Takemasa Tsuji

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.

Phase I Trial of Overlapping Long Peptides from a Tumor Self-Antigen and Poly-ICLC Shows Rapid Induction of Integrated Immune Response in Ovarian Cancer Patients

Purpose: Long peptides are efficiently presented to both CD4+ and CD8+ T cells after intracellular processing by antigen-presenting cells. To investigate the safety and in vivo immunogenicity of synthetic overlapping long peptides (OLP) from a human tumor self-antigen, we conducted a phase I clinical trial with OLP from cancer-testis antigen NY-ESO-1 in various adjuvant combinations. Experimental Design: Twenty-eight patients with advanced ovarian cancer in second or third remission were enrolled sequentially in three cohorts and received at least one vaccination. Patients in Cohort 1 (n = 4) received 1.0 mg OLP, Cohort 2 (n = 13) received OLP in Montanide-ISA-51, and Cohort 3 (n = 11) received OLP + 1.4 mg Poly-ICLC in Montanide-ISA-51 on weeks 1, 4, 7, 10, and 13. Humoral and cellular responses were evaluated by standardized immunomonitoring techniques (ELISA, ELISPOT assay, intracellular cytokine staining, and tetramer staining). Results: The vaccine was generally well tolerated with injection site reactions and fatigue that resolved. NY-ESO-1–specific antibody and CD8+ T cells were undetectable after vaccination with OLP alone, but were found in 6 of 13 (46%) and 8 of 13 (62%) patients, respectively, after vaccination with OLP+Montanide, and in 10 of 11 (91%) and 10 of 11 (91%) patients, respectively, after vaccination with OLP+Montanide+Poly-ICLC. NY-ESO-1–specific CD4+ T cells were detected in all patients with greater frequency and polyclonality when Montanide-ISA-51 was used for vaccination. Inclusion of Poly-ICLC as an adjuvant further accelerated the induction of NY-ESO-1–specific immune responses. Conclusions: The current study shows that NY-ESO-1 OLP vaccine is safe and rapidly induces consistent integrated immune responses (antibody, CD8+ and CD4+) in nearly all vaccinated patients when given with appropriate adjuvants. Clin Cancer Res; 18(23); 6497–508. ©2012 AACR.

The critical role of type-1 innate and acquired immunity in tumor immunotherapy

The discovery of a large array of tumor antigens has demonstrated that host lymphocytes can indeed recognize and destroy tumor cells as originally proposed in the cancer immunosurveillance hypothesis. Recent reports that led to the cancer immuno‐editing concept also strongly support the immunosurveillance hypothesis, and they further indicate that the host immune system plays a critical role not only in promoting host protection against cancer but also in selecting tumors that can better escape from immune attack. Thus, it is now clear that T cells have the ability to recognize and destroy spontaneously arising tumors. However, the generation of antitumor immunity is often difficult in the tumor‐bearing host because of various negative regulatory mechanisms. Here, we review our recent work on tumor immunotherapy, which utilizes the activation of type‐1 innate and/or acquired immunity as a potent strategy to overcome immunosuppression in the tumor‐bearing host. We have established a variety of tumor therapeutic protocols that aim to activate type‐1 immunity, such as tumor‐vaccine therapy with CpG encapsulated in liposomes, cell therapy using tumor‐specific Th1 cells, and gene therapy using gene‐engineered Th1 cells. We found that CpG encapsulated in liposomes stimulated IL‐12‐producing DC and induced IFN‐γ‐producing NK cells, NKT cells, and tumor‐specific CTL. Th1 cell therapy was also shown to be beneficial for acceleration of APC/Th1 cell‐cell interaction in the DLN, which was critical for inducing tumor‐specific CTL at the tumor site. Therefore, we conclude that the activation of type‐1 innate and acquired immunity is crucial for tumor immunotherapy in order to overcome strong immunosuppression in the tumor‐bearing host.

Liposome-encapsulated CpG oligodeoxynucleotides as a potent adjuvant for inducing type 1 innate immunity.

Unmethylated cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODNs) exhibit potent immunostimulating activity by binding with Toll-like receptor 9 (TLR9) expressed on antigen-presenting cells. Here, we show that CpG-ODN encapsulated in cationic liposomes (CpG-liposomes) improves its incorporation into CD11c+ dendritic cells (DCs) and induces enhanced serum interleukin (IL)-12 levels compared with unmodified CpG-ODN. CpG-liposome potently activated natural killer (NK) cells (84.3%) and NKT cells (48.3%) to produce interferon-γ (IFN-γ), whereas the same dose of unmodified CpG-ODN induced only low numbers of IFN-γ–producing NK cells (12.7%) and NKT cells (1.6%) to produce IFN-γ. In contrast with the NKT cell agonist α-galactosylceramide, which induces both IFN-γ and IL-4 production by NKT cells, CpG-liposome only induced IFN-γ production by NKT cells. Such potent adjuvant activities of CpG-liposome were absent in TLR9-deficient mice, indicating that CpG-liposome was as effective as CpG-ODN in stimulating type 1 innate immunity through TLR9. In addition to TLR9, at least two other factors, IL-12 production by DCs and direct contact between DCs and NK or NKT cells, were essential for inducing type 1 innate immunity by CpG-liposome. Furthermore, ligation of TLR9 by CpG-liposome coencapsulated with ovalbumin (OVA) caused the induction of OVA-specific CTLs, which exhibited potent cytotoxicity against OVA-expressing tumor cells. These results indicate that CpG-liposome alone or combined with tumor antigen protein provides a promising approach for the prevention or therapy of tumors.

