gp100/pmel 17 is a murine tumor rejection antigen: induction of "self"-reactive, tumoricidal T cells using high-affinity, altered peptide ligand - PubMed (original) (raw)

gp100/pmel 17 is a murine tumor rejection antigen: induction of "self"-reactive, tumoricidal T cells using high-affinity, altered peptide ligand

W W Overwijk et al. J Exp Med. 1998.

Abstract

Many tumor-associated antigens are nonmutated, poorly immunogenic tissue differentiation antigens. Their weak immunogenicity may be due to "self"-tolerance. To induce autoreactive T cells, we studied immune responses to gp100/pmel 17, an antigen naturally expressed by both normal melanocytes and melanoma cells. Although a recombinant vaccinia virus (rVV) encoding the mouse homologue of gp100 was nonimmunogenic, immunization of normal C57BL/6 mice with the rVV encoding the human gp100 elicited a specific CD8(+) T cell response. These lymphocytes were cross-reactive with mgp100 in vitro and treated established B16 melanoma upon adoptive transfer. To understand the mechanism of the greater immunogenicity of the human version of gp100, we characterized a 9-amino acid (AA) epitope, restricted by H-2Db, that was recognized by the T cells. The ability to induce specific T cells with human but not mouse gp100 resulted from differences within the major histocompatibility complex (MHC) class I-restricted epitope and not from differences elsewhere in the molecule, as was evidenced by experiments in which mice were immunized with rVV containing minigenes encoding these epitopes. Although the human (hgp10025-33) and mouse (mgp10025-33) epitopes were homologous, differences in the three NH2-terminal AAs resulted in a 2-log increase in the ability of the human peptide to stabilize "empty" Db on RMA-S cells and a 3-log increase in its ability to trigger interferon gamma release by T cells. Thus, the fortuitous existence of a peptide homologue with significantly greater avidity for MHC class I resulted in the generation of self-reactive T cells. High-affinity, altered peptide ligands might be useful in the rational design of recombinant and synthetic vaccines that target tissue differentiation antigens expressed by tumors.

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Figures

Figure 1

Figure 1

B16 melanoma-reactive T cells specifically recognize nonmutated gp100. Splenocytes from hgp100-immunized mice were cultured as described in Materials and Methods, and cocultured for 24 h with various targets shown on the ordinate, including B16 melanoma, the immortalized normal melanocyte line Melan-A, and human 293 kidney cells expressing the mouse restriction elements H-2Kb and H-2Db infected with rVV encoding mouse melanocyte differentiation antigens. Supernatants were assayed for IFN-γ by ELISA. Specific IFN-γ release was detected against targets expressing mgp100 or hgp100.

Figure 2

Figure 2

gp100 is a tumor rejection antigen. (A) A clone of bulk gp100-reactive T cells, clone no. 9, was established by limiting dilution at 0.3 cells/well. Upon coculture, clone no. 9 specifically recognized B16.WT, B16.B7-1, and JB/MS melanomas, but not control H-2b tumors EL-4, MCA205, and MC38. Clone no. 9 also recognized MC38 sarcoma infected with either rVV or rAd encoding hgp100 or mgp100, but not control rVV or rAd. (B ) Specific in vivo reactivity of gp100-specific T cell clone no. 9 was evaluated by injecting mice with 2 × 105 B16.WT cells intravenously. 3 d later, the specified numbers of clone no. 9 T cells were adoptively transferred. 3 × 105 IU rhIL-2 was then given intraperitoneally two times daily for 5 d. Mice were killed 18 d after tumor inoculation and pulmonary nodules were enumerated. Transfer of T cell clone no. 9, but not β-gal–specific T cells or rhIL-2 alone, dramatically reduced the number of pulmonary nodules in a dose-dependent manner, indicating that gp100 can function as a tumor rejection antigen in vivo. Additional independent clones yielded similar results in repeat experiments.

