Schematic representations of HPX42B and NA88-A sequences. Drawings are not to scale. (A) cDNAs; EMBL/GenBank/DDBJ accession nos. AF164963 (NA88-A) and AF068006 (HPX42B). Homologous regions of the two cDNAs are marked in black. For HPX42B cDNA, the open reading frame coding for the homeoprotein (ORF) and the 3′ UTR containing an Alu family member (Alu) are marked. For NA88-A cDNA, the positions of primers used for RT-PCR are marked (400 and 970). (B) Alignment of HPX42B and NA88-A genomic sequences, EMBL/GenBank/DDBJ accession nos. AF164964 and AC006177, respectively. The positions of two HPX42B gene introns are marked, but not their sequences. Dashes represent bases missing in one of the two sequences, and the number of bases concerned is marked. The alternative 3′ splice site of the HPX42B second exon is marked 3′SS. For the NA88-A sequence, the last base shown (g) corresponds to the first base of the cloned cDNA whose sequence is represented in Fig. 2 A. (C) Schematic alignment of the genomic sequences. For the HPX42B gene, exons are marked as boxes, and introns separating them are shown as lines. The alternative 3′ splice site is marked 3′SS. The positions of initiation codons (ATG) and stop codons (*) discussed in the text and boxed in A are marked. The NA88-A gene representation is a composite using information derived from the cosmid and the cDNA as indicated.

Identification of differences between HPX42B and NA88-A cDNAs important for their differential ability to generate a signal stimulating TNF release by M88.7 T cells. (A) Comparison of NA88-A and HPX42B nucleotide and encoded protein sequences in the region coding for antigenic peptide. Potential initiation and stop codons are indicated in bold characters. (B) Schematic representations of normal and mutated NA88-A and HPX42B cDNA sequences. Boxes marked P code for antigenic peptides and are white for the NA88-A peptide and grey for the HPX42B peptide (as marked above each box). For NA88-A Ser, a Leu codon, CTG, has been changed to a Ser codon, TCG, within the peptide-coding region. For NA88-A Trp, a stop codon tag immediately following the antigenic peptide's terminal Val codon has been changed to a Trp codon, tgg. For HPX42B Stop, a Trp codon, tgg, immediately following a Val codon has been changed to a stop codon, tag. (C) pcDNA3-based plasmids containing cDNAs as described in B were cotransfected into COS-7 cells with pHLA B*1302. The ability of transfected cells to induce TNF release by M88.7 T cells was measured. Note that all results shown here are from transfections carried out simultaneously in parallel, allowing comparison of results between samples. TNF release values for pNA88-A are higher than those shown in the separate experiment of Fig. 1 and Fig. 3, as results depend on COS-7 cell transfection efficiencies and the condition of the WEHI cells used for TNF assays, both of which vary between individual experiments.

Characterization of the antigen recognized by M88.7 T cells. (A) Restriction element determination. Melanoma cell lines were tested for their ability to induce TNF release by M88.7 T cells before (−) and after (+) transfection with an HLAB*1302 expression vector. (B) Cloning of the cDNA. COS-7 cells were transfected with plasmids as indicated and tested for their ability to induce TNF release by M88.7 T cells.

Identification of the antigenic peptide. (A) Translation products of nucleotides 674–730 of NA88-A cDNA in all three reading frames. 1, 2, and 3: peptides synthesized. (B) Production of TNF by M88.7 T cells in response to M88 cells or one of the three synthetic peptides shown in A as marked. (C and D) Determination of the minimal antigenic peptide. Production of TNF (C) and IL-2 (D) by the M88.7 T clone in response to different synthetic peptides derived from peptide 1. For A, T cell clone M88.7 was stimulated with different concentrations of each peptide. For B, T cell clone M88.7 was stimulated with 50 μM peptide. Fixed, stimulated cells were stained for cytokine and analyzed on a FACScan™. Percentages of positive cells are indicated in the dot plots.