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T cell regenerative medicine represents an emerging immunotherapeutic approach using antigen-specific Induced Pluripotent Stem Cells (iPSC) to rejuvenate CD8 + cytotoxic T lymphocytes (CTL). Here we report on an iPSC-derived therapeutic strategy targeting B-Cell Maturation Antigen (BCMA) against multiple myeloma (MM) via establishment of antigen-specific iPSC, followed by differentiation into highly functional BCMA-specific CD8 + CTL. The reprogrammed BCMA-specific iPSC displayed normal karyotypes and pluripotency potential as evidenced by expression of stem cell markers (SSEA-4, TRA1-60) and alkaline phosphatase, along with differentiation into three germ layers (Ectoderm, Mesoderm, Endoderm). During embryoid body formation, BCMA-specific iPSC further polarized into the mesoderm germ layer, evidenced by the activation of SNAI2, TBX3, PLVAP, HAND1 and CDX2 transcriptional regulators. Next, the BCMA-specific iPSC clones committed to CD8 + T cell differentiation were characterized by analyzing their hematopoietic progenitor cells (HPC; CD34 + CD43 +/CD14 - CD235a -) for specific transcriptional regulation. RNAseq analyses indicated a low variability and similar profiles of gene transcription within the iPSC clones committed to CD8 + CTL compared to increased transcriptional variability within iPSC clones committed to different cell types. The unique transcriptional profiles of the iPSC committed to CD8 + T cells included upregulation of transcriptional regulators controlling CD4/CD8 T cell differentiation ratio, memory CTL formation, NF-kappa-B/JNK pathway activation, and cytokine transporter/cytotoxic mediator development, as well as downregulation of regulators controlling B and T cell interactions, CD4 + Th cells, and inhibitory receptor development. Specifically, a major regulatory shift, indicated by upregulation of specific genes involved in immune function, was detected in HPC from the iPSC committed to CD8 + T cells. BCMA-specific T cells differentiated from the iPSC were characterized as displaying mature CTL phenotypes including high expression of CD3, CD8a, CD8b, TCRab, CD7 along with no CD4 expression (Fig. 1). In addition, the final BCMA iPSC-T cells were predominantly CD45RO + memory cells (central memory and effector memory cells) expressing high level of T cell activation (CD38, CD69) and costimulatory (CD28) molecules. Importantly, these BCMA iPSC-T cells lacked immune checkpoints (CTLA4, PD1, LAG3, Tim3) expression and regulatory T cells induction, distinct from other antigen-stimulated T cells. The rejuvenated BCMA iPSC-T cells demonstrated a high proliferative (1,000 folds increase) during the differentiation process as well as poly-functional anti-tumor activities and Th1 cytokine (IFN-g, IL-2, TNF-a) production triggered in response to MM patients' cells in HLA-A2-restricted manner (Fig. 2). Furthermore, the immune responses induced by these BCMA iPSC-T cells were specific to the parent heteroclitic BCMA 72-80 (YLMFLLRKI) peptide used to reprogram and establish the antigen-specific iPSC. Evaluation of 88 single cell Tetramer + CTL from the BCMA iPSC-T cells revealed a clonotype of unique T cell receptor (TCRa, TCRb) sequence. The BCMA-specific iPSC clones maintained their specific differentiation potential into the antigen-specific CD8 + memory T cells, following multiple subcloning in long-term cultures under feeder-free conditions or post-thaw after long-term (18 months) cryopreservation at -140 oC, which provides additional benefits to treat patients in a continuous manner. Taken together, rejuvenated CD8 + CTL differentiated from BCMA-specific iPSC were highly functional with significant (*p < 0.05) levels of anti-MM activities including proliferation, cytotoxic activity and Th-1 cytokine production. Therefore, the antigen-specific iPSC reprogramming and T cells rejuvenation process can provide an effective and long-term source of antigen-specific memory CTL lacking immune checkpoints and suppressors for clinical application in adoptive immunotherapy to improve patient outcome in MM. Figure 1 Figure 1. Disclosures Munshi: Amgen: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Legend: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Pfizer: Consultancy. Ritz: Amgen: Research Funding; Equillium: Research Funding; Kite/Gilead: Research Funding; Avrobio: Membership on an entity's Board of Directors or advisory committees; Akron: Consultancy; Biotech: Consultancy; Blackstone Life Sciences Advisor: Consultancy; Clade Therapeutics, Garuda Therapeutics: Consultancy; Immunitas Therapeutic: Consultancy; LifeVault Bio: Consultancy; Novartis: Consultancy; Rheos Medicines: Consultancy; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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.

