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Cutaneous lymph nodes and spleens from mice treated either orally or topically (treated and untreated side, respectively) were pooled and minced in DPBS/2% FBS/2 mM EDTA, filtered through 70 μM strainers and spun at 350 g for 5 min. Blood was collected in EDTA‐coated tubes and red blood cells were lysed using BD FACS lysing solution (as was red blood cells from spleen single cell suspensions). Cell differential were obtained from haematology analyzer Sysmex XE‐2100 (Sysmex Corporation, Japan). Equal cell numbers for each sample were incubated with anti‐mouse CD16/CD32 block to prevent unspecific binding and then stained for 30 min at 4°C with the following anti‐mouse antibodies (all from BD); FITC CD3e (145‐2C11), APC‐Cy7 TCRβ (H‐57‐597), BV510 or BV786 CD8α (53‐6.7), BV711 NKG2D (Cx5), BUV395 CD4 (GK1.5), BV711 or BV605 CD44 (IM7), BUV395 CD45 (30‐F11), PerCP‐Cy5.5 CD19 (1D3) and PE‐Cy7 CD62L (MEL‐14). For Ki‐67 (PE‐Cy7, B56) analysis, the cells were permabilised and fixed in eBioscience Fix/Perm buffer, followed by Ki‐67 antibody staining in eBioscience permeabilisation buffer for 1 h at room temperature. The cells were analyzed on a BD LSRII flow cytometer and analysis was performed with FlowJo (Tree Star) software.

WT-1, BIRC5, MUC-1, and TERT were purchased from MyBioSource (San Diego, CA, USA). Recombinant mGM-CSF was purchased from PeproTech (Cranbury, NJ, USA). Cisplatin and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (Burlington, MA, USA). Fluorophore-labeled monoclonal antibodies (Annexin V-FITC, 7-AAD, CD3e-FITC, CD4-PE, CD8-Pacific blue, CD11c-APC, CD80-PE, CD86-FITC, Fixable Viability Stain 780, IFN-γ-BV605, MHC I-PE, MHC II-PerCP-Cy5.5) were purchased from BD Bioscience (Franklin Lakes, NJ, USA). Cell trace violet was purchased from Invitrogen (Waltham, MA, USA). LLC1 was purchased from ATCC (Manassas, VA, USA).

Multiparameter flow cytometry was performed in accordance to BD Pharmingen™ Protocol. In brief, splenocytes were harvested at day 14 post second boost; 5 × 10⁶ cells were stimulated in a Nunc™ 96-well conical-bottom plate (Thermo Scientific™, USA) for 6 h at 37 °C with 10 μg of M2eh peptide and 1 μg of influenza virus A/Aichi/2/68 (H3N2) in the presence of 1 μg/ml of Brefeldin A (BD Bioscience, USA) and purified hamster anti-mouse CD28. The cells were then washed, and Fc receptors were blocked using CD16/CD32 antibodies (Mouse BD Fc Block, BD Pharmingen, USA) for 30 min. Next, cells were incubated with Zombie Aqua (Zombie Aqua™ Fixable Viability Kit, Biolegend, USA) to enable gating of live cells during analysis and subsequently stained with antibodies (CD3e-FITC, CD8a-APC-Cy7, CD4-PerCP, CD62L-PE-Cy7, CD44-APC) at 4 °C for 30 min (antibodies from BD Pharmingen, USA). Cells were permeabilized using the BD Cytofix/Cytoperm Plus (BD Bioscience, USA) protocol and stained with anti-TNF-α-BV421 or anti-IFN-γ-PE (BD Pharmingen, USA). Sample acquisition (50,000 live CD3+ were collected) was performed with a BD FACS Canto II flow cytometer (Becton Dickinson, USA) and analyzed using Kaluza, version 1.5 (Beckman Coulter, USA).

