Figure 4.

CTS Immune Cell SR optimises the expansion of T cells specific for the subdominant tumour-association EBV antigens, LMP1&2 and EBNA1. PBMC from healthy EBV-seropositive donors were cultured with autologous AdE1-LMPpoly infected PBMC in either RPMI–FBS or OpTmizer supplemented with SR. T-cell cultures were supplemented with 50% fresh media containing 120 IU ml⁻¹ IL-2 after 3 days and every 3–4 days after. On day 14, cell numbers were determined using trypan blue exclusion, then T-cell specificity was determined using an intracellular IFN-γ assay following recall with a pool of defined LMP1&2 and EBNA1. (a) Representative analysis from the same donor stimulated with AdE1-LMPpoly and cultured in either RPMI–FBS or OpTmizer-SR is shown. (b) Data represent the mean±s.e.m. from seven donors of the number of viable cells following AdE1-LMPpoly stimulation in RPMI–FBS or OpTmizer-SR. (c) Data represent the mean±s.e.m. from seven donors of the frequency of LMP1&2/EBNA1-specific CD8⁺ T cells following AdE1-LMPpoly stimulation in RPMI–FBS or OpTmizer-SR. (d) AdE1-LMPpoly expanded T cells were assessed for multiple cytokine production (IFN-γ, TNF and IL-2) and degranulation (CD107a) following recall with cognate peptides. Data represent the mean±s.e.m. from three donors of the proportion of EBV-specific CD8⁺ T cells producing different combinations of IFN-γ, TNF, IL-2 and CD107a in RPMI–FBS or OpTmizer-SR. (e) AdE1-LMPpoly expanded T cells, were stained with the HLA B35/HPVGEADYFEY pentamer, then assessed for the intracellular expression of granzymes B and K and perforin. Data represent the mean±s.e.m. from two donors (use average and s.d.) of the proportion of HPVGEADYFEY-specific CD8⁺ T cells producing each effector molecule in RPMI–FBS or OpTmizer-SR.