Early Notch signals control SLEC differentiation and early transcriptional events in antigen-specific CD8 ⁺ T cells. (A) Notch1 and Notch2 expression by OT-I CD8⁺ T cells was measured in naïve mice (gated on OT-I) or on days 2, 3, 5, and 7 (gated on OT-I CD44⁺) after infection with Lm-OVA. (B) The redundancy of Notch signaling via Notch1 and Notch2 receptors was tested by measuring the proportion of OVA-specific SLECs following Lm-OVA infection in WT (E8I-Cre⁻-Notch1/2^fl/fl), Notch1 KO (E8I-Cre⁺-Notch1^fl/fl), Notch2 KO (E8I-Cre⁺-Notch2^fl/fl), or both Notch1/2 KO (E8I-Cre⁺Notch1/2^fl/fl). (C) The consequence of early (days 0 and 2) or delayed (day 3.5 [d3.5]) inhibition of Notch signaling with anti-DLL1/4 antibodies on SLEC differentiation was assessed on day 7 after Lm-OVA infection. Gated on Tet-OVA⁺ cells. (D) RNA-seq was performed on day 3 after infection to compare the transcriptomes of Notch WT (E8I-Cre⁻-Notch1/2^fl/fl) and Notch KO (E8I-Cre⁺-Notch1/2^fl/fl) OT-I cells. (E) Volcano plot of differentially expressed genes (adjusted P value <0.05). (F) GSEA showing the Hallmark Notch Signaling from MSigDB on RNA-seq data comparing Notch WT and KO OT-I cells on day 3 after infection. NES, normalized enrichment score; FDR, false discovery rate. (G) Enrichment of RBPJ binding motifs at DEGs. Data are from two independent experiments (A) or three independent experiments (B and C) with a total of n = 5–9 mice per group. Error bars display means ± SEM. Ordinary one-way ANOVA with Tukey’s multiple comparison was used for multiple group comparison and unpaired two-tailed t test was used for two-group comparisons. *P < 0.05, **P < 0.01, ****P < 0.0001.