HLA DR antigens and susceptibility to insulin-dependent diabetes mellitus.

Two novel analytic methods to evaluate the roles of the HLA alleles of the human major histocompatibility complex in disease predisposition have been formulated and applied to insulin-dependent diabetes mellitus (IDDM). The HLA-DR antigens, as they are presently defined, are shown not to directly predispose individuals to IDDM. This result does not discount the possibility that subdivision of the DR antigens will yield the predisposing agents. In Caucasian populations, after consideration of the predisposing effect of the antigens DR3 and DR4, the protective effect of DR2 in predisposition is demonstrated. Additionally, DR1 and possibly DRw8 exhibit a higher than expected frequency in patients.     

The immunoglobulin heavy-chain variable region in insulin-dependent diabetes mellitus: affected-sib-pair analysis and association studies.

We have analyzed immunoglobulin heavy-chain variable-region (VH) polymorphisms and genetic susceptibility to insulin-dependent diabetes mellitus (IDDM) by using a set of polymorphic loci that span approximately 1,000 kb of the VH region on chromosome 14q32. One hundred one Finnish families with at least two children affected with IDDM were studied. Conventional RFLPs determined by hybridization were used, since no microsatellite repeat markers have been available for this gene region. No evidence for linkage between the VH genes and IDDM could be obtained from haplotype-sharing analysis among the 133 diabetic sib pairs. The frequencies of various VH genotypes were also compared between 101 familial IDDM cases and 114 controls derived from the Finnish background population. The distribution of the genotypes at the VH2-B5 locus was significantly different between these groups (P=.004), the 3.4/3.4 genotype being less common in the IDDM cases. In addition, a different genotype distribution at the VH5-B2 locus was observed in the diabetic subjects (P = .022). When the IDDM cases were stratified by presence or absence of the high-risk HLA-DQB1*0302 allele, no differences in VH genotype frequencies were observed between the 0302-positive and 0302-negative cases. In the transmission test for linkage disequilibrium (TDT), no differences were found between the expected and observed frequencies of the transmitted alleles at the VH2-B5 or VH5-B2 locus. In conclusion, significant differences in VH genotype distributions were observed between the familial IDDM cases and the controls, but the observed associations could not be confirmed by the TDT. Haplotype sharing analysis provided no evidence for genetic linkage between the VH gene region and IDDM.     

A molecular basis for genetic susceptibility to insulin-dependent diabetes mellitus

HLA-DQ molecules, which mediate immune responsiveness, may specify susceptibility and resistance to the autoimmune disease insulin-dependent diabetes mellitus. A model describing the role of HLA-DQ gene products in disease pathogenesis and in protection against the onset of diabetes is presented.     

Evolutionary dynamics of the immunoglobulin heavy chain variable region genes in vertebrates

Immunoglobulin heavy chains are polypeptides encoded by four genes: variable (IGHV), joining (IGHJ), diversity (IGHD), and constant (IGHC) region genes. The number of IGHV genes varies from species to species. To understand the evolution of the IGHV multigene family, we identified and analyzed the IGHV sequences from 16 vertebrate species. The results show that the numbers of functional and nonfunctional IGHV genes among different species are positively correlated. The number of IGHV genes is relatively stable in teleosts, but the intragenomic sequence variation is generally higher in teleosts than in tetrapods. The IGHV genes in tetrapods can be classified into three phylogenetic clans (I, II, and III). The clan III and/or II genes are relatively abundant, whereas clan I genes exist in small numbers or are absent in most species. The genomic organization of clan I, II, and III IGHV genes varies considerably among species, but the entire IGHV locus seems to be conserved in the subtelomeric or near-centromeric region of chromosome. The presence or absence of specific IGHV clan members and the lineage-specific expansion and contraction of IGHV genes indicate that the IGHV locus continues to evolve in a species-specific manner. Our results suggest that the evolution of IGHV multigene family is more complex than previously thought and that several factors may act synergistically for the development of antibody repertoire. Electronic supplementary materials The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A susceptibility locus for multiple sclerosis is linked to the T cell receptor beta chain complex

