A Cre-driven allele-conditioning line to interrogate CD4+ conventional T cells

• Published in: Immunity (Cambridge, Mass.) Mass.), 2021-10, Vol.54 (10), p.2209-2217.e6
• Main Authors: Andrews, Lawrence P., Vignali, Kate M., Szymczak-Workman, Andrea L., Burton, Amanda R., Brunazzi, Erin A., Ngiow, Shin Foong, Harusato, Akihito, Sharpe, Arlene H., Wherry, E. John, Taniuchi, Ichiro, Workman, Creg J., Vignali, Dario A.A.
• Format: Article
• Language: English
• Subjects: allele-conditioning; CD4; CD8; gene editing; T cells; Treg
• ISSN: 1074-7613; 1097-4180
• DOI: 10.1016/j.immuni.2021.08.029

CD4+ T cells share common developmental pathways with CD8+ T cells, and upon maturation, CD4+ T conventional T (Tconv) cells lack phenotypic markers that distinguish these cells from FoxP3+ T regulatory cells. We developed a tamoxifen-inducible ThPOKCreERT2.hCD2 line with Frt sites inserted on either side of the CreERT2-hCD2 cassette, and a Foxp3Ametrine-FlpO strain, expressing Ametrine and FlpO in Foxp3+ cells. Breeding these mice resulted in a CD4conviCreERT2-hCD2 line that allows for the specific manipulation of a gene in CD4+ Tconv cells. As FlpO removes the CreERT2-hCD2 cassette, CD4+ Treg cells are spared from Cre activity, which we refer to as allele conditioning. Comparison with an E8IiCreERT2.GFP mouse that enables inducible targeting of CD8+ T cells, and deletion of two inhibitory receptors, PD-1 and LAG-3, in a melanoma model, support the fidelity of these lines. These engineered mouse strains present a resource for the temporal manipulation of genes in CD4+ T cells and CD4+ Tconv cells. [Display omitted] •ThPOKCreERT2.hCD2 mice restrict Cre activity to CD4+ T cells•Foxp3Ametrine-FlpO and ThPOKF/F.iCreERT2.hCD2 mice generate a CD4conviCreERT2-hCD2 line•CD4+ Tconv cell-type restriction of Cre activity obtained using allele conditioning•CD4conviCreERT2-hCD2 selectively removed PD-1 and LAG-3 on CD4+ Tconv cells only CD4+ T cell subpopulations have been difficult to genetically interrogate because of shared pathways with CD8+ T cells during T cell development. Using an allele-conditioning strategy, Andrews et al. generate murine lines that temporally restrict Cre activity and gene deletion to CD4+ T conventional cells, while sparing CD4+ T regulatory cells.