Human tumor antigen-specific helper and regulatory T cells share common epitope specificity but exhibit distinct T cell repertoire

Abstract

CD4(+) regulatory T cells (Tregs) accumulate at tumor sites and play a critical role in the suppression of immune responses against tumor cells. In this study, we show that two immunodominant epitopes derived from the tumor Ags (TAs) NY-ESO-1 and TRAG-3 stimulate both CD4+ Th cells and Tregs. TA-specific Tregs inhibit the proliferation of allogenic T cells, act in a cell-to-cell contact dependent fashion and require activation to suppress IL-2 secretion by T cells. TRAG-3 and NY-ESO-1-specific Tregs exhibit either a Th1-, a Th2-, or a Th0-type cytokine profile and dot not produce IL-10 or TGF-beta. The Foxp3 levels vary from one Treg clone to another and are significantly lower than those of CD4+CD25high Tregs. In contrast to NY-ESO-1-specific Th cells, the NY-ESO-1-specific and TRAG-3-specific Treg clonotypes share a common TCR CDR3 Vbeta usage with Foxp3+CD4+CD25high and CD4+CD25- T cells and were not detectable in PBLs of other melanoma patients and of healthy donors, suggesting that their recruitment occurs through the peripheral conversion of CD4+CD25- T cells upon chronic Ag exposure. Collectively, our findings demonstrate that the same epitopes spontaneously stimulate both Th cells and Tregs in patients with advanced melanoma. They also suggest that TA-specific Treg expansion may be better impaired by therapies aimed at depleting CD4+CD25high Tregs and preventing the peripheral conversion of CD4+CD25- T cells.

FIGURE 1.

TRAG-3 34-48–specific and NY-ESO-1 119-143–specific CD4⁺ Treg clones inhibit the proliferation of responder T cells. The proliferation of allogenic CFSE-labeled naive CD45RA⁺ T cells obtained from blood of HDs was assessed after 5 d of stimulation with soluble anti-CD3 and IL-2 in the presence of autologous CD3-depleted APCs and TRAG-3– or NY-ESO-1–specific CD4⁺ T cell clones. Flow cytometry histograms are displayed for different ratios of responder CD4⁺ and CD8⁺ naive T cells to effector CD4⁺ T cell clones (R:E). Proliferation of naive T cells was inhibited by the presence of (A) TRAG-3 34-48–specific CD4⁺ T cell clones 61/58 (MP1) and 62/3 (HD1) and (B) NY-ESO-1 119-143–specific CD4⁺ T cell clones 30/79 and 35/67 (HD2). C, Opposite effect was observed with TRAG-3 34-48–specific CD4⁺ T cell clone 62/8 (HD1) and NY-ESO-1 119-143–specific CD4⁺ T cell clone 11/4 (MP3). Transwell experiments confirmed that cell–cell contact is required for inhibition of proliferation by Treg clones (A-C, right panels). One of three independent experiments is depicted.

FIGURE 2.

NY-ESO-1–specific CD4⁺ Tregs are isolated ex vivo from PBLs of melanoma patients. A, CD4⁺ T cells isolated from PBLs of MP2 were incubated ex vivo for 6 h with autologous APCs pulsed with peptide NY-ESO-1 119-143 or peptide HIVpol 711-725 as negative control. CD4⁺ T cells were stained with an anti–IFN-γ–PE Ab or a PE-labeled IgG isotype control Ab. IFN-γ–positive NY-ESO-1–specific CD4⁺ T cells were sorted (one cell per well) and expanded in vitro in the absence of peptide. B, NY-ESO-1–specific CD4⁺ T cell clone 103/3 inhibited the proliferation of naive T cells. C, NY-ESO-1–specific CD4⁺ T cell clones 103/4 and 107/5 enhanced the proliferation of responder cells. One of three independent experiments performed is displayed.

FIGURE 3.

TRAG-3 34-48–specific and NY-ESO-1 119-143–specific CD4⁺ Treg clones have low proliferative capacity. Proliferation of CFSE-labeled TRAG-3–specific CD4⁺ Treg clone 61/58 and NY-ESO-1–specific CD4⁺ Treg clones 30/79, 35/67, and 103/3 was assessed after 5 d of stimulation with soluble anti-CD3 Ab (0.1 μg/ml) and IL-2 (50 IU/ml) in the presence of allogenic CD3-depleted APCs obtained from PBLs of one HD. TRAG-3–specific CD4⁺ Treg clone 61/58 and NY-ESO-1–specific CD4⁺ Treg clones 30/79, 35/67, and 103/3 proliferated significantly less than TRAG-3–specific CD4⁺ Th clone 62/8, NY-ESO-1–specific CD4⁺ Th clones 11/4 and 103/4 as well as naive CD45RA⁺ T cells isolated from PBLs of one HD. One of three independent experiments is depicted. No Stim, no stimulation.

FIGURE 4.

Phenotypic analysis of TRAG-3 34-48–specific and NY-ESO-1 119-143–specific CD4⁺ Tregs and Th clones. A, TRAG-3–specific CD4⁺ Treg clones 61/58 and 62/3 expressed high levels of GITR and Foxp3 compared with Th clone 62/8 (upper panel). Two of three NY-ESO-1–specific CD4⁺ Treg clones, clones 30/79 and 103/3, expressed high levels of GITR and all Treg clones expressed high levels of Foxp3, compared with Th clones 103/4 and 107/5 (lower panel). All Treg clones expressed high levels of CD25 and variable levels of CTLA-4. B, The relative expression of Foxp3 by TRAG-3–specific and NY-ESO-1–specific CD4⁺ T cell clones was estimated by real-time quantitative PCR and is displayed using the expression of Foxp3 by CD4⁺ CD25⁻ T cells of one HD as base line (i.e., value of 1). C, The demethylation status of Foxp3 intron 1 from TRAG-3–specific and NY-ESO-1–specific CD4⁺ T cell clones was assessed by QRT-PCR. The results are expressed as percentages of unmethylated Foxp3 sequences and compared with CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells of one HD. D, The expression pattern of TLR-8 in TRAG-3–specific and NY-ESO-1–specific CD4⁺ T cell clones was determined by RT-PCR. β-actin was used as an internal control. PBLs obtained from one HD served as a positive control (HD-PBL). None of the analyzed TRAG-3 34-48–specific and NY-ESO-1 119-143–specific CD4⁺ T cell clones showed expression of TLR-8. One of three independent experiments is depicted.

