LN-CLL cells are functionally better at T-cell activation and induce superior CLL:T-cell synapses. (A) Paired LN and PB-CLL cells (irradiated for thymidine-incorporation assays) from 6 patients and B cells from 5 normal donors were mixed at a 1:1 or 1:10 ratio with allogeneic T cells in triplicate. Compared with PB-CLL cells, LN-CLL cells and normal B cells have an increased ability to stimulate T-cell activation and proliferation, as shown by increased expression of Ki67 on gated CD4⁺ (Ai) cells and CD8⁺ cells (Aii) after a 48-hour coculture, and increased thymidine-incorporation by CD3⁺ T cells in a 5-day MLR (Aiii). (B) To investigate the ability of the LN-derived CLL cells to induce autologous T-cell synapse formation compared with PB-CLL cells and normal B cells, we measured the area of filamentous actin polymerization in 90 CLL or B-cell:CD4⁺ T-cell conjugates in 4 paired patient samples and 1 paired normal sample. This representative figure from 1 patient shows the increased synapse formation induced by the LN-derived CLL cells compared with matched PB-CLL cells. (C) Representative result from 1 patient showing that synapse area was significantly greater when LN-derived CLL cells were used (mean synapse area induced by LN-CLL 6.534 μm² ± 2.7 vs PB-CLL 3.594 μm² ± 2.3; P < .0001). (D) Representative result from another patient demonstrating the synapse area generated by LN-CLL cells is comparable to that of normal B cells. ns, not significant; pTyr, phosphotyrosine.