SELECTIVE GENE TRGETING OF TCR Vδ GENES AND ANALYSIS OF THE KNOCKOUT MICE

TCR Vδ基因的选择性基因靶向及敲除小鼠分析

• 批准号: 12670305
• 负责人: KISHIHARA Kenji
• 金额: $ 2.05万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670305/
• 关键词: γδ T cell; Gene targeting; Dendritic epidermal T cell; T cell receptor; T cell development; Resident pulmonary lymphocyte; T cell repertoire; Fetal thymocyte; γδ; T細胞; レセプター; 分化; 皮膚; ノックアウトマウス

In this Research project, we produced Vδ1 gene-deficient mice by selective gene targeting with Cre-1oxP system, followed by analyzing development of γδ T cells including fetal thymocytes, dendritic epidermal T cells (DETCs) and resident pulmonary T lymphocyrtes (RPLs) in the knockout mice.Murine DETCs localized in epidermis are derived from fetal thymocytes and overwhelmingly express Vγ5/Vδ1-TCR without junctional diversity. In Vδ1- deficient mice, the development of Vγ5^+ fetal thymocytes as precursors of DETC was markedly blocked. On the other hand, γδTCR^+ DETCs with a typical dendritic morphology were observed in Vδ1-deficient mice. Moreover, DETCs from Vδ1 deficient mice were predominantly Vγ5^+ but Vγ5^- DETCs were considerably detected in them. The Vγ5^+ DETCs showed mature phenotypes and age-associated expansion, while the Vγ5^- DETCs did immature phenotypes and no expansion in epidermis. These results suggest that optimal DETC development does not require a particular Vγ/Vδ usage but a limited diversity of of γδ TCRs is required for maturation and expansion of DETCs within the epidermal microenvironment.Murine RPLs contain γδ T cell subpopulation as a minority (ca. 5 %). The γδ RPLs are also derived from fetal thymocytes and predominantly express Vγ6/Vδ1-TCR without junctional diversity. However, a novel y6RPL subset expressing Vγ4/Vδ5 generate after birth and then they expand as a major population. In Vδ1-deficient mice, Vγ6^+ RPLs were virtually undetectable and the markedly decreased expression of Vγ1, Vγ4, Vδ4 and Vδ5 gene sements in RPL was also observed in comparison with RPLs from wild type mice. Therefore, the blocked development of fetus-type γδRPLs (Vγ6/Vδ1^+) may influence the development and/or migration of adult-type γδRPL (Vγ4/Vδ5^+).

本研究利用Cre-1oxP系统进行基因打靶，获得了Vδ1基因缺陷小鼠，并分析了基因敲除小鼠体内γδT细胞的发育情况，包括胎儿胸腺细胞、树突状表皮T细胞(DETCs)和常驻肺T淋巴细胞(RPLs)。定位于表皮的DETCs来源于胎儿胸腺细胞，绝大多数表达Vγ5/Vδ1-TcR，无连接多样性。在Vδ1缺陷小鼠中，Vγ5^+胎儿胸腺细胞作为DETC前体细胞的发育明显受阻。另一方面，在Vγδ1缺陷小鼠中观察到δTcr^+DETCs具有典型的树突状形态。此外，Vδ1基因缺陷小鼠的DETCs以Vγ5^+为主，但也可检测到Vγ5^-DETCs。Vγ5^+DETCs表型成熟且随年龄增长而扩张，而Vγ5^-DETCs表型不成熟，表皮无扩张。这些结果表明，DETC的最佳发育不需要特定的Vγ/Vδ使用，但在表皮微环境中DETCs的成熟和扩张需要有限多样性的γδTCR。小鼠RPL含有少数γδT细胞亚群(约5%)。γδRPL也来源于胎儿胸腺细胞，主要表达Vγ6/Vδ1-TCR，无连接多样性。然而，一个新的表达Vγ4/Vδ5的y6RPL亚群是在出生后产生的，然后它们作为主要群体扩展。在Vδ1缺陷小鼠中，Vγ6^+RPL几乎检测不到，与野生型小鼠相比，RPL中Vγ1、Vγ4、Vδ4和Vδ5基因的表达也显著降低。因此，胎儿型γδRPL(Vγ6/Vδ1^+)的发育受阻可能影响成人型γδRPL(Vγ4/Vδ5^+)的发育和/或迁移。

项目成果

Hara H.Kishihara K.Matsuzaki G.Takimoto H.Tsukiyama T.Tigelaar RE.Nomoto K: "Development of dendritic epidermal T cells with a skewed diversity of γδTCRs in Vδ1-deficient mice"Journal of Immunology. 165(7). 3695-3705 (2000)

Hara H.Kishihara K.Matsuzaki G.Takimoto H.Tsukiyama T.Tigelaar RE.Nomoto K：“在 Vδ1 缺陷小鼠中开发具有倾斜多样性的 γδTCR 树突状表皮 T 细胞”免疫学杂志 165(7)。 -3705 (2000)

Hara H., Kishihara K., Matsuzaki G., Takimoto H., Tsukiyama T., Tigelaar RE., Nomoto K: "Development of dendritic epidermal T cells with a skewed diversity of γδTCRs in Vδ1-deficient mice"Journal of Immunology. 165(7). 3695-3705 (2000)

Hara H.、Kishihara K.、Matsuzaki G.、Takimoto H.、Tsukiyama T.、Tigelaar RE.、Nomoto K：“在 Vδ1 缺陷小鼠中开发具有 γδTCR 多样性的树突状表皮 T 细胞”免疫学杂志。 165（7）。3695-3705（2000）。

Hara H., Kishihara K., Matsuzaki G., Takimoto H., Tsukiyama T., Tigelaar RE., and Nomoto K.: "Development of dendritic epidermal T cells with a skewed diversity ofγδTCRs in Vδ1-deficient mice"Journal of Immunology. 165, 7. 3695-3705 (2000)

Hara H.、Kishihara K.、Matsuzaki G.、Takimoto H.、Tsukiyama T.、Tigelaar RE. 和 Nomoto K.：“在 Vδ1 缺陷小鼠中开发具有 γδTCR 多样性的树突状表皮 T 细胞”免疫学杂志165, 7. 3695-3705 (2000)

Hiromitsu Hara 等: "Development of dendritic epidermal T cells with a skewed diversity of γδTCRs in Vδ1-deficient mice."Journal of Immunology. 165. 3695-3705 (2000)

Hiromitsu Hara 等人：“在 Vδ1 缺陷小鼠中开发具有 γδTCR 多样性的树突状表皮 T 细胞。”免疫学杂志 165. 3695-3705 (2000)