RPMI 1640, FBS, penicillin, and streptomycin were obtained from Invitrogen (Grand Island, NY). The liver dissociation kit, recombinant murine was from Miltenyi Biotec. Phorbol 12-myristate 13-acetate (PMA), brefeldin A, ionomycin, CD3, CD28, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). We obtained the following fluorescein-conjugated anti-mouse antibodies from eBioscience (San Diego, CA), Biolegend (San Diego, CA) and BD: CD3e-APC-Cy7 (145-2C11), CD4-PerCP-Cy5.5 (RM4-5), CD8a-PE (53-6.7), ICOS-PE-Cy7 (C398.4A), CD69-BV421 (MIH5), CD19- PE-Cy5(6D5), CD138-PE-Cy7 (281-2), B220-APC-Cy7 (RA3632), CD80-PE (16-10A1), CD86-APC (GL1), CD3e-FITC (145-2C1), CD11b- PE-Cy7 (M1/70), Ly-6C-PerCP-Cy5.5 (HK1.4), F4/80- APC-Cy7 (3M8), CD192/CCR2-BV421 (SA203G11), CX3CR1-APC (SA011F11), CD135-BV421 (A2F10.1), CD11c- PerCP-Cy5.5 (HL3), Gr-1-FITC (RB6-8C5), Ly-6G- APC-Cy7 (1A8), CD287/TLR7-PE (A94B10), CD103-PE (M290), IFN-γ-APC (XMG1.2), IL-4-PE (11B11), IL-2-PE (JES6-5H4), IL-6-APC (MP5-20F3), IL-10-PE (JES5-16E3), IL-13-eFlour450 (ebio13A), IL-17-PE (TC11-18H10.1)), IL-21-APC (FFA21) and their corresponding isotype controls.

Cell proliferation was assessed by BrdU incorporation assays with a BrdU Flow Kit (BD Pharmingen) or an EdU Click Proliferation Kit (BD Pharmingen). Cell apoptosis was analyzed with a PE-Annexin V Kit (BD Pharmingen). The following antibodies were purchased from BD Biosciences: CD122-PE (TM-Bta1), CD19-FITC (1D3), CD335/NKp46-PE (29A1.4), CD3e-FITC (145-2C11), CD3e-PerCP-Cy5.5 (145-2C11), CD4-FITC (RM4-5), CD49b (DX5) Pan-NK Cells-BV421, CD8a-PE (53-6.7), Erythroid Cells-FITC (TER-119), Ly-6G/Ly-6C-FITC (RB6-8C5), NK-1.1-APC (PK136), NK-1.1-APC-Cy7 (PK136), and TCR-Bta Chain-BV421 (H57-597). Stained samples were analyzed using FACSCantoII and FACSDiva software Version 6.1.2 (BD Biosciences).

Multi-parameter flow cytometry was performed in accordance with the BD Pharmingen^TM protocol. In brief, mouse lungs were harvested at day 14 post second boost; lung lymphocytes (5x10⁶) were stimulated in Nunc^TM 96-well conical bottom plates (Thermo Scientific™, USA) for 6 h at 37°C with 10 μg of M2eh peptide, in combination with 1 μg of either the A/Aichi/2/68 (H3N2) or the A/California/07/09 (H1N1pdm09) influenza virus, in the presence of 1 μg/ml of Brefeldin A (BD Bioscience, USA) and purified hamster anti-mouse CD28. The cells were then washed, and Fc receptors were blocked using CD16/CD32 antibodies (Mouse BD Fc Block, BD Pharmingen, USA) for 30 min. Next, cells were incubated with Zombie Aqua (Zombie Aqua™ Fixable Viability Kit, Biolegend, USA) to enable gating of live cells during analysis, and they were subsequently stained with CD3e-FITC, CD8a- APC-Cy™7, CD4- PerCP, CD62L-PE-Cy™7, or CD44-APC (BD Pharmingen, USA) antibodies at 4⁰ C for 30 min. Cells were permeabilized using the BD Cytofix/Cytoperm Plus (BD Bioscience, USA) protocols and stained with anti-TNF-α-BV421 or anti-IFN-γ-PE antibodies (BD Pharmingen, USA). Sample acquisition (50,000 live CD3+ T-cells were collected) was performed with a BD FACS Canto II flow cytometer (Becton Dickinson, USA) and analyzed using Kaluza version 1.5 (Beckman Coulter, USA).