Contribution of Va24Vb11 natural killer T cells in Wilsonian hepatitis.

Wilson disease (WD) is an autosomal recessive disorder of copper transport, resulting in copper accumulation and toxicity to the liver and brain. There is no evidence that the WD patient's immune system attacks copper accumulated hepatocytes. Here we describe that the frequency and absolute number of Valpha24+Vbeta11+ natural killer T (NKT) cells were significantly increased in 3 cases of WD, whereas those of CD3+CD161+ NKT cells were within the normal range. Patients no. 1 and 2 had a presymptomatic form of WD. Their tissue specimens showed pathological changes of mild degeneration of hepatocytes with a few infiltrating mononuclear cells and a low degree of fatty change. Patient no. 3 displayed fulminant hepatitis with Coombs-negative haemolytic anaemia. The tissue specimens of patient no. 3 showed macronodular cirrhosis with thick fibrosis, inflammatory infiltrates and spotty necrosis. Human Valpha24+Vbeta11+ NKT cells are almost equal to CD1d-restricted NKT cells. Therefore we investigated CD1d-restricted NKT cells in the LEC rat as an animal model of WD. In LEC rats before hepatitis onset, the number and phenotype of liver NKT cells were normal. At about 4 months of age all LEC rats developed acute hepatitis accompanied by acute jaundice, and CD161high NKT cells developed in their livers. CD161highalphabetaTCRbright NKT cells developed in some of them. Their hepatitis was severe. CD161highalphabetaTCRbright NKT cells expressed an invariant rat Valpha14-Jalpha281 chain, which is CD1d-restricted. Furthermore, liver lymphocytes in the acute jaundiced LEC rats with CD161highalphabetaTCRbright NKT cells had significant and CD1d-specific cytotoxic activity.

The cell surface-expressed HSC70-like molecule preferentially reacts with the rat T-cell receptor Vdelta6 family.

We previously showed that the cell surface-expressed Mr 70,000 heat shock cognate (hsc70, a constitutively expressed member of the hsp70 family) protein-like molecule (#067 molecule) interacts with rat CD3+, CD4-, CD8-, T-cell receptor (TCR)alphabeta-, natural killer recetor-P1- T cells. This 70hsc-like molecule was also suggested to present cellular peptide antigens to these T cells. In the present study, we identified the genetic structure of the TCR by establishing T-cell hybridomas between these T cells and mouse BW5147 cells. Our data indicated that these T cells preferentially used TCRs with the Vdelta6 family. Analysis of the nucleotide sequence of the CDR3 junctional portion showed that there are substantial diversities, with insertion of seven to nine amino acid residues. These data provide indirect evidences for our hypothesis that an hsc70-like molecule could be presented together with cellular peptide antigens to particular T cells with TCR gammadelta chains. Since the expression of this hsc70-like #067 antigen on the cell surface is usually induced along with cell transformation by activated oncogenes, T cells with the TCR Vdelta6 family are likely to contribute to host resistance to tumor cells.

NKT cells in the rat: organ-specific distribution of NK T cells expressing distinct V alpha 14 chains.

Rat invariant TCR alpha-chains and NKT cells were investigated to clarify whether CD1d-mediated recognition by NKT cells is conserved further in evolution. Rats had multiple-copies of TRAV14 genes, which can be categorized into two types according to the diversity accumulated in the CDR2 region. Rats retained invariant TCR alpha forms with the homogeneous junctional region similar to mouse invariant TRAV14-J281. The proportion of invariant TCR among V alpha 14+ clones was 12.9% in the thymus and increased in the periphery, 31% in the spleen and 95% in hepatic sinusoidal cells. The invariant TRAV14-J281 was expressed by liver sinusoidal and splenic NKT cells with CD8, CD44high, and TCR V beta 8. Type 1 invariant TCR alpha was expressed more frequently in hepatic lymphocytes, while type 2 invariant TCR alpha was expressed predominantly in the spleen. Both types of cells cytolyzed to and were stimulated to proliferate by CD1d-expressing cells in a CD1d-restricted manner. These results suggested that rat NKT cells bearing distinct V alpha 14 chains are distributed in a tissue-specific pattern. NKT cell populations in rats were more variable than those in mice, indicating that they play novel roles in nature. The implication of the molecular interaction between the structurally diverse invariant TCR alpha and CD1d/ligand complex in different organs is discussed.

