αpd l1

For neoadjuvant and adjuvant immunotherapy, αPD-L1 (BioXcell, clone - 10F.9G2) and isotype rat IgG (BioXcell) were utilized. Intraperitoneal injections of 100 μg doses were administered every 3 days for a total of 3 doses. On day 14 post-tumor inoculation, mice were assigned to receive neoadjuvant αPD-L1, neoadjuvant isotype rat IgG, or undergo primary tumor removal (adjuvant). In the neoadjuvant groups, αPD-L1 or isotype control was given on days 14, 17, and 20. Surgery for the neoadjuvant-treated group was performed on day 24. In the adjuvant groups, αPD-L1 or isotype rat IgG was given on days 19 (5 days post-operatively), 22, and 25. The study also examined the effects of early neoadjuvant therapy (antibody administered on days 10, 13, and 17; surgery on day 21) and late adjuvant therapy (surgery on day 21; antibody administered on days 24, 27, and 30).

RAW264.7 cells were treated with PD‐L1 neutralizing antibody (αPD‐L1, 15 mg/ml, Bio X Cell) and/or interleukin‐13 (IL‐13, 30 ng/ml, Biolegend) for 48 h. Cells were cultured in serum‐free medium for 24 h after the supernatant was discarded. Then, the supernatant of RAW264.7 cells was collected and centrifuged for 20 min. The supernatant was collected as the conditioned medium for subsequent experiments. Conditioned medium was classified into control, IL‐13 and IL‐13 + αPD‐L1 groups according to different treatments.

Treatments were started when LKRM or LLC tumors reached a size of ~200 mm³. Adoptive transfer of neutrophils was carried out on LKRM tumor-bearing mice, injected with 2–5 × 10⁶ NDN or 5 × 10⁶ LDN isolated from LLC-GCSF tumor-bearing mice. The mice were injected intraperitoneally (IP) for 3 days in a row. LKRM and LLC tumor-bearing mice were injected IP with 250 μg α-PD-L1 or α-PD-1 antibodies (BioXCell (Lebanon, NH, USA), clone RMPI-14, clone 10F.9G2, respectively) every 3 days for a total of 3 injections. LKRM and LLC tumor-bearing mice were treated IP with 150µg α-Ly6G antibody (BioXCell, clone 1A8) for depletion of neutrophils every 2 days for a total of 4 injections. Depletion was confirmed using flow cytometry. Control mice were injected IP with either PBS or Isotype Rat IgG2A (BioXCell, clone 2A3) or IgG2B (BioXCell, clone LTF-2). LLC tumor-bearing mice were injected IP with MPO inhibitor 4-ABAH (4- Aminobenzoic hydrazide 95%, Sigma Aldrich) at a dose of 40 μg/g in 500 μL of HBSS (Biological Industries), twice a day, starting from day 8 following tumor injection for 12 days.

The animal experiment schedule of this study was carried out with similar to the previous studies [6 (link),27 (link)]. The Ethics Committee of Virocure approved all animal experiment protocols (IACUC No. VRC-IACUC-2208) and conducted according to the National Research Council Guide for the Care and Use of Laboratory Animals, Eight Edition. 2011. Six weeks-old immunocompetent female C57Bl6/J mice were purchased from Orient Bio (Seongnam, Republic of Korea). The C57BL6 mice were subcutaneously inoculated with 3 × 10⁵/100 μL B16F10 murine melanoma cells in the right flank. The cells were mixed with Matrigel (BD Bioscience, San Jose, CA, USA) at a 1:1 ratio. Once palpable tumors reached 100 mm³, mice were randomly divided and received PBS, 10⁷ (TCID50) of MyxV_0100, 10⁷ (TCID50) of MyxV_CD47, 10⁷ (TCID50) of MyxV_IFN-γ, and 10⁷ (TCID50) of MyxV_CD47/IFN-γ via intratumoral of in a volume of 50µL at days 1, 3, and 5. In combination treatment, from the second MyxV administration, 10 mg/kg of α-PDL1 (BioXcell, Lebanon, NH, USA) antibody was administered intraperitoneally, with a total of three times at two-day intervals. Tumor volumes and body weights were recorded every three days. For analysis of TIL, three animals from each group were sacrificed on day 9 for FACS analysis. For survival analyses, five to ten mice were used, and mice were monitored for 33 days.

Female C57BL/6, IFN-γ^−/− (B6.129S7-IFNγtm1Ts/J), CD8^−/− (B6.129S2-Cd8atm1Mak/J), Ly5.1 (B6.SJL-Ptprca Pepcb/BoyJ), and IL12R^−/− (B6.129S1-Il12rb2tm1Jm/J) mice age 6-8 weeks were purchased from Jackson Laboratories, Bar Harbor, ME. For day 35 (D35) and day 42 (D42) experiments, 1 × 10⁷ ID8-Muc16^ecto tumor cells were injected intraperitoneally (i.p.) on D0 and animals were treated with CAR T cells on D35 or D42 respectively. Mice treated with αFasL blocking antibody (R&D systems) were treated with 250 μg/mouse of antibody on D41 after tumor inoculation. Animals treated with PBS/Clodronate liposomes (Cedarlane, Amsterdam, The Netherlands) were injected with 200 μL/mouse of either liposome on D38 and D40 after tumor injection. Pretreatment with a single dose of 250 μg/mouse of αPD-L1 (BioXcell, West Lebanon, NH) was performed on D41 after tumor inoculation. All injections were i.p. All mice were monitored for survival and were euthanized when showing signs of distress or significant ascites.