Objective. T helper cells develop into discrete Th1, Th2 or Th17 lineages that selectively express IFN-gamma, IL-4/IL-5/IL-13, or IL-17, respectively and actively silence signature cytokines expressed by opposing lineages. Our objective was to compare Th1, Th2 and Th17 polarization in cell culture models using JIA patient samples. Methods. Peripheral blood mononuclear cells were isolated from JIA or healthy prepubescent children. T cell naive and memory phenotypes were assessed by flow cytometry. T cell proliferation was measured using a fluorescence-based assay. Th cell cultures were generated in vitro and IFN-gamma, IL-17, and TNF-alpha measured by ELISA and flow cytometry. Results. JIA Th1 cells produced increased IFN-gamma and inappropriately produced IL-17. JIA Th17 cells produced increased IL-17. JIA Th1 cell cultures develop dual producers of IFN-gamma and IL-17, which are Th1.17 cells. JIA Th1 cultures expressed elevated levels of both T-bet and ROR-gamma-T. RNA sequencing confirmed activation of immune responses and inappropriate activation of IL-17 signaling pathways in Th1 cultures. A subset of JIA patient samples was disproportionally responsible for the enhanced IFN-gamma and IL-17 phenotype and Th1.17 phenotype. Conclusions. This study reveals that JIA patient uncommitted T cell precursors, but not healthy children, inappropriately develop into inflammatory effector Th1.17 and Th17 cells under Th1 polarizing conditions.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein that disrupt its folding pathway. The most common mutation causing CF is a deletion of phenylalanine at position 508 (ΔF508). CFTR contains five domains that each form cotranslational structures that interact with other domains as they are produced and folded. CFTR is comprised of two transmembrane spanning domains (TMDs), two nucleotide binding domains (NBDs) and a unique regulatory region (R). The first domain translated, TMD1, forms interdomain interactions with the other domains in CFTR. In TMD1, long intracellular loops extend into the cytoplasm and interact with both NBDs via coupling helices and with TMD2 via transmembrane spans (TMs). We examined mutations in TMD1 to determine the impact on individual domain and multidomain constructs. We found that mutations in a TM span or in the cytosolic ICLs interfere with specific steps in the hierarchical folding of CFTR. TM1 CF-causing mutants, G85E and G91R, directly affect TMD1, whereas most ICL1 and ICL2 mutant effects become apparent in the presence of TMD2. A single mutant in ICL2 worsened CFTR trafficking in the presence of NBD2, supporting its role in the ICL2-NBD2 interface. Mutation of hydrophobic residues in ICL coupling helices tended to increased levels of pre-TMD2 biogenic intermediates but caused ER accumulation in the presence of TMD2. This suggests a tradeoff between transient stability during translation and final structure. NBD2 increased the efficiency of mutant trafficking from the ER, consistent with stabilization of the full-length constructs. While the G85E and G91R mutants in TM1 have immediately detectable effects, most of the studied mutant effects and the ΔF508 mutant are apparent after production of TMD2, supporting this intermediate as a major point of recognition by protein quality control.