Determining optimal stimulation round for effective EBV-specific T
cell manufacture
Third-party EBV-specific T cell banks based on LCL stimulation involve
leukapheresis-derived T cells co-cultured with irradiated autologous LCL
through several rounds to generate a relatively homogenous
virus-specific T cell product. We hypothesized that multiple rounds of
LCL-mediated antigen presentation could potentially lead to exhaustion
of virus-specific T cells. To determine this, we re-derived exemplars
from our second-generation EBV-specific T cell bank leukapheresis and
LCL stocks and analyzed cells by flow cytometry at 7-9 days following
each successive stimulation round to characterize phenotypic
development. Changes in CD4/CD8 ratio were seen through each round of
stimulation (Figure 1A), indicating that the CD4 population forms less
than 1% of the total T cells from stimulation round (SR) four onwards.
The corresponding IFN-γ/TNF-α expression in the CD8 central memory
(TCM), effector memory (TEM) and differentiated effector (TEMRA)
compartment shows the majority of cells (86%) co-express IFN-γ and
TNF-α in response to PMA/ionomycin stimulation (Figure 1A, lower panel).
From SR2 onwards this increases to over 97% IFN-γ+/TNF-α+, indicating
the CD8 compartment rapidly progresses to functional cytotoxic T cells.
However, percentage cytokine expression stabilizes after SR3, indicating
no significant benefit to functional capacity with multiple rounds of
LCL stimulation. This was quantified in six different EBV-CTL lines
(data is represented as mean ± SEM), and confirmed that outgrowth of the
CD8 population was consistent (Figure 1B). The mean percentage of
CD8+/CD45RO+ cells peaks at SR3 (Figure 1C) then starts to decrease,
indicating downregulation of CD45RO with continued stimulations.
Expression of the activation / senescence marker CD57 also increases
throughout each stimulation round. While the mean percentage of
TNF-α/IFN-γ-expressing CD8+ T cells cytokine does not vary significantly
throughout, it is clear that the mean fluorescence intensity corrected
to negative control (cMFI) of TNF-α and IFN-γ peaks at SR3 or 4 and then
starts to decrease (Figure 1D), indicating that extending stimulation
beyond four rounds potentially compromises functional cytokine levels.
Further cytometric analysis of exhaustion markers (PD-1 / LAG-3 / Tim-3)
demonstrated no significant co-expression of these indicators of loss of
function (data not shown).The results from Figure 1 suggest that
EBV-specific T cell cultures reach peak product quality by SR3/4, after
which functional parameters decline.