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Blood, Vol. 112, Issue 12, 4694-4698, December 1, 2008
Clinical improvement by farnesyltransferase inhibition in NK large granular lymphocyte leukemia associated with imbalanced NK receptor signaling
Blood Epling-Burnette et al.
112: 4694
Supplemental materials for: Epling-Burnette et al
Files in this Data Supplement:
- Figure S1. Extensive phenotype analysis of CD56+/CD16+ NK cells from the LGL leukemia patient (JPG, 55.2 KB)
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(A) The phenotype was determined by flow cytometry and dot plots are shown on CD56+/CD16+/CD3− NK-cells (R2) from the NK-LGL patient in Fig. 1a. The reactivity of NK-cells to anti-NKG2D (a), anti-NKp30 (b), anti-NKp46 (c), anti-NKp44 (d), anti-NKB2 (KIR3DL1) (e), anti-244 (2B4) (f), anti-CD161 (g), anti-NKG2A (h), and anti-CD57 (i).
- Figure S2. Blockade of 721.221 lysis by anti-CD158a and CD16 antibodies and overexpression of Cw3 (JPG, 44.5 KB)
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(A) NK-cells from the LGL patient were exposed to medium, isotype control antibody, anti-NKp46, anti-CD158a (KIR2DL1/KIR2DS1), anti-CD158b (KIR2DL2/KIR2DL3/KIR2DS2/KIR2DS3), and anti-CD16 antibodies prior to the interaction with 721.221 target cells in 51-Cr release assays. (B) NK-cells from the LGL patient were used as effector cells in 51-Cr release assays when K562, 721.221 parental cells expressing no HLA-class I molecules, and 721.221 cells transfected with Cw3 (721.Cw3) or Cw4 (721.Cw4). HLA-Cw3 represents a C1 antigen that binds KIR2DL2/2DL3/2DS2/2DS3. In this patient, HLA-Cw07 that is also a C1 antigen was present. In contrast, HLA-Cw4 represents a C2 antigen that binds KIR2DL1/KIR2DS1 and the HLA-C1 antigen expressed by this patient is HLA-Cw15. These results show that the KIR2DL1 interaction with the Cw4-C2 antigen was defective and failed to protect 721.221 cells from lysis.
- Figure S3. Lysis of CRL-2598 cells mediated by NKG2D and KIR2DS1 (JPG, 41.4 KB)
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NK-cells from the LGL patient were used at a 50:1 effector-to-target (E:T) ratio with CRL-2598 cells as a target in 51-Cr release cytotoxicity assays. Blockade in cytotoxicity was examined with medium, isotype control antibody (Iso), anti-NKG2D, anti-NKp30, anti-NKp46, anti-NKp44, anti-CD158a (KIR2DS1/KIR2DL1), anti-CD158b (KIR2DS2/KIR2DL2/KIR2DL3), and anti-CD244 (2B4), anti-HLA-Class I added to the target cells.
- Figure S4. Perforin granule mobilization blocked by dnDAP proteins (JPG, 45.5 KB)
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NK-cells from the LGL leukemia patient were treated under mock conditions, infected with recombinant CD56 vaccinia virus (CD56, control), dnDAP10 alone, dnDAP12 alone, or a combination of dnDAP10 plus dnDAP12 recombinant vaccinia viruses. These groups of effector cells were admixed at 6:1, 12:1, 25:1 and 50:1 effector to target ratios with the CRL-2598 target cells. A proportion of the cells were examined for cytotoxicity in 5-hr 51-Cr release assays shown in Fig. 1. A portion of cells were stained and perforin granule redistribution examined. Perforin is shown after staining with anti-perforin-FITC (green) (a). CRL-2598 cells were stained with cell tracker orange (red) and the nucleus stained with DAPI (blue) (b). Cell treatments are indicated in c–h.
- Figure S5. Dose-dependent apoptosis after tipifarnib in vitro (JPG, 14.3 KB)
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Results of annexin-V/7-actinomycin D staining of PBMC from the LGL leukemia patient (dark blue) or three healthy controls treated with DMSO (drug solvent control), 100 nM, 500 nM, 1µM, and 5µM tipifarnib in vitro.
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