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DOCK2 Is Required in T Cell Precursors for Development of V14 NK T Cells¹

Yuya Kunisaki^*, Yoshihiko Tanaka^*, Terukazu Sanui^*, Ayumi Inayoshi^*,, Mayuko Noda^*,, Toshinori Nakayama, Michishige Harada, Masaru Taniguchi, Takehiko Sasazuki^¶ and Yoshinori Fukui^2,*,

^* Division of Immunogenetics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan; Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan; Laboratory for Immune Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan; and ^¶ International Medical Center of Japan, Tokyo, Japan

	Abstract

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Mouse CD1d-restricted V14 NKT cells are a unique subset of lymphocytes, which play important roles in immune regulation, tumor surveillance and host defense against pathogens. DOCK2, a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster myoblast city, is critical for lymphocyte migration and regulates T cell responsiveness through immunological synapse formation, yet its role in V14 NKT cells remains unknown. We found that DOCK2 deficiency causes marked reduction of V14 NKT cells in the thymus, liver, and spleen. When -galactosylceramide (-GalCer), a ligand for V14 NKT cells, was administrated, cytokine production was scarcely detected in DOCK2-deficient mice, suggesting that DOCK2 deficiency primarily affects generation of V14 NKT cells. Supporting this idea, staining with CD1d/-GalCer tetramers revealed that CD44^–NK1.1^– V14 NKT cell precursors are severely reduced in the thymuses of DOCK2-deficient mice. In addition, studies using bone marrow chimeras indicated that development of V14 NKT cells requires DOCK2 expression in T cell precursors, but not in APCs. These results indicate that DOCK2 is required for positive selection of V14 NKT cells in a cell-autonomous manner, thereby suggesting that avidity-based selection also governs development of this unique subset of lymphocytes in the thymus.

	Introduction

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Natural killer T cells are a unique subset of lymphocytes, which are characterized by the coexpression of NK cell surface markers and TCR. The majority of mouse NKT cells are selected in the thymus through the interaction with CD1d molecules on CD4⁺CD8⁺ double-positive (DP)³ thymocytes and express a semi-invariant TCR composed of a V14-J18 rearrangement that preferentially associates with the V8, V7, or V2 gene segment (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). These V14 NKT cells are found in peripheral tissues, including the liver, and are almost uniformly reactive to the marine sponge-derived glycolipid -galactosylceramide (-GalCer) (11). Several lines of evidence indicate that V14 NKT cells play important roles in immune regulation, tumor surveillance, and host defense against pathogens (12).

The developmental pathway of V14 NKT cells in the thymus can be defined with CD1d/-GalCer tetramers. After positive selection, CD44^–NK1.1^–tetramer⁺ precursors appear, and differentiate into CD44⁺NK1.1⁺ mature V14 NKT cells through CD44⁺NK1.1^– intermediates (13, 14). This process is accompanied by cell division and expansion (13). Thus far, several molecules have been identified in generation, expansion, and/or maintenance of V14 NKT cells. These include cathepsin L, adaptor protein complex 3, and saposins, which are critical for Ag presentation by CD1d molecules (15, 16, 17), and IL-15 and RelB, both of which are stromal factors that regulate expansion and/or maintenance of V14 NKT cells (18, 19, 20, 21). The Src family tyrosine kinase Fyn, its signaling lymphocyte activation molecule (SLAM)-associated protein (SAP), and IB kinase 2 (IKK2) have also been reported to be required for development of V14 NKT cells, but not development of conventional T cells (22, 23, 24, 25, 26, 27, 28).

DOCK2 is a new member of the CDM protein family, Caenorhabditis elegans CED-5, human DOCK180, and Drosophila melanogaster myoblast city, which is specifically expressed in hemopoietic cells (29). We had earlier reported that DOCK2-deficient (DOCK2^–/–) mice exhibit a migration defect of lymphocytes in response to various chemokines (30). More recently, we have shown that Ag-induced translocation of TCR and lipid rafts, but not PKC- and LFA-1, to the APC interface is severely impaired in DOCK2^–/– T cells (31). Thus, in conventional T cells, DOCK2 critically regulates migration and immunological synapse formation by functioning downstream of chemokine receptors and TCRs. However, DOCK2 deficiency did not affect the chemotactic response of monocytes (30), although it has been reported that DOCK2 is expressed in tissue macrophages (32). Therefore, the function of DOCK2 may be relatively limited to lymphoid cell lineages. NKT cells possess properties of both T cells (lymphoid) and NK cells (myeloid). In this respect, it would be interesting to examine how DOCK2 functions in this unique subset of lymphocytes.

