Junko Matsuzaki

Tumor-infiltrating NY-ESO-1–specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer

NY-ESO-1 is a “cancer-testis” antigen frequently expressed in epithelial ovarian cancer (EOC) and is among the most immunogenic tumor antigens defined to date. In an effort to understand in vivo tolerance mechanisms, we assessed the phenotype and function of NY-ESO-1–specific CD8+ T cells derived from peripheral blood lymphocytes (PBLs), tumor-infiltrating lymphocytes (TILs), and tumor-associated lymphocytes (TALs) of EOC patients with NY-ESO-1-expressing tumors, with or without humoral immunity to NY-ESO-1. Whereas NY-ESO-1–specific CD8+ T cells were readily detectable ex vivo with tetramers in TILs and TALs of seropositive patients, they were only detectable in PBLs following in vitro stimulation. Compared with PBLs, tumor-derived NY-ESO-1–specific CD8+ T cells demonstrated impaired effector function, preferential usage of dominant T-cell receptor, and enriched coexpression of inhibitory molecules LAG-3 and PD-1. Expression of LAG-3 and PD-1 on CD8+ T cells was up-regulated by IL-10, IL-6 (cytokines found in tumor ascites), and tumor-derived antigen-presenting cells. Functionally, CD8+LAG-3+PD-1+ T cells were more impaired in IFN-γ/TNF-α production compared with LAG-3+PD-1− or LAG-3−PD-1− subsets. Dual blockade of LAG-3 and PD-1 during T-cell priming efficiently augmented proliferation and cytokine production by NY-ESO-1–specific CD8+ T cells, indicating that antitumor function of NY-ESO-1-specific CD8+ T cells could potentially be improved by therapeutic targeting of these inhibitory receptors.

Generation and regulation of human CD4+ IL-17-producing T cells in ovarian cancer

Despite the important role of Th17 cells in the pathogenesis of many autoimmune diseases, their prevalence and the mechanisms by which they are generated and regulated in cancer remain unclear. Here, we report the presence of a high percentage of CD4+ Th17 cells at sites of ovarian cancer, compared with a low percentage of Th17 cells in peripheral blood mononuclear cells from healthy donors and cancer patients. Analysis of cytokine production profiles revealed that ovarian tumor cells, tumor-derived fibroblasts, and antigen-presenting cells (APCs) secreted several key cytokines including IL-1β, IL-6, TNF-α and TGF-β, which formed a cytokine milieu that regulated and expanded human IL-17-producing T-helper (Th17) cells. We further show that IL-1β was critically required for the differentiation and expansion of human Th17 cells, whereas IL-6 and IL-23 may also play a role in the expansion of memory Th17 cells, even though IL-23 levels are low or undetectable in ovarian cancer. Further experiments demonstrated that coculture of naïve or memory CD4+ T cells with tumor cells, APCs, or both could generate high percentages of Th17 cells. Treatment with anti-IL-1 alone or a combination of anti-IL-1 and anti-IL-6 reduced the ability of tumor cells to expand memory Th17 cells. Thus, we have identified a set of key cytokines secreted by ovarian tumor cells and tumor-associated APCs that favor the generation and expansion of human Th17 cells. These findings should accelerate efforts to define the function of this important subset of CD4+ T cells in the human immune response to cancer.

Vaccination with an NY-ESO-1 peptide of HLA class I/II specificities induces integrated humoral and T cell responses in ovarian cancer.

