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IT-901 is a novel and selective NF-κB inhibitor with promising activity in pre-clinical models. Here we show that treatment of chronic lymphocytic leukemia cells (CLL) with IT-901 effectively interrupts NF-κB transcriptional activity. CLL cells exposed to the drug display elevated mitochondrial reactive oxygen species, which damage mitochondria, limit oxidative phosphorylation and ATP production, and activate intrinsic apoptosis. Inhibition of NF-κB signaling in stromal and myeloid cells, both tumor-supportive elements, fails to induce apoptosis, but impairs NF-κB-driven expression of molecules involved in cell-cell contacts and immune responses, essential elements in creating a pro-leukemic niche. The consequence is that accessory cells do not protect CLL cells from IT-901-induced apoptosis. In this context, IT-901 shows synergistic activity with ibrutinib, arguing in favor of combination strategies. IT-901 is also effective in primary cells from patients with Richter syndrome (RS). Its anti-tumor properties are confirmed in xenograft models of CLL and in RS patient-derived xenografts, with documented NF-κB inhibition and significant reduction of tumor burden. Together, these results provide pre-clinical proof of principle for IT-901 as a potential new drug in CLL and RS.

NF-κB plays a variety of roles in oncogenesis and immunity that may be beneficial for therapeutic targeting, but strategies to selectively inhibit NF-κB to exert antitumor activity have been elusive. Here, we describe IT-901, a bioactive naphthalenethiobarbiturate derivative that potently inhibits the NF-κB subunit c-Rel. IT-901 suppressed graft-versus-host disease while preserving graft-versus-lymphoma activity during allogeneic transplantation. Further preclinical assessment of IT-901 for the treatment of human B-cell lymphoma revealed antitumor properties in vitro and in vivo without restriction to NF-κB–dependent lymphoma. This nondiscriminatory, antilymphoma effect was attributed to modulation of the redox homeostasis in lymphoma cells resulting in oxidative stress. Moreover, NF-κB inhibition by IT-901 resulted in reduced stimulation of the oxidative stress response gene heme oxygenase-1, and we demonstrated that NF-κB inhibition exacerbated oxidative stress induction to inhibit growth of lymphoma cells. Notably, IT-901 did not elicit increased levels of reactive oxygen species in normal leukocytes, illustrating its cancer selective properties. Taken together, our results provide mechanistic insight and preclinical proof of concept for IT-901 as a novel therapeutic agent to treat human lymphoid tumors and ameliorate graft-versus-host disease. Cancer Res; 76(2); 377–89. ©2016 AACR.

Therapeutic T-cell engineering is emerging as a powerful approach to treat refractory hematological malignancies. Its most successful embodiment to date is based on the use of second-generation chimeric antigen receptors (CARs) targeting CD19, a cell surface molecule found in most B-cell leukemias and lymphomas. Remarkable complete remissions have been obtained with autologous T cells expressing CD19 CARs in patients with relapsed, chemo-refractory B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. Allogeneic CAR T cells may also be harnessed to treat relapse after allogeneic hematopoietic stem cell transplantation. However, the use of donor T cells poses unique challenges owing to potential alloreactivity. We review different approaches to mitigate the risk of causing or aggravating graft-versus-host disease (GVHD), including CAR therapies based on donor leukocyte infusion, virus-specific T cells, T-cell receptor–deficient T cells, lymphoid progenitor cells, and regulatory T cells. Advances in CAR design, T-cell selection and gene editing are poised to enable the safe use of allogeneic CAR T cells without incurring GVHD.

