On the future of HLA.

As the immunobiological function of the HLA (human leucocyte antigen) class I and II molecules was revealed, we have seen an explosive development of the HLA field. Today, the HLA complex occupies a central position in basic and clinical immunology. In this Opinion article, I will briefly discuss some challenges which in my opinion are more important than others in the near future of HLA, with a focus on products of the classical HLA class I and II genes. Matching for HLA antigens will continue to be of importance in organ and hematopoietic stem cell transplantations. In the latter field, induction of graft-versus-leukemia effects will receive greater attention, where HLA will play a central role. It is anticipated that we will see an extensive development in our knowledge of the etiology and pathogenesis of autoimmune diseases, where some HLA class I and II genes by far are the strongest predisposing genes. To predict and prevent autoimmune diseases will be a major challenge for the HLA field in the future. HLA will also be of increasing importance in pharmacogenomics, vaccinations and anthropology. Together, this will leave the HLA field with many new challenges and opportunities, which in the future will require more focus on functional aspects of the immunogenetics of HLA.

No extreme genetic risk for type 1 diabetes among DR3/4-DQ8 siblings sharing both extended HLA haplotypes with their diabetic proband.

An extreme genetic risk for type 1 diabetes (T1D) was reported for DR3/4-DQ8 siblings sharing both extended human leukocyte antigen (HLA) haplotypes identical-by-descent (IBD) with their diabetic proband. We attempted to replicate this finding in our prospective Dutch T1D cohort and in families from the Type 1 Diabetes Genetics Consortium (T1DGC). Only 2 of the 14 Dutch siblings, sharing both DR3-DQ2/DR4-DQ8 haplotypes IBD with their diabetic proband, developed T1D in a 12-year follow-up period. No differential sharing of HLA haplotypes or significant transmission distortion in parents homozygous for HLA risk alleles was found in T1DGC material. Therefore, we could not confirm the reported extreme risk for T1D, suggesting that the risk conferred by other HLA complex loci is moderate.

Antigen-presenting properties of human vascular endothelial cells.

Human endothelial cells were obtained by collagenase digestion of the umbilical vein wall, explanted into tissue culture, and grown as monolayers of cells. Endothelial cells extracted from these monolayers were specifically lysed by anti-HLA-DR alloantisera. Moreover, the stimulating capacity of these endothelial cells toward allogeneic peripheral blood mononuclear lymphocytes was specifically and significantly inhibited by the presence of relevant anti-HLA-DR antisera. Endothelial cells that expressed HLA-DR determinants were also capable of substituting for macrophages in the lymphoproliferative response of purified T cells to soluble protein antigens. Moreover, in concordance with the results previously reported in which macrophages were employed, the endothelial cell donor should share HLA-DR determinants with the T cell donor for an optimal response to occur.

Clonal distribution of HLA-restricted antigen-reactive T cells in man.

The [3H]thymidine suicide technique was used to test the hypothesis that the response of immune human T cells from HLA-D/DR heterozygous donors to the soluble protein antigen purified protein derivative (PPD) is clonally expressed and consists of the concurrent proliferation of at least two separable subpopulations of lymphocytes. The results showed that each of the two subpopulations react to one or the other of the HLA-D/DR antigens presented together with PPD by allogeneic monocytes. In addition, using in vitro priming techniques of in vivo sensitized lymphocytes from heterozygous donors, it was possible to generate specific memory cells capable of recognizing the priming soluble protein antigen together with the HLA-D/DR determinant present in the initial sensitizing culture.

Inhibition of the Fc receptor of human lymphoid cells by antisera-recognizing determinants of the hla system.

The inhibitory effect of HLA antisera on Fc receptors of human lymphoid cells was investigated. The ability of lymphoid cells to form rosettes (FcRFC) with antibody-coated sheep red blood cells and to function as effector cells (K cells) in antibody-dependent cell-mediated cytotoxicity were used as assay systems. We found that antisera recognizing determinants of the HLA-A, -B, and -C series had no effect on FcRFC, while a specific inhibitory effect was observed of antisera probably reacting with determinants identical to or closely associated with those of the HLA-D series. This inhibitory effect was retained in the F(ab')2 fragments. Specific inhibition of K cells was observed with all HLA antisera, but this effect was lost in the F(ab')2 fragments. We conclude that the Fc receptor of rosette-forming lymphocytes may be closely associated with products of the HLA-D region. This is analogous to the association between the Fc receptor and the Ia antigens on murine rosette-forming B lymphocytes.