In the present study, we have analyzed the role of DOCK2 in NKT cells. We demonstrate that DOCK2 expression in T cell precursors is required for early development of V14 NKT cells in the thymus.

	Materials and Methods

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Mice

DOCK2^–/– and CD1d-deficient (CD1d^–/–) mice have been described elsewhere (30, 33). Both mouse lines were backcrossed with C57BL/6 (B6) mice for more than eight generations before use in this study. In some experiments, DOCK2^+/– littermates obtained by crossing DOCK2^–/– males and DOCK2^+/– females were used as controls. DOCK2^–/– mice lacking MHC class I expression were developed by crossing with ₂-microglobulin (₂m)-deficient (₂m^–/–) mice. For construction of bone marrow chimeric mice, 10 Gy irradiated ₂m^–/– mice were reconstituted with T cell-depleted bone marrow cells. All animals were kept under specific pathogen-free conditions. All experiments were done in accordance with the guidelines of the committee of Ethics of Animal Experiments, Faculty of Medical Sciences (Kyushu University).

Reagents

-GalCer was synthesized by the Pharmaceutical Research Laboratory, Kirin Brewery. CD1d/-GalCer tetramers were generated as previously described (34).

Flow cytometric analysis

The following mAbs were obtained from BD Pharmingen: biotinylated or FITC-conjugated anti-NK1.1 (PK136), PE-conjugated anti-TCR- (H57-597), PE- or FITC-conjugated anti-CD4 (RM4-5), FITC anti-CD8 (53-6.7), FITC anti-B220 (RA3-6B2), FITC anti-CD11b (M1/70), FITC anti-Gr-1 (RB6-8C5), CyChrome anti-CD44, CyChrome anti-CD3 (145-2C11), FITC-CD1d (1B1), anti-heat-stable Ag (HSA, J11d), and anti-CD16 (2.4G2). Cells were treated with anti-CD16 mAb to eliminate nonspecific staining, and then stained with relevant mAbs. For ontogeny analysis, HSA^low thymocytes were enriched by treatment with the mAb to HSA and complement. In some experiments, cells negative for B220, CD11b, Gr-1, CD8, and/or CD4 were analyzed for NKT cell populations. Analysis was performed on a FACSCalibur flow cytometer (BD Biosciences).

Measurement of -GalCer-induced cytokine production and proliferation

Spleen cells (2 x 10⁵/well) were cultured with -GalCer (100 ng/ml) or vehicle alone for 84 h, and 1 µCi of [³H]thymidine was added during the final 12 h of culture. Cytokine production was quantified with ELISA kits (Pierce). To examine cytokine production in vivo, mice were injected both i.v. and i.p. with either vehicle alone or with 2 mg of -GalCer, and serum IL-4 and IFN- levels were measured at 2.5 or 17 h later, respectively. For T cell hybridoma stimulation, DN32.D3 cells (3 x 10⁴/well) were cultured with or without purified DP thymocytes (1 x 10⁶/well), and IL-2 production was quantified by the proliferative response of CTLL cells (5 x 10³/well).

Con A-induced hepatitis

Con A (Sigma-Aldrich) was dissolved in PBS and injected i.v. into mice (0.5 mg/mouse). Serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured with Fuji DRI-CHEM slides.

Statistical analysis

Two-tailed Student’s t test was used for comparison of mean values. A value of p < 0.05 was considered statistically significant.

