Yts 169

For CD8⁺ T and NK cells depletion experiments, 200 μL PBS containing 50 µg of anti-CD8 (YTS-169.4, BioXcell, Cat. BE0117) or anti-NK (PK136, Biolegend, Cat. 108701) monoclonal antibodies or not was injected into tumor-bearing mice intraperitoneally (i.p.) 2 days before initiation of adoptive cell transfer and every subsequent 2 days for a total of five injections. For depletion of macrophages, mice were intranasally administrated with 50μL of Clodronate Liposomes (5 mg/ml; LIPOSOMA, Cat.CP-005-005) or PBS control on Day -2 of adoptive cell transfer and every 2 days post-transfer for a total of four doses. For ICB experiments, i.p. injections for 150 μg of anti-PD-L1 (RMP1-14, BioXCell) monoclonal antibody or PBS control were performed every 2 days after adoptive cell transfer, four doses in total. FTY720 treatment was given i.p. of 25 μg in PBS every 2 days from Day 1 to Day 7 post-cell transfer.

Vaccines were administered via intramuscular injections in the quadriceps by delivering a volume of 50 µl per side at 5 × 10⁸ vp. For efficacy studies, α-hPD-L1-mIgG1 (InvivoGgen, Cat. Number: hpdl1-mab9) and α-mPD1 (BioXcell, clone RMP114, Cat. Number: BE0146) were administered twice a week until day 16 post treatment start. To deplete T-cell subsets, α -mCD8 (BioXcell, clone YTS169.4, Cat. Number: BE0117) and α-mCD4 (BioXcell, clone YTS191, Cat. Number: BE0119) were used. Each antibody was administered i.p. at a dosage of 200 μg.

Mouse experiments were performed according to Good Scientific Practice-principles and approved by the Ethical Committee (EC) of the Faculty of Veterinary Medicine, Ghent University (EC 2020-030 and 2021-018).
Eight-week (w)-old female and male BALB/c mice were mated and pups were weaned 12-14 days (d) post-parturition. One hour (h) after weaning, lactating females were intraductally inoculated in the third mammary gland pair with 5 × 10⁴ 4T1-luc cells suspended in a 100 μl mixture of PBS and Matrigel^® (1:10; Corning, Bedford, MA, USA) under inhalation anesthesia and analgesia as described previously (15 (link)). For intravenous (i.v.) treatment, eMSCs or eEpSCs were dissolved in DMEM at 3x10⁵ per 100 μl and injected through the tail vein using a 29G insulin needle. I.v. injections with DMEM only were used as a negative (sham) control. Antibody treatments for depletion of CD4⁺ (clone GK1.5) and CD8α⁺ T-cells (clone YTS169.4) or rat IgG2b isotype controls (clone LTF-2) were purchased from BioXCell (West Lebanon, NH, USA) and diluted (200 μg/100 μl) in InVivoPure dilution buffer (BioXCell) at pH6.5 (for anti-CD4) or pH7 (for anti-CD8α and isotype control) for i.p. administration.

We performed in vivo CD8 + T-cell depletion studies using intraperitoneal (IP) injection of monoclonal anti-mouse CD8 (YTS 169.4, BioXCell, Lebanon, NH, USA) in addition to treatment with either IgG or ICI. IP injections of anti-CD8 (BioXCell, 300 µg/animal) or IgG2b (BioXcell, 300 µg/animal) were performed at day 0 and day 3 prior to IgG1 and 2 (IgG) or anti-CTLA4 and anti-PD-L1 (ICI) injections, and once a week thereafter. The four treatment groups in this experiment were anti-CD8 + ICI, anti-CD8 + IgG, IgG2b + ICI, and IgG2b + IgG. CD8 + T-cell depletion was confirmed by FACS analysis of primary splenocytes of treated mice.

For antibody treatments, mice received 100 μg of anti-PD-L1 (10 F.9G2: BioXCell) and/or 100 μg anti-CTLA-4 (BE0131: BioXCell) intraperitoneally on days 7, 10, and 13 after tumor engraftment. For depletion studies, mice received 200 μg of either anti-CD8 (YTS 169.4: BioXCell) and/or anti-NK1.1 (PK136: BioXCell) or a combination of anti-CD8+ anti-NK1.1 2 days before tumor engraftment and once every 7 days after. Depletion of the relevant cell populations was confirmed by flow cytometry analysis of blood 3 days after antibody administration and in the tumor at endpoint.

For TIM4 blocking studies healthy or tumor-bearing mice were treated with anti-TIM4 antibody (RMT-53, BioXCell) or isotype control (2A3, BioXCell) at the concentration of 250 µg/100 µL intravenously or intraperitoneally. The protocol for TIM4 blockade (number of injections and timing) are described in each specific experiment.
For CD8 depletion C57BL/6 mice were injected with anti-mouse CD8 (YTS 169.4, BioXCell) or isotype control (2A3, BioXCell) at the concentration of 200 µg/100 µL intraperitoneally at day −1, +4, 8, 11 after KP-OVA implantation. Mice were sacrificed 15 days after tumor inoculation to assess tumor burden and validate CD8⁺ T cell depletion by FACS.
KP tumor-bearing mice were either left untreated or received chemotherapy intraperitoneally (i.p.) at day 7 and 14 after tumor engraftment (Oxaliplatino, 2.5 mg/kg; Cyclophosphamide, 50 mg/kg, Tocris Bioscience). In these experiments, mice were treated with anti-TIM4 antibodies or isotype control the day before chemotherapy administration.