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Blood, Vol. 110, Issue 2, 606-615, July 15, 2007
Antigen-activated human T lymphocytes express cell-surface NKG2D ligands via an ATM/ATR-dependent mechanism and become susceptible to autologous NK- cell lysis
Blood Cerboni et al.
110: 606
Supplemental materials for Cerboni et al, Vol 110, Issue 2, 606-615
Files in this Data Supplement:
- Table S1. MFI of NKG2D ligands on SEB-activated CFSEhigh , CFSEmedium and CFSElow cells (gate on CD4+ T cells) (PDF, 169 KB) -
Analysis was performed on SEB-activated T cells as described in the legends of Figures 1,2. CFSEhigh , CFSEmedium and CFSElow cells were identified by the sequential loss of CFSE fluorescence intensity: CFSEhigh , cells that had not divided (both quadrants to the right of the R1 gate; Figure 1); CFSElow , cells at the very left of the R1 gate, corresponding to the last 1-2 cellular divisions; CFSEmedium , all cells between CFSEhigh and CFSElow cells. Numbers represent the average and the range of the MFI values obtained from several donors.
- Table S2. MFI of NKG2D ligands on SEB-activated CFSEhigh , CFSEmedium and CFSElow cells (gate on CD8+ T cells) (PDF, 170 KB) -
Analysis was performed on SEB-activated T cells as described in the legends of Figures1,2. CFSEhigh , CFSEmedium and CFSElow cells were identified by the sequential loss of CFSE fluorescence intensity, as described in Table S1. Numbers represent the average and the range of the MFI values obtained from several donors.
- Figure S1. Unstimulated T cells did not express NKG2D ligands on their cell surface (JPG, 89.7 KB)
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PBMCs were CFSE-labelled and stained with anti-CD3 and antibodies specific for NKG2D ligands (MICA, ULBP1, 2 and 3), and their expression was evaluated on CD3+ T cells at day 0 and after 3, 5 and 7 days of culture in medium without SEB. The percentage of cells in all quadrants is indicated. Representative dot plots from 1 out of 5 donors are shown.
- Figure S2. MICA, ULBP1, and ULBP3 staining was blocked by the specific NKG2D ligand fusion protein (NKG2DL-Fc) (JPG, 49.7 KB)
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MICA-Fc, ULBP1-Fc, ULBP2-Fc and ULBP3-Fc or a control-Fc (2 µg of each Fc) were preincubated with the specific mAb directed against the relative ligand (eg, MICA-Fc with the anti-MICA mAb) for 30 minutes on ice, and then added to CFSE-labelled, SEB-activated PBMCs. Binding was detected using GAM-PE, followed by anti-CD3 staining. Control-Fcs were as follows: anti-MICA, antiULBP1, and anti-ULBP3 mAbs were preincubated with ULBP2-Fc, while anti-ULBP1 mAb was preincubated with MICA-Fc. MFI values relative to each ligand and calculated on cells gated in R1 (which includes both quadrants to the left), are shown. Representative dot plots from 2 out of 4 donors are shown.
- Figure S3. NKG2D ligands were mainly expressed on proliferating (CFSElow ) cells (JPG, 47.3 KB)
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PBMCs were activated, harvested, and stained as described in the legend of Figure 1. Dot plots were divided in four quadrants, and percentages of NKG2DL+ cells were calculated among the upper-right (UR) and upper-left (UL) quadrants, corresponding to not proliferating-NKG2DL+ (CFSEhigh ) and to proliferating-NKG2DL+ (CFSElow ) cells, respectively. The sum of all NKG2DL+ cells (ie, UR+UL) was considered as 100%. The percentage of proliferating-NKG2DL+ cells increased with time, while the percentage of NKG2DL+ but not proliferating cells (UR) decreased, suggesting that some of the latter cells entered cell division with time (ie, moved from the UR to the UL quadrant).
- Figure S4. NKG2DLs were expressed on up to 50% of proliferating (CFSElow ) cells (JPG, 46.3 KB)
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PBMCs were activated, harvested and stained as described in the legend of Figure 1. Dot plots were divided in four quadrants, and percentages of NKG2DL+ and NKG2DL− cells were calculated among all proliferating (CFSElow ) cells. The sum of proliferating cells (ie, UL+LL) was considered as 100%. The analysis shows that among proliferating cells, 10 to 50% of them expressed NKG2DLs. UL, upper-left quadrant (proliferating-NKG2DL+ cells). LL, lower-left quadrant (proliferating-NKG2DL− cells).
- Figure S5. Kinetics of MICA expression on SEB activated T cells (JPG, 42.8 KB)
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PBMCs were activated as described in the legend of fig. 1. Cells were stained with mAb specific for CD3, MICA (thick line) or control Ig isotype (filled histogram). MFI values relative to MICA and evaluated on CD3+ T cells are shown.
- Figure S6. The ATM specific pharmacological inhibitor KU-55993 blocks MICA expression on PHA-activated T cells (JPG, 41.4 KB)
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(A) PBMCs were pretreated with caffeine (5mM) or KU-55933 (10µM) and then stimulated with PHA for 18 hours. Cells were stained with mAbs specific for CD3, MICA (thick line) or control Ig isotype (filled histogram). Expression of MICA was evaluated on CD3+ T cells. (B) PBMCs were prepared as described in A. Data are represented as the mean (± SD) of the percentage of CD3+MICA+ cells of three different healthy donors. Significant differences, as calculated by paired t-test, are indicated: **P<0.01;*** P<0.001.
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