	Results

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DOCK2 deficiency causes severe reduction of V14 NKT cells in the thymus, liver, and spleen

Although the majority of NKT cells in the thymus and liver are V14 NKT cells, the spleen includes considerable numbers of CD1d-independent NKT cells (34, 35). To assess the role of DOCK2 in NKT cells, we first analyzed the NK1.1⁺ TCR⁺ population in the thymus, liver, and spleen. In the thymus, the numbers of both CD4⁺ and CD4^–CD8^– double negative (DN) NKT cells were markedly reduced in DOCK2^–/– mice, compared with cells of DOCK2^+/– littermates (Fig. 1A). Similar results were obtained when the numbers of CD4⁺ and DN NKT cells in the liver were compared between DOCK2^+/– and DOCK2^–/– mice (Fig. 1B). Although DOCK2 deficiency also reduced the NKT cell population in the spleen, this effect was modest compared with the effect on NKT cells in the thymus and liver (Fig. 1). In contrast, the numbers of NK cells in the liver and spleen were comparable between DOCK2^+/– and DOCK2^–/– mice (Fig. 1, B and C), indicating that DOCK2 deficiency does not affect generation and migration of NK cells.

View larger version (39K): [in this window] [in a new window]   FIGURE 1. DOCK2–/– mice exhibit reduction of NKT cells in the thymus, liver and spleen. A, The number of CD4+ or DN NKT cells in the thymus was compared between DOCK2+/– () and DOCK2–/– () littermates. FACS profiles show the expression of NK1.1 and TCR on cells negative for B220, CD11b, Gr-1, CD8, and CD4 (DN NKT). The values (inset upper quadrants) indicate the percentage of each subset of the cells. B, The number of CD4+ NKT, DN NKT, or NK cells in the liver was compared between DOCK2+/– () and DOCK2–/– () littermates. FACS profiles show the expression of NK1.1 and TCR on cells negative for B220, CD11b, Gr-1, and CD8 (CD4+ NKT and DN NKT). The values (inset upper quadrants) indicate the percentage of each subset of the cells. C, The number of CD4+ NKT, DN NKT, or NK cells in the spleen was compared between DOCK2+/– () and DOCK2–/– () littermates. FACS profiles show the expression of NK1.1 and TCR on cells negative for B220, CD11b, Gr-1, and CD8 (CD4+ NKT and DN NKT). The values (inset upper quadrants) indicate the percentage of each subset of the cells. Data represent at least three mice analyzed at 9–10 wk of age.

View larger version (45K): [in this window] [in a new window]   FIGURE 2. Phenotypic and functional analysis of V14 NKT cells in the spleen and liver. A, Spleen cells (2 x 105/well) from DOCK2+/– () and DOCK2–/– () mice were cultured with -GalCer (100 ng/ml) or vehicle alone (control) for 84 h, and [3H]thymidine uptake (left) and cytokine production (middle and right) were analyzed. B, The number of V14 NKT cells in the spleen was compared among B6 (), DOCK2–/– (), and CD1d–/– () for n = 3 mice in each group at 6 wk of age. FACS profiles show the reactivity of CD1d/-GalCer tetramers and anti-CD3 mAb for cells negative for B220, CD11b, Gr-1, and CD8. The value (upper right quadrant) indicates the percentage of tetramer+CD3+ cells. C, The number of V14 NKT cells in the liver was compared among B6 (), DOCK2–/– (), and CD1d–/– () for n = 3 mice in each group at 12 wk of age. FACS profiles show the reactivity of CD1d/-GalCer tetramers and anti-CD3 mAb for cells negative for B220, CD11b, Gr-1, and CD8. The value (upper right quadrant) indicates the percentage of tetramer-positive (tet+) CD3+ cells.