Background Even though hematopoietic stem cell transplantation can be curative in patients with severe combined immunodeficiency, there is a need for additional strategies boosting T cell immunity in individuals suffering from genetic disorders of lymphoid development. Here we show that image-guided intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγnull mice is feasible and facilitates the generation of functional T cells conferring protective immunity. Methods Hematopoietic stem and progenitor cells were isolated from the bone marrow of healthy C57BL/6 mice (wild-type, Luciferase+, CD45.1+) and injected intravenously or intrathymically into both male and female, young or aged NOD-scid IL2rγnull recipients. The in vivo fate of injected cells was analyzed by bioluminescence imaging and flow cytometry of thymus- and spleen-derived T cell populations. In addition to T cell reconstitution, we evaluated mice for evidence of immune dysregulation based on diabetes development and graft-versus-host disease. T cell immunity following intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγnull mice was assessed in a B cell lymphoma model. Results Despite the small size of the thymic remnant in NOD-scid IL2rγnull mice, we were able to accomplish precise intrathymic delivery of hematopoietic stem and progenitor cells by ultrasound-guided injection. Thymic reconstitution following intrathymic injection of healthy allogeneic hematopoietic cells was most effective in young male recipients, indicating that even in the setting of severe immunodeficiency, sex and age are important variables for thymic function. Allogeneic T cells generated in intrathymically injected NOD-scid IL2rγnull mice displayed anti-lymphoma activity in vivo, but we found no evidence for severe auto/alloreactivity in T cell-producing NOD-scid IL2rγnull mice, suggesting that immune dysregulation is not a major concern. Conclusions Our findings suggest that intrathymic injection of donor hematopoietic stem and progenitor cells is a safe and effective strategy to establish protective T cell immunity in a mouse model of severe combined immunodeficiency. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0478-z) contains supplementary material, which is available to authorized users.

Preventing unfavorable GVHD without inducing broad suppression of the immune system presents a major challenge of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We developed a novel strategy to ameliorate GVHD while preserving graft-versus-tumor (GVT) activity by small molecule–based inhibition of the NF-κB family member c-Rel. Underlying mechanisms included reduced alloactivation, defective gut homing, and impaired negative feedback on interleukin (IL)-2 production, resulting in optimal IL-2 levels, which, in the absence of competition by effector T cells, translated into expansion of regulatory T cells. c-Rel activity was dispensable for antigen-specific T-cell receptor (TCR) activation, allowing c-Rel–deficient T cells to display normal GVT activity. In addition, inhibition of c-Rel activity reduced alloactivation without compromising antigen-specific cytotoxicity of human T cells. Finally, we were able to demonstrate the feasibility and efficacy of systemic c-Rel inhibitor administration. Our findings validate c-Rel as a promising target for immunomodulatory therapy and demonstrate the feasibility and efficacy of pharmaceutical inhibition of c-Rel activity. Significance: Chemical inhibition of c-Rel diminishes alloactivation while preserving antigen-specific TCR activation, revealing the redundancy of c-Rel in T cell–mediated antitumor activity of both mouse and human T cells. Our study provides a highly innovative immunomodulatory approach that has true potential for drug development and clinical application with broad therapeutic implications, including allo-tolerance induction after allo-HSCT, as well as antitumor therapies. Cancer Discov; 4(5); 578–91. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 495

Velardi et al. show that sex steroids regulate thymopoiesis by directly modulating Notch signaling, and provide a novel clinical strategy to boost immune regeneration., Paradoxical to its importance for generating a diverse T cell repertoire, thymic function progressively declines throughout life. This process has been at least partially attributed to the effects of sex steroids, and their removal promotes enhanced thymopoiesis and recovery from immune injury. We show that one mechanism by which sex steroids influence thymopoiesis is through direct inhibition in cortical thymic epithelial cells (cTECs) of Delta-like 4 (Dll4), a Notch ligand crucial for the commitment and differentiation of T cell progenitors in a dose-dependent manner. Consistent with this, sex steroid ablation (SSA) led to increased expression of Dll4 and its downstream targets. Importantly, SSA induced by luteinizing hormone-releasing hormone (LHRH) receptor antagonism bypassed the surge in sex steroids caused by LHRH agonists, the gold standard for clinical ablation of sex steroids, thereby facilitating increased Dll4 expression and more rapid promotion of thymopoiesis. Collectively, these findings not only reveal a novel mechanism underlying improved thymic regeneration upon SSA but also offer an improved clinical strategy for successfully boosting immune function.

Keratinocyte growth factor (KGF), which is given exogenously to allogeneic bone marrow transplantation (allo-BMT) recipients, supports thymic epithelial cells and increases thymic output of naive T cells. Here, we demonstrate that this improved T-cell reconstitution leads to enhanced responses to DNA plasmid tumor vaccination. Tumor-bearing mice treated with KGF and DNA vaccination have improved long-term survival and decreased tumor burden after allo-BMT. When assayed before vaccination, KGF-treated allo-BMT recipients have increased numbers of peripheral T cells, including CD8+ T cells with vaccine-recognition potential. In response to vaccination, KGF-treated allo-BMT recipients, compared with control subjects, generate increased numbers of tumor-specific CD8+ cells, as well as increased numbers of CD8+ cells producing interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). We also found unanticipated benefits to antitumor immunity with the administration of KGF. KGF-treated allo-BMT recipients have an improved ratio of T effector cells to regulatory T cells, a larger fraction of effector cells that display a central memory phenotype, and effector cells that are derived from a broader T-cell–receptor repertoire. In conclusion, our data suggest that KGF can function as a potent vaccine adjuvant after allo-BMT through its effects on posttransplantation T-cell reconstitution.

