Toshihiro Nagato

Nasal natural killer (NK)/T-cell lymphoma: clinical, histological, virological, and genetic features

Nasal natural killer (NK)/T-cell lymphoma (NNKTL) is a clinical illness characterized by progressive unrelenting ulceration and necrosis of the nasal cavity and midline facial tissues. Histological features of the lymphoma include angiocentric and polymorphous lymphoreticular infiltrates, called polymorphic reticulosis. Surface antigens and the NK-cell marker, CD56, as well as pan-T antigen CD2, cytoplasmic CD3 (CD3ɛ), and CD45 are expressed in the lymphoma cells. The origin of the lymphoma is thought to be either NK-cell linkage without T-cell receptor (TCR) rearrangement or γδT-cell linkage with γδTCR rearrangement. Since the authors of this study first demonstrated the presence of Epstein Barr virus (EBV)-DNA and EBV oncogenic proteins in NNKTL, the lymphoma has been classified as one of the EBV-associated malignancies. The NNKTL cells produce interleukin (IL)-9, IL-10, and interferon-γ-inducible protein-10 (IP-10), possibly due to EBV-oncogenic proteins in the lymphoma cells, and such cytokines take an important part in the cell proliferation and invasion, acting in an autocrine manner. Clinically, the serum EBV-DNA copy number is useful as a specific tumor marker and a predictive prognostic factor. Even in early clinical stages, the lymphoma shows poor prognosis caused by the rapid progression of the lesion into distinct organs. Our newly designed arterial infusion chemotherapy, from the superficial temporal artery, in combination with radiotherapy, has shown a favorable outcome in patients with NNKTL. In this article, the clinical, pathological, and virological characteristics of the lymphoma are reviewed, along with a report of our investigations.

Expression of Interleukin-9 in Nasal Natural Killer/T-Cell Lymphoma Cell Lines and Patients

Purpose: Nasal natural killer (NK)/T-cell lymphoma is associated with EBV and has distinct clinical and histologic features. However, little is known about its genetic features. In this study, we examined the genes expressed by SNK-6 and SNT-8 cells, which were established from nasal NK/T-cell lymphomas, and found that interleukin (IL)-9 was specifically expressed in these two cell lines. Experimental Design: cDNA array was used to examine the genes expressed by SNK-6 and SNT-8 cells. Expression of IL-9 and IL-9 receptor was investigated by reverse transcription-PCR, ELISA, and flow cytometry. Cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Immunohistologic staining and ELISA were used to examine IL-9 expression in biopsies and sera from patients, respectively. Results: In cDNA array, expression of IL-9 mRNA was much higher in SNK-6 and SNT-8 cells than in NK-92 cells from non-nasal NK-cell lymphoma and peripheral blood mononuclear cells from healthy volunteers. Furthermore, IL-9 was specifically expressed by SNK-6 and SNT-8 cells but not by other NK-cell, NK-like T-cell, and T-cell lymphoma/leukemia cell lines. IL-9 receptor was also expressed on the surfaces of SNK-6 and SNT-8 cells. An IL-9-neutralizing antibody inhibited the growth of these two cell lines, whereas recombinant human IL-9 enhanced their growth. Most significantly, IL-9 was present in biopsies and sera from patients with this lymphoma. Conclusions: These results suggest that IL-9 plays an important role in nasal NK/T-cell lymphoma possibly via an autocrine mechanism.

Defining MHC class II T helper epitopes for WT1 tumor antigen.

The product of Wilms‘ tumor gene 1 (WT1) is overexpressed in diverse human tumors, including leukemia, lung and breast cancer, and is often recognized by antibodies in the sera of patients with leukemia. Since WT1 encodes MHC class I-restricted peptides recognized by cytotoxic T lymphocytes (CTL), WT1 has been considered as a promising tumor-associated antigen (TAA) for developing anticancer immunotherapy. In order to carry out an effective peptide-based cancer immunotherapy, MHC class II-restricted epitope peptides that elicit anti-tumor CD4+ helper T lymphocytes (HTL) will be needed. In this study, we analyzed HTL responses against WT1 antigen using HTL lines elicited by in vitro immunization of human lymphocytes with synthetic peptides predicted to serve as HTL epitopes derived from the sequence of WT1. Two peptides, WT1124–138 and WT1247–261, were shown to induce peptide-specific HTL, which were restricted by frequently expressed HLA class II alleles. Here, we also demonstrate that both peptides-reactive HTL lines were capable of recognizing naturally processed antigens presented by dendritic cells pulsed with tumor lysates or directly by WT1+ tumor cells that express MHC class II molecules. Interestingly, the two WT1 HTL epitopes described here are closely situated to known MHC class I-restricted CTL epitopes, raising the possibility of stimulating CTL and HTL responses using a relatively small synthetic peptide vaccine. Because HTL responses to TAA are known to be important for promoting long-lasting anti-tumor CTL responses, the newly described WT1 T-helper epitopes could provide a useful tool for designing powerful vaccines against WT1-expressing tumors.

