OP0315 EFFECTOR CD4 T CELLS REQUIRE SURVIVIN FOR REGULATION OF GLUCOSE METABOLISM AND IFNg PRODUCTION

Abstract

Background:Interferon-gamma (IFNg) producing effector T cells play the leading role in triggering and perpetuation of inflammation in rheumatoid arthritis. Inflammation leads to metabolic reprogramming of T cells and high energy consumption supporting proliferation and IFNg production. Being a part of chromosomal passenger complex, oncoprotein survivin is essential for cell proliferation. It has also been identified as a marker of severe therapy-resistant rheumatoid arthritis. Thus, we aimed to explore the association between survivin and IFNg producing phenotype of CD4 T cells.Objectives:We study if survivin mediates the glucose dependent mechanism of IFNg production in CD4 T cells.Methods:CD4 cells were sorted from the peripheral blood of RA patients and healthy controls, activated with aCD3, cultured in presence of survivin inhibitor YM155 and subjected to RNA sequencing (Illumina, Life Science). IFNg levels in supernatants were measured by ELISA. To study glucose uptake in presence of YM155, CD4 cells were treated with IFNg+aCD3 overnight followed by 2NBD-glucose challenge for 30 min. Uptake of fluorescent 2NBD-glucose probe was measured by flow cytometry. Statistical analysis of RNAseq was performed in R-studio using the Bioconductor package DESeq2.Results:Comparison of the whole-genome transcription profile of CD4 cells different by levels of BIRC5, coding for survivin, demonstrated that the BIRC5hi group expressed significantly higher levels of IFNg (mRNA, p=10-26 and protein, p=10-4). Also, BIRC5hi CD4 cells had higher expression of glucose transporter GLUT1 (SLC2A1, p=0.0064) and of glycolytic enzymes glucose-6-phosphate dehydrogenase (G6PD, p=10-6), pyruvate kinase (PFKP, p=10-6), and lactate dehydrogenase (LDHA, p=10-14). On the contrary, expression of the key regulator of glycolysis 6-phosphofructo-2-kinase (PFKFB3) was significantly lower in the BIRC5hi group (p=4.4x10-5). Notably, expression of glycolytic enzymes G6PD and PFKFB3 correlated strongly to IFNg (r=0.880 and -0.698, respectively), TBX21 (r=0.811 and -0.698) and perforin (r=0.781 and -0.698). To demonstrate functional relevance of the connection between BIRC5 and glucose metabolism, survivin was inhibited in CD4 cell cultures. Survivin inhibition resulted in significant increase of PFKFB3 (p=7x10-6) and LDHA (p=0.0089), leading to inhibition of phosphoglycerate mutase PGAM1 and ATF citrate lyase ACLY (p=0.021 and p=0.0074, respectively), which dignify the restoration of aerobic glycolysis. Importantly, inhibition of survivin decreased 2NBD-glucose uptake by CD4 cells (p=0.031) and reduced expression of GLUT1 (p=0.034). These changes in glucose metabolism were followed by decreased IFNg production in supernatants (p=0.037).Conclusion:The study demonstrates a strong connection between IFNg production and glucose metabolism in CD4 cells. Survivin emerges as an important regulator of glycolysis acting through expression of glycolytic enzymes and glucose transport.Disclosure of Interests:None declared.