them were restricted by epitope(s) on HLA-DR molecules present only on DR2-short/DwFJO cells but not on DR2-negative or DR2-long positive (Dw2, Dw12, Dw-) cells. Two clones were restricted by epitopes borne by DQ products. Here again, these epitopes were present on DR2-short/DwFJO but not on DR2-long, DQw1 (Dw2, Dw12) cells, indicating that the DQwl molecules of DR2-long and DR2-short haplotypes are different. Taken together, these results indicate that the DR2-short, DwFJO haplotype is characterized by both HLA-DR- and DQ-specific molecules. Finally, one clone was restricted by an epitope shared by DR products from DR2 short/DwFJO, DRw11, and DRw13 haplotypes. This latter functional determinant has never been described until now.Abbreviations used in this paper APC antigen-presenting cells - HAU hemagglutinin units of influenza virus - HLA human leukocyte antigens - HTC homozygous typing cells - IL-2 interleukin 2 - mAb monoclonal antibody - MHC major histocompatibility complex - MLR mixed lymphocyte reactions - PBM peripheral blood mononuclear cells - %RR relative response percent     

HLA-DR typing using DNA amplification by the polymerase chain reaction and sequential hybridization to sequence-specific oligonucleotide probes

A series of sequence-specific oligonucleotides (SSOs) have been used to type alleles at the HLA-DRB1 locus. Genomic DNA was amplified to high copy number by the polymerase chain reaction (PCR) and hybridizations of the dot-blotted, amplified DNA to a series of 14 SSOs enabled the identification of the major specificities DR1-DRw14. Certain alleles (DR3 and DR4) could be rapidly and accurately identified by running the products of allele-specific amplification of genomic DNA on agarose gels. This approach facilitated the typing of serological specificities such as subtypes of DR3 (DRw17 and DRw18) as well as alleles previously detected by the mixed lymphocyte reaction including subtypes of DR4 (Dw4, Dw10, Dw13, Dw14, and Dw15). The HLA-DR types obtained by SSO probing conformed to rules of Mendelian inheritance when they were applied to a series of 75 families. A full DR type could be obtained from many individuals simultaneously without needing to separate or store viable lymphocytes. Thus, this technique may have considerable implications for the analysis of disease associations with HLA class II alleles, particularly in circumstances where facilities for the initial preparation and storage of the samples may be limited.     

Heterogeneity of HLA genetic factors in IDDM susceptibility

The association of certain HLA-D alleles with insulin-dependent diabetes mellitus (IDDM) is well known. One hundred and sixty-one non-related diabetic individuals and 142 non-related healthy controls were typed for the HLA DR-DQw-Dw association, using a restriction fragment length polymorphism (RFLP) typing method that combines three probe/enzyme systems: DRB/Taq I, DQB/Taq I, and DQB/Bam HI. Comparison of frequencies in both diabetics and controls confirms previous results in terms of HLA class II and IDDM association. Moreover, we have found that DR3/4 heterozygous individuals are more susceptible to IDDM when they are also Dw25 (associated with B18) than when they are Dw24 (associated with B8). Using oligonucleotide dot-blot hybridizations we analyzed the HLA-DQB1 sequence of DR3, Dw24 and DR3, Dw25 homozygous individuals, and we found no difference at position 57 between these two DR3-carrying haplotypes. This observation points to the heterogeneity of HLA genetic factors in IDDM susceptibility. Offprint requests to: D. Cohen.     

Mapping and characterization of the DQ subregion of the ovine MHC

A map of the ovine MHC class II DQ subregion has been constructed from overlapping cosmid clones. This region consists of two loci linked on a linear tract of 130 kb DNA. Each locus consists of a DQA and a DQB gene in a tail-to-tail orientation. The genes in each locus are transcribed but only those designated DQ1 express class II molecules at the surface of mouse L cells following DNA-mediated gene transfection. The DQA1 and DQB1 genes are separated by 11kb while the DQA2 and B2 genes are 25 kb apart. The loci are separated by 22 kb.     

Analysis of the molecular specificities of anti-class II monoclonal antibodies by using L cell transfectants expressing HLA class II molecules

Expressible HLA class II alpha- and beta-chain cDNA were used for DNA-mediated gene transfer to produce L cell transfectants expressing single types of human class II molecules. Cloned transfectants expressing nine different class II molecules were isolated: DR alpha: DR1 beta I, DR alpha: DR4 beta I, DR alpha: DR5 beta I, DR alpha: DR5 beta III (DRw52), DR alpha: DR7 beta I, DR alpha: DR4/7 beta IV (DRw53), DQ7 alpha: DQw2 beta, DQ7 alpha: DQw3 beta, and DPw4 alpha: DPw4 beta. These class II-expressing transfectants were used to analyze by flow cytometry the molecular specificities of 20 anti-class II mAb. These analyes indicate that some mAb are more broadly reactive than was previously thought based on immunochemical studies. In contrast, the narrow molecular specificities of other anti-class II mAb were confirmed by this approach. Transfectants expressing human class II molecules should be valuable reagents for studies of B cell and T cell defined epitopes on these molecules.     