Inheritance of T cell receptor beta chain (TCR beta) genes was analyzed in families of 40 sibling pairs concordant for the relapsing-remitting form of multiple sclerosis (MS). TCR beta haplotypes were determined by segregation analysis of polymorphic markers within the TCR beta complex. The mean proportion of TCR beta haplotypes identical by descent (IBD) inherited by MS sibling pairs was significantly increased compared with expected values (means test, p less than 0.004), whereas the distribution of haplotype sharing was random when MS patients were compared with their unaffected siblings. Furthermore, one allelic form of a TCR beta variable region gene segment was overrepresented on MS chromosomes compared with those parental chromosomes not transmitted to MS offspring both in the MS sibling pair families and in a second group of families containing only one individual affected with MS. These results demonstrate that a gene within the TCR beta complex or a closely linked locus influences susceptibility to MS.     

Variability in T cell receptor V beta gene usage in human peripheral blood lymphocytes. Studies of identical twins, siblings, and insulin-dependent diabetes mellitus patients.

Recent studies focused on the diversity and molecular organization of the human TCR-beta complex have begun to establish the genetic basis for the potential repertoire of V beta specificities in T cells. The scope and variability of the actual repertoire derived from this potential repertoire, however, remains to be clarified. In this study, V beta usage by human peripheral T cells derived from serial samples of the same individual, identical twins, and the members of three nuclear families that include four members with insulin-dependent diabetes mellitus (IDDM) was assessed by both quantitative polymerase chain reaction and Northern blotting with V beta subfamily-specific probes. Samples taken from the same individual over a period of 21 months and analyzed in separate experiments indicated stability in the peripheral repertoire, whereas the similarity in peripheral V beta usage in a pair of identical twins suggested a strong role for genetics in shaping the peripheral T cell repertoire. In contrast, V beta usage in siblings and in unrelated individuals was observed to differ substantially. In particular, peripheral expression of V beta 3 and V beta 20 differed by more than sixfold among members of two different families. Segregation analysis of TCR and HLA haplotypes in these families suggested that variation in V beta 20 expression was TCR haplotype specific. Subsequent nucleotide sequence analysis of the V beta 20 gene segment in multiple members of these families revealed the presence of a null allele for V beta 20 expression. No consistent significant differences in V beta usage were observed in IDDM patients relative to their siblings or between identical twins discordant for IDDM. These results suggest that the repertoire of peripheral T cell specificities present in different individuals in human populations varies dramatically because of the effects of multiple factors, including TCR germ-line polymorphism.     

Abnormalities of immunoregulatory T cell subsets in patients with insulin-dependent diabetes mellitus

Patients with insulin-dependent diabetes mellitus (IDDM) have autoantibodies that react with cells in the islets of Langerhans. To determine whether these patients suffer from a more generalized immunoregulatory disorder, the ratio of phenotypic helper to suppressor cells was evaluated by specific monoclonal antibodies. Our experiments showed that the helper/suppressor cell ratio was significantly increased in patients with IDDM of less of 2 mo duration and then gradually returned to normal. Despite the alteration in the helper/suppressor cell ratio, there was no evidence for polyclonal activation as measured by the number of immunoglobulin-secreting plaque-forming cells in the peripheral blood. There was, however, a significant increase in the number of spontaneous plaque-forming cells in patients suffering from both IDDM and Hashimoto's thyroiditis (HT). Nonetheless, immunoglobulin production after stimulation with pokeweed mitogen was not different in diabetics with or without HT when compared to normal controls. These findings suggest that subtle changes in the immunoregulatory system occur during the early stages of IDDM.     

