Background Checkpoint blockade therapies have transformed the landscape of cancer care. Durable clinical responses have been observed in a subset of patients. However, many patients do not respond, and understanding the mechanisms that determine tumor resistant to checkpoint blockade drugs could potentially benefit more patients. Ferroptosis is a relatively newly described form of regulated cell death distinct from apoptosis and necroptosis. Recently, T cell-promoted tumor ferroptosis was shown to be an anti-tumor mechanism and targeting this pathway could be a potential therapeutic approach.

Methods To identify genes critical to immunotherapy resistance, B16.SIY cells were transduced with a genome-scale gRNA lentivirus to generate loss of function mutants. In vitro-primed CD8+ T cells isolated from 2C/Rag2–/– TCR transgenic mice specific for the SIY antigen were co-cultured with transduced B16.SIY tumor cells. Resistant mutants were identified by sequencing the gRNAs of survival clones. The gene encoding Decr2, a peroxisomal 2,4-dienoyl-CoA reductase, was identified. To investigate the role of Decr2 in tumor growth and immune responses in vivo, the Decr2 knock-down or Decr2 overexpressed tumors were transplanted into B6 mice and the mice were subsequently treated with anti-PD-L1 antibody. The tumor microenvironments were analyzed by flow cytometry. To understand the resistance mechanism of Decr2 knock-down tumors, RNA-seq was performed and analyzed. The CD8+ T cell mediated tumor ferroptosis in vitro and in vivo was analyzed for lipid reactive oxygen species.

Results Decr2 mutants were relatively resistant to CD8+ T cell killing in vitro. Consistent with this resistance to CD8+ T cell killing, Decr2 knock-down tumors showed minimal response to anti-PDL1 therapy in vivo. RNA-seq analysis of Decr2 knock-down B16.SIY tumors revealed upregulation of ferroptosis-related genes, including slc7a11. Further mechanistic studies showed that Decr2 knock-down tumors displayed defects in ferroptosis in vitro and in vivo.

Conclusions Decr2-deficient tumors were relatively resistant to CD8+ T cell killing in vitro and anti-PD-L1 immunotherapy in vivo by modulating CD8+ T cell-induced ferroptosis.