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)
P2A-TCRb P14/TCRwt
10
. The vector pMX-opt pmel-1
11
9
and pMP71-TCRa-
was provided by T.
Schumacher (The Netherlands Cancer Institute, Amsterdam, The Netherlands). pMFGhgp100-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.
1
SUPPLEMENTARY MATERIALS AND METHODS, Engels et. al.
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 = 110fold over MC57).
2
SUPPLEMENTARY MATERIALS AND METHODS, Engels et. al.
Supplementary References
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3
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4