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. 2003 May 19;197(10):1279–1289. doi: 10.1084/jem.20030044

Figure 2.

Figure 2.

Identification of the antigenic epitope encoded by UGT2B17. (A) Location of the epitope-containing region by transfection of truncated UGT2B17 constructs. Alignment of 4A2 cDNA with the UGT2B17 cDNA (NM_001077), the nucleotide numbering of the constructs used for transfection corresponds to that provided for the UGT2B17 GenBank sequence. Constructs I, II, and III contained the indicated UGT2B17 sequences and were transfected with HLA-A*2902 into COS cells. IFN-γ production by PL8 CTL was measured after coculture with COS transfectants and is indicated by (+) or (−) in the right-hand column. (B) UGT2B17 493–564 encodes a decamer peptide with anchor residues for HLA-A*2902. The amino acid sequence encoded by UGT2B17 493–564 is shown and a putative epitope for PL8 CTL is boxed. (C) CTL recognition of donor B-LCL cultured with synthetic peptides corresponding to UGT2B17 sequences. The concentration of peptide that elicited half-maximal lysis was ∼0.7 pM for AELLNIPFLY, ∼6 pM for ELLNIPFLY, ∼50 pM for LAELLNIPFLY, and ∼80 nM for AELLNIPFL. Specific lysis is shown as the mean of triplicate cultures at an E:T ratio of 5:1. (D) Transfection of minigene constructs define a requirement for tyrosine at the COOH terminus of the naturally processed UGT2B17 epitope. Donor B-LCL were transfected by electroporation with either UGT2B17 493–564 that encodes the 24 amino acid polypeptide described in B or UGT2B17 493–561 that encodes 23 amino acids with the COOH-terminal tyrosine deleted. Transfected B-LCL were selected for 3 d with puromycin (0.6 μg/ml). The lysis of UGT2B17 493–564-transfected donor B-LCL (solid circles), UGT2B17 493–561-transfected donor B-LCL (triangles), recipient B-LCL (squares), and untransfected donor B-LCL (open circles) is shown as the mean of triplicate cultures at various E:T ratios.