Identification of a major histocompatibility complex class I-restricted T-cell epitope in the tumour-associated antigen, 5T4

Abstract

5T4 is a surface glycoprotein expressed on placental trophoblasts and also on a wide range of human carcinomas. Its highly restricted expression on normal tissues and broad distribution on many carcinomas make 5T4 a promising target for cancer immunotherapy. In the current study, we set out to investigate whether a 5T4-specific cytotoxic T lymphocyte (CTL) repertoire exists in healthy individuals. CD4-depleted peripheral blood mononuclear cells (PBMCs) from blood donors were screened using an ex vivo interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay. A panel of overlapping peptides, spanning the full length of the 5T4 protein, was used as a source of antigen. In the process of screening, one out of 30 blood donors demonstrated a positive ex vivo IFN-gamma ELISPOT response to a single 5T4 peptide. A polyclonal T-cell line was derived from this donor by culturing PBMCs with autologous peptide-pulsed dendritic cells (DCs). The resulting polyclonal T-cell line and clones were tested in a 51Cr-release assay and by ELISPOT and were shown to be peptide specific. Furthermore, antigen-presenting cells (APCs), infected with a viral vector expressing 5T4, were able to stimulate IFN-gamma production by the peptide-specific T-cell clones. A minimal CD8 epitope, PLADLSPFA, has been identified and found to be restricted through human leucocyte antigen (HLA) Cw7. Subsequently, we have demonstrated that HLA-Cw7-positive colorectal cancer patients vaccinated with a recombinant vaccinia viral vector encoding 5T4 (TroVax) are capable of mounting a strong IFN-gamma ELISPOT response to this novel CTL epitope. These findings have potential application in cancer immunotherapy in terms of subunit vaccine design and the monitoring of immune responses induced in patients by 5T4-based therapies.

Figures

Figure 1

Ex vivo enzyme-linked immunospot (ELISPOT) assay of OB8 CD4-depleted peripheral blood mononuclear cells (PBMCs) with 5T4 peptides of pool 8. The cells were incubated for 24 hr with the peptide pool 8, previously defined as positive, and with the individual peptides constituting peptide pool 8 at a concentration of 2 µg/ml in triplicate wells. Peptide 8.7 induced interferon-γ (IFN-γ) production of the same magnitude as the pool. This experiment was repeated three times, with similar results obtained on each occasion. No Ag cont., no antigen control; SFC, spot forming cells.

Figure 2

An in vitro-generated OB8 polyclonal T-cell line was tested for the presence of 5T4-reactive T cells in functional assays. Cytotoxic T lymphocytes (CTLs), expanded using autologous dendritic cells (DCs) pulsed with p8.7, demonstrated killing of autologous and human leucocyte antigen (HLA)-matched target LCL, expressing 5T4 protein, in a 51Cr-release assay at a effector:target (E:T) ratio of 20 : 1 (a). These data are representative of three experiments. The CTLs also produced interferon-γ (IFN-γ) in response to autologous LCL exogenously loaded with p8.7 and expressing 5T4 from TroVax, when tested by enzyme-linked immunospot (ELISPOT) assay (b). APC, antigen-presenting cells; SFC, spot forming cells.

Figure 3

A generated OB8 T-cell clone was tested by 51Cr-release assay against target cells loaded with p8.7 and its variants [effector:target (E:T) = 5 : 1]. T-cell clones recognized original 10-mer peptide p8.7 and a 9-mer variant, p8.7A, but not other variants of p8.7 (a). Titration of peptides p8.7 (PLADLSPFAF) and p8.7A (PLADLSPFA) showed that the lowest concentration at which target cells were recognized was 100 nm (b).

Figure 4

The human leucocyte antigen (HLA) restriction element of p8.7A was identified by a 51Cr-release assay (a) and enzyme-linked immunospot (ELISPOT) (b) using a 5T4-specific T-cell clone as effector cells and partially HLA-matched LCLs as targets and antigen-presenting cells (APCs), respectively. The T-cell clone recognized autologous LCL and GS LCL matched with OB8 by the Cw7 allele. These data are representative of three experiments. No Ag cont., no antigen control; SFC, spot forming cells.