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. 2003 Jul;109(3):343-50.
doi: 10.1046/j.1365-2567.2003.01654.x.

Characterization of extrathymic CD8 alpha beta T cells in the liver and intestine in TAP-1 deficient mice

Chika Tsukada  1 , Chikako Miyaji, Hiroki Kawamura, Ryoko Miyakawa, Hisashi Yokoyama, Yuiko Ishimoto, Shinobu Miyazawa, Hisami Watanabe, Toru Abo
Affiliations

Characterization of extrathymic CD8 alpha beta T cells in the liver and intestine in TAP-1 deficient mice

Chika Tsukada et al. Immunology. 2003 Jul.

Abstract

TAP-1 deficient (-/-) mice cannot transport MHC class I antigens onto the cell surface, which results in failure of the generation of CD8+ T cells in the thymus. In a series of recent studies, it has been proposed that extrathymic T cells are generated in the liver and at other extrathymic sites (e.g. the intestine). It was therefore investigated whether CD8+ extrathymic T cells require an interaction with MHC class I antigens for their differentiation in TAP-1(-/-) mice. Although CD8+ thymically derived T cells were confirmed to be absent in the spleen as well as in the thymus, CD8 alpha beta+ T cells were abundant in the livers and intestines of TAP-1(-/-) mice. These CD8+ T cells expanded in the liver as a function of age and were mainly confined to a NK1.1-CD3int population which is known to be truly of extrathymic origin. Hepatic lymphocytes, which contained CD8+ T cells and which were isolated from TAP-1(-/-) mice (H-2b), responded to neither mutated MHC class I antigens (bm1) nor allogeneic MHC class I antigens (H-2d) in in vitro mixed lymphocyte cultures. However, the results from repeated in vivo stimulations with alloantigens (H-2d) were interesting. Allogeneic cytotoxicity was induced in liver lymphocytes in TAP-1(-/-) mice, although the magnitude of cytotoxicity was lower than that of liver lymphocytes in immunized B6 mice. All allogeneic cytotoxicity disappeared with the elimination of CD8+ cells in TAP-1(-/-) mice. These results suggest that the generation and function of CD8+ extrathymic T cells are independent of the existence of the MHC class I antigens of the mouse but have a limited allorecognition ability.

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Figures

Figure 1
Figure 1
Presence of CD8+ T cells at extrathymic sites in TAP-1(−/−) mice. (a) Number of lymphocytes yielded by the spleen, liver, intestine (IEL = intraepithelial lymphocytes) and thymus (n = 4). (b) Phenotypic characterization of lymphocytes. Lymphocytes were obtained from both B6 and TAP-1(−/−) mice at the age of 12 weeks. Two-colour staining for CD4 and CD8α and for TCRαβ and TCRγδ was conducted. Numbers in the figure represent the percentages of fluorescence-positive cells in corresponding areas. The data shown here are representative of three experiments. *P < 0·05.
Figure 2
Figure 2
Further phenotypic characterization of lymphocytes in the livers and spleens of B6 and TAP-1(−/−) mice. (a) Two-colour staining for CD3 and IL-2Rβ and for CD3 and NK1·1. (b) Two-colour staining for CD4 and CD8α and for CD4 and CD8β. (c) Two-colour staining for TCRαβ and TCRγδ. In these experiments, young (12-week-old) and old (40-week-old) B6 and TAP-1(−/−) mice were used to show the expansion of CD8+ extrathymic T cells in the liver. Numbers in the figure represent the percentages of fluorescence-positive cells in corresponding areas. The data shown here are representative of three experiments.
Figure 3
Figure 3
Age-associated increase in the number of total lymphocytes and CD8+ extrathymic T cells in the spleens and livers of B6 and TAP-1(−/−) mice. (a) Total lymphocytes. (b) CD8αβ+ T cells. Young (12-week-old) and old (40-week-old) B6 and TAP-1(−/−) mice were used. The mean (± 1 SD) is for four mice. *P < 0·05.
Figure 4
Figure 4
CD8α expression on various lymphocyte subsets in the livers of young and old B6 and TAP-1(−/−) mice. Three-colour staining for CD3, IL-2Rβ (or NK1·1) and CD8α was conducted. By gated analysis, CD8α expression on lymphocyte subsets (R1 and R2) was estimated. The data shown here are representative of three experiments.
Figure 5
Figure 5
Lack of alloreactivity in in vitro mixed lymphocyte cultures. Splenic or liver lymphocytes of B6 or TAP-1(−/−) mice were stimulated with various syngeneic or allogeneic lymphocytes. In some experiments, CD4+ T cells were depleted by complement-mediated cell lysis in conjunction with anti-CD4 mAb. The mean (± 1 SD) is for triplicate cultures. The data shown here are representative of two experiments. cpm, count per minute.
Figure 6
Figure 6
Allorecognition in in vivo stimulation with allogeneic lymphocytes. (a) Two-colour staining for CD3 and IL-2Rβ and for CD3 and NK1·1. (b) Two-colour staining for CD4 and CD8α. Allogeneic stimulation was carried out twice in the course of 10 days. A cytotoxicity assay against Con A-lymphoblasts of BALB/c (H-2d) origin was conducted (n = 4). TAP-1(−/−) or B6 mice were i.p. injected with 3 × 107 splenic lymphocytes of TAP-1(−/−) or BALB/c origin. Phenotypic and cytotoxicity assays were conducted on day 10. In some experiments, CD8+ T cells were eliminated by in vivo injection of anti-CD8 mAb (3 and 5 days before cell preparation).
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