Combinatorial immunotherapy of polyinosinic-polycytidylic acid and blockade of programmed death-ligand 1 induce effective CD8 T-cell responses against established tumors.

Purpose: Epitope-based cancer vaccines capable of inducing CD8 T-cell responses to tumor-associated antigens (TAA) expressed by tumor cells have been considered as attractive alternatives for the treatment of some types of cancer. However, reliable TAAs have not been identified for most malignant diseases, limiting the development of epitope-based vaccines. Herein, we report that the combinatorial therapy of polyinosinic–polycytidylic acid (poly-IC) and antiprogrammed death-ligand 1 (PD-L1) monoclonal antibody (mAb) can be implemented with good results for tumors where no known TAAs have been identified. Experimental Design: Three cancer mouse models (melanoma, lung, and colon) were used to evaluate therapeutic efficacy and examine the immunologic mechanisms of the poly-IC/anti–PD-L1 mAb therapy. Results: The combined administration of poly-IC and anti–PD-L1 mAb into tumor-bearing mice generated potent immune responses resulting in the complete eradication or remarkable reduction of tumor growth. In some instances, the poly-IC/anti–PD-L1 mAb therapy induced long-lasting protection against tumor rechallenges. The results indicate that CD8 T cells but not CD4 T cells or NK cells mediated the therapeutic efficacy of this combinatorial therapy. Experiments using genetically deficient mice indicate that the therapeutic efficacy of this combinatorial therapy depended in part by the participation of type-I IFN, whereas IFN-γ did not seem to play a major role. Conclusions: The overall results suggest that immunotherapy consisting of the combination of poly-IC/anti–PD-L1 mAb could be a promising new approach for treating patients with cancer, especially those instances where no reliable TAAs are available as a therapeutic vaccine. Clin Cancer Res; 20(5); 1223–34. ©2014 AACR.

Induction of EBV–Latent Membrane Protein 1–Specific MHC Class II–Restricted T-Cell Responses against Natural Killer Lymphoma Cells

EBV-encoded latent membrane protein 1 (LMP1) has oncogenic potential and is expressed in many EBV-associated malignancies. Although LMP1 is regarded as a potential tumor-associated antigen for immunotherapy and several LMP1-specific MHC class I-restricted CTL epitopes have been reported, little is known regarding MHC class II-restricted CD4 helper T-lymphocyte (HTL) epitopes for LMP1. The goal of the present studies was to determine whether MHC class II-restricted CD4 T-cell responses could be induced against the LMP1 antigen and to evaluate the antitumor effect of these responses. We have combined the use of a predictive MHC class II binding peptide algorithm with in vitro vaccination of CD4 T cells using candidate peptides to identify naturally processed epitopes derived from LMP1 that elicit immune responses against EBV-expressing tumor cells. Peptide LMP1(159-175) was effective in inducing HTL responses that were restricted by HLA-DR9, HLA-DR53, or HLA-DR15, indicating that this peptide behaves as a promiscuous T-cell epitope. Moreover, LMP1(159-175)-reactive HTL clones directly recognized EBV lymphoblastoid B cells, EBV-infected natural killer (NK)/T-lymphoma cells and naturally processed antigen in the form of LMP1+ tumor cell lysates presented by autologous dendritic cells. Because the newly identified epitope LMP1(159-175) overlaps with an HLA-A2-restricted CTL epitope (LMP1(159-167)), this peptide might have the ability to induce simultaneous CTL and HTL responses against LMP1. Overall, our data should be relevant for the design and optimization of T-cell epitope-based immunotherapy against various EBV-associated malignancies, including NK/T cell lymphomas.

