RNA expression of reactivated Antigen-specific memory CD4 T cells previously exposed to tolerogenic or immunogenic stimulation. RNA expression of reactivated Antigen-specific memory CD4 T cells previously exposed to tolerogenic or immunogenic stimulation

Activated and memory CD4 T cells play important roles in many autoimmune diseases often acting as upstream co-ordinators of inflammation and tissue destruction. A major therapeutic strategy is to turn off or tolerize these CD4 T cells thereby providing a cure. While much is understood about inducing tolerance in naïve CD4 T cells, little is known about whether or how to induce tolerance in previously activated or memory CD4 T cells. Here, we use RNA-sequencing to investigate the consequences of activating mouse CD4 memory T cells with antigen delivered in the absence of adjuvant, a classic tolerance-inducing strategy for naïve T cells. To examine the long term consequences of exposing memory CD4 T cells to tolerizing signals, we first reactivated them with antigen delivered with (control) or without (experimental) adjuvant, rested the cells and then reactivated them with antigen and adjuvant 5 days prior to isolating RNA for gene expression analysis. Overall design: We used a triple transgenic reporter mouse, TRACE, to identify antigen specific CD4 T cells. In TRACE mice, T cell activation drives expression of rtTA via the interleukin 2 promoter. Reporter mice are supplied with a diet containing doxycycline for a total of 7 days starting 2 days prior to immunisation. Binding of rtTA to doxycycline activates the tet-ON promoter that drives expression of Cre recombinase which removes a stop codon blocking EYFP expression from the Rosa locus. Thus, CD4 T cells activated in the presence of doxycycline become permanently EYFP+. TRACE mice were immunized subcutaneously in the scuff with OVA protein, conjugated to 200nm polyethylene beads delivered with anti-CD40 and polyIC. EYFP+ CD4 T cells were isolated from TRACE mice 8 days post-immunisation using a CD4 T cell enrichment kit (EasySep, StemCell Technologies) before further purification via FACS-sorting. EYFP+ CD4 T cells were transferred intravenously to sex-matched, naïve C57BL/6 recipients and administered with OVA peptide alone, or OVA peptide with LPS 14 days later. A further 30 days later, all mice were immunized with OVA protein with alum intraperitoneally. EYFP+ CD4 T cells from lymphoid organs were FACS-sorted 5 days later. Each sorted sample was then processed to extract total RNA using the RNeasy Micro kit (Qiagen).

活化及记忆性CD4 T细胞在诸多自身免疫性疾病中发挥关键作用，常作为炎症与组织破坏的上游调控枢纽。当前主流治疗策略之一便是抑制或诱导这些CD4 T细胞产生免疫耐受，以此实现疾病治愈。尽管学界对诱导初始CD4 T细胞(naïve CD4 T cells)免疫耐受的机制已有较多认知，但对于如何在已活化或记忆性CD4 T细胞中诱导免疫耐受，目前仍知之甚少。本研究利用RNA测序(RNA-sequencing)技术，探究在无佐剂存在的情况下通过抗原刺激小鼠CD4记忆性T细胞所产生的生物学效应——这是诱导初始T细胞免疫耐受的经典策略。为探究记忆性CD4 T细胞接受耐受诱导信号后的长期效应，研究团队首先分别使用携带佐剂（对照组）与不携带佐剂（实验组）的抗原对细胞进行再活化，待细胞静息后，于分离RNA进行基因表达分析前5天，再次使用抗原与佐剂对细胞进行活化。

实验整体设计：
本研究使用三重转基因报告小鼠TRACE来鉴定抗原特异性CD4 T细胞。在TRACE小鼠中，T细胞活化会通过白细胞介素2(interleukin 2, IL-2)启动子驱动rtTA的表达。于免疫接种前2天开始，向报告小鼠投喂含有多西环素(doxycycline)的饲料，持续共计7天。rtTA与多西环素结合后，会激活tet-ON启动子，进而驱动Cre重组酶(Cre recombinase)的表达，后者可切除Rosa基因座上阻断EYFP表达的终止密码子。因此，在多西环素存在下活化的CD4 T细胞将永久表达EYFP。
将TRACE小鼠于足垫皮下免疫结合了200nm聚乙烯微球的卵清蛋白(OVA)蛋白，免疫佐剂为抗CD40抗体与polyIC。免疫接种后8天，使用CD4 T细胞富集试剂盒（EasySep, StemCell Technologies）从TRACE小鼠体内分离EYFP阳性CD4 T细胞，随后通过荧光激活细胞分选(FACS-sorting)进行进一步纯化。
将EYFP阳性CD4 T细胞经静脉注射移植至性别匹配的初始C57BL/6受体小鼠体内，并于移植后14天分别单独给予OVA肽段，或联合脂多糖(LPS)给予OVA肽段。再经过30天后，对所有受体小鼠腹腔注射含有明矾(alum)的OVA蛋白进行免疫接种。免疫接种后5天，从淋巴器官中分离EYFP阳性CD4 T细胞并进行FACS分选。每份分选得到的样本均使用RNeasy Micro试剂盒（Qiagen）提取总RNA。