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

本研究利用Cre-1oxP系统，通过选择性基因靶向培养Vδ1基因缺陷小鼠，分析了敲除小鼠中γδ T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）。小鼠表皮的detc来源于胎儿胸腺细胞，绝大多数表达v γ - 5/Vδ1-TCR，没有连接多样性。在Vδ1-缺陷小鼠中，作为DETC前体的Vγ5^+胎儿胸腺细胞的发育被明显阻断。另一方面，在v δ1缺陷小鼠中观察到典型树突状形态的γδTCR^+ detc。此外，Vδ1缺陷小鼠的detc主要为Vγ5^+，但也有相当数量的Vγ5^- detc。Vγ5^+ DETCs表现为成熟表型和年龄相关扩增，而Vγ5^- DETCs表现为不成熟表型，表皮无扩增。这些结果表明，最佳的DETC发育不需要特定的Vγ/Vδ使用，但在表皮微环境中，DETC的成熟和扩展需要有限的γδ tcr多样性。小鼠RPLs中含有γδ T细胞亚群，占少数（约5%）。γδ RPLs也来源于胎儿胸腺细胞，主要表达v - γ6/ v - δ1- tcr，没有连接多样性。然而，一个新的表达Vγ4/Vδ5的y6RPL子集在出生后产生，然后它们扩展为主要群体。在v δ1缺陷小鼠中，几乎检测不到v γ γ6^+ RPL，与野生型小鼠的RPL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年型γδRPL （Vγ4/Vδ5^+）的发育和/或迁移。

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^+).