We present a strategy for adoptive immunotherapy using T-lineage committed lymphoid precursor cells generated by Notch1-based culture. We found that allogeneic T-cell precursors can be transferred to irradiated individuals irrespective of major histocompatibility complex (MHC) disparities and give rise to host-MHC restricted and host-tolerant functional allogeneic T cells, improving survival in irradiated recipients as well as enhancing anti-tumor responses. T-cell precursors transduced to express a chimeric receptor targeting hCD19 resulted in significant additional anti-tumor activity, demonstrating the feasibility of genetic engineering of these cells. We conclude that ex vivo generated MHC-disparate T-cell precursors from any donor can be used universally for 'off-the-shelf' immunotherapy, and can be further enhanced by genetic engineering for targeted immunotherapy.

Background Neurotrophin receptor signaling regulates proliferation, differentiation and death of neuronal cells. Expression of Trk receptors has been implicated in the pathogenesis and prognosis of embryonal tumors, including neuroblastoma, nephroblastoma, and medulloblastoma. Procedure We analyzed TrkA, TrkB, TrkC, and p75 expression using semi-quantitative RT-PCR in 23 retinoblastomas and 8 retinoblastoma cell lines. Comparison of mRNA expression with clinical variables as well as the proliferation (PI) and apoptotic index (AI) of the tumor, was performed by Pearson correlation analysis and two-sample t-test. Results Almost all tumor samples and cell lines demonstrated high expression of all Trk receptors. Expression of TrkB and its ligand, BDNF, was most pronounced, suggesting TrkB to be the major Trk receptor involved in retinoblastoma biology. In contrast, p75 expression was substantially reduced in a subset of tumors and cell lines, in particular compared to its expression in normal retina. Tumors with infiltrative growth demonstrated significantly lower relative levels of TrkC expression than localized tumors (P = 0.004). High expression of TrkA was associated with a higher AI (P = 0.04), and high expression of TrkC was associated with a younger age of the patients (P = 0.03). Inhibition of Trk signaling by K252a resulted in marked growth inhibition of retinoblastoma cells in vitro. Conclusions Our findings suggest a role for neurotrophin signaling in the biology of retinoblastoma. General Trk inhibitors are effective in decreasing growth rates of retinoblastoma cells in vitro, and should be evaluated in in vivo studies. Pediatr Blood Cancer 2008;50:218–222. © 2007 Wiley-Liss, Inc.

Hematopoietic stem cells in the bone marrow give rise to lymphoid progenitors, which subsequently differentiate into B and T lymphocytes. Here we show that the proto-oncogene LRF plays an essential role in the B versus T lymphoid cell-fate decision. We demonstrate that LRF is key for instructing early lymphoid progenitors in mice to develop into B lineage cells by repressing T cell–instructive signals produced by the cell-fate signal protein, Notch. We propose a new model for lymphoid lineage commitment, in which LRF acts as a master regulator of the cell's determination of B versus T lineage.

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell-depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1-derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1-derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1-derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1-derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.

T-cell deficiency related to disease, medical treatment, or aging represents a major clinical challenge and is associated with significant morbidity and mortality in cancer and bone marrow transplantation recipients. This study describes several innovative and clinically relevant strategies to manipulate thymic function based on an interventional radiology technique for intrathymic injection of cells or drugs. We show that intrathymic injection of multipotent hematopoietic stem/progenitor cells into irradiated syngeneic or allogeneic young or aged recipients resulted in efficient and long-lasting generation of functional donor T cells. Persistence of intrathymic donor cells was associated with intrathymic presence of cells resembling long-term hematopoietic stem cells, suggesting a self-renewal capacity of the intrathymically injected cells. Furthermore, our approach enabled the induction of long-term antigen-specific T-cell–mediated antitumor immunity following intrathymic injection of progenitor cells harboring a transgenic T-cell receptor gene. The intrathymic injection of interleukin-7 prior to irradiation conferred radioprotection. In addition, thymopoiesis of aged mice improved with a single intrathymic administration of low-dose keratinocyte growth factor, an effect that was sustained even in the setting of radiation-induced injury. Taken together, we established a preclinical framework for the development of novel clinical protocols to establish lifelong antigen-specific T-cell immunity.

