Materials:

• T2cell line (can be purchased from the American Type Culture Collection as CRL 1992 see (www.atcc.org)
• HLA-A*0201 restricted peptides
• Anti CD3 antibody
• PBMC or T cells

1. Add peptide of interest to T2 cells and incubate at least 2 hours at 37°C, preferably overnight. We have had success using peptides at concentrations of approximately 1 uM.
2. Prepare positive control wells by coating wells of a 96 well round bottom plate with anti-CD3 monoclonal antibody. Be sure to use antibody that is sterile and does not contain preservatives. Dilute antibody to 0.5 ug/ml in PBS and add 100ul per well. Incubate at room temperature for 2 hours or at 2-8oC overnight. Remove antibody before addition of cells.
3. The next day, collect T2 cells and adjust concentration to 5 x 10⁵/ml in Iscove’s DMEM containing 10% human AB serum or other suitable medium.
4. Prepare cells to be tested. Use PBMC at 5 x 10⁶ per mL. If you are using T cells that have been stimulated with the peptide, adjust cell concentration to 5 x 10⁵/ml in Iscove’s DMEM, 10% human AB serum.
5. Add both T2 cells and PBMC or T cells at 100 ul per well in round bottom 96 well plates. Incubate 48 hours at 37°C in 5% CO₂.
6. After incubation, centrifuge plate at 2000 rpm for 5 minutes, no brake.
7. Use a multi channel pipettor to collect 150-180 ul of culture supernatant per well.
8. Measure interferon gamma present in the culture supernatant by ELISA.

Controls should include wells containing PBMC or T cells alone, wells with T2 cells lacking antigen, and wells coated with anti CD3 as a positive control.