PBMC culture assays
PBMCs were cultured in 200 µL of RPMI 1640 medium (Sigma-Aldrich) containing 10% heat-inactivated fetal calf serum (Thermo Fisher Scientific), 100 U/ml penicillin (Thermo Fisher Scientific), 100 μg/ml streptomycin (Thermo Fisher Scientific), and 2 mM L-glutamine (Thermo Fisher Scientific). 96-well Plates (Corning Inc.) were used for all of the PBMC cultures.
To evaluate the immunosuppressive functions of estradiol in vitro , PBMCs (1 × 106 cells/well) were incubated with 2 μg/ml of 17β-Estradiol (Cayman Chemical) and optimal antigen stimulations as shown below. PBMCs from BLV-uninfected cattle were cultured in the presence of 1 μg/ml anti-CD3 monoclonal antibody (mAb) (MM1A; WSU Monoclonal Antibody Center, Pullman, WA, USA) and 1 μg/ml anti-CD28 mAb (CC220; Bio-Rad, Hercules, CA, USA) for 24 hours. PBMCs from BLV-infected cattle were cultured in the presence of fetal lamb kidney (FLK)-BLV antigen (2% heat-inactivated culture supernatant of FLK-BLV cells) for 6 days. As described in a previous paper, FLK-BLV supernatant contained BLV-antigens to activate BLV-specific T cells in PBMC cultures [20]. Dimethyl sulfoxide (DMSO, Nacalai Tesque Inc., Kyoto, Japan) was used as a vehicle. After incubation, culture supernatants were collected and IFN-γ concentrations were quantitated by ELISA, as described above.
To examine the effects of estradiol on the gene expression ofCOX2 , PBMCs (1 × 106 cells/well) from BLV-uninfected cattle were incubated with 2 μg/ml 17β-Estradiol for 24 hours Then, cells were harvested and the expression of COX2 was quantitated by real-time PCR as described above. In addition, culture supernatants were collected after incubation, and then, PGE2 concentrations were measured by ELISA as described above.
To assess the effects of the inhibition of PGE2receptors in the presence of estradiol, PBMCs (1 × 106cells/well) from BLV-uninfected cattle were pretreated with each EP antagonist (EP1: SC-19220, EP2: AH 6809, EP3: L-798,106, EP4: ONO-AE3-208; Cayman Chemical) for 1 hour. After preincubation, PBMCs were washed, and then, cultured with 2 μg/ml of 17β-Estradiol in the presence of 1 μg/ml anti-CD3 mAb and 1 μg/ml anti-CD28 mAb for 24 hours. After cultivation, culture supernatants were collected, and IFN-γ concentrations were quantitated by ELISA as described above.
To evaluate BLV-specific Th1 responses in BLV-infected pregnant cattle, PBMCs (4 × 105 cells/well) from BLV-infected cattle were incubated with 0.1 μg/ml BLV gp51 peptide mix [21] for 6 days. After incubation, IFN-γ concentrations in culture supernatants were quantitated by ELISA, as described above.