The genetic polymorphism of the fourth component of human complement: methodological aspects and a presentation of linkage and association data relevant to its localization in the HLA region.

The C4 polymorphism in man has been studied by immunofixation electrophoresis, crossed immunoelectrophoresis, and functional detection after agarose gel electrophoresis. It has so far not been possible to reveal this polymorphism by isoelectric focusing and functional detection of C4 bands. Three common alleles and one less frequently occuring allele have been identified. In a small population sample studied by all the different techniques and verified by family segregation, the following gene frequencies have been found: C4F: 0.46, C4S: 0.32, C4F1: 0.20, and C4M: 0.02. By linkage and association studies in a family material it has been shown that a structural C4 locus is situated in the HLA region of chromosome 6 very close to the HLA-B and Bf loci.

Identification, at the genomic level, of an HLA-DR restriction element for cloned antigen-specific T4 cells.

Two T4 cell clones (TLC) specific for antigenic epitopes on Chlamydia trachomatis were studied. Using a panel of allogeneic antigen-presenting cells (APC), both TLC were found to be restricted by HLA class II elements closely associated with, but not identical to the DRw5S specificity, as determined by highly selected alloantisera, a monoclonal antibody (mAb), 109d6, and confirmed on the DNA level by determination of restriction fragment length polymorphisms (RFLP) with a DR beta probe. Furthermore, HLA-DR-specific mAb, including 109d6, but not other HLA class II- or class I-specific antibodies inhibited the two TLC, strongly suggesting that the restriction element is expressed by a DR molecule. Using digestion with Hind III restriction enzyme and a DR beta probe, we found a complete concordance between the appearance of a 9.3 kilobase band and the ability of allogeneic APC to restimulate the T cell clones. Thus, the restriction element for these T cell clones appear to be expressed by DR molecules, but can, at present, only be detected at the genomic level.

Evidence for a primary association of celiac disease to a particular HLA-DQ alpha/beta heterodimer.

Typing of DNA from 94 unrelated children with celiac disease (CD) with HLA-DQA1 and -DQB1 allele-specific oligonucleotide probes revealed that all but one (i.e., 98.9%) may share a particular combination of a DQA1 and a DQB1 gene. These genes are arranged in cis position on the DR3DQw2 haplotype and in trans position in DR5DQw7/DR7DQw2 heterozygous individuals. Thus, most CD patients may share the same cis- or trans-encoded HLA-DQ alpha/beta heterodimer.

Gliadin-specific, HLA-DQ(alpha 1*0501,beta 1*0201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients.

Celiac disease (CD) is most probably an immunological disease, precipitated in susceptible individuals by ingestion of wheat gliadin and related proteins from other cereals. The disease shows a strong human HLA association predominantly to the cis or trans encoded HLA-DQ(alpha 1*0501,beta 1*0201) (DQ2) heterodimer. T cell recognition of gliadin presented by this DQ heterodimer may thus be of immunopathogenic importance in CD. We therefore challenged small intestinal biopsies from adult CD patients on a gluten-free diet in vitro with gluten (containing both gliadin and other wheat proteins), and isolated activated CD25+ T cells. Polyclonal T cell lines and a panel of T cell clones recognizing gluten were established. They recognized the gliadin moiety of gluten, but not proteins from other cereals. Inhibition studies with anti-HLA antibodies demonstrated predominant antigen presentation by HLA-DQ molecules. The main antigen-presenting molecule was established to be the CD-associated DQ(alpha 1*0501, beta 1*0201) heterodimer. The gluten-reactive T cell clones were CD4+, CD8-, and carried diverse combinations of T cell receptor (TCR) V alpha and V beta chains. The findings suggest preferential mucosal presentation of gluten-derived peptides by HLA-DQ(alpha 1*0501, beta 1*0201) in CD, which may explain the HLA association.

Norwegian Sami differs significantly from other Norwegians according to their HLA profile.

