Hiroshi Fujiwara

Improved Expression and Reactivity of Transduced Tumor-Specific TCRs in Human Lymphocytes by Specific Silencing of Endogenous TCR

Adoptive T-cell therapy using lymphocytes genetically engineered to express tumor antigen-specific TCRs is an attractive strategy for treating patients with malignancies. However, there are potential drawbacks to this strategy: mispairing of the introduced TCR alpha/beta chains with the endogenous TCR subunits and competition of CD3 molecules between the introduced and endogenous TCRs can impair cell surface expression of the transduced TCR, resulting in insufficient function and potential generation of autoreactive T cells. In addition, the risk of tumor development following the infusion of cells with aberrant vector insertion sites increases with the vector copy number in the transduced cells. In this study, we developed retroviral vectors encoding both small interfering RNA constructs that specifically down-regulate endogenous TCR and a codon-optimized, small interfering RNA-resistant TCR specific for the human tumor antigens MAGE-A4 or WT1. At low copy numbers of the integrated vector, the transduced human lymphocytes exhibited high surface expression of the introduced tumor-specific TCR and reduced expression of endogenous TCRs. In consequence, the vector-transduced lymphocytes showed enhanced cytotoxic activity against antigen-expressing tumor cells. Therefore, our novel TCR gene therapy may open a new gate for effective immunotherapy in cancer patients.

HLA Class I Binding of HBZ Determines Outcome in HTLV-1 Infection

CD8+ T cells can exert both protective and harmful effects on the virus-infected host. However, there is no systematic method to identify the attributes of a protective CD8+ T cell response. Here, we combine theory and experiment to identify and quantify the contribution of all HLA class I alleles to host protection against infection with a given pathogen. In 432 HTLV-1-infected individuals we show that individuals with HLA class I alleles that strongly bind the HTLV-1 protein HBZ had a lower proviral load and were more likely to be asymptomatic. We also show that in general, across all HTLV-1 proteins, CD8+ T cell effectiveness is strongly determined by protein specificity and produce a ranked list of the proteins targeted by the most effective CD8+ T cell response through to the least effective CD8+ T cell response. We conclude that CD8+ T cells play an important role in the control of HTLV-1 and that CD8+ cells specific to HBZ, not the immunodominant protein Tax, are the most effective. We suggest that HBZ plays a central role in HTLV-1 persistence. This approach is applicable to all pathogens, even where data are sparse, to identify simultaneously the HLA Class I alleles and the epitopes responsible for a protective CD8+ T cell response.

HBZ is an immunogenic protein, but not a target antigen for human T-cell leukemia virus type 1-specific cytotoxic T lymphocytes.

Recently, HBZ has been reported to play an important role in the proliferation of adult T-cell leukaemia (ATL) cells and might be a target of novel therapy for ATL. To develop a novel immunotherapy for ATL, we verified the feasibility of cellular immunotherapy targeting HBZ. We established an HBZ-specific and HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) clone. Detailed study using this CTL clone clearly showed that HBZ is certainly an immunogenic protein recognizable by human CTLs; however, HBZ-specific CTLs could not lyse ATL cells. Failure of HBZ-specific CTLs to recognize human T-cell leukemia virus type 1 (HTLV-1)-infected cells might be due to a low level of HBZ protein expression in ATL cells and resistance of HTLV-1-infected cells to CTL-mediated cytotoxicity. Although HBZ plays an important role in the proliferation of HTLV-1-infected cells, it may also provide a novel mechanism that allows them to evade immune recognition.

Gemcitabine enhances Wilms’ tumor gene WT1 expression and sensitizes human pancreatic cancer cells with WT1-specific T-cell-mediated antitumor immune response

