Identification of NY-ESO-1 epitopes presented by human histocompatibility antigen (HLA)-DRB4*0101-0103 and recognized by CD4(+) T lymphocytes of patients with NY-ESO-1-expressing melanoma

Abstract

NY-ESO-1 is a member of the cancer-testis family of tumor antigens that elicits strong humoral and cellular immune responses in patients with NY-ESO-1-expressing cancers. Since CD4(+) T lymphocytes play a critical role in generating antigen-specific cytotoxic T lymphocyte and antibody responses, we searched for NY-ESO-1 epitopes presented by histocompatibility leukocyte antigen (HLA) class II molecules. Autologous monocyte-derived dendritic cells of cancer patients were incubated with recombinant NY-ESO-1 protein and used in enzyme-linked immunospot (ELISPOT) assays to detect NY-ESO-1-specific CD4(+) T lymphocyte responses. To identify possible epitopes presented by distinct HLA class II alleles, overlapping 18-mer peptides derived from NY-ESO-1 were synthetized and tested for recognition by CD4(+) T lymphocytes in autologous settings. We identified three NY-ESO-1-derived peptides presented by DRB4*0101-0103 and recognized by CD4(+) T lymphocytes of two melanoma patients sharing these HLA class II alleles. Specificity of recognition was confirmed by proliferation assays. The characterization of HLA class II-restricted epitopes will be useful for the assessment of spontaneous and vaccine-induced immune responses of cancer patients against defined tumor antigens. Further, the therapeutic efficacy of active immunization using antigenic HLA class I-restricted peptides may be improved by adding HLA class II-presented epitopes.

Figure 1

ELISPOT assays showing CD4⁺ T cell recognition of APCs pulsed with NY-ESO-1. Numbers of IFN-γ spots/100,000 CD4⁺ selected T lymphocytes are shown. CD4⁺ T cells were tested against autologous APCs, either alone or after pulsing with NY-ESO-1–derived peptides or recombinant NY-ESO-1 protein. Peptides tested were different NY-ESO-1–derived 18-mer peptides. Patients NW29 and NW37, who share the HLA-DRB4*0101–0103 alleles, show the same pattern of peptide recognition, whereas the HLA-DRB4* 0101–0103⁻ patient NW690 shows recognition of NY-ESO-1 protein only, suggesting that peptides 21, 22, and 25 are presented by HLA-DRB4* 0101–0103.

Figure 2

ELISPOT assays showing the blocking effect of HLA-DR–specific antibody L243 on CD4⁺ T cell recognition of NW29 APCs pulsed with the DRB4*0101–0103-binding NY-ESO-1 peptide 25. Experiments were performed as in the legend to Fig. 1, with L243 added at 2 μg/well to one set of experiments. HLA class I–binding antibody W6/32 was used at 2 μg/well as a control. Results showed a decrease of positive spots by >50% in the presence of L243 (gray bars), whereas W6/32 (white bars) did not affect the specific recognition of peptide 25.

Figure 3

Standard [³H]TdR proliferation assay showing specific proliferative response of peptide-stimulated NW29 CD4⁺ T cells to autologous APCs alone or pulsed with the respective DRB4*0101–0103-restricted NY-ESO-1 peptides (21, 22, and 25).

Figure 4

ELISPOT assay with autologous APCs pulsed with NY-ESO-1–derived peptide 22, and the recombinant NY-ESO-1 and SSX proteins. The CD4⁺ T cell line NW37 CD4/22 was used as effectors. Bars indicate the number of spots/10⁴ NW37 CD4/22 cells. Values represent the results of three independent experiments.

Figure 5

ELISPOT assay with autologous APCs derived from patients NW29 and NW37 pulsed with NW-MEL-38 lysate and tested for specific recognition with the CD4⁺ T cell lines NW29 CD4/25 and NW37 CD4/22. Bars indicate the number of spots/10⁴ NW37 CD4/22 cells.