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Blood, Vol. 108, Issue 8, 2712-2719, October 15, 2006
B-chronic lymphocytic leukemia cells and other B cells can produce granzyme B and gain cytotoxic potential after interleukin-21-based activation
Blood Jahrsdörfer et al.
108: 2712
Supplemental materials for: Jahrsdörfer et al
Files in this Data Supplement:
- Figure S1. IL-21 receptor is expressed on B-CLL cells and is upregulated by CpG ODN (JPG, 33.9 KB)
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PBMC from 9 subjects with B-CLL were isolated, suspended in AIM-V medium and cultured for 2 hours, 4 days or 7 days in the presence or absence of CpG ODN or control ODN at 2.5 µg/ml. Gene expression of the IL-21 receptor (n=9) was assessed with two different probes after 2 hours (Figure 1A), IL-21 receptor protein expression on B-CLL cells (n=3) was measured by flow cytometry (median fluorescence intensity, MFI) on days 4 and 7 (Figure 1B). Error bars indicate SEM.
- Figure S2. DNA fragmentation in B-CLL cells is detectable as early as 12 hours after start of incubation with IL-21 and CpG ODN (JPG, 29.5 KB)
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B-CLL cells were isolated, purified to a percentage of at least 99.9% based on CD19 staining and cultured in the presence of IL-21 and CpG ODN for 12 hours. Then cells were harvested, fixed, permeabilized and FITC-labeled dUTP was enzymatically linked to fragmented DNA. Histograms show the percentages of B-CLL cells positive for dUTP.
- Figure S3. B-CLL cells secrete granzyme B in response to IL-21 (JPG, 56.2 KB)
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B-CLL cells were purified based on CD19 expression to a percentage of >99.9%. The cells were then cultured at 37°C on 96-well EliSpots plates for granzyme B detection at the indicated cell number per well and in the presence of different B cell activators alone or with IL-21. After 16 hours plates were developed and dots counted. Every condition was run in triplicates. Panels A shows one representative experiment with decreasing cell numbers of purified B-CLL cells. Panel B shows PHA controls with whole PBMC and purified B-CLL cells.
- Figure S4. Granzyme B produced by B-CLL cells is able to cleave specific substrate and is accompanied by the occurrence of functionally active caspase 6 (JPG, 11 KB)
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PBMC from 2 subjects with B-CLL were isolated and cultured for 4 days in the presence of IL-21 (100 ng/ml) and CpG ODN (2.5 µg/ml). Activity of caspase 6 in B-CLL cells was determined using specific cell-permeable fluorogenic substrates and staining of CD19. The percentages of B-CLL cells positive for activated caspase 6 are shown, averaged from two independent experiments.
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