Siinfekl

The E.G7 T-cell lymphoma line was kindly provided by Dr. Jacques Galipeau (University of Wisconsin–Madison, WI, USA). All cell culture media and reagents were purchased from Wisent Bioproducts (St Jean-Baptiste, QC, Canada). The antibodies used in the flow cytometry, including I-Ab and CD47 antibodies, were purchased from BD Biosciences (San Jose, CA, USA). The IL-2 Quantikine kit was purchased from R&D Systems (Minneapolis, MN, USA). The chicken egg white ovalbumin (OVA) protein was purchased from Sigma-Aldrich (St-Louis, MI, USA). Recombinant murine IFNγ, GM-CSF and IL-21 were purchased from Peprotech (Rocky Hill, NJ, USA). The SIINFEKL and ISQAVHAAHAEINEAGR peptides were synthesized by Genscript (Piscataway, NJ, USA). The CD4 T-cell isolation kit was purchased from StemCell Technologies (Vancouver, BC, Canada). Liposomes and liposome-clodronate were purchased from Liposoma Research (Amsterdam, The Netherlands). XenoLight D-Luciferin K+ Salt was purchased from PerkinElmer (MA, USA). An Annexin-V staining kit was purchased from Cedarlane (Burlington, ON, Canada).

Before the treatment day, Raw264.7-H2Kb cells were pretreated with recombinant murine GMCSF (400 U/mL = 20 ng/mL, Peprotech, US) overnight. GMCSF was also present in the treatment media. On the treatment day, cells (0.1 × 10⁶ per well in 96-well plates) were incubated with 1000 μg/mL ovalbumin (Ovalbumin EndoFit, Invivogen, France) or 10 μg/mL OT1 long peptides (Agenus, US) or 10 μg/mL E7 peptides (Agenus, US) or 10 μg/mL SIINFEKL (GenScript, China) and the indicated adjuvants as well as inhibitors for 6 h in a 37°C incubator. Then media was removed. Cells were washed and stained with an anti-mouse H-2Kb bound to SIINFEKL antibody (Clone 25-D1.16, Cat 141604, BioLegend, US) as well as LIVE/DEAD Fixable Near-IR Dead Cell Stain reagent (ThermoFisher, US) for flow cytometry analysis.

MHC were prepared as described.^{42 (link)} The
constructs for the heavy chains (HLA-A2,
H-2Kb) and human beta-2-microglobulin (hβ2m) were generously
provided by M. Toebes and T.N. Schumacher from the NKI. They were
produced as inclusion bodies in E.coli BL21(DE3)pLysS using T7 RNA
polymerase/promoter system.^{43 (link)} Isolated
inclusion bodies were solubilized in a denaturing buffer (8 M urea/100
mM Tris·Cl, pH 8). Hβ2m was prefolded in dialysis against
10 mM Tris·Cl (pH 7) in PBS. To prepare the final MHC complex,
hβ2m and heavy chains were dissolved to final concentrations
of 6 and 3 mM, respectively, in folding buffer (100 mM Tris·Cl,
pH 8; 400 mM l-arginine; 2 mM EDTA; 5% glycerol; 5 mM reduced
glutathione; 0.5 mM oxidized glutathione; Protease Inhibitor Cocktail,
Roche Diagnostics) with a 60 mM template peptide (NY-ESO-V: SLLMWITQV,
which is a higher affinity analogue of the natural NY-ESO-1_157–165 peptide where the terminal cysteine (C) amino acid is replaced by
valine (V),^{44 (link)} SIINFEKL, SIITFEKL; GenScript).
The folding reaction mixture was incubated at 10 °C for 5 days.
After filtration, concentration, and buffer change to PBS, the complexes
were purified via size exclusion chromatography using a HiLoad 16/600
Superdex 75pg column (Cytiva). Ready MHC was analyzed using SDS-PAGE
and NanoDrop, concentrated, snap-frozen, and stored at −80
°C until further use.

Ultrapure LPS Escherichia coli 0111:B4 was purchased from InvivoGen. Low endotoxin grade OVA (<0.01 EU/μg protein) was used for immunization (Hyglos). MHC-I H-2Kb immunodominant peptides OVA_257–264 (SIINFEKL) and TRP-2_180–188 (SVYDFFVWL) were ordered from GenScript with the TG-sequence at their N-termini followed with N-terminal extension from the native protein OVA (SGLEQLE)36 (link) allowing proteolytic processing to liberate the epitopes (NQEQVSPLSGLEQLESIINFEKL, NQEQVSPLSGLEQLESVYDFFVWL). PE-labeled H-2Kb/OVA_257–264 pentamer was purchased from ProImmune. Pentamer staining was performed according to the manufacturer's instructions.

Spleens were isolated from OT-I (CD8⁺) and OT-II (CD4⁺) transgenic mice (obtained from the Walter and Elizabeth Hall Institute of Medical Research, Australia). Splenocytes were stimulated with either SIINFEKL (OT-I) or OVA_323-339 (OT-II) peptide (GenScript; 300 nM) for 3–4 days and supplemented with daily IL-2 (100 units/ml). AT3ova cells were either treated with 10–100 nM of trametinib for 12 h prior to co-culture (pre-treated 12 h MEKi) or trametinib was added directly to the co-culture for 24 h without prior pre-treatment (in culture 24 h MEKi). OT-I and OT-II cells were then co-cultured 1:1 with AT3ova cells (1 × 10⁵) for 24 h in the absence or presence of trametinib. Cells were collected and analyzed by FACS for expression of MHC-I, 4-1BB, and OX-40. Supernatants were collected for IFNγ (pg/ml) cytokine production by CBA.