Diabetes resistance/susceptibility in T cells of nonobese diabetic mice conferred by MHC and MHC-linked genes

Polymorphism of MHC and MHC-linked genes is tightly associated with susceptibility to type 1 diabetes (T1D) in human and animal models. Despite the extensive studies, however, the role of MHC and MHC-linked genes expressed by T cells on T1D susceptibility remains unclear. Because T cells develop from TCR(-) thymic precursor (pre-T) cells that undergo MHC restriction mediated by thymic stroma cells, we reconstituted the T cell compartment of NOD.scid-RIP-B7.1 mice using pre-T cells isolated from NOD, NOR, AKR, and C57BL/6 (B6) mice. T1D developed rapidly in the mice reconstituted with pre-T cells derived from NOD or NOR donors. In contrast, most of the NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells from AKR or B6 donors were free of T1D. Further analysis revealed that genes within MHC locus of AKR or B6 origin reduced incidence of T1D in the reconstituted NOD.scid-RIP-B7.1 mice. The expression of MHC class I genes of k, but not b haplotype, in T cells conferred T1D resistance. Replacement of an interval near the distal end of the D region in T cells of B6 origin with an identical allele of 129.S6 origin resulted in T1D development in the reconstituted mice. These results provide evidence that the expression of MHC class I and MHC-linked genes in T cells of NOD mice indeed contributes to T1D susceptibility, while expression of specific resistance alleles of MHC or MHC-linked genes in T cells alone would effectively reduce or even prevent T1D.     

Cutting edge: impaired MHC class I expression in mice deficient for Nlrc5/class I transactivator

MHC class I and class II are crucial for the adaptive immune system. Although regulation of MHC class II expression by CIITA has long been recognized, the mechanism of MHC class I transactivation has been largely unknown until the recent discovery of NLRC5/class I transactivator. In this study, we show using Nlrc5-deficient mice that NLRC5 is required for both constitutive and inducible MHC class I expression. Loss of Nlrc5 resulted in severe reduction in the expression of MHC class I and related genes such as β(2)-microglobulin, Tap1, or Lmp2, but did not affect MHC class II levels. IFN-γ stimulation could not overcome the impaired MHC class I expression in Nlrc5-deficient cells. Upon infection with Listeria monocyogenes, Nlrc5-deficient mice displayed impaired CD8(+) T cell activation, accompanied with increased bacterial loads. These findings illustrate critical roles of NLRC5/class I transactivator in MHC class I gene regulation and host defense by CD8(+) T cell responses.     

Through regulation of TCR expression levels, an Idd7 region gene(s) interactively contributes to the impaired thymic deletion of autoreactive diabetogenic CD8+ T cells in nonobese diabetic mice

When expressed in NOD, but not C57BL/6 (B6) genetic background mice, the common class I variants encoded by the H2g7 MHC haplotype aberrantly lose the ability to mediate the thymic deletion of autoreactive CD8+ T cells contributing to type 1 diabetes (T1D). This indicated some subset of the T1D susceptibility (Idd) genes located outside the MHC of NOD mice interactively impair the negative selection of diabetogenic CD8+ T cells. In this study, using both linkage and congenic strain analyses, we demonstrate contributions from a polymorphic gene(s) in the previously described Idd7 locus on the proximal portion of Chromosome 7 predominantly, but not exclusively, determines the extent to which H2g7 class I molecules can mediate the thymic deletion of diabetogenic CD8+ T cells as illustrated using the AI4 TCR transgenic system. The polymorphic Idd7 region gene(s) appears to control events that respectively result in high vs low expression of the AI4 clonotypic TCR alpha-chain on developing thymocytes in B6.H2g7 and NOD background mice. This expression difference likely lowers levels of the clonotypic AI4 TCR in NOD, but not B6.H2g7 thymocytes, below the threshold presumably necessary to induce a signaling response sufficient to trigger negative selection upon Ag engagement. These findings provide further insight to how susceptibility genes, both within and outside the MHC, may interact to elicit autoreactive T cell responses mediating T1D development in both NOD mice and human patients.     

