Identification of CD4+ T cell epitopes from NY-ESO-1 presented by HLA-DR molecules

Abstract

In previous studies, the shared cancer-testis Ag, NY-ESO-1, was demonstrated to be recognized by both Abs and CD8+ T cells. Gene expression of NY-ESO-1 was detected in many tumor types, including melanoma, breast, and lung cancers, but was not found in normal tissues, with the exception of testis. In this study, we describe the identification of MHC class II-restricted T cell epitopes from NY-ESO-1. Candidate CD4+ T cell peptides were first identified using HLA-DR4 transgenic mice immunized with the NY-ESO-1 protein. NY-ESO-1-specific CD4+ T cells were then generated from PBMC of a patient with melanoma stimulated with the candidate peptides in vitro. These CD4+ T cells recognized NY-ESO-1 peptides or protein pulsed on HLA-DR4+ EBV B cells, and also recognized tumor cells expressing HLA-DR4 and NY-ESO-1. A 10-mer peptide (VLLKEFTVSG) was recognized by CD4+ T cells. These studies provide new opportunities for developing more effective vaccine strategies by using tumor-specific CD4+ T cells. This approach may be applicable to the identification of CD4+ T cell epitopes from many known tumor Ags recognized by CD8+ T cells.

Figures

FIGURE 1

A, Purification of full-length NY-ESO-1 protein using a Ni2+ chromatography column. SDS polyacrylamide gel showed the crude extract from E. coli strain BL21(DE3) bearing pET28 vector (lane 1), pNY-ESO-1 (lane 2), the purified NY-ESO-1 protein (lane 3), bacterial extract encoding the truncated NY-ESO-1 (lane 4), and the purified truncated NY-ESO-1 protein, ESO1–74 (lane 5). B, Western blot to confirm the specificity of Abs against NY-ESO-1. Sera at 1/2000 dilution from two representative patients, one with (lanes 1–3) and one without (lanes 4–6) detectable NY-ESO-1 Abs by ELISA, were used against bacterial extract encoding the vector only (lanes 1 and 4), encoding NY-ESO-1 (lanes 2 and 5), and the purified NY-ESO-1 protein (lanes 3 and 6). C, Patient TE was among the melanoma patients who had Abs against NY-ESO-1 protein. ELISA was performed using sera from 88 patients for the presence of Abs against NY-ESO-1 (BSA as control protein). Values of OD 450 at 1/25, 1/250, and 1/2500 of sera dilutions were plotted. Sera from normal donors were used as controls, and their mean OD value was also plotted (ND).

FIGURE 2

Testing of putative NY-ESO-1 epitopes using HLADR4-Tg mice immunized with NY-ESO-1 protein. Eight peptides based on the predicted binding affinity to HLA-DR4 were used for in vitro sensitization of lymphocytes from immunized mice. Murine lymphocytes were tested for IFN-γ production against either medium alone, 1359EBV B (HLA-DR4+) cells alone, or 1359EBV B cells pulsed with the peptide used for in vitro stimulation.

FIGURE 3

Characterization of the TE4-1 CD4+ T cell line. A, TE4-1 specifically recognized 1088 EBV B cells (HLA-DR4+) pulsed with ESO p116–135 peptide or purified NY-ESO-1 protein, but not ESO1–74 protein, which lacked the putative epitope. B, HLA-DR4 restriction was required for the recognition of NY-ESO-1 by TE4-1. Two overlapping peptides, ESO p111–130 and p116–135, were recognized when pulsed onto 293IMDR4 cells. A total of 1 × 105 target cells was cocultured with 4 × 104 TE4-1 cells overnight before GM-CSF secretion was measured. C, Recognition of 293IMDR4 pulsed with ESO p116–135 peptide was specifically inhibited by the anti-HLA-DR Ab (HB55), but not by the anti-class I Ab (HB95). The amount of GM-CSF secreted by TE4-1 in the absence of Abs was used as the reference, against which the percentage of GM-CSF release in the presence of Abs was calculated. Inhibition by the control (mouse IgG2a) and the anti-MHC class I Abs (HB95) had little effect. CTL C3G1 (courtesy of C. Macalli, Surgery Branch, National Cancer Institute) was a gp100-specific CD8+ T cell line that recognized 624.38 mel, and was used as a control for the activity of HB95. T3-80 was a CD4+ T cell line that recognized 1362 mel and was used as the control for the activity of HB55.

FIGURE 4

Recognition of tumor cells by the CD4+ T cell line TE4-1. All melanoma lines used as targets for TE4-1 were analyzed for the expression of HLA-DR4 and NY-ESO-1 by FACS and RT-PCR, respectively. TE4-1 was able to recognize NY-ESO-1+ tumor lines constitutively expressing the HLA-DR4 molecule (1359 mel and F049 mel). F050 mel expressing DR1 and NY-ESO-1 was also recognized by T cells. There was no reactivity against control targets 526 mel (DR4 positive and NY-ESO-1 negative), 397 mel, 624.38 mel (DR negative and NY-ESO-1 positive), or 1300 mel (DR1 positive and NY-ESO-1 weakly positive).

FIGURE 5

Characterization of the NY-ESO-1 peptide epitope recognized by TE4-1. A, Determination of the anchor positions for the HLA-DR4-restricted NY-ESO-1 epitope. The 1088 EBV B cells were pulsed with 20 μM of the indicated peptides. TE4-1 cells were cocultured with the target cells overnight before GM-CSF was measured. B, Peptide titration experiment using ESO p119–130. ESO p119–130 was chosen based on its recognition shown in A. ESO p119–130 diluted at the indicated concentrations were pulsed onto 1088 EBV B cells, which were used as targets for recognition by TE4-1. Recognition of a control peptide, ESO p91–110, was measured only at the highest concentration of 33 μM.