Hek blue il 12

We cloned and expressed canine-ized cytokines IL-2 and IL-12 fused to the collagen-binding domain LAIR1 as detailed in the Supplementary Methods S1 (sequences in Supplementary Table S1). To assess IL-2 bioactivity, 10,000 CTLL-2 cells (ATCC Cat# TIB-214, RRID:CVCL_0227) were plated per well in incomplete T-cell media (RPMI 1640, 10% fetal bovine serum, 2mM L-glutamine, 1mM sodium pyruvate) and incubated for 48h at 37°C with dilutions of the IL-2 fusion protein. After incubation, cell viability was assessed using the CellTiter-Glo 2.0 assay (Promega). The HEK Blue IL-12 (Invivogen; RRID:CVCL_UF31) assay was performed according to vendor instructions using dilutions of the collagen-binding IL-12 cytokine. Collagen-binding of both cytokines was evaluated through ELISA with rat collagen I-coated plates and an anti-hexahistidine detection antibody (Abcam Cat# ab1269, RRID:AB_299333) as previously reported(16 (link)). Low endotoxin levels (<5 EU/kg/dose) were confirmed for each cytokine batch using the Endosafe Nexgen-PTS system (Charles River Labs) with typical values <1 EU/mg protein.

The cell lines HEK293-F (Gibco; RRID:CVCL_D603), B16F10 (ATCC Cat# CRL-6475, RRID:CVCL_0159), CTLL-2 (ATCC Cat# TIB-214, RRID:CVCL_0227), and HEK Blue IL-12 (Invivogen; RRID:CVCL_UF31) were cultured according to vendor instructions. All cells were maintained in 5% CO₂ at 37°C and tested negative for mycoplasma. B16F10 cells were confirmed to be negative for rodent pathogens (IMPACT I test, IDEXX) prior to murine tumor studies. Six- to eight-week old female C57BL/6 mice (Taconic) were purchased and used for murine Nanostring and combination therapy efficacy experiments. All mouse studies were conducted under approval of the MIT Committee on Animal Care in accordance with federal, state, and local guidelines. Additional details on study designs and dosing can be found in the Supplementary Methods S1.

Recombinant human (rh) cytokines were obtained from the following: IL12p70 (Biolegend), rhIL18 (InvivoGen or R&D Systems), rhIL15 (Miltenyi or NCI), and rhIL2 (Proleukin, Clinigen). CHO-K1 cells (ATCC, CCL-61) have been validated for GMP production of recombinant proteins as outlined in (21 ). Daudi cells (ATCC, CCL-213) (18 (link)), Raji cells (ATCC, CCL-86) (18 (link)), K562 cells (ATCC, CCL-243) (CBReGFP) (7 ), and 32Dβ cells (ATCC,CRL 11346) transfected with pREP9 (Invitrogen) encoding human IL15Rβ were cultured as described previously (22 (link)). All cell lines obtained from ATCC were cultured and expanded per ATCC recommendations, viably cryopreserved, and stored in liquid nitrogen. Once thawed, cultures were maintained for less than two months of continuous culture according to ATCC instructions. All cell lines were verified Mycoplasma-free by the MycoAlert Plus Mycoplasma Detection Kit (Lonza) (performed by the Washington University Tissue Culture Support Service or ATCC Universal Mycoplasma Detection Kit (ATCC, 30–1012K). HEK-Blue IL12 and HEK-Blue IL18 reporter cell lines were from InvivoGen and cultured as recommended and verified mycoplasma free using the ATCC Universal Mycoplasma Detection Kit. Antibodies for flow cytometry and CyTOF are described in Supplemental Tables S1 and S2.