Anti–third-party Tcm eradicate lymphoma cells and artificially activated B cells in vitro but do not significantly impair the humoral response in vivo. (A) ICAM-1 expression on the different B-cell types was analyzed by flow cytometry. A representative experiment is shown out of 3 performed. Isotype controls (Isotype, nonfilled histograms), naive B cells (Naive B, light gray), activated B cells (Activated B, black), and A20 lymphoma cells (Lymphoma, dark gray). (B) In vitro killing assay. Naive B cells purified from BALB/c spleen using magnetic beads, activated B cells, obtained by further stimulation with 2 mg/mL LPS for 24 hours, or A20 cells were incubated for 16 hours with syngeneic anti–third-party Tcm. The number of viable B cells was determined by flow cytometry. Results shown represent average ± SD of triplicates in 1 representative experiment out of 3 performed. ***P value < .001 compared with percent killing displayed using naive B cells as targets. (C) Lethally irradiated BALB/c mice received 3 × 106 syngeneic nude BM cells in the absence (BM control n = 6) or presence of 10 × 106 syngeneic Tcm (BM+Tcm n = 6). At 5 weeks posttransplant, recipient mice were vaccinated with TNP-KLH in CFA as described in “Methods.” Anti-TNP antibodies in the serum were measured by ELISA. Results represent average serum levels of anti-TNP antibodies in each group, reflected by optical density (O.D) values at 630 nm. CTRL, control.
