HLA-A2-restricted T-cell epitopes specific for prostatic acid phosphatase

Abstract

Prostatic acid phosphatase (PAP) has been investigated as the target of several antigen-specific anti-prostate tumor vaccines. The goal of antigen-specific active immunotherapies targeting PAP would ideally be to elicit PAP-specific CD8+ effector T cells. The identification of PAP-specific CD8+ T-cell epitopes should provide a means of monitoring the immunological efficacy of vaccines targeting PAP, and these epitopes might themselves be developed as vaccine antigens. In the current report, we hypothesized that PAP-specific epitopes might be identified by direct identification of pre-existing CD8+ T cells specific for HLA-A2-restricted peptides derived from PAP in the blood of HLA-A2-expressing individuals. 11 nonamer peptides derived from the amino acid sequence of PAP were used as stimulator antigens in functional ELISPOT assays with peripheral blood mononuclear cells from 20 HLA-A2+ patients with prostate cancer or ten healthy blood donors. Peptide-specific T cells were frequently identified in both groups for three of the peptides, p18-26, p112-120, and p135-143. CD8+ T-cell clones specific for three peptides, p18-26, p112-120, and p299-307, confirmed that these are HLA-A2-restricted T-cell epitopes. Moreover, HLA-A2 transgenic mice immunized with a DNA vaccine encoding PAP developed epitope-specific responses for one or more of these three peptide epitopes. We propose that this method to first identify epitopes for which there are pre-existing epitope-specific T cells could be used to prioritize MHC class I-specific epitopes for other antigens. In addition, we propose that the epitopes identified here could be used to monitor immune responses in HLA-A2+ patients receiving vaccines targeting PAP to identify potentially therapeutic immune responses.

Fig. 1

PAP-derived peptide-specific T cells identified in HLA-A2-expressing individuals by ELISPOT. a Representative ELISPOT assay using peripheral blood mononuclear cells (PBMC) from a 49-year-old patient with stage D2 prostate cancer. PBMC were stimulated once in vitro with 10 μg/ml of the indicated peptides or tetanus toxoid protein (1 LFU/ml, as a positive control), and assayed for peptide-specific IFNγ release upon restimulation with peptide, as described. In this assay, IFNγ spots in response to tetanus toxoid stimulation and PAP peptide p18–26 were highly significant (P < 0.001) compared with media only (no antigen). b ELISPOT assays were performed in identical fashion using PBMC from multiple individuals. T-cell frequencies were calculated as spot-forming units (sfu) per 10⁶ PBMC. Open circles (N) show data for the ten volunteer blood donors (n = 10), and closed circles (C) show data for the patients with prostate cancer (n = 20)

Fig. 2

Peptide-specific T-cell clones specific for p18–26, p112–120, and p299–307, but not p135–143, can lyse PAP-expressing cells in an HLA-A2-restricted fashion. Clonal T-cell lines specific for p18–26, p112–120, p135–143, or p299–307 were assessed for their ability to lyse peptide-pulsed T2 target cell lines (column a), HLA-A2-expressing TK6 cell lines expressing PAP (TK6-PAP) versus control TK6 cells expressing GFP (TK6-GFP, column b), or prostate cancer cell lines (column c). Cytotoxicity was measured as LDH release after 4-h co-culture of effector cells with target cells. Assays were performed in triplicate at multiple effector-to-target ratios as indicated. Shown is the mean and standard deviation of percent specific lysis at each effector-to-target ratio, determined from triplicate assessments, of each clonal line and target cell line. column a: solid squares represent T2 cells pulsed with the relevant peptide, and open squares represent T2 target cells pulsed with an irrelevant HLA-A2-binding peptide. column b: solid diamonds represent TK6-PAP target cells, and open diamonds represent TK6-GFP target cells. column c: closed triangles represent HLA-A2-transduced LNCaP target cells (expressing PAP), and open triangles represent HLA-A2-transduced LNCaP target cells in the presence of an HLA-A2 blocking antibody. Open circles represent HLA-A2-transduced DU145 target cells (not expressing PAP). Results are representative of multiple independent experiments

Fig. 3

PAP-specific epitopes can be used for monitoring immune responses following DNA immunization. HHD-II mice were immunized six times at 14-day intervals with plasmid DNA encoding PAP (pTVG-HP, n = 7) or vector control (pTVG4, n = 6). Splenocytes obtained 2 weeks after the final immunization were cultured with all peptides in bulk and assessed after 7 days by IFNγ ELISPOT using individual peptides, an HLA-A2-specific influenza control peptide (pFlu), PAP protein (PAP), or concanavalin A (ConA) as stimulatory antigens. The frequency of IFNγ sfu per 10⁶ is shown for each animal. Closed circles represent data from vector control-immunized mice, and open circles represent data from PAP DNA-immunized mice. Comparisons of means (shown as lines) between groups are made with an unpaired t test, and the P values are shown for the relevant comparisons. a Analysis of IFNγ ELISPOT responses by individual stimulatory peptide antigens. b The same data but pooled with respect to the PAP-derived non-epitope peptides or PAP-specific HLA-A2 epitopes