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Blood, Vol. 111, Issue 3, 1004-1012, February 1, 2008
TACI, unlike BAFF-R, is solely activated by oligomeric BAFF and APRIL to support survival of activated B cells and plasmablasts
Blood Bossen et al.
111: 1004
Supplemental materials for: Bossen et al
Files in this Data Supplement:
- Document 1. Supplemental methods (PDF, 72.1 KB)
- Figure S1. Schematic representation of the different recombinant BAFF used in the study (JPG, 86.6 KB)
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Recombinant BAFF were characterized previously.1
- Figure S2. A non-cleavable form of full length mouse BAFF (JPG, 49.4 KB)
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293T cells were transfected with full length wild type mouse BAFF (BAFF wt) or full length mouse BAFF with mutations R125A and R126A in the consensus furin processing site (BAFF uncleav.). Cells and conditioned medium were harvested and analyzed by western blotting using a cross-reacting anti-mouse BAFF mAb kDa (Buffy-2).
- Figure S3. BAFF 60-mer is a potent activator of BCMA-dependent NF-κB activation (JPG, 67.1 KB)
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293T cells transiently co-transfected with full length human BCMA and NF- B reporter plasmids were stimulated with BAFF 3-mer or 60-mer at the indicated concentrations. NF¬B activity is shown as fold induction compared to BCMA-transfected, but non-stimulated cells. BCMA transfection by itself induced a 3-fold NF-B activation compared to control (ctrl; grey squares).
- Figure S4. BAFF promotes survival, but not proliferation, in co-stimulation assays (JPG, 76.9 KB)
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CFSE-labelled B cells were cultured for 3 days plus or minus anti-IgM, and in the presence of the indicated ligands. Cells were analyzed by FACS (3 × 104 events). Histograms show the CFSE profile of cells in the live lymphocyte gate.
- Figure S5. BAFF oligomers but not BAFF 3-mer co-stimulate TACI-dependent thymidine incorporation (JPG, 55.8 KB)
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Purified wild type B splenocytes were cultured under BCR-stimulating conditions, with or without anti-BAFF-R B9C11, and with the indicated concentrations of BAFF 3-mer and oligomers. After 48 h, cells were pulsed for 16 h with 3H-thymidine, harvested and counted. Points represent means ± SD of triplicate cultures.
- Figure S6. BAFF 60-mer signaling through TACI can be inhibited by an excess of BAFF 3-mer (JPG, 49 KB)
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Purified BCMA−/− B splenocytes were cultured under BCR-stimulating conditions with anti-BAFF-R B9C11, in the presence of fixed concentrations of BAFF 60-mer N242Q (0, 4, 20 or 100 ng/ml), and with the indicated concentrations of BAFF 3-mer H218A. After 48 h, cells were pulsed for 16 h with 3H-thymidine, harvested and counted. The specific effect of BAFF 60-mer is shown (Specific effect of BAFF 60-mer = thymidine incorporation with BAFF 60-mer − thymidine incorporation without BAFF 60-mer. Thymidine incorporation without BAFF 60-mer varied form 13 to 15 × 103 cpm). Points represent means ± SD of triplicate cultures. Similar results were obtained with wild type B cells (data not shown).
- Figure S7. BAFF oligomers but not BAFF 3-mer signal MHC class II (JPG, 51.2 KB)
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Purified wild type B splenocytes were stimulated with the indicated BAFF preparations and surface expression of MHC class II was monitored by FACS. MFI: mean fluorescence intensity. This experiment was performed in the absence of IL4 and IL6.
- Figure S8. Enhanced sensitivity of multimeric BAFF detection using cycloheximide and Jurkat BCMA:Fas cells (JPG, 47.4 KB)
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Jurkat BCMA:Fas cells were incubated with the indicated volume of plasma from BAFF transgenic mice, in a final volume of 50 µl, and in the presence or absence of 1.5 µg/ml of cycloheximide and of 2.5 µg/ml of BCMA-Fc. Cell viability was measured after 16 h incubation with the PMS/MTS assay.
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