Bovine CD4+ T Cell Activation Assay

Anti-bovine CD3 (Clone #MM1A, WSU, Pullman, WA) was added to 24-well plates at 10 µg/mL in 250 µL 1X PBS (Hyclone, Logan, UT), as in previous reports^{93 (link),94 (link)}. Sorted, CFSE-labeled bovine CD4+ T cells were seeded at 2 × 10⁵ per well, and autologous neutrophils (from the same cattle) were added to the wells of T cells at either 1:1 or 10:1 (2 × 10⁶ neutrophils) ratios. In experiments using transwell plates^{95 (link)}, CD4+ T cells were seeded in the lower chamber to interact with plate-coated anti-bovine CD3 Ab, and neutrophils were placed in a transwell insert with a pore size of 0.4 µm (Greiner Bio-one, Monroe, NC) in 100 µL medium. Neutralizing Ab against bovine IL-10 (CC320, BioRad, Hercules, CA) was added to the medium at 10 µg/mL^{59 (link),94 (link)}, and the same amount of purified mouse IgG1 (Biolegend, San Diego, CA) was supplemented in the controls. Plates were incubated at 37 °C in an atmosphere of 5% CO₂ for 3.5 days and then analyzed for CFSE dilution and CD25 expression in CD4+ T cells using flow cytometry.