Anti mhc class 2

Immature bone marrow-derived dendritic cells (BMDCs) from the bone marrow of C57BL/6 mice were induced by in vitro culture for 5 to 6 days in complete RPMI 1640 medium (supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, penicillin, streptomycin, and 2-ME) with 10% FCS and 20 ng/mL GM-CSF (R&D Systems, Minneapolis, MN, USA).
Immature BMDCs were transfected with 0.5 μg of plasmid DNA (pcDNA, pOVAv, pOVAy) using Nucleofector II and Mouse Dendritic Cell Nucleofector Kit with program Y-001 (Amaxa Biosystems, Cologne, Nordrhein-Westfalen, Germany). The cells were then cultured with or without EPO (at a final concentration of 1 μM) for 5 h, fixed with 0.5% paraformaldehyde/PBS for 10 min, placed in 1 M glycine/PBS for 15 min, washed with RPMI1640, and used as antigen-presenting cells (APCs). OT-I CD8⁺ T cells were isolated from the spleen by negative sorting using a CD8α⁺ T cell isolation kit (CD4, CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, Anti-MHC-class II, TER119, and TCRγδ) (Miltenyi Biotec, Bergisch Gladbach, Germany). APC (1 × 10⁴ cells) and OT-I CD8⁺ T cells (1 × 10⁵ cells) were cultured in a 96-well plate for 24 h. Then, culture supernatants were collected to measure IFN-γ. An ELISA kit for mouse IFN-γ (BioLegend, San Diego, CA, USA) was used in this study.

Splenocytes were isolated at 48 hours after the last administration of polyplex micelles to subcutaneous tumor models. The cells were then subjected to flow cytometric analysis using PE-conjugated anti-CD11c (Miltenyi Biotec Gmbh, Bergisch Gladbach, Germany), FITC-conjugated anti-CD11b (Abcam), anti-CD80, anti-CD86 (Beckman Coulter, Fullerton, CA), and anti-MHC-Class II (Miltenyi Biotec Gmbh) monoclonal antibodies.