Circulating tumor antigen-specific regulatory T cells in patients with metastatic melanoma

Abstract

Although it is accepted that regulatory T cells (T regs) contribute to cancer progression, most studies in the field consider nonantigen-specific suppression. Here, we show the presence of tumor antigen-specific CD4(+) T regs in the blood of patients with metastatic melanoma. These CD4(+) T regs recognize a broad range of tumor antigens, including gp100 and TRP1 (melanoma tissue differentiation antigens), NY-ESO-1 (cancer/testis antigen) and survivin (inhibitor of apoptosis protein (IAP) family antigen). These tumor antigen-specific T regs proliferate in peripheral blood mononuclear cells (PBMC) cultures in response to specific 15-mer peptides, produce preferentially IL-10 and express high levels of FoxP3. They suppress autologous CD4(+)CD25(-) T cell responses in a cell contact-dependent manner and thus share properties of both naturally occurring regulatory T cells and type 1 regulatory T cells. Such tumor antigen-specific T regs were not detected in healthy individuals. These tumor antigen-specific T regs might thus represent another target for immunotherapy of metastatic melanoma.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

NY-ESO-1 peptides promoting IL-10 secretion in PBMC cultures induce the proliferation of FoxP3high CD4+ T cells. (A) Two NY-ESO-1 peptides promoted IL-10 secretion. PBMCs (Pt#094-004) were incubated with 15-mer overlapping NY-ESO-1 single peptides for 2 days. The secreted IL-10 in the culture supernatant was measured. (B) Proliferation of CD4+ T cells in response to the identified NY-ESO-1 peptide. CFSE-labeled PBMCs were cultured with NY-ESO-1 p#23, and CFSE dilution was analyzed at day 6. Data are representative of 11 experiments. (C) The proliferation of peptide-specific CD4+ T cells depends on MHC-class II molecules. The frequencies of CFSE-diluted CD4+ T cell population responding to NY-ESO-1 p#23in the presence or absence of anti-HLA class II blocking mAb are shown. (D) NY-ESO-1-responding CD4+ T cells contain cells with high levels of FoxP3. CFSE-labeled PBMCs cultured with NY-ESO-1 p#23 or TT for 6 days were analyzed for the expression of FoxP3. (E) FoxP3 levels in CFSE-diluted CD4+ T cell populations responding to NY-ESO-1p#23 or TT are shown in the histogram (a representative of 6).

Fig. 2.

NY-ESO-1p#23-responding FoxP3high CD4+ T cells maintain FoxP3 expression. (A) PBMCs from Pt# 094-004 were cultured with NY-ESO-1 p#23 or TT for 10 d, and FoxP3 expression in CD4+ T cells was analyzed. (B) Mean fluorescent intensity of FoxP3 levels in CFSE-diluted FoxP3high and FoxP3low CD4+ T cells reacting with NY-ESO-1 p23, and in CFSE-diluted CD4+ T cells reacting with TT, were analyzed at days 6, 8, and 10 of cultures.

Fig. 3.

Expanded CFSE-diluted CD4+ T cells produce IL-10 in response to NY-ESO-1 p#23 stimulation. (A and B) NY-ESO-1 p#23-responding CD4+ T cells secrete higher amounts of IL-10 than TT-responding CD4+ T cells. CFSE-diluted CD4+ T cells in response to IL-10-inducing peptides or TT were sorted into 96-well plates at 3–10 cells per well, and cultured for 21 d with irradiated ICOS-ligand transfectant and anti-CD3 mAb in the presence of rhIL-2 (1,000 units/ml). IL-10 levels secreted during the cell expansion in each well were measured by ELISA. (B is a representative result of 12). (C) Peptide-specific IL-10 secretion. CD4+ T cells pooled from wells containing >400 pg/ml IL-10 at 3 weeks of culture were stimulated overnight with autologous DCs incubated with NY-ESO-1 p#23 or an irrelevant peptide, and secreted cytokines were measured (a representative result of 8).

Fig. 4.

Expanded NY-ESO-1 p#23-specific IL-10high CD4+ T cells have suppressive functions. (A) Inhibition of CD4+CD25− T cell proliferation. The pooled IL-10high CD4+ T cells were cultured alone or together with autologous CD4+CD25− T cells (1:1 ratio) in the presence of CD3/CD28 stimulation. [3H]Thymidine incorporation was analyzed on day 3 (a representative of >10). (B) Titration of pooled IL-10high CD4+ T cells for suppressive function. Indicated number of the pooled cells was added to the autologous CD4+CD25− T cell culture stimulated with CD3/CD28 mAb. (C) The pooled CD4+ T cells abrogate cytokine production from CD4+CD25− T cells. Culture supernatants were harvested at day 2 of the suppression assay shown in A, and secreted cytokines were measured (a representative of 8).

Fig. 5.

NY-ESO-1-specific IL-10high T regs require cell-to-cell contact for their suppression. (A) Soluble factor is not responsible for the suppression. CD4+CD25− T cells were separated from the pooled IL-10high CD4+ T cells by transwell in suppression assays (a representative of 6). (B) IL-10 plays only a minor role in the suppression. An IL-10 Rα blocking mAb was added in the suppression assays. Each dot represents an independent experiment. (C) The pooled IL-10high CD4+ T cells contain FoxP3highCTLA-4high T cells. Pooled IL-10high CD4+ T cells and IL-10low cells were examined for their expression of FoxP3 and CTLA-4.

Fig. 6.

Identification of other tumor antigen-specific T regs. Survivin54–67 (Survivin p#15) and TRP-1449–463 (TRP-1 p#113) were found as CD4+ T reg epitopes in Pt#094–004, whereas gp100369–383 (gp100 p#93) was found as a CD4+ T reg epitope in Pt#094–005. IL-10 levels at 48 h PBMC cultures with the identified peptides are shown on the left. The FoxP3 expression of CD4+ T cells in CFSE-labeled PBMCs cultured for 6 days with the indicated T reg epitopes is shown (Middle) (representatives of 6 on each). Suppression assays with pooled IL-10-secreting oligoclonal CD4+ T cell lines (IL-10high CD4+ T cells) are shown on the right (representatives of >10 on each). There, the pooled IL-10high CD4+ T cells were cultured alone or together with autologous CD4+CD25− T cells (1:1 ratio) in the presence of CD3/CD28 stimulation. [3H]thymidine incorporation was analyzed on day 3.

Fig. 7.

CD4+ T reg epitopes of NY-ESO-1. CD4+ T reg epitopes identified in this study are shown in green. Previously identified CD8+ T cell epitopes are shown in red, and previously identified CD4+ helper T cell epitopes are shown in blue. Refs: a, Cancer Immunity peptide database, www.cancerimmunity.org/peptidedatabase/Tcellepitopes.htm, and refs. and 36); b, ref. .