Abstract 2097: Characterization of mouse and cellular phenotype of ABI1 KO T-cells

Abstract

Background: The immune system plays a critical part in cancer development and progression. The increase in our understanding of mechanisms regulating immune-mediated cancer eradication has led to the development of immune-based therapies which have revolutionized treatment of some previously lethal malignancies. T-cell activation requires proper intercellular interactions mediated by cytoskeletal rearrangements and adhesion molecules, along with intracellular signal transduction via phosphorylation by tyrosine kinases (Lck and Fyn). Abelson interactor-1 (ABI1) is an adaptor protein known to regulate actin polymerization, adhesion molecules and tyrosine kinases. Previously, ABI1 has been shown to localize to the immunological synapse in an antigen-dependent manner and has been linked to regulation of IL-2 secretion. Published data from our lab shows that ABI1 has high affinity binding to SH2 domains of Src family kinases, Fyn and Lck, both of which are essential in T-cell activation. Additionally, ABI1 has been linked to hyperactivation of SFKs/STAT3/NF-kB signaling. In this study, we aim to delineate the role of ABI1 in T-cell activation, differentiation and signaling. Methods: We have generated a novel conditional knock-out mouse model to evaluate the effect of a T-cell specific ABI1 KO at the organismal level (ABI1(fl/fl)CD4Cre (ACD4). T-cells isolated from control (Abi1(fl/fl)CD4Cre(-)) and ABI1 KO mice (Abi1(fl/fl)CD4Cre(+)) were used for ex-vivo analysis and further characterization of ABI1 KO cellular phenotype with: Flow Cytometry (CD3+ subpopulations in immune organs), Immunofluorescence (actin polymerization), Western blotting (TCR-mediated T-cell activation) and RNAseq (transcriptional reprogramming). Results: In vitro studies of ABI1 KO primary murine CD3+ T-cells showed increased levels of NF-kB and STAT3. Flow cytometry analysis of CD3+ T cells subpopulations showed a difference in the percentage of CD4+ and CD8+ T cells; percentage of CD8+ T cells was increased, while percentage of CD4+ T cells was decreased in ABI1 KO vs. WT control mice. Furthermore, RNA seq analysis of CD8+ ABI1 KO T cells showed a significant decrease in the mRNA expression of Thpok (Zbtb7b) compared to WT. Conclusion: Our preliminary studies show that ABI1 is a potential modulator of T-cell activation through regulation of Lck/Fyn. This suggests ABI1 may play as a molecular switch negatively regulating T-cell activation by connecting phosphorylation-dependent signal transduction and actin cytoskeletal dynamics. Suggesting ABI1 as a new therapeutic to improve efficacy and longevity of T cell-based cancer immunotherapies. In addition, ABI1 appears to be implicated in the regulation of the percentage of CD4 and CD8 T-cells, a novel discovery which warrants further investigation. Citation Format: Maria Altagracia Ortiz, Andras Perl, Marcin Kortylewski, Leszek Kotula. Characterization of mouse and cellular phenotype of ABI1 KO T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2097.