Acidic pH attenuates immune killing through inactivation of perforin

Abstract Cytotoxic lymphocytes are pivotal effectors in our immune system. Their most potent way to delete virus infected or cancerous target cells is by perforin/granzyme dependent killing. Perforin is secreted into the immunological synapse and forms transmembrane pores in the target cell plasma membrane, allowing granzymes to enter the target cell cytosol and trigger apoptosis. The prowess of cytotoxic lymphocytes to efficiently eradicate target cells has been widely harnessed in immunotherapies against haematological cancers. Despite efforts to achieve a similar outcome against solid tumours, the immunosuppressive and acidic tumour microenvironment poses a persistent obstacle. Using different types of effector cells, including therapeutically relevant anti-CD19 CAR T cells, we demonstrate that the acidic pH typically found in solid tumours hinders the efficacy of immune therapies by impeding perforin pore formation within the immunological synapse. Nanometre-scale study of purified recombinant perforin undergoing oligomerization reveals that pore formation is inhibited specifically by preventing the formation of a transmembrane β-barrel. The absence of perforin pore formation directly prevents target cell death. This finding uncovers a novel layer of immune effector inhibition that must be considered in the development of effective immunotherapies for solid tumours. One Sentence Summary Our study reveals that an acidic environment hinders the efficacy of immune therapies by impeding the action of perforin, a critical protein in immune-mediated cell killing, directly within the immunological synapse.