In allogeneic hematopoietic cell transplantation (HSCT), donor T lymphocytes mediate the graft-versus-leukemia (GVL) effect, but induce graft-versus-host disease (GVHD). Suicide gene therapy—that is, the genetic induction of a conditional suicide phenotype into donor T cells—allows dissociating the GVL effect from GVHD. Genetic modification with retroviral vectors after CD3 activation reduces T-cell alloreactivity. We recently found that alloreactivity is maintained when CD28 costimulation, IL-7, and IL-15 are added. Herein, we used the minor histocompatibility (mH) antigens HA-1 and H-Y as model alloantigens to directly explore the antileukemia efficacy of human T cells modified with the prototypic suicide gene herpes simplex virus thymidine kinase (tk) after activation with different stimuli. Only in the case of CD28 costimulation, IL-7, and IL-15, the repertoire of tk+ T cells contained HA-1– and H-Y–specific CD8+ cytotoxic T cells (CTL) precursors. Thymidine kinase–positive HA-1– and H-Y–specific CTLs were capable of self-renewal and differentiation into potent antileukemia effectors in vitro, and in vivo in a humanized mouse model. Self-renewal and differentiation coincided with IL-7 receptor expression. These results pave the way to the clinical investigation of T cells modified with a suicide gene after CD28 costimulation, IL-7, and IL-15 for a safe and effective GVL effect.

Phenotypically described hematopoietic stem cells (HSCs) represent a functionally heterogeneous pool of primitive cells with conceivable potential to replenish and maintain the whole hematopoietic system. The diverse lineage potential of HSCs is supposed to play a significant role in the response to different kinds of hematopoietic stress. Since subcategorization of HSCs biased towards specific lineage(s) highly relies on the retrospective information, e.g. transplantation assay, exploring additional markers will allow us to understand further molecular mechanisms of HSC regulation such as activation and lineage choice but also the degree of correlation between them. Here, we show that the cell surface protein Junctional adhesion molecule 2 (Jam2) serves as an amplifier of the Notch/Delta signal thereby representing the higher T cell potential of HSCs. Flow cytometry analyses revealed that a subset of CD150+CD48-KSL cells in mouse bone marrow (BM) were positive for Jam2 (Jam2+HSC, 36.6 ±13.0%), while other Jam family member, Jam1 (F11r), was expressed on all HSCs and Jam3 was not detected. Transplantation assay using 30 Jam2+ or Jam2-HSCs revealed that Jam2+HSCs reconstituted lethally irradiated mice more efficiently than Jam2-HSCs (77.5 ±15.9 and 51.7 ±29.3% in peripheral blood, respectively). Lineage analyses revealed that Jam2+HSCs have a greater potential in lymphoid cell reconstitution, particularly T cells, whereas the chimerism in myeloid cells was not significantly different from Jam2-HSCs. This tendency of higher contribution to the T cell development was even more pronounced in the secondary transplantation experiments, where the contribution of Jam2+HSCs in T cells was close to 100%. Of note, most of Jam2+HSCs were in a dormant state, suggesting that the T cell potential of Jam2+HSCs is independent of the cell cycle progression. Jam2 has been reported to mediate the Notch signaling through an interaction with Jam1 (Kobayashi et al., Nature, 2014). In addition, Jam2+HSCs express Notch1 at a higher level than Jam2-HSCs (23.6 ±6.7 and 9.05 ±5.8%, respectively). We therefore analyzed the functional role of Jam2 in the Notch/Delta-oriented T cell production using a competitive feeder-free T cell culture system. At a low concentration of DLL1, that is insufficient to promote T cell production by itself, Jam2+HSCs effectively produced T cell lineages only in the presence of recombinant Jam1 protein, but not Jam2 or Jam3. In contrast, Jam2+HSCs did not require Jam1 protein with a higher concentration of DLL1. These differences were not observed with Jam2-HSCs, indicating that Jam2/Jam1 interaction amplifies Notch signal transduction and is crucial for the subsequent T cell specification of Jam2+HSCs. To elucidate the molecular signature of Jam2+HSCs, gene expression profiling was performed using a microarray analysis. Gene set enrichment analysis (GSEA) observed that Jam2+HSCs were significantly enriched for common lymphoid progenitor (CLP) and early T cell gene expression. Of note, Jam2+HSCs were also enriched for E2F target genes, G2M checkpoint genes and glycolysis related genes, which potentially explains the reason why Jam2+HSCs display a bivalent phenotype: being more dormant compared to Jam2-HSCs at the steady state but at the same time having the capacity to reconstitute more actively upon engraftment. Since Jam2 positivity correlates to T cell potential, we asked if altered T lymphopoietic environment affects the proportion of Jam2+HSCs. In vivo T cell depletion resulted in significantly higher frequency of Jam2+HSCs but not upon other stress inducers, such as 5-FU treatment, suggesting that the increase in Jam2+HSC pool was specifically due to the T cell deficiency. These findings indicate that the lack of T cells, which also means a requirement for immediate T cell replenishment, leads to an increase of Jam2+HSC fraction. Our findings suggest that Jam2 is the key protein that controls T lymphopoiesis by enhancing the Notch/Delta signal transduction via interaction with Jam1. It also means that the lineage balance particularly towards T lymphopoiesis might be regulated at a higher stage of hematopoietic hierarchy than currently understood. Thus, Jam2 is a new marker representing the T lymphocyte potential of HSCs, as the frequency of Jam2+HSCs sensitively reflects the state of the T cell environment. Disclosures No relevant conflicts of interest to declare.

