Autoreactive diabetogenic T-cells in NOD mice can efficiently expand from a greatly reduced precursor pool.

A broad repertoire of pancreatic beta-cell autoreactive T-cells normally contributes to the development of type 1 diabetes in NOD mice. However, it has been unknown if a large reduction in the precursor pool from which autoreactive T-cells are drawn would inhibit the development of type 1 diabetes. To address this issue, we reduced the precursor frequency of autoreactive T-cells in NOD mice through allelic exclusion induced by transgenic expression of an H2-Db class I-restricted T-cell receptor (TCR) specific for a pathologically irrelevant lymphocytic choriomeningitis virus (LCMV) peptide. TCR allelic exclusion greatly reduced the pool of T-cells from which diabetogenic effectors could be derived in these NODxLCMV TCR Tg mice. Surprisingly, this did not impair their type 1 diabetes susceptibility. Furthermore, a diabetogenic CD8 T-cell population that is prevalent in standard NOD mice was present at essentially equivalent levels in pancreatic islets of NODxLCMV TCR Tg mice. Other data indicated that the antigenic specificity of these CD8 T-cells is primarily the function of a shared TCR-alpha chain. Although the percentage of TCR transgenic T-cells decreased in NOD versus B6,D2 control mice, much higher total numbers of both the TCR transgenic and the nontransgenic T-cells accumulated in the NOD strain. This transgenic T-cell accumulation in the absence of the cognate peptide indicated that the NOD genetic background preferentially promotes a highly efficient antigen-independent T-cell expansion. This might allow diabetogenic T-cells in NOD mice to undergo an efficient expansion before encountering antigen, which would represent an important and previously unconsidered aspect of pathogenesis.

Unusual resistance of ALR/Lt mouse beta cells to autoimmune destruction: role for beta cell-expressed resistance determinants.

Genetic analysis of autoimmune insulin-dependent diabetes mellitus (IDDM) has focused on genes controlling immune functions, with little investigation of innate susceptibility determinants expressed at the level of target beta cells. The Alloxan (AL) Resistant (R) Leiter (Lt) mouse strain, closely related to the IDDM-prone nonobese diabetic (NOD)/Lt strain, demonstrates the importance of such determinants. ALR mice are unusual in their high constitutive expression of molecules associated with dissipation of free-radical stress systemically and at the beta-cell level. ALR islets were found to be remarkably resistant to two different combinations of beta-cytotoxic cytokines (IL-1beta, tumor necrosis factor alpha, and IFN-gamma) that destroyed islets from the related NOD and alloxan-susceptible strains. The close MHC relatedness between the NOD and ALR strains (H2-Kd and H2-Ag7 identical) allowed us to examine whether ALR islet cells could survive autoimmune destruction by NOD-derived Kd-restricted diabetogenic cytotoxic T lymphocyte clones (AI4 and the insulin-reactive G9C8 clones). Both clones killed islet cells from all Kd-expressing strains except ALR. ALR resistance to diabetogenic immune systems was determined in vivo by means of adoptive transfer of the G9C8 clone or by chimerizing lethally irradiated ALR or reciprocal (ALR x NOD)F1 recipients with NOD bone marrow. In all in vivo systems, ALR and F1 female recipients of NOD marrow remained IDDM free; in contrast, all of the NOD recipients became diabetic. In conclusion, the ALR mouse presents a unique opportunity to identify dominant IDDM resistance determinants expressed at the beta cell level.

Identification of a CD8 T cell that can independently mediate autoimmune diabetes development in the complete absence of CD4 T cell helper functions.

Previous work has indicated that an important component for the initiation of autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse model entails MHC class I-restricted CD8 T cell responses against pancreatic beta cell Ags. However, unless previously activated in vitro, such CD8 T cells have previously been thought to require helper functions provided by MHC class II-restricted CD4 T cells to exert their full diabetogenic effects. In this study, we show that IDDM development is greatly accelerated in a stock of NOD mice expressing TCR transgenes derived from a MHC class I-restricted CD8 T cell clone (designated AI4) previously found to contribute to the earliest preclinical stages of pancreatic beta cell destruction. Importantly, these TCR transgenic NOD mice (designated NOD.AI4alphabeta Tg) continued to develop IDDM at a greatly accelerated rate when residual CD4 helper T cells were eliminated by introduction of the scid mutation or a functionally inactivated CD4 allele. In a previously described stock of NOD mice expressing TCR transgenes derived from another MHC class I-restricted beta cell autoreactive T cell clone, IDDM development was retarded by elimination of residual CD4 T cells. Hence, there is variability in the helper dependence of CD8 T cells contributing to the development of autoimmune IDDM. The AI4 clonotype represents the first CD8 T cell with a demonstrated ability to progress from a naive to functionally activated state and rapidly mediate autoimmune IDDM development in the complete absence of CD4 T cell helper functions.

