ALBERT

Description: Background TET2 is known as an enzyme which converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Loss-of-function mutations in TET2 are frequent in angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS). Normal counterpart of AITL is thought to be follicular helper T cells (Tfh). PTCL-NOS is likely to consist of heterogeneous groups. Some of the PTCL-NOS cases also have features of Tfh. In the last annual meeting, we reported that aged homozygous Tet2 gene-trap mice (Tet2gt/gt), which showed 80% reduction in the Tet2 mRNA level in various hematopoietic cells, developed T-cell lymphoma. To further investigate the mechanism of T-lymphomagenesis, we analyzed methylome, hydroxymethylome, and transcriptome in the lymphoma cells. Material and Method Tet2 gt mice have a trapping vector inserted into the second intron of the Tet2 locus. In all the analyses, we used CD4+ T cells prepared from lymphoma cells developed in these mice, as well as CD4+ T cells prepared from spleen of wild-type mice as a control. To investigate genome-wide methylation and hydroxymethylation statuses, we performed MeDIP and hMeDIP sequencing, and bisulfite sequencing. To examine comprehensive gene expression, we performed microarray-based analysis, followed by Gene Set Enrichment Analysis (GSEA). Result After observation over a year (median, 67 weeks), 5 out of 7 Tet2gt/gt mice developed T-cell lymphomas with Tfh-like immunostaining pattern. No differences were found in the average levels of 5mC between lymphoma and control CD4+ cells throughout the regions around transcription start sites (TSS) +/- 5 kb. In contrast, when the same regions were analyzed for 5hmC levels, those in the lymphoma cells were significantly lower at the regions around TSS +/- 1 kb. When focused on regions having high 5mC contents (MACS score〉5.0), lymphoma cells demonstrated a significant enrichment at regions around TSS +/- 1 kb, intragenic regions, and CpG islands (p=0.013, 0.006, 0.022 respectively). On the other hand, 5hmC was significantly decreased at regions around TSS +/- 1 kb in lymphoma cells than control cells (p=0.018). In a set of genes whose expression was higher in lymphoma cells than control cells, 5hmC levels were significantly lower in lymphoma cells. GSEA analysis revealed upregulation of Tfh-associated genes such as Bcl6 and cMaf (FDR q value=0.0004), key transcription factors for Tfh differentiation, in lymphoma cells compared with control cells. The expression of upregulated Tfh-associated genes was validated by real-time PCR. We focused on the epigenetic change of Bcl6 because it is among the most important transcription factors for Tfh development. It was reported that hypermethylation at intron 1 of Bcl6 upregulated its transcriptional activity in B cell lymphomas. Bisulfite sequencing revealed that the CpG sites in the intron 1 of Bcl6 were massively methylated/hydroxymathylated in lymphoma cells, whereas those in control cells were mostly at an unmodified status. MeDIP sequencing indicated that intron 1 of Bcl6 had more methylated status in lymphoma cells than control cells. We also found that CpG sites in the same region were densely methylated/hydroxymathylated in EL4, mouse T-cell lymphoma cell line, and that the decitabine treatment converted them into unmodified CpG along with the decrease in the Bcl6 expression levels. Discussion and Conclusion Tet2 gt/gt mice developed T-cell lymphoma with both Tfh-like immunohistological character and gene expression pattern. We found distinct changes in methylome, hydroxymethylome, and transcriptome. We also found a tight linkage between the increased methylation of intron 1 of Bcl6 and increased expression of its mRNA level in lymphoma cells developed in Tet2 knockdown mice. The same scenario is indicated in a T-cell lymphoma cell line. These observations imply that, in normal CD4+ T cells, reduced Tet2 function might increase the methylation status of the CpG sites in intron 1 of Bcl6, which may result in upregulation of Bcl6 expression and deviated Tfh generation. These processes might be an initiating event for the development of T-cell lymphoma with the Tfh features. Because of the long latency before lymphoma development in Tet2gt/gtmice, it is likely that additional hits are necessary. Disclosures: No relevant conflicts of interest to declare.

