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Blood, Vol. 110, Issue 12, 4037-4046, December 1, 2007
Cooperation of the proapoptotic receptor agonist rhApo2L/TRAIL with the CD20 antibody rituximab against non-Hodgkin lymphoma xenografts
Blood Daniel et al.
110: 4037
Supplemental materials for: Daniel et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 11 KB)
- Figure S1. Flow cytometry analysis of DR4 and DR5 expression on NHL cell lines (JPG, 69.9 KB)
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A panel of NHL cell lines were stained with PE conjugated antibodies to DR4 and DR5. Each panel shows the cells (20,000 cells) stained with a PE conjugated DR4 or DR5 antibody (black line) or PE conjugated isotype control (dotted line).
- Figure S2. Kinetic analysis of rhApo2L/TRAIL induced caspase-3 cleavage in Ramos RA1 cells treated with rituximab (JPG, 32.5 KB)
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Ramos RA1 cells were treated with vehicle (x), 1 microgram/ml rhApo2L/TRAIL (open circle), 20 micrograms/ml rituximab (T0, closed triangle), rhApo2L/TRAIL and rituximab simultaneously (T0, closed square), rituximab (T-24, open triangle), or rhApo2L/TRAIL and rituximab added 24 hr prior to addition of rhApo2L/TRAIL (T-24, open square). Cells were analyzed for active caspase-3 by flow cytometry at the indicated number of hours after addition of rhApo2L/TRAIL.
- Figure S3. Effect of rituximab on pro-apoptotic receptor expression, DISC assembly and Bcl-2 family member expression in Ramos RA1 cells (JPG, 74.8 KB)
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(A) Ramos RA1 cells were left untreated (red line) or treated with 20 micrograms/ml rituximab (green line) for 24 hours and analyzed for changes in expression of DR4 and DR5 by flow cytometry. Isotype control staining is indicated in blue. (B) Ramos RA1 cells were treated with 1 microgram/ml rhApo2L/TRAIL (A) with and without 20 micrograms/ml rituximab (A + R) for the indicated amount of time. Lysates were generated and DISC components immune precipitated with an antibody to rhApo2L/TRAIL. The precipitated DISC was analyzed by immunoblot for FADD and caspase-8 (C-8). (C) Effect of rituximab and rhApo2L/TRAIL on the levels of pro- and anti-apoptotic Bcl-2 family members and XIAP in Ramos RA1 cells. Cells were treated with rhApo2L/TRAIL (1 microgram/ml) and/or rituximab (20 micrograms/ml) for 24 hours and cell lysates immunoblotted for the indicated proteins. (D) Generation of a Ramos RA1 cell line over-expressing Bcl-2. Ramos RA1 cells were infected with retrovirus containing a plasmid conferring stable expression of human Bcl-2, and analyzed by Bcl-2 immunoblot. Immunoblot analysis of human Bcl-2 and actin from uninfected Ramos RA1 cells (U), Ramos RA1 cells stably expressing hBcl-2 (hBcl-2) and Ramos RA1 vector control cells (V).
- Figure S4. Activity of rhApo2L/TRAIL and rituximab against subcutaneous Raji xenografts (JPG, 49.6 KB)
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CB17 ICR SCID mice bearing established Raji tumors (∼200 mm3 in size; n = 8 mice/group) were treated with vehicle (black x), 60 mg/kg rhApo2L/TRAIL (open blue circle), 4 mg/kg rituximab (closed green triangle), or rhApo2L/TRAIL and rituximab (closed red square). rhApo2L/TRAIL was administered daily for 5 consecutive days followed by a two day break and a second 5 days treatment as indicated by the blue bar. Rituximab was administered once a week as indicated by the green arrow.
- Figure S5. Contribution of ADCC and CDC to the cooperative activity of rhApo2L/TRAIL and rituximab (JPG, 68.7 KB)
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(A) BJAB cells were analyzed for antibody dependent cellular cytotoxicity by LDH release after xx hours of treatment with rituximab alone (triangles), rhApo2L/TRAIL alone (circles; 1 µg/ml) or rhApo2L/TRAIL (1 µg/ml) and rituximab (squares) in the presence of purified human NK cells. The cells were incubated at an effector:target ratio of 10:1. Data is expressed as percent cytotoxicity. (B) Confirmation of NK depletion by anti-asialo GM1 (aGM1) antibodies as determined by flow cytometry analysis of CD49b+ NK cells from the spleens of treated mice. The mean NK cell numbers are shown for each group (n=5/group) with untreated mice (solid bars) and aGM1 treated (hatched bars). Untreated (black), rhApo2L/TRAIL (blue), rituximab (green) and rhApo2L/TRAIL + rituximab (red) are shown. (C) Confirmation of complement depletion. Ramos RA1 xenograft bearing mice were treated with CVF. Serum from the mice was tested for the presence of C3 by immunoblot with a goat anti-mouse C3 IgG.
- Figure S6. Bioluminescence imaging of mice with disseminated BJAB-Luc tumors xenografts (JPG, 99.4 KB)
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BJAB-Luc cells were injected intravenously into CB17 ICR SCID mice. The mice were randomly distributed into treatment groups on day 13 (d13, starting tumor burden) at which time treatment with vehicle, rituximab, rhApo2L/TRAIL and rhApo2L/TRAIL + rituximab was initiated. After completing 2 cycles of treatment, mice were imaged on day 26 (d26) to evaluate therapeutic efficacy. Five paired false color luminescence images per treatment group are shown. The images are ventral views shown as an overlay of the reference image of the mouse at two time points (d13 and d26).
- Figure S7. CD55 and CD59 expression on Ramos RA1 and Ramos T1 cells (JPG, 53.2 KB)
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Ramos RA1 and Ramos T1 (rituximab refractory) were stained with a FITC conjugated antibody to CD55 and a PE conjugated antibody to CD59. Each panel shows the expression of CD55 and CD59 and the percentage of cells in each quadrant.
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