Abstract 4843: Characterization of T-cell metabolic defects in chronic lymphocytic leukemia preclinical model
Authors: Gamal Wael , Mediavilla-Varela Melanie , Maharaj Kamira , Uriepero Angimar , Kunta Vishaal , Sahakian Eva , Pinilla-Ibarz Javier 
Published: June 2023

Abstract
Chronic lymphocytic leukemia (CLL) is the most predominant leukemia affecting elderly population in western countries. T-cell dysfunction is a CLL hallmark contributing to the low success rates of adoptive cell therapy in patients. Generally, exhausted states of tumor infiltrating lymphocytes are associated with defects in metabolic and mitochondrial adaptation, preventing normal cytotoxic functions. A handful of recent reports have demonstrated metabolic defects in CLL patients’ T-cells. Nevertheless, the field is still lacking sufficient studies to validate these metabolic changes in the preclinical Eµ-TCL1 murine model which is the most established model for CLL. Our aim is to uncover T-cell mitochondrial and metabolic defects in the Eµ-TCL1 splenic microenvironment. In this study, CLL was induced in mice via the adoptive transfer of leukemic splenocytes from transgenic Eµ-TCL1 mice into wild type (WT) recipients. Multiparameter flow cytometry was used to investigate mitochondrial properties and cellular glucose uptake of splenic CLL CD8+ T-cells. Moreover, we measured the expression levels of PGC-1α, a master regulator of mitochondrial function, and exhaustion-related markers in CD8+ T-cells. T-cell mitochondrial function was measured using mitochondrial stress test following in vitro CD3/CD28 stimulation for 72 hours. Nanostring gene expression analysis was done to compare metabolic gene expression profiles of CD8+ T-cells from either WT or Eµ-TCL1 mice. Probing Eµ-TCL1 splenocytes with mitotracker green and tetramethylrhodamine methyl ester revealed accumulation of depolarized mitochondria in CD8+ T-cells compared to WT counterparts. Our results also showed significant changes in mitochondrial and cellular reactive oxygen species levels as indicated by mitosox and H2DCFDA staining, respectively. Additionally, marked reduction of glucose uptake by Eµ-TCL1 CD8+ T-cells was noted following incubation with 2-NBDG glucose analog. Observed mitochondrial abnormalities in the Eµ-TCL1 CD8+ T-cells were associated with a significant downregulation of PGC-1α expression. Flow cytometry analysis demonstrated a significant increase in the expression of exhaustion markers in Eµ-TCL1 CD8+ T-cells and reduction in self-renewal marker, TCF-1. Interestingly, the abnormal mitochondrial properties were directly correlated with PD-1 expression in Eµ-TCL1 T-cells. Nanostring significance score analysis of Eµ-TCL1 versus WT CD8+ T-cell RNA revealed upregulation of glycolysis, mitochondrial respiration, T-cell receptor and costimulatory pathways. On the other hand, there was a downregulation in autophagy, AMPK and amino acid transporter pathways. In conclusion, our work characterizes for the first time the metabolic and mitochondrial defects of CD8+ T-cells from Eµ-TCL1 murine model and outlines the correlation of these defects with T-cell exhaustion in CLL.
Citation Format: Wael Gamal, Melanie Mediavilla-Varela, Kamira Maharaj, Angimar Uriepero, Vishaal Kunta, Eva Sahakian, Javier Pinilla-Ibarz. Characterization of T-cell metabolic defects in chronic lymphocytic leukemia preclinical model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4843.