V14 NKT cells are known to play a major role in Con A-induced hepatitis (36, 37). Having found that V14 NKT cells were remarkably reduced in the liver of DOCK2^–/– mice, we next examined how DOCK2 deficiency affects Con A-induced hepatitis. Although DOCK2^+/– mice exhibited an increase in the serum concentration of ALT and AST to 1988 U/L and 2445 U/L on average, respectively, at 12 h after i.v. injection of Con A (Fig. 3A), no increase was found in DOCK2^–/– mice (Fig. 3A). Thus, DOCK2 deficiency makes B6 mice completely resistant to Con A-induced hepatitis. Interestingly, the serum concentration of ALT and AST in DOCK2^–/– mice was significantly lower vs the concentration in CD1d^–/– mice at 12 h after Con A injection (109.7 ± 12.0 U/L vs 266.3 ± 35.9 U/L for ALT, p < 0.01; 26.0 ± 1.0 U/L vs 151.0 ± 56.3 U/L for AST, p < 0.05) (Fig. 3B). These results suggest that DOCK2 deficiency suppresses Con A-induced hepatitis not only by reducing V14 NKT cells in the liver, but also by attenuating activation of conventional T cells (31, 38).

View larger version (25K): [in this window] [in a new window]   FIGURE 3. DOCK2–/– mice are resistant to Con A-induced hepatitis. A, Serum activities of ALT and AST were measured for n = 4 each DOCK2+/– () and DOCK2–/– (•) mice at the indicated time points after i.v. injection of 0.5 mg of Con A. B, Serum activities of ALT and AST at 12 h after Con A injection were compared among B6 (), DOCK2–/– (), and CD1d–/– () for n = 3 mice/group.

DOCK2^–/– mice do not produce cytokines in response to systemic administration of -GalCer

Because DOCK2 is critical for lymphocyte homing (30, 39), the reduction of V14 NKT cells in the spleen and liver of DOCK2^–/– mice may represent abnormal localization of this lymphocyte subset. To address this possibility, DOCK2^+/– and DOCK2^–/– mice were injected both i.v. and i.p. with -GalCer, and the serum IL-4 and IFN- levels were compared between these mice. As shown in Fig. 4, DOCK2^+/– mice produced large amounts of IL-4 and IFN- in response to -GalCer. However, such cytokine production was not elicited in DOCK2^–/– mice even though -GalCer was systemically administrated (Fig. 4). These results, together with the data that NKT cells are markedly reduced in DOCK2^–/– thymus (Fig. 1A), suggest that DOCK2 deficiency affects either development or maintenance/survival of V14 NKT cell in the thymus.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Systemic administration of -GalCer does not induce cytokine production in DOCK2–/– mice. DOCK2+/– () and DOCK2–/– () mice were injected both i.v. and i.p. with either vehicle alone (control) or 2 mg of -GalCer, and serum IL-4 and IFN- levels were measured at 2.5 or 17 h, respectively.

DOCK2 is required for early development of V14 NKT cells in the thymus

To directly examine whether DOCK2 deficiency affects development of V14 NKT cells in the thymus, HSA^low thymocytes were prepared from B6, DOCK2^–/–, and CD1d^–/– mice at the age of 3, 6, or 12 wk and stained with CD1d/-GalCer tetramers. Although tetramer⁺ thymocytes were scarcely detected in the CD1d^–/– thymus, 4–5% of B6 thymocytes were stained with the tetramers (Fig. 5A). As expected, DOCK2^–/– mice, compared with B6 controls, exhibited a 30- to 50-fold reduction of tetramer⁺ thymocytes at any time point tested (Fig. 5B). When tetramer⁺ thymocytes were analyzed for the expression of NK1.1 and CD44 in 3-wk-old B6 mice, CD44^–NK1.1^–, CD44⁺NK1.1^–, and CD44⁺NK1.1⁺ thymocytes were found at similar frequencies (Fig. 5A). However, the ratio of CD44^–NK1.1^– to CD44⁺NK1.1⁺tetramer⁺ thymocytes decreased with age (Fig 5), which supports a developmental sequence from CD44^–NK1.1^– to CD44⁺NK1.1⁺ via CD44⁺NK1.1^– thymocytes (13, 14). Interestingly, the number of tetramer⁺CD44^–NK1.1^– thymocytes in 3-wk-old DOCK2^–/– mice was 180-fold less than thymocytes in age-matched B6 controls (Fig. 5B). Although CD44⁺NK1.1^– and CD44⁺NK1.1⁺tetramer⁺ thymocytes were also reduced in DOCK2^–/– mice, the effect of DOCK2 deficiency on these thymocyte subsets was modest, compared with that on tetramer⁺CD44^–NK1.1^– thymocytes (Fig. 5B). Taken together, these results indicate that DOCK2 is required for early development, but not expansion and survival, of V14 NKT cells in the thymus.