Figure 3

Figure 3

B16-specific T cells recognize a Db-restricted 9-AA peptide from gp100. In experiment 1, human 293 kidney cells stably transfected with Kb and Db were transfected with human and mouse gp100 pDNA and cocultured for 24 h with gp100-specific T cells. Supernatants were assayed for IFN-γ by ELISA. Specific IFN-γ release was detected when T cells were cocultured with transfected 293 KbDb, but not 293 Kb, indicating that gp100 recognition was predominantly Db-restricted. In experiment 2, human 293 kidney cells stably transfected with Kb and Db were transfected with 3′ exonuclease truncated constructs of hgp100 pDNA and cocultured for 24 h with gp100-reactive T cells. Supernatants were assayed for IFN-γ by ELISA. Specific IFN-γ release was detected with each truncated construct, including the 300-bp cDNA fragment, indicating that a specific immunogenic peptide was located in the 100 NH2-terminal AAs of the gp100 molecule. In experiment 3, peptides corresponding to the binding motif for Db were identified in the first 100 residues of the hgp100 molecule and synthesized together with the corresponding sequences from mgp100. Individual peptides were added to gp100-reactive T cells, and supernatants were assayed for GM-CSF by ELISA. Specific GM-CSF release was detected with peptide pair gp10025–33, suggesting it was the 9-AA epitope responsible for the gp100-reactivity of the T cells.

Figure 4

Figure 4

Recognition of mgp10025–33 and hgp10025–33 peptides at limiting concentrations. EL-4 thymoma cells were incubated with the mgp10025–33 peptide EGSRNQDWL and hgp10025–33 peptide KVPRNQDWL at the concentrations shown on the abscissa for 2 h at 37°C, washed twice, and cocultured for 24 h with gp100-reactive T cells. Supernatants were assayed for IFN-γ by ELISA, which was expressed as percentage of the maximal release (at 1 μM peptide, as shown on the abscissa). Half-maximal recognition of hgp10025–33 was reached at a concentration ∼1,000-fold lower than that needed for mgp10025–33. Data shown is an average of two independent experiments.

Figure 5

Figure 5

Fine specificity of gp100-reactive T cells: EL-4 thymoma cells were incubated with either alanine-substituted peptide variants (A) or NH2-terminally deleted peptide variants (B) for 2 h at 37°C, washed twice, and cocultured for 24 h with gp100-reactive T cells. Supernatants were assayed for GM-CSF by ELISA.

Figure 6

Figure 6

The TAP-deficient cell line RMA-S, expressing “empty” MHC class I molecules, was incubated with the concentrations shown on the abscissa with the mgp10025–33 and hgp10025–33 peptides, as well as control peptides, for 45 min at 25°C, then incubated for 4 h at 37°C. Cells were then stained with FITC-conjugated H-2Db–specific mAb KH95 for 1 h at 4°C, and staining was assessed using flow cytometric analysis and expressed as percentage of the maximal stabilization at 100 μM. The hgp10025–33 peptide stabilized H-2Db molecules at a 100-fold lower concentration than did the mgp10025–33 peptide.

Figure 7

Figure 7

rVVhgp100-induced T cells are tumor specific and can treat established melanoma. (A) Mice were vaccinated with 107 rVVhgp100 intravenously 3 wk before splenocytes were isolated and cultured with 1 μM of mgp10025–33 peptide for 6 d, after which the cells underwent two more cycles of restimulation with mgp10025–33-pulsed splenocytes. The resulting CD4−CD8+ T cells were cocultured for 24 h with various tumor targets, as well as MC38 sarcoma infected with indicated rVV or MC38 sarcoma pulsed with 1 μM of peptide, at an E/T ratio of 1:1, and IFN-γ release was measured by ELISA. rVVhgp100-induced T cells were mgp10025–33– and hgp10025–33–specific, and specifically recognized mgp100+ melanomas, as well as MC38 sarcoma infected with rVVhgp100 or rVVmgp100 but not rVVβ-gal, and MC38 sarcoma pulsed with hgp10025–33 or mgp10025–33 peptide, but not OVA257-264 peptide. (B) Mice were injected intravenously with 2 × 105 B16.WT and treated 3 d later with 5 × 106 of the T cells that are shown in A, or with 5 × 106 β-gal–specific T cells, followed by 3 × 105 IU rhIL-2 intraperitoneally twice daily for 5 d. 18 d after tumor inoculation, the number of pulmonary nodules was specifically reduced by mgp 10025–33–specific T cells. (C) Mice were injected intravenously with 4 × 106 of the T cells that are shown in A, or 4 × 106 β-gal–specific T cells. 5 d later, mice were challenged intravenously with 2 × 105 B16.WT. 18 d after tumor inoculation, the number of pulmonary nodules was dramatically lower in mice receiving mgp 10025–33–specific T cells, indicating in vivo survival of functional, autoreactive T cells. Two repeat experiments yielded similar results.

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