In the search for genes expressed in hematopoietic stem cells, we identified that the expression of Gfi-1B (growth factor independence-1B) is highly restricted to hematopoietic stem cells, erythroblasts, and megakaryocytes. Gfi-1 and Gfi-1B are zinc finger proteins that share highly conserved SNAG and 6 zinc finger domains. Gfi-1 has been characterized as an oncogene involved in lymphoid malignancies in mice. In contrast, role of Gfi-1B in hematopoiesis has not been well characterized. In this study, we analyzed its function in human hematopoiesis. Enforced expression of Gfi-1B in human CD34(+) hematopoietic progenitors induced a drastic expansion of erythroblasts in an erythropoietin-independent manner. Expression of Gfi-1B did not promote erythroid commitment, but enhanced proliferation of immature erythroblasts. Erythroblasts expanded by exogenous Gfi-1B, however, failed to differentiate beyond proerythroblast stage and showed massive apoptosis. These biologic effects of Gfi-1B were mediated through its zinc finger domain, but not by the SNAG or non-zinc finger domain. Proliferation of erythroblasts was associated with sustained expression of GATA-2 but not of GATA-1, indicating a potential link between Gfi-1B and GATA family regulators. Importantly, the function of Gfi-1B to modulate transcription was dependent on promoter context. In addition, activation of transcription of an artificial promoter was mediated through its zinc finger domain. These findings establish Gfi-1B as a novel erythroid regulator and reveal its specific involvement in the regulation of erythroid cell growth through modulating erythroid-specific gene expression.

Phenotypically well-characterized hematopoietic stem cells (HSCs) still represent a heterogeneous pool of primitive cells regarding to their functionality. In particular, different lineage potential of HSCs have been considered as one of key features of the HSC heterogeneity. The lineage output of HSCs is often coupled with cell cycle status or long-term reconstitution potential, however molecular mechanisms of the mutuality are unclear and other type of the regulation may exist. In addition, prospective isolation of such HSCs biased towards specific lineage(s) is still problematic, as many of categorizations highly rely on retrospective information, e.g. transplantation assay. Although several markers have been reported to be able to subdivide HSCs into subcategories, exploration of additional markers will allow us understanding further molecular mechanisms of HSC regulations including activation and lineage choice. Here, we show that cell surface expression of Junctional adhesion molecule 2 (Jam2) represents higher reconstitution capacity of HSCs and the T cell potential. Flow cytometry analyses revealed that a subset of CD150+CD48-KSL cells in mouse bone marrow (BM) were positive for Jam2 (Jam2+HSC, 36.6 ±13.0 %), while other Jam family member Jam1 (F11r) was expressed on all HSCs and Jam3 was not detected. To examine functional differences of Jam2+ and Jam2-HSCs, 30 cells were separately transplanted into lethally irradiated mice. Peripheral blood analyses revealed that Jam2+HSCs reconstituted more efficiently than Jam2-HSCs (77.5 ±15.9 and 51.7 ±29.3 %, respectively). In case of transplantation using 5 cells, the frequency of reconstituted mice was higher in Jam2+HSCs (7 in 11) compared to Jam2-HSCs (4 in 11), indicating that Jam2+ population is more enriched for functional HSCs. The expression of Jam2 on HSC is reversible, but not hierarchical, as both Jam2+ and Jam2-HSCs reconstituted opposite population in the BM.Lineage analyses revealed that Jam2+HSCs have a greater potential in lymphoid cell reconstitution, particularly T cells, whereas the chimerism in myeloid cells was not significantly different from Jam2-HSCs. This tendency of higher contribution to the T cell development was even more pronounced in the secondary transplantation experiments, where the contribution of Jam2+HSCs in T cells was close to 100 %. Of note, most of Jam2+HSCs were in a dormant state, suggesting that the T cell (or lymphoid) potential of Jam2+HSCs is independent of cell cycle progression. Jam2 has been reported to interact with Jam1, which mediates the Notch signaling (Kobayashi et al., Nature, 2014). Competitive co-culture of Jam2+ vs Jam2-HSCs on OP9-DL1 showed that Jam2+HSCs dominated the T cell production, whereas no difference was seen in B cell production upon OP9 co-culture. Since Jam2 positivity correlates to T cell potential, we asked if altered T lymphopoiesis environment affects the cell surface Jam2 expression. Comparison of C57BL/6, NOD, NOD-Scid and NOD-Scid Il2rγ KO (NSG) mice showed that HSCs of NSG mice have significantly higher frequency of Jam2+HSCs, suggesting that cell surface Jam2 expression might be regulated by specific cytokine(s) binding to IL2Rγ. Our findings suggest Jam2 is a new marker for a subset of HSCs that preferentially generate T cells. In addition, this work uncouples the lineage choice and cell cycle status, which proposes a novel model to the lineage-determining machineries. Since efficient and immediate generation of T cells in transplantation therapy is important to minimize infectious risks, understanding the molecular basis of the Jam-Notch cooperation would contribute to establish safer and more efficient treatment. Disclosures No relevant conflicts of interest to declare.

