Structural similarity of the HLA-DQ region in DQ3 and DQ4 haplotypes and structural diversity of the HLA-DQ region in HLA-DR7 haplotypes.

Genomic DNA obtained from a B lymphoblastoid cell line was digested with appropriate restriction endonuclease and hybridized with several probes specific for genes encoding HLA-DQ. Southern hybridization with a DQA1 3'untranslated (UT) region probe showed DQ2-type hybridization pattern in DR7DQ3 haplotype. On the contrary, DQB1 3'UT probe showed DQ3-type pattern in the same haplotype. Gene cloning and DNA sequencing analysis revealed a repetitive sequence, (TG)19, between DQA1 and DQB1 gene in the DR7DQ3 haplotype. These results suggest that a recombination event has occurred near this potential Z-DNA structure in the haplotype, DR7DQ3. The 3'UT region probes of DQA1 and DQB1 genes failed to detect restriction fragment length polymorphism (RFLP) differences between DR4DQ3 and DR4DQ4 haplotypes in this experiment, suggesting that the gene structure between DQA1 and DQB1 is conserved in these haplotypes.     

Associations of HLA DR and DQ molecules with Lyme borreliosis in Latvian patients

ABSTRACT: BACKGROUND: Many autoimmune diseases are associated with variants of HLA genes such as those encoding the MHC complex. This correlation is not absolute, but may help in understanding of the molecular mechanism of disease. The purpose of this study was to determine HLA-DR,-DQ alleles in Latvian patients with Lyme borreliosis and control (healthy) persons. Case patients and control subjects were similar in age, gender and ethnic heritage and differed only as regards the presence of Borrelia burgdorferi infection. The study included 20 patients with clinical stage - erythema migrans and 25 control (healthy) persons. HLA genotyping was performed by PCR with sequence-specific primers. RESULTS: The results show difference in HLA-DRB1 alleles distribution between patients and control subjects. The frequencies of HLA-DRB1 *04 (OR 8.65; p<0.022) and HLA-DRB1 *17 (03) (OR 7.00; p<0.048) were increased in the Lyme disease patients. And the frequency of allele DRB1*13 (OR 0.13; p<0.033) was lower in Borreliosis patients and higher in control group. But, significant differences in frequencies of HLA-DQ alleles we did not detect. CONCLUSIONS: HLA predisposition to Lyme borreliosis appears not to be limited to HLA molecules, but some HLA-DR alleles also have a significant influence, and, may have implications in our understanding of pathogenesis of this disease. In particular, HLA-DRB1*04 and DRB1 *17 (03) may contribute to the Lyme borreliosis development in Latvian population KEYWORDS: Lyme borreliosis, HLA alleles, PCR.     

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.     

HLA class II DR and DQ genotypes and haplotypes associated with rheumatic fever among a clinically homogeneous patient population of Latvian children

The HLA system is being paid more and more attention because it is very significant in polymorphous immunological reactions. Several studies have suggested that genetic susceptibility to rheumatic fever (RF) and rheumatic heart disease (RHD) is linked to HLA class II alleles. We hypothesized that HLA class II associations within RHD may be more consistent if analysed amongst patients with a relatively homogeneous clinical outcome. A total of 70 RF patients under the age of 18 years were surveyed and analysed in Latvia. HLA genotyping of DQA1, DQB1 and DRB1 was performed using PCR with amplification with sequence-specific primers. We also used results from a previous study of DQB1 and DRB1 genotyping. In the RF patients, HLA class II DQA1*0401 was found more frequently compared to DQA1*0102. In the RF homogeneous patient groups, DQA1*0402 has the highest odds ratio. This is also the case in the multivalvular lesion (MVL) group, together with DQA1*0501 and DQA1*0301. In the chorea minor patients, DQA1*0201 was often found. Significant HLA DQA1 protective genotypes were not detected, although DQA1 genotypes *0103/*0201 and *0301/*0501 were found significantly and frequently. In the distribution of HLA DRB1/DQA1 genotypes, *07/*0201 and *01/*0501 were frequently detected; these also occurred significantly often in the MVL group. The genotype *07/*0201 was frequently found in Sydenhamn's chorea patients that had also acquired RHD, but DRB1*04/DQA1*0401 was often apparent in RF patients without RHD. In the distribution of HLA DQA1/DQB1 genotypes, both in RF patients and in the homogeneous patient groups, the least frequent were *0102/*0602-8. The genotype DQA1*0501 with the DQB1 risk allele *0301 was often found in the MVL group. The genotype *0301/*0401-2 was frequently found in the RF and Sydenhamn's chorea patient groups. The haplotype *07-*0201-*0302 was frequently found in RF and homogeneous patient groups, including the MVL group. In addition, haplotypes *04-*0401-*0301 and *04-*0301-*0401-2 were frequent amongst patients with Sydenhamn's chorea. The protective alleles DQA1*0102 and DQB1*0602-8 in the haplotype DRB1*15 were less frequently found in RF patients. The results of the present study support our hypothesis and indicate that certain HLA class II haplotypes are associated with risk for or protection against RHD and that these associations are more evident in patients in clinically homogeneous groups.     

