To determine the HLA locus restriction of identified epitopes, Ab inhibition assays were performed. After 14 d of stimulation with Timothy grass pollen extract (50 μg/ml) or specific peptide (10 μg/ml), PBMCs were incubated with 10 μg/ml of Abs (Strategic Biosolutions, Windham, ME) against HLA-DR (LB3.1), DP (B7/21), or DQ (SVPL3) 30 min prior to peptide addition. Cytokine production in response to positive peptides was then measured in ELISPOT assays as described above. The pan MHC class I Ab (W6/32) was used as a control.
To determine the specific HLA allele restriction, donor-derived T cells were expanded for 10 d using a single epitope peptide and were then subsequently incubated with peptide-pulsed EBV cell lines and/or fibroblasts expressing known HLA molecules also expressed in the donor from whom T cells were derived. Cytokine-specific ELISPOT assays were performed as described above to determine cytokine production and allele restriction determined by analyzing a matrix of negative and positive cytokine responses with the HLA-expressing EBV lines and fibroblasts used.
To determine the specific HLA allele restriction, donor-derived T cells were expanded for 10 d using a single epitope peptide and were then subsequently incubated with peptide-pulsed EBV cell lines and/or fibroblasts expressing known HLA molecules also expressed in the donor from whom T cells were derived. Cytokine-specific ELISPOT assays were performed as described above to determine cytokine production and allele restriction determined by analyzing a matrix of negative and positive cytokine responses with the HLA-expressing EBV lines and fibroblasts used.