Wilms’ tumor gene (WT1), which is expressed in human pancreatic cancer (PC), is a unique tumor antigen recognized by T-cell-mediated antitumor immune response. Gemcitabine (GEM), a standard therapeutic drug for PC, was examined for the regulation of WT1 expression and the sensitizing effect on PC cells with WT1-specific antitumor immune response. Expression of WT1 was examined by quantitative PCR, immunoblot analysis, and confocal microscopy. Antigenic peptide of WT1 presented on HLA class I molecules was detected by mass spectrometry. WT1-specific T-cell receptor gene–transduced human T cells were used as effecter T cells for the analysis of cytotoxic activity. GEM treatment of human MIAPaCa2 PC cells enhanced WT1 mRNA levels, and this increase is associated with nuclear factor kappa B activation. Tumor tissue from GEM-treated MIAPaCa2-bearing SCID mice also showed an increase in WT1 mRNA. Some human PC cell lines other than MIAPaCa2 showed up-regulation of WT1 mRNA levels following GEM treatment. GEM treatment shifted WT1 protein from the nucleus to the cytoplasm, which may promote proteasomal processing of WT1 protein and generation of antigenic peptide. In fact, presentation of HLA-A*2402-restricted antigenic peptide of WT1 (CMTWNQMNL) increased in GEM-treated MIAPaCa2 cells relative to untreated cells. WT1-specific cytotoxic T cells killed MIAPaCa2 cells treated with an optimal dose of GEM more efficiently than untreated MIAPaCa2 cells. GEM enhanced WT1 expression in human PC cells and sensitized PC cells with WT1-specific T-cell-mediated antitumor immune response.

Human herpesvirus 6 infection impairs Toll-like receptor signaling

Human herpesvirus 6 (HHV-6) has a tropism for immunocompetent cells, including T lymphocytes, monocytes/macrophages, and dendritic cells (DCs) suggesting that HHV-6 infection affects the immunosurveillance system. Toll-like receptor (TLR) system plays an important role in innate immunity against various pathogens. In the present study, we investigated the effect of HHV-6 infection on the expression and intracellular signaling of TLRs in DCs. Although expression levels of TLRs were not decreased or slightly elevated following HHV-6 infection, the amounts of cytokines produced following stimulation with ligands for TLRs appeared to be dramatically decreased in HHV-6-infected DCs as compared to mock-infected DCs. Similarly, phosphorylation levels of TAK-1, IκB kinase, and IκB-α following stimulation of HHV-6-infected DCs with lipopolysaccharide, which is the ligand for TLR4, appeared to be decreased. These data show that HHV-6 impairs intracellular signaling through TLRs indicating the novel mechanism of HHV-6-mediated immunomodulation.

Application of Adoptive T-Cell Therapy Using Tumor Antigen-Specific T-Cell Receptor Gene Transfer for the Treatment of Human Leukemia

The last decade has seen great strides in the field of cancer immunotherapy, especially the treatment of melanoma. Beginning with the identification of cancer antigens, followed by the clinical application of anti-cancer peptide vaccination, it has now been proven that adoptive T-cell therapy (ACT) using cancer antigen-specific T cells is the most effective option. Despite the apparent clinical efficacy of ACT, the timely preparation of a sufficient number of cancer antigen-specific T cells for each patient has been recognized as its biggest limitation. Currently, therefore, attention is being focused on ACT with engineered T cells produced using cancer antigen-specific T-cell receptor (TCR) gene transfer. With regard to human leukemia, ACT using engineered T cells bearing the leukemia antigen-specific TCR gene still remains in its infancy. However, several reports have provided preclinical data on TCR gene transfer using Wilms tumor gene product 1 (WT1), and also preclinical and clinical data on TCR gene transfer involving minor histocompatibility antigen, both of which have been suggested to provide additional clinical benefit. In this review, we examine the current status of anti-leukemia ACT with engineered T cells carrying the leukemia antigen-specific TCR gene, and discuss the existing barriers to progress in this area.

Aurora-A kinase: a novel target both for cellular immunotherapy and molecular target therapy against human leukemia

Background: Although cellular immunotherapy still remains in its infancy, it is one of the important treatment options against cancer. The marked improvement of its clinical efficacy requires a ‘better’ target antigen, which is well recognized by cancer-cell-specific cytotoxic T lymphocytes. We have recently demonstrated the potential of Aurora-A kinase (Aurora-A) as such a ‘better’ target for cellular immunotherapy against human leukemia. Aurora-A is a member of the serine/threonine kinase family that properly regulates the cell division process, and has recently been implicated in tumorigenesis. On the other hand, small-molecule inhibitors targeting Aurora-A have recently been developed and preliminary but promising observations from Phase I clinical trials have been reported. These facts highlight the attractiveness of Aurora-A as an important target of comprehensive cancer therapies. Objective/methods: In this review, we cover Aurora-A in the areas of immunotherapy and small-molecule inhibitor therapy against cancers. Results/conclusions: Aurora-A kinase is an attractive molecule not only as a target for small-molecule inhibitors, but also as a potential target for immunotherapy against cancer.

Activation of T-cell receptor signaling in peripheral T-cell lymphoma cells plays an important role in the development of lymphoma-associated hemophagocytosis.

