The upregulation of PD-1 levels in vaccinated mice supports our strategy to combine the pDom-M/F vaccine with an anti-PD-1 antibody and this can potentially be applied in the clinic as anti-PD-1 antibodies have now been approved for the treatment of recurrent/metastatic HNSCC. preclinical model, antigen-specific T cells were induced by DNA vaccines and increased T cell infiltration into the tumor, but not MDSC or regulatory T cells. The vaccines inhibited tumor growth and improved the outcome alone and upon combination with anti-PD1 and resulted in tumor clearance in approximately 75% of mice. Pre-existence of MAGED4B and FJX1-reactive T cells in HNSCC patients suggests that these widely expressed antigens are highly immunogenic and could be further expanded by vaccination. The DNA vaccines targeting these antigens induced robust T cell responses and with the anti-PD1 antibody conferring excellent tumor control. This opens BTZ043 (BTZ038, BTZ044) Racemate up an opportunity for combination immunotherapy that might benefit a wider population of HNSCC patients in an antigen-specific manner. stimulation (IVS) ELISpot assay. All peptides were produced by BTZ043 (BTZ038, BTZ044) Racemate JPT Peptide Technologies (Germany) with 70% purity by high-performance liquid chromatography (HPLC). Stimulation of T Cells PBMCs were thawed and rested at high density (1×107 cells per well of a 24-well plate) for two days Rabbit Polyclonal to TAS2R10 before T cells were isolated using CD4+ and CD8+ microbeads (Miltenyi, UK). The remaining T-cell depleted PBMCs were transfected with mRNA encoding individual antigens. DNA fragments encoding each antigen (MAGED4B, FJX1 or CEFT) were synthesised through the Invitrogen GeneArt service (ThermoFisher, UK) and inserted into the pcDNA3. The DNA sequence synthesised for each antigen consisted of the antigen sequence, flanked at the N-terminus with an Ig signalling peptide and at the C terminus with an MHC-I trafficking signal (MITD) (29). mRNA for each antigen was produced using the HiScribe T7 ARCA mRNA kit with tailing from linearized plasmids (New England Biolabs, UK). Five micrograms of mRNA encoding CEFT or 10g of MAGED4B or FJX1 mRNA was used to transfect T-cell depleted PBMC using the Human Dendritic Cell Nucleofector BTZ043 (BTZ038, BTZ044) Racemate kit (Lonza, UK) to serve as antigen-presenting cells (APC). Purified T cells were incubated with transfected and irradiated (15 Gy) APC (ratio1:1) in OpTmizer? medium (Thermo, UK) supplemented with 20 U/ml IL-2 and 5 ng/ml IL-15 (both PeproTech, US) at 37C for 14 days. Half of the medium was changed to fresh OpTmizer medium supplemented with 20 U/ml IL-2 every 2-3 days. Stimulated T cells were subjected to ELISpot. Human IFN ELISpot Assay 1 x 104 stimulated T cells were incubated with peptide-loaded dendritic cells (DCs) at 1:30 ratio on a 96 well ELISpot plates pre-coated with anti-human IFN antibody (mAb 1-DIK, Mabtech, UK) overnight at 37C. DC were differentiated from autologous CD14+ monocytes, according to Miltenyi manufacturers protocol in ImmunoCult? DC differentiation media (Stemcell Technologies, UK) for two weeks. DCs were loaded with 1 g/ml CEFT, MAGED4B or FJX1 OPP. Samples were plated in duplicate or triplicate. Spots were detected with a biotin-conjugated human IFN antibody (mAb 7-B6-1, Mabtech UK) BTZ043 (BTZ038, BTZ044) Racemate followed by incubation with Streptavidin ALP (Mabtech, UK) and BCIP/NBT (Thermo, UK). Plates were scanned using ImmunoSpots reader (AID, UK). The mean values were represented as spot-forming cells (SFCs) per 1 x 104 T cells. Levels were considered positive if at least two times above medium control. Flow Cytometry Analysis of Human Samples To detect antigen-specific T cells, PBMC from Malaysian HLA-A2 patients were first incubated with FVS780 (fixable viability stain) (BD Biosciences, US) for 15 min at room temperature (RT), washed and further incubated with MAGED4B501-509/HLA-A2 tetramer-PE for 30 min at RT. After incubation, cells were stained with anti-CD3+ (FITC: SK7), CD4+ (PerCP Cy5.5:SK3) and CD8+ (BV510: RPA-T8) for 30 min at RT. For UK cohort, similar protocol was used to detect antigen-specific T cells. PBMCs were first stained with Live/dead violet stain (Invitrogen, US), followed by MAGED4B501-509/HLA-A2 tetramer-PE, anti-CD3+ (FITC: OKT3), CD4+ (APC: OKT4), CD8+ (PE-Cy7: SK1) for 30 min at RT. MAGED4B501-509/HLA-A2 tetramer-PE was generated in house by University of Southampton Protein Core Facility. Gating strategy is detailed in Figure S1A . All stained cells were analyzed using LSR Fortessa flow cytometer or FACSCanto (both BD Biosciences, US) and gated against fluorescence minus one control (FMO) or unstained controls. The flow panels were designed to accommodate different lasers and FACS machine capacities in both institutions. Details.