S Ul-lfuTlary A novel subset ofT ceils characterized by the expression of an invariant T cell antigen receptor (TC1L) encoded by V0t24J~xQ gene segments was investigated in patients with systemic sclerosis (SSc). Polymerase chain reaction analysis demonstrated that the Vot24 TClL repertoire was selectively used in CD4-CD8-double-negative T cells both in patients and in healthy individuals, while almost all families of TCR.Vot were expressed in single-positive T cell fractions. The Vor + double-negative T cells were increased by approximately fivefold in patients. However, sequence analysis clearly showed significant differences in the Vo~24 TC1L repertoire dominating in patients and healthy donors. In healthy individuals, the invariant Vot24JoLQ was expanded and comprised 20-50% of the total TCR-ot, while their selective reduction was observed in SSc patients who also showed expansion of invariant Vex24 TCR other than Vot24JcxQ. Analogous to murine invariant V~14JoL281 TC1L, these results suggest that T cells with invariant V&24JotQ TCR would function as regulatory T cells, whereas T cells bearing other invariant Vex24 TCR in SSc patients could be autoaggressive T cells in nature. Recent studies by Porcelli et al. (6) and Dellabona et al. (7,8) indicate that the invariant Vcx24J~xQ sequence is preferentially expressed on DN ot/]3 T cells from healthy individuals. The homology of the nucleotide sequences is found to be 75% in the V0t24 and 90% in the CDR3 regions compared with murine Vo~24 TCR (9). Therefore, the human V&24 sequence is a homologue of murine V&14 TCP,. Another striking similarity to murine invariant V~14 T cells is that human peripheral DN T cells also express a limited TCR.-[3 repertoire including V [32, V[38, VI311,10). Interestingly, the decrease in invariant Vod4Jo~281 TCR expression in autoimmune prone mice correlates with disease development. It is thus likely that Vot14 § NK T cells play a role in the regulation ofautoimmune disease development.

A novel T cell subset characterized by cell surface NK1.1+ TCR alpha beta+ expression was investigated for its TCR alpha usage, particularly that of invariant V alpha 14 TCR, which was found to be preferentially used in peripheral CD4-CD8- T cells developed at extrathymic sites. We found that NK+ alpha beta T cell subsets account for 0.4% in thymocytes, 5% in the splenic T cells and 40.5% in the bone marrow T cells. Among these NK+ alpha beta T cells, two distinct subsets were detected; cell surface TCR V alpha 14+ and V alpha 14- subpopulations. Almost all of NK+ alpha beta thymocytes express V alpha 14 mRNA; however, only < 20% were positive, while > 80% were negative or undetectable for V alpha 14 TCR expression on the cell surface in the thymus. Similarly, approximately 50% of NK+ alpha beta T cells in spleen and bone marrow are V alpha 14+ as revealed by FACS. TCR repertoire analysis by nucleotide sequences on inverse PCR products demonstrated that most NK+ alpha beta T cells express an invariant TCR encoded by the V alpha 14J alpha 281 gene with a 1 base N-region in all tissues. Thus, invariant V alpha 14 TCR is uniquely expressed on NK T cells, and can be a marker to distinguish NK, NK T and T cells.

ABSTRACT:Cytochrome P450 (P450) 2B6 is a hepatic enzyme of potential importance for the metabolism of clinically used drugs and environmental or abused toxicants. Genetic polymorphisms of CYP2B6 (CYP2B6*2, CYP2B6*3, CYP2B6*4, CYP2B6*5, CYP2B6*6 and CYP2B6*7; wild-type, CYP2B6*1) were found previously in white and Japanese populations. In the present study, the goal was to investigate the effects of amino acid substitutions on CYP2B6 function. Wild-type (CYP2B6.1) and all of the known variants of CYP2B6 (CYP2B6.2, CYP2B6.3, CYP2B6.4, CYP2B6.5, CYP2B6.6, and CYP2B6.7) were transiently expressed in COS-1 cells, and their 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activities were determined. The levels of the variant CYP2B6 proteins were relatively low compared with that of CYP2B6.1, although the differences were not significant. The activities of 7-ethoxy-4-trifluoromethylcoumarin O-deethylation on the basis of the CYP2B6 protein level at low (0.5 M) and high (50 M) substrate concentrations varied among wild-type and variant CYP2B6 proteins. All CYP2B6 enzymes showed typical Michaelis-Menten kinetics. The K m value of CYP2B6.6 was significantly higher than that of CYP2B6.1. Those CYP2B6 variants having a Lys262Arg substitution (CYP2B6.4, CYP2B6.6, and CYP2B6.7) showed increased values for V max and V max /K m , whereas the kinetic parameters of CYP2B6.2 and CYP2B6.3 were not affected by the corresponding amino acid substitution. These results may mean that Lys262 in combination with other amino acid residues such as Gln172 and Arg487 is associated with the CYP2B6 function and that the genetic polymorphism of CYP2B6 leads to interindividual differences in xenobiotic metabolism.

Summal~It is known that rearrangement of the T cell antigen receptor (TCIL) gene occurs in the thymus during T cell development and consequently results both in the deletion of DNA between the variable (V) and diversity/joining segments and in the formation of a circular DNA with recombination signal sequences. Here, we provide evidence that Vot14 + TCR gene rearrangements take place in extrathymic sites, such as bone marrow, liver, and intestine, but not in spleen, because we were able to detect frequent productive and nonproductive Vod4 + coding and signal sequences as a result of TCR. rearrangements in extrathymic sites. Similar findings were also detected in athymic mice. Quantitative analysis shows that the relative amounts of Vot14 gene-mediated signal sequences in extrathymic tissues are higher than those in thymus. On the contrary, TCR rearrangements of Veal.1 T cells, which are known to develop in the thymus, were mainly detected in the thymus, Peyer's patch, and spleen, but not in other extrathymic tissues, showing patterns distinct from Vot14 TCR rearrangements. These findings are evidence of extrathymic development of Veal4 + T cells. Differential characteristic TCR rearrangement patterns also indicate that distinct TCR repertoires are generated in different lymphoid tissues.

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