Follicular lymphoma induces defective actin cytoskeletal signaling in previously healthy T cells, and FL TILs exhibit defective downstream functional responses. (A) Healthy CD3⁺ T cells were cocultured (primary coculture) as described in Figure 2A with either healthy allogeneic B cells (B, top image panels) or allogeneic FL cells (bottom image panels) and subsequently used in conjugation assays with sAg-pulsed third-party allogeneic healthy donor B cells (APCs, blue). T-cell conjugates formed were analyzed by immunofluorescence and confocal microscopy (F-actin was stained red using rhodamine phalloidin). Images shown are representative of evaluation of 250 conjugates from 5 independent experiments for each protein analyzed (stained green), including CD11a/CD18 (LFA-1), Lck, tyrosine-phosphorylated protein (P-Tyr), Itk, Filamin-A, and Rab27A (CD8⁺ T cells). White arrows denote protein localization. Colocalization of proteins is shown in yellow. (B-D) Quantitative analysis of protein accumulation (green) at the synapse site is shown for (B) P-Tyr, (C) Filamin-A, (D) Rab27A, and LFA-1, Lck, and Itk in supplemental Figure 1. Data represent means ± SD from 5 independent experiments (50 conjugates analyzed per experiment). (E) Mitogenic activity of PB T cells or TILs from FL patients was assessed by thymidine incorporation using PMA and ionomycin, or anti-CD3 and anti-CD28 mAbs. Results are shown as counts per minute. For mixed lymphocyte (MLR), T cells were stimulated with irradiated allogeneic Epstein-Barr virus–transformed lymphoblastoid cell line as stimulator and results shown as the stimulation index. The results shown are the mean + SD of 8 paired (LN and PB) patient samples studied.