Genetic analysis of autoimmune sialadenitis in nonobese diabetic mice: a major susceptibility region on chromosome 1

The nonobese diabetic (NOD) mouse strain provides a good study model for Sj?gren's syndrome (SS). The genetic control of SS was investigated in this model using different matings, including a (NOD x C57BL/6 (B6))F(2) cross, a (NOD x NZW)F(2) cross, and ((NOD x B6) x NOD) backcross. Multiple and different loci were detected depending on parent strain combination and sex. Despite significant complexity, two main features were prominent. First, the middle region of chromosome 1 (chr.1) was detected in all crosses. Its effect was most visible in the (NOD x B6)F(2) cross and dominated over that of other loci, including those mapping on chr.8, 9, 10, and 16; the effect of these minor loci was observed only in the absence of the NOD haplotype on chr.1. Most critically, the chr.1 region was sufficient to trigger an SS-like inflammatory infiltrate of salivary glands as shown by the study of a new C57BL/6 congenic strain carrying a restricted segment derived from NOD chr.1. Second, several chromosomal regions were previously associated with NOD autoimmune phenotypes, including Iddm (chr.1, 2, 3, 9, and 17, corresponding to Idd5, Idd13, Idd3, Idd2, and Idd1, respectively), accounting for the strong linkage previously reported between insulitis and sialitis, and autoantibody production (chr.10 and 16, corresponding to Bana2 and Bah2, respectively). Interestingly, only two loci were detected in the (NOD x NZW)F(2) cross, on chr.1 in females and on chr.7 in males, probably because of the latent autoimmune predisposition of the NZW strain. Altogether these findings reflect the complexity and heterogeneity of human SS.     

Genetic analysis of diabetes in the nonobese diabetic mouse. I. MHC and T cell receptor beta gene expression

Backcross nonobese diabetic (NOD) ((NOD x SWr)F1 x NOD) mice (108 females and 105 males) were typed for MHC, TCR V beta, and monitored for 350 days for the onset of diabetes. The presence of "antipolar" antibodies in the sera and the occurrence of insulitis was examined in a proportion of these backcross mice. There was no difference in the incidence of diabetes in mice heterozygous for TCR V beta b/a vs those homozygous for TCR V beta b/b. Among the 17 diabetics (all female) detected in this backcross, 14/17 were H-2nod/nod but 3/17 were H-2nod/q. This supports a previous observation suggesting that the MHC-linked diabetogenic gene originally thought to be recessive may rather be dominant but have a low penetrance in the heterozygous state. Antipolar autoantibodies were found in both female and male backcross mice, and were similarly distributed in diabetic and nondiabetic mice. There appeared to be no correlation between the level of these auto-antibodies and development of diabetes. The incidence and severity of insulitis was linked to MHC but no influence of TCR genes on insulitis nor an association between insulitis and antipolar antibodies could be demonstrated in this study. Further analyses of H-2nod/nod intercross mice homozygous for TCR V beta a or TCR V beta b are currently underway.     

Protection of nonobese diabetic mice from diabetes by gene(s) closely linked to IFN-gamma receptor loci

Nonobese diabetic (NOD) mice carrying a segment of chromosome flanking the disrupted IFN-gamma receptor gene from original 129 ES cells are resistant to development of diabetes. However, extended backcrossing of this mouse line to the NOD mouse resulted in a segregation of the IFN-gammaR-deficient genotype from the diabetes-resistant phenotype. These results indicate that the protection of NOD mice from the development of diabetes is not directly linked to the defective IFN-gamma receptor gene but, rather, is influenced by the presence of a diabetes-resistant gene(s) closely linked to the IFN-gammaR loci derived from the 129 mouse strain.     

Cutting edge: V alpha 14-J alpha 281 NKT cells naturally regulate experimental autoimmune encephalomyelitis in nonobese diabetic mice

Although deficiencies in the NKT cell population have been observed in multiple sclerosis and mouse strains susceptible to experimental autoimmune encephalomyelitis (EAE), little is known about the function of these cells in CNS autoimmunity. In this work we report that TCR Valpha14-Jalpha281 transgenic nonobese diabetic mice, which are enriched in CD1d-restricted NKT cells, are protected from EAE. The protection is associated with a striking inhibition of Ag-specific IFN-gamma production in the spleen, implying modulation of the encephalitogenic Th1 response. This modulation is independent of IL-4 because IL-4-deficient Valpha14-Jalpha281 mice are still protected against EAE and independent of NKT cell-driven Th1 to Th2 deviation, because no increased autoantigen-specific Th2 response was observed in immunized Valpha14-Jalpha281 transgenic mice. Our findings indicate that enrichment and/or stimulation of CD1d-dependent NKT cells may be used as a novel strategy to treat CNS autoimmunity.     

