Defective development of NK1 Defective development of NK1.1+ T-cell antigen receptor αβ+ cells in zeta-associated protein 70 null mice with an accumulation of NK1.1+CD3− NK-like cells in the thymus by Kazuya Iwabuchi, Chikako Iwabuchi, Saori Tone, Daisuke Itoh, Noriko Tosa, Izumi Negishi, Kazumasa Ogasawara, Toshimitsu Uede, and Kazunori Onoé Blood Volume 97(6):1765-1775 March 15, 2001 ©2001 by American Society of Hematology

NK1.1 expression on thymocytes and splenocytes from control (ZAP-70+/−) and ZAP-70−/− mice.Thymocytes and splenocytes were stained with PE–anti-NK1.1 and FITC–anti-CD3, or FITC–anti-TCRαβ, or with PE–anti-NK1.1 and biotin–anti-TCRγδ/streptavidin-FITC. NK1.1 expression on thymocytes and splenocytes from control (ZAP-70+/−) and ZAP-70−/− mice.Thymocytes and splenocytes were stained with PE–anti-NK1.1 and FITC–anti-CD3, or FITC–anti-TCRαβ, or with PE–anti-NK1.1 and biotin–anti-TCRγδ/streptavidin-FITC. Expressions of NK1.1 (vertical) and CD3, TCRαβ, or TCRγδ (horizontal) are illustrated. Proportions of NK1.1+ CD3− and NK1.1+ CD3+ (left column), NK1.1+TCRαβ− and NK1.1+ TCRαβ+(middle column), and NK1.1+ TCRγδ−, NK1.1+ TCRγδ+, and NK1.1−TCRγδ+ (right column) cells are indicated in the figures. Results are representative of 6 independent experiments. Kazuya Iwabuchi et al. Blood 2001;97:1765-1775 ©2001 by American Society of Hematology

Cell number of NK1. 1+TCRαβ+, NK1. 1+ TCRαβ−, NK1. 1+ TCRγδ+, and NK1 Cell number of NK1.1+TCRαβ+, NK1.1+ TCRαβ−, NK1.1+ TCRγδ+, and NK1.1−TCRγδ+ subpopulations in the thymus and spleen of control and ZAP-70−/− mice.Mean cell numbers of each subpopulation in the thymus and spleen of control (▪, n = 6) and ZAP-70−/− (■, n = 5)... Cell number of NK1.1+TCRαβ+, NK1.1+ TCRαβ−, NK1.1+ TCRγδ+, and NK1.1−TCRγδ+ subpopulations in the thymus and spleen of control and ZAP-70−/− mice.Mean cell numbers of each subpopulation in the thymus and spleen of control (▪, n = 6) and ZAP-70−/− (■, n = 5) mice were calculated and shown as means and SD. Kazuya Iwabuchi et al. Blood 2001;97:1765-1775 ©2001 by American Society of Hematology

Phenotype of NK1. 1+ thymocytes from control and ZAP-70−/− mice Phenotype of NK1.1+ thymocytes from control and ZAP-70−/− mice.Thymocytes were stained with PE–anti-NK1.1 or FITC–anti-NK1.1 and various cell surface markers as described in “Materials and methods.” Expressions of NK1.1 (vertical) and other markers (horizon... Phenotype of NK1.1+ thymocytes from control and ZAP-70−/− mice.Thymocytes were stained with PE–anti-NK1.1 or FITC–anti-NK1.1 and various cell surface markers as described in “Materials and methods.” Expressions of NK1.1 (vertical) and other markers (horizontal) on thymocytes are illustrated: expressions of NK1.1 and T-cell surface markers (A), NK surface markers (B), and stem cell markers (C). Results are representative of 6 independent experiments. Kazuya Iwabuchi et al. Blood 2001;97:1765-1775 ©2001 by American Society of Hematology

DX5 expressions on NK1.1+ thymocytes and splenocytes from control (ZAP-70+/−) and ZAP-70−/− mice.Thymocytes and splenocytes were stained with PE–anti-NK1.1, FITC–anti-TCRαβ, and biotinylated anti-DX5 followed by streptavidin–Red 670. DX5 expressions on NK1.1+ thymocytes and splenocytes from control (ZAP-70+/−) and ZAP-70−/− mice.Thymocytes and splenocytes were stained with PE–anti-NK1.1, FITC–anti-TCRαβ, and biotinylated anti-DX5 followed by streptavidin–Red 670. NK1.1+ TCRαβ+ or NK1.1+ TCRαβ− cells in the thymus and spleen were electronically gated and analyzed for the expression of DX5 on FACScan. Dead cells were electronically gated out with propidium iodide staining. FACS profiles were shown in contour with NK1.1 versus DX5 staining. Kazuya Iwabuchi et al. Blood 2001;97:1765-1775 ©2001 by American Society of Hematology