The narcolepsy-associatedDRw15, DQw6, Dw2 haplotype has no uniqueHLA-DQA or -DQB restriction fragments and does not extend to theHLA-DP subregion

Almost all patients with cataplectic narcolepsy are DR2-positive. It has been suggested that thenon-DR2 allele/haplotype might not be neutral with respect to disease susceptibility. It has also been reported thatTaq IDQA andBam HI,Eco RI,Eco RV, andPst IDQB restriction fragments might differentiate between narcoleptic and healthy DR2-positive individuals. In the present study,HLA class II gene polymorphisms were investigated by restriction fragment length polymorphism (RFLP) analysis in 47 Swedish patients with cataplectic narcolepsy, 100 random controls, and DR2-associated homozygous cell lines. All patients hadTaq IDRBDQA-DQB patterns corresponding to theDRw15,DQw6, Dw2 haplotype. The non-DR2 haplotype was found to be neutral. This genotyped group of patients allows firm rejection of a recessive mode of inheritance and supports a dominant or additive model. NoDQA orDQB RFLPs were found that could differentiate between DR2-positive narcoleptics, DRw15,DQw6,Dw2-positive controls, orDw2-homozygous cell lines. No significantMsp IHLA-DP association was found. No linkage disequilibrium was observed between theDRw15,DQw6,Dw2 haplotype and alleles of theDP subregion in patients or controls. Thus, theHLA-D region-associated narcolepsy susceptibility gene may be located telomeric to theHLA-DP subregion. No RFLPs have been observed that can locate the narcolepsy susceptibility gene closer to theDQ than to theDR subregion.     

The polymorphisms of HTC-defined HLA specificities in the Shanghai Chinese population

With reference sera and homozygous typing cells (HTCs) of 3rd Asia-Oceania Histocompatibility Workshop Conference, 56 healthy unrelated subjects in Shanghai were typed for HLA-A, B, C, DR, DQ, and Dw. This paper presents the results of HLA-Dw typing, its relationship to serological class II antigens, and the distribution of Dw in the population. The polymorphism patterns of Chinese Dw specificities were quite different from those in Caucasoids and Japanese. The predominant Dw phenotypes detected in Shanghai Chinese were Dw 2, Dw 3, DKT 2, Dw 7 c, (Dw7 + Dw 17) and Dw 23 (DB 5). And significant correlations were observed between Dw 1 and DR 1, Dw 2 and DR 2, Dw 3 and DR 3, Dw 7 c and DR 7, DB 7 and DRw 8, as well as Dw 23 and DR 9. SMY 129, a novel Dw specificity defined by local HTCs and co-studied by the laboratories joined for Dw typing in 3rd AOHWC showed its correlation with DR 5. Nevertheless, more than fifty percent of Dw specificities could not be assigned in the four correspondent designated serological antigens, DR 2, DR 5, DRw 8 and DR 9, respectively, which, together with other blank Dw specificities, gave a total blank Dw gene frequency as high as 43.2% in the population. It was suggested by further analysis that novel Dw specificities might be identified more effectively if efforts would be concentrated on DR 5 and DR 9, two antigen families which, in some way, might represent the characteristics of HLA system in Chinese. Besides, certain HTC-defined antigens, e.g. Dw 3 and the DR 4-related Dw specificities, have been revealed to be in linkage disequilibrium with other DR antigens in addition with the correspondent designated ones, resulting in some unique haplotype combinations in Shanghai Chinese. It seems to us that the particular patterns of polymorphisms of serum- and cell-defined HLA class II antigens would be helpful to elucidate the mechanisms by which certain diseases are in association with HLA in Chinese in a different manner as compared with that in Caucasoids.     