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.

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.

Generation and Targeting of Human Tumor-Specific Tc1 and Th1 Cells Transduced with a Lentivirus Containing a Chimeric Immunoglobulin T-Cell Receptor

CD4+ Th cells, in particular IFN-γ-producing Th1 cells, play a critical role in the activation and maintenance of Tc1 cells that are essential for tumor eradication. Here, we report the generation of artificial tumor-specific Th1 and Tc1 cells from nonspecifically activated T cells using a lentiviral transduction system. Anti-CD3-activated T cells from healthy human donors were transduced with a lentivirus containing a chimeric immunoglobulin T-cell receptor gene composed of single-chain variable fragments derived from an anticarcinoembryonic antigen (CEA)-specific monoclonal antibody fused to an intracellular signaling domain derived from the cytoplasmic portions of membrane-bound CD28 and CD3ζ. These artificial tumor-specific Tc1 and Th1 cells, termed Tc1- and Th1-T bodies, respectively, could be targeted to CEA+ tumor cells independently of MHC restriction. Specifically, Tc1-T bodies demonstrated high cytotoxicity and produced IFN-γ in response to CEA+ tumor cell lines but not CEA− tumors. Although Th1-T bodies exhibited low cytotoxicity, they secreted high levels of IFN-γ and interleukin-2 in response to CEA+ tumor cells. Such CEA+ tumor-specific activation was not observed in mock gene-transduced nonspecific Tc1 and Th1 cells. Moreover, Tc1- and Th1-T bodies exhibited strong antitumor activities against CEA+ human lung cancer cells implanted into RAG2−/− mice. Furthermore, combined therapy with Tc1- and Th1-T bodies resulted in enhanced antitumor activities in vivo. Taken together, our findings demonstrate that Tc1- and Th1-T bodies represent a promising alternative to current methods for the development of effective adoptive immunotherapies.

A Critical Role for Mouse CXC Chemokine(s) in Pulmonary Neutrophilia During Th Type 1-Dependent Airway Inflammation

Ag-specific Th1 and Th2 cells have been demonstrated to play a critical role in the induction of allergic diseases. Here we have investigated the precise mechanisms of Th1-induced airway inflammation. Airway inflammation was induced in BALB/c mice by transfer of freshly induced OVA-specific Th1 or Th2 cells followed by OVA inhalation. In this model, both Th1 and Th2 cells induced airway inflammation. The former induced neutrophilia in airways, whereas the latter induced eosinophilia. Moreover, we found that Th1 cells induced more severe airway hyperresponsiveness (AHR) than Th2 cells. The eosinophilia induced by Th2 cell infusion was almost completely blocked by administration of anti-IL-5 mAb, but not anti-IL-4 mAb. In contrast, Th1-induced AHR and pulmonary neutrophilia were inhibited by the administration of anti-human IL-8R Ab, which blocks the function of mouse CXC chemokine(s). These findings reveal a critical role of mouse CXC chemokine(s) in Th1-dependent pulmonary neutrophilia and AHR.

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.

Influence of CD4+CD25+ Regulatory T Cells on Low/High-Avidity CD4+ T Cells following Peptide Vaccination

We have recently reported that NY-ESO-1-specific naive CD4+ T cell precursors exist in most individuals but are suppressed by CD4+CD25+ regulatory T cells (Tregs), while memory CD4+ T cell effectors against NY-ESO-1 are found only in cancer patients with spontaneous Ab responses to NY-ESO-1. In this study, we have analyzed mechanisms of CD4+ T cell induction following peptide vaccination in relation to susceptibility to Tregs. Specific HLA-DP4-restricted CD4+ T cell responses were elicited after vaccination with NY-ESO-1157–170 peptide (emulsified in IFA) in patients with NY-ESO-1-expressing epithelial ovarian cancer. These vaccine-induced CD4+ T cells were detectable from effector/memory populations without requirement for in vitro CD4+CD25+ T cell depletion. However, they were only able to recognize NY-ESO-1157–170 peptide but not naturally processed NY-ESO-1 protein and had much lower avidity compared with NY-ESO-1-specific pre-existing naive CD4+CD25− T cell precursors or spontaneously induced CD4+ T cell effectors of cancer patients with NY-ESO-1 Ab. We propose that vaccination with NY-ESO-1157–170 peptide recruits low-avidity T cells with low sensitivity to Tregs and fails to modulate the suppressive effect of Tregs on high-avidity NY-ESO-1-specific T cell precursors.