View larger version (60K): [in this window] [in a new window]   FIGURE 5. Early development of V14 NKT cells is impaired in DOCK2–/– mice. A, The HSAlow thymocytes prepared from B6, DOCK2–/–, and CD1d–/– mice at 3 wk (top panels), 6 wk (middle panels), and 12 wk (bottom panels) of age were stained with CD1d/-GalCer tetramers (left panels), and the expression of CD44 and NK1.1 was analyzed for tetramer+ thymocyte population for B6 and DOCK2–/– mice (right panels). The values (inset upper quadrants) indicate the percentage of each subset of cells. B, The numbers of total tetramer+ (tet+), CD44–NK1.1–tetramer+, CD44+NK1.1–tetramer+, and CD44+NK1.1+tetramer+ thymocytes were compared among B6 (), DOCK2–/– (), and CD1d–/– () for n = 3 mice in each group at the indicated time points.

DOCK2 expression is required in T cell precursors, but not in APCs, for V14 NKT cell development

The development of V14 NKT cells requires the interaction with CD1d molecules expressed on DP thymocytes (4, 6). To examine whether DOCK2 deficiency affects Ag presentation by DP thymocytes, V14 NKT cell hybridoma DN32.D3 cells (4) were cultured with DP thymocytes with or without DOCK2 expression, and IL-2 production was measured with the IL-2-sensitive T cell line CTLL. As shown in Fig. 6A, DP thymocytes from DOCK2^+/– and DOCK2^–/– mice comparably stimulated DN32.D3 cells. In addition, the cell surface expression of CD1d molecules in T and B cells was unchanged between these mice (Fig. 6B). These results indicate that DOCK2 is not required for expression of and Ag presentation by CD1d molecules.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. CD1d-mediated lipid Ag presentation is intact in DOCK2–/– mice. A, V14 NKT cell hybridoma DN32.D3 cells (3 x 104/well) were cultured with (DP+) or without (DP–) purified DP thymocytes (1 x 106/well) from DOCK2+/– () and DOCK2–/– () mice, and IL-2 production was quantified by the proliferative response of CTLL cells (5 x 103/well). B, The expression levels of CD1d on T and B cells were compared between DOCK2+/– and DOCK2–/– mice. Profile staining with anti-CD11d mAb (solid histogram) or control Ab (dotted histogram) is indicated.

View larger version (35K): [in this window] [in a new window]   FIGURE 7. DOCK2 expression is required in T cell precursors, but not in APCs, for development of V14 NKT cells. A, MHC class I-negative NKT cells in the thymus (top panels) and liver (bottom panels) were analyzed in the irradiated 2m–/– chimeric mice reconstituted with a mixture of 2m–/– bone marrow cells (0.3 x 107) and CD1d-expressing DOCK2+/– () or DOCK2–/– () bone marrow cells (1.5 x 107). FACS profiles show the expression of NK1.1 and TCR on cells negative for B220, CD11b, Gr-1, and CD8 (CD4+ NKT and DN NKT). Values (inset upper quadrant) indicate the percentage of each subset of the cells. B, MHC class I-negative NKT cells in the thymus (top panels) and liver (bottom panels) were analyzed in the irradiated 2m–/– chimeric mice reconstituted with a mixture of DOCK2+/– 2m–/– () or DOCK2–/– 2m–/– () bone marrow cells (0.3 x 107) and B6 bone marrow cells (1.5 x 107). FACS profiles show the expression of NK1.1 and TCR on cells negative for B220, CD11b, Gr-1, and CD8 (CD4+ NKT and DN NKT). Values (inset upper quadrant) indicate the percentage of each subset of cells. C, MHC class I-negative V14 NKT cells in the thymus (top panels) and liver (bottom panels) were analyzed with CD1d/-GalCer tetramers in the irradiated 2m–/– chimeric mice reconstituted with a mixture of DOCK2+/+ 2m–/– () or DOCK2–/– 2m–/– () bone marrow cells (0.3 x 107) and B6 bone marrow cells (1.5 x 107). FACS profiles were obtained for B220-, CD11b-, Gr-1-, and CD8-negative cells. Values (inset upper quadrant) indicate the percentage of each subset of cells.