Involvement of class II MHC molecules in the LPS-induction of IL-1/TNF secretions by human monocytes. Quantitative differences at the polymorphic level

Anti-class II ag mAb (DR and DQ) inhibited, in a dose-dependent manner, LPS-induced IL-1 and TNF secretions from human monocytes (34 to 95% inhibition). The potentiating effect of IFN-gamma on LPS-induced TNF secretion (15.3 +/- 0.7 to 44 +/- 0.6 ng/ml) was also blocked by anti-class II ag mAb (44 +/- 0.6 to 0.3 +/- 0.03 ng/ml). We also report a relationship between interindividual differences in monocyte IL-1 and TNF secretions and the HLA-D-encoded genetic polymorphism. Heterozygotes were, in general, higher secretors of those cytokines than homozygotes. Analysis of these secretions in heterozygotes demonstrated a differential effect of certain haplotype combinations (i.e., DR2-DR4 vs DR2-DR3) that could be arbitrarily characterized as being "low" or "high" secretors (6,230 +/- 2,950 vs 13,029 +/- 6,541 cpm for IL-1, and 12 +/- 10 vs 25 +/- 15 ng/ml for TNF, p = 0.006 and 0.048). DR-associated Dw subtypes appeared to account for differences within certain haplotype combinations (Dw18 vs Dw19 in DRw13/DR4) (11,227 +/- 3,648 vs 17,166 +/- 3,176 cpm for IL-1, and 13 +/- 9 vs 25 +/- 10 ng/ml for TNF, p = 0.02 and 0.047). Interindividual differences were better explained by differences in LPS sensitivity than by differences in the kinetics of secretion and related not to the secretory process itself but to the rate of cytokine synthesis. Finally, there were no relationships between high secretor genotypes and IDD high risk genotypes. Thus, we conclude that, a) LPS-induced IL-1 and TNF secretions are, at least in part, regulated by class II MHC molecules, b) that HLA-D region-encoded genetic polymorphism accounts for interindividual differences in these secretions, and c) that the HLA-associated risk to develop IDD is not explained by these cytokine secretory differences as previously proposed.     

HLA-DQ molecules form alpha-beta heterodimers of mixed allotype

Retroviral vectors with an internal cytomegalovirus major immediate-early gene enhancer/promoter regulating HLA class II gene expression were used to transfer HLA cDNA into human EBV-transformed B-lymphoblastoid cell lines. HLA-DQ2 beta and DQ3.2 beta cDNA were transferred into DQ3.2 and DQ2 homozygous lymphoblastoid cell lines, respectively. Serologic analysis of the infected cell lines with allospecific mAb demonstrated surface expression of these exogenous DQ molecules implying that DQ alpha-chains from DR3, DQ2-positive cells can pair with DQ3.2 beta-chains and, similarly, DQ alpha-chains from DR4, DQ3.2-positive cells can pair with DQ2 beta-chains. Immunoprecipitation of the introduced DQ3.2 beta molecule resulted in co-purification of the allotype-mismatched endogenous DQ2 alpha polypeptide. We also show that vectors with a cytomegalovirus major immediate-early gene enhancer/promoter result in higher levels of expression of the transduced gene compared to previously described HLA vectors with either the SV-40 early enhancer/promoter or the lymphotropic papovavirus-enhanced SV-40 promoter. Although deletion of HLA cDNA did sometimes occur in the process of generating virus-producing clones, the HLA cDNA is stably maintained in virus-producing clones, once it is generated. This retroviral expression system is a highly efficient way to study HLA gene function.     

Restriction fragment length polymorphism of the major histocompatibility complex of the dog

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D-homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (Bam HI, Eco RI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I, and Bgl II), separated by agarose gel electrophoresis, and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabeled HLA cDNA probes corresponding to DR, DQ, DP, and DO beta genes. The autoradiograms for all nine enzyme digests displayed multiple bands with the DRb, DQb, and DPb probes while the DOb probe hybridized with one to two bands. The RFLP patterns were highly polymorphic but consistent within each DLA-D type. Standard RFLP patterns were established for nine DLA-D types which could be discriminated from each other by using two enzymes (Rsa I and Pst I) and the HLA-DPb probe. Cluster analysis of the polymorphic restriction fragments detected by the DRb probe revealed four closely related supertypic groups or DLA-DR families: Dw3+Dw4+D1, Dw8+D10, D7+D16+D9, and Dw1. This study provides the basis for DLA-D genotyping at a population level by RFLP analysis. These results also suggest that the genetic organization of the DLA-D region may closely resemble that of the HLA complex.     