Regenerative therapies that use allogeneic cells are likely to encounter immunological barriers similar to those that occur with transplantation of solid organs and allogeneic hematopoietic stem cells (HSCs). Decades of experience in clinical transplantation hold valuable lessons for regenerative medicine, offering approaches for developing tolerance-induction treatments relevant to cell therapies. Outside the field of solid-organ and allogeneic HSC transplantation, new strategies are emerging for controlling the immune response, such as methods based on biomaterials or mimicry of antigen-specific peripheral tolerance. Novel biomaterials can alter the behavior of cells in tissue-engineered constructs and can blunt host immune responses to cells and biomaterial scaffolds. Approaches to suppress autoreactive immune cells may also be useful in regenerative medicine. The most innovative solutions will be developed through closer collaboration among stem cell biologists, transplantation immunologists and materials scientists.

Previous studies demonstrated selective inhibition of the BCR-ABL (breakpoint cluster region-Abelson murine leukemia oncogene) tyrosine kinase by RNA interference in leukemic cells. In this study, we evaluated the effect of BCR-ABL small interfering RNA (siRNA) and GFI1B siRNA silencing on chronic myeloid leukemia (CML) cells in myeloid blast crises. The GFI1B gene was mapped to chromosome 9 and is, therefore, located downstream of the BCR-ABL translocation in CML cells. Co-transfection of BCR-ABL siRNA and GFI1B siRNA dramatically decreased cell viability and significantly induced apoptosis and inhibited proliferation in K562 cells (P

BACKGROUND: Tumor cell survival critically depends on heterotypic communications with non-malignant cells in the microenvironment. Most of these signals converge on the activation of the transcription factor NF-κB that regulates complex cellular functions, including apoptosis, cell survival and proliferation. Even if NF-kB is constitutively active in most malignancies, including chronic lymphocytic leukemia (CLL), and plays a major role in tumorigenesis, there are no currently approved drugs to target it. IT901 has been recently reported as a novel NF-kB inhibitor, showing efficacy in a non-tumor context1. AIM OF THE WORK: The aim of this work is to test the efficacy of IT901 in CLL and in its more aggressive transformation, Richter syndrome (RS), which represents an unmet therapeutic need. The molecular mechanisms of action of IT901 in leukemic cells are studied, alongside its effects on cells belonging to CLL microenvironment. RESULTS: IT901 induces apoptosis in primary leukemic cells in a dose- and time-dependent manner, showing significant efficacy after 24h of treatment. The apoptotic response is independent of the prognostic subgroup. Conversely, IT901 has minimal impact upon normal B cells. Treatment of CLL cells with IT901 interferes with NF-kB transcriptional activity, resulting in a diminished binding of both p50 and p65 to DNA. Moreover, biochemical analyses indicate a diminished expression of these subunits in the nucleus, as well as of the whole NF-kB complex in the cytoplasm. At the molecular level, compromised expression of NF-kB triggers activation of the Caspase-3 apoptotic pathway, with increased expression of pro-apoptotic proteins (e.g., Bim), paralleled by a diminished expression of the anti-apoptotic ones (e.g., XIAP). Concomitantly, a prominent increase in mitochondrial ROS is evident, providing a link between IT901 effects and induction of apoptosis. Recent data reported the involvement of NF-kB as a transcriptional controller of metabolic pathways promoting oxidative phosphorylation in cancer cells. In line with NF-kB constitutive activation in CLL, dynamic measurement of the energetic profile, indicates a reliance on oxidative phosphorylation, with limited glycolytic capacity. After IT901 treatment, there is a dramatic drop in mitochondrial respiration, with compromised ATP production and a net increase in proton leak, suggesting that primary CLL cells are trying to compensate impaired respiration by shifting to glycolysis. This metabolic response is mediated at the transcriptional levels, as IT901 induces a down-modulation of the genes involved in mitochondrial respiration (e.g., ATP5A1) and a concomitant up-modulation of the ones involved in glucose uptake and lactate transport (e.g., GLUT1). The CLL microenvironment is critical for disease progression and for providing protection from drug-induced apoptosis. Therefore it is important to consider the effects of novel drugs also on non-neoplastic bystander elements. Nurse-like cells (NLC) are a population of monocyte-derived activated macrophages that nurtures CLL cells via soluble and cell contact dependent mechanisms. These interactions are known to activate NF-kB signaling in both partners. Consistently, IT901 inhibited nuclear localization of the p65 subunit in NLC and shifted their polarization towards an M1-phenotype. These results are confirmed using a xenograft model. The Mec-1 cell line was injected into NSG mice and left to engraft for 2 weeks before beginning treatment. Animals treated with IT901 are characterized by decreased tumor growth and leukemic cells diffusion compared to controls, as shown by a diminished number of leukemic cells in kidneys, liver and spleen. Finally, IT901 shows promising effects in a small cohort of leukemic cells obtained from RS patients, inducing significant apoptosis by interfering with the expression and nuclear localization of NF-kB. CONCLUSIONS: Altogether, these results indicate that IT901 blocks NF-kB transcriptional activity. This effect is followed by rapid and marked decrease in genes supporting oxidative phosphorylation, causing mitochondrial damage, ROS release and induction of intrinsic apoptosis. Moreover, IT901 interrupts the support that CLL obtains from the microenvironment. Thus, targeting NF-kB by means of IT901 may be effective for CLL, and possibly even RS patients. 1. Y. Shono et al., Cancer Res76, 377 (Jan 15, 2016). Disclosures Furman: Genentech: Consultancy; Janssen: Consultancy; Abbvie: Consultancy, Honoraria; Gilead Sciences: Consultancy; Pharmacyclics: Consultancy, Speakers Bureau.

