Polyinosinic polycytidylic acid sodium salt poly 1 c

Other vaccine adjuvants such as Montanide ISA 720 (70% v/v final, Tall Bennett Group, USA), Polyinosinic–polycytidylic acid sodium salt (Poly I:C; 25 μg/mouse final, Sigma Aldrich, St. Louis, MO, USA) were also used in this study. The adjuvant effect of these vaccine formulations were tested in vivo by immunization of mice and measuring for IFN-γ production by ELISpot assay (Xiang et al., 2006 (link)). Briefly, adjuvant mixed KI formulations (e.g., KI + Montanide; KI + PolyI:C; KI + PSNPs) at the desired final concentrations (∼25 μg KI/mouse) and nanoparticle conjugated KI (PSNPs-KI at 1% solid of PSNPs) formulations were injected into mice intradermally (i.d.) at the base of the tail. 14 days after the last immunization, mice were sacrificed and splenocytes were isolated and assayed for IFN-γ production via ELISpot assay.

Double-stranded RNA (dsRNA) was synthesized using T7 RNA polymerase on a 400 bp luciferase template in pCRII (InVitrogen) with flanking T7 RNA promoters and purified according to manufacturer's recommendations (Ambion). 4 µg of dsRNA was electroporated into hAECs cells at about 5×10⁵ dsRNA units per cell. To ensure experimental reproducibility, in later experiments of dose-response, synthetic dsRNA (polyinosinic–polycytidylic acid sodium salt [poly (I:C)], Sigma [St. Louis, MO]) was substituted for enzymatically synthesized dsRNA. The treated cells were harvested at 18 hr following electroporation and the total RNA of the cells was extracted for further measurements.

For cytotoxicity assay, lactate dehydrogenase (LDH) activity was measured using a Cytotoxicity detection kit (Roche Diagnostics) according to manufacturer’s protocol. For pinocytosis assay cells were incubated with 0.25 mg/ml FITC-Dextran (Sigma-Aldrich) at 37°C for 20 minutes and subsequently analysed using flow cytometry. TLR3 agonist Polyinosinic-polycytidylic acid sodium salt (Poly(I:C)) (20 μg/ml) (Sigma-Aldrich) was added to Mo-M cultures on day 5. For migration assay of T cells, T_regs and B cells, a SPLInsert™ Hanging 3μm pore size (SPL Life Sciences) migration chamber was used. 2x10⁵ isolated T cells, T_regs or B cells were allowed to migrate towards conditioned media from M2 or M2/IFN treated CD169⁺ Mo-M for 18h h, or serum as positive control, with subsequent 4% PFA fixation of transmigrated cells and subsequent Cytospin with H/E staining was performed prior to counting. For cell lines, MDA-MB-231 (ATCC) and SUM159 (a kind gift from Professor S. Ethier (27 (link)) and bought from BioIVT, NY, US) TNBC breast cancer cells were used.

Vaccines consisted of the following formulations in PBS; SYIPSAEKI (final 5 μg/ml) pulsed DCs (1 × 10⁶ cells/mouse in 100 μl PBS) with or without the addition of PSNPs (50 μl at 2% solids mixed in); SYIPSAEKI (final 25 μg/mouse) mixed with the adjuvants Montanide ISA 720 (70% v/v with PBS; Tall Bennet Group, USA), or Polyinosinic-polycytidylic acid sodium salt (Poly I:C; 25 μg/mouse final, Sigma–Aldrich). Nanovaccines contained either SYIPSAEKI (~25 μg/mouse) simply mixed with PSNPs (~1% final solids), or peptides covalently conjugated to the PSNPs, including SYIPSAEKI or the variant peptides SYIPSAERI, SYIPSAEVI, SYIPSAEAI, SYIPSAEEI, and SYIPSAEDI (target 25 μg/mouse, ~0.65–1.47% final solids to achieve target peptide loading per mouse). Mice were immunized with respective formulations, either once, or twice, 2 weeks apart, intradermally at the base of tail. Approximately 13–14 days after the last immunization (unless otherwise stated) mice were humanely euthanized and splenocytes harvested and assessed for IFN-γ production by ELISpot assay.