ObjectivesImmunodeficient mice are widely utilized not only for basic research but also for translational research in various fields. The recent rapid progress in the humanized mouse technology has been based on the development of novel immunodeficient mouse strains like NOG or NSG mice. Due to the massive deficiency of the endogenous murine immune systems arising from the combination of the scid mutation and the lack of the IL-2 receptor γ (IL-2Rγ) gene, those mice cannot invoke protective immune responses and accept even human-derived tissues. Particularly, normal human hematopoiesis from human hematopoietic stem cells (HSCs) is possible and the developed human leucocytes are sustained for long term. Furthermore, the reconstituted immune systems are functional and mediate immune reactions to some extent.Extensive genetic modification of the immunodeficient mice has been attempted for enabling better engraftment, development, and differentiation of human cells, since some human cell linages are poorly developed from HSCs and maintained. For example, mature human erythrocytes (RBCs) or platelets hardly develop from human HSCs. In addition, even after infusion of an enormous number of human RBCs (up to 5x109, equivalent 40 % of total RBC in peripheral blood), they persist no more than 96hr in the mouse peripheral blood. This is quite striking considering that human HSCs less than 5x104 successfully engraft NOG mice. Although several proceeding results have suggested that mouse macrophages (MΦs) are responsible for the elimination of human RBCs, the molecular mechanisms have remained to be clarified. Considering that macrophages recognize and phagocytose various foreign substances through a wide range of receptors, especially for glycoproteins, we focused on identifying the receptors responsible for the elimination of human RBCs by mouse MΦs. In addition, we explored whether it is possible to improve the function of the reconstituted human immune systems in humanized mice by genetic manipulations of such receptors. Methods1) Screening of mouse lectin-like receptors and scavenger receptors Mononuclear cells were isolated from liver, lung, spleen, and bone marrow (BM) of NOG mice. Mouse macrophages were purified by fluorescence-activated cell sorting (FACS) and total RNA was isolated for RNA sequence (RNAseq). Lectin-like receptors and scavenger receptors highly expressed in the tissue-specific mouse MΦs have been identified from the RNAseq results. Subsequently, we constructed the cDNA for expressing Fc-fusion proteins, which consisted of mouse Fc and the lectin domain of the aforementioned receptors. The recombinant proteins were produced in 293 T cells by transfection of the cDNA constructs in vitro. The culture supernatants were used for examining their binding capacity to human RBCs and white blood cells (WBCs) after confirming the production of the recombinant proteins by Western-blot analysis.2) Production of NOG mice deficient for adapter moleculesMany receptors including several lectin-like receptors utilize adapter molecules for transmitting the signals from exogenous stimuli. We have produced a NOG strain deficient for Fcγ receptors (FcεrIγ and FcγRIIb). FcεrIγ is also called Fc common γ chain (FcRγ) and known to work as a subunit of various receptors, which include several c-type lectin-like receptors (CLRs) such as Dectin2 or Mincle etc. We performed characterization of this novel mouse strain by transplanting human HSCs.Results1) Discovery of mouse clec(X) as a hRBC-binding proteinWe have examined twenty-six different CLR molecules in total (including 6 out of 8 molecules in liver macrophages, 3 out of 4 molecules in splenic macrophages, 3 in lung macrophages, and 3 out of 5 in BM macrophages) and 13 scavenger receptors for the capability to bind human RBCs. The binding of the recombinant proteins to the surface of human RBCs was detected by flow cytometry. In this screening, we found that one molecule, mouse clec(x), can bind human RBCs (manuscript in preparation). 119Improved human xenograft in NOG mice by manipulating receptors for glycansTakeshi TakahashiIkumi Katano, Takuya Yamaguchi, Motohito Goto and Iyo OtsukaCentral Institute for Experimental Animals (CIEA)
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