Thymic and postthymic regulation of diabetogenic CD8 T cell development in TCR transgenic nonobese diabetic (NOD) mice

Natural development of diabetes in nonobese diabetic (NOD) mice requires both CD4 and CD8 T cells. Transgenic NOD mice carrying alphabeta TCR genes from a class I MHC (Kd)-restricted, pancreatic beta cell Ag-specific T cell clone develop diabetes significantly faster than nontransgenic NOD mice. In these TCR transgenic mice, a large fraction of T cells express both transgene derived and endogenous TCR beta chains. Only T cells expressing two TCR showed reactivity to the islet Ag. Development of diabetogenic T cells is inhibited in mice with no endogenous TCR expression due to the SCID mutation. These results demonstrate that the expression of two TCRs is necessary for the autoreactive diabetogenic T cells to escape thymic negative selection in the NOD mouse. Further analysis with MHC congenic NOD mice revealed that diabetes development in the class I MHC-restricted islet Ag-specific TCR transgenic mice is still dependent on the presence of the homozygosity of the NOD MHC class II I-Ag7.     

Evolution by recombination and transspecies polymorphism in the MHC class I gene of Xenopus laevis

The patterns of major histocompatibility complex (MHC) evolution involve duplications, deletions, and independent divergence of loci during episodes punctuated by natural selection. Major differences in MHC evolution among taxa have previously been attributed to variation in linkage patterns of class I and class II MHC genes. Here we characterize patterns of evolution in the MHC class Ia gene of Xenopus laevis in terms of polymorphism, recombination, and extent of transspecies polymorphism. We also compare these patterns to see if a correlation exists with linkage or separation of the MHC class I and class II regions as seen in amphibians and teleost fishes. In X. laevis, we find high levels of polymorphism. Also, genetic exchange is relatively frequent and occurs in intron II, reshuffling allelic forms of exons 2 and 3. Evolutionary relationships among class I alleles show an intermingling of alleles from divergent Xenopus species rather than a species-specific clustering. Results indicate that the patterns of evolution are similar to those found in salmonid fishes and are different from the mode of evolution seen in primates. Similar patterns of class Ia evolution in salmonid fishes and X. laevis suggest that nonlinkage of class I and class II regions alone is insufficient to explain some patterns of MHC evolution in salmonids.     

Lipolysis is altered in MHC class I zinc-alpha(2)-glycoprotein deficient mice

Non-conventional major histocompatibility complex class I molecules are involved in a variety of physiological functions, most at the periphery of the immune system per se. Zinc-alpha(2)-glycoprotein (ZAG), the sole soluble member of this superfamily has been implicated in cachexia, a poorly understood yet life-threatening, severe wasting syndrome. To further ascertain the role of ZAG in lipid metabolism and perhaps the immune system, we inactivated both ZAG alleles by gene targeting in mice. Subjecting these ZAG deficient animals to standard or lipid rich food regimens led to increased body weight in comparison to identically treated wild-type mice. This phenotype appeared to correlate with a significant decrease in adipocytic lipolysis that could not be rescued by several pharmacological agents including beta(3)-adrenoreceptor agonists. Furthermore, in contrast to previously reported data, ZAG was found to be ubiquitously and constitutively expressed, with an especially high level in the mouse liver. No overt immunological phenotype was identified in these animals.     

Allelic variants of the human MHC class I chain-related B gene (MICB)

 The human major histocompatibility complex (MHC) is located within a 4 megabase segment on chromosome 6p21.3. Recently, a highly divergent MHC class I chain-related gene family, MIC was identified within the class I region. The MICA and MICB genes in this family have unique patterns of tissue expression. The MICA gene is highly polymorphic, with more than 20 alleles identified to date. To elucidate the extent of MICB allelic variations, we sequenced exons 2 (α1), 3 (α2), 4 (α3), and 5 (transmembrane) as well as introns 2 and 4 of this gene in 46 HLA homozygous B-cell lines. We report the identification of eleven alleles based on seven non-synonymous, two synonymous, and four intronic nucleotide variations. Interestingly, one allele has a nonsense mutation resulting in a premature termination codon in the α2 domain. Thus, MICB appears to have fewer alleles than MICA, not unlike the allelic ratio between the HLA-C and -B loci. A preliminary linkage analysis of the MICB alleles with those of the closely located MICA and HLA-B genes revealed no conspicuous linkage disequilibrium between them, implying the presence of a potential recombination hotspot between the MICB and MICA genes. Received: 16 April 1997 / Revised: 19 May 1997     