The mechanism underlying the cytotoxicity mediated by a human CD4+ cytotoxic T-lymphocyte (CTL) clone directed against a peptide derived from the acute myelogenous leukemia-associated fusion protein, DEK-CAN, was investigated. A DEK-CAN fusion peptide-specific CD4+ Th0 CTL clone, designated HO-1, was established from the peripheral blood lymphocytes of a healthy individual. HO-1 exerted direct but not “innocent bystander” cytotoxicity within 2 hours. The cytotoxicity mediated by HO-1 was completely Ca2+-dependent. Because HO-1 lysed peptide-loaded Fas-deficient target cells derived from a patient with a homozygousFas gene mutation, its cytotoxicity appeared to be mediated by a Fas-independent pathway. In addition, its cytotoxicity was only partially inhibited by treatment with concanamycin A and strontium ions, which are inhibitors of the perforin-based cytotoxic pathway. Although membrane-bound type of tumor necrosis factor- (TNF-) was expressed on HO-1, an anti–TNF- antibody had no effect on HO-1–mediated cytotoxicity. HO-1 expressed mRNA for apoptosis-inducing mediators, including perforin, granzyme B, Fas ligand, TNF-, and lymphotoxin; however, no DNA fragmentation was detected in target cells incubated with HO-1 by 5-[125I]Iodo-2′-deoxyuridine release assay and agarose gel electrophoresis of DNA. Although it has been suggested that the Fas/Fas ligand system is the main pathway by which CD4+ CTL-mediated cytotoxicity is exerted in murine systems, HO-1 produced peptide-specific and HLA-restricted cytotoxicity via a Fas-independent and nonapoptotic pathway. The present study thus describes a novel mechanism of cytotoxicity mediated by CD4+ CTL.

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder caused by BCR-ABL fusion protein that has constitutively active tyrosine kinase activity. Although the prognosis of the patient with CML in chronic phase has markedly improved by the advent of tyrosine kinase inhibiters, the management of the patients with CML in advanced phase remains to be the major challenge. Immunotherapy is considered to be one of the promising treatment strategies for refractory CML. BCR-ABL fusion region, b3a2 peptide, represents a neo-epitope that can induce CML-specific immune responses. The activation of b3a2 peptide-specific CD4+ T helper (Th) cells and their interaction with dendritic cells (DCs) can induce a robust cytotoxic T lymphocyte (CTL)-mediated anti-leukemic immunity through epitope spreading. However, current vaccination strategies cannot effectively induce the proliferation of antigen-specific Th cells in vivo, presumably due to the tumor-induced immunosuppressive milieu. In addition, ex vivo expansion of antigen-specific Th cells attenuates their effector functions by expansion-related cell senescence, and the procedure to establish antigen-specific Th cells for each patient's treatment is too complicated for the clinical application. The purpose of the present study is to establish a method to generate large amounts of functional b3a2-specific CD4+ Th cells enough for the treatment of the patients with refractory CML by using induced pluripotent stem cell (iPSC) technology. First, we established b3a2-specific CD4+ Th clone from peripheral blood mononuclear cells of a healthy donor positive for HLA-DRB1*09:01 and HLA-A*24:02. The Th clone recognized b3a2 peptide in the context of HLA-DR9 and exhibited a Th1 profile. Second, we established iPSCs from the Th clone and differentiated them into T cell lineage by coculture with OP9 stromal cells expressing Notch ligand Delta-like 1. The iPSC-derived T cells (b3a2-iPS-T cells) expressed the same T cell antigen receptor (TCR) as the original Th clone but not CD4 molecule. Because CD4 acts as a co-receptor in the TCR-mediated Th responses, we transduced b3a2-iPS-T cells with CD4 gene. The CD4-expressing b3a2-iPS-T cells (CD4+ b3a2-iPS-T cells) recognized b3a2 peptide in the context of HLA-DR9 as the original Th clone. Moreover, CD4+ b3a2-iPS-T cells activated by b3a2 peptide induced DC maturation, as indicated by the upregulation of CD86 on DCs. In the additional presence of HLA-A24-restricted Wilms tumor 1 (WT1) peptide, the mature DCs stimulated primary expansion of WT1-specific CTLs. The CTLs exerted cytotoxicity against WT1 peptide-loaded HLA-A24 positive cell lines. These data suggest that the CD4+ b3a2-iPS-T cells have a potential to induce effective anti-leukemic immunity via DC maturation and subsequent CTL responses. The current approach enable to provide large amounts of b3a2 specific CD4+ Th-like cells that would augment CTL-mediated anti-leukemic responses via DC maturation, which may contribute to the treatment of patients with refractory CML. Disclosures Kiyoi: Yakult Honsha Co.,Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Eisai Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; MSD K.K.: Research Funding; Japan Blood Products Organization: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Teijin Ltd.: Research Funding. Naoe:Celgene K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemical CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties; FUJIFILM Corporation: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Astellas Pharma Inc.: Research Funding. Kaneko:AsTlym Co., Ltd: Other: founder, shareholder and scientific adviser.

