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. 2021 Apr 15;118(16):e2014553118. doi: 10.1073/pnas.2014553118

Fig. 1.

Fig. 1.

IRF4 is not required for maintenance of CD8+ memory T cells in vitro. (A) Scheme of genetic modifications of tamoxifen-inducible Irf4 knockout. (B–E) Irf4fl/+×CreERT2 and Irf4fl/−×CreERT2 mice were treated on 5 consecutive d with tamoxifen. Following 1 wk after the last treatment, spleen cells were analyzed. (B) Deletion of exons 1 and 2 of Irf4 in sorted CD8+ T cells. Primers are located upstream of the 5′ loxP site and within exon 3 and amplify a 2 kb fragment in the Cre-modified gene locus of the Irf4fl allele. (C) Expression of Irf4 exon 2 coding mRNA in sorted CD8+ T cells quantified by RT-qPCR. Normalized to 18S rRNA and to the Irf4 expression of GFP− Irf4fl/+×CreERT2 cells. (D) IRF4 expression in CD8+ T cells. Spleen cells were stimulated with PMA/ionomycin and after anti-IRF4 staining, analyzed with FACS. Representative FACS plots for activated CD8+ T cells. (E) Mean fluorescence intensity (MFI) values for stimulated (Left) and nonstimulated cells (Right; green symbols: GFP+ cells). Mean ± SEM, n = 5 per group, paired t test. **P ≤ 0.01, ***P ≤ 0.001. (F and G) CD8+ T cells purified from three to four pooled spleens from either Irf4fl/+×CreERT2 or Irf4fl/−×CreERT2 mice after tamoxifen treatment were stimulated for 3 d with anti-CD3 mAb, anti-CD28 mAb, and IL-2 and then eFluor670 labeled and incubated with IL-7 (SI Appendix, Fig. S1A). (F) % GFP+ of CD8+ T cells at indicated time points. (G) eFluor670 staining. The numbers give the MFI. (H and I) CD8+ T cells purified from three to four pooled spleens from either Irf4fl/+×CreERT2 or Irf4fl/−×CreERT2 mice were stimulated for 3 d with anti-CD3 mAb, anti-CD28 mAb, and IL-7, and then incubated with 4-hydroxytamoxifen and IL-7 (SI Appendix, Fig. S1C). After 6 d, cells were washed, labeled with eFluor670, and incubated for indicated time periods with IL-7. (H) % GFP+ of CD8+ T cells. (I) eFluor670 staining. (B) Result of one experiment with two pooled spleens of Irf4fl/+×CreERT2 and Irf4fl/−×CreERT2 mice, respectively. (C) Result of one experiment with three Irf4fl/+×CreERT2 or Irf4fl/−×CreERT2 mice. (D–I) Representative result of two or three independent experiments.