A high-throughput RNAi screen for detection of immune-checkpoint molecules that mediate tumor resistance to cytotoxic T lymphocytes.
Bottom Line: Tumor-immune resistance is mediated by cell surface ligands that engage immune-inhibitory receptors on T cells.These ligands represent potent targets for therapeutic inhibition.So far, only few immune-suppressive ligands have been identified.
Affiliation: Division of Translational Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany email@example.com firstname.lastname@example.org.Show MeSH
Related in: MedlinePlus
Mentions: To translate the Luc-CTL assay to a high-throughput RNAi screening approach, we focused on a library of 520 genes coding for transmembrane and cell surface proteins as these are suitable targets for therapeutic function-blocking antibodies. The candidate identification procedure is outlined in Fig2A. We conducted the screen not only with MCF7luc cells (screen 1) but also with transiently Luc-transfected cells (screen 2) in parallel, as the latter approach can be easily employed for screening of various tumor cell lines. Moreover, given the limitations in expanding antigen-specific T cells, we also tested the feasibility of using polyclonal, pre-activated CTLs from healthy donors as effector T cell population with the aid of bi-specific antibody. Using the latter approach, we could not only identify tumor-associated immune suppressors that suppress T cell function, but also targets that could further aid in better clinical success of the anti-EpCAM × CD3 bi-specific antibody. Each screen contained a set of 4 replicates, two of which were exposed to CTLs (toxicity set) and two were incubated without CTLs (viability set). Finally, we employed data from an additional screen (based on CTG assay) in which cell viability was determined independent of luciferase activity by measuring intracellular ATP levels (screen 4) and genes that impacted cell viability in this assay were also excluded from the final hit list (Supplementary Fig S1A).
Affiliation: Division of Translational Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany email@example.com firstname.lastname@example.org.