Rat igg2a

B16-OVA tumor-bearing mice received 200 μg of αPD-1 (clone RMP-1–14, Rat IgG2a, BioXCell) or 2A3 isotype control (Rat IgG2a, BioXCell) on days 9, 12, and 15 post tumor engraftments.

To investigate gene expression after αCD40 stimulation, murine splenic B cells were plated at 2.5 × 10⁶/ml in 24-well plates in B cell medium: RMPI 1640 + 10% FBS (Life Technologies, Carlsbad, CA, USA) + penicillin-streptomycin (100 U/ml, Thermo Fisher Scientific, Waltham, MA, USA). Cells were incubated with 10 μg/ml of αCD40 (clone: FGK4.5) (BioXCell, Lebanon, NH, USA) or rat IgG2a (clone: 2A3, BioXCell) for 6, 24, 48 h, and collected for gene expression analysis.
To investigate whether CD40 stimulation or IL-10 affected CD11b expression, murine splenic B cells were plated at 2.0 × 10⁶ cells/ml in B cell medium + 5 mM of Mg²⁺ in 96-well plates. Cells were incubated for 48 h with (a) medium alone, (b) 2 µg/ml of lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO, USA), (c) 2 µg/ml of LPS + 10 μg/ml of rat IgG2a (clone: 2A3, BioXCell), (d) 2 µg/ml of LPS + 10 μg/ml of αCD40 (clone: FGK4.5, BioXCell), (e) 2 µg/ml of LPS + 50 ng/ml of IL-10 (Recombinant mouse IL-10, Biolegend, San Diego, CA, USA). After 48 h, cells were collected and stained to assess CD11b surface expression by flow cytometry.

EMT6-hHER2 and EMT6-WT cells were injected into the mammary gland of female BALB/c mice (8–10 weeks old). Once tumors reached an average volume of 80 mm³ (day0), mice were treated with T-PNU (1 mg/kg) and/or α-mouse PD1 (12.5 mg/kg) (RPM1–14, rat IgG2a, Biox Cell), T-DM1 (15 mg/kg), trastuzumab (20 mg/kg) or mitoxantrone (2 μg/kg) or as indicated in the figures or legends. Tumor volume was measured three times per week as 1 alone or in combination with α-PD1 at day 0, 2, 4, 7. For FACS analysis and CD45-positive cell isolation, T-PNU, trastuzumab or T-DM1 were given as single treatment once the tumors reached 80 mm³ at the concentration indicated above. Animals were euthanized 10 days after treatments, when tumors in the T-PNU cohort reached a volume of 30 mm³ suitable for tumor harvest and cell processing. For T cell depletion experiments, α-CD8 depleting antibodies (53–6.72, rat IgG2a, Bio X Cell) were given at day − 2, 0, and once a week for the next 4 weeks at 10 mg/kg. Mice were euthanized once tumors reached a volume of 1200 mm³. For re-challenge experiments, T-PNU-cured animals received EMT6-hHER2 (10⁶), EMT6-WT (2.5 × 10⁵) or T/SA Thy1.1 (2.5 × 10⁵) tumor cells by mammary fat pad injection and orthotopic tumor growth was measured for a further 70 days.