Sequencing of HLA-D in responders and nonresponders to short ragweed allergen,Amb a V

We investigated the molecular basis for the striking association between HLA-DR2,Dw2 and human immune responsiveness to the Ambrosia artemisiifolia (short ragweed) pollen allergen Amb a V by sequencing the second exons of the DRB and DQBI genes of 17 selected ragweed-allergic Caucasoid subjects. We also studied the DQA1 allelic polymorphic regions (APRs) in these patients by dot-blotting using sequence-specific oligonucleotides (SSOs). The deduced amino acid sequences of the respective class II and polypeptides were compared, with particular emphasis on residues in the APRs that are implicated in antigen binding. No evidence for new HLA-DRB or DQB sequences unique to Amb a V responders were found on sequencing seven Dw2⁺ subjects. This suggests that the presence of a particular Dw2-associated class II molecule usually provides a necessary, but not always sufficient condition for responsiveness to Amb a V. The HLA phenotypes of three subjects suggest that they possess novel recombinant haplotypes containing either DRB1 ^* 1501 and DRB5 ^* 0101 (Dr2.2-associated) or DQB1 ^* 0602 (DQ1.2-associated) sequences. In these subjects, responsiveness to Amb a V was associated with the DR2.2 but not the DQ1.2 sequences, suggesting that DR_I or DR_V class II molecules are involved in antigen presentation. We investigated whether there may be shared HLA-D-encoded responder sequences present in all responders, including some exceptional DR2^– Amb a V responders. The 13 subjects producing antibody (Ab) responses to Amb a V [either from natural exposure and/or after ragweed immunotherapy (Rx)] possessed DRB1 ^* 1501, 1601, 1602, 0103, 0402, 0404, 0801 or 1101 sequences, which share the majority of their aa residues in the APRs 2–4. Some of these shared residues might be important for the binding of a common Amb a V agretope prior to presentation of the class II Amb a V complex to the T-cell receptor (Tcr). An alternative postulate is that the recognition of two different Amb a V agretopes may be determined by the _I polypeptides of molecules having the DR2 and DQw3 specificities.     

HLA transgenic mice provide evidence for a direct and dominant role of HLA class II variation in modulating the severity of streptococcal sepsis

Our epidemiologic studies on invasive Group A Streptococci (GAS) infections identified specific HLA class II haplotypes/alleles conferring high-risk or protection from streptococcal toxic shock syndrome with a strong protection conferred by the DRB1*15/DQB1*06 haplotype. We used HLA-transgenic mice to provide an in vitro and in vivo validation for the direct role of HLA class II allelic variation in streptococcal toxic shock syndrome. When splenocytes from mice expressing the protective HLA-DQB1*06 (DQ6) allele were stimulated with a mixture of streptococcal superantigens (SAgs), secreted by the prevalent M1T1 strain, both proliferative and cytokine responses were significantly lower than those of splenocytes from mice expressing the neutral DRB1*0402/DQB1*0302 (DR4/DQ8) alleles (p < 0.001). In crisscross experiments, the presentation of SAgs to pure T cells from either the DQ6 or the DR4/DQ8 mice resulted in significantly different levels of response depending on the HLA type expressed on the APCs. Presentation by HLA-DQ6 APCs elicited significantly lower responses than the presentation by HLA-DR4/DQ8 APCs. Our in vitro data were supported by in vivo findings, as the DQ6 mice showed significantly longer survival post-i.v. infection with live M1T1 GAS (p < 0.001) and lower inflammatory cytokine responses as compared with the DR4/DQ8 mice (p < 0.01). The data presented here provide evidence for a direct role of HLA class II molecules in modulating responses to GAS SAgs and underscore the dominant role of HLA class II allelic variation in potentiating the severity of GAS systemic infections.     

HLA-DR/DQ Transgenic, class II deficient mice as a novel model to select for HSP T cell epitopes with immunotherapeutic or preventative vaccine potential

Protective immunity against mycobacteria is dependent on antigen/MHC class II specific, CD4⁺ Th1 cells. HLA-DR3-restricted Th1 cells respond to a subset of mycobacterial antigens, including the immunodominant hsp65, and recognize a single epitope in hsp65, notably p1-20. Altered peptide ligands (APL) of p1-20 can inhibit p1-20/hsp65-induced proliferation of DR3-restricted T cells in an allele specific mannerin vitro. In order to develop a preclinical model in which p1-20 APL can be testedin vivo in the context of HLA, we have used murine class II deficient, HLA transgenic (Ab⁰) mice, in which all CD4⁺ T cells are restricted by the tg HLA molecule. BCG-immunized DR3.Ab⁰ and DQ8.Ab⁰ mice both responded well to hsp65. Furthermore, DR3.Ab⁰ mice recognized precisely the same p1-20 epitope as DR3-restricted human T cells, whereas DQ8.Ab⁰ mice responded to a different set of hsp65 peptides. This shows that (i) the same immunodominant protein and peptide epitope are recognized by T cells from DR3.Ab⁰ mice and DR3⁺ humans and (ii) indicates the major role of HLA-polymorphism in controlling the human T cell response to mycobacterial antigens. Thus, HLA-transgenic, Ab⁰ mice provide a novel, preclinical model system to analyze APL and vaccines in the context of HLA polymorphism.