Phenotype of NK1. 1+CD3−thymocytes from ZAP-70−/−, RAG-1−/−, and B6 Phenotype of NK1.1+CD3−thymocytes from ZAP-70−/−, RAG-1−/−, and B6.Whole thymocytes (B6-RAG-1−/−), thymocytes enriched for CD8− HSA− population with MACS (ZAP-70−/−), and thymocytes enriched for CD8− HSA− with MACS and electronic gating for CD3− thymocytes ... Phenotype of NK1.1+CD3−thymocytes from ZAP-70−/−, RAG-1−/−, and B6.Whole thymocytes (B6-RAG-1−/−), thymocytes enriched for CD8− HSA− population with MACS (ZAP-70−/−), and thymocytes enriched for CD8− HSA− with MACS and electronic gating for CD3− thymocytes (B6 mice) were stained with PE–anti-NK1.1 and various cell surface markers (grouped in panels A-C) as described in “Materials and methods.” Expressions of NK1.1 (vertical) and other markers (horizontal) on either enriched or whole thymocytes are indicated. Kazuya Iwabuchi et al. Blood 2001;97:1765-1775 ©2001 by American Society of Hematology

Production of IFN-γ by thymocytes and splenocytes from control and ZAP-70−/− mice.Sorted NK1.1+ TCRαβ− splenocytes from control mice (open bar), NK1.1+TCRαβ− splenocytes (shaded bar), and NK1.1+ TCRαβ− thymocytes (closed bar) from ZAP-70−/− mice were cultur... Production of IFN-γ by thymocytes and splenocytes from control and ZAP-70−/− mice.Sorted NK1.1+ TCRαβ− splenocytes from control mice (open bar), NK1.1+TCRαβ− splenocytes (shaded bar), and NK1.1+ TCRαβ− thymocytes (closed bar) from ZAP-70−/− mice were cultured in medium alone (medium) or with immobilized anti-NK1.1 mAb (NKR-P1) in the presence of rhIL-2 (1000 U/mL). Then, IFN-γ in the culture supernatants were quantified by enzyme-linked immunosorbent assay. Results are representative results of 3 separate experiments. Kazuya Iwabuchi et al. Blood 2001;97:1765-1775 ©2001 by American Society of Hematology

Cytotoxicity of thymocytes and splenocytes from control and ZAP-70−/− mice.Thymocytes and splenocytes from control (●, ▪) and ZAP-70−/− (○, ■) mice administered tilorone 24 hours before collecting cells were cultured for 7 days in the presence of rhIL-2 (10... Cytotoxicity of thymocytes and splenocytes from control and ZAP-70−/− mice.Thymocytes and splenocytes from control (●, ▪) and ZAP-70−/− (○, ■) mice administered tilorone 24 hours before collecting cells were cultured for 7 days in the presence of rhIL-2 (1000 U/mL). Cells were harvested and cultured with51Cr-labeled YAC-1 (●, ○) or P815 (▪, ■) cells at indicated effector:target ratios for 4 hours. Percent specific lysis was calculated as described in “Materials and methods.” Kazuya Iwabuchi et al. Blood 2001;97:1765-1775 ©2001 by American Society of Hematology

Gene rearrangement on TCRβ loci in NK1. 1+TCRαβ− thymocytes Gene rearrangement on TCRβ loci in NK1.1+TCRαβ− thymocytes.A PCR was performed with genomic DNA from B6 thymocytes (Thy; left lane), B6 ear skin (middle lane), or the NK1.1+CD3− thymocytes of ZAP-70−/− mice (right lane) with a primer pair of a coding region... Gene rearrangement on TCRβ loci in NK1.1+TCRαβ− thymocytes.A PCR was performed with genomic DNA from B6 thymocytes (Thy; left lane), B6 ear skin (middle lane), or the NK1.1+CD3− thymocytes of ZAP-70−/− mice (right lane) with a primer pair of a coding region of Dβ2 (Dβ2-5′) and 3′-downstream region of Jβ2.7 (Jβ2-3′) to detect rearrangements of Dβ2 to Jβ2 cluster as described in “Materials and methods.” Germline bands (G) and a rearranged band (R) are indicated. Kazuya Iwabuchi et al. Blood 2001;97:1765-1775 ©2001 by American Society of Hematology

