Figure 3

Egr2-independent induction of PD-L1 expression in CD4⁺ T cells by ectopic expression of Klf1. (a) Venn diagram representing the genomic overlap between LAG3⁺ Tregs specific transcription factors from microarray analysis of CD4⁺ T cell subsets from ArrayExpress database (E-MEXP-1343) and PD-L1-inducible candidate genes predicted by JASPAR database. (b) Gene expression levels of Klf1 in CD4⁺ T cells. Klf1 gene expression in indicated T cell subsets relative to unstimulated naïve CD4⁺ cells using micro array data set as in (a) (left) and relative to Actb mRNA of each indicated T cell subset confirmed by qRT-PCR (right). *p < 0.001 (Bonferroni’s multiple comparison test) (c) Evaluation of PD-L1 expression in Klf1 gene-transduced CD4⁺ T cells. Klf1 cDNA was inserted into the retrovirus vector, pMIG with a GFP reporter gene. pMIG-Mock or pMIG-Klf1 was transfected into CD4⁺ T cells. MFI ratio represents the PD-L1 MFI signals of GFP negative versus GFP positive. (d) GFP⁺ and GFP^- cells in (c) were fractionated and mRNA expression of Cd274 (encoding PD-L1) was evaluated by qRT-PCR (n = 3). (e,f) Egr2 and Klf1 mRNA expression was evaluated by qRT-PCR using the cells transfected in (c) and Fig. 1d. (g) Klf1 and Cd274 mRNA expression in Klf1 gene-transduced CD4⁺ T cells from Egr2^fl/flCD4Cre⁺ mice was evaluated by qRT-PCR (n = 3). *p < 0.05; n.s.: not significant (unpaired two-tailed Student’s t-test) Plots are representative of 3 independent experiments.