Abstract 703 Patient-specific, induced pluripotent stem cells (iPSCs) enable us to study disease mechanisms and drug screening. To clarify the phenotypic alterations caused by the loss of c-MPL, the thrombopoietin (TPO) receptor, we established iPSCs derived from skin fibroblasts of a patient who received curative bone marrow transplantation for congenital amegakarycytic thrombocytopenia (CAMT) caused by the loss of the TPO receptor gene, MPL. The resultant CAMT-iPSCs exhibited mutations corresponding to the original donor skin. Then using an in vitro culture system yielding hematopoietic progenitor cells (HPCs), we evaluated the role of MPL on the early and late phases of human hematopoiesis. Although CAMT-iPSCs generated CD34+ HPCs, per se, their colony formation capability was impaired, as compared to control CD34+ HPCs. Intriguingly, both Glycophorin A (GPA)+ erythrocyte development and CD41+ megakaryocyte yields from CAMT-iPSCs were also impaired, suggesting that MPL is indispensable for MEP (megakaryocyte erythrocyte progenitors) development. Prospective analysis along with the hematopoietic hierarchy revealed that, in CAMT-iPSCs but not control iPSCs expressing MPL, mRNA expression and phosphorylation of putative signaling molecules downstream of MPL are severely impaired, as is the transition from CD34+CD43+CD41-GPA- MPP (multipotent progenitors) to CD41+GPA+ MEP. Additional analysis also indicated that c-MPL is required for maintenance of a consistent supply of megakaryocytes and erythrocytes from MEPs. Conversely, complimentary transduction of MPL into CAMT-iPSCs using a retroviral vector restored the defective erythropoiesis and megakaryopoiesis; however, excessive MPL signaling appears to promote aberrant megakaryopoiesis with CD42b (GPIba)-null platelet generation and impaired erythrocyte production. Taken together, our findings demonstrate the usefulness of CAMT-iPSCs for validation of functionality in the human hematopoiesis system. For example, it appears that MPL is not indispensable for the emergence of HPCs, but is indispensible for their maintenance, and for subsequent MEP development. Our results also strongly indicate that an appropriate expression level of an administered gene is necessary to achieve curative gene correction / therapy using patient-derived iPSCs. Disclosures: No relevant conflicts of interest to declare.

Abstract 2984 T lymphocytes play central roles in cellular immunity, exerting their proliferative and effector activities when they recognize antigens via T-cell receptors (TCRs) in HLA-restricted and antigen-specific manner. Adoptive cell transfer therapy (ACT), the administration of ex vivo-activated and -expanded autologous tumor-reactive T lymphocytes, is currently one of the effective methods for immunotherapy, especially for treatment of metastatic solid tumors including melanoma. However, the successful applications of this method are currently limited for tumor therapies. To broaden the range of the application of ACT, we endeavored to develop easier method to obtain cells that carry antigen-specific TCR genes. For the purpose, generation of induced pluripotent stem (iPS) cells from an antigen-reactive single T lymphocyte is attractive and rewarding way. iPS cells have a capacity for unlimited self-renewal while maintaining pluripotency. These features may enable us to induce an unlimited number of T lymphocytes, especially high proliferative naïve / central memory-type T lymphocytes, showing reactivity to specific antigens. If they retain properties of naïve T lymphocytes, they may proliferate for a longer period and achieve better therapeutic effects than their peripheral blood counterparts expanded in vitro. Peripheral T lymphocytes were isolated from healthy volunteers. Then reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) were transduced into fresh or frozen / thawed T lymphocytes. T lymphocyte-derived iPS-like colonies were observed within 3 weeks and they were isolated and clonally expanded. They exhibited standard ES-like morphology, cell surface marker expression, alkaline phosphatase activity, as well as differentiation potential into various tissues related to all three germ layers. Human TCRs are encoded in four genes (TCRA, TCRB, TCRG, TCRD), which should be genetically assembled in an irreversible manner during T-lymphocyte development. This feature allowed us to retrospectively confirm that the iPS cells were generated from T lymphocyte. The TCR gene rearrangements encoded in an iPS colony were clonal for all iPS lines, indicating that each iPS colony was derived from a single T lymphocyte. Sequence analyses of TCR genes revealed whether the rearrangements were productive, and the productivity might promise the conservation of TCR genes rearrangement during the reprogramming process. Next, we tried to re-differentiate T lymphocyte derived-iPS (T-iPS) cells into T cells by co-culturing them with murine stromal cell layers (OP9 and OP9-DL1). T-cell differentiation was evidenced by the expression of T-cell markers, such as CD5, CD7, CD27, CD4, CD8, TCR α β and CD3. We obtained 33.5 ± 17.9% CD4+ CD8+ double positive (DP) cells, 6.51 ± 5.40% CD4+ CD8− single positive (SP) cells and 3.80 ± 1.28% CD4− CD8+ SP cells. They could be activated via TCR stimulation, and produce cytokines as functionally matured T lymphocytes do. The re-differentiation efficiency of T-iPS cells was higher than those of other pluripotent stem cells, such as embryonic stem (ES) cells, fibroblasts derived-iPS cells, or cord blood derived-iPS cells. Transcribed TCR mRNA sequences in re-differentiated T cells were analyzed, and they were revealed to be identical to that engraved in the pre-differentiated T-iPS cells genome in CD4+ CD8+ DP phase. However, fully matured CD4+ CD8− or CD4− CD8+ SP phase cells had several TCRA gene rearrangement patterns distinct from the original T-iPS cell's. On the other hand, TCRB gene maintained identity with the original. The variance of the sequences, especially antigen-recognition site sequences, indicated that the antigen-specificity in the original T lymphocyte might be converted during DP to SP transition process in vitro. These data indicate that functionally matured T cells were generated by re-differentiating T-iPS cells in vitro, and that re-assemble of TCRA genes could take place during SP T cell maturation process. In order to fulfill the T-iPS-mediated immunotherapy, we need to overcome the obstacle of further TCRA gene rearrangements. We think the solution lies in refinement of the re-differentiation method for controlling the expression of RAG1 and RAG2 recombinases or for inhibiting their activities. Disclosures: No relevant conflicts of interest to declare.

