Seiji Nagano

Regeneration of CD8αβ T Cells from T-cell–Derived iPSC Imparts Potent Tumor Antigen-Specific Cytotoxicity

Although adoptive transfer of cytotoxic T lymphocytes (CTL) offer a promising cancer therapeutic direction, the generation of antigen-specific CTL from patients has faced difficulty in efficient expansion in ex vivo culture. To resolve this issue, several groups have proposed that induced pluripotent stem cell technology be applied for the expansion of antigen-specific CTL, which retain expression of the same T-cell receptor as original CTL. However, in these previous studies, the regenerated CTL are mostly of the CD8αα+ innate type and have less antigen-specific cytotoxic activity than primary CTL. Here we report that, by stimulating purified iPSC-derived CD4/CD8 double-positive cells with anti-CD3 antibody, T cells expressing CD8αβ were generated and exhibited improved antigen-specific cytotoxicity compared with CD8αα+ CTL. Failure of CD8αβ T-cell production using the previous method was found to be due to killing of double-positive cells by the double-negative cells in the mixed cultures. We found that WT1 tumor antigen-specific CTL regenerated by this method prolonged the survival of mice bearing WT1-expressing leukemic cells. Implementation of our methods may offer a useful clinical tool. Cancer Res; 76(23); 6839-50. ©2016 AACR.

Blastic Plasmacytoid Dendritic Cell Neoplasm Expressing the CD13 Myeloid Antigen

Blastic plasmacytoid dendritic cell neoplasm (BPDCN), currently considered to originate from immature plasmacytoid dendritic cells (DC), is a rare and aggressive CD4+CD56+ neoplasm that frequently involves the skin and bone marrow. We present a case of an 80-year-old man with a CD4+CD56+ BPDCN that affected the orbital cavity and bone marrow. Although BPDCN has not been reported to express any lineage-specific markers, the neoplastic cells strongly expressed the CD13 antigen. Therefore, in addition to pathological examination, we attempted to induce in vitro morphological and surface marker changes with IL-3 and CD40 ligand. After treatment with these cytokines, the tumor cells enlarged markedly, acquired many fine dendrites, similar to mature DC, and showed enhanced expression of antigens specific to DC or antigen-presenting cells, such as CD40, CD80, CD83 and CD86. To the best of our knowledge, this is the first report of BPDCN expressing a myeloid antigen, CD13, although CD33 expression has been described in some cases. The present patient received 2 courses of combination chemotherapy consisting of cytarabine and etoposide, which resulted in complete remission. Given that the cellular origin of plasmacytoid DC is still controversial, myeloid antigen expression involving CD13 may not exclude a diagnosis of BPDCN.

NK cell alloreactivity against KIR-ligand-mismatched HLA-haploidentical tissue derived from HLA haplotype-homozygous iPSCs.

Summary HLA haplotype-homozygous (HLA-homo) induced pluripotent stem cells (iPSCs) are being prepared to be used for allogeneic transplantation of regenerated tissue into recipients carrying an identical haplotype in one of the alleles (HLA-hetero). However, it remains unaddressed whether natural killer (NK) cells respond to these regenerated cells. HLA-C allotypes, known to serve as major ligands for inhibitory receptors of NK cells, can be classified into group 1 (C1) and group 2 (C2), based on their binding specificities. We found that the T cells and vascular endothelial cells regenerated from HLA-homo-C1/C1 iPSCs were killed by specific NK cell subsets from a putative HLA-hetero-C1/C2 recipient. Such cytotoxicity was canceled when target cells were regenerated from iPSCs transduced with the C2 gene identical to the recipient. These results clarify that NK cells can kill regenerated cells by sensing the lack of HLA-C expression and further provide the basis for an approach to prevent such NK cell-mediated rejection responses.

Successful treatment of γ-heavy-chain disease with rituximab and fludarabine.

