Bat-mouse bone marrow chimera: a novel animal model for dissecting the uniqueness of the bat immune system

Yong, Kylie Su Mei; Ng, Justin Han Jia; Her, Zhisheng; Hey, Ying Ying; Tan, Sue Yee; Tan, Wilson Wei Sheng; Irac, Sergio Erdal; Liu, Min; Chan, Xue Ying; Gunawan, Merry; Foo, Randy Jee Hiang; Low, Dolyce Hong Wen; Mendenhall, Ian Hewitt; Chionh, Yok Teng; Dutertre, Charles-Antoine; Chen, Qingfeng; Wang, Lin-Fa

Due to their immunodeficiency, NSG mice are permissive for the engraftment of foreign cells 30 . To investigate their ability to support the engraftment of bat cells, we created bat-mice by transferring 1 × 10 6 whole bat BM cells intravenously into eight-week old adult NSG mice. Ten weeks post-transplantation, blood samples were collected and analyzed for bat and mouse genes with species-specific primers (Supplementary Table 1 ) by quantitative polymerase chain reaction (qPCR). Untreated NSG mice were also kept in parallel as controls. Wild bat (E. spelaea) and C57BL/6 mice were used as both positive and negative controls, depending on the species-specific primers used. The results showed the presence of bat housekeeping genes (GAPDH and 18 S) within bat-mice, but not in control NSG mice (Fig. 1a) . To further dissect the immune cell composition in blood, peripheral blood from both NSG and bat-mice were analyzed via flow cytometry. Due to the lack of bat-specific antibodies, antibodies such as anti-mouse CD11b, CD44, and major histocompatibility complex class II (MHC-II), which showed cross reactivity with bats previously 20, 42 and are able to specifically bind to E. spelaea cells ( Supplementary Fig. 1) were used. As shown in Fig. 1b and c, mouse-specific CD45.1 and Ter119 antibodies were used to gate out majority of the mouse leukocytes and erythroid lineage cells. CD45.1 − Ter119 − population was further separated into two populations by CD44 and CD11b antibodies. Within the CD44 + CD11b + population, monocytes and dendritic cells (DCs) were gated out by further staining with CD44 and MHC-II. From CD44 + CD11b − cell population, CD44 and MHC-II were used to distinguish between T/natural killer (NK) and B cells. All of the major bat immune cell populations, such as monocytes, T/NK cells, B cells and DCs were found within the peripheral blood of bat-mice (Fig. 1c) . Bat chimerism in bat-mouse peripheral blood was calculated by analyzing the cells, which stained negative for both mouse CD45.1 and Ter119, using the formula: