Innate and adaptive Treg in Immune tolerization of RA

描述（由申请人提供）：已经提出两类Treg可以通过表型和功能特征来识别。“先天”Treg和“适应性”Treg会合作限制潜在的有害炎症过程。这种调节功能在自身免疫中可能受损。它的修复可以提供新的治疗方法。该项目旨在揭示Treg在类风湿关节炎（RA）中诱导对抗原肽（dnaJP1）耐受性的作用。已经在治疗开始时和每月间隔收集了105个来自接受dnaJP1或安慰剂治疗的RA患者的1期（已完成）和II期（正在进行）临床试验的样本。我们将测试粘膜对肽的耐受性是否与具有调节表型的细胞的出现有关。我们还将确定Treg中是否有一部分是dnajp1特异性的，以及这些细胞的数量和功能特征是否会因免疫治疗而改变。具体目的：具体目的1：从类风湿关节炎耐受性试验中获得的一系列样本中表征“先天”和“适应性”Treg，并探索它们在耐受性过程中的功能作用。SA1a：外周血单核细胞（PBMC）、滑液单核细胞（SFMC）和从RA患者滑膜中获得的T细胞将通过FACS分析来评估T调节性T细胞特征的表型标记。特别是CD25， CD4， CTLA4和CCR4的水平将被研究。“先天”和“适应性”T细胞将根据一系列表型和功能变量进行区分，包括CD25的表达水平，以及通过real time PCR （TaqMan）对分选细胞中几种被认为参与Treg功能的分子的基因表达进行定量。这些基因包括IL-10、TGF β、IL-4、FOXP3、CTLA。获得的样品还将在体外研究中进行测试，以探索“先天”和“适应性”Treg对T细胞回忆抗原以及推定参与致病过程的抗原（gp39， dnaJP1和p205）的调节特性。SA1b：临床信息将与免疫学数据进行比较。特异性目的2：研究某些Treg是否对dnajp1具有特异性，以及dnajp1特异性Treg的功能和表型特征是否与耐受性过程有关。分选细胞的功能特性将由TaqMan进行评估。

DESCRIPTION (provided by applicant): It has been suggested that two categories of Treg can be identified by phenotypical and functional characteristics. "Innate" and "adaptive" Treg would cooperate in limiting potentially noxious inflammatory processes. This regulatory function may be impaired in autoimmunity. Its restoration could provide novel therapeutic approaches. This project aims to unravel the role, which may pertain to Treg function in induction of tolerance to an antigenic peptide (dnaJP1) in rheumatoid arthritis (RA). 105 samples from RA patients treated in the context of a Phase 1 (completed) and a Phase II (ongoing) clinical trial with dnaJP1 or placebo have already been collected at the beginning of treatment and at monthly intervals. We will test whether mucosal tolerization to a peptide is associated with emergence of cells with a regulatory phenotype. We will also determine if a proportion of Treg are dnaJP1-specific, and if numbers and functional characteristics of these cells change as a consequence of immunotherapy. The specific aims are: Specific Aim 1: To characterize "innate" and "adaptive" Treg in serial samples obtained from a tolerization trial in rheumatoid arthritis and to explore their functional role in the tolerization process. SA1a: Peripheral blood mononuclear cells (PBMC), synovial fluid mononuclear cells (SFMC) and T cells obtained from synovial membranes of RA patients will be evaluated by FACS analysis for phenotypical markers characteristic of T regulatory T cells. In particular, levels of CD25, CD4, CTLA4 and CCR4 will be studied. "Innate" and "adaptive" T cells will be differentiated based on a set of phenotypical and functional variables, including levels of CD25 expression and quantification on sorted cells by real time PCR (TaqMan) gene expression of several molecules putatively involved in Treg function. These genes will include IL-10, TGF beta, IL-4, FOXP3, CTLA. Samples obtained will be also tested in in vitro studies to explore regulatory properties of "innate" and "adaptive" Treg on T cell responses to recall antigens as well as to antigens (gp39, dnaJP1 and p205) putatively involved in the pathogenic process. SA1b: Clinical information will be compared with immunological data Specific Aim 2: To investigate whether some Treg have specificity for dnaJpl and whether functional and phenotypical characteristics of dnaJP1-specific Treg are associated with the course of the tolerization process. Functional characteristics of sorted cells will be evaluated by TaqMan.