This study reports extensive genomic data for both human leukocyte antigen (HLA) class I and II loci in Norwegian Sami, a native population living in the northwest of Europe. The Sami have a distinct culture and their own languages, which belong to the Uralic linguistic family. Norwegian Sami (n = 200) were typed at the DNA level for the HLA-A, -C, -B, -DRB1 and -DQB1 loci, and compared with a non-Sami Norwegian population (n = 576). The two populations exhibited some common genetic features but also differed significantly at all HLA loci. The most significantly deviating allele frequencies were an increase of HLA-A*03, -B*27, -DRB1*08 and -DQB1*04 and a decrease of HLA-A*01, C*01, -DRB1*04 and -DQB1*02 among Sami compared with non-Sami Norwegians. The Sami showed no deviation from Hardy-Weinberg equilibrium. The hypothesis of selective neutrality was rejected at all loci except for the A- and C- loci for the Sami. HLA haplotype frequencies also differed between the two populations. The most common extended HLA haplotypes were A*02-B*27-C*01-DR*08-DQB1*04 in the Sami and A*01-B*08-C*07-DR*03-DQB1*02 in the other Norwegians. Genetic distance analyses indicated that the Norwegian Sami were highly differentiated from other Europeans and were most closely related to Finns whose language also belongs to the Uralic linguistic family. In conclusion, the Norwegian Sami and the non-Sami Norwegians were significantly different at all HLA loci. Our results can be explained by the fact that the two populations have different origins and that the Sami population has remained smaller and more isolated than its neighbors.

Genetic variants of the HLA-A, HLA-B and AIF1 loci show independent associations with type 1 diabetes in Norwegian families.

The main genetic predisposition to type 1 diabetes (T1D) is known to be conferred by the HLA-DRB1, -DQA1 and -DQB1 genes in the major histocompatibility complex (MHC). Other genetic factors within this complex are known to contribute, but their identity has often been controversial. This picture is shared with several other autoimmune diseases (AIDs). Moreover, as common genetic factors are known to exist between AIDs, associations reported with other AIDs may also be involved in T1D. In this study, we have used these observations in a candidate gene approach to look for additional MHC risk factors in T1D. Using complementary conditional methods (involving conditional logistic regression and family-based haplotype tests) and analyses of linkage disequilibrium (LD) patterns, we confirmed association for alleles of the HLA-A and HLA-B genes and found preliminary evidence for a novel association of a single-nucleotide polymorphism (rs2259571) in the AIF1 gene, independent of the DRB1-DQA1-DQB1 genes and of each other. However, no evidence of independent associations for a number of previously suggested candidate polymorphisms was detected. Our results illustrate the importance of a comprehensive adjustment for LD effects when performing association studies in this complex.

Reproducible association with type 1 diabetes in the extended class I region of the major histocompatibility complex.

The high-risk human leukocyte antigen (HLA)-DRB1, DQA1 and DQB1 alleles cannot explain the entire type 1 diabetes (T1D) association observed within the extended major histocompatibility complex. We have earlier identified an association with D6S2223, located 2.3 Mb telomeric of HLA-A, on the DRB1(*)03-DQA1(*)0501-DQB1(*)0201 haplotype, and this study aimed to fine-map the associated region also on the DRB1(*)0401-DQA1(*)03-DQB1(*)0302 haplotype, characterized by less extensive linkage disequilibrium. To exclude associations secondary to DRB1-DQA1-DQB1 haplotypes, 205 families with at least one parent homozygous for these loci, were genotyped for 137 polymorphisms. We found novel associations on the DRB1(*)0401-DQA1(*)03-DQB1(*)0302 haplotypic background with eight single nucleotide polymorphisms (SNPs) located within or near the PRSS16 gene. In addition, association at the butyrophilin (BTN)-gene cluster, particularly the BTN3A2 gene, was observed by multilocus analyses. We replicated the associations with SNPs in the PRSS16 region and, albeit weaker, to the BTN3A2 region, in an independent material of 725 families obtained from the Type 1 Diabetes Genetics Consortium. It is important to note that these associations were independent of the HLA-DRB1-DQA1-DQB1 genes, as well as of associations observed at HLA-A, -B and -C. Taken together, our results identify PRSS16 and BTN3A2, two genes thought to play important roles in regulating the immune response, as potentially novel susceptibility genes for T1D.

A short history of HLA.

In 1958, just a little more than 50 years ago, an alloantigen present on human leucocytes was detected, which was to become the 'first' human leucocyte antigen (HLA); HLA-A2. Since then, we have seen a tremendous development of the HLA field, which has moved from histocompatibility to become one of the most central fields in basic and clinical immunology. This development is briefly reviewed in this article, focusing on some highlights of the history of HLA class I and II molecules and their role in immune responses. It is emphasized that the quick and extensive development of the HLA field is the result not only of excellent individual contributions by outstanding pioneers in the field, but also of an extensive international collaboration, in particular through the many international histocompatibility workshops. Admitting that it is too late to change the name now, it is concluded that instead of calling the HLA complex and similar complexes in other species the major histocompatibility complex, these gene complexes should better have been named the major immune response complex, the MIRC.