Generation of NK1. 1+ TCRαβ+cells from NK1 Generation of NK1.1+ TCRαβ+cells from NK1.1+ CD3− thymocytes of ZAP-70−/− mice in neonatal thymic organ culture.(A) A representative flow cytometric profile of inductive generation of the NK1.1+ TCRαβ+ cells in the dGuo-treated thymi in the presence of PMA ... Generation of NK1.1+ TCRαβ+cells from NK1.1+ CD3− thymocytes of ZAP-70−/− mice in neonatal thymic organ culture.(A) A representative flow cytometric profile of inductive generation of the NK1.1+ TCRαβ+ cells in the dGuo-treated thymi in the presence of PMA plus ionomycin. The sorted NK1.1+ TCRαβ− cells for the culture were demonstrated in the square of the left panel. Proportions of sorted NK1.1+ TCRαβ− cells were 98.8% to 99.5%. Collected cells from cultures were stained with either PE-mouse IgG2a (isotype control of PK136)/FITC–anti-TCRαβ/propidium iodide (middle panel) or PE–anti-NK1.1/FITC–anti-TCRαβ/propidium iodide (right panel) and analyzed with FACScan. The proportions of NK1.1+ TCRαβ+ cells are indicated. (B) Mean proportion of NK1.1+ TCRαβ+ cells. The NK1.1+ CD3− (about 5 × 103/lobe to 8 × 103/lobe) were seeded to the dGuo-treated neonatal thymic lobes and cultured in hanging-drop setup in the absence (left column) or presence (right column) of PMA and ionomycin. Five days later, total cells were stained as described for panel A. The net proportion of induced NK1.1+ TCRαβ+ cells were calculated as follows: [percentage of NK1.1+TCRαβ+ cells minus percentage of control IgG2a+ TCRαβ+ cells]. The data indicate mean net proportion and SD. Kazuya Iwabuchi et al. Blood 2001;97:1765-1775 ©2001 by American Society of Hematology

Detection of Vα14Jα281 transcripts with RT-PCR in inductive cultures Detection of Vα14Jα281 transcripts with RT-PCR in inductive cultures.Total RNA was extracted from the thymic lobes (RAG-1−/−in C57BL/6 background) alone (lane 1) or lobes with sorted NK1.1+ TCRαβ− cells in the presence (lane 3) or absence (lane 2) of PMA pl... Detection of Vα14Jα281 transcripts with RT-PCR in inductive cultures.Total RNA was extracted from the thymic lobes (RAG-1−/−in C57BL/6 background) alone (lane 1) or lobes with sorted NK1.1+ TCRαβ− cells in the presence (lane 3) or absence (lane 2) of PMA plus ionomycin, and RT-PCR was performed as described in “Materials and methods” with primer pairs Vα14 Leader/Cα-rev1 and Vα14/Jα281, RAG-1 5′/3′ and RAG-1 5′ nest/3′ nest, or EF-1α 5′/EF-1α 3′ for positive control. RT-PCR was also performed without RNA (lane 4) as control. Results are representative of 3 separate experiments. Kazuya Iwabuchi et al. Blood 2001;97:1765-1775 ©2001 by American Society of Hematology

Detection of NK1.1+ TCRαβdim thymocytes in ZAP-70−/−/DO10 TCR transgenic mouse.(A) Thymocytes obtained from ZAP-70+/−, ZAP-70−/−, or ZAP- 70−/−/DO10 mice were stained with PE–anti-NK1.1/FITC–anti-TCRαβ (i), PE–anti-NK1.1/FITC–anti-CD4 (ii), PE–anti-NK1.1/FI... Detection of NK1.1+ TCRαβdim thymocytes in ZAP-70−/−/DO10 TCR transgenic mouse.(A) Thymocytes obtained from ZAP-70+/−, ZAP-70−/−, or ZAP- 70−/−/DO10 mice were stained with PE–anti-NK1.1/FITC–anti-TCRαβ (i), PE–anti-NK1.1/FITC–anti-CD4 (ii), PE–anti-NK1.1/FITC–anti-CD8 (iii), and PE–anti-CD4/FITC–anti-CD8 (iv), and analyzed. Proportions of NK1.1+ TCRαβ+, NK1.1+TCRαβ− (upper and middle panels), or NK1.1+ TCRαβdim populations (lower panel) are indicated in the leftmost panels on the top of each squared region. Results are representative of 3 separate experiments. (B) Flow cytometric analysis and RT-PCR detection of transgenic TCR in NK1.1+ TCRαβdim thymocytes in DO10/ZAP-70−/− mice. Thymocytes of DO10/ZAP-70−/− mice were stained with PE–anti-NK1.1 antibody and biotinylated KJ1-26 followed by streptavidin-FITC. Dead cells were electronically gated out with propidium iodide staining. The NK1.1+ TCRαβdim thymocytes (NKT) and NK1.1− TCRαβ+ thymocytes (T) (demarcated with squares in the left panel) were sorted, and the expressions of DO10-specific TCRα chain (VαJαDO) and Cα were examined with RT-PCR. Kazuya Iwabuchi et al. Blood 2001;97:1765-1775 ©2001 by American Society of Hematology