To initially evaluate the IgT+ B cell depletion effect of anti-trout IgT mAbs and TAs in vivo, fish (~2-3 g) were intraperitoneally injected with two different doses (2 or 10 μg) of mouse anti-trout IgT mAbs (clone 41.8; IgG2b) (23 (link)) or 10 μg of mouse IgG2b as isotype control antibody (Biolegend). After 24 hours, half of the fish from each group were further injected with 30 μl of TAs (titer ≥ 1:204,800), whereas the other half were injected with TCs. Seven days after antibody injection, blood leukocytes were obtained and the IgT+ and IgM+ B cell populations were stained and evaluated by flow cytometry as described in the Supplementary Methods. After confirmation that effective IgT+ B cell depletion was achieved with the treatment of 10 μg of anti-trout IgT mAbs and TAs, we used for the rest of experimental procedure 2.5 times the amount of the anti-trout IgT mAb (25 μg/fish) to ensure a consistent and high level of IgT+ B cell depletion in all fish. A time course of IgT+ B cell depletion from blood, head kidney and gill were thereafter performed using the 25 μg/fish dose of anti-trout IgT mAb or its corresponding isotype control, in combination with the subsequent injection of TAs in all groups. Concentration of IgT, IgM and IgD in the serum and gill mucus of these fish were measured as described in the Supplementary Methods.