Innate and adaptive Treg in Immune tolerization of RA

描述（由申请人提供）：已表明可通过表型和功能特征鉴定两类Treg。“先天性”和“适应性”Treg将合作限制潜在有害的炎症过程。这种调节功能可能在自身免疫中受损。它的恢复可以提供新的治疗方法。该项目旨在阐明Treg在类风湿关节炎（RA）中诱导对抗原肽（dnaJP 1）耐受的作用。在治疗开始时和每月间隔时，已经收集了来自在使用dnaJP 1或安慰剂的1期（已完成）和II期（正在进行）临床试验的背景下治疗的RA患者的105个样本。我们将测试对肽的粘膜耐受是否与具有调节表型的细胞的出现相关。我们还将确定Treg的比例是否是dnaJP 1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。具体目标是：具体目标1：在类风湿性关节炎耐受化试验中获得的系列样本中表征“先天性”和“适应性”Treg，并探索其在耐受化过程中的功能作用。SA1a：将通过FACS分析评价从RA患者滑膜获得的外周血单核细胞（PBMC）、滑液单核细胞（SFMC）和T细胞的T调节性T细胞的表型标志物特征。具体而言，将研究CD 25、CD 4、CTLA 4和CCR 4的水平。“先天”和“适应性”T细胞将基于一组表型和功能变量来区分，包括CD 25表达水平和通过真实的PCR（TaqMan）对分选的细胞进行的几种参与Treg功能的分子的基因表达的定量。这些基因将包括IL-10、TGF β、IL-4、FOXP 3、CTLA。还将在体外研究中测试所获得的样品，以探索“先天性”和“适应性”Treg对T细胞对回忆抗原以及对致病过程中涉及的抗原（gp 39、dnaJP 1和p205）应答的调节特性。SA1b：具体目的2：研究一些Treg是否对dnaJPl具有特异性以及dnaJPl特异性Treg的功能和表型特征是否与耐受化过程的过程相关。将通过TaqMan评价分选细胞的功能特征。

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.