Regulation of Cutaneous Inflammation by local gd T Cells

描述（由申请人提供）：在鸡、小鼠和人类等多种物种中，大量 T 细胞与体表上皮细胞（如肠道、泌尿生殖道和皮肤）组成性相关。这种上皮内淋巴细胞 (IEL) 通常富含 T 细胞受体 (TCR) γ/δ 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（也称为树突状表皮 T 细胞 (DETC)）表达极其同质的 Vgamma5/Vdelta1 TCR，因其缺乏连接（CDR3 区域）多样性而引人注目。联合使用不同品系的 TCRd-/-（敲除）小鼠并通过过继转移选择性重建此类小鼠表明，Vgamma5 DETC（而非其他 γ/δ 细胞）是多种生理相关皮肤炎症反应的有效下调剂，包括局部、遗传依赖性、TCRα/β T 细胞依赖性环境依赖性慢性皮炎，其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个强大的实验模型来定义局部 T 细胞调节局部组织内系统免疫反应影响的机制。该项目的具体目标是： 1. 使用以下方法详细表征通常被 DETC 下调的皮肤炎症： a) 免疫组织学、离体流式细胞术和微阵列分析； b) 易感 delta-/- 小鼠与同样通过细胞因子拮抗剂或阻断抗体治疗或第二次“敲除”基因突变而缺乏选定促炎细胞和/或分子的 delta-/- 小鼠的表型比较 2. 为了通过基因表达系列分析 (SAGE) 来表征体外“静息”DETC 和“激活”DETC 表达的基因，通过 DETC 在各种体内和体外的定量 RT-PCR 验证此类分析。体外激活状态，并将此类基因表达模式与肠道相关的 γ/δ 和 α/β IEL 以及系统性 CD8 α/β“初始”和“记忆”T 细胞的基因表达模式进行比较。 3.通过用缺乏候选抗炎分子的胎儿胸腺DETC前体重建δ-/-受体来研究选定的候选DETC抗炎细胞因子/效应分子。 4.利用全基因组微卫星作图来识别控制对某些（但不是其他）d-/-小鼠中发生的自发性皮炎的易感性/抵抗力的遗传区间，随后进行额外的研究（同源系的开发、该区间内包含的基因的差异表达分析），旨在最终鉴定调节皮肤炎症的基因。

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.