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.