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Blood, Vol. 112, Issue 10, 4128-4138, November 15, 2008
Natural killer T-cell autoreactivity leads to a specialized activation state
Blood Wang et al.
112: 4128
Supplemental materials for: Wang et al
Files in this Data Supplement:
- Figure S1. Flow cytometric analysis of CD1d expression by human myeloid DCs (left panel) and CD1d transfected 3023 human lymphoblastoid cells (JPG, 48.6 KB)
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Human monocytes and DCs express similar levels of CD1d (not shown).
- Figure S2. CD4− NKT cell clones show hierarchical cytokine production in response to increasing TCR stimulation (JPG, 91.6 KB)
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(A) A CD4− NKT cell clone (GL1.5) was stimulated by CD1d transfected APCs that were pulsed with low (open bars) or high (filled bars) doses of α-GalCer. (B) A CD4− NKT cell clone (GL1.1) was stimulated by plate-bound CD1d-Fc fusion protein pulsed with titrated concentrations of α-GalCer. Cytokine secretion was quantitated by ELISA. Data are represented as means and SDs; the “#” symbol indicates not tested.
- Figure S3. Enhancement of cytokine production by IL-12 and IL-18 co-stimulation (JPG, 37.9 KB)
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NKT cells were exposed to CD1d transfected APCs alone (“Auto”) or in the presence of recombinant IL-12 and IL-18 (“Auto+IL-12&18”), and secretion of the indicated cytokines was assayed by ELISA.
- Figure S4. Control experiments for NKT cell pre-exposure analysis (JPG, 81.9 KB)
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(A) The left plot shows flow cytometric analysis of CD1d transfected APCs that were surface labeled with biotin and fluorescently labeled with CFSE mixed with untreated APCs. The right plot shows flow cytometric analysis of the population after biotinylated cells were depleted by magnetic sorting. The frequencies of the CFSE labeled cells in the starting sample and after depletion are shown above the respective gate markers. (B) NKT cells were pre-exposed to biotinylated CD1d+ APCs, then these cells were incubated with untransfected APCs either without added cytokines (“NT”), in the presence of IL-12 and IL-18 (“IL-12&18”), or in the presence of IL-12 and IL-18 and an anti-CD1d mAb (“IL-12&18+CD1d59”). (C) NKT cells were mixed at the indicated ratios with CD1d+ APCs either without added cytokines (open bars) or with IL-12 and IL-18 (filled bars). (D) NKT cells were pre-exposed to the U0126 MEK inhibitor or mock treated for 4 hours, then washed twice and after two hours the NKT cells were co-incubated with CD1d transfected APCs alone (“Auto”) or in the presence of IL-12 and IL-18. IFNγ secretion was quantitated by ELISA.
- Video 1. Autoreactive interaction: close up view of NKT (green) + untreated DC (red) (MOV, 84.2 KB)
- Video 2. α-GalCer interaction: close up view of NKT (green) + α-GalCer pulsed DC (red) (MOV, 85.3 KB)
- Video 3. α-GalCer interaction: close up view of NKT (green) + a-GAlCer pulsed CD1d transfectant (red) (MOV, 25.7 KB)
- Video 4. Autoreactive interaction: close up view of NKT (green) + untreated CD1d transfectant (red) (MOV, 62.0 KB)
- Video 5. No CD1d interaction: close up view of NKT (green) + CD1d-negative APCs (red) (MOV, 23.4 KB)
- Video 6. α-GalCer interaction: large field view of NKT cells (green) with α-GalCer pulsed DCs (red) (MOV, 286 KB)
- Video 7. Autoreactive interaction: large field view of NKT cells (green) with untreated DCs (red) (MOV, 621 KB)
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