Vac pic

To measure T cell phenotype in metLNs, we set up four arms: saline, PR8, 4T1, and 4T1+PR8. In the saline group, we subcutaneously injected 10 ml saline into the mice thigh of tumor-naive mice. In the PR8 group, we inoculated 1 × 10⁵ PFU UV-inactivated PR8 influenza virus + 2 μg anti-CD40 (Cat: BE0016-2; BioXCell) + 2 μg poly I:C (Cat: VAC-PIC; InvivoGen) via subcutaneous injection into the thigh of tumor-naive mice. 9–10 d later, we collected the draining lymph nodes in both groups for flow cytometry. In the 4T1 and 4T1+PR8 groups, we implanted 4T1 cancer cells into the fourth mammary fat pad. When the tumor reached 250 mm³, we dissected the primary tumor and randomized the mice into two groups. 5 d later, we inoculated 1 × 10⁵ PFU UV-inactivated PR8 influenza virus + 2 μg anti-CD40 (Cat: BE0016-2; BioXCell) + 2 μg poly I:C (Cat: VAC-PIC; InvivoGen) in half of the mice (4T1+PR8 group) and saline in the other half of the mice (4T1 group) via thigh injection. Another 9 d later, draining lymph nodes were collected for flow cytometry.

Peptides (Peptide 2.0) were dissolved in DMSO at 10 mM and stored at −20°C. For vaccination, 50 μg of each SLP and 100 μg of poly(I:C) (Invivogen #vac-pic) were mixed with 100 μl of sterile 0.9% NaCl physiological saline and injected subcutaneously at the tail base. Vaccine injection regions were shaved at least 3 d before the cell inoculation to prevent inflammation. The mICAM1 (TVYNFSAL (wild type ICAM1: TVYNFSAP)) and p15E (KSPWFTTL) MHC-I epitope peptides were used for ELISA and CD8⁺ ELISPOT analysis. The original mICAM1 SLP (DQILETQRTLTVYNFSALVLTLSQLEVS), altered-1 mICAM1 SLP (DQILETQRTLTVYNFSALVFFLSQLEVS), altered-2 mICAM1 SLP (DQILETQRTLTVYNFSALVDDLSQLEVS) SLP and p15E SLP (GLFNKSPWFTTLISTIMGPLIILLLILL) were used for CD4⁺ ELISPOT analysis and vaccination. Underlined sequences represent altered amino acids.