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

本研究采用Cre-1 oxP系统选择性基因打靶法制备Vδ1基因敲除小鼠，并对小鼠胚胎胸腺细胞、树突状表皮T细胞（Dendritic epidermis T cells，DETC）和肺T淋巴细胞（resident pulmonary T lymphocyte，RPLs）等γδ T细胞的发育进行分析。在Vδ1缺陷小鼠中，作为DETC前体的Vγ5^+胎儿胸腺细胞的发育明显受阻。另一方面，在Vδ1缺陷小鼠中观察到具有典型树突状形态的γδTCR^+ DETC。此外，来自Vδ1缺陷小鼠的DETC主要是Vγ5^+，但在它们中也检测到相当多的Vγ5^-DETC。Vγ5^+ DETC表现为成熟的表型和与年龄相关的扩增，而Vγ5^-DETC表现为不成熟的表型，在表皮中没有扩增。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的成熟和扩增需要有限的γδ TCR多样性。5%）。γδ RPL也来源于胎儿胸腺细胞，主要表达Vγ6/Vδ1-TCR，没有连接多样性。然而，表达Vγ4/Vδ5的新的γ 6 RPL亚群在出生后产生，然后它们作为主要群体扩增。在Vδ1缺陷小鼠中，几乎检测不到Vγ6^+ RPL，并且与野生型小鼠的RPL相比，还观察到RPL中Vγ1、Vγ4、Vδ4和Vδ5基因的表达显著降低。因此，胎儿型γδ RPL（Vγ6/Vδ1^+）发育受阻可能影响成人型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^+).