Complete sequence of the HLA DQ alpha and DQ beta cDNA from a DR5/DQw3 cell line

The HLA-D region is composed of three subregions termed DR, DQ, and DP. We previously reported the sequence of a DR5 beta I and two DR5 beta III cDNA from the DR5 cell line Swei. We now report on the nucleotide and deduced amino acid sequence of the DQ alpha and DQ beta cDNA from the same DR5 cell line, which also types as DQw3. Comparison with other available DQ sequences indicates that DQ alpha has one region of major variability, whereas DQ beta appears to have four regions of variability. In addition, these comparisons indicate that DQw3 alpha from DR5 is different from DQw3 alpha from DR4, but identical to DQw2 alpha from DR3. In contrast, DQw3 beta from DR5 is very similar to DQw3 beta from DR4. These data indicate that at least for DQw2 and DQw3 it is the DQ beta chain that is responsible for DQ typing. Most sequence differences in DQ alleles can be attributed to point mutations; however, codon additions/deletions in the DQ alpha chain may contribute to variability. In addition, regions of possible gene conversion in the DQ alpha and DQ beta chains is suggested by the presence of a chi-like sequence in each chain. Finally, comparison of available haplotypes suggest recombination events may take place between DQ beta and DQ alpha, between DQ alpha and DR beta I, and between DR beta I and DR beta III.     

Structural relationships between the DR beta 1 and DR beta 2 subunits in DR4, 7, and w9 haplotypes and the DRw53 (MT3) specificity

The class II molecules of DR4, DR7, and DRw9 haplotypes were analyzed by immunoprecipitation, followed by two-dimensional gel electrophoresis and N-terminal amino acid sequencing. By using HLA-DR chain-specific monoclonal antibodies, two distinct DR beta-chains were identified. One beta-chain, designated DR beta 2, had a characteristic acidic mobility. In all three DR types the DR beta 2-chains were indistinguishable by two-dimensional gel electrophoresis and partial N-terminal sequencing. A second DR beta-chain designated beta 1 had a more basic mobility on two-dimensional gel electrophoresis, and differed from the DR beta 2-chains by the consistent presence of phenylalanine at position 18. In contrast to the DR beta 2-chains, the DR beta 1-chains were clearly polymorphic, with specific amino acid sequence differences characteristic of each DR type. The monoclonal antibodies 109d6 and 17-3-3S, recognizing distinct polymorphic epitopes similarly correlated with the DRw53 allospecificity, were found to react with different DR beta-chains. The epitope recognized by monoclonal antibody 109d6 was identified on the DR beta 2-chain in the prototypic DR4, DR7, and DRw9 cell lines. However, the DR7, Dw11, DQw3 cell line BEI was unreactive with antibody 109d6 by either immunofluorescence or immunoprecipitation despite the presence of the DRw53 allodeterminant on this cell line. The other DRw53-like monoclonal antibody, 17-3-3S, reacted with the DR beta 1-rather than the DR beta 2-chain in all DR4 and DR7 cell lines tested, including the cell line BEI. However, antibody 17-3-3S did not react with the DRw53-positive DRw9 cell line ISK. These studies suggest that the DRw53 allospecificity is more complex than previously thought and may comprise a number of distinct epitopes encoded by two different DR beta loci.     

