Wild-Type vs BTP2 Treated Th1 and Pathogenic Th17 cells_ 96h

Transicriptome comparison between murine WT and BTP2 Treated Th1 and pTH17cells at 96 hours following differentiation Overall design: Naive CD4+ T-cells were isolated and purified from wild-type mice. Naïve CD4+ T cells were enriched by magnetic sorting from single-cell suspensions generated by mechanical disruption of spleens and lymph nodes of adult mice using MagniSort naïve CD4+ T cell enrichment kit (catalog # 8804-6824-74,ThermoFisher Scientific). For effector T cell differentiation, cells were stimulated with anti-CD3 antibody (1452C11, Bio X Cell) and anti-CD28 antibody (Clone 37.51, Bio X Cell) for 48 hours on a plate coated with rabbit anti-hamster antibody (MP Biomedicals). CD4 + CD25 - T cells were cultured with anti-IL-4 antibody (Peprotech) and 10 µg/ml anti-IFN-? antibody, 30 ng/ml IL-6 (Peprotech), 10 ng/ml IL-23 (R&D Systems) and 10 ng/ml IL-1ß (R&D Systems) for pathogenic Th17 differentiation and cultured in T cell medium. For Th1 cell differentiation, CD4 + CD25 - T cells were cultured with anti-IL-4 antibody (Peprotech) and IL-12 for Th1 differentiation. In the BTP2 treatment groups, BTP2 was applied transiently between 24-48 hrs during differentiation. Cells were collected at 96h and prepared for bulk RNA sequencing analysis.