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Blood, Vol. 113, Issue 20, 4875-4884, May 14, 2009
Bromohydrin pyrophosphate enhances antibody-dependent cell-mediated cytotoxicity induced by therapeutic antibodies
Blood Gertner-Dardenne et al.
113: 4875
Supplemental materials for: Gertner-Dardenne et al
Files in this Data Supplement:
- Figure S1. PAg and RTX induce phospho-ZAP70 and phospho-ERK1/2 signaling cascades in freshly isolated TCRVγ9+ PBMCs (JPG, 21.6 KB)
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Western blots of 106 purified, freshly isolated TCRVγ9+ γδ T cells harvested at the specified time points after stimulation with PAg (400nM BrHPP) and/or cross-linked RTX (10 µg/ml) showed increased phosphorylation of ZAP70 (on Y319) and ERK1/2 (on T202/Y204). RTX was cross-linked with goat anti-human IgG (GAH). The data are from one representative experiment out of more than five independent experiments, each using cells from a different donor.
- Figure S2. TCRVγ9+ lymphocytes directly bind RTX and TTZ through cell surface FcγRIIIA (JPG, 22.5 KB)
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Anti-CD16 mAb (clone 3G8) was progressively out-competed by increasing concentrations of RTX (gray bars) and TTZ (dashed bars) IgG1 for binding to the cell surface of TCRVγ9+ γδ T lymphocytes. Means + 1 s.d. from triplicates, nt: not tested, *: P < 0.05 for difference to the ‘none’ group by one-way ANOVA on ranks. The data are from one representative experiment out of three performed.
- Figure S3. TCRVγ9+ lymphocyte-mediated cell binding requires live target cells (JPG, 42.2 KB)
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TCRVγ9+ γδ T lymphocytes obtained as in Fig.1A were co-incubated in vitro for 0 min. or 60 min. with PKH67+ RAJI cells that were either alive (left) or killed (by treatment with 0.1% saponin for 10 min at room temperature and then washed) and stained with 7-Amino-actinomycin D (7-AAD) to identify dead cells. TCRVγ9+ γδ T lymphocytes were gated for analysis. The increased PKH67 fluorescence of γδ T lymphocytes (MESF, inset) indicates binding to live (7-AADlow) but not to dead (7-AADhigh) RAJI cells, ruling out the possibility that PKH67 passively leaks from dead cells to the γδ cell surface during the co-incubation.
- Figure S4. TCRVγ9+ lymphocyte-mediated cell binding to MCF7 mammary carcinoma cells (JPG, 31 KB)
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TCRVγ9+ γδ T-cell binding to CD20−HER2/Neu+ MCF7 mammary carcinoma cells in the presence of BrHPP (400 nM), TTZ (20 µg/ml) and RTX (10 µg/ml), as indicated. The increase in PKH67 fluorescence from 0 min to 60 min of co-incubation are shown. The data are from one representative experiment out of at least three independent experiments, each with PBMCs from a different donor.
- Figure S5. Primary CLL B cells from patients CLL016 and CLL017 and the MEC-2 CLL cell line are resistant to killing by RTX alone (JPG, 15.3 KB)
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In 51Cr-release assays, labeled CLL B cells (10,000 /well) did not spontaneously release the cytosolic 51Cr tracker when kept alone for 4 h in complete culture medium in the presence of RTX (10 µg/ml). Data are means + 1 s.d. from triplicates. The data are from one representative experiment out of more than twenty performed.
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