Functional reactivity of WT31 monoclonal antibody with T cell receptor-gamma expressing CD3+4-8- T cells

About 3% of normal peripheral blood T lymphocytes have the phenotype CD3+4-8-. The vast majority of these cells lack the conventional TCR-alpha-, beta complex but express the recently identified TCR-gamma, delta/CD3 receptor complex. These TCR gamma+/CD3+ cells were initially discovered by using as a criterion the lack of reactivity with WT31 mAb. This mAb has been reported to recognize a "framework" epitope on the TCR-alpha, beta/CD3 complex. However, using high concentrations of WT31 mAb, low levels of reactivity with the cell membrane of TCR-gamma+ cells can be observed. This reactivity was significantly increased upon removal of sialic acid residues by neuraminidase. In addition, WT31 mAb is capable to induce lysis by TCR-gamma+ clones. Moreover, immunoprecipitation with WT31 by using cell lysates prepared with the mild detergent digitonin resulted in the isolation of the intact TCR-gamma/CD3 complex. Thus, in contrast to what was previously assumed, WT31 mAb also reacts with a functional epitope present on gamma, delta/CD3 T cells, and therefore lack of reactivity with WT31 mAb is not always a proper hallmark for TCR-gamma-expressing cells.     

L3T4+ T-cell-independent reactivity of Lyt2+ T cells in vivo

The aim of this study was to analyze in vivo the L3T4+ T-cell-subset-independent reactivity of Lyt2+ T cells toward transplantation alloantigens. To this end, we depleted normal mice of L3T4+ T cells by injection of monoclonal antibodies to the L3T4 antigen. This procedure not only led phenotypically to a disappearance of L3T4+ T cells, but also effectively abolished reactivity toward class II MHC antigens in vitro and in vivo. However, L3T4+ T-cell-depleted mice still reacted to class I MHC alloantigens in vivo: after immunization with class I MHC alloantigens Il-2 receptor-bearing T cells appeared in the draining lymph nodes, and developed antigen-specific cytolytic activity. Moreover, upon in vivo priming the frequencies of class I MHC-specific precursors of Il-2-producing and cytolytic Lyt2+ T lymphocytes increased up to 20-fold. L3T4+ T-cell-depleted mice rejected class I MHC-bearing skin grafts promptly. We conclude that not only in vitro but also in vivo Lyt2+ T cells remain reactive toward class I MHC antigens in the absence of L3T4+ T helper cells.     

Primate DRB genes from the DR3 and DR8 haplotypes contain ERV9 LTR elements at identical positions

The HLA-DRB genes of the human major histocompatibility complex constitute a multigene family with a varying number of DRB genes in different haplotypes. To gain further knowledge concerning the evolutionary relationship, the complete nucleotide sequence was determined for a region spanning introns 4 and 5 of the three DRB genes (DRB1*0301, DRB2 and DRB3*0101) from a DR52 haplotype and the single DRB gene (DRB1*08021) in the DR8 haplotype. These analyses identified an endogenous retroviral long terminal repeat element (ERV9 LTR3), inserted at identical positions in intron 5 of the functional DRB genes in these two haplotypes. Comparison of the nucleotide sequence from introns 4 and 5 including the ERV9 LTR elements revealed a strong similarity between the three expressed DRB genes. The DRB3*0101 and DRB1*08021 genes were most similar in this comparison. These findings provide further evidence for a separate duplication in a primordial DR52 haplotype followed by a gene contraction event in the DR8 haplotype. A homologous element was found in a chimpanzee DRB gene from a DR52 haplotype. This represents the first characterized ERV9 LTR element in a nonhuman species. The corresponding introns of the DRB genes in the DR4 haplotype contain no ERV9 LTRs. In contrast, these genes have insertions of distinct Alu repeats, implying distinct evolutionary histories of DR52 and DR53 haplotypes, respectively. Phylogenetic analyses of DRB introns from DR52, DR53, and DR8 haplotypes showed a close relationship between the DRB2 and DRB4 genes. Thus, the ancestral DR haplotype that evolved to generate the DR52 and DR53 haplotypes most likely shared a primordial common DRB gene.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X82660–X82663     

Antibodies to the L3T4 and Lyt-2 molecules interfere with antigen receptor-driven activation of cloned murine T cells