An 84-year-old Japanese man was admitted because of pancytopenia. The bone marrow was hypoplastic with a predominance of abnormal small lymphocytes and grape cells, which were positive for CD19 and CD20, and partially for the surface ĸ-light chain. Systemic CT scanning showed neither lymph node swelling nor hepatosplenomegaly. Serum immunoelectrophoresis and rocket immunoselection assays showed the presence of monoclonal IgG protein without a corresponding light chain and faint IgMĸ monoclonal protein. Histologic analysis of the clot preparation of the bone marrow aspirate facilitated a diagnosis of lymphoplasmacytic lymphoma (LPL). PCR analysis of the marrow cells demonstrated a clonal rearrangement of the immunoglobulin heavy-chain gene. From these results, we made a final diagnosis of γ-heavy-chain disease (γ-HCD) with underlying LPL localized in the bone marrow. We performed only a single course of immunochemotherapy (rituximab and fludarabine) in view of severely impaired hematopoiesis, which resulted in marked reduction of lymphoma cells and improvement of hematopoiesis. This report suggests the efficacy of rituximab plus fludarabine therapy for LPL-associated γ-HCD.

Abstract B126: Regeneration of tumor antigen specific CTLs utilizing iPSC technology

Current cancer immunotherapies mainly aim to activate and expand tumor antigen specific cytotoxic T lymphocytes (CTLs) in vitro or in vivo, but these methods are not so successuful because of the difficulty in preparing sufficient number of CTLs. We are trying to overcome this problem by utilizing the induced pluripotent stem cell (iPSC) technology. Our strategy is based on the concept that i) when iPSCs are produced from antigen specific T cells, the rearranged configurations of T cell receptor (TCR) genes are inherited to the iPSCs (which we call T-iPSCs), and that ii) when T cells are regenerated from such T-iPSCs, all of them should come to express the same TCR as the original one. Therefore it will become possible to obtain de novo generated tumor specific CTLs almost unlimitedly, because it is possible to make a mass-culture of cells at the iPSC level. In line with this concept, we have succeeded in establishing T-iPSCs from CTLs specific for the melanoma antigen MART-1 derived from tumor infiltarating lymphocytes, and in regenerating MART-1 specific T cells from such T-iPSCs (Vizcardo et al, Cell Stem Cell, 2013). The strategy mentioned above has been conducted in the category of autologous transplantation. Autologous transplantation setting, however, has some problems; i) high cost to establish T-iPSCs from each patients, ii) heterogeneneity of the quality as iPSCs, iii) difficulty in getting high affinity TCR. To solve these problems we are now planing to apply this method to the allogeneic transplantation setting. In allogeneic setting, we can select high quality T-iPSC clone and such a clone can be used to many other patients. It is preferable to produce T-iPSCs from a HLA haplotype homo donor, since CTLs generated from these T-iPSCs can be administerd to the patients carrying the same HLA haplotype on one allele. Thus by utilizing allogenic T-iPSCs the cost would be reduced and the quality of T-iPSCs can be ensured. Now we are trying to establish T-iPSCs from CTLs specific for various cancer antigen from healthy donors. We succeded in establishing T-iPSCs from CTLs specific for EB virus related antigen LMP2 and cancer testis antigen WT1, both of which are restricted to HLA A2402, the commonest HLA-A in the Japanese. We improved in vitro culture condition and have succeded in regenerating CD8 single positive cells expressing CD8 alpha-beta heterodimers. These regenerated CTLs exhibited very high antigen specific killing activity comparable to the original CTLs. The CTLs derived from WT1 specific T-iPSCs were able to kill some leukemia cell lines which express endogenous WT1 protein. They can be expanded more than ten thousand fold by repeated TCR stimulation without reducing their killing activity. We are now examining in vivo killing activity of these CTLs using zenograft model. This method could bring about a breakthrough in cancer immunotherapy. Citation Format: Takuya Maeda, Seiji Nagano, Hiroshi Ichise, Kyoko Masuda, Toshio Kitawaki, Akifumi Kondo-Takaori, Hiroshi Kawamoto. Regeneration of tumor antigen specific CTLs utilizing iPSC technology. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B126.