New Single-Cell Screening Method Finds Immunotherapy Response Modulator

Understanding mechanisms of intrinsic resistance to immune checkpoint inhibitors (ICI) is critical to the development of methods to circumvent this obstacle. To address this, Frangieh, Melms, Thakore, Geiger-Schuller, and colleagues developed a method dubbed Perturb-CITE-seq, which combines the systematic CRISPR–Cas9- based single-cell transcriptomic genetic screening technique Perturb-seq with the single-cell approach CITE-seq, which enables transcriptomic profiling alongside measurement of cell-surface proteins. Using Perturb-CITE-seq, 248 genes associated with intrinsic ICI resistance were systematically targeted with 744 CRISPR–Cas9- based knockouts (KO), and then single-cell transcriptomic profiling and measurement of 20 relevant cell-surface proteins, with the final number of cells included in the analysis being approximately 218,000. In coculture experiments combining patient-derived melanoma cells and ex vivo expanded tumor-infiltrating lymphocytes, a viability screen (to assess the knockouts' effects on T-cell mediated antitumor immunity) was combined with Perturb-CITE-seq (to ascertain the mechanisms underlying effects seen in the viability screen). In addition to identifying genes anticipated to be involved in immune evasion based on clinical and preclinical data, B2M, HLA-A, JAK1, JAK2, STAT1, IFNGR1, and IFNGR2 conferred resistance to T cell–mediated killing. Interestingly, genetic perturbation of CD58 (also known as LFA3)—encoding a cell-surface protein typically expressed on antigen-presenting cells that acts as a ligand for T-cell and natural killer–cell CD2—was also a hit in the Perturb-CITE-seq screen. Mechanistically, IFNγ signaling did not affect CD58 protein expression, and MHC expression was not altered by CD58 perturbation, implying that CD58 loss-of-function mutations do not promote immune escape via the same mechanisms as other known mutations. Notably, patient data showed that levels of CD58 mRNA were lower in ICI-resistant melanomas than in ICI-naïve melanomas. Collectively, this study demonstrates the utility of Perturb-CITE-seq for biological research, including on cancer, and identifies CD58 as a potential modulator of immunotherapy response.

Frangieh CJ, Melms JC, Thakore PI, Geiger-Schuller KR, Ho P, Luoma AM, et al. Multimodal pooled Perturb-CITE-seq screens in patient models define mechanisms of cancer immune evasion. Nat Genet 2021;53:332–41.

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