Description: Background The transcription factor Hairy enhancer of split1 (Hes1) is well characterized as a downstream target of Notch signaling. Hes1 is a basic helix-loop-helix-type protein, and represses target gene expression. Notch signaling has been proposed to play both pro- and anti-tumorigenic roles; it promotes development of T-cell acute lymphoblastic leukemia (T-ALL), while serves as a tumor suppressor for acute myeloid leukemia (AML). Hes1 has been proven as an essential mediator of Notch signaling in T-ALL development. In contrast, we reported, in the last annual meeting, that Hes1 functions as a tumor suppressor against AML development, using a mouse model of AML induced by the MLL-AF9 fusion protein. We further explored the mechanism of Hes1-mediated suppression of AML development. Methods Common myeloid progenitors (CMPs) purified from RBP-Jf/f mouse bone marrow (BM) were serially transduced with MLL-AF9 and Cre recombinase (iCre) using retroviral vectors, and transplanted into lethally irradiated syngenic mice. CMPs from Hes1-/- mouse fetal liver were also retrovirally transduced with MLL-AF9 and transplanted after multiple rounds of replating, and then, expression levels of downstream targets were evaluated by cDNA array. Next Hes1 was retrovirally re-expressed in MLL-AF9/Hes1-/- cells and these cells were transplanted. MLL-AF9-transduced cells were treated with a hamster anti-mouse Notch agonistic antibody (Notch Ab). Results Mice transplanted with MLL-AF9/RBP-J-/- cells developed leukemia at shorter latencies than those with MLL-AF9/RBPJ+/+ cells. MLL-AF9-transduced Hes1-/- cells formed the higher number of colonies at third replating compared with MLL-AF9-transduced Hes1+/+ cells. When infused into irradiated syngenic mice, MLL-AF9/Hes1-/- cells developed leukemia at shorter latencies than MLL-AF9/ Hes1+/+ cells (MLL-AF9/Hes1-/-, 7-10 weeks, n=18 vs MLL-AF9/Hes1+/+, 10-14 weeks, n=18; p

Description: Abstract 118 Background: The transcription factor Hairy enhancer of split1 (Hes1) is well characterised as a downstream target of Notch signaling. Hes1 is a basic helix-loop-helix-type protein, and represses target gene expression. Notch signaling has been proposed to play bivalent roles in tumorigenesis, depending on the cell types. Notch/Hes1 pathway promotes leukemogenesis in T-cell acute lymphoblastic leukemia, and Hes1 also plays an oncogenic role in a mouse model of blastic transformation of chronic myelogenous leukemia. Recent studies, however, demonstrated that Notch signaling serves as a tumor suppressor for chronic myelomonocytic leukemia. Meanwhile, the role of Notch signaling in acute myeloid leukemia (AML) remains to be elucidated. To explore whether Hes1 could possibly play oncongenic or tumor suppressive roles in AML, we focused on a mouse leukemia model established by retroviral transduction with MLL-AF9. Methods: Common myeloid progenitors (CMPs), purified from Hes1−/− or Hes1+/+mouse fetal liver, were retrovirally transduced with MLL-AF9 cDNA. MLL-AF9-transduced cells were subjected to a colony-forming assay, with multiple rounds of replating. These cells were transplanted into lethally irradiated syngenic mice. CMPs from wild-type mouse bone marrow (BM-CMPs) were transduced with MLL-AF9 and dominant negative Hes1 (dnHes1), which lacks WRPW domain, and these cells were transplanted into irradiated mice. Secondary transplantation was performed by infusing leukemic bone marrow cells obtained from the primary recipients. Expression levels of downstream targets were evaluated by cDNA array and quantitative RT-PCR. Result: MLL-AF9-transduced Hes1−/− CMPs formed higher number of colonies at third replating compared to MLL-AF9-transduced Hes1+/+ CMPs, while colony numbers were not different in first and second plating. When infused to irradiated syngenic mice, MLL-AF9/Hes1−/− cells developed leukemia at shorter latencies than MLL-AF9/Hes1+/+ cells (MLL-AF9/Hes1−/−, 7–10 weeks, n=18 vs MLL-AF9/Hes1+/+, 10–14 weeks, n=18; p

