Engineering a Novel In Vivo Circuit to Uncover Cell-intrinsic Resistance Mechanisms to Killer Lymphocyte-mediated Cytotoxicity

Abstract

Cytotoxic lymphocytes, such as CD8+ T cells and natural killer (NK) cells, play an essential role in the elimination of cancer and infected cells. Cancer cells evade detection and clearance by cytotoxic lymphocytes via several mechanisms. These include immune-evasive mechanisms such as orchestrating an immunosuppressive niche, as well as resistance to mediators of cytotoxicity. Granule-mediated cytotoxicity is an essential mechanism by which killer lymphocytes directly lyse their targets. This process involves the release of perforin and granzymes into the immunological synapse, where perforin oligomerization at the target cell membrane facilitates the delivery of granzymes, such as granzyme B (GzmB) into the target cell to induce cell death. Recent reports have shown that cancer cells can resist GzmB-induced cell death via expression of the GzmB inhibitor Serpin B9, autophagy or downregulation of gasdermin E. Identifying the mechanisms by which cancer cells survive cytotoxic lymphocyte attacks is crucial to improving immunotherapy and prevent disease relapse. To identify and characterize cancer cells that are resistant to mediators of cytotoxicity, we have developed a reporter system that allows cytotoxic lymphocytes to irreversibly label the target cells they attack. To build this system, we took advantage of the perforin-GzmB pathway and fused a Cre recombinase to GzmB, which is delivered into the target cell during the cytotoxic attack. Target cells were engineered to express a Cre-inducible mCherry. Thus, cancer cells that survive a cytotoxic attack would be irreversibly labeled with mCherry. This reporter system allows for convenient identification, isolation and characterization of cancer cells that survived a cytotoxic attack to uncover their mechanisms of resistance, potentially paving the way for improved immunotherapies.