Chemical chaperones reverse early suppression of regulatory circuits during Unfolded Protein Response in B cells from Common Variable Immunodeficiency patients

Rare B cell samples from two CVID patients (P1 and P2) and a healthy donor (C) were used in controlled experiments analyzing the early UPR with and without the presence of chemical chaperones. As ER stressors we used Thapsigargin (Tg) and Tunicamycin (Tm). As exogenous chaperones we used DMSO, PBA, and TUDCA. For quantification of specific transcripts of interest we used a high-throughput platform based on TaqMan technology (Applied Biosystems TaqMan OpenArray Real-Time PCR System). Samples containing 3 x 10e5 EBV-B cells that received treatments with ER stressors and/or chemical chaperones for 0, 1, 2, 3, or 4 hr. Purified RNA was quali- and quantified by spectrometry in a Nanodrop and by fluorimetry in a Qubit, respectively. cDNA was generated using 300 ng of total RNA, and amplified in triplicate reactions for quantification of specific transcripts using a QuantStudio 12K Flex Real-Time PCR System. TaqMan OpenArray primers were custom-made by Thermo Fisher Scientific. Genes access numbers can be found in Supplementary Table 2 of the respective publication. Target DCq values were normalized against Gapdh DCq levels. Replicates were averaged and filtered for SE < 0.5 for removal of low-abundance measurements. A ratio of Treated / Untreated was calculated, log2-transformed, and a T-test was applied. Only those ratios whose SE were < 0.5 and that were significantly different (p ≤ 0.05) from untreated controls in at least two time-points were considered relevant. Regulatory circuits were built using BioTapestry 7.1 software. Networks show only those elements assayed in this study. Inputs and outputs of indicated genes are color coded according to their upstream origin (yellow for ATF6, red for PERK, blue for IRE1a, and green for sXBP1). Orange lines indicate those elements whose expression depends on inputs from both ATF6 and PERK pathways. Linkages substantiated by cis-regulatory data are indicated by diamonds colored according to strength of the experimental evidence: blue diamonds for expression studies using gain/loss of function, pink diamonds for binding affinity assays, and orange diamonds for promoter analysis in vivo. A green bar represents post-transcriptional modification of Xbp1 mRNA. A yellow bar represents post-translational modification of ATF6 protein. # (OR) and & (AND) are Boolean rules governing input elements in specific promoters. Bold gene = active expression. Bold gene + thick line = upregulated expression compared to untreated control. Grey gene + grey line = downregulated expression compared to untreated control. For visualization of each patient’s experimental regulatory network, refer to Supplementary Movies (A-G) for average [CVID patients minus healthy control], (H-N) for healthy control, (O-U) for patient P1, and (V-Y) for patient P2.

本研究采用来自2例常见可变免疫缺陷症（Common Variable Immunodeficiency, CVID）患者（P1、P2）及1名健康供者（C）的稀有B细胞样本，开展对照实验以分析存在/不存在化学分子伴侣时的早期未折叠蛋白反应（unfolded protein response, UPR）。实验所用内质网应激诱导剂为毒胡萝卜素（Thapsigargin, Tg）与衣霉素（Tunicamycin, Tm），外源性分子伴侣则为二甲基亚砜（DMSO）、4-苯基丁酸（PBA）与牛磺熊去氧胆酸（TUDCA）。针对目标特异性转录本的定量分析，采用基于TaqMan技术的高通量平台——应用生物系统公司（Applied Biosystems）TaqMan OpenArray实时荧光定量PCR系统。实验样本包含3×10^5个EB病毒转化B细胞（EBV-B cells），分别经内质网应激诱导剂和/或化学分子伴侣处理0、1、2、3或4小时。纯化后的RNA分别通过Nanodrop超微量紫外分光光度计进行质量鉴定、通过Qubit荧光定量仪进行浓度定量。以300 ng总RNA反转录合成cDNA，随后采用QuantStudio 12K Flex实时荧光定量PCR系统进行三次重复反应，以定量目标转录本。TaqMan OpenArray引物由赛默飞世尔科技（Thermo Fisher Scientific）定制合成。基因登录号可参见对应发表论文的补充表2。目标循环阈值（DCq）值以甘油醛-3-磷酸脱氢酶（GAPDH）的DCq值作为内参进行归一化处理。对重复实验结果取平均值，并过滤掉标准误（Standard Error, SE）≥0.5的低丰度检测结果。计算处理组与未处理组的比值（Treated / Untreated），并进行以2为底的对数转换，随后采用t检验进行显著性分析。仅当比值的标准误<0.5，且至少在两个时间点与未处理对照组差异具有统计学意义（p ≤ 0.05）时，该比值才被认定为具有生物学相关性。本研究采用BioTapestry 7.1软件构建基因调控回路，网络图仅包含本研究中检测的基因调控元件。指定基因的输入与输出调控关系根据其上游信号来源进行颜色编码：黄色代表ATF6（激活转录因子6）通路，红色代表PERK（蛋白激酶R样内质网激酶）通路，蓝色代表IRE1α（肌醇需求酶1α）通路，绿色代表sXBP1（剪接型X盒结合蛋白1）通路。橙色线条代表其表达同时依赖ATF6与PERK通路信号输入的基因调控元件。由顺式调控实验数据证实的调控关联以菱形标记，菱形颜色根据实验证据可靠程度区分：蓝色菱形代表基于功能获得/缺失实验的表达分析结果，粉色菱形代表结合亲和力检测结果，橙色菱形代表体内启动子分析结果。绿色条带代表Xbp1 mRNA的转录后修饰过程，黄色条带代表ATF6蛋白的翻译后修饰过程。符号#（代表逻辑或OR）与&（代表逻辑与AND）为调控特定基因启动子输入元件的布尔逻辑规则。其中，加粗字体的基因代表处于活跃表达状态的基因；加粗字体基因搭配粗线条代表相较于未处理对照组表达上调的基因；灰色字体基因搭配灰色线条代表相较于未处理对照组表达下调的基因。若需查看各患者实验调控网络的可视化结果，请参考补充视频：[A-G]为CVID患者与健康供者的平均差值数据，[H-N]为健康供者组结果，[O-U]为患者P1组结果，[V-Y]为患者P2组结果。