Description: Abstract 1299 Background: Loss-of-function mutations in TET2 are frequent in human myeloid and lymphoid malignancies. Especially, TET2 mutations were found in 30–47% of angioimmunoblastic T-cell lymphoma (AITL) and 10–38% of peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS). The tumor origin of AITL is thought to be follicular helper T cells (Tfh). PTCL, NOS is a group of heterogenous T-cell-derived lymphomas, whereas some cases of PTCL, NOS also appear to be derived from Tfh. Several recent papers reported that Tet2 knockout mice demonstrated premalignant status of myeloid lineages, implicating that TET2 mutations have a driver's role in myeloid malignancies; however, there are no clues to how impaired Tet2 function provokes T-cell lymphomas. To address this issue, we analyzed Tet2 gene trap (Tet2gt) mice. Materials and methods: Tet2gt mice (Transgenic Res 17:599, 2008) have a trapping vector inserted into the second intron of Tet2 locus. Lineage-, Sca1+, and c-kit+ (LSK) cells were sorted from Tet2gt E15.5 fetal liver (FL) cells and transplanted into lethally irradiated Ly5.1 wild-type (WT) mice. Bone marrow, spleen, and tumors when developed were analyzed in the recipient mice as well as adult Tet2gtmice by flow cytometory and immunohistochemical staining. Result: Adult Tet2gt mice were obtained under the following ratio; Tet2+/+:Tet2+/gt:Tet2gt/gt=48:52:25. TET2 mRNA expression level in Tet2gt/gt Lin- FL cells was reduced to 20% of that in WT Lin- FL cells, while the 5-hydroxymethyl cytosine level was reduced to around a half of that in WT cells. At 40 weeks of age, the proportion of CD4+/PD1+/Cxcr5+ cells, immunophenotypically similar to Tfh cells, significantly increased in Tet2gt/gt mice compared to WT mice (P=0.022). Two out of 8 Tet2gt/gt mice developed marked splenomegaly (more than 300 mg) at 40 weeks, and 4 out of 6 developed marked splenomegaly with swollen lymph nodes and multi-nodular tumors in liver and lungs at 60 weeks or older (median, 67 weeks). Histological examination of the enlarged spleen and swollen lymph nodes in these 4 mice demonstrated completely destroyed follicular structures. The spleen, lymph nodes, and nodular tumors showed infiltration of morphologically abnormal lymphocytes, which were proven to be CD4+/PD1+ T cells. These cells were also weakly positive for Cxcr5. CD4+ cells purified from the tumors revealed restricted rearrangement patterns of T-cell receptor genes, implying T-cell lymphoma. There were no increased vascular structures representing AITL. CD4+/PD1+ cells purified from the tumors expressed Bcl6 and cMaf, two key transcription factors of Tfh, at significantly higher levels compared to CD4+/PD1+ cells purified from spleen of WT mice. Meanwhile, one of 6 mice transplanted with Tet2gt/gt LSK cells also developed lymphoma with similar pattern of CD4+/PD1+/CXCR5 expression at 40 weeks after transplantation. Discussion: Our observations indicate that reduced expression of TET2 may cause skewed differentiation into Tfh, which eventually develop tumors that pathologically recapitulate PTCL, NOS in human, while the tumor-comprising cells show characteristics of Tfh. The long latency period required for tumor development indicates secondary genetic hits in addition to the downregulation of TET2. These secondary events might determine the specificity of the hematologic malignancies, particularly AITL and PCTL, NOS with propensity for AITL. Disclosures: No relevant conflicts of interest to declare.