Abstract 490 T lymphocytes play central roles in cellular immunity, exerting their proliferative and effector activities when they recognize antigens, in HLA-restricted and antigen-specific manner, via T-cell receptors (TCRs). Successful treatment of leukemias/cancers with T-lymphocytes infusions is a direct proof that human immunity has the potential to eradicate cancers. However, continuous exposure to tumor/self antigens drives T lymphocytes into a highly exhausted state, with loss of potentials for long-term survival, proliferation, and effector functions that can end up with deletion of antigen-responding T-lymphocyte pools. Several workers have endeavored to develop clinical protocols for expanding antigen-responding T cells from the few naïve T-cell pools remaining in the patient. However, highly expanded T cells in such protocols have not proved fully effective so far, because functional losses like those in the patient occur during ex vivo manipulation. To overcome this obstacle to T-lymphocyte based immunotherapy, we endeavored to induce antigen-specific TCR-expressing T lymphocytes from induced pluripotent stem (iPS) cells, which were derived from antigen-reactive single T lymphocytes. iPS cells have a capacity for unlimited self-renewal while maintaining pluripotency. These features enabled us to induce an unlimited number of T lymphocytes, especially naïve T lymphocytes, showing reactivity to specific antigens. If they retain properties of naïve T lymphocytes, they may proliferate for a longer period and achieve better therapeutic effects than their peripheral blood counterparts expanded in vitro. Peripheral T lymphocytes were isolated from healthy volunteers. Then three reprogramming factors (OCT4, SOX2, and KLF4) and additional factors (c-MYC and/or NANOG) were transduced into fresh or frozen/thawed T lymphocytes using a retrovirus. The virus-infected T lymphocytes were then transferred onto mouse embryonic fibroblasts (MEFs) in the presence of cytokines and chemicals favorable for T-lymphocyte survival/proliferation. iPS-like colonies were observed within 3 weeks after infections. Single T lymphocyte-derived colonies were isolated and clonally expanded. They exhibited standard ES-like morphology, cell surface markers and alkaline phosphatase activity, as well as differentiation potential into various tissues related to all three germ layers. Human TCRs are encoded in four genes (TCRA, TCRB, TCRG, TCRD), which should be genetically assembled in an irreversible manner during T-lymphocyte development. This feature allowed us to retrospectively confirm the iPS cells were generated from T lymphocyte. The TCR genes rearrangement encoded in an iPS colony was single in all iPS lines, indicating that the iPS colony was derived from single T lymphocyte. Sequence analyses of TCR genes revealed whether the rearrangements were productive, and the productivity might promise the conservation of TCR genes rearrangement during the reprogramming process. Next, we tried to re-differentiate T-lymphocyte derived-iPS (T-iPS) cells into T-lineage cells by co-culturing them with murine stromal cell layers (OP9 and OP9-DL1). These T-lineage committed cells were expressed TCRab heterodimer and T-cell surface markers such as CD3. They could activate via TCR stimulation, and produce IL-2 and IFN-g as maturing T lymphocytes. The re-differentiation efficiency of T-iPS cells was higher than those of embryonic stem cells, fibroblasts derived-iPS cells, or cord blood derived-iPS cells. mRNA sequence of TCRs transcribed in re-differentiated T-lineage cells was identical to that engraved in the pre-differentiated T-iPS cells genome. The invariance of the sequence, especially antigen-recognition site sequence, indicated that the antigen-specificity in original T lymphocyte was conserved during re-differentiation process. Here we show that the conservation of the antigen-specificity encoded in TCR genes throughout induction of T-iPS cells and re-differentiation into T-lineage cells. These data suggest that further optimization of these processes for clinical application could open the door to the development of novel T-lymphocyte therapy, repeatedly supplying patient-compatible and disease-specific naïve T lymphocytes. Disclosures: No relevant conflicts of interest to declare.