Alloreactive T cells are crucial for graft-versus-host disease (GVHD) pathophysiology, and modulating their trafficking patterns has been efficacious in ameliorating experimental disease. We report in this paper that P-selectin, a glycoprotein found on resting and inflamed endothelium, is important for donor alloreactive T cells trafficking into GVHD target organs, such as the intestines and skin. Compared with wild-type (WT) recipients of allogeneic bone marrow transplantation, P-selectin−/− recipients exhibit decreased GVHD mortality and decreased GVHD of the skin, liver, and small bowels. This was associated with diminished infiltration of alloactivated T cells into the Peyer’s patches and small bowels, coupled with increased numbers of donor T cells in the spleen and secondary lymphoid organs (SLOs). Surprisingly, however, donor T cells deficient for P-selectin glycoprotein ligand 1, the most well described P-selectin ligand, mediated GVHD similar to WT T cells and accumulated in SLO and target organs in similar numbers as WT T cells. This suggests that P-selectin may be required for trafficking into inflamed tissues but not SLO and that donor T cells may use multiple P-selectin ligands apart from P-selectin glycoprotein ligand 1 to interact with P-selectin and traffic into inflamed tissues during GVHD. We conclude that targeting P-selectin may be a viable strategy for GVHD prophylaxis or treatment.

Graft-versus-host disease (GVHD) is a serious complication of allogeneic bone marrow transplantation, and donor T cells are indispensable for GVHD. Current therapies have limited efficacy, selectivity, and high toxicities. We used a novel flow cytometry technique for the analysis of intracellular phosphorylation events in single cells in murine BMT models to identify and validate novel GVHD drug targets.1-7 This method circumvents the requirement for large numbers of purified cells, unlike western blots. We defined a signaling profile for alloactivated T cells in vivo and identified the phosphorylation of ERK1/2 and STAT-3 as important events during T-cell (allo)activation in GVHD. We establish that interference with STAT-3 phosphorylation can inhibit T-cell activation and proliferation in vitro and GVHD in vivo. This suggests that phospho-specific flow cytometry is useful for the identification of promising drug targets, and ERK1/2 and STAT-3 phosphorylation in alloactivated T cells may be important for GVHD.

