Fig 2: CTLA4-FasL effect on pro and anti apoptotic signals. (A) Sk-Hep1 (left) and A498 (right) cell lines were pre-incubated with or without caspase inhibitors (Z-VAD-FMK (general), Z-LEHD-FMK (caspase 9), Z-IETD-FMK (caspase 8)) for 1 hour followed by incubation with his6CTLA4-FasL at different concentrations for 24 hours. Cells’ viability was tested by the MTS assay. The results represent the average of three independent experiments. +/- SE (*p = 0.05). (B) Sk-Hep1 cells were incubated with CTLA4-FasL, sFasL, CTLA4-Ig or combination of the later two for 24 hours. Cell viability was tested by the MTS assay. The results represent the average of four independent experiments. +/- SE (*p = 0.05). (C) CTLA4-FasL effects on the expression of apoptotic and anti-apoptotic proteins in B cell lymphoma cell lines (left) and RCC (right). Raji and A498 cell lines were incubated with indicated concentrations of CTLA4-FasL, sFasL, CTLA4-Ig or the combination of the later two for 2 h. Whole cell lysates were analyzed by Western blot. These are representative results of the three independent experiments. (D) Effect of B7 blockade on CTLA-FasL’s effect on pro and anti apoptotic signals – Raji cell lines were incubated for 1 h with 1 µg/ml of anti CD80, anti CD86 or both prior to the addition of CTLA4-FasL (50 ng/ml). Cells were collected after 2 h. Whole cell lysates were analyzed by Western blot. (E) CTLA4-FasL effect on NF?B pathway - A498 (lower panel) and Raji (upper panel) cell lines were incubated with indicated concentrations of CTLA4-FasL, sFasL, CTLA4-Ig or the combination of the later two for 2 h (Raji) or 6 h (A498). Whole cell lysates were analyzed by Western blot.

Fig 3: CTLA4-FasL molecular structure. (A) SDS-PAGE analysis of the CTLA4-FasL under reducing conditions, coomassie G-250 stain. (B) Western blot analysis using anti CTLA4 antibody following enzymatic removal of the N-glycan chains from the protein. (C) Iso-Electric Focusing analysis of CTLA4-FasL at pH3-7 and pH3-10.

Fig 4: Schematic model of the CTLA4-FasL homo-hexamer. Monomer represents one domain of the CTLA-4 connected to one domain of the FasL. Trimer is composed of the FasL trimer and three domains of the CTLA-4, while two of them are connected by a disulfige bridge. Two CTLA4-FasL trimers may form a homo-hexamer, made of dimer of trimmers or trimer of dimers, resulting in a shift towards a small fraction of a dodecamer state.

Fig 5: CTLA4-FasL inhibits tumor growth in vivo . Ex vivo JY cells were injected subcutaneously to irradiated NUDE mice. At day 5 after JY injection, tumor volume was calculated and mice were treated daily with subcutaneous injections of CTLA4-FasL or PBS for 5 days. (A) Tumor volume. Results represent the mean +/- SE volume of tumors, (n = 10 for each group). *P < 0.05 between PBS group and 10 ug*2 or PBS vs 25 ug*2. **P <0.01 respectively. (B) The survival curve of the experimental groups and control.