* Correspondence: Ralf Dressel: 1 Supplementary Figures and Tables. - Supplementary Tables 1 to 3. - Supplementary Figures 1 to 6
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1 s Efficient killing of murine pluripotent stem cells by natural killer (NK) cells requires activation by cytokines and partly depends on the activating NK receptor NKG2D Carina Gröschel, Daniela Hübscher, Jessica Nolte, Sebastian Monecke, André Sasse, Leslie Elsner, Walter Paulus, Claudia Trenkwalder, Bojan Polić, Ahmed Mansouri, Kaomei Guan, Ralf Dressel* * Correspondence: Ralf Dressel: rdresse@gwdg.de 1 Supplementary Figures and Tables - Supplementary Tables 1 to 3 - Supplementary Figures 1 to 6
2 1.1 Supplementary Tables Supplementary Table 1. Antibodies for immunofluorescence (IF) and immunoblotting (IB) Antigen Isotype Clone Supplier Label Assay Dilution KLF4 rabbit IgG polyclonal (ab34814) LIN28 goat IgG polyclonal (YFC01) NANOG goat IgG polyclonal (AF2729) OCT4 rabbit IgG polyclonal (ab18976) SALL4 rabbit IgG polyclonal (ab29112) SOX2 rabbit IgG polyclonal (ab59776) Abcam, Cambridge, United Kingdom R&D Systems, Wiesbaden, Germany R&D Systems, Wiesbaden, Germany Abcam, Cambridge, United Kingdom Abcam, Cambridge, United Kingdom Abcam, Cambridge, United Kingdom - IB 1: IF 1:100 - IF 1:200 - IB 1: IB 1: IB 1:1000 SSEA1 mouse IgM MC480 Developmental Studies Hybridoma Bank (DSHB), Iowa City, Iowa, USA - IF undiluted hybridoma supernatant α- Tubulin mouse IgG1 B Sigma, Darmstadt, Germany - IB 1:10000 ZFP206 rabbit IgG polyclonal kindly provided by L.W. Stanton, Singapore - IB 1:2000 goat IgG monkey anti-goat IgG polyclonal ( ) Jackson Laboratories, via Dianova, Hamburg, Germany Cy3 IF 1:600 mouse IgM Goat antimouse IgG+IgM polyclonal ( ) Jackson Laboratories, via Dianova, Hamburg, Germany Cy3 IF 1:600 2
3 mouse IgG Horse antimouse IgG polyclonal (#7076) Cell Signaling Technology, Danvers, Massachusetts, USA HRP IB 1:
4 Supplementary Table 2. Primers used for or qpcr Gene Sequence 5'-3' Assay Afp F: CCC ACC CTT CCA GTT TCC R: TAC TGA GCA GCC AAG G F: CCT ACC CCA CAC ATT ACA TGG Flk1 R: TTT TCC TGG GCA CCT TCT ATT F: GCA GTG GCA AAG TGG AGA TT Gapdh R: TCT CCA TGG TGG TGA AGA CA F: AGC CCC AAA ATG GTT AAG GTT GC Hprt R: TTG CAG ATT CAA CTT GCG CTC AT qpcr F: TCA GGT ACC CCT CTC TCT TCT TTC Klf4 R: CGC TTC ATG TGA GAG AGT TCC T qpcr Lin28 F: TCC TCC TGT GTC TCC CAT TC R: AGA GTG AGG CCC TGT CTC AA Mash1 (Ascl1) F: CTC GTC CTC TCC GGA ACT GAT G R: CGA CAG GAC GCC GCG CTG AAA G F: CTG CTG GAG AGG TTA TTC CTC G Myh6 R: GGA AGA GTG AGC GGC GCA TCA AGG Nanog F: AGG GTC TGC TAC TGA GAT GCT CTG R: CAA CCA CTG GTT TTT CTG CCA CCG F: TTA CAA GGG TCT GCT ACT GAG ATG Nanog R: CAG GAC TTG AGA GCT TTT GTT TG qpcr Oct4 F: GGC GTT CTC TTT GGA AAG GTG TTC R: CTC GAA CCA CAT CCT TCT CT F: CGG AAG AGA AAG CGA ACT AGC Oct4 R: GCC TCA TAC TCT TCT CGT TGG qpcr F: GGC GGC AAC CAG AAG AAC AG Sox2 R: GCT TGG CCT CG TCG ATG AAC F: GAG AGG AGG TGG TAC AGC TAT TG Zfp206 R: AGG TGG AGG TAA CTC ATT CAG TG qpcr 4
5 Supplementary Table 3. Antibodies and isotype controls used for flow cytometry Antigen Isotype Clone Label Supplier CD3 rat IgG2b 17A2 FITC BioLegend, Fell, Germany CD49b rat IgM DX5 PE BioLegend, Fell, Germany CD112 rat IgG2a Santa Cruz, Heidelberg, Germany CD155 rat IgG2a TX56 - BioLegend, Fell, Germany CD314 (NKG2D) mouse IgG PE R&D Systems, Wiesbaden, Germany H2K b mouse IgG2a AF6-885 PE BioLegend, Fell, Germany H2D b mouse IgG2b KH95 PE BioLegend, Fell, Germany H60 rat IgG2a MULT-1 rat IgG2a R&D Systems, Wiesbaden, Germany R&D Systems, Wiesbaden, Germany RAE-1 rat IgG2a R&D Systems, Wiesbaden, Germany rat IgG goat IgG polyclonal ( ) FITC Jackson Laboratories, via Dianova, Hamburg, Germany - mouse IgG1 MOPC-21 PE BioLegend, Fell, Germany - mouse IgG2a MOPC-173 PE BioLegend, Fell, Germany - mouse IgG2b MPC-11 PE BioLegend, Fell, Germany - rat IgG2b RTK4530 FITC BioLegend, Fell, Germany - rat IgM RTK2118 PE BioLegend, Fell, Germany The following abbreviations are used: FITC, fluorescein isothiocyanate, and PE, phycoerythrin. 5
