br Materials and methods br

Materials and methods
Results
Discussion ENU mutagenesis is a powerful approach to identify novel genes or DPNI-caged-GABA receptor to dissect the molecular basis of mammalian immune system (Siggs, 2014). T cells are critical components in the host defense against pathogens, and their activity has to be precisely calibrated. In this perspective, point mutations from ENU mutagenesis or knockin alleles could provide more precise information of the gene function (Ordonez-Rueda et al., 2012). It is well-established that CD4 plays an important role in T cell development in the thymus (Wang et al., 2001b). The early thymocyte precursors do not express the co-receptors CD4 and CD8, and they are low in numbers and represent only a few percent of the total thymocytes (Starr et al., 2003). Full expansion of thymocytes occurs when the cells reach the double positive stage, where MHC and T cell receptor ligation take place and transduce cell proliferation signals (Carpenter and Bosselut, 2010). CD4 functions as co-receptor to ligate MHCII which is essential for T cell development. Structural studies provide insights into the interaction between these two molecules but in vivo experimental data is still limited. In our experiments, we first obtained a mutant mouse line that was absent in CD4 T cell on C57BL/6 background. The methods exome capture and next generation sequencing in our study facilitated identification of the mutant allele responsible for the mutant phenotype, which was shown in previous studies (Andrews et al., 2012). Strikingly, the mutant gene coding the CD4I99N protein was a complete loss of function, which was supported by the defect of T cell development in the thymus. Studies of CD4 positive T cells usually involve adoptive transfer experiments, to determine the cell intrinsic mechanisms. For example, the CD4 T cells have distinct lineages, including various types of helper T cells and a critical immune suppressive cell type termed regulatory T cells that express a master transcription factor Foxp3. In the adoptive transfer setting, such different type of CD4 T cells can be assessed in terms of homeostasis, differentiation and function in the recipient model without CD4 T cells. Both Rag recombinase deficient mice and T cell receptor deficient mice are used as recipient, however such models may still introduce undesirable impacts blurring outcome of the donor cells, such as the “side effects” from CD8 T cell deficiency and B cell deficiency. In the Rag recombinase deficient mice, the donor CD4 T cells lose contact with B cells and CD8 T cells, which are important for CD4 T cell to functional properly (Wei et al., 2005). In the T cell receptor deficient model such as CD3e knockout mice, the donor cells are deprived of contact with CD8 T cells. Such limits can be overcome in the recipient mice that are only deficient in CD4 T cells. We used CD4 T cells as donor cells and purified them from a knockin model in which regulatory T cells or Tregs carry GFP reporter, and transferred them to both CD3e knockout mice and CD4I99N mutant mice. The homogeneous distribution of the GFP positive proportion indicated that CD4I99N mutant mice absent only in CD4 T cells, were an optimal recipient model to perform CD4 T cell adoptive transfer experiments. This data suggests that adoptive transfer to analyze CD4 T cell especially the regulatory T cells, more desirable model as recipient mice could be those only deficient in CD4 T cells such as CD4 knockout mice and our newly isolated I99N mutant line. The point mutation model aids in elucidating the function of critical domains, even though in this study we were not able to analyze the signaling defects originating from the mutant CD4 co-receptor for T cell activation. For the CD4I99N mutant mouse, it remains interesting to investigate on the structural basis how the mutant protein could affect the contact with MHC molecule. This CD4I99N mutant line develop normally but the long term immunological consequence of this point still needs to be further analyzed. In addition, the CD4I99N mutant mice could be a valuable tool to study T cell biology.