SUPPLEMENTARY MATERIALS and METHODS, Engels Et. Al

SUPPLEMENTARY MATERIALS AND METHODS, Engels et. al.

Supplementary Materials and Methods

Retroviral Vectors

The 2C T cell clone, from which the 2C TCR was isolated, was derived from a BALB.B (C.B10-H2b/LilMcd) mouse immunized with the DBA/2 (H-2d) mastocytoma line, P815 and restimulated with BALB/c splenocytes in vitro 1. The nanomolar-affinity TCR m33 was generated from the wild-type TCR 2C by yeast display 2. Both TCR -P2A- and CD8 -P2A- expression cassettes 3 were optimized for expression in Mus musculus at Geneart GmbH (Regensburg, Germany). The optimized TCR sequences also contained point mutations for an additional disulfide bond in the constant regions (C Ser57Cys and C Thr48Cys) 4, 5 and a point mutation to stabilize the V-V interface (V Leu43Pro) 6. The entire cassette was cloned into pMP71-GFP 7, 8 using the unique NotI and EcoRI restriction sites (all cloning enzymes New England Biolabs, Ipswich, MA). W. Uckert (Max-Delbrück-Center for Molecular Medicine, Berlin, Germany) kindly provided the retroviral vectors pMP71-GFP (pMP71GPRE) 9 and pMP71-TCRa-P2A-TCRb P14/TCRwt 10. The vector pMX-opt pmel-1 11 was provided by T. Schumacher (The Netherlands Cancer Institute, Amsterdam, The Netherlands). pMFG-hgp100-EGFP and pMFG-dEV-8-EGFP were constructed by inserting annealed oligonucleotides (IDT, Coralville, IA) encoding triple KVPRNQDWL-AAY or EQYKFYSV-AAY repeats, respectively, into the NcoI-linearized pMFG-EGFP vector. All constructs were verified by sequence analysis (University of Chicago Cancer Research Center DNA Sequencing Facility). Sequences of genes and oligonucleotides will be provided on request.

Cell lines

Plat-E 12 and Phoenix-ampho 13 cells were cultured in DMEM (Mediatech, Manassas, VA), 10% non-heat inactivated FCS (Sigma-Aldrich, St. Louis, MO) at 37ºC in a 5% CO2 humidified incubator. T2-Kb cells, TAP-deficient lymphoblastoid cells transfected with murine Kb, were cultured in RPMI, 10% FCS (Gemini Bio-Products, West Sacramento, CA). Cancer cells lines were cultured in DMEM, 5% FCS (Gemini Bio-Products) at 37ºC in a 10% CO2 dry incubator. P. Ohashi (University of Toronto, Toronto, Ontario, Canada), with permission of H. Hengartner (University Hospital Zurich, Zurich, Switzerland), provided the MC57G methylcholanthrene-induced, C57BL/6-derived fibrosarcoma (MC57). Its transfectant MC57-SIY has been described previously (MC57-SIY-Hi in 14). MC57-dEV-8 was generated by transductions with pMFG-dEV-8-EGFP. Briefly, Phoenix-ampho cells were transfected with pMFG vector, using the CalPhos Mammalian Transfection Kit (Clontech, Mountain View, CA). Supernatants were then used to transduce MC57 cells. Repeated rounds of transductions and FACS derived the highly dEV-8/EGFP-expressing line MC57-dEV-8 (MFI = 110-fold over MC57).

Supplementary References

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