Toshiki Ochi

Generation of functional human T-cell subsets with HLA-restricted immune responses in HLA class I expressing NOD/SCID/IL2rγnull humanized mice

Whereas humanized mouse models have contributed significantly to human immunology research, human T cells developing in mouse thymic environment fail to demonstrate HLA-restricted function. To achieve HLA-restricted human immune response, we created an immune-compromised non-obese diabetic/SCID/IL2rgnull strain (NSG) with homozygous expression of HLA class I heavy chain and light chain (NSG-HLA-A2/HHD). Transplantation of purified Lin−CD34+CD38− human hematopoietic stem cells into NSG-HLA-A2/HHD newborns resulted in the development of human CD4+ and CD8+ TCRαβ+ T cells and CD4−CD8− and CD8+ TCRγδ+ cells in recipient bone marrow and spleen. Human cytotoxic T lymphocytes (CTLs) become functionally mature, as evidenced by the production of granzyme corresponding to phenotypic transition from naïve to effector memory CTLs. In these recipients, human Th17 cells developed along with Th1 and Th2 cells. Epstein–Barr virus (EBV) infection in the humanized NSG-HLA-A2/HHD recipients resulted in the formation of lymphoproliferative lesions consisting mainly of human B cells with scattered human T cells. Human CTLs developing in the recipients recognized EBV-derived peptides in an HLA-restricted manner and exerted HLA-restricted cytotoxicity against EBV-infected human B cells. The HLA-expressing humanized mouse with functional HLA-restricted T cells and consistent representation of rare T-cell subsets overcomes a major constraint in human immunology, and serves as a useful model for investigation of human immune responses against pathogens and for the development of therapeutic strategies against human diseases.

Aurora-A kinase: A novel target of cellular immunotherapy for leukemia

Aurora-A kinase (Aur-A) is a member of the serine/threonine kinase family that regulates the cell division process, and has recently been implicated in tumorigenesis. In this study, we identified an antigenic 9-amino-acid epitope (Aur-A(207-215): YLILEYAPL) derived from Aur-A capable of generating leukemia-reactive cytotoxic T lymphocytes (CTLs) in the context of HLA-A*0201. The synthetic peptide of this epitope appeared to be capable of binding to HLA-A*2402 as well as HLA-A*0201 molecules. Leukemia cell lines and freshly isolated leukemia cells, particularly chronic myelogenous leukemia (CML) cells, appeared to express Aur-A abundantly. Aur-A-specific CTLs were able to lyse human leukemia cell lines and freshly isolated leukemia cells, but not normal cells, in an HLA-A*0201-restricted manner. Importantly, Aur-A-specific CTLs were able to lyse CD34+ CML progenitor cells but did not show any cytotoxicity against normal CD34+ hematopoietic stem cells. The tetramer assay revealed that the Aur-A(207-215) epitope-specific CTL precursors are present in peripheral blood of HLA-A*0201-positive and HLA-A*2402-positive patients with leukemia, but not in healthy individuals. Our results indicate that cellular immunotherapy targeting Aur-A is a promising strategy for treatment of leukemia.

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.

BET bromodomain inhibition enhances T cell persistence and function in adoptive immunotherapy models

Adoptive immunotherapy is a potentially curative therapeutic approach for patients with advanced cancer. However, the in vitro expansion of antitumor T cells prior to infusion inevitably incurs differentiation towards effector T cells and impairs persistence following adoptive transfer. Epigenetic profiles regulate gene expression of key transcription factors over the course of immune cell differentiation, proliferation, and function. Using comprehensive screening of chemical probes with defined epigenetic targets, we found that JQ1, an inhibitor of bromodomain and extra-terminal motif (BET) proteins, maintained CD8+ T cells with functional properties of stem cell-like and central memory T cells. Mechanistically, the BET protein BRD4 directly regulated expression of the transcription factor BATF in CD8+ T cells, which was associated with differentiation of T cells into an effector memory phenotype. JQ1-treated T cells showed enhanced persistence and antitumor effects in murine T cell receptor and chimeric antigen receptor gene therapy models. Furthermore, we found that histone acetyltransferase p300 supported the recruitment of BRD4 to the BATF promoter region, and p300 inhibition similarly augmented antitumor effects of the adoptively transferred T cells. These results demonstrate that targeting the BRD4-p300 signaling cascade supports the generation of superior antitumor T cell grafts for adoptive immunotherapy.

Subtypes of familial hemophagocytic lymphohistiocytosis in Japan based on genetic and functional analyses of cytotoxic T lymphocytes.

