The expression of the inhibitor receptors PD-1, CD244, CD160, Lag3 were determined by flow cytometry on day 5 of in vitro culture of OT-I CD8+ T cells. Cells were cultured either without peptide (no peptide), one time OVA peptide stimulation (single) or daily peptide stimulations (repeat peptide). (A) Representative flow cytometry plots depicting the frequency of live CD8+ T cells expressing inhibitory receptors. (B) Pooled data of frequency of CTL expressing individual inhibitory receptors within the different culture conditions. (C) SPICE figures depicting the frequency of CD8+ T cells expressing one, two, three or four inhibitor receptors (PD-1, CD244, CD160, Lag3) simultaneously. Each symbol representative one animal (n = 7–11), with 7–9 independent experiments performed. Line depicts mean ± SE. Between the groups, Wilcoxon signed rank test was performed with exception of % Tim-3+ and % CD160+ for which a Student’s t test with Welch’s correction was used. *<0.05, **P<0.01, ***P<0.001, ****P<0.0001.