Fig. 1

T helper cells upregulate the IRE1a-XBP1 pathway during activation. a Schematic representation of the hypothesis. In this study, we are asking what role does the IRE1a-XBP1 pathway play during T helper cell activation. T helper cell activation is a dramatic transformation from a quiescent cell state to a rapidly proliferative and highly protein productive/secretive cellular state. b Overview of the experiment. Splenic naïve T cells were purified by negative selection and activated in anti-CD3e/C28 antibody-coated plates under Th2 differentiation conditions (i.e., in the presence of anti-IFNγ neutralizing antibody, IL2, and IL4) for 72 h, rested for 42 h, and restimulated on anti-CD3e/CD28 antibody-coated plate. Restimulated Th2 cells were used in RNA sequencing, ChIPmentation (ChIP sequencing), Western blot, qPCR, and flow cytometry. To perturb IRE1a-XBP1 pathway, we used 15-μM 4μ8c that specifically blocks the pathway by inhibiting IRE1a endonuclease activity. The drug was added to the culture media at the beginning of the culture and during passage from the activation plate to the resting plate. c Naïve T helper cells and in vitro differentiated Th2 lymphocytes were analyzed for IRE1a mRNA expression by qRT-PCR (left panel), protein expression by Western blot (middle panel), and phosphorylated IRE1a (P-IRE1a) by Western blot (right panel). The density of Western blot bands from five independent experiments of IRE1a and three independent experiments of phospho-IRE1a were measured and displayed on top of each Western blot panel. d Naïve T cells were cultured under Th2 differentiation conditions in the presence or absence of IRE1a inhibitor (4μ8c). In vitro reactivated Th2 lymphocytes were analyzed by RT-PCR using a pair of primers that discriminate the cDNA derived from spliced and unspliced form of XBP1 mRNA. Tunicamycin-treated Th2 cells were used as a positive control. e Naïve T helper cells (N) and in vitro differentiated and restimulated Th2 cells (differentiated in the presence or absence of 4μ8c) were stained with fluorescent dye-conjugated anti-XBP1s-specific antibody and analyzed by flow cytometry. Gating: singlets > live cells > XBP1s. One representative FACS profile is displayed (left panel), and the graph containing all results (n = 5) is shown in the “right panel”. Tunicamycin-treated Th2 cells were used as a positive control