MHC class I and MHC class II DRB gene variability in wild and captive Bengal tigers (Panthera tigris tigris)

Bengal tigers are highly endangered and knowledge on adaptive genetic variation can be essential for efficient conservation and management. Here we present the first assessment of allelic variation in major histocompatibility complex (MHC) class I and MHC class II DRB genes for wild and captive tigers from India. We amplified, cloned, and sequenced alpha-1 and alpha-2 domain of MHC class I and beta-1 domain of MHC class II DRB genes in 16 tiger specimens of different geographic origin. We detected high variability in peptide-binding sites, presumably resulting from positive selection. Tigers exhibit a low number of MHC DRB alleles, similar to other endangered big cats. Our initial assessment—admittedly with limited geographic coverage and sample size—did not reveal significant differences between captive and wild tigers with regard to MHC variability. In addition, we successfully amplified MHC DRB alleles from scat samples. Our characterization of tiger MHC alleles forms a basis for further in-depth analyses of MHC variability in this illustrative threatened mammal.     

A peptide binding motif for I-Eg7, the MHC class II molecule that protects E alpha-transgenic nonobese diabetic mice from autoimmune diabetes.

The nonobese diabetic (NOD) mouse, a model of spontaneous insulin-dependent diabetes mellitus (IDDM), fails to express surface MHC class II I-Eg7 molecules due to a deletion in the E alpha gene promoter. E alpha-transgenic NOD mice express the E alpha E beta g7 dimer and fail to develop either insulitis or IDDM. A number of hypotheses have been proposed to explain the mechanisms of protection, most of which require peptide binding to I-Eg7. To define the requirements for peptide binding to I-Eg7, we first identified an I-Eg7-restricted T cell epitope corresponding to the sequence 4-13 of Mycobacterium tuberculosis 65-kDa heat shock protein (hsp). Single amino acid substitutions at individual positions revealed a motif for peptide binding to I-Eg7 characterized by two primary anchors at relative position (p) 1 and 4, and two secondary anchors at p6 and p9. This motif is present in eight of nine hsp peptides that bind to I-Eg7 with high affinity. The I-Eg7 binding motif displays a unique p4 anchor compared with the other known I-E motifs, and major differences are found between I-Eg7 and I-Ag7 binding motifs. Analysis of peptide binding to I-Eg7 and I-Ag7 molecules as well as proliferative responses of draining lymph node cells from hsp-primed NOD and E alpha-transgenic NOD mice to overlapping hsp peptides revealed that the two MHC molecules bind different peptides. Of 80 hsp peptides tested, none bind with high affinity to both MHC molecules, arguing against some of the mechanisms hypothesized to explain protection from IDDM in E alpha-transgenic NOD mice.     

The enlarged population of marginal zone/CD1d(high) B lymphocytes in nonobese diabetic mice maps to diabetes susceptibility region Idd11

The NOD mouse is an important experimental model for human type 1 diabetes. T cells are central to NOD pathogenesis, and their function in the autoimmune process of diabetes has been well studied. In contrast, although recognized as important players in disease induction, the role of B cells is not clearly understood. In this study we characterize different subpopulations of B cells and demonstrate that marginal zone (MZ) B cells are expanded 2- to 3-fold in NOD mice compared with nondiabetic C57BL/6 (B6) mice. The NOD MZ B cells displayed a normal surface marker profile and localized to the MZ region in the NOD spleen. Moreover, the MZ B cell population developed early during the ontogeny of NOD mice. By 3 wk of age, around the time when autoreactive T cells are first activated, a significant MZ B cell population of adult phenotype was found in NOD, but not B6, mice. Using an F2(B6 x NOD) cross in a genome-wide scan, we map the control of this trait to a region on chromosome 4 (logarithm of odds score, 4.4) which includes the Idd11 and Idd9 diabetes susceptibility loci, supporting the hypothesis that this B cell trait is related to the development of diabetes in the NOD mouse.     

Cutting edge: TCR alpha beta+ CD8 alpha alpha+ T cells are found in intestinal intraepithelial lymphocytes of mice that lack classical MHC class I molecules.