NY-ESO-1 is a “cancer-testis” antigen expressed in epithelial ovarian cancer (EOC) and is among the most immunogenic tumor antigens defined to date. The NY-ESO-1 peptide epitope, ESO157–170, is recognized by HLA-DP4-restricted CD4+ T cells and HLA-A2- and A24-restricted CD8+ T cells. To test whether providing cognate helper CD4+ T cells would enhance the antitumor immune response, we conducted a phase I clinical trial of immunization with ESO157–170 mixed with incomplete Freunds adjuvant (Montanide ISA51) in 18 HLA-DP4+ EOC patients with minimal disease burden. NY-ESO-1-specific Ab responses and/or specific HLA-A2-restricted CD8+ and HLA-DP4-restricted CD4+ T cell responses were induced by a course of at least five vaccinations at three weekly intervals in a high proportion of patients. There were no serious vaccine-related adverse events. Vaccine-induced CD8+ and CD4+ T cell clones were shown to recognize NY-ESO-1-expressing tumor targets. T cell receptor analysis indicated that tumor-recognizing CD4+ T cell clones were structurally distinct from non-tumor-recognizing clones. Long-lived and functional vaccine-elicited CD8+ and CD4+ T cells were detectable in some patients up to 12 months after immunization. These results confirm the paradigm that the provision of cognate CD4+ T cell help is important for cancer vaccine design and provides the rationale for a phase II study design using ESO157–170 epitope or the full-length NY-ESO-1 protein for immunotherapy in patients with EOC.

Efficacy of vaccination with recombinant vaccinia and fowlpox vectors expressing NY-ESO-1 antigen in ovarian cancer and melanoma patients

Recombinant poxviruses (vaccinia and fowlpox) expressing tumor-associated antigens are currently being evaluated in clinical trials as cancer vaccines to induce tumor-specific immune responses that will improve clinical outcome. To test whether a diversified prime and boost regimen targeting NY-ESO-1 will result in clinical benefit, we conducted two parallel phase II clinical trials of recombinant vaccinia-NY-ESO-1 (rV-NY-ESO-1), followed by booster vaccinations with recombinant fowlpox-NY-ESO-1 (rF-NY-ESO-1) in 25 melanoma and 22 epithelial ovarian cancer (EOC) patients with advanced disease who were at high risk for recurrence/progression. Integrated NY-ESO-1-specific antibody and CD4+ and CD8+ T cells were induced in a high proportion of melanoma and EOC patients. In melanoma patients, objective response rate [complete and partial response (CR+PR)] was 14%, mixed response was 5%, and disease stabilization was 52%, amounting to a clinical benefit rate (CBR) of 72% in melanoma patients. The median PFS in the melanoma patients was 9 mo (range, 0–84 mo) and the median OS was 48 mo (range, 3–106 mo). In EOC patients, the median PFS was 21 mo (95% CI, 16–29 mo), and median OS was 48 mo (CI, not estimable). CD8+ T cells derived from vaccinated patients were shown to lyse NY-ESO-1-expressing tumor targets. These data provide preliminary evidence of clinically meaningful benefit for diversified prime and boost recombinant pox-viral-based vaccines in melanoma and ovarian cancer and support further evaluation of this approach in these patient populations.

Efficacy of Levo-1-Methyl Tryptophan and Dextro-1-Methyl Tryptophan in Reversing Indoleamine-2,3-Dioxygenase-Mediated Arrest of T-Cell Proliferation in Human Epithelial Ovarian Cancer

It has been reported that levo-1-methyl tryptophan (L-1MT) can block indoleamine-2,3-dioxygenase (IDO) expressed by human dendritic cells (DC), whereas dextro-1-methyl tryptophan (D-1MT) is inefficient. However, whether L-1MT or D-1MT can efficiently reverse IDO-induced arrest of human T-cell proliferation has not been clarified. Here, we show a marked immunosuppressive effect of IDO derived from INDO-transfected 293 cell, IDO+ ovarian cancer cells, and monocyte-derived DCs on CD4+ Th1 cells, CD8+ T cells, and natural killer cells derived from peripheral blood, ascites, and tumors of ovarian cancer patients. We found that, whereas L-1MT and D/L-1MT can restore proliferation of tumor-derived and peripheral blood T-cell subsets, D-1MT does not effectively restore IDO-induced arrest of T-cell proliferation. Although D-1MT inhibited kynurenine production at high concentrations, L-1MT was more effective in abrogating kynurenine generation and tryptophan depletion, whereas tryptophan was completely depleted by IDO even in the presence of high amounts of D-1MT. Together, the results indicate that, whereas the generation of tryptophan metabolites (kynurenines) by IDO is important in mediating suppression of T-cell proliferation, the degree to which tryptophan depletion is restored by 1MT is also critical in overcoming IDO-induced arrest of T-cell proliferation.

