2.5. Dog PBMC Isolation, Thawing, Stimulation and Suppression

Whole blood from canine healthy donors and cancer patients was diluted with DPBS at a ratio of 1:1, and layered over 6 mL of Ficoll^® Paque Plus (GE17-1440-02, Sigma, St. Louis, MO, USA) in V-bottom 15 mL tubes. The samples were centrifuged at 900 g for 25 min at room temperature with brakes at lowest setting. The PBMC layer was transferred to a clean V-bottom 15 mL tube and washed with DPBS. The cells were counted and resuspend in ice cold freezing medium (5:4:1 RPMI1640: FBS: DMSO) on ice at 2 × 10⁶ cells/mL. The samples were initially preserved in a freezing container at −80 °C for 2 days and then transferred to −150 °C for long-term storage.

For analysis or stimulation of cryopreserved PBMCs, thawed aliquots were slowly diluted in warm complete medium containing RPMI1640 (21875034, Thermo Fisher), 10% FBS (P30-2602, Pan Biotech), GlutaMAX (2 mM, 35050061, Thermo Fisher), penicillin-streptomycin (100 units, 15140122, Thermo Fisher), sodium pyruvate (1 mM, 11360070, Thermo Fisher), 5 mL of non-essential amino acids (NEAA, 0.1 mM, 11140050, Thermo Fisher) and 12.5 mL HEPES buffer (25 mM, 15630080, Thermo Fisher). PBMCs were washed with warm medium and used in the assays at described concentrations.

For the polyclonal stimulation of PBMCs and assessment of PD-1 expression 2 × 10⁵ healthy donor derived PBMCs were stimulated with 2.5 μg/mL of concanavalin A (C2010, Sigma) and incubated for 48 h.

For the assessment of functional activity of human ICIs on canine PBMCs, 2 × 10⁵ healthy donor or cancer patient PBMCs were distributed/well, stimulated with 50 ng/mL of staphylococcal enterotoxin B (BT202, Toxin Technology, Sarasota, FL, USA) on U-bottom 96-well plates (353077, Corning, New York, NY, USA) and incubated for 72 h. For extra suppression of stimulation PBMCs were cultured with 10 μg/mL of scPD-L1Fc [9]. To reverse the suppression 10 μg/mL of indicated ICIs were added. Responders (R) were defined as the samples with >5% increase in cIFN-γ production over durvalumab (control), while non-responders (NR) were defined as <5% increase or a decrease in cIFN-γ production. To establish if durvalumab can be used as a non-binding IgG1 isotype control for atezolizumab and avelumab it was tested against a commercially available human IgG1 isotype control (#BE0297, Bio X Cell, Lebanon, NH, USA).