Genomic organization and chromosome location of the rat CD3zeta locus (Cd3z).

The structure and pattern of expression of the CD3zeta chain have apparently been conserved throughout mammalian evolution. The organization of the rat CD3zeta locus was determined by genomic cloning and nucleotide sequencing. Most of the rat CD3zeta coding region was similar to mouse and human CD3zeta sequences. Whereas the 3' region involving alternative splicing was relatively well conserved at the nucleotide level, the deduced amino acid sequences were different in rats, mice and humans due to the presence of deletion and insertion mutations. Alternative splicing products of the CD3zeta locus, such as CD3eta, CD3θ and CD3tau, which have been reported for mice, were not expressed by rat T cells. By using fluorescence in situ hybridization, we have localized rat CD3zeta to chromosome 13q22-->q23.

A novel monoclonal antibody against rodent common Thy-1.1 epitope.

Here we report a new anti-rat Thy-1 monoclonal antibody (MAb) 12C5 that recognized a novel epitope on a GPI-anchored Thy-1 antigen. In rat thymocytes, MAb 12C5 reacted with 25 kDa Thy-1 antigen purified with MAb OX7. In rats, MAb 12C5 was expressed on 94% of thymocytes, 46% of spleen cells, 31% of mesenteric lymphnode cells, 76% of bone marrow cells, and 24% of splenic T cells. Furthermore, MAb 12C5 reacted with the hematopoietic cells in Mongolian gerbils, hamsters, and AKR mice (Thy-1.1) as in rats. In AKR mice, almost all bone marrow cells reacted with MAb 12C5. Thus, MAb 12C5 recognized a novel epitope of Thy-1 that was widespread in hematopoietic cells of rats and mice, as well as other rodents.

Deviated overexpression of TCR-beta, TCR-gamma, CD4, and CD8 on thymic lymphomas induced by 1-propyl-1-nitrosourea: destruction of the allelic exclusion of TCR-beta and expression of functional TCR-betagamma heterodimer on a lymphoma, cFTL53.

Thymic lymphomas (FTLs) induced by the chemical carcinogen 1-propyl-1-nitrosourea (PNU) in F344 rats showed deviated overexpression of TCR-beta, TCR-gamma, CD4, and CD8. Even though most FTLs were in the CD4+ CD8+ stage, all FTLs expressed TCR-beta mRNA with TCR-gamma mRNA, but without TCR-alpha mRNA or TCR-delta mRNA. One of the FTLs, cFTL53, expressed two kinds of TCR-beta mRNA and two kinds of TCR-gamma mRNA, but did not express any mRNA of TCR-alpha or TCR-delta. Both alleles of TCR-beta loci were rearranged on cFTL53. cDNA cloning and sequencing analysis showed that one TCR-gamma mRNA, Vgamma4-Jgamma1-Cgamma1, and both TCR-beta mRNA, Vbeta2-Dbeta2-Jbeta2.1 and Vbeta19-Dbeta2-Jbeta2.1-Cbeta2, on cFTL53 were in the productive form, while the other TCR-gamma mRNA, Vgamma1-Jgamma4-Cgamma4L, was not. Both TCR beta-chains and a TCR gamma-chain were expressed on cFTL53, making a novel set of TCR-betagamma heterodimer. Cross-linking of TCR-betagamma heterodimer on cFTL53 resulted in a calcium flux, indicating that TCR-betagamma works as a signal transduction receptor. Thus, there are four strange phenomena on FTLs; CD4 and CD8 are expressed without TCR-alphabeta or TCR-gammadelta, TCR-beta mRNA and TCR-gamma mRNA were expressed simultaneously without TCR-alpha and TCR-delta mRNA on FTLs, the allelic exclusion of TCR-beta was destroyed in cFTL53, and a novel set of functional TCR-betagamma heterodimer was expressed on cFTL53.

