Avadomide activates previously exhausted CLL patient T cells and induces expression of PD-L1. (A) Intercellular autologous CD4⁺ or CD8⁺ T-cell:tumor cell conjugates formed from vehicle- or avadomide-treated CLL patient samples (n = 25). Image analysis data presented as mean % T cell:CLL conjugates ± SEM. Representative confocal images show patient T cell:CLL cell (blue) conjugate F-actin (red) interactions after treatment. Original magnification, ×63 (scale bars: 10 μm). (B) Representative medial optical section (scale bars: 5 μm) and 3D volume rendered images of CD8⁺ T-cell conjugates with increased F-actin (red) immune synapse formation with CLL tumor cells (arrow) following avadomide treatment. Relative recruitment index, RRI image analysis of (C) F-actin (red) and (D) tyrosine-phosphorylated protein polarization in autologous CD4⁺ or CD8⁺ T cell:CLL conjugates following vehicle or avadomide treatment (n = 30). (E) Representative 3D volume rendered images of CD8⁺ T-cell conjugates showing increased phosphotyrosine signal (white, pTyr) at synapses with avadomide. (F) Representative flow cytometric histograms of CD25 and CD69 (top) and HLA-DR (bottom) expression on stimulated patient CD8⁺ T cells with treatment (n = 5). CD86 expression on treated CD5⁺ CD19⁺ CLL cells is also shown. Frequency of (G) PD-1- and (H) PD-L1-expressing cells (stimulated CD4⁺, blue; CD8⁺, red; or CD5⁺ CD19⁺ CLL cells, black) following avadomide treatment (n = 19). (I) Representative 3D volume rendered images of CD8⁺ T cell:tumor (blue) conjugates showing reduced and increased expression of PD-1 (white) and PD-L1 (green), respectively, with both molecules polarizing at synapses with avadomide. (J) Representative immunoblots of pretreated (as indicated), stimulated patient T cells subsequently conjugated with MEC-1 tumor cells (T cell:tumor cell conjugates, 5 and 15 minutes for early and late conjugation times, respectively) probed for the phospho (p)-proteins p-ZAP-70, p-LAT, p-MAPK, and p-AKT (n = 3). **P < .01 using Wilcoxon signed-rank tests (A,C-D,G-H). Data presented as mean ± SEM. RRI, relative recruitment index.