IMiD3 costimulates T cells and overcomes CTLA-4–Ig blockade.(A) CD3⁺ T cells were negatively selected from healthy donors and incubated with IMiD3 alone (10 μM), anti-CD3 alone (200 ng/mL), IMiD3 and anti-CD3, or DMSO alone as a negative control. Cells were washed in phosphate-buffered saline (PBS), fixed and permeabilized in 70% ethanol, treated with 10 μg/mL DNase-free Rnase, and stained with propidium iodide. Cell cycle profile was determined by flow cytometry using Coulter EPICS XL-MCL (Coulter, Birmingham, United Kingdom). Panel A presents the proportion of cells in different stages of the cell cycle from 2 of 3 healthy donors. (B) CD3⁺ T cells were stimulated with IMiD3 alone (10 μM), anti-CD3 alone (200 ng/mL), IMiD3 and anti-CD3, or DMSO as a control. IFN-γ was detected using a cytokine secretion assay, and cells were counterstained for CD4 or CD8 FITC-conjugated antibody. The numbers indicate the proportion of cells staining positive for IFN-γ. (C) CD3⁺ T cells were incubated for 72 hours in the presence or absence of IMiD3 (10 μM), anti-CD3 (200 ng/mL), immature or mature dendritic cells, and CTLA-4–Ig (10 μg/mL), as indicated. Cells were pulsed with [³H]TdR for the last 12 hours, harvested, and [³H]TdR uptake was measured using a liquid scintillation counter. Results represent the mean ± standard deviation of the mean cpm from a triplicate assay. These results are representative of 3 independent experiments (*P < .05). (D) CD3⁺ T cells from HLA-A2 donors were incubated in ELISPOT plates with PBMCs for 40 hours in the presence or absence of IMiD3 (10 μM) or CTLA-4–Ig (10 μg/mL), as indicated. Cells were stimulated with EBV-, influenza-, or HTLV-1–specific peptides (10 μg/mL). Plates were washed and stained for IFN-γ. ▤ indicates EBV; ▧, influenza; and ▨, HTLV-1. Results represent mean ± standard deviation of the mean IFN-γ spot forming cells (SFC) from 3 independent experiments (*P < .05).