TCR alpha beta+ intestinal intraepithelial lymphocytes (IEL) can express either the typical CD8 alpha beta heterodimer or an unusual CD8 alpha alpha homodimer. Both types of CD8+ IEL require class I molecules for their differentiation, since they are absent in beta2m-/- mice. To gain insight into the role of class I molecules in forming TCR alpha beta+ CD8+ IEL populations, we have analyzed the IEL in mice deficient for either TAP, beta 2m, CD1, or K and D. We find that K-/-D-/- mice have TCR alpha beta+ CD8 alpha alpha+ IEL, although they are deficient for TCR alpha beta+ CD8 alpha beta+ cells. This indicates that at least some TCR alpha beta+ CD8 alpha alpha+ IEL require only nonclassical class I molecules for their development. Surprisingly, the TCR alpha beta+ CD8 alpha alpha+ IEL are significantly increased in K-/-D-/- mice, suggesting a complex interaction between CD8+ IEL and class I molecules that might include direct or indirect negative regulation by K and D, as well as positive effects mediated by nonclassical class I molecules.     

Mapping of the rabbit MHC reveals that class I genes are adjacent to the DR subregion and defines an insertion/deletion-related polymorphism in the class II region.

Molecular analyses of genes in the rabbit MHC (RLA) by pulsed field gel electrophoresis have shown that the relative order of class II genes (DP, DO, DQ, DR) is identical to that in humans and similar to that in the mouse. However, a major difference from either HLA or H-2 was observed at the DR end of the RLA class II complex: class I genes are located in close proximity to DR with no interposed class III sequences. A MluI fragment of 180 kb and a 210-kb SalI fragment both hybridized with the DR probe as well as with different class I probes including that for pR27, a class I gene with T cell-limited pattern of expression. Comparison of two different RLA haplotypes, A and B, indicated that the distance between the DQ and DR subregions differs by approximately 700 kb in the two haplotypes. Testing other unrelated rabbits suggested that this difference segregates within the rabbit population and presumably derives from an insertion/deletion event in different haplotypes. A further difference between the A and B haplotypes included variable distance between genes encoding DO beta and DP; the DR end of the complex and the class I genes linkage was conserved in the two haplotypes.     

Cutting edge: Membrane nanotubes in vivo: a feature of MHC class II+ cells in the mouse cornea

Membrane nanotubes are a recently discovered form of cellular protrusion between two or more cells whose functions include cell communication, environmental sampling, and protein transfer. Although clearly demonstrated in vitro, evidence of the existence of membrane nanotubes in mammalian tissues in vivo has until now been lacking. Confocal microscopy of whole-mount corneas from wild-type, enhanced GFP chimeric mice, and Cx3cr1(gfp) transgenic mice revealed long (>300 microm) and fine (<0.8 microm diameter) membrane nanotube-like structures on bone marrow-derived MHC class II(+) cells in the corneal stroma, some of which formed distinct intercellular bridges between these putative dendritic cells. The frequency of these nanotubes was significantly increased in corneas subjected to trauma and LPS, which suggests that nanotubes have an important role in vivo in cell-cell communication between widely spaced dendritic cells during inflammation. Identification of these novel cellular processes in the mammalian cornea provides the first evidence of membrane nanotubes in vivo.     

Variations in the cytoplasmic region account for the heterogeneity of the chicken MHC class I (B-F) molecules

Molecular variation among major histocompatibility complex (MHC) class I (B-F) proteins from B-homozygous chickens is apparently caused by C-terminal variation. Analysis of the total B-F protein pool revealed substantial heterogeneity with two or three molecular mass constituents, each being comprised by several isoelectric focusing variants. This heterogeneity could not be reduced by enzymatic deglycosylation. By contrast, proteolytic removal of a small (M _r 1000–4000) fragment from the chain resulted in the generation of a M _r 36 000 fragment, common to all the molecular mass variants. Unlike the parent proteins, the M _r 36 000 fragment derived from isolated variants yielded identical, simple patterns in two-dimensional gel electrophoresis and identical finger prints in peptide mapping. This, together with N-terminal amino acid sequencing, as well as comparison of hydrophobicity properties of fragments obtained by gradual proteolytic digestion, indicated that the small peptide responsible for the major B-F heterogeneity was situated in the intracellular, C-terminal part.