Regulation of Cutaneous Inflammation by local gd T Cells

描述(申请人提供)：在各种物种中，如鸡、小鼠和人类，大量的T细胞与体表上皮细胞构成相关，如肠道、泌尿生殖道和皮肤。这类上皮内淋巴细胞(IEL)通常富含T细胞受体(TCR)γ/Delta细胞，通常具有有限的组织相关抗原受体多样性；因此，小鼠皮肤中的大多数IEL，也被称为树突状表皮T细胞(DETC)，表达显著同质的VGamma5/Vdelta1 TCRs，以其缺乏连接(CDR3区)多样性而闻名。联合使用不同品系的TCRd-/-(基因敲除)小鼠并通过过继转移选择性地重建此类小鼠表明，VGamma5 DETC，而不是其他伽马/德尔塔细胞，对几种生理相关的皮肤炎反应具有强大的下调作用，包括一种局部的、遗传依赖的、TCRα/βT细胞依赖的环境依赖型慢性皮炎，它具有人类特应性皮炎的几个特征。该项目的长期目标是利用这一强大的实验模型来确定局部T细胞调节局部组织内系统免疫反应的机制。这个项目的具体目标是：1.利用免疫组织学、体外流式细胞术和基因芯片分析，详细描述DETC通常下调的皮肤炎症的特征；B)通过细胞因子拮抗剂或阻断抗体的治疗，或第二个“敲除”基因突变2.为了通过基因表达序列分析(SAGE)来表征在体外“静止的”DETC和DETC“激活”的基因表达，通过DETC在体内和体外的不同激活状态的定量RT-PCR来验证这样的分析，并将这些基因表达模式与肠道相关的Gamma/Delta和α/βIELs的基因表达模式进行比较，将易感的Delta-/-小鼠与同样缺乏选定的促炎细胞和/或分子的Delta-/-小鼠进行表型比较。以及系统性CD8α/β“幼稚”和“记忆”T细胞。3.通过将缺乏候选抗炎分子的胎儿胸腺DETC前体重组为Delta型/-受体，研究选定的候选DETC抗炎细胞因子/效应分子。4.利用全基因组微卫星作图来确定控制某些d-/-小鼠(而不是其他d-/-小鼠)对自发性皮炎的易感性/抵抗力的遗传区间(S)，随后进行其他研究(同源基因系的建立，包含在该区间内的基因的差异表达分析)，以确定调控皮肤炎症的基因(S)。

DESCRIPTION (provided by applicant): In species as diverse as chickens, mice, and humans, substantial numbers of T cells are constitutively associated with body surface epithelia, such as the gut, genitourinary tract, and the skin. Such intraepithelial lymphocytes (IELs) are commonly enriched in T cell receptor (TCR)gamma/delta+ cells, frequently with limited tissue associated antigen receptor diversity; thus, most IELs in mouse skin, also known as dendritic epidermal T cells (DETC), express strikingly homogeneous Vgamma5/Vdelta1 TCRs notable for their lack of junctional (CDR3 region) diversity. Combined use of different strains of TCRd-/- (knockout) mice and selective reconstitution of such mice via adoptive transfer has shown that Vgamma5+ DETC, but not other gamma/delta cells, are potent down-regulators of several physiologically relevant, cutaneous inflammatory responses, including a localized, genetically-dependent, TCRalpha/beta+ T cell-dependent environmentally-dependent, chronic dermatitis that shares several features of human atopic dermatitis. The long-term goal of this project is to utilize this powerful experimental model to define the mechanisms by which local T cells regulate the effects of systemic immune responses within local tissues. The specific aims of this project are: 1. To characterize in detail the cutaneous inflammation normally down-regulated by DETC) using: a) immunohistology, ex vivo flow cytometry, and microarray analysis; b) phenotypic comparisons of susceptible delta-/- mice with delta-/- mice also deficient in selected pro-inflammatory cells and/or molecules by virtue either of treatment with cytokine antagonists or blocking antibodies, or of a second "knockout" genetic mutation 2. To characterize the genes expressed by "resting" DETC and DETC "activated" in vitro by serial analysis of gene expression (SAGE), validate such analyses by quantitative RT-PCR of DETC in various in vivo and in vitro activation states, and compare such gene expression patterns with those both of gut-associated gamma/delta+ and alpha/beta+ IELs, and of systemic CD8+ alpha/beta+ "naive" and "memory" T cells. 3. To investigate selected candidate DETC anti-inflammatory cytokines/effector molecules by reconstituting delta-/- recipients with fetal thymic DETC precursors rendered deficient in candidate anti-inflammatory molecules. 4. To utilize genome-wide microsatellite mapping to identify the genetic interval(s) controlling susceptibility/resistance to the spontaneous dermatitis that develops in some, but not other d-/- mice, followed by additional studies (development of congenic lines, differential expression analyses of genes contained within this interval) directed at definitive identification of the gene(s) that regulate cutaneous inflammation.