Antibody-Targeted NY-ESO-1 to Mannose Receptor or DEC-205 In Vitro Elicits Dual Human CD8+ and CD4+ T Cell Responses with Broad Antigen Specificity

Immunization of cancer patients with vaccines containing full-length tumor Ags aims to elicit specific Abs and both CD4+ and CD8+ T cells. Vaccination with protein Ags, however, often elicits only CD4+ T cell responses without inducing Ag-specific CD8+ T cells, as exogenous protein is primarily presented to CD4+ T cells. Recent data revealed that Ab-mediated targeting of protein Ags to cell surface receptors on dendritic cells could enhance the induction of both CD4+ and CD8+ T cells. We investigated in this study if these observations were applicable to NY-ESO-1, a cancer-testis Ag widely used in clinical cancer vaccine trials. We generated two novel targeting proteins consisting of the full-length NY-ESO-1 fused to the C terminus of two human mAbs against the human mannose receptor and DEC-205, both internalizing molecules expressed on APC. These targeting proteins were evaluated for their ability to activate NY-ESO-1–specific human CD4+ and CD8+ T cells in vitro. Both targeted NY-ESO-1 proteins rapidly bound to their respective targets on APC. Whereas nontargeted and Ab-targeted NY-ESO-1 proteins similarly activated CD4+ T cells, cross-presentation to CD8+ T cells was only efficiently induced by targeted NY-ESO-1. In addition, both mannose receptor and DEC-205 targeting elicited specific CD4+ and CD8+ T cells from PBLs of cancer patients. Receptor-specific delivery of NY-ESO-1 to APC appears to be a promising vaccination strategy to efficiently generate integrated and broad Ag-specific immune responses against NY-ESO-1 in cancer patients.

Epigenetic Potentiation of NY-ESO-1 Vaccine Therapy in Human Ovarian Cancer

Odunsi and colleagues show that the DNA methyltransferase inhibitor decitabine augmented the efficacy of the NY-ESO-1 vaccine and doxorubicin treatment of patients with refractory epithelial ovarian cancer, demonstrating the potential of the combined chemo-immunotherapy regimen. The cancer–testis/cancer germline antigen, NY-ESO-1, is a vaccine target in epithelial ovarian cancer (EOC), but its limited expression is a barrier to vaccine efficacy. As NY-ESO-1 is regulated by DNA methylation, we hypothesized that DNA methyltransferase inhibitors may augment NY-ESO-1 vaccine therapy. In agreement, global DNA hypomethylation in EOC was associated with the presence of circulating antibodies to NY-ESO-1. Preclinical studies using EOC cell lines showed that decitabine treatment enhanced both NY-ESO-1 expression and NY-ESO-1–specific CTL-mediated responses. On the basis of these observations, we performed a phase I dose-escalation trial of decitabine, as an addition to NY-ESO-1 vaccine and doxorubicin liposome chemotherapy, in 12 patients with relapsed EOC. The regimen was safe, with limited and clinically manageable toxicities. Both global and promoter-specific DNA hypomethylation occurred in blood and circulating DNAs, the latter of which may reflect tumor cell responses. Increased NY-ESO-1 serum antibodies and T-cell responses were observed in the majority of patients, and antibody spreading to additional tumor antigens was also observed. Finally, disease stabilization or partial clinical response occurred in six of ten evaluable patients. On the basis of these encouraging results, evaluation of similar combinatorial chemo-immunotherapy regimens in EOC and other tumor types is warranted. Cancer Immunol Res; 2(1); 37–49. ©2014 AACR.

