The EBV-specific CD8⁺ T-cell response restricted through individual HLA-class I alleles. (A) An EBV-specific T-cell culture was raised from the peripheral blood lymphocytes of the HLA-A*01⁺, EBV-seropositive healthy donor B19 (HLA-A*01, A*03, B*08, B*44) by stimulation (responder-stimulator ratio of 10:1) with an irradiated autologous LCL, generated earlier by infection of B lymphocytes with the B95-8 strain of EBV. After 3 days of culture, the tissue culture medium (10% fetal bovine serum/RPMI 1640) was supplemented with recombinant interleukin-2 (20 units/mL), and on day 10 an ICS assay was performed to assess the number of EBV-specific T cells restricted through individual HLA alleles. This involved flow cytometric detection of interferon-γ–producing T cells after overnight incubation at 37°C in medium supplemented with brefeldin A (BD Pharmingen) and with the HLA-class-I–negative 721.221 cell line that had been transfected to express a single HLA-class-I allele. These cell lines were established as follows: cDNA for each HLA allele was amplified using sequence-specific primers and cloned into the expression vector pEGFP-N1 (Clontech, Palo Alto, CA), which contains a green fluorescent protein (GFP) tag and an antibiotic resistance gene to allow for positive selection. The HLA-EGFP-N1 expression constructs were transfected into 721.221 cells using the Bio-Rad Gene Pulser, and transfectants were cultured in the presence of G418 antibiotic (800 μg/mL) for 3 weeks. GFP-positive cells were sorted using a FACSVantage, and the purified cells were maintained in the presence of G418 (800 μg/mL). HLA class I expression on the transfectants was confirmed using an anti–HLA-class-I monoclonal antibody (W6/32) and flow cytometry. The antibody staining for the ICS flow cytometry analysis involved incubation of the responder-stimulator cell mixtures at 4°C for 30 minutes with peridinin chlorophyll protein–conjugated anti-CD8, and allophycocyanin-conjugated anti-CD3. Cells were then fixed and permeabilized with Cytofix/Cytoperm (BD Pharmingen) at 4°C for 20 minutes, washed and incubated with phycoerythrin (PE)–conjugated anti-interferon-γ (BD Pharmingen) at 4°C for 30 minutes. Gated CD8⁺ cells are shown, and the numbers on the figure show the percentage of CD8⁺ cells coexpressing interferon-γ and CD3. (B) EBV-specific T-cell cultures were raised from another 7 HLA-A*01⁺, EBV-seropositive healthy donors and were analyzed, as described, to assess the number of EBV-specific T cells restricted through individual HLA alleles. Data are expressed as the percentage of CD8⁺ cells stimulated to produce interferon-γ. Note that Donors B19, S3C and K6J responded more strongly to some stimulator cells compared with the other 5 donors and so the graph scales have been adjusted accordingly. The HLA-A/B types of the donors are as follows: Donor P2H: A*01, A*02, B*08, B*44; Donor K6J: A*01, A*02, B*08, B*57; Donor B3: A*01, A*02, B*08, B*40; Donor B13: A*01, A*02, B*27, B*57; Donor B17: A*01, A*02, B*07, B*08; Donor S3C: A*01, B*08; M4W: A*01, A*03, B*08, B*35. (C) Control experiment demonstrating that the 721.221 cell line transfected with HLA-A*01 does express functional HLA molecules on the cell surface. A CD8⁺ T-cell clone was raised from a healthy HLA-A*01⁺ human cytomegalovirus–exposed donor by in vitro stimulation with the YSEHPTFTSQY peptide from the pp65 antigen which has been shown to be immunogenic in the context of HLA-A*01. An ICS assay, performed as described, showed that the clone failed to be stimulated by the HLA-A*01-transfected 721.221 cells unless these cells were preincubated with the YSEHPTFTSQY peptide (1 μg/mL) and washed 5 times.