Fig. 4

In vitro priming of cells with the ERV-K-Env epitope did not result in antigen-specificity. a. Magnetic selection is used to separate PBMCs from healthy donors into CD14 + and CD14- fractions. The CD14 + cells were cultured in the presence of GM-CSF and IL-4 to promote development into DCs. Then DCs were loaded with various amounts of peptide and matured with a cocktail of activation cytokines. The peptide-loaded DCs were then used to stimulate the cryopreserved CD14- fraction along with T cell activation cytokines. Additional CD14 isolations were performed to stimulate the ongoing T cell cultures for a total of 2—3 stimulations. Cells were harvested a week after the final stimulation for functional assays. b. Fold-expansion of T cell lines from each donor over the course of the expansion towards the indicated amounts of the ERV-K-Env epitope. c. Representative images of developed wells of T cells from each donor expanded against the ERV-K-Env peptide. Images shown are for T cells from stimulation 3, 100 ng of peptide for background IFN-γ secretion (media), IFN-γ secretion in the presence of 50 µg of ERV-K-Env peptide, and IFN-γ secretion in the presence of the positive control. Number of spots in each well are indicated to the bottom right of each image. Number of IFN-γ spots per well normalized to the positive control (expressed as % of PHA) of T cells from each round of stimulation (increasing shades of blue) for the indicated conditions: Media = background level of IFN-γ secretion from T cells, actin = negative control, ERV-K-Env = ERV-K-Env 12mer/15mer, + PHA = positive control. Each donor is indicated by a different symbol. Open symbols indicate T cells expanded against 100 ng of peptide. Closed symbols indicate CD14- cells or T cells expanded against 500 ng of peptide. Error bars represent SEM of all data points for all donors. ELISpot conditions were plated in duplicate or triplicate as cell numbers allowed. The Kruskal Wallis test was used to compare the number of IFN-γ spots between T cells in the presence of each antigen. Any Kruskal Wallis test that had p < 0.05 was followed by post-hoc pairwise Wilcoxon rank sum tests and adjusted for multiple comparisons using the Bonferroni correction. *p < 0.05, **p < 0.005, ***p < 0.0005. e. Representative flow plots for intracellular IFN-γ and TNF-α of cells from each round of stimulation from Donor 180557 in the presence of media (background), actin (negative control), ERV-K-Env epitiope, or PMA/ionomycin (positive control). Cells were cultured for 6 hours in the presence of 1X protein transport inhibitors (media, actin, ERV-K-Env epitope) or 1X stimulation cocktail (PMA/ionomycin). Flow gating strategy is in Figure S10. N = 1 technical replicate for each donor. f. Quantification of CD8 + T cells from 3 healthy donors that stained positively for TNF-α. Increasing shades of blue indicate T cells from each round of peptide stimulation. Each donor is indicated by a different symbol. Open symbols indicate T cells expanded against 100 ng of peptide. Closed symbols indicate CD14- cells or T cells expanded against 500 ng of peptide. Error bars represent SEM of all data points for all donors. DC: dendritic cell; GM-CSF: granulocyte–macrophage colony stimulating factor; LPS: lipopolysaccharide; ELISpot = Enzyme-linked immunosorbent spot; PHA = phytohemagglutinin; SEM = standard error of the mean