Abstract 3900 Poster Board III-836 Background Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofiblosis (PMF) are pathologically related and now classified under myeloproliferative neoplasm (MPN). Subsequent studies revealed that MPN is a group of clonal hematopoietic stem cell disorders characterized by proliferation of one or more of the myeloid lineages. The somatic activating mutation in the JAK2 tyrosine kinase, JAK2V617F, is now broadly recognized as a mutation responsible for MPN (Levine R.L. and Gilliland D.G. Blood 2008). Indeed, Most of PV patients, and half of patients with ET or PMF possess this mutation. Recent studies revealed that PV phenotype can be generated in homozygous JAK2V617F transgenic mice, while ET or atypical CML-like marked leukothrombocytosis with mild myelofibrosis can be observed in heterozygous JAK2V617F mice (Tiedt et al, Blood 2008, Shide et al Leukemia 2008). These results indicate that expression levels of JAK2V617F may influence PV and ET phenotypes. On the other hand, typical PMF phenotype has not been generated by the introduction of JAK2V617F. According to the WHO criteria, PMF could be defined as “spent phase of hematopoiesis” with fibrosis formation followed by increased bone marrow cellularity as consequences of granulocytic proliferation and megakaryocyte changes with ineffective hematopoiesis. In this study, we focused on STAT5a, a direct downstream molecule of JAK2, because we previously reported that upon transplantation, purified CD34- lineage- sca-1+ c-Kit+ (CD34-KSL) hematopoietic stem cells (HSCs) transduced with constitutive active form of STAT5A acted as MPN initiating cells causing granulocytosis without erythrocytosis/thrombocytosis (Kato Y. et al, J Exp Med 2005). Based on these observations, we attempted to make PMF model through mimicking typical PMF dynamics; hyper proliferation of HSCs by the introduction of constitutive active STAT5a and following early HSC exhaustion. Materials and Methods CD34-KSL HSCs or CD34+KSL hematopoietic progenitor cells (HPCs) were purified from bone marrow (BM) of C57BL/6 (B6)-Ly5.1 mice. Then, the cells were retrovirally transduced with STAT5a wild-type (wt) or its constitutive active mutant, STAT5a(1*6). The prepared cells were used for methylcellulose assay and were transplanted into lethally irradiated B6-Ly5.2 recipient mice together with 5 × 105 B6-Ly5.1/5.2 competitor BM cells. Peripheral blood (PB) of transplanted mice was monitored biweekly for donor chimerism and lineage deviation using flow cytometry. Subsequently, histrogical analyses of bone marrow and spleen were performed to determine myelofiblosis grade and detecting extramedullar hematopoiesis. Finally, immunohistochemical staining of bone marrow with anti-TGF-b antibody was performed to detect effector cells of myelofibrosis. Results Transplantation of STAT5a (1*6) transduced HSCs resulted in generation of 57 MPN mice (total 83 mice), while no MPN mouse was obtained by STAT5a (1*6) transduced HPCs (total 12 mice). Pathological analysis revealed that majority (70%) of MPN mice had PMF phenotype as defined by leukoerythroblastosis and dacryocytosis without leukothrombocytosis. These mice with PMF phenotype showed marked splenomegaly with extramedullary hematopoiesis, and granulocytic proliferation with megakaryocyte change. In BM, granulocytic proliferation advanced to severe myelofibrosis and osteomyelosclerosis in very short period of time (4 to 8 weeks). Those mice died of hemorrhage induced by pancytopenia within a few months, much faster than the mice with JAK2V617F based PV/ET models. Immunohistological analysis revealed that dominance of Gr-1 / Mac-1 positive granulocytes and CD41 positive small megakaryocytes strongly expressing TGF-beta, a putative inducer of fibroblastosis in BM of PMF mice. Conclusion By transplanting STAT5a(1*6) transduced HSCs, we were able to develop mice with phenotype closely resembling human PMF. Because PMF is rare disease, this animal model should be useful for understanding etiology of PMF, for evaluating existing treatment, and for developing therapeutics targeting STAT5a or its downstream pathway. Disclosures: No relevant conflicts of interest to declare.

Alloantigen targeting adoptive immunotherapy is a powerful therapeutic approach for the treatment of hematologic malignancies. A compelling example is represented by donor lymphocyte infusions after allogeneic hematopoietic stem cell transplantation. The clinical efficacy of T-cell based therapy relies not only on the ability of T cells to mediate a direct anti-tumor effect (GvT) and a protective immune response against pathogens, but also in their capacity to persist and expand in vivo, providing a long-term protection from disease relapse. Despite undeniable efficacy, the extensive exploitation of donor lymphocytes is limited by the risk of a severe and potentially life-threatening complication: Graft-versus-host disease (GvHD). To solve this double bind, we investigated the therapeutic potential of donor lymphocytes retrovirally transduced to express the suicide gene thymidine kinase of Herpes Simplex virus (TK) in patients affected by hematologic malignancies, and showed that the suicide machinery controls severe GvHD. To maximize the extent and persistence of GvT activity mediated by TK cells, we exploited the positive effects of IL-7 in maintaining the homeostasis of memory lymphocytes. We observed that while stimulation with anti-CD3 antibodies and culture in the presence of high doses of IL-2 generates mainly CD45RA−CD62L− effector memory (EM) TK cells with a limited ability to engraft and persist in vivo, stimulation with anti-CD3/CD28 conjugated cell sized beads and culture with low doses of IL-7 result in the generation of CD45RA−/CD62L+ TK cells with central memory (CM) functional phenotype, producing high levels of IL-2 upon stimulation, and expressing persistent high levels of IL-2R alpha and IL-7R alpha, a molecule associated to activation and long term survival memory T-cells, respectively. In mixed lymphocytes cultures (MLR), CM TK cells showed higher proliferative potential and lower sensitivity to apoptosis than EM TK cells, and such alloreactive CM TK cells kept CCR7 and IL-7R alpha expression even after 2ndary MLR. Accordingly, CM cells were more potent than EM cells in mediating allogeneic GvHD, after infusion in NOD/Scid mice, previously transplanted with allogeneic human skin. Newly developed homeostatic CM TK cells combine a high alloreactive potential with the selective sensitivity to GCV-mediated cell death, providing a tool for maximal anti-tumor activity with control of GvHD.

