A standardized protocol for assessing immunodeficiency in mouse models.

Immunodeficient mouse strains are widely used in several fields of biomedical research. Despite that, no standardized system for evaluating immunodeficiency in mice currently exists, and an unbiased comparison of various immunodeficient mouse strains is difficult. The aim of our study was to develop a standardized multi-disciplinary protocol for the morpho-phenotypical assessment of immunodeficient mouse models. We selected 4 immunodeficient strains of mice (Cd40l^-/-, Was^-/-, Rag1^R972Q/R972Q, and Rag1^-/-) on a C57BL/6J genetic background and a group of control C57BL/6J wild-type mice. The lymphoid organs were harvested, weighed, and analyzed by histology, immunohistochemistry, and flow cytometry. Hematology and bone marrow cytology were also performed. The main immune cell populations were investigated, including lymphocytes, monocytes/macrophages, neutrophils, and natural killer cells. Relative organ weights were lower in the strains with the highest level of immunodeficiency (Rag1^R972Q/R972Q and Rag1^-/-). Histology revealed overall lower cellularity in the same strains, particularly in Rag1^-/- mice. Tissue spatial distributions of the immune cell populations were confirmed by immunohistochemistry, while flow cytometry allowed for their relative quantification. Likewise, hematology detected moderate lymphopenia in the Rag1^R972Q/R972Q mice and more severe lymphopenia in Rag1^-/- mice. Our protocol has proven itself effective for the morpho-phenotypical assessment of the immunodeficient mouse models under investigation and was useful in characterizing the type and severity of the defects. The different laboratory techniques were consistent in the characterization and confirmation of immunodeficiency in the different strains, providing different complementary insights.