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. 2021 Jul 7;12:683680. doi: 10.3389/fimmu.2021.683680

Figure 2.

Figure 2

Overexpression of BHLHE40 induces co-expression of CD49b and LAG3 in naïve CD4+ T cells. (A) Validation of a dox inducible system for transgene overexpression in CD4+ T cells. pCW57.1 construct used for expressing GFP or BHLHE40 (top). GFP expression in CD4+ T cells is induced after addition of dox. Transgene expression is maintained during activation. Dox was incubated for 4 hours and cells were immediately collected and checked for GFP expression by flow cytometry (Bottom-left). BHLHE40 overexpression confirmed by qPCR. GFP and BHLHE40 transduced CD4+ T cells were incubated with dox for 4 hours and RNA was collected. BHLHE40 RNA was measured using qPCR Taqman probes along with housekeeping genes: RPLPO, GAPDH. n = 3 (Bottom-right). (B) Timeline of dox-inducible BHLHE40 in naïve CD4+ T cells. CD4+ naïve T cells were cultured with IL-10 and then expanded on allo-feeders with IL-2 for 14 days before being characterized. (C, D) Surface phenotype of transduced cells after 2 rounds of allo-feeder expansion. Gated on live, CD3+, CD4+ cells (C) CD49b and LAG3 expression. Representative plots of CD49b and LAG3 in one donor (Left). Summarized data of CD49b and LAG3 expression on BHLHE40 overexpressing cells compared to GFP expressing cells. (Right). n = 6. (D) Co-expression of LAG3 and PD1. Representative plots of PD1 and LAG3 in one donor (Left). Summarized data of PD1 and LAG3 expression on BHLHE40 overexpressing cells compared to GFP expressing cells (Right). n = 6. (E, F) Increased Th1 phenotypic cytokines in BHLHE40 overexpressing cells. (E) Increased intracellular IFN-γ+/IL4- cells in BHLHE40 overexpressing cells. Intracellular cytokine staining of IFN-γ and IL-4 in BHLHE40 overexpressing cells after 5 hours of PMA/I and Brefeldin A n = 6. Ratio paired t-test. (F) Increased secretion of IFN-γ. Cells are stimulated for 48 hours with plate-bound αCD3 and soluble αCD28.Cytokines in the supernatant were quantified by ELISA. IFNγ/IL-4 ratios (Left). Absolute IL-10 concentrations (Right). n = 5, Ratio paired t test. PMA/I, phorbol-myristate-acetate/ionomycin, Allo-feeders = allogenic-PBMC feeders; Dox, doxycycline; dpf, days post feeder; puro, puromycin. *p value < 0.05.