Reevaluation of the major histocompatibility complex genes of the NOD-progenitor CTS/Shi strain.

The common Kd and/or Db alleles of NOD mice contribute to the development of autoimmune diabetes, but their respective contributions are unresolved. The major histocompatibility complex (MHC) of the CTS/Shi mouse, originally designated as H2ct, shares MHC class II region identity with the H2g7 haplotype of NOD mice. However, CTS mice were reported to express distinct but undefined MHC class I gene products. Because diabetes frequency was reduced 56% in females of a NOD stock congenic for H2ct, this partial resistance may have derived from the MHC class I allelic differences. In the present report, we use a combination of serologic analysis and sequencing of MHC class I cDNAs to establish that NOD/Lt and CTS/Shi share a common H2-Kd allele but differ at the H2-D end of the MHC complex. The H2-D allele of CTS/Shi was identified as the rare H2-Ddx recently described in ALR/Lt, another NOD-related strain. These results in mouse model systems show that multiple MHC genes confer diabetes resistance and suggest that at least one of the protective MHC or MHC-linked genes in CTS mice may be at the H2-D end of the complex.

Major histocompatibility complex class I-restricted T cells are required for all but the end stages of diabetes development in nonobese diabetic mice and use a prevalent T cell receptor alpha chain gene rearrangement.

Nonobese diabetic (NOD) mice develop insulin-dependent diabetes mellitus due to autoimmune T lymphocyte-mediated destruction of pancreatic beta cells. Although both major histocompatibility complex class I-restricted CD8(+) and class II-restricted CD4(+) T cell subsets are required, the specific role each subset plays in the pathogenic process is still unclear. Here we show that class I-dependent T cells are required for all but the terminal stages of autoimmune diabetes development. To characterize the diabetogenic CD8(+) T cells responsible, we isolated and propagated in vitro CD8(+) T cells from the earliest insulitic lesions of NOD mice. They were cytotoxic to NOD islet cells, restricted to H-2Kd, and showed a diverse T cell receptor beta chain repertoire. In contrast, their alpha chain repertoire was more restricted, with a recurrent amino acid sequence motif in the complementarity-determining region 3 loop and a prevalence of Valpha17 family members frequently joined to the Jalpha42 gene segment. These results suggest that a number of the CD8(+) T cells participating in the initial phase of autoimmune beta cell destruction recognize a common structural component of Kd/peptide complexes on pancreatic beta cells, possibly a single peptide.

Cloning and characterization of a 70 kd heat shock cognate (hsc70) gene from the zebrafish (Danio rerio).

The heat shock 70 family of proteins is one of the most highly conserved among all species. The genes encoding these proteins have been cloned and sequenced from bacterial species to humans with a high degree of homology preserved throughout evolution. Here we describe the cloning and characterization of a cDNA encoding a 70 kd heat shock cognate (hsc70) gene from the zebrafish (Danio rerio). A high degree of conservation is observed among hsc70 genes of other species as shown by phylogenetic analysis. The characterization of a hsc70 gene in the zebrafish provides a marker for studying the role of a constitutively expressed member of the hsp70 family in an important developmental and evolutionary model system.

Molecular cloning of major histocompatibility complex class II B gene cDNA from the Bengalese finch Lonchura striata.

The only avian major histocompatibility complex (Mhc) genes thus far identified are from species of the relatively small order of Galliformes, while by far the largest order of Passeriformes (songbirds), containing some 60% of extant bird species, has not been studied at all in this regard. The Galliformes emerged more than 55 million years (my) ago, the Passeriformes some 25 my ago. Because of the potential for the use of Mhc genes as markers in the study of songbird populations, an attempt was made to clone class II B genes of a passeriform species, the Bengalese finch Lonchura striata acuticauda. Using a set of primers designed on the basis of known sequences, a probe corresponding to part of exon II was obtained by the polymerase chain reaction. The probe was then used to screen a Bengalese finch cDNA library and to isolate and sequence two nearly full-length clones. The sequences reveal the presence of one presumably functional class II B locus in this bird species.