ANTIGEN PRESENTING CELLS OF THE SKIN

本项目将研究皮肤抗原呈递细胞， 确定CD 1a的功能，CD 1a是一种非多态性分子， Langerhans细胞（LC）。 CD 1a与I类专业的相似之处 组织相容性（MHC）分子表明，CD 1a可以结合肽 抗原的 与I类MHC分子一样，通过T细胞介导细胞毒性 受体（TCR）的抗原，但添加的抗原在这方面的作用 相互作用尚未确定。 第一个具体目标是使用两个 确定CD 1a特异性T细胞是否识别抗原的策略 与CD 1a结合或仅识别CD 1a的不同部分。 首先，氨基 由CD 1a形成的假定抗原结合沟底部的酸 将通过定点诱变来改变，以取代任何 通常结合在沟内的肽。 如果一个或两个氨基酸的变化 改变多个T细胞克隆对CD 1a的识别， 假设TCR与结合于CD 1a肽抗原相互作用， 支持，因为这些氨基酸不应直接用于 与TCR接触。 接下来，来自致敏供体的T细胞将被 筛选用于识别由表达以下蛋白的细胞呈递的特异性抗原： 转染的CD 1a，但带有经典的MHC分子。 T细胞仅反应 在同时存在CD 1a和抗原的情况下， 由CD 1a提供。 对于第二个具体目标， 将通过扩增和 对来自CD 1a特异性T细胞的TCR mRNA序列进行测序。 第三 特异性目的将测试CD 1a在细胞中呈递特异性抗原的能力。 体内通过开发表达人CD 1a的转基因小鼠， 鼠I类MHC基因调控序列。 由于转基因小鼠可以 诱导表达功能性人I类MHC分子， 预期转基因人CD 1a也将具有功能。 小鼠 将进行免疫接种，并检测使用CD 1a作为抗原的能力 呈递分子 因为非- 多态性人类分子以前没有被证明，这些 研究将提供关于TCR识别抗原的新信息， 关于表皮LC的特殊性质

This project will investigate cutaneous antigen presenting cells by determining the function of CD1a, a non-polymorphic molecule expressed by Langerhans cells (LC). Similarities between CD1a and class I major histocompatibility (MHC) molecules suggest that CD1a can bind peptide antigen. Like class I MHC molecules, mediates cytotoxicity via the T-cell receptor (TCR) for antigen, but the role of added antigen in this interaction has not been determined. The first specific aim uses two strategies to determine whether CD1a specific T-cells recognize antigen bound to CD1a or recognize only distinct portions of CD1a. First, amino acids in the floor of the putative antigen binding groove formed by CD1a will be changed by site directed mutagenesis in order to displace any peptide normally bound int he groove. If changes of one or two amino acids alter the recognition of CD1a by multiple T-cell clones, then the hypothesis that TCRs interact with peptide antigen bound to CD1a will be supported since these amino acids should not be accessible for direct contact with the TCR. Next, T-cells from sensitized donors will be screened for recognition of specific antigens presented by cells expressing transfected CD1a but lacing classical MHC molecules. T-cells reacting only in the presence of both CD1a and antigen must be recognizing the antigen presented by CD1a. For the second specific aim, an estimate of the number of antigens which could be presented by CD1a will be made by amplifying and sequencing TCR mRNA sequences from CD1a specific T-cells. The third specific aim will test the ability of CD1a to present specific antigens in vivo by developing transgenic mice that express human CD1a controlled by murine class I MHC gene regulatory sequences. Since transgenic mice can be induced to express functional human class I MHC molecules, it is anticipated that transgenic human CD1a will also be functional. The mice will be immunized and tested for the ability to use CD1a as an antigen presenting molecule. Because presentation of specific antigens by non- polymorphic human molecules has not been previously demonstrated, these studies will provide new information about TCR recognition of antigens and about special properties of epidermal LC.