Survey of naturally occurring CD4+ T cell responses against NY-ESO-1 in cancer patients: correlation with antibody responses

Abstract

NY-ESO-1 is one of the most immunogenic proteins described in human cancers, based on its capacity to elicit simultaneous antibody and CD8+ T cell responses in vivo. Although HLA class II restricted epitopes from NY-ESO-1 have been identified, no broad survey has yet established the status of natural CD4+ T cell responses in cancer patients in relation to CD8+ and antibody responses. We used a recently developed general strategy for monitoring CD4+ responses that overcomes the need for prior knowledge of epitope or HLA restriction to analyze a series of 31 cancer patients and healthy donors for the presence of CD4+ T cells to NY-ESO-1, and related this response to NY-ESO-1 expression in tumor cells and serum antibodies to NY-ESO-1. None of the 18 patients that tested seronegative for NY-ESO-1 had detectable CD4+ T cell responses. On the contrary, 11 of 13 cancer patients with serum antibodies to NY-ESO-1 had polyclonal CD4+ T cell responses directed against various known and previously undescribed NY-ESO-1 epitopes. NY-ESO-1 peptide 80-109 was the most immunogenic, with 10 of 11 patients responding to this peptide. We show here that 12-mer determinants from NY-ESO-1 eliciting a CD4+ T cell response were peptide 87-98 with promiscuous HLA class II presentation, peptide 108-119 restricted by HLA-DP4, and peptides 121-132 and 145-156, both shorter epitopes from previously described HLA-DR4 peptides, also presented by HLA-DR7. This study represents the next step in compiling a comprehensive picture of the adaptive immune response to NY-ESO-1, and provides a general strategy for analyzing the CD4+ T cell response to other tumor antigens eliciting a humoral immune response.

Fig. 1.

Specificity controls of CD4⁺ T cell presensitization from melanoma patient NW29. CD4⁺ T cells were presensitized with indicated peptides or recombinant vectors and tested by ELISPOT at d23 against autologous T-APC pulsed with peptides NP206-229, ESO115-132, ESO80-109, or ESO145-174. Results represent the mean number of spots per 50,000 effector CD4⁺ T cells in duplicate wells, with error bars indicating standard deviation.

Fig. 2.

(A) Titration of peptide ESO80-109 recognized by CD4⁺ T cells from patient NW29. CD4⁺ T cells were presensitized with peptide ESO80-109 and tested by ELISPOT against autologous T-APC pulsed with peptide ESO80-109 in concentrations ranging from 100 μM to 1 nM, or not pulsed. (B) Blocking of ESO80-109 recognition by anti-HLA-DR antibodies. The same effectors were tested by ELISPOT against targets pulsed with ESO80-109 or irrelevant control peptide NP206-229 in the presence or absence of anti-HLA-DR (10 μg/ml) or anti-HLA class I (10 μg/ml) monoclonal antibodies. Results represent the mean number of spots per 50,000 effector CD4⁺ T cells in duplicate wells, with error bars indicating standard deviation.

Fig. 3.

Presensitization of CD4⁺ T cells from ovarian cancer patient NW1558. CD4⁺ T cells were presensitized with AdESO or with peptides ESO100-129 or ESO108-119 and tested by ELISPOT at d19 against autologous T-APC pulsed with ESO98-105, ESO108-119, or ESO100-129, or infected with FP-NP or FP-ESO. Results represent the mean number of spots per 50,000 effector CD4⁺ T cells in duplicate wells, with error bars indicating standard deviation.

Fig. 4.

Mapping of epitopes recognized by CD4⁺ T cells from different cancer patients. (A) CD4⁺ T cells from patient NW1558 were presensitized with adenovirus recombinant for NY-ESO-1 (AdESO) and tested by ELISPOT against hsistocompatible EBV-B cells pulsed with indicated 18-mer NY-ESO-1 peptides, or infected with recombinant fowlpox. (B) CD4⁺ T cells from patient NW903 were presensitized with ESO-CHP and tested by ELISPOT against histocompatible EBV-B cells pulsed with indicated NY-ESO-1 peptides, or infected with recombinant fowlpox. (C) CD4⁺ T cells from patient NW634 were presensitized with ESO-CHP and tested by ELISPOT against histocompatible EBV-B cells pulsed with indicated NY-ESO-1 peptides. (D) CD4⁺ T cells from patient NW29 were presensitized with AdESO and tested by ELISPOT against histocompatible EBV-B cells pulsed with indicated NY-ESO-1 peptides, or infected with recombinant fowlpox.

Fig. 5.

HLA restriction usage for presentation of NY-ESO-1 to CD4⁺ T cells specific for ESO80-109 and ESO100-129. (A) CD4⁺ T cells from patient NW1454 (HLA-DRB1*07,15; -DQB1*02,06; -DPB1*04,09) were presensitized with peptide ESO80-109 and tested by ELISPOT against partially histocompatible EBV-B cells infected with NY-ESO-1 or NP recombinant fowlpox vectors. HLA class II alleles shared between effectors and targets are indicated. *DPnt: targets not tested for HLA-DP compatibility with effectors. (B) Similar analysis with CD4⁺ T cells from patient NW1558 (HLA-DRB1*03,12; -DQB1*02,03; -DPB1*04,10) presensitized with peptide ESO80-109. (C) Similar analysis with CD4⁺ T cells from patient NW1558 presensitized with peptide ESO100-129. Results represent the mean number of spots per 50,000 effector CD4⁺ T cells in duplicate wells with error bars indicating standard deviation.

Fig. 6.

Epitope distribution along the NY-ESO-1 sequence. Above the sequence, defined HLA class I-restricted peptides; below the sequence, HLA class II-restricted peptides. Italics indicate peptides defined by other groups (10, 11, 13).