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This report is for researcher, MD, Ph.D , not for general.

Results
Establishment of NK cell lines expressing functional KIR Studies of mutant KIR are hampered by the inability to transfect
human NK cell lines or primary clones with standard mammalian expression vectors. Therefore, we sought to establish a retroviral transduction system to introduce exogenous cDNAs and still maintain functional attributes of the cell line. To this end, we used the bicistronic retroviral vector, pBMN-IRES-EGFP, in combination with Phoenix Amphotropic packaging cells to generate recombinant retrovirus. pBMN-IRES-EGFP is capable of generating retrovirus that can coordinately express a cloned cDNA and EGFP simultaneously in transduced cell lines through the incorporation of an intervening IRES sequence. In experiments testing retroviral transduction of several transformed human NK-like cell lines, the human NK-92 lymphoma line demonstrated the highest susceptibility to transduction (5–12% of IL-2-stimulated cells).
The NK-92 cell line is an IL-2-dependent NK-like cell line.
Notably, NK-92 does not express FcRIII (CD16), allowing the use of intact Abs instead of F(ab)2 to manipulate surface receptors.
NK-92 serves as an excellent model system to study KIR functions, because it also exhibits strong target cell cytotoxicity
and expresses no endogenous 3DL1, allowing the 3DL1 cDNAtransduced cells to be specifically stained with anti-3DL1 mAb,DX9, by flow cytometry (as in Fig. 1C). Importantly, transduction of NK-92 cells using this system does not affect growth or cytotoxicity responses (data not shown). The transduced NK-92 cells stably expressed cDNA products for at least 1 mo using pBMNIRES-EGFP and for 3 mo using retrovirus generated with a modified version of the plasmid that lacks the IRES and EGFP elements (data not shown), which was used in some studies.
No differences in biological or biochemical results were observed in the presence or absence of EGFP.
To examine the potential inhibitory function of the 2DL4 cytoplasmic domain in isolation from this receptor’s charged transmembrane segment, we generated a chimeric receptor construct in which the cytoplasmic domain of 2DL4, including the single ITIM, was fused with the extracellular and transmembrane domains of 3DL1 (Fig. 1, A and B). This allowed us to directly compare inhibitory properties of the 2DL4 cytoplasmic domain with those of 3DL1 and avoid any positive influences from the 2DL4 transmembrane domain. Several additional constructs weregenerated to study the effect of ITIM mutation (Y277F) and deletion of various portions of the cytoplasmic domain (Y277F/282H, 272P, 255E) on the inhibition (Fig. 1, A and B). These receptors were expressed on the surface at similar levels in retrovirus-transduced NK-92 cells (Fig. 1C) and another IL-2-dependent human NK cell line, NK3.3 (Fig. 1D).
FIGURE 1. Map of gene constructs used in these studies and establishment of stable transduced NK cell lines. A, Schematic representations of the various chimeric constructs between 3DL1 (open boxes) and 2DL4 (shaded boxes) that were generated. D0, D1, and D2 are highly conserved Ig-like domains in the KIR family. TM and CY represent transmembrane and cytoplasmic domains, respectively. Tyrosine-based ITIM motifs (YAQL and YTEL) are marked in the cytoplasmic domains. Some of the constructs possess the mutant ITIM tyrosine (Y277F). These genes were introduced into a retrovirusbased plasmid vector, pBMN-IRES-EGFP. B, Amino acid sequence of the cytoplasmic domains of the KIR constructs. The 3DL1 and 2DL4 were fused at the common methionine in the cytoplasmic domain by introducing a BspHI restriction site (underlined), which encodes the amino acids VMN. The dots indicate identity of either 3DL1 or 2DL4 to the chimera. Boldface sequences represent the positions of ITIMs. The last amino acids of KIR3DL1/255E, 3DL1/L4/272P, and 3DL1/L4/282H are labeled with #. C, Flow cytometric analysis of 3DL1-positive transduced cells. The 3DL1-negative NK cell line,
NK-92, was introduced with those various constructs shown as in A by retroviral transduction. The data show staining for 3DL1 with PE-labeled DX9 mAb and background staining of the 3DL1-negative parent cell line (thin lines). D, Flow cytometric analysis of NK3.3 cells transduced with either 3DL1 or 3DL1/L4 chimera cDNA. The data show staining with PE-labeled DX9 mAb.....