Description: BACKGROUND: Large number of rejuvenated antigen-specific T cells generated from iPS cells (iPSCs) may have a large impact on the T-cell immunotherapy field. We previously reported the generation of functional CD8 single positive (SP) cytotoxic lymphocytes (CTLs) from iPSCs (Minagawa etal. CellStemCell, 23: 850-858. (2018)), and a regenerative CTL-based immunotherapy is about to begin in clinical trials. However, these two-dimensional differentiation protocols using the OP9DL1 murine feeder cell line or DLL4 recombinant proteins could differentiate iPSCs into CD8SP CTLs but not into robust CD4SP helper T (Th) cells. It is clear that, we could better control immune reactions if we could produce each Th cell fraction. For example, we could enhance antitumor immunity if we could specifically induce Th1 cells to command cellular immunity. The drastic therapeutic effect of CD19 CAR modified T cells including both CD8SP CTLs and CD4SP Th cells is clearly based on the role of CD4SP Th cells in helping CD8SP CTLs prolong the therapeutic effect against B-cell malignancy. This led us to hypothesize that Th cell induction from iPSCs is essential for efficient immunotherapy. In this situation, a three-dimensional (3D) method called artificial thymic organoid (ATO) was reported to support robust differentiation of both CD4SP and CD8SP TCRαβ cells from primary hematopoietic stem and progenitor cells (Seet etal. NatureMethods, 14: 521-530. (2017)) and hematopoietic progenitor cells derived from ES cells and iPSCs (Montel-Hagen etal. CellStemCell, 24: 376-389. (2019)). Here, we evaluated the advantages and unsolved tasks of the 3D method to induce antigen-specific and functional CD4SP Th cell subsets from iPSCs. METHODS and RESULTS: By applying the ATO methods, we cultured mixed pellets of iPSC-derived hematopoietic progenitor cells (HPCs) and Notch ligand-expressing MS5 feeder cells on cell culture inserts for up to 9 to 12 weeks. Next, we analyzed differentiated T cells in the ATOs. We used iPSCs derived from antigen-specific Th cells containing HLA class II restricted TCR genes corresponding to an original Th cell clone. Most importantly, the ATO method supported robust differentiation of CD4SP T cells as expected even from these iPSCs. These CD4SP T cells showed high expression of Th-POK as a master regulator of Th cells, and high secretion ability of several key cytokines produced by Th cells-IL-2, IFN-γ, and IL-4. However, the majority of iPSC-derived CD4SP T cells showed simultaneous secretion of both IFN-γ and IL-4 unlike normal peripheral CD4SP Th cells. We tried to make the regenerated CD4SP T cells separately produce the Th1 (IFN-γ) and Th2 (IL-4) cytokines by optimizing culture conditions, but we failed to achieve separated Th1/2 differentiation. To understand the reason for the bipolarized cytokine production profile of regenerated CD4SP T cells, we checked the master regulator expression profile. We found that a majority of the CD4SP T cell population highly expressed T-bet, the master regulator of Th1. Contrary to our expectation, GATA3's expression levels were not high in most CD4SP T cells. And, GATA3 is master regulator not only of Th2 but of T cell differentiation itself, so we guessed knock out (KO) of GATA3 led to failure of differentiation into T cell. To "polarize" the CD4SP T cells to have Th1 or Th2 functions, we knocked out TBX21 (coding T-bet) or the Th2 "master cytokine" IL4 of undifferentiated iPSCs using CRISPR-Cas9 to obtain TBX21KO/KO iPSCs or IL4KO/KO iPSCs, respectively. Those iPSCs were successfully differentiated into HPCs, and ATOs were then prepared using these cells. After 9 to 12 weeks, mature CD4SP T cells and CD8SP T cells were observed in both ATOs with the same surface marker profile as T cells from wild type iPSCs, and TBX21KO/KO CD4SP T cells or IL4KO/KO CD4SP T cells from iPSCs with selective production of IL-4 or IFN-γ, respectively (Figure1). These results also suggested the potential utility of ATO-based invitro T cell differentiation from genome-edited iPSC for understanding of human developmental immunology. CONCLUSIONS: "Polarized" CD4SP Th cells were successfully obtained from master regulator or cytokine gene-knockout iPSCs in ATO-based invitro differentiation. We are now investigating the actual helper function of these "polarized" iPS-Th cells that could be induced by the target peptide on HLA Class II molecules of antigen presenting cells. Disclosures Shinohara: Takeda Pharmaceutical Company Limited: Employment. Koga:Takeda Pharmaceutical Company Limited: Employment. Kassai:Takeda Pharmaceutical Company Limited: Employment. Kiyoi:Zenyaku Kogyo Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Pfizer Japan Inc.: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding; Perseus Proteomics Inc.: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding. Kaneko:KIRIN holdings Co.,Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Other: Scientific adviser, Research Funding; TERUMO Co., Ltd.: Research Funding; TOSOH Co., Ltd.: Research Funding; Thyas Co., Ltd.: Other: Founder, Shareholder, Chief Science Officer, Research Funding.