Further evidence of an Amerindian contribution to the Polynesian gene pool on Easter Island.

Available evidence suggests a Polynesian origin of the Easter Island population. We recently found that some native Easter Islanders also carried some common American Indian (Amerindian) human leukocyte antigen (HLA) alleles, which probably were introduced before Europeans discovered the island in 1722. In this study, we report molecular genetic investigations of 21 other selected native Easter Islanders. Analysis of mitochondrial DNA and Y chromosome markers showed no traces of an Amerindian contribution. However, high-resolution genomic HLA typing showed that two individuals carried some other common Amerindian HLA alleles, different from those found in our previous investigations. The new data support our previous evidence of an Amerindian contribution to the gene pool on Easter Island.

The FCRL3 -169T>C polymorphism is associated with rheumatoid arthritis and shows suggestive evidence of involvement with juvenile idiopathic arthritis in a Scandinavian panel of autoimmune diseases.

BACKGROUND AND OBJECTIVES: The Fc receptor-like 3 (FCRL3) gene -169T>C single nucleotide polymorphism (SNP) has been reported to be associated with several autoimmune diseases (AIDs) in Japanese populations. However, association results in other populations have been conflicting. Therefore, we investigated this SNP in a Scandinavian panel of AIDs. METHODS: We genotyped patients with rheumatoid arthritis (RA; n = 708), juvenile idiopathic arthritis (JIA; n = 524), systemic lupus erythaematosus (SLE; n = 166), ulcerative colitis (UC; n = 335), primary sclerosing cholangitis (PSC; n = 365), Crohn disease (CD; n = 149), a healthy control group (n = 1030) and 425 trio families with type 1 diabetes (T1D). Statistical analysis consisted of case-control and family-based association tests. RESULTS: RA was associated with the C allele (odds ratio (OR) = 1.16, 95% CI 1.01 to 1.33) and the CC genotype (OR = 1.30, 95% CI 1.01 to 1.67) of the FCRL3 -169T>C SNP in our material. Suggestive evidence for association was also found for JIA (CC genotype: OR = 1.30, 95% CI 0.99 to 1.70), and clinical subgroup analysis indicated that this was connected to the polyarticular subgroup. No significant association was found with SLE, UC, CD, PSC or T1D. In patients with RA, we found no significant interaction between the FCRL3 -169T>C and PTPN22 1858C>T SNPs, nor between the FCRL3 -169CC genotype and IgM-rheumatoid factor or anti-cyclic citrullinated peptide titre levels. CONCLUSION: We found an association between the FCRL3 -169T>C SNP and RA, and suggestive evidence for involvement with JIA, in a Norwegian population. These findings lend support for a role for this SNP in RA across ethnically diverse populations, and warrant follow-up studies in JIA.

HLA complex genes in type 1 diabetes and other autoimmune diseases. Which genes are involved?

The predisposition to develop a majority of autoimmune diseases is associated with specific genes within the human leukocyte antigen (HLA) complex. However, it is frequently difficult to determine which of the many genes of the HLA complex are directly involved in the disease process. The main reasons for these difficulties are the complexity of associations where several HLA complex genes might be involved, and the strong linkage disequilibrium that exists between the genes in this complex. The latter phenomenon leads to secondary disease associations, or what has been called 'hitchhiking polymorphisms'. Here, we give an overview of the complexity of HLA associations in autoimmune disease, focusing on type 1 diabetes and trying to answer the question: how many and which HLA genes are directly involved?

Particular HLA-DQ alpha beta heterodimer associated with IDDM susceptibility in both DR4-DQw4 Japanese and DR4-DQw8/DRw8-DQw4 whites.

Insulin-dependent diabetes mellitus (IDDM) susceptibility is associated with the DR4-DQw4 haplotype in Japanese and the DR4-DQw8/-Drw8-DQw4 genotype (among others) in whites. We investigated whether these Japanese and white individuals encode the same or a similar DQ alpha beta heterodimer, which may be an IDDM-susceptibility molecule in both populations. First, we carried out genomic DQA1 and DQB1 typing with sequence-specific oligonucleotide probes. The results revealed that Japanese DR4-DQw4 and white DR4-DQw8/DRw8-DQw4 IDDM patients carried the DQA1*0301 allele and the DQB1*0401 or DQB1*0402 allele, either in the cis (Japanese DR4-DQw4 individuals) or trans (white DR4-DQw8/DRw8-DQw4 individuals) position. Because the DQB1*0401 and DQB1*0402 alleles differ only at residue 23, these DQB1 genes are very similar. We next tested cells from these individuals with a particular DQ-specific T-lymphocyte clone, HH58. The clone was only restimulated with cells from Japanese individuals who carried the DQA1*0301 and DQB1*0401 alleles in the cis position or white individuals who carried the DQA1*0301 and DQB1*0402 alleles in the trans position. Thus, particular cis- or trans-encoded DQ alpha beta heterodimers, which in both cases are recognized by T lymphocytes, may confer susceptibility to IDDM in both ethnic groups.

