ROLE OF PEPTIDE IN ALLOREACTIVITY

肽在异反应性中的作用

• 批准号: 2179727
• 负责人: NED S BRAUNSTEIN
• 金额: $ 26.18万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179727
• 关键词: MHC class II antigen; T lymphocyte; clone cells; epitope mapping; heart cell; heart transplantation; high performance liquid chromatography; histocompatibility; isoantigen; laboratory mouse; leukocyte activation /transformation; membrane reconstitution /synthesis; peptides; tissue /cell culture; transplant rejection

A large proportion of naive T lymphocytes respond to cells expressing allogeneic MHC molecules. The magnitude of this reaction underlies the observation that, in the absence of pharmacologically induced immunosuppression, MHC disparate allografts are rapidly rejected. The molecular basis of T cell recognition of allogeneic MHC and, in particular, the role of peptide in this recognition process, is controversial. To investigate this, we have taken advantage of a cell line (T2) that fails to process antigen for presentation by either class I or class II MHC. As a result, T2 transfectants express class II MHC exclusively in the SDS-stable MHC conformer and are therefore mostly peptide-dependent. We also show that MHC molecules on T2 transfectants can be loaded with peptide, converted into the SDS-stable form, and stimulate T cells. Peptide-loaded T2 transfectants are therefore unique reagents that express monomorphic functional complexes of a single class II MHC molecule and a single peptide. In specific aim #1, we will use these cells to determine the percent of alloreactive T cells that are not peptide-specific. This will be accomplished by priming mice with allogeneic, B6, splenocytes and assaying the primed CD4+ T cells for the ability to respond to T2-I-Ab cells that express I-Ab-peptide complexes not expressed by B6 cells. This will be further investigated by cloning T cells with this reactivity pattern and assaying for their responses to T2-I-Ab transfectants loaded with other peptides. In aim #2, we will determine the frequency of alloreactive T cells specific for the most prevalent I-Ab-peptide complexes. Examples of such complexes include Ealpha-peptide-I-Ab and invariant chain peptide-I-Ab. Mice will be primed with B10.A(5R) spleen cells. B10.A(5R) cells express both of these I-A-peptide complexes. The primed T cells will be assayed using B6 spleen cells or methA-I-Ab (an invariant chain negative cell) in the presence or absence of peptide. In aim #3, we will apply these methods to an in vivo allograft system and determine the corresponding frequencies of graft-infiltrating T cells that are either not peptide- specific or specific for these common I-Ab-binding peptides. We will extend these in vivo studies to identify allograft-reactive T cells that are specific for cardiac myocytes. We will then use these T cells to screen HPLC fractions of peptides purified from the I-Ab expressed by myocardial cells. If the myocardial peptides involved in graft rejection are expressed at relatively high levels, we hope to sequence them. Lastly, in aim #4, we will determine if T cells recognize MHC-peptide differently in the context of self MHC vs. allo MHC. We will generate variant Ealpha peptides and compare the dose response curves of allo T cells specific for Ealpha-peptide-I-Ab vs. syngeneic T cells that recognize the same complex. In these same experiments, we hope to identify peptides that can inhibit T cell responses for Ealpha-peptide-I- Ab. Such peptides may be useful in altering the course of in vivo graft rejection.

大部分幼稚的T淋巴细胞对表达细胞的反应 同种异体MHC分子。 这种反应的大小是 观察到，在没有药理诱导的情况下 免疫抑制，MHC不同的同种异体移植被迅速拒绝。 这 T细胞识别同种异体MHC的分子基础，在 特别是，肽在此识别过程中的作用是 有争议的。 为了调查这一点，我们已经利用了一个单元 线（T2）未能处理两类呈现的抗原以进行抗原 我或II类MHC。 结果，T2转染物表达II类MHC 仅在SDS稳定的MHC构象异构体中，因此主要是 肽依赖性。 我们还表明T2转染物上的MHC分子 可以加载肽，转换为SDS稳定形式，然后 刺激T细胞。因此，负载肽的T2转染剂是独特的 表达单层单态功能复合物的试剂 II MHC分子和单个肽。 在特定的目标＃1中，我们将使用 这些细胞确定不是 特定于肽的。 这将通过用小鼠与 同种异体，B6，脾细胞和分析启动的CD4+ T细胞 能够响应表达I-AB肽复合物的T2-I-AB细胞 B6细胞不表达。 克隆将进一步研究 具有这种反应性模式的T细胞，并分析了它们对 带有其他肽的T2-I-AB转染物。 在AIM＃2中，我们将 确定最特定的同种异体T细胞的频率 普遍的I-AB肽复合物。 这种复合物的例子包括 Ealpha肽-I-AB和不变链肽-I-AB。 老鼠会 用B10.A（5R）脾细胞启动。 B10.A（5R）细胞表达两个 这些I-A肽复合物。 启动的T细胞将使用 B6脾细胞或Metha-i-AB（不变链负细胞） 存在或不存在肽。 在AIM＃3中，我们将应用这些方法 到体内同种异体移植系统并确定相应的 不肽 - 对于这些常见的I-AB结合肽的特定或特异性。 我们将 扩展这些体内研究，以鉴定同种异体反应性T细胞 特定于心肌细胞。 然后，我们将使用这些T单元 从I-AB纯化的肽筛选肽的筛选HPLC级分。 心肌细胞。如果涉及移植物排斥的心肌肽 我们希望以相对较高的水平表示它们。 最后，在AIM＃4中，我们将确定T细胞是否识别MHC肽 在自我MHC与Allo MHC的背景下，其方式有所不同。 我们将生成 变体Ealpha肽并比较Allo T的剂量反应曲线 针对Ealpha肽-I-I-AB与合成型T细胞的细胞 识别相同的复合物。 在这些相同的实验中，我们希望 鉴定可以抑制T细胞反应的肽 - 肽 - 肽-I- ab。 这种肽可能有助于改变体内移植物的过程 拒绝。