Humanized mouse models have become indispensable tools for investigating human gene function and disease modeling. However, conventional transgenic approaches carry the risk of unforeseen biological consequences. Here, to address this concern, we developed a novel human CD4 knock-in (hCD4 KI) mouse model using CRISPR-Cas9 gene-editing technology. We replaced the region encoding the first two major extracellular domains of the mouse Cd4 gene, which are critical for interaction with major histocompatibility complex (MHC) class II, with the corresponding human CD4 sequence. Subsequently, we conducted comprehensive physiological and immune system analyses on hCD4 KI mice, including both heterozygous (CD4m/h) and homozygous (CD4h/h) genotypes. Our investigations revealed a dosage-dependent impact of the hCD4 KI, resulting in a decreased percentage of CD4+ single-positive cells, accompanied by a corresponding increase in CD8+ single-positive cells within the thymus. These developmental alterations, evident in the thymus, were also observed in the peripheral lymphatic system such as the spleen and in the peripheral blood, exhibiting an increased population of mature CD8+ T cells and a decreased proportion of mature CD4+ T cells. Despite these changes, hCD4 KI mice exhibited normal biological characteristics, including T cell activation and proliferation functions, blood composition, tissue structure and body weight, closely resembling those of wild-type (CD4m/m) mice. Our study underscores hCD4 KI mice as a valuable tool for exploring CD4 and MHC class II interactions, with potential for future integration with humanized MHC class II KI mice, offering insights into immune disease mechanisms.