Insulin gene region-encoded susceptibility to IDDM maps upstream of the insulin gene.

The gene region on chromosome 11p15.5 known to be involved in insulin-dependent diabetes mellitus (IDDM) susceptibility was recently mapped to a 4.1-kilobase region including the insulin gene. The region contains 10 candidate polymorphisms that are in strong linkage disequilibrium. By genotyping 7 of these 10 polymorphisms and the tyrosine hydroxylase microsatellite in Finnish Caucasoid IDDM patients and control subjects, we demonstrate that many of the polymorphisms found to be associated with IDDM in other Caucasoid populations do not show any association in this Finnish population. Of the polymorphisms typed, only those at -23 Hph I and the variable number of tandem repeats (VNTR) sites confer significant relative risk. Furthermore, we have demonstrated that the -23 Hph I polymorphism cannot explain the association. Comparison of the genotypic patterns observed here and previously suggests that the VNTR is the most likely candidate for IDDM2. The VNTR is located adjacent to defined regulatory DNA sequences affecting insulin gene expression, which suggests a possible effect on expression of insulin or one of the neighboring genes, tyrosine hydroxylase or insulin-like growth factor 2.

HLA-encoded genetic predisposition in IDDM: DR4 subtypes may be associated with different degrees of protection.

Recent studies have shown that the risk conferred by the high-risk DQA1*03-DQB1*0302 (DQ8) haplotype is modified by the DRB1*04 allele that is also carried by this haplotype. However, many of these studies suffer from lack of sufficient numbers of DQ-matched control subjects, which are necessary because there is a strong linkage disequilibrium between genes in the HLA complex. In the present study, using a large material of IDDM patients and DQ-matched control subjects, we have addressed the contribution of DR4 subtypes to IDDM susceptibility. Our data, together with recent data from others, clearly demonstrate that some DR4-DQ8 haplotypes are associated with disease susceptibility, while others are associated with protection, depending on the DRB1*04 allele carried by the same haplotype. In particular, our data demonstrate that DRB1*0401 confers a higher risk than DRB1*0404. Based on combined available data on the genetic susceptibility encoded by various DR4-DQ8 haplotypes and the amino acid composition of the involved DRbeta*04 chains as well as the ligand motifs for these DR4 subtypes, we have developed a unifying hypothesis explaining the different risks associated with different DR4-DQ8 haplotypes. We suggest that disease susceptibility is mainly conferred by DQ8 while DR4 subtypes confer different degrees of protection. Some DR4 subtypes (i.e., DRB1*0405, 0402, and 0401) confer little or no protection, while others (i.e., DRB1*0404, 0403, and 0406) cause an increasing degree of protection, possibly by binding a common protective peptide. Features of a protective peptide that fit such a model are briefly discussed.

No independent associations of LMP2 and LMP7 polymorphisms with susceptibility to develop IDDM.

Results from a recent study suggested that polymorphisms within the HLA class II genes LMP2 and LMP7 were associated with the susceptibility for developing IDDM, and that this association could not be explained by linkage disequilibrium to HLA-DR or -DQ genes. We typed 285 IDDM patients and 337 HLA-DRB1-DQA1-DQB1 genotypically matched control subjects from an ethnically homogeneous population for both the G/T polymorphism in intron 6 of the LMP7 gene and the Arg-His polymorphism in the LMP2 gene. In addition, we typed IDDM families in which at least one parent was homozygous for a DRB1-DQA1-DQB1 haplotype and performed a transmission/disequilibrium test of these LMP polymorphisms. Our data suggest that none of these LMP2 or LMP7 polymorphisms are independently associated with IDDM susceptibility, in contrast to what has been previously reported by others. Further, our results suggest that one partial explanation for the previously reported independent association between IDDM and these LMP polymorphisms may have been that patients and control subjects were not matched for DRB1*04 subtypes. Our results emphasize the need for a complete matching for DRB1, DQA1, and DQB1 alleles between patients and control subjects when attempting to detect independent effects of other polymorphisms in the HLA complex on IDDM susceptibility or protection.