Delayed activation in CLL-derived T cells and diminished effector function after restimulation. (A-B) PBMCs from HDs and patients with CLL were thawed and T cells were stimulated once with soluble stimulating CD3/CD28 antibodies and kept in culture for 16 days. (A) Expression of CD25 and PD-1 was measured daily (HD, n = 6; CLL, n = 10-12). (B) On day 14, all cells were washed, stained with Cell Trace Violet (CTV), and resuspended in culture medium with or without a single dose of CD3/CD28 antibodies. After 48 hours, T-cell activation was measured by analyzing CD25 expression. (C) In addition, the proliferation and replication index was calculated (using FlowJo) based on CTV intensity 5 days after restimulation (HD, n = 6; CLL, n = 10). (D) In addition, after an initial 14 days of single stimulation with CD3/CD28 antibodies, T cells were restimulated and kept for 2 days, and IFN-γ, TNFα, IL-2, and degranulation (CD107a) were measured on CD8 T cells (HD, n = 4; CLL, n = 3). (E) In a repeated stimulation experiment, PBMCs from HDs and patients with CLL were thawed and at start and on day 5 and day 10, PBMCs were washed and resuspended with the addition of CD3/CD28 antibodies. Every 5 days, the proliferation (population doublings) of T cells was analyzed using counting beads (HD, n = 3; CLL, n = 3). P values were calculated using a t test (A), a Welch t test (B), a Mann-Whitney test (C), a 2-way analysis of variance (ANOVA) that corrected for multiple comparisons using a Tukey test (D), or a 1-way ANOVA (E). Data are presented as mean ± standard error of the mean (SEM); ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001.