	Discussion

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DOCK2 is expressed not only in T cell precursors, but also in DP thymocytes responsible for Ag presentation by CD1d molecules. In this respect, it is formally possible that the Ag-presenting capacity of DP thymocytes might be impaired in DOCK2^–/– mice. However, we found that DP thymocytes from DOCK2^+/– and DOCK2^–/– mice comparably stimulated V14 NKT cell hybridoma DN32.D3 cells, which indicates that lipid Ag presentation by DP thymocytes is intact in DOCK2^–/– mice. In addition, studies using bone marrow chimeras clearly indicated that development of V14 NKT cells requires DOCK2 expression in T cell precursors, but not in APCs. Therefore, we conclude that DOCK2 is required for early development of V14 NKT cells in a cell-autonomous manner.

Several molecules have been reported to play an important role in early development of V14 NKT cells (40). These include Fyn, SAP, and IKK2 (22, 23, 24, 25, 26, 27, 28). Because Fyn, SAP, and the membrane receptor SLAM make up a trimolecular complex and regulate TCR-induced NF-B activation (41, 42, 43, 44), one may argue that DOCK2 might function in this signaling pathway. However, this possibility seems unlikely because, unlike the case in SAP- or Fyn-deficient T cells (44), in DOCK2^–/– T cells Ag-induced PKC- translocation to the APC interface and TCR-mediated NF-B activation are intact (31). Therefore, it is suggested that DOCK2 regulates early development of V14 NKT cells, independently of the signaling pathway involving Fyn, SAP, and NF-B activation.

How DOCK2 is involved in early development of V14 NKT cells remains to be determined. However, DOCK2 regulates T cell responsiveness through immunological synapse formation in conventional T cells (31). Although the development of conventional T cells is apparently normal in DOCK2^–/– mice, the effect of DOCK2 deficiency on positive selection becomes visible in TCR transgenic mice where selecting self-peptides would be limited (31). This situation would be similar to that of V14 NKT cells because V14 NKT cells express highly restricted TCRs and endogenous lipid ligands mediating positive selection of V14 NKT cells are also extremely limited (45). In addition, positive selection of V14 NKT cells is considered to require higher TCR avidity than that of conventional T cells (12). Therefore, it seems likely that DOCK2 deficiency impairs early development of V14 NKT cells by affecting the strength of TCR signaling and altering the threshold for positive selection. In contrast to the case of V14 NKT cells, CD1d-independent NKT cells express diverse TCRs (12, 34). This finding may explain why DOCK2 deficiency preferentially affects the development of V14 NKT cells.

In conclusion, we have shown that DOCK2 expression is required in T cell precursors for early development of V14 NKT cells in the thymus. Our findings thus reveal a novel function of DOCK2 in the immune system and suggest that avidity-based selection also governs development of this unique subset of lymphocytes.

	Acknowledgments

 
We thank Dr. Mitchell Kronenberg for CD1d tetramer construct, Dr. Albert Bendelac for DN32.D3, and Dr. Laurie M. Erickson for comments on this manuscript.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan, the Japan Science and Technology Agency, and The Sumitomo Foundation.

² Address correspondence and reprint requests to Dr. Yoshinori Fukui, Division of Immunogenetics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan. E-mail address: fukui{at}bioreg.kyushu-u.ac.jp

³ Abbreviations used in this paper: DP, CD4⁺CD8⁺ double positive; -GalCer, -galactosylceramide; IKK2, IB kinase 2; ₂m, ₂-microglobulin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DN, CD4^–CD8^– double negative; HSA, heat-stable Ag; SAP, SLAM-associated protein.

Received for publication May 10, 2005. Accepted for publication February 2, 2006.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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