Potentiation of Tumor Eradication by Adoptive Immunotherapy with T-cell Receptor Gene-Transduced T-Helper Type 1 Cells

Adoptive immunotherapy using antigen-specific T-helper type 1 (Th1) cells has been considered as a potential strategy for tumor immunotherapy. However, its application to tumor immunotherapy has been hampered by difficulties in expanding tumor-specific Th1 cells from tumor-bearing hosts. Here, we have developed an efficient protocol for preparing mouse antigen-specific Th1 cells from nonspecifically activated Th cells after retroviral transfer of T-cell receptor (TCR)-α and TCR-β genes. We demonstrate that Th1 cells transduced with the TCR-α and -β genes from the I-Ad-restricted ovalbumin (OVA)323–339-specific T-cell clone DO11.10 produce IFN-γ but not interleukin-4 in response to stimulation with OVA323–339 peptides or A20 B lymphoma (A20-OVA) cells expressing OVA as a model tumor antigen. TCR-transduced Th1 cells also exhibited cytotoxicity against tumor cells in an antigen-specific manner. Moreover, adoptive transfer of TCR-transduced Th1 cells, but not mock-transduced Th1 cells, exhibited potent antitumor activity in vivo and, when combined with cyclophosphamide treatment, completely eradicated established tumor masses. Thus, TCR-transduced Th1 cells are a promising alternative for the development of effective adoptive immunotherapies.

CXCR3+ T Regulatory Cells Selectively Accumulate in Human Ovarian Carcinomas to Limit Type I Immunity

Antitumor type I T-cell responses involving IFN-γ production are critical to control cancer, but the efficacy of this response is limited by a variety of immunosuppressive mechanisms that promote tumoral immune escape. One critical mechanism involves the accumulation of FOXP3(+) T regulatory cells (Treg), a class of suppressive T cells that prevent excessive tissue destruction caused by unchecked immune responses. Recent studies have revealed that FOXP3(+) Treg include distinct subsets specifically controlling over the corresponding effector subset. In particular, CXCR3(+) Treg have been described as a subset specialized in the control of type I T-cell responses in vivo. Here, we show that CXCR3(+) Treg are highly enriched in human ovarian carcinomas, particularly in solid tumor masses, where they represent the majority of Treg. Tumor-associated CXCR3(+.) Treg coexpress T-bet but do not secrete IFN-γ ex vivo and suppress proliferation and IFN-γ secretion of T effectors. In addition, they coexpress Helios, suggesting that they originate from natural Treg. Finally, we show that the proportion of CXCR3(+) Treg at tumor sites is directly correlated with that of CXCR3(+) T effectors, consistent with expression of CXCR3 ligands. Together, our findings support the concept that natural CXCR3(+) T-bet(+) Treg selectively accumulate in ovarian tumors to control type I T-cell responses, resulting in the collateral limitation of efficient antitumor immunity.

Critical role of the Th1/Tc1 circuit for the generation of tumor-specific CTL during tumor eradication in vivo by Th1-cell therapy

Th1 and Th2 cells obtained from OVA‐specific T cell receptor transgenic mice completely eradicated the tumor mass when transferred into mice bearing A20‐OVA tumor cells expressing OVA as a model tumor antigen. To elucidate the role of Tc1 or Tc2 cells during tumor eradication by Th1‐ or Th2‐cell therapy, spleen cells obtained from mice cured of tumor by the therapy were restimulated with the model tumor antigen (OVA) for 4 days. Spleen cells obtained from mice cured by Th1‐cell therapy produced high levels of IFN‐γ, while spleen cells from mice cured by Th2‐cell therapy produced high levels of IL‐4. Intracellular staining analysis demonstrated that a high frequency of IFN‐γ‐producing Tc1 cells was induced in mice given Th1‐cell therapy. In contrast, IL‐4‐producing Tc2 cells were mainly induced in mice after Th2‐cell therapy. Moreover, Tc1, but not Tc2, exhibited a tumor‐specific cytotoxicity against A20‐OVA but not against CMS‐7 fibrosarcoma. Thus, immunological memory essential for CTL generation was induced by the Th1/Tc1 circuit, but not by the Th2/Tc2 circuit. We also demonstrated that Th1‐cell therapy is greatly augmented by combination therapy with cyclophosphamide treatment. This finding indicated that adoptive chemoimmuno‐therapy using Th1 cells should be applicable as a novel tool to enhance the Th1/Tc1 circuit, which is beneficial for inducing tumor eradication in vivo.