When L3T4+ cloned murine helper T lymphocytes (HTL) are stimulated with antigen or immobilized anti-T cell receptor (TCR) monoclonal antibodies (mAb) at concentrations which are optimal for proliferation, anti-L3T4 mAb inhibits activation as measured by proliferation and lymphokine production. Under similar conditions, IL 2-independent proliferation of Lyt-2+ cloned murine cytolytic T lymphocytes (CTL) stimulated by anti-TCR mAb is inhibited by anti-Lyt-2 antibodies. Proliferation of cloned HTL and CTL cells stimulated by IL 2 is not affected by the anti-L3T4 and anti-Lyt-2 mAb. The inhibition of TCR-induced activation of the T cell clones is not due to interference with the binding of the anti-TCR mAb. Stimulation of the TCR has been proposed to induce lymphokine secretion and proliferation by T cells through a pathway involving the activation of protein kinase C and the stimulation of an increase in the concentration of intracellular free calcium. However, proliferation of T cells stimulated by PMA (which activates protein kinase C) plus the calcium ionophore A23187 (which increases the concentration of intracellular free calcium) is not affected by mAb reactive with the Lyt-2 or L3T4 structures. If TCR stimulation does indeed activate T cells by activating protein kinase and increasing intracellular free calcium, then our data suggest that anti-L3T4 and anti-Lyt-2 mAb inhibit TCR-driven proliferation at some step before the activation of protein kinase C and the stimulation of a rise in intracellular free calcium concentration. Our results suggest that anti-L3T4 and anti-Lyt-2 mAb interfere with early biochemical processes induced by stimulation of the TCR. In HTL, which proliferate via an autocrine pathway, anti-L3T4 mAb appears to inhibit proliferation by interfering with signaling events involved in lymphokine production. Inhibition of IL 2-independent proliferation of Lyt-2+ cells by anti-Lyt-2 mAb appears to occur by a different mechanism. The precise molecular basis for the interference of each cell type has not yet been characterized.     

Primary structural variation among serologically indistinguishable DS antigens: the MB3-bearing molecule in DR4 cells differs from the MB3-bearing molecule in DR5 cells

HLA-DS molecules bearing the MB3 supertypic specificity have been isolated from two DR4 and two DR5 homozygous cell lines by using the monoclonal antibody IVD12 . Limited amino-terminal amino acid sequence analysis of these molecules demonstrates polymorphism of the HLA-DS subregion. Although the distribution of amino-terminal tyrosine residues in the alpha-chains of all IVD12 -reactive molecules was identical, amino-terminal amino acid sequence differences existed between DS beta-chains isolated from these two groups of cell lines bearing different DR specificities. These studies indicate that two DS molecules bearing the same serologic determinant ( MB3 ), although similar to one another, may be structurally distinct.     

Linkage disequilibrium of HLA-SB1 with the HLA-A1, B8, DR3, SCO1 and of HLA-SB4 with the HLA-A26, Bw38, Dw10, DR4, SC21 extended haplotypes

Homozygous typing cells from 13 normal HLA-A1, B8, Dw3, DR3 and five normal HLA-A26, Bw38, Dw10, DR4 individuals were typed for the following markers: HLA-SB, MB, MT; complement proteins BF, C2, C4A, C4B; and GLO. Ninety-one percent of A1, B8, Dw3, DR3 homozygous individuals (HI) tested were homozygous for BF ^* S, C2 ^* C, C4A ^* QO, and C4B ^*1 (SCO1 complotype), which indicates that the SCO1 complotype is in linkage disequilibrium with the A1, B8, DR3 haplotype in randomly selected normal populations. Sixty-seven percent of HLA-A1, B8, Dw3, DR3, SCO1 positive HI also expressed SB1; since the frequency of SB 1 in random Caucasian populations is 11.2%, this finding indicates that SB1 is in linkage disequilibrium with the A1, B8, DR3, SCO1 extended haplotype. All HI with the A26, Bw38, Dw10, DR4 haplotype were homozygous for both SC21 and SB4, suggesting that SC21 and SB4 should be included in the A26, Bw38, Dw10, DR4 extended haplotype. On the other hand, neither of the GLO markers were found in association with either haplotype. The results of this study indicate that HLA-SB is included in some extended haplotypes and may be important in these markers for diseases such as insulin-dependent diabetes mellitus. This study also demonstrated an apparent influence of HLA-SB on primary mixed lymphocyte culture (MLC) responses. The mean relative response of primary MLCs between individuals matched for HLA-A, B, D, DR, MB and MT but not SB was 40% of that for the MLCs with mismatched HLA-D, significantly higher than the MLCs matched for all HLA and complotypes.     

