96-well polyvinylidene difluoride backed plates (MAIP S 45; Millipore , Bedford, MA) were coated with 15 μg/ml of anti–IFN-γ mAb 1-D1K (Mabtech, Stockholm, Sweden) overnight at 4°C. Plates were then washed 6 times with RPMI-1640 and blocked with RPMI supplemented with l -glutamine, penicillin, and 10% heat-inactivated pooled human AB serum (R10) for 1 h. PBMCs were separated from heparinized whole blood on LYMPHOPREP (Nycomed Pharma AS, Oslo, Norway), washed 3 times, and resuspended in R10. PBMCs were added in 100 μl R10/well to the precoated plates. Input cell numbers were 5 × 105/well, in duplicate wells. For assays performed in parallel with limiting dilution analyses (LDAs), duplicate wells with 5 x 105 and 2.5 × 105 PBMCs/well were used.
Detection of peptide-specific T cells from freshly isolated PBMCs is complicated by the fact that the target cells used for peptide presentation elicit responses from T cells of other specificities. Heterologous B cell lines (BCLs) register strong responses from alloreactive T cells, whereas autologous BCLs result in potent EBV-specific responses. Allo-specific and EBV-specific responses were circumvented by using the autologous fresh PBMCs themselves to present peptide. Peptides were added to a final concentration of 2 μM. Where the cell line CIR-A2.01 was used to present the M1 58–66 peptide to fresh PBMCs, the cell line was prepulsed with a 2 μM concentration of peptide in R10 for 1 h, and then washed 3 times.
Assays were incubated for 6 h at 37°C, 5% CO2, but some experiments were run overnight (14 h) for convenience. Assays were arrested by shaking off the contents and washing 6 times with PBS 0.05% Tween 20 (Sigma Chemical Co. , St. Louis, MO). Next, 100 μl of 1 μg/ml of the biotinylated anti–IFN-γ mAb 7-B6-1 biotin (Mabtech, Stockholm, Sweden) was added. After 3 h of incubation, plates were washed six times more and a 1:1,000 dilution of streptavidin alkaline phosphatase conjugate (Mabtech) was added to the wells and the plates incubated at room temperature for a further 2 h. Next, wells were again washed 6 times and 100 μl of chromogenic alkaline phosphatase substrate (Bio Rad Labs., Hercules, CA), diluted 1:25 with deionized water, was added. After 30 min, the colorimetric reaction was terminated by washing with tap water and plates were air dried.
Detection of peptide-specific T cells from freshly isolated PBMCs is complicated by the fact that the target cells used for peptide presentation elicit responses from T cells of other specificities. Heterologous B cell lines (BCLs) register strong responses from alloreactive T cells, whereas autologous BCLs result in potent EBV-specific responses. Allo-specific and EBV-specific responses were circumvented by using the autologous fresh PBMCs themselves to present peptide. Peptides were added to a final concentration of 2 μM. Where the cell line CIR-A2.01 was used to present the M1 58–66 peptide to fresh PBMCs, the cell line was prepulsed with a 2 μM concentration of peptide in R10 for 1 h, and then washed 3 times.
Assays were incubated for 6 h at 37°C, 5% CO2, but some experiments were run overnight (14 h) for convenience. Assays were arrested by shaking off the contents and washing 6 times with PBS 0.05% Tween 20 (