6 1.2 Supplementary Figures Supplementary Figure 1. The average percentage and SD of CD49b + CD3 - NK cells among splenocytes (C57BL/6: n=26 and Klrk1-/-: n=25) and MACS-purified cells (MACS+, n=10) of C57BL/6 and Klrk1 -/- mice is shown. Splenocytes of two to three mice were used for one MACS separation. 6
7 A B C Supplementary Figure 2. The ipsc lines used for autologous transplantation are pluripotent. (A) The expression of pluripotency marker genes (Oct4, Sox2, Nanog, and Lin28) and the housekeeping gene Gapdh was determined by in fibroblasts and ipsc clones derived from these fibroblasts. This is exemplified here for the fibroblasts F6 and F8 of two donor mice and in two ipsc clones derived from these fibroblasts (6-4, 6-5 and 8-6, 8-7). (B) The ipscs (d0) were differentiated in hanging drops and in suspension for 5 days (d5) and subsequently cultured on 0.1% gelatin-coated dishes for further 5, 15, or 25 days (d5+5, d5+15, d5+25). The expression of marker genes for endoderm (Afp), ectoderm (Mash1), and mesoderm (Flk) was analyzed by as illustrated here for the ipsc line 0-3. Expression of alpha-mhc (Myh6) indicates a differentiation into cardiomyocytes. Gapdh was amplified as housekeeping gene and MEFs served as negative control for the marker genes. (C) Cells of the ipsc lines were subcutaneously injected into immunodeficient RAG2 -/- mice and teratomas were obtained after 35 to 91 days. For ipsc line 1-2, the mesodermal differentiation into cartilage (a), endodermal differentiation into intestinal epithelium (b), and ectodermal differentiation into neural rosettes (c) is exemplified here. The scale bar indicates 100 µm. 7
8 Supplementary Figure 3. The newly generated ESC line BTL1 expresses pluripotency markers. (A) The expression of pluripotency marker genes (Oct4, Nanog, Klf1, and Zfp206) was determined in parallel to the housekeeping gene Hprt by qpcr in the ESC line BTL1. The mean relative expression of two biological replicates is shown compared to the long established ESC line R1. (B) The expression of the pluripotency marker proteins OCT4, SALL4, SOX2, KLF4, and ZFP206 in ESC BTL1 cells is demonstrated by immunoblotting. The expression of α-tubulin is shown as loading control. 8
9 Supplementary Figure 4. Cells of the stem cell lines ipsc 129Sv, ipsc C57BL/6, and ESC BTL1 were subcutaneously injected into immunodeficient SCID/beige mice and resulting tumors were sectioned and stained with H&E. For each cell line, an ectodermal differentiation (keratinized epithelium), endodermal differentiation (intestinal epithelium), and mesodermal differentiation (cartilage or muscle cells) is shown. The black scale bars indicate 50 µm and the white scale bar 20 µm. 9
10 Supplementary Figure 5. A summary of means and SEM of specific lysis of (A) ESCs, (B) ipscs, and (C) magscs by freshly purified NK cells (day 0) or IL-2-activated NK cells (day 4) from C57BL/6 wild type mice is shown as determined by 51 Cr-release assays. P-values for the comparisons (2-way- ANOVA adjusted for E:T ratios) are indicated for the comparison of killing by resting and IL-2- activated NK cells. 10
11 Supplementary Figure 6. A summary of means and SEM of specific lysis of (A) ESC BTL1 cells, (B) ESC MPI-II cells, (C) ipsc 129Sv cells, (D) ipsc C57BL/6 cells, (E) magsc 129Sv cells, and (F) magsc C57BL/6 cells by freshly purified NK cells (day 0) or IL-2-activated NK cells (day 4) from C57BL/6 wild type (wt) or Klrk1 -/- mice (ko) is shown as determined by 51 Cr-release assays. P-values (2-way-ANOVA adjusted for E:T ratios) are indicated for the comparison of killing by resting and wild type NK cells (wt day) as well as by resting wild type and NKG2D-deficient NK cells (day 0 killer) and IL-2-activated wild type and NKG2D-deficient NK cells (day 4 killer). 11
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