Immune synapse formation between B and T cells according to PD-1 expression. Spleen cells from mice with CLL after AT (n = 5) and age-matched WT mice (n = 3) were debulked of CLL/B cells using magnetic beads; stained for CD3, 8, and PD-1; and flow-sorted into CD3⁺CD8⁺, CD3⁺CD8⁺PD-1^high, and CD3⁺CD8⁺PD-1^low cells. Cells from WT mice were sorted on CD3⁺CD8⁺ only. (A) Comparison of median areas of immune synapses (µm²) between normal B and CD8⁺ T cells (hBC-hTC), normal B cells and CLL T cells (hBC-CLL TC), normal B cells and PD-1^lowCD8⁺ T cells (hBC-PD-1^low), and normal B cells and PD-1^highCD8⁺ T cells (hBC-PD-1^high). ns, nonsignificant. *P < .05, **P < .001, ***P < .0001. (B) Representative confocal images of synapses taken with a ×63 objective between hBC-hTC, hBC- PD-1^high, and hBC-PD-1^low cells. Blue, amino-4-chloromethylcoumarin–labeled B cells; red, rhodamine phalloidin staining actin cytoskeleton.