Optimization of T cell: LCL ratio for EBV antigen-driven
stimulation
Conventionally, ex vivo expansion of EBV-specific cytotoxic T
cell lines (CTLs) has been performed using a ratio of 40 PBMC : 1 LCL
for the initial stimulation, and subsequent stimulation rounds at a
ratio of 1 T cell : 5 LCL in G-Rex rapid expansion protocol which could
potentially drive anergy, senescence or activation-induced cell death
(AICD). We determined whether modulation of the stimulation ratio
conferred benefits in terms of functional subpopulation profile.
T cell cultures from four EBV-positive donors were initiated using an
initial concentration of 40 PBMC : 1 LCL as per standard protocol, then
at SR2 or SR3 cultures were split and co-cultured at standard (1 T cell
: 5 LCL) or at lower intensity (1 T cell : 1 LCL). At day 30 (the end of
SR3), a direct comparison of each stimulation ratio was assessed. Mean
harvest cell counts indicated a 28.1 ± 13.63-fold expansion for
1 T cell : 5 LCL cultures, and
21.68 ± 9.24-fold expansion for 1 T cell : 1 LCL cultures (Figure 4A).
Samples taken at day 30 were comparable for surface marker analysis,
with ~90% CD8+ cells and equivalent levels of each
surface marker subset (Figure 4B). While the percentage of T cells with
co-expression of IFN-γ / TNF-a was equivalent between the two groups
(Figure 4D), the cMFI of IFN-γ (Figure 4C) was significantly higher
(p=0.00218) in 1 T cell : 1 LCL
cultures (mean = 162.5 ± 16.03) than in 1 T cell : 5 LCL cultures (mean
= 77.65 ± 25.50). Similarly, TNF-α cMFI was significantly increased
(p=0.0202) in 1 T cell : 1 LCL cultures (mean = 112.18 ± 18.56) than in
1 T cell : 5 LCL cultures (mean = 53.69 ± 12.2). The increased cytokine
cMFI indicates co-culture with a lower intensity LCL stimulation result
in EBV-specific T cells with an enhanced capacity for functional
cytokine secretion. These data clearly demonstrate the robustness of the
current LCL-based method for EBV-specific T cell stimulation and
expansion, as most parameters are unaffected by the modification of LCL
ratios. However, the finding that reducing LCL dose corresponds with
increased secretion of IFN-γ and TNF-α which may enhance effector
functions of these cells in vivo , suggests that the EBV-specific
T cell culture product can be optimized. We have therefore demonstrated
that our optimised culture approach provides a significant improvement
in retention of TCM cells with increased cytokine expression. The
expanded phenotyping approach allows for a better characterisation of
start material and final products, and we assessed how to analyse the
data from this multi-parameter flow cytometric approach more
effectively.