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 ThPOK^CreERT2.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.