Inhibiting glycolysis skews T cells towards a more memory phenotype
Upon activation, T cells undergo a metabolic switch from oxidation of fatty acids to glycolysis to enable effector function(Chang & Pearce, 2016; Edwards-Hicks, Mitterer, Pearce, & Buescher, 2020). The switch into effector function is critical for immune surveillance however is undesirable during ex vivo manufacturing due to the loss of memory function of T cell and its association with reduced therapeutic efficacy and persistence(Delgoffe et al., 2021; Eyles et al., 2019). We hypothesized that limiting glucose intake could sustain the memory phenotype of T cells and potentially lead to increased efficacy and persistence. Cells were activated and expanded in the presence of a glycolysis inhibitor, 2-DG, at 2mM on day0 until harvest on day9 or gradually removed at day6(Pelicano, Martin, Xu, & Huang, 2006) through semi-continuous perfusion (1L/day) until harvest. No inhibition of cell growth was observed in the presence of 2-DG independent of when 2-DG was removed (Fig. 4A) . While cells still continuously differentiated from Tn to Tscm and eventually enriched to a mixed pool of Tcm and Tem, glycolysis inhibition by 2-DG noticeably increased the %Tscm and Tcm at different timepoints during expansion with data shown for two representative donors. (Fig. 4B) . % Tscm was higher in conditions with 2-DG on day4 which led to 10% to 20% higher of % Tcm + Tscm on day 7 or day9 compared to the condition lacking 2-DG(Fig. 4B) . In addition to enriching total memory T cells, glycolysis inhibition by 2-DG also increased TCR expression by 10% at time of harvest. (Fig. 4C) .
Mitochondrial membrane potential (MMP) and mitochondrial mass were also quantified based on the mean fluorescent intensity (MFI) of DilC1(5) and MitoTracker Red acquired by flow cytometry to investigate whether glycolysis inhibition improved metabolic fitness of TCR-T cells. After encountering target cells, TCR-T cells from all conditions exhibited elevated mitochondrial metabolism as MMP became more polarized and mitochondrial mass increased in order to sustain energetic metabolism for effector function (Fig. 4D & 4E) . Interestingly, MMP and mitochondrial mass were less elevated for TCR-T cells activated and expanded in the presence of 2-DG than those cultured without 2-DG. TCR-T cells generated in the presence of 2-DG had similar lysis capacity (E:T=1) (Fig. 4F) with lower MMP and mitochondrial mass than cells cultured in the absence of 2-DG. Thissuggests that 2-DG cells have a greater mitochondrial capacity and fitness to meet the increased energy demand for effector function (Sukumar et al., 2016; van der Windt et al., 2012).