Mouse Splenocytes CD4+ T Cells

Genetic Dissection of Eomes Function in CD4⁺ T Cells Using a Novel Tbx21ᴱ Allele

To delineate whether Eomesodermin (Eomes) mirrors or diverges from T-bet in CD4⁺ T cells, Thelen et al. employed a novel Tbx21ᴱ knock-in allele that replaces T-bet with Eomes and an mCherry reporter. Combining this with wild-type (Tbx21⁺/⁺) and knockout (Tbx21⁻/⁻) alleles generated five mouse lines with titrated T-box transcription factor expression (Fig. 1A). This design uncouples Eomes induction from chronic antigen exposure, allowing direct assessment of its lineage-specific effects under Th1-polarizing conditions.

Transcriptional profiling confirmed allele fidelity: Tbx21⁻/ᴱcells lacked Tbx21mRNA, while Tbx21⁺/ᴱand Tbx21⁻/ᴱcells exhibited robust Eomes expression (Fig. 1B). Loss of Tbx21alone caused only marginal increases in endogenous Eomes, indicating that Eomes is not a default backup for T-bet. Flow cytometry validated these findings, showing reduced T-bet protein in haploin sufficient cells and complete absence in Tbx21⁻/⁻and Tbx21⁻/ᴱ CD4⁺ T cells (Fig. 1C, 1D). Strikingly, the Tbx21ᴱ allele drove uniform, high-level Eomes expression paralleled by mCherry fluorescence, confirming that Eomes is transcribed from the engineered locus rather than the endogenous Eomes promoter. IL-12 further amplified Eomes and mCherry expression, demonstrating full transcriptional control by the Tbx21 regulatory elements. These results establish that Eomes expression from the Tbx21ᴱ allele faithfully recapitulates the dynamics of wild-type T-bet, providing a powerful genetic system to dissect Eomes-specific transcriptional programs in CD4⁺ T cells independent of environmental cues.