Human igg1

Using a FACSAria II cell sorter (BD), LSK cells were separated based on their high expression of SCA-1 and c-KIT from lineage-depleted bone marrow, as described in Dallas et al. (2005) (link). Independent LSK isolates were cultured on immobilized Delta1 (Varnum-Finney et al., 2000 (link)), as previously described (Varnum-Finney et al., 2003 (link)). In brief, non-tissue culture-treated flasks (Nunc) were incubated overnight at 4°C with Delta1^ext-IgG (0.75 or 5 μg/mL) or human IgG₁ (5 μg/mL, Sigma I4506) diluted in PBS, together with 5 μg/mL Retronectin (Takara). Flasks were washed extensively with PBS. Isolated LSK cells were added to prepared flasks in Iscove’s modified Dulbecco’s medium supplemented with 20% fetal bovine serum and 4GF (100 ng/mL murine SCF, human FLT-3 ligand, and human IL-6; 10 ng/mL human IL-11). Each Delta1^ext-IgG culture was independently treated with IL-7 (100 ng/mL) at day 14 to drive cells further into T cell development. From each biological LSK replicate, DN1 (KIT⁺⁺CD44⁺CD25⁻) cells were isolated from 4GF conditions, whereas DN2a (KIT⁺⁺CD44⁺CD25⁺) and DN2b (KIT⁺CD44^loCD25⁺) subpopulations were isolated from 4GF + IL-7 conditions using anti-mouse monoclonal antibodies CD25-APC-Cy7 (clone PC61, BD catalog no. 557658), CD44-APC (clone IM7, BD catalog no. 559250), and c-KIT-PE-Cy5 (clone 2B8, eBioscience catalog no. 15-1171-82).

Siglec ligand expression on paraffin-embedded glioma tissue samples (4 μm) was assessed using human Siglec-Fc chimeras. Sections were deparaffinized and rehydrated with Xylene, graded ethanol and water. Sections were heated at 96 °C for 30 min in target retrieval solution, citrate pH 6.0 (Dako, Agilent Technologies, Santa Clara, CA). After cooling down to room temperature, sections were treated with 3% H₂O₂ in PBS for 15 min, washed with PBS and blocked with 20% goat serum. Siglec-7 and -9 ligands were stained with 50 nM Siglec-Fc chimeras, pre-complexed with 20 nM horseradish peroxidase-conjugated anti-human Fc antibody (Thermo Scientific, Waltham, MA) in HBSS at 4 °C. Human IgG1 (Sigma-Aldrich) was used as isotype control. Siglec-Fc binding was detected with a DAB peroxidase substrate kit (Vector Laboratories). All tissue sections were counterstained with hematoxylin, washed with water, dehydrated and mounted with KP-mounting medium (Klinipath, Olen, Belgium). Alternatively, sections were treated with 250 mU/ml Clostridium perfringens sialidase (Sigma-Aldrich) in HBSS for 2.5 h at 37 °C and washed with PBS before staining with Siglec-Fc chimeras. Images were acquired using a Leica DM6000 system (Leica, Wetzlar, Germany).

GT-00AxIL15, its parental anti-TA-MUC1 mAb GT-00A and an untargeted MOPCxIL15 isotype control lacking TA-MUC1 binding were generated by Glycotope as described in Supplemental methods. rh-IL15 (Miltenyi, Bergisch Gladbach, Germany), human IgG1 (Sigma (St. Louis, MO, USA) or Biolegend (San Diego, CA, USA)), αEGFR mAb cetuximab (Erbitux, Eli Lilly and Merck KGaA, Darmstadt, Germany), αPD-L1 mAb avelumab (Bavencio, Merck Serono and Pfizer) and αPD-L1 clone 10F.9G2 (ratIgG2bκ, BioXCell, Lebanon, NH, USA) were purchased from commercial sources. For in vitro binding studies using primary human cells, GT-00AxIL15 was labelled with Alexa Fluor 647 according to manufacturer´s instructions (Molecular Probes, Invitrogen, Waltham, MA, USA). For in vivo biodistribution studies, GT-00AxIL15 and MOPCxIL15 were radiolabeled with the radionuclide zirconium-89 (⁸⁹Zr) by conjugation of p-SCN-Bn-Deferoxamine to lysine amino acid residues on the antibodies via a thiourea linkage and purified by size-exclusion chromatography. Radiopurity and specific activity was comparable for both samples (>99% and 60 MBq/mg).

Human IgG1 Fc-fused recombinant human DLL1 (DLL1-Fc), DLL4 (DLL4-Fc), and JAG1 (JAG1-Fc), and Flag-tagged human JAG2 (JAG2-Flag) were as described [27] (link), [28] (link). Human IgG1 (Sigma, St Louis, MO) was used as a control for recombinant Notch ligands. EGTA, sodium azide (NaN₃), and DMSO were purchased from Wako Pure Chemical Industries (Osaka, Japan). A γ-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was purchased from the Peptide Institute (Osaka, Japan).