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

Abstract

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.

Figure 1

Melanoma-specific CD4⁺ TIL 1558 are HLA-DR restricted. TIL 1558 were coincubated with whole autologous 1558-mel cells for 20 h, in the absence or presence of anti-MHC mAb, and specific Ag recognition was measured by GM-CSF secretion. As a control for mAb blocking, the MART-1–specific, HLA-A2–restricted CD8⁺ TIL 1088 line was cocultured with its autologous 1088-mel. CD4⁺ TIL 1558 were inhibited by mAbs directed against HLA-DR (L243) and a monomorphic class II determinant (IVA12), whereas the CD8⁺ TIL were inhibited only by an anti–class I mAb (W6/32).

Figure 2

CD4⁺ TIL 1558 recognize a cytosolic Ag. THP-1 cells (1.5 × 10⁵ cells/well) were used as APCs for an unfractionated freeze–thaw lysate of 1558-mel, or for various subcellular fractions (2 × 10⁵ cells equivalents/well) in a microtiter assay. CY1, unfractionated cytosol; CY2, cytosol precipitated at 50% (NH₄)2SO₄ saturation; CY3, cytosol precipitated at 75% (NH₄)2SO₄; CY4, proteins solubilized in 0.5 M KCl from a polyethyleneimine (PEI) precipitate of CY1; M1, membrane-bound proteins solubilized in 0.5 M KCl; M2, membrane proteins solubilized in 2% CHAPS detergent; N, nuclear fraction. T cell reactivity against protein-pulsed APCs was measured by GM-CSF secretion.

Figure 3

Purification of the 1558-mel Ag by size exclusion chromatography. Gel filtration chromatography was used, after anion exchange chromatography, to separate proteins derived from the 1558-mel cytosol according to size. SDS-PAGE of the eluted protein fractions is shown (gel stained with Coomassie blue), as well as the results of the corresponding bioassay. T cell recognition of APCs pulsed with individual protein fractions (4 × 10⁷ cell equivalents/well) is shown as stimulation index, the fold specific secretion of GM-CSF above background secretion (T cells plus unpulsed APCs).

Figure 4

Purification of the 1558-mel Ag by hydrophobic interaction chromatography. Individual eluate fractions (5 × 10⁷ cell equivalents/ well) were pulsed onto APCs for T cell recognition. SDS-PAGE (silver stain) shows that bioactive fractions 4 and 6 appear to contain a single protein band of ∼30 kD. The protein in fraction 6 was sequenced via mass spectrometric analysis, identifying it as TPI. This was confirmed by NH₂-terminal Edman degradation sequencing of fraction 4.

Figure 5

Enhanced recognition of a mutated TPI peptide by CD4⁺ TIL 1558. 15-mer peptides containing a wild-type (open circles) or mutated (filled circles) sequence were pulsed onto THP-1 cells for recognition by T cells. T cell secretion of GM-CSF in response to peptide stimulation was measured by ELISA. Background secretion of GM-CSF by TIL cultured with THP-1 cells in the absence of peptide was undetectable (<8 pg/ml).