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.