Transcriptional program of ex vivo expanded T cells

描述(申请人提供)：体外细胞免疫疗法 扩增的T细胞(经过或不经过基因改造)是很有前途的工具 用于治疗其他无法治愈的癌症和病毒感染， 包括医源性病毒(如Epstein-Barr病毒或巨细胞病毒 感染)。在所有情况下，免疫细胞都是从患者或 同种异体供体，必要时转基因，体外扩增 在大量细胞(10e9-10e11)被注入患者之前。 因此，培养参数的优化与细胞增殖和 功能是免疫治疗成功的关键。目前的大部分 方案在5%二氧化碳和环境空气(~20%二氧化碳张力，P02)中培养T细胞。 然而，在体内，造血和淋巴器官组织中的平均PO2接近40毫米汞(或气体大气中O2的5%)。我们已经证明了 PO2影响T细胞增殖(4.8至13倍)、受体表达和 密度、细胞凋亡率和代谢率。同样，我们发现自体 血浆对总培养物有深远的影响(是非血浆培养的43到74倍) 细胞扩张。然而，人们对潜在的分子却知之甚少。 这些深远的生物学影响，特别是关于大规模基因 激活和扩张过程中的表达模式(转录组) T细胞的体外实验。因此，我们建议研究大规模转录组。 使用DNA阵列体外扩增人类原代T细胞，从而使多达8500个 将检查基因的差异表达。选择的T细胞和 未经选择的外周血单核细胞(PBMC)将用于检查： (1)培养条件下T细胞的时间差异转录程序 5%或20%PO2，有或没有自体血浆；(2)“亲属” 在5%和20%pO2条件下培养的细胞的差异转录程序， 有无自体血浆；(3)差异转录 在有无抗氧化剂的情况下培养T细胞的程序； 以及(4)所选T细胞的差异转录程序与。 低PO2和高PO2条件下的未选择细胞。DNA阵列数据的分析将 用于测试以下假设，即这些影响是否会导致 大转录效应对：(I)细胞周期机制；(Ii) 细胞凋亡机制；(Iii)信号转导/转录因子网络； (4)糖酵解/三氯乙酸循环；(5)应激和氧化损伤反应。在……里面 为了实现这些目标，我们将寻求先进的聚类方法 以及新的生物信息学方法，可能会让我们检查收集到的 DNA阵列表达数据与现有途径和调控相一致 网络模型。

DESCRIPTION (provided by applicant): Cellular immunotherapies with ex vivo expanded T cells (with or without genetic modifications) are promising tools for the treatment of otherwise untreatable cancers and viral infections, including iatrogenic ones (such as Epstein-Barr virus or Cytomegalovirus infections). In all cases, immune cells are isolated from the patient or an allogeneic donor, genetically modified if necessary, and expanded ex vivo before a large number of cells (10e9 -10e11) are infused into the patient. Thus, optimization of culture parameters with respect to cell proliferation and function is critical for successful immunotherapy. The majority of current protocols culture T cells in 5% C02 and ambient air (~20% 02 tension, p02). However, in vivo the mean pO2 in hematopoietic and lymphoid-organ tissues is closer to 40 mmHg (or 5% of the 02 in the gas atmosphere). We have shown that pO2 affects T-cell proliferation (by 4.8 to 13-fold), receptor expression and density, apoptosis, and metabolic rates. Similarly, we found that autologous plasma has a profound effect (43 to 74-fold over non-plasma cultures) on total cellular expansion. However, little is known about the underlying molecular biology of these profound effects, and specifically, about the large-scale gene expression patterns (transcriptome) during the activation and expansion process of T cells ex vivo. Thus, we propose to examine the large-scale transcriptome of ex vivo expanded human primary T cells using DNA arrays, whereby up to 8,500 genes will be examined for differential expression. Both T-cell selected and unselected peripheral blood mononuclear cells (PBMC) will be used to examine: (1) the temporal differential transcriptional program of T cells cultured under 5% or 20% pO2, and with or without autologous plasma; (2) the "relative" differential transcriptional program of cells cultured under 5% versus 20% pO2, and with versus without autologous plasma; (3) the differential transcriptional program of T cells cultured in the presence versus absence of an antioxidant; and (4) the differential transcriptional program of selected T cells vs. unselected cells under both low and high pO2. Analysis of DNA-array data will be used to test the following hypotheses as to whether these effects result from large transcriptional effects on: (i) The cell-cycle machinery; (ii) the apoptosis machinery; (iii) signal transduction/transcription factor networks; (iv) glycolysis/TCA cycle; and (v) stress and oxidative damage responses. In order to achieve these objectives, we will purse advanced clustering methods and new bioinformatic approaches that may allow us to examine if the collected DNA array expression data are consistent with existing pathway and regulatory networks models.