Organization and evolution of variable region genes of the human immunoglobulin heavy chain

We have isolated 23 different cosmid clones of the heavy-chain variable region genes (VH) of human immunoglobulin. These clones encompass about 1000 X 10(3) base-pairs of DNA containing 61 VH genes. Characterization of the 23 clones by Southern blot hybridization showed that VH genes belonging to different families were physically linked in many regions. Cluster 71, which was analyzed in detail, comprised seven VH segments arranged in the same orientation with different intervals. This clone contained internal homology regions, each carrying two VH segments of different families. Comparison of the nucleotide sequences of VH segments within each family showed that profiles of accumulation of mutations in framework (FR) and complementarity-determining (CDR) regions were different. CDR had more mutations at amino-acid-substituting positions than at silent positions, whereas FR had the reverse distribution of mutations. Five out of seven VH segments of this cluster were pseudogenes containing various mutations. VH pseudogenes were classified into two distinct groups; one with a few replacement mutations (conserved pseudogenes), and the other with rather extensive mutations (diverged pseudogenes). The possibility that conserved pseudogenes serve as a reservoir of VH segments is discussed.     

T cell tolerance to Mlsa encoded antigens in T cell receptor V beta 8.1 chain transgenic mice.

To study T cell tolerance, transgenic mice were generated that expressed the Mlsa-reactive T cell receptor (TCR) beta chain V beta 8.1 (cDNA) under the control of the H-2Kb promoter/immunoglobulin heavy chain enhancer on approximately 90% of peripheral T cells. In transgenic mice bearing Mlsa, thymocytes expressing the TCR at a high density were deleted and the percentage of Thy 1.2+ lymph node cells was reduced. The CD4/CD8 ratio of mature T cells was reversed in Mlsa and Mlsb transgenic mice independent of the H-2. RNA analysis and immunofluorescence with TCR V beta-specific antibodies revealed that expression of endogenous TCR beta genes was suppressed. Both Mlsa and Mlsb TCR beta chain transgenic mice mounted a T-cell-dependent IgG response against viral antigens, whereas the capacity to generate alloreactive and virus-specific cytotoxic T cells was impaired in TCR beta chain transgenic Mlsa, but not in transgenic Mlsb mice.     

A novel family of variable region genes of the human immunoglobulin heavy chain

We isolated and sequenced six variable-region (V) gene segments of the human immunoglobulin heavy-chain (H) using the V71-2 segment as probe. These VH segments were more than 90% homologous to each other and less than 65% homologous to members of the three known VH families. The VH fragments hybridized to an identical set of restriction fragments on Southern blots of human placenta DNA. The new family was designated as the VH-IV family. The complexity of the VH-IV family was estimated to be at least nine genes, of which the sequenced seven were functional genes. The VH-IV family is homologous (76%) to the mouse Vh36-60 family.     

DRB genotyping supports recessive inheritance of DR3-associated susceptibility to insulin-dependent diabetes mellitus.

The mode of inheritance of HLA-associated susceptibility to insulin-dependent diabetes mellitus was investigated by the antigen genotype frequency among patients method in a white Caucasian population and a North Indian Asian population. DR genotypes were determined by DRB/DQB RFLP analysis. In white Caucasians, simple recessive and simple additive inheritance of a single HLA-associated disease susceptibility allele were rejected (P less than .025 and P less than 10(-6), respectively). The data were compatible with a three-allele model of disease susceptibility. In North Indian Asians, simple additive inheritance was rejected (P less than 10(-6)). The observed genotype frequencies were compatible with a single DR3-associated disease susceptibility allele which is inherited recessively. These data show that study of DR genotypes in populations of different ethnic origins may further the understanding of inherited susceptibility to insulin-dependent diabetes mellitus.     

Rearrangements and deletions of immunoglobulin heavy chain genes in the double-producing B cell lymphoma I.29.

