Figure 4. Infection and antigen affinity drive the largest changes in transcriptome of responding CD8⁺ T cells.

Naïve CD8⁺ T cells were sorted from spleens of OT-1/ Rag^−/− (WT) or Itk^−/−/OT-1/Rag^−/−/ (Itk^−/−) mice for RNA-sequencing on D0. Three biological replicates were used for WT and Itk^−/− groups. For D7 analysis, donor CD8⁺ T cells were isolated from spleens and sorted (CD45.2⁺CD45.1⁻) on D7 post infection with LM-expressing N4 or LM-expressing T4 OVA peptide. Three technical replicates were used for each condition. Principal component analysis was performed on all three conditions with a log₂ fold change and p<0.05 (A) with PC3 at 5.96% and PC4 at 4.01%. (B) Volcano plot of significantly up and down-regulated genes under high (N4) and low (T4) antigen affinity condition and (C) under differential TcR signal strength based on log₂ fold change. (D) The ratio of the log transformed values for Itk^−/− N4: Itk^−/− T4 to WTN4:WTT4 were calculated (referred to as IWR). GSEA revealed that the inflammatory response was significantly upregulated in the Itk^−/− cells compared to WT cells. (E) Custom heatmap displaying the enriched inflammatory response genes compared to other conditions: N4 (Itk^−/−:WT cells infected with LM-N4), T4 (Itk^−/−:WT cells infected with LM-T4) was generated.