N-linked carbohydrates in tyrosinase are required for its recognition by human MHC class II-restricted CD4(+) T cells.

Glycosylation of mammalian proteins is known to influence their intracellular trafficking, half life, and susceptibility to enzymatic degradation. Rare instances of natural T cell epitopes dependent upon glycosylation for recognition have been described. We report here on human CD4(+) T lymphocyte cultures and clones from two melanoma patients that recognize the melanoma-associated Ag tyrosinase in the context of HLA-DR4 and -DR8. These T cells recognize tyrosinase, normally a heavily glycosylated molecule, when expressed constitutively in melanoma cells or in COS-7 transfectants pulsed as lysates onto autologous APC. However, these T cells fail to recognize tyrosinase expressed in bacteria, nor do they react with overlapping peptides covering full-length tyrosinase, suggesting a critical role for glycosylation in the processing and / or composition of the stimulatory epitopes. The requirement for glycosylation was demonstrated by the failure of tyrosinase-specific CD4(+) T cells to recognize tyrosinase synthesized in the presence of glycosylation inhibitors, or deglycosylated enzymatically. Site-directed mutagenesis of each of seven potential N-glycosylation sites showed that four sites were required to generate forms of tyrosinase that could be recognized by individual T cell clones. These data indicate that certain carbohydrate moieties are required for processing the tyrosinase peptides recognized by CD4(+) T cells. Post-translational modifications of human tumor-associated proteins such as tyrosinase could be a critical factor for the development of antitumor immune responses.

Identification and functional characterization of neo-poly(A) polymerase, an RNA processing enzyme overexpressed in human tumors.

Poly(A) polymerase (PAP) plays an essential role in polyadenylation of mRNA precursors, and it has long been thought that mammalian cells contain only a single PAP gene. We describe here the unexpected existence of a human PAP, which we call neo-PAP, encoded by a previously uncharacterized gene. cDNA was isolated from a tumor-derived cDNA library encoding an 82.8-kDa protein bearing 71% overall similarity to human PAP. Strikingly, the organization of the two PAP genes is nearly identical, indicating that they arose from a common ancestor. Neo-PAP and PAP were indistinguishable in in vitro assays of both specific and nonspecific polyadenylation and also endonucleolytic cleavage. Neo-PAP produced by transfection was exclusively nuclear, as demonstrated by immunofluorescence microscopy. However, notable sequence divergence between the C-terminal domains of neo-PAP and PAP suggested that the two enzymes might be differentially regulated. While PAP is phosphorylated throughout the cell cycle and hyperphosphorylated during M phase, neo-PAP did not show evidence of phosphorylation on Western blot analysis, which was unexpected in the context of a conserved cyclin recognition motif and multiple potential cyclin-dependent kinase (cdk) phosphorylation sites. Intriguingly, Northern blot analysis demonstrated that each PAP displayed distinct mRNA splice variants, and both PAP mRNAs were significantly overexpressed in human cancer cells compared to expression in normal or virally transformed cells. Neo-PAP may therefore be an important RNA processing enzyme that is regulated by a mechanism distinct from that utilized by PAP.

Biochemical identification of a mutated human melanoma antigen recognized by CD4(+) T cells.

CD4(+) T cells play a critical role in generating and maintaining immune responses against pathogens and alloantigens, and evidence suggests an important role for them in antitumor immunity as well. Although major histocompatibility complex class II-restricted human CD4(+) T cells with specific antitumor reactivities have been described, no standard method exists for cloning the recognized tumor-associated antigen (Ag). In this study, biochemical protein purification methods were used in conjunction with novel mass spectrometry sequencing techniques and molecular cloning to isolate a unique melanoma Ag recognized by a CD4(+) tumor-infiltrating lymphocyte (TIL) line. The HLA-DRbeta1*0101-restricted Ag was determined to be a mutated glycolytic enzyme, triosephosphate isomerase (TPI). A C to T mutation identified by cDNA sequencing caused a Thr to Ile conversion in TPI, which could be detected in a tryptic digest of tumor-derived TPI by mass spectrometry. The Thr to Ile conversion created a neoepitope whose T cell stimulatory activity was enhanced at least 5 logs compared with the wild-type peptide. Analysis of T cell recognition of serially truncated peptides suggested that the mutated amino acid residue was a T cell receptor contact. Defining human tumor Ag recognized by T helper cells may provide important clues to designing more effective immunotherapies for cancer.

Melanoma-specific CD4+ T cells recognize nonmutated HLA-DR-restricted tyrosinase epitopes.

Tyrosinase was the first melanoma-associated antigen shown to be recognized by CD4+ T cells. In this study, we have identified two HLA-DRB1*0401-restricted peptides recognized by these T cells: Ty 56-70 and Ty 448-462. As with many of the MHC class I-restricted melanoma epitopes, both are nonmutated self peptides that have intermediate and weak MHC binding affinities, respectively. Mutated and truncated versions of these peptides were used to define their MHC binding anchor residues. Anchor residues were then modified to derive peptides with increased MHC binding affinities and T cell stimulatory properties. Ty 56-70 and Ty 448-462 enhance the list of immunogenic HLA-A2-, A24-, and B44-restricted tyrosinase peptides already described. Thus, tyrosinase provides a model for anti-melanoma vaccines in which a single molecule can generate multivalent immunization incorporating both CD4+ and CD8+ T cell responses.