Induction of T cell anergy by combined treatment with LTβR-Ig and anti-CD40L mAb. (a) Sublethally irradiated BDF1 (filled circles) or B6 (open squares) mice were infused with B6.Ly5.1 splenocytes together with anti-CD40L and LTβR-Ig on day 0. On day 9, B6.Ly5.1⁺ cells were purified and stimulated with irradiated DBA/2 splenocytes for 5 days. CTL activity against P815 and EL4 was assessed. The same recipient mice were injected with splenocytes from BDF1 mice as the controls (open circles). (b) CTL activity of splenocytes from recipients that survived GVHDmore than60 days was assessed against indicated targets without in vitro culture (filled circles). Splenocytes from BDF1 recipients that had received B6 (open circles) or BDF1 splenocytes (open squares) for 10 days were used as controls. (c) Splenocytes from recipients survived more than 60 days (filled circles) were stimulated for 5 days as described above, and subsequently examined for CTL activity against indicated targets. As controls, splenocytes from naive B6 mice (open squares) or B6 BM–reconstituted BDF1 mice (filled squares) were used as responder cells. (d) Splenocytes from recipients that survived (more than 60 days) were stimulated as described above in the absence (dark gray bars) or presence (black bars) of IL-2. Naive B6 splenocytes were similarly stimulated in the absence (white bars) or presence (light gray bars) of IL-2. After 5 days, CTL activity against P815 cells was examined. Results are expressed as the mean ± SD of triplicate wells. E/T ratio: ratio of effector cells to target cells in CTL assay.