T-cell depletion of allogeneic hematopoietic stem cell graft (HSCT) represents the most powerful approach to prevent graft-versus-host disease (GvHD), thus allowing to overcome HLA barriers in patients with high risk malignancies, lacking a conventional donor. We hypothesized that early add-back of suicide-gene transduced donor lymphocytes (TK cells) to leukemic patients undergoing haploidentical HSCT (haplo-HSCT) could provide early immune-reconstitution and selective control of GvHD. In a phase II clinical trial (protocol MMTK007), 17 of 29 enrolled pts, (median age 52), received add-backs of 10^7/kg TK cells 42 days after haplo-HSCT. TK cells engraftment, observed in 14 patients, was necessary and sufficient for a rapid and effective immunereconstitution (IR), with a median of 144 (101–336) CD3+, 59 (28–149) CD4+ and 86 (52–279) CD8+ cells/mcl at day 100 after HSCT. Accordingly, engraftment of TK cells was tightly correlated with clinical outcome: while 3/3 pts who failed TK cells engraftment died of infections, only 1/14 TK engrafted patients died from infections (last event at day +166). As shown in Table I, the immune repertoire of treated patients improved significantly at 6 months post transplant and normalized completely in 12 months. High numbers of T cell precursors specific for CMV and EBV were detected at immune reconstitution (median of 86 and 69 gIFN specific spots/10^5 PBMC respectively) and predicted subsequent freedom from viral reactivation (p=0.002). Six pts developed acute (GvHD), (grade I to IV) that was always completely abrogated by the suicide system. Overall survival of TK cells treated patients is 50% at three years. These results indicate that TK-DLI abolish late mortality after CD34+ haplo-SCT in adults. A phase III multicentric study will start in 2007 to validate prospectively the advantage of TK-DLI in haplo-SCT.

Suicide gene therapy (SGT) is a powerful approach to exploit anti-host reactivity following allogeneic hemopoietic cell transplantation (allo-HCT) for a full graft-versus-leukemia (GvL) effect, while controlling graft-versus-host disease (GvHD). This is accomplished through genetic modification of donor lymphocytes with a suicide gene. The herpes simplex thymidine kinase (TK) suicide gene converts at a cellular level the pro-drug ganciclovir (GCV) into tri-phosphate toxic derivatives, thence conferring selective sensitivity. Clinical studies as well as animal models have substantiated the concept that a time-wise GCV administration is able to actually separate the therapeutic GvL effect from life-threatening GvHD. Genetic modification of lymphocytes with TK is currently pursued through retroviral vectors (RV). In vitro RV genetic modification requires proliferation, which is easily accomplished by polyclonal stimulation. Polyclonal stimulation with anti-CD3 antibodies (aCD3) has been show to reduce anti-host reactivity of gene-modified lymphocytes. This possibly limits the impact of the suicide gene strategy in allo-HCT. In this study we tackled the rules governing anti-host reactivity of TK+ human lymphocytes. We found that TK+ lymphocytes generated with aCD3 are mainly CD45RA−CCR7− effector memory (EM) cells (84,6±6,6%). Upon re-stimulation they produce interferon-γ, perforin B and granzyme A, but fail to up-regulate CD40L. EM TK+ lymphocytes have a mixed phenotype in regard to CD28/CD27 co-expression and displayed a limited ability to engraft and cause GvHD in a xenogeneic model using conditioned NOD/scid mice (take: 11%). In sharp contrast, TK+ lymphocytes generated with novel protocols taking advantage of anti-CD3 and anti-CD28 antibodies conjugated to para-magnetic cell-sized beads are enriched for CD45RA−CCR7+ central memory (CM) cells (65,3±6,2%) that are able to produce IL-2 and to strongly up-regulate CD40L. CM TK+ lymphocytes are homogenously CD28+CD27+ (91,1,3±2,5%). When infused in conditioned NOD/scid mice CM TK+ lymphocytes persistently engrafted and caused lethal GvHD in a significant fraction of mice (take: 55%, P=0,0017 vs EM TK+ cells). GCV administration to diseased animals resulted in the elimination of TK+ cells in blood and in target organs. Treated animals were rescued with survival up to 120 days (P=0,009 vs saline-treated mice). These results demonstrate that CM TK+ lymphocytes retain significant anti-host reactivity and provide a clue to their in vitro generation. CM TK+ lymphocytes are promising candidates for safe and effective allo-HCT for the cure of hematological malignancies.