Recognition of Prostate and Breast Tumor Cells by Helper T Lymphocytes Specific for a Prostate and Breast Tumor-Associated Antigen, TARP

Purpose: T cell–based immunotherapy via the in vitro or in vivo expansion of prostate tumor-associated antigen (TAA)–specific T lymphocytes is one of the most promising therapeutic approaches to treat prostate cancer. T-cell alternate reading frame protein (TARP) is a mitochondrial protein that is specifically expressed in prostate epithelial cells. We have done experiments aimed at identifying helper T lymphocyte (HTL) epitopes for TARP for the design of T cell–based immunotherapy for prostate cancer. Experimental Design: Dendritic cells from normal donors were pulsed with synthetic peptides derived from TARP, which were predicted to serve as HTL epitopes. These dendritic cells were used to stimulate CD4+ T cells in vitro to trigger HTL responses against TARP. T-cell responses to these peptides were also studied with lymphocytes from prostate cancer patients. Results: The two peptides, TARP1-14 and TARP14-27, were shown to elicit effective in vitro HTL responses using lymphocytes from both normal volunteers and prostate cancer patients. Peptide TARP1-14-reactive HTLs were found restricted by HLA-DR53 and could recognize naturally processed protein antigen derived from tumor cells, which was presented by autologous dendritic cells. Most significantly, stimulation with peptide TARP14-27 generated four HTL lines restricted by HLA-DR1, HLA-DR9, HLA-DR13, and HLA-DR15, some of which capable of recognizing naturally processed antigens presented by dendritic cell or directly by TARP-positive tumor cells. Conclusions: Our results show that TARP constitutes a TAA that can be recognized by tumor-reactive HTL. The newly described TARP epitopes could be used to optimize and improve T-cell epitope–based immunotherapy against prostate and other tumors expressing TARP.

Production of Interferon-γ–Inducible Protein-10 and Its Role as an Autocrine Invasion Factor in Nasal Natural Killer/T-Cell Lymphoma Cells

Purpose: Nasal natural killer (NK)/T-cell lymphoma is associated with Epstein-Barr virus and has poor prognosis because of local invasion and/or multiple dissemination. Recently, the role of chemokines/chemokine receptors in tumor proliferation and invasion has been shown. In this study, we examined whether the specific chemokines were related to the tumor behaviors in nasal NK/T-cell lymphoma. Experimental Design: A chemokine protein array was used to examine specific chemokines produced by SNK-6 and SNT-8 (Epstein-Barr viruspositive nasal NK/T-cell lymphoma lines). The expression of interferon inducible protein 10 (IP-10) and the IP-10 receptor CXCR3 was investigated by ELISA and flow cytometry. Cell growth and invasion were assessed by the MTT and Matrigel invasion assays, respectively. Immunohistologic staining and ELISA were used to examine IP-10 expression in biopsies and sera from patients, respectively. Results: IP-10 was specifically produced by SNK-6 and SNT-8. Moreover, CXCR3 was expressed on the NK cell lines. Functionally, IP-10 did not affect cell proliferation but enhanced cell invasion. In biopsy samples, IP-10 and CXCR3 expressions were detected in the lymphoma cells. Serum IP-10 levels in the patients were much higher than those of healthy controls and the levels were decreased during the complete remission phase after treatments. Conclusions: These results suggest that IP-10 may play an important role in cell invasion in nasal NK/T-cell lymphoma through an autocrine mechanism. (Clin Cancer Res 2009;15(22):67719)

