Induction of functional exhaustion in REP over time. (A) Left: CD45RO, CD62L and CCR7 expression on HA-1 multimer-positive cells in REP and right: increase of the percentage of CD8+ HA-1+ CCR7+ for each donor over time. (B) PD-1 and KLRG-1 expression over time on HA-1 multimer-specific CD8+ T cells; For A and B; n = 4, histograms represent means ± SEM. (C) Cells in each culture steps were harvested and stimulated in vitro with HA-1 peptide. Specific IFNγ spot forming cells (SFC) were measured and compared to non-stimulated cells with the ELISpot assay; dotted line represent detection threshold, n = 4, data show average ± SEM. (D) Cells from REP cultures were harvested over time and submitted to intracellular staining for flow cytometry analysis of TNFα, IFNγ and IL-2 production after HA-1 peptide restimulation; left: representative plots and right: representation in each culture. (E) Proportion of TNFα secretion in IFNγ-positive cells in every cell lines. (F) left: one representative and right: percentage of CD8+ Ki67+ for each donor in the REP over time. (G) comparison of Ki67 expression between the IFNγ-positive and negative CD8+ T-cell fractions within the cultures after HA-1 peptide restimulation. *, P < 0.05.