Table S1. Antibodies and recombinant proteins used in this study

Transcription

1 Table S1. Antibodies and recombinant proteins used in this study Labeled Antibody Clone Cat. no. Streptavidin-PerCP BD Biosciences Biotin anti-mouse CD25 7D4 BD Biosciences PE anti-mouse CD25 PC61 BD Biosciences Biotin anti-mouse TCRβ H BD Biosciences DimerXI:soluble dimeric mouse CD1d:Ig fusion protein BD Biosciences Biotin anti-mouse IgG1 A85-1 BD Biosciences PE anti-mouse IgG1 A85-1 BD Biosciences APC anti-mouse IgG1 X56 BD Biosciences Purified mouse IgG1, λ isotype control (anti-tnp) A111-3 BD Biosciences Pacific Blue anti-mouse CD44 IM7 ebioscience PE-Cy7 anti-mouse CD11b M1/70 BD Biosciences APC-Cy7 anti-mouse CD45 30-F11 BD Biosciences APC anti-mouse c-kit 2B8 BD Biosciences FITC anti-mouse CD24 M1/69 ebioscience FITC anti-mouse TCRβ H ebioscience PE-Cy7 anti-mouse CD8α BD Biosciences APC-Cy7 anti-mouse CD4 GK1.5 BD Biosciences Pacific Blue anti-mouse CD3ε 500A2 BD Biosciences APC anti-mouse NK1.1 PK136 BD Biosciences PE anti-mouse CD3ε 145-2C11 BD Biosciences PE anti-mouse CD4 RM4-5 BD Biosciences PE anti-mouse CD8α BD Biosciences PE anti-mouse CD24 M1/69 BD Biosciences PE anti-mouse CD34 RAM34 ebioscience PE anti-mouse CD44 IM7 BD Biosciences PE anti-mouse CD62L MEL-14 BD Biosciences PE anti-mouse CD69 H1.2F3 ebioscience PE anti-mouse Thy BD Biosciences PE anti-mouse CD122 TM-β1 BD Biosciences PE anti-mouse CD131 bil-3/bc BD Biosciences PE anti-mouse CD135 A2F10.1 BD Biosciences

2 Labeled Antibody Clone Cat. no. PE anti-mouse NKG2D C7 ebioscience PE anti-mouse Sca-1 D7 ebioscience PE anti-mouse NK1.1 PK136 BD Biosciences FITC anti-mouse IFN-γ XMG1.2 ebioscience Table S2. Primers for genotyping used in this study Primer1 Primer2 Primer3 Primer4 Primer5 Primer6 5 -ccaatctgacttgagaaagctgggg-3 5 -gccactttcctgcataccaccc-3 5 -cccgtgtggcgctcttcatg-3 5 -gctgaggctgtggtccagttga-3 5 -cacacaggaagggagtgtac-3 5 -cgccctcgccctcgccggacacg-3

3 Table S3. Primers for RT-PCR used in this study CD34-F CD34-R RAG1-F RAG1-R RAG2-F RAG2-R Vα14-Jα18-F Vα14-Jα18-R ptα-f ptα-r TCR-Cβ1-F TCR-Cβ1-R GM-CSFRα-F GM-CSFRα-R IL-3Rα-F IL-3Rα-R Commonβ-F Commonβ-R HPRT-F HPRT-R 5 -ccaatctgacttgagaaagctgggg-3 5 -gccactttcctgcataccaccc-3 5 -cccgtgtggcgctcttcatg-3 5 -gcccaaagggtcccctaagg-3 5 -gtctgtaaccggctactggataacatg-3 5 -cctgagtctgaggggcttttgc-3 5 -gacccaagtggagcagagtcct-3 5 -cagctccaaaatgcagcctccctaa-3 5 -acctccagccaccaccctca-3 5 -tctcaccagcccgacatgcc-3 5 -ccttctggcacaatcctcgcaac-3 5 -ccctagcaggatctcatagagg-3 5 -gtgacagcctgcgcagtcct-3 5 -ggcggggctatagaggaggat-3 5 -ccttggctccacccccaaca-3 5 -ggctgtcttcacaggcatcacc-3 5 -gctctccggtggtgaaggag-3 5 -ctcttcgctccacttgctccag-3 5 -ctgtgtgctcaaggggggct-3 5 -ggactcctcgtatttgcagattcaacttg-3

