Improved human xenograft in NOG mice by manipulating receptors for glycansTakeshi TakahashiTo examine the involvement of clec(x) in the elimination process of human RBCs in vivo, we disrupted the mouse clec(x) gene by CRISPR/Cas9-mediated genome editing. Thus, NOG mice deficient for clec(x) were established and subsequently transferred with human RBCs. Unexpectedly, the retention of human RBCs in NOG-clec(x) KO mice was not different from that in NOG mice (data not shown). Then, we crossed the NOG-clec(x) KO mice with another NOG strain deficient for mouse C3 (NOG-C3 KO), which show better capability of sustaining human RBCs1). Human RBCs were transferred to the NOG-C3/clec(x) double deficient (DKO) mice and the survival was analyzed. The additional deficiency of clec(x) significantly prolonged the survival of human RBCs about 20% compared to that in NOG-C3 KO mice (Figure 1, manuscript in preparation). Furthermore, another additional deficiency of the siglec-1 gene, which is known to bind xenogeneic RBCs, has no effects on the survival of human RBCs as shown in the results using C3/clec(x)/siglec-1 triple deficient mice (TKO) (Figure 1).Histological analysis of the mouse tissues by Prussian blue staining demonstrated that there were differences in the places where human RBCs were captured. In NOG mice, the liver is the major place for the phagocytosis of human RBCs. However, in NOG-C3/clec(x) DKO mice, engulfment of many human RBCs was detected in the spleen (data not shown). Those results suggested that mouse immune systems employ multiple mechanisms for recognizing human cells and some of them were mediated through CLRs. The clarification of those mechanisms will accelerate the development of better immunodeficient mice. Figure 1. Survival of human RBC in various mouse strains2) Characterization of NOG mice deficient for FcγR moleculesNOG-FcγR deficient mice (NOG-FcγR KO mice) were originally developed for minimizing the activity of antibody-dependent cellular cytotoxicity (ADCC) from mouse innate immune systems2). We used this mouse strain in HSC-transplantation experiments, since FcRγ transmit the signals of various lectin receptors as an adapter molecule as mentioned above. Hence, we speculated that the disruption may influence a broad range of biological process, which includes the recognition of human cells by mouse innate immune cells. We performed characterization of this novel mouse strain by transplanting human HSCs. Interestingly, NOG-FcγR deficient mice (NOG-FcγR KO mice) showed higher engraftment level of various lineages of human hematopoietic cells than that in NOG mice, when human HSCs were transplanted3). Those include CD33+ myeloid cells, CD19+ B cells, and CD3+ T cells.The distinct immunological features in NOG-FcγR KO mice became evident when they were used in the experiments for evaluating the anti-tumor activity of Nivolumab (anti-PD-1) antibody. When NOG mice reconstituted with human hematopoietic cells (huNOG mice) were inoculated with various human tumor cell lines, the growth of those human tumor was not suppressed by Nivolumab treatment. We could not detect the therapeutic effects of Nivolumab in all the cell lines we have tested in NOG mice (more than 10 tumor lines including patient-derived xenograft cell lines (PDXs)). In contrast, the growth of some tumor cell lines was strongly suppressed or rejected in huNOG-FcγR KO mice by Nivolumab treatment (Figure 2)3). 120
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