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.