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Blood, Vol. 112, Issue 12, 4655-4664, December 1, 2008
Leukemic challenge unmasks a requirement for PI3K in NK cell–mediated tumor surveillance
Blood Zebedin et al.
112: 4655
Supplemental materials for: Zebedin et al
Files in this Data Supplement:
- Document 1. Supplemental methods and materials (PDF, 135 KB)
- Table S1. Expression of MHC I, Rae-1, and Mult-1 on cell lines (PDF, 15.3 KB)
- Table S2. Expression of activating receptors on IL-2 expanded NK-cells derived from PI3Kδ+/− and PI3Kδ−/− mice (PDF, 41.9 KB)
- Table S3. Characterization of PI3Kδ+/− and PI3Kδ−/− NK-cells in cytotoxicity- and degranulation-assays (PDF, 74.3 KB)
- Figure S1. Characterization of PI3Kδ−/− NK-cells (JPG, 76.4 KB)
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(A) Comparable maturation and development of PI3Kδ−/− NK-cells in vivo. NK-cells can be separated into distinct functional subsets according to the expression of the surface markers DX5, Mac-1 and CD27.1 (B, C) Characterization of in vitro IL-2 expanded NK-cell cultures ten days after the initial isolation. No differences in the expression of surface markers were detected. (C) In vitro expanded NK-cells were analyzed for the expression of perforin, granzymes, NKG2D and FasL by semi-quantitative RT-PCR. No significant differences were detected.
1Hayakawa Y, Watt SV, Takeda K, Smyth MJ. Distinct receptor repertoire formation in mouse NK cell subsets regulated by MHC class I expression. J Leukoc Biol. 2008; 83:106–111.
- Figure S2. Analyzing R-ADCC - Redirected antibody dependent cellular cytotoxicity (JPG, 42.6 KB)
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(A) R-ADCC assay was performed according to Caraux et al.2 (FcR+) Daudi cells were lysed by PI3Kδ+/− NK-cells in an antibody-dependent manner. PI3Kδ−/− NK-cells did not induce significant lysis irrespective of the stimulus used. Activating stimuli included antibodies directed against Ly49D, Ly49H, CD16 and CD244.
2Caraux A, Kim N, Bell SE, Zompi S, Ranson T, Lesjean-Pottier S, Garcia-Ojeda ME, Turner M, Colucci F. Phospholipase C-gamma2 is essential for NK cell cytotoxicity and innate immunity to malignant and virally infected cells. Blood. 2006;107:994–1002.
- Figure S3 PI3Kδ−/− NK-cells fail to secret IFN-γ (JPG, 115 KB)
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(A) Reduced IFN-γ secretion of PI3Kδ−/− NK-cells (ELISA). Stimulation of INF-γ secretion by co-incubation with YAC-1, wt v-abl target cells and Eµ-myc-derived tumor cells. IFN-γ was analyzed by ELISA in the supernatants and is illustrated for YAC-1 targets. IFN-γ was also measured at higher levels in the supernatants of PI3Kδ+/− NK-cells (174 pg/ml for wt v-abl targets and 237 pg/ml for Eµ-myc-derived target cells) when compared to PI3Kδ−/− NK-cells (67 pg/ml for wt v-abl target cells and 60 pg/ml for Eµ-myc-derived target cells). (B) Intracellular IFN-γ protein levels determined by FACS. Following co-incubation with tumor targets (exemplified by co-incubation with YAC-1) lower amounts of secreted IFN-γ in the supernatant of PI3Kδ−/− NK-cells were reflected reciprocally in significantly more PI3Kδ−/− NK-cells that were positive for intracellular IFN-γ (p=0.015, right hand panel). In the absence of target cells the basal IFN-γ protein levels were comparable (p=0.11, left hand panel). (C) Comparable basal mRNA levels of IFN-γ as determined by semi-quantitative RT-PCR. Taken together, these results suggest a secretion defect of PI3Kδ−/− NK-cells resulting in an accumulation of intracellular IFN-γ. (D) To ensure the specificity of the IFN-γ signal NK-cells derived from INF-γ−/− animals were included in the determination of IFN-γ by FACS.
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