1. Arrested natural killer cell development associated with transgene insertion into the Atf2 locus
by Sungjin Kim, Yun-Jeong Song, Darryl A. Higuchi, Hyunseok P. Kang, Jennifer R. Pratt, Liping Yang, Caron M. Hong, Jennifer Poursine-Laurent, Koho Iizuka, Anthony R. French, John B. Sunwoo, Shunsuke Ishii, Andreas M. Reimold, and Wayne M. Yokoyama
Blood
Volume 107(3):
February 1, 2006
©2006 by American Society of Hematology

2. Accumulation of NK cells in the BM of Tg mice.
Accumulation of NK cells in the BM of Tg mice. BM and spleen cells from WT and Tg mice were stained for NK1.1 and CD3 as indicated. Only viable cells are shown, and the numbers represent the percentages of cells in each quadrant. Data are representative of analysis of at least 5 Tg and WT mice. There was no change in total number of spleen or BM cells in Tg mice (data not shown).
Sungjin Kim et al. Blood 2006;107:
©2006 by American Society of Hematology

3. Functional immaturity of Tg NK cells.
Functional immaturity of Tg NK cells. (A) Freshly isolated BM cells from WT and Tg mice pretreated with poly-I:C were used in standard cytotoxicity assays against the indicated targets. (B) BM and spleen cells were stimulated with IL-2 plus IL-12 and analyzed for intracellular IFNγ. Gated NK1.1+ CD3- cells are shown, and the numbers represent the percentages of IFNγ-producing cells. (C) Spleen cells from indicated mice were co-incubated with YAC-1 tumor cells and analyzed for intracellular IFNγ. Gated NK1.1+ CD3- cells are shown, and the numbers represent the percentages of IFNγ-producing cells. (D) Splenocytes from indicated mice were stimulated by anti-NK1.1 cross-linking then analyzed for intracellular IFNγ. Gated NK1.1+ CD3- cells are shown, and the numbers represent the percentages of IFNγ-producing cells. All data are representative of analysis of at least 3 WT and Tg mice.
Sungjin Kim et al. Blood 2006;107:
©2006 by American Society of Hematology

4. Immature phenotype of Tg NK cells.
Immature phenotype of Tg NK cells. (A) BM and spleen cells from WT and Tg mice were stained for Mac-1, NK1.1, and CD3. Shown are profiles of Mac-1 expression on gated CD3- cells, and the numbers represent the percentages of cells in each quadrant. (B) BM and spleen cells were stained for CD43, DX5, NK1.1, and CD3. Gated NK1.1+ CD3- cells are shown, and the numbers represent the percentages of CD43hi cells. (C) BM and spleen cells were stained for integrin αv, DX5, NK1.1, and CD3. Gated NK1.1+ CD3- cells are shown, and the numbers represent the percentages of cells in each quadrant. More than 5 mice were analyzed for each marker.
Sungjin Kim et al. Blood 2006;107:
©2006 by American Society of Hematology

5. Integration of Tg construct into ATF-2 gene.
Integration of Tg construct into ATF-2 gene. (A) Schematic representation of WT and Tg ATF-2 allele with Tg construct integration. White vertical boxes represent exons, and the arrowheads (a1, a2, b1, and b2) represent primers amplifying the junction sequences, parts of which are shown. Atf2 genomic sequence information was obtained from C57BL/6J mouse BAC clone RP (accession number AL ), and granzyme A genomic sequence information was obtained from C57BL/6J mouse chromosome 13 genomic contig (accession number NT_039590). (B) PCR products amplified by the indicated primer sets (panel A) are present only in Tg genomic DNA samples. WT DNA did not prime amplification. (C) RT-PCR of abnormal Atf2 transcripts in Tg NK cells. RT-PCR products from the indicated RNA samples are shown. Primers were derived from intron 10 (c1) and exon 12 of Atf2 (c2) (A). The c1:c2 product represents RNA, which has spliced out intron11. Primers from HPRT (bottom panel) indicate equivalent template availability. RNA was prepared from LAK cells of indicated mice or from EL-4 cell line.
Sungjin Kim et al. Blood 2006;107:
©2006 by American Society of Hematology

6. Intrinsic defect in the NK-cell lineage derived from Tg mice.
Intrinsic defect in the NK-cell lineage derived from Tg mice. (A) Spleen cells from indicated BM chimeric mice were stained for Mac-1, NK1.1, and CD3. Gated CD3- cells are shown, and the numbers represent the percentages of cells in each quadrant. Similar numbers of splenocytes were found in each chimeric mouse (data not shown). Two mice were analyzed for each group, and similar results were obtained. (B) Freshly isolated spleen cells from indicated control and chimeric mice pretreated with poly-I:C were used in standard cytotoxicity assays against the indicated targets. The bars represent standard deviation of triplicate wells for each point. Data are representative of 2 independent experiments with similar results.
Sungjin Kim et al. Blood 2006;107:
©2006 by American Society of Hematology