Functional Analysis of Birch Pollen Allergen Bet v 1-Specific Regulatory T Cells

Allergen-specific immunotherapy using peptides is an efficient treatment for allergic diseases. Recent studies suggest that the induction of CD4+ regulatory T (Treg) cells might be associated with the suppression of allergic responses in patients after allergen-specific immunotherapy. Our aim was to identify MHC class II promiscuous T cell epitopes for the birch pollen allergen Bet v 1 capable of stimulating Treg cells with the purpose of inhibiting allergic responses. Ag-reactive CD4+ T cell clones were generated from patients with birch pollen allergy and healthy volunteers by in vitro vaccination of PBMC using Bet v 1 synthetic peptides. Several CD4+ T cell clones were induced by using 2 synthetic peptides (Bet v 1141–156 and Bet v 151–68). Peptide-reactive CD4+ T cells recognized recombinant Bet v 1 protein, indicating that these peptides are produced by the MHC class II Ag processing pathway. Peptide Bet v 1141–156 appears to be a highly MHC promiscuous epitope since T cell responses restricted by numerous MHC class II molecules (DR4, DR9, DR11, DR15, and DR53) were observed. Two of these clones functioned as typical Treg cells (expressed CD25, GITR, and Foxp3 and suppressed the proliferation and IL-2 secretion of other CD4+ T cells). Notably, the suppressive activity of these Treg cells required cell-cell contact and was not mediated through soluble IL-10 or TGF-β. The identified promiscuous MHC class II epitope capable of inducing suppressive Treg responses may have important implication for the development of peptide-based Ag-specific immunotherapy to birch pollen allergy.

Monocytes enhance cell proliferation and LMP1 expression of nasal natural killer/T‐cell lymphoma cells by cell contact‐dependent interaction through membrane‐bound IL‐15

Nasal natural killer (NK)/T‐cell lymphoma (NNKTL) is an Epstein‐Barr virus (EBV)‐related malignancy with poor prognosis and has distinct histological features characterized by angiocentric and polymorphous lymphoreticular infiltrates including inflammatory cells such as granulocytes, monocytes, macrophages and lymphocytes. Here, we show that the monocytes enhance proliferation as well as LMP1 expression of NNKTL cells by cell contact‐dependent interaction through membrane‐bound interleukin (IL)‐15. We used two EBV‐positive NK‐cell lines, SNK6 and KAI3, which originated from two patients—SNK6 from a patient with NNKTL and KAI3 from a patient with a severe mosquito allergy. We cocultured the cell lines with granulocytes or monocytes and examined whether proliferation, survival and LMP1 expression of the cells changed. Although cocultured granulocytes did not affect proliferation, survival or LMP1 expression of the cells, cocultured monocytes enhanced both proliferation and LMP1 expression in a dose‐dependent manner. These phenomena were not seen when monocytes were placed in a separate chamber. Moreover, the monocyte‐inducible proliferation and LMP1 expression were inhibited by treatment with an antibody against IL‐15. Furthermore, production of interferon‐gamma‐inducible protein (IP)‐10 were enhanced by coculture with monocytes and were inhibited by the antibody. Immunohistological studies confirmed that a number of infiltrating CD14‐positive monocytes contacted CD56‐positive lymphoma cells in all of 20 NNKTL tissues tested. These results suggest that monocytes enhance cell growth as well as LMP1 expression of NNKTL cells by cell contact‐dependent interaction through membrane‐bound IL‐15. In the microenvironment of NNKTL tissue, a positive feedback loop of interaction between lymphoma cells and monocytes may be present and contribute to lymphoma progression.

Expression and functional role of MDL-1 (CLEC5A) in mouse myeloid lineage cells

Myeloid DNAX activation protein 12 (DAP12)‐associating lectin‐1 (MDL‐1), also known as C‐type lectin domain family 5, member A, is a type II transmembrane protein belonging to the C‐type lectin family and associates with DAP12 (also called KARAP or TYROBP). It has been reported that two isoforms of MDL‐1—long form (MDL‐1L) and short form (MDL‐1S)—exist in mice. Previously, we observed the marked induction of MDL‐1 mRNA expression during the pulmonary mycobacterial infection in mice. The data suggested that the MDL‐1‐expressing cells were involved in immune responses against mycobacterial infection; however, little is known about the function of MDL‐1 as yet. In this study, we demonstrated the significant protein expression of MDL‐1L and MDL‐1S in mouse neutrophils and macrophages. MDL‐1L was highly glycosylated by N‐linked glycan and sialic acid. Interestingly, the expression pattern of MDL‐1 was different between neutrophils and macrophages. MDL‐1 expression was notably induced during the differentiation of the mouse myeloid cell line 32Dcl3 into neutrophils. Additionally, we observed that MDL‐1 stimulation induced a significant amount of RANTES and macrophage‐derived chemokine production in 32Dcl3 cells in cooperation with signaling through TLR. MDL‐1 stimulation also up‐regulated CD11b expression and maintained cell survival. Our findings indicate that MDL‐1, therefore, plays an important role in immune defense as a result of an innate immunity, which involves neutrophils and macrophages.