Fig. 3

OM9.2 T cells were activated by the ERV-K-Env peptide when presented by HLA-A*03:01 + B LCLs. a., d. Diagram of the ELISpot assay designs used in b.-c. and e.-f., respectively, Representative images of developed wells for OM9.2 T cell levels of background IFN-γ secretion (media) and IFN-γ secretion co-cultured with unpulsed and peptide-pulsed OM9-derived or non OM9-derived B LCLs for the ELISpot assays quantified in c. and f. Spot numbers are indicated to the bottom right of each image. Images shown for OM9.2 T cells. c., f. Number of IFN-γ spots per well normalized to the positive control (expressed as % of PHA) of untransduced T cells (black) and OM9.2 T cells (red) for the indicated conditions: Media = background level of IFN-γ secretion from T cells, B LCLs = background level of IFN-γ secretion from T cells co-cultured with unpulsed B LCLs, actin-pulsed B LCLs = negative control-pulsed B LCLs, ERV-K-Env-pulsed B LCLs = ERV-K-Env epitope-pulsed B LCLs, pp65-pulsed B LCLs = HLA-B*35 off-target control pulsed B LCLs, PHA = positive control. Each donor is indicated by a different symbol. ELISpot conditions were plated with the indicated peptide concentrations in duplicate or triplicate as cell numbers allowed for n = 2 donors (c) or n = 3 donors (f). g. Representative flow plots of intracellular IFN-γ and TNF-α staining from transduced T cells (gated on GFP + or GFP- cells) from Donor 272768 for the indicated conditions: T cells alone = background level of cytokine production from T cells, T cells + peptide = level of cytokine production from T cells in the presence of 50 µg peptide; various E:T ratios = level of cytokine production from T cells in the presence of decreasing numbers of unpulsed or peptide-pulsed non-OM9 B LCLs. B LCLs were pulsed with 10 µg peptide per million cells for 1 – 2 hours prior to plating with T cells. B LCLs and T cells were co-cultured for 6 hours in the presence of 1X protein transport inhibitors (cells alone, cells + peptide, or E:T conditions) or 1X stimulation cocktail (PMA/ionomycin). Flow gating strategy is in Figure S6c. h. Quantification of transduced T cells (GFP- = open red symbols/bars, GFP +  = closed red symbols/bars) that stained positively for TNF-α alone for the indicated conditions. Each donor is indicated by a different symbol. Error bars represent SEM of all data points for all donors. The Wilcoxon rank sum test was used to compare values between untransduced and transduced T cells (indicated by *). The Kruskal Wallis test was used to compare the number of IFN-γ spots between each antigen condition for untransduced or transduced cells. Any Kruskal Wallis test that had p < 0.05 was followed by post-hoc pairwise Wilcoxon rank sum tests and adjusted for multiple comparisons (indicated by #). All statistical comparisons for ELISpot data are in Tables S1 and S2 and for intracellular cytokine staining in Table S4. *p < 0.05, **p < 0.005, ***p < 0.0005. ELISpot = Enzyme-linked immunosorbent spot; B LCL = B lymphoblastoid cell line; PMA = phorbol 12-myristate 13-acetate; SEM = standard error of the mean