Background Familial hemophagocytic lymphohistiocytosis (FHL) is a rare disease of infancy or early childhood. To clarify the incidence and subtypes of FHL in Japan, we performed genetic and functional analyses of cytotoxic T lymphocytes (CTLs) in Japanese patients with FHL. Design and Methods Among the Japanese children with hemophagocytic lymphohistiocytosis (HLH) registered at our laboratory, those with more than one of the following findings were eligible for study entry under a diagnosis of FHL: positive for known genetic mutations, a family history of HLH, and impaired CTL-mediated cytotoxicity. Mutations of the newly identified causative gene for FHL5, STXBP2, and the cytotoxicity and degranulation activity of CTLs in FHL patients, were analyzed. Results Among 31 FHL patients who satisfied the above criteria, PRF1 mutation was detected in 17 (FHL2) and UNC13D mutation was in 10 (FHL3). In 2 other patients, 3 novel mutations of STXBP2 gene were confirmed (FHL5). Finally, the remaining 2 were classified as having FHL with unknown genetic mutations. In all FHL patients, CTL-mediated cytotoxicity was low or deficient, and degranulation activity was also low or absent except FHL2 patients. In 2 patients with unknown genetic mutations, the cytotoxicity and degranulation activity of CTLs appeared to be deficient in one patient and moderately impaired in the other. Conclusions FHL can be diagnosed and classified on the basis of CTL-mediated cytotoxicity, degranulation activity, and genetic analysis. Based on the data obtained from functional analysis of CTLs, other unknown gene(s) responsible for FHL remain to be identified.

Development of a novel redirected T-cell–based adoptive immunotherapy targeting human telomerase reverse transcriptase for adult T-cell leukemia

Although adult T-cell leukemia (ATL) has a poor prognosis, successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) in some cases suggests that a cellular immune-mediated strategy can be effective. So far, however, no effective target for anti-ATL immunotherapy has been defined. Here we demonstrated for the first time that human telomerase reverse transcriptase (hTERT) is a promising therapeutic target for ATL, and we developed a novel redirected T-cell-based immunotherapy targeting hTERT. hTERT messenger RNA was produced abundantly in ATL tumor cells but not in steady-state normal cells. Rearranged human leukocyte antigen-A*24:02 (HLA-A*24:02) -restricted and hTERT461-469 nonameric peptide-specific T-cell receptor (TCR) α/β genes were cloned from our previously established cytotoxic T lymphocyte clone (K3-1) and inserted into a novel retroviral TCR expression vector encoding small interfering RNAs for endogenous TCR genes in redirected T cells (hTERT-siTCR vector). Consequently, allogeneic or autologous gene-modified CD8(+) T cells prepared using the hTERT-siTCR vector successfully killed ATL tumor cells, but not normal cells including steady-state hematopoietic progenitors, in an HLA-A*24:02-restricted manner both in vitro and in vivo. Our experimental observations support the development of a novel hTERT-targeting redirected T-cell-based adoptive immunotherapy for ATL patients, especially those for whom suitable allo-HSCT donors are lacking.

Identification of functional and expression polymorphisms associated with risk for anti-neutrophil cytoplasmic autoantibody-associated vasculitis

To identify risk alleles relevant to the causal and biologic mechanisms of antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV).

Aurora kinase A-specific T-cell receptor gene transfer redirects T lymphocytes to display effective antileukemia reactivity

Aurora kinase A (AURKA) is overexpressed in leukemias. Previously, we demonstrated that AURKA-specific CD8(+) T cells specifically and selectively lysed leukemia cells, indicating that AURKA is an excellent target for immunotherapy. In this study, we examined the feasibility of adoptive therapy using redirected T cells expressing an HLA-A*0201-restricted AURKA(207-215)-specific T-cell receptor (TCR). Retrovirally transduced T cells recognized relevant peptide-pulsed but not control target cells. Furthermore, TCR-redirected CD8(+) T cells lysed AURKA-overexpressing human leukemic cells in an HLA-A*0201-restricted manner, but did not kill HLA-A*0201(+) normal cells, including hematopoietic progenitors. In addition, AURKA(207-215)-specific TCR-transduced CD4(+) T cells displayed target-responsive Th1 cytokine production. Finally, AURKA(207-215)-specific TCR-transduced CD8(+) T cells displayed antileukemia efficacy in a xenograft mouse model. Collectively, these data demonstrate the feasibility of redirected T cell-based AURKA-specific immunotherapy for the treatment of human leukemia.

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.