Abstract 3269: Ablation of Cbl-b gene function leads to increased activation and resistance to immunosuppression in human primary T lymphocytes

Abstract

Abstract Purpose: The role of Cbl-b as an essential modulator of T cell activation has been widely documented, particularly in mouse studies. To substantiate the effects of Cbl-b ablation in human T cells, gene editing conditions were optimized, and gene KO efficiency was evaluated along with T cell response to stimulatory antibodies. Experimental design: Non-preactivated human primary T cells obtained from several donors were electroporated with the CRISPR/Cas9-gRNA ribonucleoprotein complex targeting CBLB gene or controls. Cells were then activated in presence of anti-CD3 and anti-CD28 antibodies with or without the immunosuppressive factors PGE2 or TGFβ. Gene editing efficiency was measured using targeted NGS technology and cytokine release (IL2, IFNγ, TNFα), membrane activation markers (CD25, CD69) and cell proliferation were evaluated at various timepoints. Results: Initial studies showed moderate gene editing efficiencies with on average 45% and 25% of out-of-frame mutations using 2 gRNAs targeting different regions of CBLB gene and were correlated with increased IL-2 release. By optimizing gene editing experimental conditions, out-of-frame mutations in CBLB gene reached more than 70% and were associated with higher levels of secreted IL2, IFNγ and TNFα early after T cell stimulation and increased CD25 membrane expression at later timepoints, compared to controls. Furthermore, CBLB gene-edited T cells were less susceptible to PGE2- and TGFβ-mediated inhibition of cell proliferation. Conclusion: The efficient gene editing in human non-preactivated T cells, through the delivery of the CRISPR/Cas9-gRNA ribonucleoprotein complex, confirms that Cbl-b acts as a negative regulator of T cell activation and further validates its role in transducing immunosuppressive environmental cues. Citation Format: Eric Parmantier, Georges Kalouche, Clara Soulard, Christophe Lanneau, Sophie Boisrobert-Blais, Céline Nicolazzi, Cécile Orsini. Ablation of Cbl-b gene function leads to increased activation and resistance to immunosuppression in human primary T lymphocytes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3269.