FIGURE 5.

TA-specific CD4⁺ Tregs produce either Th1-, Th2-, or Th0-type cytokines and suppress IL-2 production by responder T cells. A, TA-specific CD4⁺ Treg and Th clones were stimulated in vitro in the presence of cognate peptide-pulsed APCs prior to measuring cytokine production in supernatants after a 24-h incubation. TRAG-3–specific clone 61/58 secreted Th1-type cytokines. NY-ESO-1–specific CD4⁺ Treg clones 103/3 displayed a Th2-type cytokine profile; whereas, NY-ESO-1–specific CD4⁺ Treg clones 30/79 and 35/67 displayed a Th0-type cytokine-profile. NY-ESO-1–specific CD4⁺ Th clone 107/5 and TRAG-3–specific CD4⁺ Th clone 62/8 displayed a Th1-type and a Th0-type cytokine profile, respectively. They produced IL-2 and higher amounts of IFN-γ and TNF-α than TA-specific Treg clones. B and C, TRAG-3 34-48–specific CD4⁺ Treg clone 61/58 or NY-ESO-1 119-143–specific CD4⁺ Treg clone 103/3 were cocultured with CFSE-labeled or unstained MART-1 27-35–specific CD8⁺ T cell clone 4/43 or NY-ESO-1 119-143–specific CD4⁺ Th clone 11/4 for 24 h in the presence of APCs pulsed with relevant or irrelevant peptides to Tregs. APCs pulsed with relevant or irrelevant peptides to CD8⁺ or Th cells were added after 24 h incubation. Supernatants were collected and analyzed for IL-2 by ELISA after an additional 24 h incubation (B) or CFSE-labeled CD4⁺ Th or CD8⁺ T cell clones were stained intracellularly for IL-2 and analyzed by flow cytometry after an additional 6 h incubation (C). Upon cognate Ag activation, TRAG-3-specific CD4⁺ Treg clone 61/58 inhibited IL-2 production by CD8⁺ and CD4⁺ Th cell clones stimulated with relevant peptides, and NY-ESO-1-specific CD4⁺ Treg clone 103/3 inhibited IL-2 secretion by CD8⁺ T cell clones. The strategy for gating on CFSE-labeled clones 11/4 and 4/43 as well as on CD4⁺ Treg clones 61/58 and 103/3 is shown. As controls, Treg clones 61/58 and 103/3 did not produce IL-2 upon Ag specific-stimulation, and TRAG-3-specific Th clone 62/8 and NY-ESO-1–specific CD4⁺ Th clone 107/5 did not inhibit IL-2 production by clones 11/4 and 4/43, respectively, upon recognition of their cognate Ag (C). One of three independent experiments is depicted. The mean ± SD of IL-2 secretion is shown.

FIGURE 6.

Inhibition of IL-2 production by TRAG-3 34-48–specific CD4⁺ Treg clone correlates with decreased proliferation of TA-specific Th clones. The proliferation of CFSE-labeled NY-ESO-1–specific CD4⁺ Th clone 11/4 was assessed after 5 d of incubation with TRAG-3–specific Treg clone 61/58 in the presence of cognate-peptide–pulsed APCs. TRAG-3 34-48–specific Treg clone 61/58 inhibited proliferation of NY-ESO-1 119-143–specific Th clone 11/4 upon recognition of its cognate Ag but not in the presence of the irrelevant peptide. As control, TRAG-3–specific Th clone 62/8 did not inhibit proliferation of clone 11/4 upon recognition of its cognate Ag. One of two independent experiments is depicted.

FIGURE 7.

NY-ESO-1–specific and TRAG-3–specific Tregs but not Th cells are detected in both CD4⁺CD25^high and CD4⁺CD25⁻ fractions of PBLs isolated from patients with advanced melanoma. A, CD4⁺ T cells were isolated ex vivo from PBLs of MP1 and MP2. CD4⁺CD25^high and CD4⁺CD25⁻ fractions were sorted by flow cytometry prior to RNA extraction. B and C, The relative expression levels of the TCR CDR3β regions of NY-ESO-1-specifc CD4⁺ Treg clone 103/3, TRAG-3–specific Treg clone 61/58 and NY-ESO-1–specific Th clones 103/4 and 107/5 in total PBLs of four MPs and four HDs and in CD4⁺CD25^high and CD4⁺ CD25⁻ cells of MP2 or MP1 were evaluated with quantitative real-time RT-PCR and were correlated with different dilution ratios of each CD4⁺ T cell clone in PBLs from one HD as described in Materials and Methods. Detectable levels of NY-ESO-1–specific Treg clone 103/3 (from MP2) and TRAG-3–specific Treg clone 61/58 (from MP1) CDR3p gene expression were found only in MP2 PBLs and MP1 PBLs, respectively, and in both the CD4⁺CD25^high and CD4⁺CD25² T cell compartments of MP2 and MP1, respectively (B). NY-ESO-1–specific Th clones, 103/4 and 107/5 (from MP2), were detectable only in the CD4⁺CD25⁻ T cell compartment of MP2 (C). One of three independent experiments is depicted.