4 Figure S1. Generation of a mouse GM-CSFRα specific rat mab (A) Expression and purification of mouse GM-CSFRα-Ig fusion protein. SDS-PAGE analysis of the culture supernatant and subsequent purification steps after transfection of the GM-CSFRα-Ig expression vector into HEK293 cells. Lane 1, culture supernatant, Lanes 2 and 3, flow through and eluate, respectively, from a Protein A-Sepharose column. (B) FACS analysis of transfectant using a mouse GM-CSFRα specific rat mab, 3A6D1. FACS profiles of HEK293 transfectants expressing Mock, GM-CSFRα, commonβ, or GM-CSFRα/commonβ stained with F(ab ) 2 rat IgG2a (shadowed) or F(ab ) 2 3A6D1 (black line). (C) FACS analysis of spleen cells using a mouse GM-CSFRα specific rat mab, 3A6D1. Anti-CD11c MACS beads enriched or whole spleen cells were prepared and CD11c + B220 conventional dendritic cells (CDC), CD11c + B220 + plasmacytoid dendritic cells (PDC), CD4 + T cells and CD8 + T cells were further analyzed surface expression of GM-CSFRα by staining with F(ab ) 2 rat IgG2a (shadowed) or F(ab ) 2 3A6D1 (black line). (D) RT-PCR analysis on GM-CSFRα related genes. FACS-sorted cells gated on (C) ( for GM-CSFRα, IL-3Rα or Commonβ; for HPRT) were analyzed for mrna levels by RT-PCR. 3A6D1 is specific for GM-CSFRα, because it was reactive to CDC positive for GM-CSFRα mrna, but not to PDC, CD4 + T cells nor CD8 + T cells, which are negative for GM-CSFRα mrna. Figure S2. Analysis of cells generated in NKT-ES cell cultures NKT-ES cells were cultured on OP9/Dll-1 or OP9/control for 20 days in the presence of IL-7 and Flt3L. Development of NKT cells (α-galcer/cd1d dimer + TCRβ + ), αβt cells (α-galcer/cd1d dimer TCRβ + ), γδt cells (TCRγδ + TCRβ ), DP or DN T cells (CD4/CD8), myeloid cells (CD11b + ), B cells (CD19 +, B220 + ), and NK cells (NK1.1 + ) was analyzed. Figure S3. FACS analysis of cells generated from wild type ES cells in vitro Cells developed from wild type ES cells on day 20 of culture with OP9/Dll-1 under the same condition as shown in Figure 2A were analyzed for the expression of the indicated markers, α-galcer/cd1d dimer + vs. TCRβ +, NK1.1 vs. CD3ε and CD4 vs. CD8. Note that a fraction of cells generated was DP CD3 + T cells, while no α-galcer/cd1d dimer + cells were detected. Figure S4. Detection of NKT cells in NKT-ES cell culture NKT-ES cells were cultured on OP9/Dll-1 under the same conditions as shown in Figure 2A and then analyzed at the indicated time points of culture by flow cytometry. The populations generated on the indicated day were analyzed in the expression of α-galcer/cd1d dimer vs. TCRβ and CD4 vs. CD8.

5 Figure S5. Surface phenotypes of DP thymocytes and liver NKT cells from B6 mice DP thymocytes and liver NKT cells were analyzed for the expression of the indicated markers for the comparison with FACS profiles of Th1-like and Th2-like NKT cells generated on day 20 of culture from NKT-ES shown in Figure 4B. Shadowed profiles indicate isotype-matched control staining. Figure S6. Stability of Th1-like and Th2-like NKT cells in the production of cytokines (A, B) Th1-like (switch culture on OP9/control at day14) or Th2-like (OP9/Dll-1) NKT cells obtained at day20 of culture demonstrated in Figure 4 were further co-cultured with OP9/Dll-1 (A) or OP9/control (B), respectively, for 5 days in the presence of IL-7 and Flt3L. The cells ( ) before or after further co-culturing for 5 days on OP9/Dll-1 (A) or OP9/control (B) were stimulated with α-galcer-pulsed BM-DCs ( ) for 3 days to measure IFN-γ and IL-4 in the supernatant by CBA. N.D.: not detected. Average of triplicate and standard deviation is shown. One representative of three experiments is shown.

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