Regnase-1和Roquin-1双基因敲除改变休息工程T细胞的激活配置文件。(一)人类健康供者T细胞受到CAR-T细胞扩张和低温贮藏。解冻CAR-T细胞休息一夜之间在媒体补充与CD4和CD8细胞因子在流式细胞术分析。淘汰赛Regnase-1和/或Roquin-1 CD4的表达增加,激活标记,CD8 T细胞。图所示是代表三个独立体。国际安全和发展理事会(B)表达式,从CAR-T costimulatory受体细胞与细胞因子休息一夜。离开流图所示是代表三个独立体。正确的图表显示这个理事会+ T细胞比例总结在至少三个独立的捐助者。顶部和底部的数字指CD4和CD8 T细胞,分别。误差线代表SD。 One-way ANOVA was used for analysis followed by Tukey’s multiple comparisons test. (C) Expression of activation markers CD25 and CD69 on CD4 (red) or CD8 (blue) CAR-T cells and UTD controls after coculture with mesothelin-negative K562 cells overnight. Plots shown are representative of two independent experiments from two independent donors each performed in triplicate. (D) Secretion of Th1 and inflammatory cytokines (IL2, IFNg, TNFa, IL6, and GM-CSF) measured via Luminex assay. CAR-T cells and untransduced (UTD) T cell controls were cocultured with mesothelin-negative K562 cells overnight, and supernatants were collected for analysis. Data shown are pooled from two independent experiments from two independent donors each performed in triplicate. Error bars represent SD. One-way ANOVA was used for analysis followed by Tukey’s multiple comparisons test. (E) Volcano plots showing differentially expressed genes between Regnase-1, Roquin-1, and double knockout versus mock engineered T cells (pooled n = 2 CAR-T and n = 1 TCR-T for total n = 3 independent donors) after thaw and overnight rest with cytokines. Genes that are statistically significant (adjusted P ≤ 0.005) and have a Log2FC ≥ 1 are shown in red. Statistical significance was calculated using the Wald test with Benjamini–Hochberg multiple testing correction. (F) Expression levels of various inflammatory genes, including Th1 cytokines (IL2, IFNg, TNFa), activation markers (CD25, CD69, CD40LG, PD1, LAG3), and costimulatory receptors (CD28, ICOS). Values shown are log normalized transcript counts of bulk RNA sequencing data from three independent donors (n = 2 CAR-T and n = 1 TCR-T). Box plots show medians, interquartile ranges, minimum, and maximum values. Not shown = not significant, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Credit:美国国家科学院院刊》上(2023)。DOI: 10.1073 / pnas.2218632120
先前的观察性研究暗示炎症调节器Regnase-1作为一个潜在的目标,间接地克服T细胞疲劳的影响,因为它会导致hyperinflammation T cells-reviving中断时产生抗肿瘤反应。研究小组,包括主要作者大卫•麦生物工程研究生在工程和应用科学学院和共同通讯作者尼尔·谢泼德DPhil, CCI T细胞工程实验室负责人推测,针对相关但独立Roquin-1同时监管机构可能会进一步刺激反应。
