Expression of Cas9 in a Syngeneic Model of Primary Central Nervous System Lymphoma Induces Intracerebral NK and CD8 T Cell-Mediated Lymphoma Cell Lysis Via Perforin

Abstract

The development of clustered regulatory interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR-Cas9)-mediated gene modification has opened an exciting avenue of targeting genes to study the pathogenesis of diseases and to develop novel therapeutic concepts. However, as the effector protein Cas9 is of bacterial origin, unwanted side effects due to a host immune response against Cas9 need to be considered. Here, we used the syngeneic model of BAL17^CNS-induced primary lymphoma of the central nervous system (PCNSL, CNS) in BALB/c mice to address this issue. Surprisingly, stable expression of Cas9 in BAL17^CNS (BAL17^CNS/Cas9) cells rendered them unable to establish PCNSL on intracerebral transplantation. Instead, they induced a prominent intracerebral immune response mediated by CD8 T cells, which lysed BAL17^CNS/Cas9 cells via perforin. In addition, B cells contributed to the immune response as evidenced by serum anti-Cas9 antibodies in BALB/c mice as early as day 8 after transplantation of BAL17^CNS/Cas9 cells. In athymic BALB/c^nu/nu mice, NK cells mounted a vigorous intracerebral immune response with perforin-mediated destruction of BAL17^CNS/Cas9 cells. Thus, in the CNS, perforin produced by NK and CD8 T cells was identified as a mediator of cytotoxicity against BAL17^CNS/Cas9 cells. These observations should be taken into account when considering therapeutic CRISPR-Cas9-mediated tumor cell manipulation for PCNSL.