Immortalization of splenic and peripheral blood macrophages using a multi-cistronic v-Raf/v-Myc lentivirus.

Abstract

Abstract We have created an improved system for immortalizing mature macrophages by modifying the original v-raf/v-myc (J2) virus. Previously, these J2 viruses required a producer co-culture condition and maintained replicating virus. This provided more complexity, reagents, and biosafety risks. Our improved virus is replication deficient. Its lentiviral genome contains the v-raf/v-myc oncogenes and a thy1.1 surface marker, each of which is separated by a self-cleaving peptide. This virus, therefore, eliminates the issues described above. We transduced our modified virus into total murine PBMC isolated from murine peripheral blood or spleen. We found the transduction and successful cell line production can be done with as few as 1×105 PBMC. The resulting cytokine independent macrophage cell line from both spleen and blood expressed mature macrophage markers (F4-80+CD11b+) but lacked early myeloid progenitor markers (Sca1−c-kit−). In addition, we found these macrophages were functional as demonstrated by their ability to undergo phagocytosis and ability to upregulate MHCII in response to IFNγ. These findings demonstrate that the v-raf and v-myc oncogenes in our new system can immortalize macrophages from spleen but uniquely, and perhaps more importantly, can immortalize macrophages from blood. This advance provides a non-invasive method to obtain the starting tissue needed to make these macrophage cell lines. Having the v-raf/v-myc oncogenes in a lentivirus means it is likely this method will work on blood from humans and other species. Additionally, the cytokine independence associated with cell lines made with our virus make it ideal for creating macrophage cell lines from mammals without commercially available growth factors.