The B cell lymphoma I.29 consists of a mixture of cells expressing membrane-bound immunoglobulin M (IgM) (lambda) and IgA (lambda) of identical idiotypes. Whereas most of the cells express either IgM or IgA alone, 1 to 5% of the cells in this tumor express IgM and IgA simultaneously within the cytoplasm and on the cell membrane (R. Sitia et al., J. Immunol. 127:1388-1394, 1981; R. Sitia, unpublished data). When IgM+ cells are purified from the lymphoma and passaged in mice or cultured, a portion of the cells convert to IgA+. These properties suggest that some cells of the I.29 lymphoma may undergo immunoglobulin heavy chain switching, although it is also possible that the mixed population was derived by a prior switching event in a clone of cells. We performed Southern blotting experiments on genomic DNAs isolated from populations of I.29 cells containing variable proportions of IgM+ and IgA+ cells and on a number of cell lines derived from the lymphoma. The results were consistent with the deletion model for heavy chain switching, as the IgM+ cells contained rearranged mu genes and alpha genes in the germ line configuration on both the expressed and nonexpressed heavy chain chromosomes, whereas the IgA+ cells had deleted both mu genes and contained one rearranged and one germ line alpha gene. In addition, segments of DNA located within the intervening sequence 5' to the mu gene, near the site of switch recombination, were deleted from both the expressed and the nonexpressed chromosomes. Although mu genes were deleted from both chromosomes in the IgA+ cells, the sites of DNA recombination differed on the two chromosomes. On the expressed chromosome, Smu sequences were recombined with S alpha sequences, whereas on the nonexpressed chromosome, Smu sequences were recombined with S gamma 3 sequences.     

Organization and polymorphism of rabbit immunoglobulin heavy chain genes

Germline genes encoding C mu, C gamma, C alpha, and C epsilon heavy chains of rabbit immunoglobulins have been isolated from recombinant phage and cosmid libraries. The JH, C mu, C gamma, and C epsilon are found in a 5'-JH-C mu-C gamma-C epsilon-3' orientation on a 90kb stretch of DNA. Four C alpha genes have been cloned and presumably reside 3' to the other CH genes. Southern blot analysis of rabbit sperm DNA indicates that the rabbit genome contains a single C gamma gene, one C mu gene, and as many as 10 C alpha genes. Restriction site polymorphism is found for C mu, C gamma, and C alpha genes of rabbits of various heavy chain haplotypes. The organization of the rabbit CH genes differs from that of mouse and human CH genes in that the rabbit has multiple C alpha genes, whereas mouse and human have one or two C alpha genes, respectively. In addition, mouse and human have four C gamma genes, whereas rabbit has only one C gamma gene. The presence of a single C gamma gene indicates that at least in the rabbits examined, no germline gene encoding latent or unexpected, C gamma allotypes is present. The genetic control of the expression of latent C gamma allotypes is discussed.     

T cell allotypic determinants encoded by genes linked to the immunoglobulin heavy chain locus. I. Establishment of monoclonal antibodies against allotypic determinants

Monoclonal alloantibodies for T cell allotypic determinants were obtained by hybridizing SP-2 tumor cells with BALB/c (H-2d, Igh-1a) spleen cells, which had been repeatedly immunized with Con A-stimulated CB-20 (H-2d, Igh-1b) spleen cells. It was found that these monoclonal anti-CB-20 antibodies detect the new allotypic determinants (distinct from the B cell Igh-C region determinant) expressed only on the augmenting or suppressor T cells. Genetic analysis of these antigenic determinants revealed these antibodies react with the gene products located on the telomeric side chromosome of the Igh variable region gene (Igh-V) cluster. These antibodies when given in vivo caused a modification of antibody production. The antibody activity was absorbed by Con A-stimulated B10.BR (H-2b, Igh-1b), C57BL/6 (H-2b, Igh-1b), CWB (H-2b, Igh-1b), CB-20 (H-2d, Igh-1b), and BAB-14 (H-2d, Igh-1b) spleen cells, but not by Con A-stimulated C3H.SW (H-2b, Igh-1j), BALB/c (H-2d, Igh-1a), A/Sn (H-2a, Igh-1e), and C.AL-20 (H-2d, Igh-1d) spleen cells. In addition, in vivo these monoclonal antibodies modified anti-SRBC antibody production only in Igh-1b allotype-bearing mice. One monoclonal antibody reacted with 4-hydroxy-3-nitrophenyl acetyl- (NP) hapten-specific augmenting T cells, and the other two batches of monoclonal antibodies reacted with NP-specific suppressor T cells of NP-mediated cutaneous responses. A mapping study with these recombinants limits the gene coding for the T cell-specific determinants to a gene within the variable region to the telomeric side of NP-VH and to the centromeric side of prealbumin. This segment is inclusive of all immunoglobulin genes, the region Owen named IgT-C, and a histocompatibility gene (H-Ig).     

Complete structure and organization of immunoglobulin heavy chain constant region genes in a phylogenetically primitive vertebrate

Immunoglobulin (Ig) gene organization in Heterodontus francisci (horned shark), a phylogenetically primitive vertebrate, is unique. Homologous Ig heavy chain variable (VH) and constant region (CH) specific probes were used to screen a spleen cDNA library constructed in lambda gt11. Both secretory (SEC) and transmembrane (TM) cDNA clones were recovered; the latter were identified by a negative selection strategy. The complete sequence of the CH portion of a Heterodontus genomic DNA-lambda clone also was determined. The sequences of the individual CH genes differ from each other in all exons. When compared to mammalian prototypes, similarities in exon and intron organization as well as conservation of sequences involved with differential splicing of SEC and TM mRNA indicate that Heterodontus heavy chain genes are of the mu type, although intron lengths are uniformly longer in Heterodontus. Heterodontus genes are not associated, however, with the family of DNA sequences that have been implicated in heavy chain class switching in mammals. Spleen cDNA library screening and RNA blot analyses indicate that mRNAs encoding TM Ig are exceedingly rare. The relationship between this quantitative difference and the distribution of polyadenylation signal sequences suggests that regulation of Ig gene expression in Heterodontus may be highly dependent on position effects.     

Retention and loss of immunoglobulin heavy chain alleles in helper T cell hybridoma clones

Retention or loss of immunoglobulin heavy chain genes was studied in 20 functional T cell hybridoma clones. DNA probes representing C mu, C alpha and JH genes, as well as VH subgroups II and III were hybridized with restriction enzyme fragments of hybridoma DNA by the Southern filter hybridization technique. Parental alleles of the hybridoma cells were distinguished on the basis of polymorphism of the lengths of restriction enzyme fragments. All clones retained the alleles of the lymphoma parent cell BW-5147 at all four loci. Thirteen clones lost both CH and VH alleles of the immune partner cell, whereas seven retained both VH alleles, and at least C alpha of the antigen-specific partner. Hence, T cell function in these cells is compatible with the loss of most immunoglobulin heavy chain alleles. This is interpreted to indicate either gene rearrangement and deletion, or chromosome loss. Accordingly, the T cell receptor is either controlled by two split gene loci in chromosome 12, at the two respective (5' and 3') ends of the mouse heavy chain gene family, or by a gene(s) outside chromosome 12.     

Rearranged beta T cell receptor genes in a helper T cell clone specific for lysozyme: no correlation between V beta and MHC restriction

The helper T cell clone 3H.25 is specific for hen egg white lysozyme and the class II MHC molecule I-Ab. This TH cell has three rearrangements in the beta-chain gene family-a V beta-D beta-J beta 1 and a D beta 2-J beta 2 rearrangement on one homolog and a D beta 1-J beta 2 rearrangement on the other. These observations demonstrate that this functional T lymphocyte expresses only a single V beta gene segment and, accordingly, exhibits allelic exclusion of beta-chain gene expression. The rearranged 3H.25 V beta gene segment is the same as that expressed in a T helper cell specific for cytochrome c and an I-Ek MHC molecule. Thus, there is no simple correlation between the V beta gene segment and antigen specificity or MHC restriction.