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Blood, Vol. 110, Issue 6, 1960-1969, September 15, 2007
Bone marrow CD8 cells down-modulate membrane IL-7R expression and exhibit increased STAT-5 and p38 MAPK phosphorylation in the organ environment.
Blood Cassese et al.
110: 1960
Supplemental materials for Cassese et al, Vol. 110, Issue 6, 1960-1969
Files in this Data Supplement:
- Document 1. Supplemental methods (PDF, 72.6 KB)
- Figure S1. Proliferative response to IL-15 and IL-7 by purified CD8 cells from spleen and BM (PDF, 16.9 KB) -
Purified CD8+ cells (5 ×104 cells/well) from spleen and BM of untreated B6 mice were cultured for 5 days in the presence of either medium or the indicated concentrations of cytokines. H3-thymidine incorporation was determined after a 12-hour pulse. (A) IL-15. (B) IL-7. In both panels, results are representative of at least 3 independent experiments. For each experiment, cells were pooled from 8 to 10 mice.
- Figure S2. Bcl-2 expression and survival response to IL-15 and IL-7 of spleen and BM CD8 cells (PDF, 65.6 KB) -
(A and B) Bcl-2 staining. Single cell suspensions were prepared from spleen and BM of untreated B6 mice. Cells from individual mice were stained with anti-TCR Alexa 647, anti-CD8 PerCP-Cy5.5, anti-CD44 FITC, plus either control PE or anti-Bcl-2 PE mAb and analyzed by flow cytometry. After gating on either CD44high or CD44int/low TCR+CD8+ cells, the mean fluorescence intensities (MFI) of Bcl-2 mAb and control mAb were determined. Representative staining profiles (A) and Bcl-2 MFI individual values and averages (B). In A, the scale on x-axis is logarithmic, in arbitrary units, and the scale on the y-axis is linear (maximum value 30 for CD44high and 80 for CD44int/low panels). The numbers represent the MFI of Bcl-2 mAb (thick line) and control mAb (thin line). In B, the results obtained in 4 independent experiments are reported, and the average is shown with the horizontal bar. (C and D) Survival response to either IL-15 or IL-7. Purified CD8+ cells (5 ×104 cells/well) from spleen and BM of untreated B6 mice were cultured for 3 days in the presence of either medium alone or the indicated concentrations of cytokines. At the end of incubation, the percentage of propidium iodide (PI) + dead cells was determined for each sample. Examples of PI staining profiles (C) and comparison of spleen and BM CD8 cell survival in response to either 10 ng/ml of IL-15 or graded concentrations of IL-7 (D). In C, the scales on the x- and y-axes are logarithmic, in arbitrary units. The numbers represent the percentage of PI+ cells in the gated region. As a control, cells were incubated with PBS. One representative experiment of 3 is shown. For each experiment, cells were pooled from 8 to 10 mice. Differences between BM and spleen were similar in the three experiments and not statistically significant (average difference never >3% for any cytokine concentration).
- Figure S3. Modulation of CD122 after culture with IL-15 and IL-7 (PDF, 11.7 KB) -
Purified CD8+ cells (5 ×104 cells/well) from BM and spleen of untreated B6 mice were cultured for 3 days in the presence of medium, IL-15, or IL-7, as indicated. At the end of incubation, the percentages of CD122high cells within PI− CD44high and CD44int/low cells were determined. One representative experiment is shown out of 3. For each experiment, BM and spleen cells were pooled from 8-10 mice.
- Figure S4. Phopsho-STAT-5 Western blotting (PDF, 51 KB) -
CD8+ cells were purified from BM and spleen of untreated B6 mice. Cells were lysed and Western blot analysis was performed with specific anti-phospho-STAT-5 mAb. Spleen cells stimulated in vitro for 15 minutes with IL-15 at 25 ng/ml were used as positive control. The panels show typical results from one experiment (lysates from 8 × 105 cells/lane). Since total STAT-5 levels were lower in BM than in spleen CD8 cells (see manuscript results and data not shown), loading control was performed with actin. Densitometric analysis showed that the ratio between phospho-STAT-5 and actin was 0.091 for BM and 0.026 for spleen. One representative experiment is shown out of 3. For each experiment, BM and spleen cells were pooled from 10 to 13 mice.
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