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Blood, Vol. 113, Issue 13, 2955-2964, March 26, 2009
Detuning CD8+ T lymphocytes by down-regulation of the activating receptor NKG2D: role of NKG2D ligands released by activated T cells
Blood Cerboni et al.
113: 2955
Supplemental materials for: Cerboni et al
Files in this Data Supplement:
- Figure S1. Analysis of different receptors on SEB-activated CD8+ T cells (JPG, 49.8 KB)
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Total or CD4-depleted PBMCs were CFSE-labeled and stimulated with SEB. After 5 days, cells were harvested and stained with anti-CD8, anti-CD3, and with mAbs specific for NKG2D, DNAM, CD28 or LFA-1 (thick line) as indicated, or control IgG isotype (filled histogram). Data shown are from CD3+CD8+ T cells. Numbers represent the MFI values relative to each receptor, analyzed as described in the legend of Fig. 1. One representative donor out of 5 tested is shown. In the right column, expression of CD3 is reported and MFI values are indicated.
- Figure S2. Standardization of the ULBP3 sandwich ELISA (JPG, 35.3 KB)
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(A) Serial dilutions of recombinant ULBP3-Fc were analyzed in a sandwich ELISA (see Materials and methods). The means of values ± SE obtained from five different experiments are shown. (B) Different dilutions of supernatants from BAF cells and the respective ULBP3 transfectant were tested for soluble ULBP3 by the specific sandwich ELISA. The means of triplicates ± SD are shown.
- Figure S3. Release of TGF-b1 and IL-21 by SEB-activated PBMCs (JPG, 39.3 KB)
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Total (black circles) or CD4-depleted (white circles) PBMCs were activated with SEB and cell culture supernatants were harvested after 3, 5 and 7 days and tested in ELISA. Each circle represents one donor, tested in triplicates, on both cell populations. The number of donors analyzed were 11 for TGF-b1 and 6 for IL-21. Solid lines indicate the average of all measurements in the respective group. Statistical analysis with paired Student’s t-test. Medium supplemented with fetal calf serum was positive for soluble TGF-b1 and its value (1 ng/ml) was subtracted.
- Figure S4. Fixation of CD4+ cells only partially restores NKG2D expression on NKG2D low cells (JPG, 24.9 KB)
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CFSE-labelled and CD4-depleted, or unlabelled total PBMCs were cultured in the presence of SEB. After 3 days, CD4+ cells were purified from total PBMCs, fixed or not in paraformaldehyde and plated with CD4¬depleted CFSE+ PBMCs at different ratio (with an excess of purified CD4+ cells). Cells were harvested 2 days later, and NKG2D or CD48 (used as a control) expression was evaluated by gating on proliferating CFSE+CD3+CD8+ cells. Data are from one out of 2 donors. Fixation did not alter binding of the ligands to the recombinant receptor, as evaluated by comparing the staining of NKG2D-Fc protein to fixed and not fixed cells (data not shown).
- Figure S5. Recovery of NKG2D expression (JPG, 23.2 KB)
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Total PBMCs were harvested after 5 days of SEB stimulation, washed and then cultured for additional 3 days with fresh medium, or with medium collected from day 5 SEB-cultured cells (SEB medium). Cells were stained at day 5 and for the additional 3 days, with anti-CD3, anti-CD8, and anti-NKG2D mAbs. MFI NKG2D values are referred to CD8+CD3+ T cells. Data are representative of one out of 3 donors.
- Figure S6. Statistical analysis of the proliferative and cytotoxic response (JPG, 43.6 KB)
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NKG2D low and NKG2D high cells were obtained as described in the legend of Fig. 6. (A–B) Analysis of the proliferative response. Mean values from 4 donors ± SE are shown. (C–D) Analysis of the degranulation. At 5 mg of anti-CD3 mAb, no effect of NKG2D costimulation was observed. Mean values from 3 donors ± SD are shown. Statistical analysis with paired Student’s t-test.
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