Peripheral T-cell lymphoma (PTCL) is a biologically diverse lymphoid malignancy. The clinical aggressiveness associated with hemophagocytic syndrome (HS) is a characteristic of PTCL, being more distinctive in CD8+ PTCL. However, the underlying mechanism of PTCL-associated HS has not yet been fully investigated. We newly established a novel IL-2-dependent CD8+ PTCL lymphoma cell line (T8ML-1) from a patient with CD8+ PTCL who suffered recurrent HS accompanying disease flare-up. Focusing on the lymphoma cell T-cell receptor (TCR), we examined the lymphoma cell functions responsible for such clinical manifestations. First, T8ML-1.1 in which endogenous TCR-α/β chains were silenced by siRNAs, and T8ML-1.2 in which endogenous TCR-α/β chains were replaced with HLA-A*24:02-restricted and WT1235–243-specific TCR-α/β, were established. T8ML-1 exerted phytohemagglutinin (PHA)-dependent cytotoxicity via granular exocytosis. Additionally, soluble factors produced by PHA-stimulated T8ML-1, which included INF-γ and TNF-α, but not by simple-cultured T8ML-1, caused human monocytes to exhibit erythrophagocytosis and thrombophagocytosis in vitro. PHA binding induced phosphorylation of CD3ζ chain. Furthermore, both cytotoxicity and hemophagocytosis were completely inhibited by T8ML-1.1, but eventually restored by T8ML-1.2. These data suggest that exogenous activation of TCR signaling in PTCL cells might play an important role in the formation of PTCL-associated HS.

Requisite considerations for successful adoptive immunotherapy with engineered T-lymphocytes using tumor antigen-specific T-cell receptor gene transfer

Introduction: Although engineered T-cell-based antitumor immunotherapy using tumor-antigen-specific T-cell receptor (TCR) gene transfer is undoubtedly a promising strategy, a number of studies have revealed that it has several drawbacks. Areas covered: This review covers selected articles detailing recent progress in this field, not only for solid tumors, but also for leukemias. In terms of achieving uniform therapeutic quality of TCR gene-modified T cells as an ‘off-the-shelf’ product, the authors abstract and discuss the requisite conditions for successful outcome, including: i) the optimal target choice reflecting the specificity of the introduced TCR, ii) the quality and quantity of expressed TCRs in gene-modified T cells, and additional genetic modification reflecting enhanced antitumor functionality, and iii) ‘on-’ and ‘off-target’ adverse events caused by the quality of the introduced TCRs and other adverse events related to genetic modification itself. Readers will be able to readily abstract recent advances in TCR gene-transferred T-cell therapy, centering notably on efforts to obtain uniformity in the therapeutic functionality of engineered T cells Expert opinion: Harmonizing the functionality and target specificity of TCR will allow the establishment of clinically useful adoptive immunotherapy in the near future.

Relapse of renal cell carcinoma with disappearance of HLA class I following hTERT peptide vaccination

A 74-year-old woman who had undergone right nephrectomy due to renal cell carcinoma (RCC) was found to have developed multiple lung metastasis of RCC 5 years after surgery. The patient was treated with interferon-a, but she was unable to tolerate this therapy because of fever and severe malaise. At this time, as molecular-targeted therapy, such as the multikinase inhibitor sorafenib, was not available, the patient was enrolled in a phase I/II clinical study of human telomerase reverse transcriptase (hTERT) peptide vaccination, as reviewed and approved by the Institutional Review Board of Ehime University Hospital. She received s.c. injection of 1 mg hTERT324–332 (VYAETKHFL) peptide [1] in Montanide ISA51 adjuvant biweekly. During the following 2 years, she received hTERT peptide vaccination a total of 20 times with some discontinuations. The protocol was well tolerated; only local erythema occurred at the peptide vaccine injection sites. As shown in Figure 1A, during vaccination with hTERT peptide for 2 years, the sizes of the RCC metastases did not change significantly. The results of enzyme-linked immunospot (ELISPOT) assay for monitoring hTERT324–332-specific CD8 + cytotoxic T lymphocytes (CTLs) in peripheral blood are shown in Figure 1B. The hTERT peptide-specific CTLs were not detectable in peripheral blood of the patient before hTERT peptide vaccination and became detectable after the fifth vaccination. The frequency of hTERT peptide-specific CTLs in peripheral blood appeared to increase as the vaccinations were repeated. letters to the editor Annals of Oncology