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

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 Foxp3(Ametrine-FlpO) strain, expressing Ametrine and FlpO in Foxp3(+) cells. Breeding these mice resulted in a CD4conv(iCreERT2-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 E8I(iCreERT2.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.