Identification of a TAP-independent, immunoproteasome-dependent CD8+ T-cell epitope in Epstein-Barr virus latent membrane protein 2

Abstract

We have identified an HLA-A2-restricted CD8(+) T-cell epitope, FLYALALLL, in the Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2), an important target antigen in the context of EBV-associated malignancies. This epitope is TAP independent, like other hydrophobic LMP2-derived epitopes, but uniquely is dependent upon the immunoproteasome for its generation.

FIG. 1.

Identification and mapping of an LMP2-specific CTL clone directed against a novel HLA-A*0201-restricted epitope, FLYALALLL. (A) CTL clone donor A c30, derived by stimulation of PBMCs with irradiated autologous LCLs, was tested in a chromium release assay against autologous (auto) or partially HLA-matched LCL target cells infected with recombinant vaccinia viruses expressing either LMP2 (vLMP2) or, as a control, EBNA3A (vE3A), and A*0201-positive targets preexposed to the known LMP2-derived A*0201-restricted epitope peptides CLGGLLTMV (CLG) or LLWTLVVLL (LLW), or to dimethyl sulfoxide (DMSO) solvent control. (B) donor A c30 was tested in a chromium release assay against autologous LCL target cells preincubated with the synthetic peptides shown, representing LMP2 amino acids 353 to 370, at the indicated molar concentration. The minimal epitope was defined as FLYALALLL (FLY; LMP2 356 to 364). Results are expressed as the percentage of specific lysis observed in a standard 5-h chromium release assay at an effector/target ratio of 5:1.

FIG. 2.

The LMP2 FLY epitope is not presented to CTLs in the T2 cell background. (A) Chromium release assays carried out with CTLs specific for the FLY/A*0201 epitope as effectors. Targets include T2 cells infected with a recombinant vaccinia virus expressing LMP2 (vLMP2) or control virus vTK− and T3 cells (rat TAP1/TAP2-transfectants of T2) infected with either vLMP2 or control virus vTK−. Both target cell lines were also preexposed to either the cognate epitope peptide or to dimethyl sulfoxide (DMSO) solvent control. (B) Assays conducted with effector CTLs specific for the TAP-independent CLG/A*0201 epitope on the same targets as those described above. (C) Assays conducted using as effectors CTLs specific for the TAP-dependent RRR/B*2704 epitope on the same targets described above, but in this case also infected with a vaccinia virus, vB*2704, expressing the HLA-B*2704 allele. Results are expressed as the percentage of specific lysis observed in a standard 5-h chromium release assay at effector/target ratios of 5:1 (black bars) and 2:1 (white bars).

FIG. 3.

Presentation of the LMP2 FLY epitope requires IFN-γ induction of the immunoproteasome. (A) The melanoma cell line MEL-275 was infected with a recombinant vaccinia virus expressing LMP2 (vLMP2) or a control virus, vTK−, or preexposed to the cognate epitope peptide or to dimethyl sulfoxide (DMSO) solvent control and then used as targets in chromium release assays with CLG-specific CTL effectors (left panel). MEL-275 cells were either untreated or pretreated with IFN-γ for 48 h prior to exposure to the vaccinia viruses or peptide described above and then used as targets for FLY-specific CTL effectors (right panel). IFN-γ-induced MEL-275 cells were also incubated with lactacystin for 1 h prior exposure to the vaccinia viruses or peptide described above and then used as targets for FLY-specific CTL effectors. Results of chromium release assays are shown as the percentage of specific lysis observed in a standard 5-h chromium release assay at effector/target ratios of 5:1 (black bars) and 2:1 (white bars). (B) Western blot analysis of MEL-275, both before and after IFN-γ induction, for expression of immunoproteasome subunits ip-lmp2 and ip-lmp7. A standard LCL was used as a positive control.

FIG. 4.

Presentation of the LMP2 FLY epitope is TAP-independent. (A) TAP-positive HLA-A*0201-positive donor B fibroblasts were infected with a recombinant vaccinia virus expressing LMP2 (vLMP2) or a control virus vTK− or preexposed to the cognate epitope peptide or to dimethyl sulfoxide (DMSO) solvent control and then used as targets in chromium release assays with CLG-specific CTL effectors (left panel). The same cells were either untreated or pretreated with IFN-γ for 48 h prior to exposure to the vaccinia viruses or peptide described above and then used as targets for FLY-specific CTL effectors (right panel). (B) The TAP2-negative HLA-A*0201-positive patient C fibroblast cells were infected with a recombinant vaccinia virus expressing LMP2 (vLMP2) or a control virus vTK− or preexposed to the cognate epitope peptide or to dimethyl sulfoxide solvent control and then used as targets in chromium release assays with CLG-specific CTL effectors (left panel). Patient C fibroblasts were either untreated or pretreated with IFN-γ for 48 h prior to exposure to the vaccinia viruses or peptide described above and then used as targets for FLY-specific CTL effectors (right panel). Results of chromium release assays are shown as the percentage of specific lysis observed in a standard 5-h chromium release assay at an effector/target ratio of 5:1 (black bars) and 2:1 (white bars). (C) Western blot analysis of patient C fibroblasts (fibros), both before and after IFN-γ induction, for expression of immunoproteasome subunits ip-lmp2 and ip-lmp7 and the TAP1 and TAP2 subunits. A standard LCL line was used as a positive control.