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-DRβ1*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.

Recognition of a New ARTC1 Peptide Ligand Uniquely Expressed in Tumor Cells by Antigen-Specific CD4+ Regulatory T Cells

“…Beside direct presentation of an Ag to T cells by tumor cells as previously suggested (33), cross-presentation of Ags by B cells or DCs has also been documented for the activation of both CD4 ϩ and CD8 ϩ effector cells (34). In previous studies, we noted that TIL1359 and TIL1087 cells can recognize 1359mel and 1087mel tumor cells respectively, but not autologous tumor lysates presented by EBV B cells (25,35), whereas TIL1363 and TIL1558 effector T cells recognize both whole tumor cells and tumor lysates presented by EBV B cells (27,28). In experiments to determine whether CD4 ϩ TIL164 Treg cells could recognize tumor cell lysates pulsed on B cells, we found that 164mel tumor cells expressing the ARTC1 ligand could specifically activate the corresponding CD4 ϩ Treg cells, but exogenous presentation of 164mel tumor cell lysate by the MHC class II-matched EBV-B cells lacked any stimulating effect on TIL164-C1 cells (Fig.…”

“…Although all T cell clones secreted GM-CSF and IFN-␥ after exposure to target cells, TIL164-derived T cell clones secreted GM-CSF, IFN-␥, and IL-10, but no IL-2, IL-4, or TGF-␤. To compare with the cytokine profile of CD4 ϩ effector cell clones, we generated CD4 ϩ effector T cell clones from TIL1363 or TIL1558 cell lines (25,27,28). These T cell clones (TIL1363-C10, -C12, and TIL1558-C3, -C4) recognized the same corresponding target Ags/ peptides as the parental cell lines TIL1363 or TIL1558 (data not shown), and showed their ability to enhance the proliferation of naive CD4 ϩ T cells purified from fresh human PBMCs, as demonstrated in Fig.…”

Identification of tumor-associated, MHC class II-restricted phosphopeptides as targets for immunotherapy

“…Of interest, although D7-pM CD4 ϩ T cells specifically reacted against intact DR1 ϩ MART-1 ϩ tumor cells, they failed to recognize DR1 ϩ EBV-B cells pulsed with lysates of MART-1 ϩ tumors. In the same experiment, CD4 ϩ TIL 1558 recognized processed 1558-mel lysate and provided a positive control for exogenous pathway processing (39). Thus, although MART-1 is a transmembrane protein, the pMART-1 epitope did not appear to be processed through the exogenous/endosomal route in these preliminary experiments.…”

“…3, D7-pM T cells recognized all of the 3 allogeneic melanomas tested that expressed both HLA-DR␤1 * 0101 and MART-1, but they failed to recognize DR1-negative or MART-1-negative cells. In the same experiment, DR1-restricted CD4 ϩ tumor infiltrating lymphocytes (TIL) 1558, specific for the TPImut antigen unique to 1558-mel, recognized 1558-mel but not the other tumors (39). Two repeat experiments yielded similar results.…”

“…Long-term CD4 ϩ T cell cultures were maintained in 120 IU/mL IL-2 and 20% conditioned medium from lymphokine-activated killer cell cultures (B-Rafspecific T cells) or 120 IU/mL IL-2, 25 ng/mL IL-7, and 25 ng/mL IL-15 (pMART-1-specific T cells). CD4 ϩ TIL from melanoma patient number 1558, recognizing a unique HLA-DR1-restricted mutant TPI epitope, were used as controls in some experiments (39). …”