Molecular analysis of the MHC class II region inDR4, DR7, andDR9 haplotypes

Within the class 11 region of the major histocompatibility complex (MHC) the amount of DNA in theDR-DQ interval has been shown to be haplotype dependent, with those carrying the DR4, DR7, and DR9 specificities having been reported to contain 110–160 kilobases (kb) more DNA than haplotypes carrying the DR3 specificity. Certain subtypes of haplotypes carrying particular DR specificities are more closely associated with autoimmune diseases than others. With the prospect of the DNA perhaps containing a disease susceptibility locus, we have mapped eight DR4 and two DR7 homozygous cell lines and a DR7/9 heterozygous cell line together with a control DR3 cell line using pulsed field gel electrophoresis (PFGE) with the enzymesBss H II,Pvu I, andNot I/Nru I. Our results, however, show that the presence and amount of the extra DNA is constant irrespective of the subtype. We have also tried to narrow down the position of insertion of the extra DNA using eight further rare-cutting enzymes but, due to the polymorphic nature of sites and/or differences in methylation in this region, it was not possible to refine it further than between DRA and DQA1/B1. This polymorphic nature of theDR-DQ region is unusual, considering the uniformity of rare cutter sites that has been observed within the rest of the class II, and class III, regions. The presence of this, and other, haplotype dependent variations in the DNA content of theDR subregion may be important with respect to recombination and will be particularly interesting if the additional DNA is found to contain novel genes.     

The beta-chains of DP4 molecules from different haplotypes are encoded by the same gene

The nucleotide sequence of a complete cDNA gene from a DP4-positive HLA-homozygous cell line, PGF, has been determined. This sequence is identical to the exon sequences in a genomic clone derived from another DP4-positive cell line, Priess. In contrast, our DP cDNA sequence shares only limited homology with partial cDNA sequences obtained from clones of three DP4-negative cell lines. On the basis of these results, we conclude that the phenotypic variation of DP alleles is directly attributable to the nucleotide sequence heterogeneity of DP-beta genes. That is, each phenotypic allelic form of DP antigen corresponds to a distinctly different DP-beta gene. Furthermore, this correspondence is found to be unaffected by the markers present at the DQ and DR loci, since the haplotypes of the PGF and Priess cell lines are, respectively, DR2,DQw1,DP4 and DR4,DQw3,DP4.     

Immunological memory: contribution of memory B cells expressing costimulatory molecules in the resting state.

Traditionally, emphasis has been placed on the roles of Th cells in generating and amplifying both cellular and humoral memory responses. Little is known about the potential contributions of B cell subsets to immunological memory. Resting memory B cells have generally been regarded as poor APC, attributed in part to the relative paucity of costimulatory molecules identified on their surface. We describe a novel subpopulation of human memory B cells that express CD80 in their resting state, are poised to secrete particularly large amounts of class switched Igs, and can efficiently present Ag to and activate T cells. This functionally distinct B cell subset may represent an important mechanism by which quiescent human B cells can initiate and propagate rapid and vigorous immune memory responses. Finally, these studies extend recent observations in the murine system and highlight the phenotypic and functional diversity that exists within the human B cell memory compartment.     

An influenza A virus-specific and HLA-DRw8-restricted T cell clone cross-reacting with a transcomplementation product of the HLA-DR2 and DR4 haplotypes

The clone TA10 is a T3+ T4+ T8- proliferative and cytolytic human T cell clone. This clone has been shown to be specific for the hemagglutinin of influenza A Texas virus and restricted by an HLA class II molecule associated with the DRw8-Dw8.1 phenotype. Here we show that TA10 and all of its subclones can also react with eight HLA-DRw8 negative, Epstein-Barr virus (EBV)-transformed cell lines or phytohemagglutinin blasts in the absence of influenza antigens. All of these cell lines are HLA-DR2/DR4 with a classic DR2 long haplotype. The only nonreactive HLA-DR2/DR4 cell line observed bears a DR2 short haplotype. Only heterozygous HLA-DR2/DR4 but not parental DR2 or DR4 EBV-transformed cell lines can be recognized by TA10, indicating that the cross-reacting determinant is a transcomplementation product between HLA-DR2 and HLA-DR4 haplotypes. DR-specific, but not DQ- or DP-specific monoclonal antibodies, inhibit in the proliferation assay and in the chromium release test both the DRw8-Dw8.1-restricted and the anti-DR2/DR4 reactions. These results show that HLA-DR-restricted, anti-viral human T cell clone can evidence cross-reactivity for allospecific class II molecules of the major histocompatibility complex, and human CTL can recognize transcomplementation products of class II HLA genes. In addition, the results suggest that a beta-chain coded for by an HLA-DR gene and associated with an alpha-chain coded for by a still unidentified but possibly HLA-DQ gene constitute this functional transcomplementation product.