RT1.P, rat class Ib genes related to mouse TL: evidence that CD1 molecules but not authentic TL antigens are expressed by rat thymus.

CD1 and TL were once thought to be genetic homologues because of their thymus-specific expression. We investigated their equivalents in the rat to clarify whether their structure and pattern of expression are conserved in rodents. Two rat class Ib genes, containing 3' sequences very similar to mouse TL, were identified and designated RT1.P. Neither of them, however, can encode ordinary class I molecules due to the accumulation of harmful mutations in the 5' regions that are unique to RT1.P, while the 3' TL-like regions still retain protein-coding capacity. Comparison of the structural organization of three types of TL family genes, which include mouse T3/T18-encoding TL antigens, mouse T1/T16, and rat RT1. P1/P2 pseudogenes, revealed the presence of a clear demarcation between the type-specific and TL-specific sequences at intron 3. This finding suggests that recombination plays an important role in creating the TL family genes in rodents. Characteristic features of TL, such as a low level of polymorphism and linkage to the major histocompatibility complex, were also observed in the rat. On the other hand, rat CD1 molecules were expressed at a high level on the surface of thymocytes. Absence of authentic TL antigens and thymic expression of CD1d molecules in the rat suggest the plasticity and conservation of class Ib genes in rodent evolution. Functions of TL may be substituted with CD1 or other class Ib molecules expressed by rat thymus.

Structural analysis of the CD3 zeta/eta locus of the rat. Expression of zeta but not eta transcripts by rat T cells.

We analyzed the structure and pattern of expression of rat TCR zeta- and eta-chains to investigate if these components function in activation and development of rat T cells. The rat zeta cDNA contained the complete open reading frame coding for a polypeptide of 164 amino acids and the 5' and 3' noncoding sequences. Comparison of the amino acid sequence to those of mouse and human counterparts revealed a high degree of similarity, more than 85% homology among all three species except for the signal peptide, which was especially high in the cytoplasmic domain including the nucleotide binding site and the possible tyrosine phosphorylation sites. Furthermore, we determined the nucleotide sequences of a rat genomic eta-like sequence located in the 3' region of the rat zeta-gene. Although it showed a high level of nucleotide similarity to mouse and human counterparts, 90.4 and 78.9%, respectively, the deduced polypeptide was very short (only 28 residues) and markedly divergent from the mouse and human eta-specific polypeptides due to frameshift mutations. Transcription of rat zeta was shown to be highly restricted to T cells; abundantly in thymocytes and scarcely in peripheral T cells. Surprisingly, the rat eta transcript could not be detected in any rat tissues so far tested by Northern blot analysis and even by the sensitive reverse transcription-polymerase chain reaction method, whereas it was readily detected in mouse thymus. These findings suggest that the zeta-chain has conserved roles in the TCR assembly and TCR-mediated signaling. However, the eta-chain seems not to be indispensable because of its structural diversity among these three species characterized to date and the apparent lack of eta expression in the rat.

A novel cell surface antigen involved in thymocyte and thymic epithelial cell adhesion.

A murine mAb, 7D3, was produced by fusion of spleen cells obtained from mice immunized with a rat thymic epithelial cell line, Tu-D3 and NS/1 myeloma cells. 7D3 antibody reacted with approximately 95% thymocytes, 17% spleen cells, less than 9% of mesenteric lymph node cells and 32% of bone marrow cells of rat origin. 7D3 also reacted with two rat thymic epithelial cell lines but not with a rat fibroblastic cell line. Immunochemical analysis demonstrated that 7D3 antibody recognized a single polypeptide with molecular weight of 80,000 in FTE cells and 80,000 to 96,000 in thymocytes. 7D3 antibody strongly inhibited the thymocyte binding to thymic epithelial cells. In addition, 7D3 antibody inhibited TPA-induced thymocyte aggregation. 7D3 negative rat thymic lymphoma cells bound to 7D3 positive thymic epithelial cells and this binding was inhibited by 7D3 antibody, indicating that a part of thymocyte-thymic epithelial cell binding was mediated by the interaction of 7D3 Ag and undefined ligand to 7D3.