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).