In this study, we employed CRISPR/Cas9 technology to generate hCD4 KI mice and performed a comprehensive physiological analysis. The results confirmed successful expression of chimeric mouse/human CD4 protein in mice. During our investigation, we observed altered T lymphocyte development in the thymus of hCD4 KI mice, characterized by an increase in DN cells and altered ratios of CD4+ and CD8+ SP cells. These findings align with observations made in mCD4-deficient mouse model29, 30, suggesting that humanized CD4-D1 and D2 can influence CD4+ T cell lineage selection. Previous research has indicated that hCD4 might exhibit greater affinity for certain mouse MHC class II molecules compared to mCD417. However, replacing mCD4’s D3 and D4 with those from hCD4 resulted in reduced binding efficiency to TCR, presumably due to a decreased affinity of this chimeric CD4 for mouse MHC class II molecules31. Additionally, it has been shown that driving the expression of hCD4, driven by mouse Cd4 gene enhancer, can compensate for mCD4-deficiency, restoring hCD4+ SP cells in the thymus and hCD4+ T cells in the peripheral blood32. In our model, the hCD4 KI mice retained all native regulatory elements of mouse Cd4 gene, yet exhibited thymic development similar to mCD4-deficient mice. Therefore, we hypothesize that the observed developmental discrepancies in the thymus are likely due to the differing affinities of mCD4 and the chimeric mouse/human CD4 for mouse MHC class II molecules. This difference in affinity may lead to variations in signal transduction intensity, influencing the differentiation of DP cells33. Consequently, hCD4 KI mice tend to favor the production of mouse CD8+ T cells rather than the chimeric mouse/human CD4+ T cells during T cell lineage selection. As a result, the decrease in CD4+ SP cells and the corresponding increase in CD8+ SP cells are associated with the introduction of hCD4. Our study provides a valuable foundation for further exploration of human CD4 application in mouse models. These insights into T cell development and lineage selection might have important implications for the use of hCD4 KI mice as a research tool in future studies.
The abnormal T cell development observed in the thymus of hCD4 KI mice has subsequent impact on the peripheral lymphoid immune system. We found that this disruption led to a decrease in CD4+ T cells and an increase in CD8+ T cells in the spleen and PBMC of hCD4 KI mice, while the total number of T cells remained unchanged. These findings suggest that compensatory mechanisms may exist to maintain a balance in the number of T lymphocytes and to preserve normal immune function in vivo34, 35. However, we also observed an increase in the proportion of naive CD4+ and CM CD4+ cells in the spleen of hCD4 KI mice, indicating that they had been exposed to specific pathogens in the past. Despite the partial compensatory mechanisms, immunodeficiency may still increase susceptibility to pathogens and other health issues, as it is restricted by immune responses34, 36. Therefore, these findings highlight the importance of understanding the impact of disrupted T cell development on the immune system and overall health of humanized mouse models. Further studies are needed to explore the potential long-term consequences of these compensatory mechanisms and their implications for translational research.
CD4+ T cells are essential for normal B cell activation and function2. In the absence of CD4+ T cells, the stimulatory signals required for proper B cell activation are weakened, leading to abnormal B cell proliferation37, 38. Our study observed a reduction in the percentage of B cells in the spleen of hCD4 KI mice, which we attribute to the lower total number of CD4+ T cells in these mice, resulting in weaker signal stimulation. We also hypothesize that the chimeric mouse/human CD4 may have reduced efficiency in interaction with MHC class II molecules of mouse B cells39, which requires further investigation to validate.
Our study shows that the chimeric mouse/human CD4 in hCD4 KI mice preserves most of the function of mouse CD4. The T cell function of hCD4 KI mice was evaluated and found to be comparable to CD4m/m mice. The physiological analyses of hCD4 KI mice also showed no significant differences from CD4m/m mice in terms of body weight, CBC reports, and tissue staining results. These results suggest that the chimeric mouse/human CD4 can play a similar role when mice partially or completely lack mouse CD4 protein, which is consistent with previous studies on humanized transgenic CD4 mice30, 32, 40. In addition, our hCD4 KI mouse model not only offers an advantage by exclude potential disruptions from endogenous mouse Cd4 genes, enhancing the accuracy and applicability of investigations of disease mechanisms and therapeutic approaches, but also provides a thorough characterization of hCD4 KI mice, establishing a foundation for their application in immunologically related research.
Previous studies have shown normal T and B cell development in humanized mice expressing HLA class II, chimeric mouse/human CD4, T-cell receptor genes, chimeric mouse/human B2M and chimeric mouse/human CD841. This suggests that hCD4 KI mice can be used with HLA class II KI mice, if available in the future, to improve abnormal thymocyte development and splenic B cell compartment. This approach can deepen our understanding of the affinity between chimeric mouse/human CD4 protein and HLA class II molecule, as well as its regulatory mechanism on T and B cells, thus advancing immune molecular biology. Combining these two models will also increase the reliability of hCD4 KI mice in studying immune-related diseases associated with MHC class II and CD4. According to this previous study, the physiological effect of multiple humanized mouse gene has revealed. However, our study uniquely focuses on the specific impact of a single humanized mouse gene on mouse physiology, which provides an important reference basis for future exploration of the role of other related genes.
In summary, this study presents a sophisticated design strategy to develops hCD4 KI mice based on precise gene editing using CRISPR/Cas9 technology. The resulting hCD4 KI mice exhibited similar overall biological and immunological activity to CD4m/m mice, albeit with altered thymocyte development manifested with changed proportion of lymphocyte subtypes in thymus, spleen and peripheral blood. Our study highlights the potential of hCD4 KI mice as a valuable research platform for investigating the dynamic interaction between CD4 and MHC class II molecules. Furthermore, a combined humanized CD4 and humanized MHC mice in the future might offer the opportunity to shed light on the pathogenic mechanisms of human immune-related diseases.