Strategies to enhance T cell recovery are of increasing clinical importance to overcome long lasting T cell deficiencies, which occur in association with infections, autoimmunity and chemo/radiotherapy as well as aging of the immune system. In this review we discuss those strategies that are close to or in the clinic. Interleukin-7, sex steroid modulation, keratinocyte growth factor, growth hormone and cellular therapies using ex vivo generated T-cell precursors are currently being tested in recipients of a hematopoietic stem cell transplantation and patients with malignancies or HIV/AIDS.

Hematopoietic stem cell transplantation (HSCT) is associated with a period of immune incompetence that particularly affects the T cell lineage. Strategies to enhance T cell reconstitution could significantly improve the survival of HSCT recipients by decreasing the incidence of fatal infectious complications and by enhancing graft-versus-tumor activity. In recent years, a variety of promising strategies have been established in preclinical models to improve T cell recovery in particular after allogeneic T cell-depleted HSCT, without aggravating graft-versus-host disease while preserving or even improving graft-versus-tumor activity. These therapies include treatment with keratinocyte growth factor (KGF), growth hormone (GH), LHRH agonists, interleukin 7 (IL-7) and interleukin 15 (IL-15). Thanks to the establishment of Notch-based culture systems, adoptive cellular therapies with T lineage-committed precursor cells have become feasible, since early T cell progenitors can now easily be generated in vitro in large quantities and have been proven to be very effective in enhancing T cell reconstitution and anti-tumor activity after allogeneic T cell-depleted HSCT. The translation of most of these strategies into clinical trials is likely and in some cases Phase I/II studies are already underway.

To investigate the molecular effects of growth factor independence 1B (Gfi-1B), a transcription factor essential for the development of hematopoietic cells and differentiation of erythroid and megakaryocytic lineages, the naturally Gfi-1B overexpressing cell line K562 was cultured in the presence of Gfi-1B target-specific small interfering RNA (siRNA). SiRNA treatment significantly knocked down Gfi-1B expression with an efficiency of nearly 90%. Analysis of the siRNA silencing protocol by colony-forming units ensured that it was not cytotoxic. Samples from Gfi-1B overexpressing cells and cells with knocked-down Gfi-1B were analyzed by oligonucleotide microarray technology and based upon rigorous statistical analysis of the data; relevant genes were chosen for confirmation by reserve transcriptase-polymerase chain reaction, including MYC/MYCBP and CDKN1A. Interestingly, transcripts within components of the signalling cascade of immune cells (PLD1, LAMP1, HSP90, IL6ST), of the tyrosine kinase pathway (TPR, RAC3) and of the transcription factors (RAC3, CEP290, JEM-1, ATR, MYC, SMC3, RARA, RBBP6) were found to be differentially expressed in Gfi-1B overexpressing cells compared to controls. Individual genes such as ZDHHC17, DMXL1, ZNF292 were found to be upregulated in Gfi-1B overexpressing cells. In addition, down-regulated transcripts showed cell signaling transcripts for several chemokine gene members including GNAL, CXCL5, GNL3L, GPR65, TMEM30, BCL11B and transcription factors (GTF2H3, ATXN3). In conclusion, several essential cell signalling factors, as well as transcriptional and post-translational regulation genes were differentially expressed in cells that overexpressed Gfi-1B compared to control cells with knocked-down Gfi-1B. Our data indicate that Gfi-1B signalling is important for commitment and maturation of hematopoietic cell populations.

Strategies to enhance post-transplant immune reconstitution without aggravating graft-vs-host disease (GVHD) can improve the outcome of allogeneic hematopoietic stem cell transplantation. Recent preclinical studies demonstrated that the use of T cell depleted allografts supplemented with committed progenitor cells (vs stem cells only) allows enhanced immune reconstitution of specific hematopoietic lineages including myeloid, B, T, and natural killer lineages in the absence of GVHD. This novel adoptive therapy resulted in significantly improved resistance to microbial pathogens and could, in some cases, even mediate tumor immunity. Clinical protocols using adoptive transfer of committed hematopoietic progenitor cells are currently being evaluated.

Growth factor-independent 1B (GFI1B) is a transcription factor essential for the development and differentiation of erythroid and megakaryocytic lineages. We evaluated the GFI1B expression in erythroleukaemia and megakaryocytic leukaemia, as well as in patients with other subtypes of acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), myelodysplastic syndrome (MDS), severe aplastic anaemia (SAA), myelofibrosis with myeloid metaplasia (MMM) and in healthy volunteers. GFI1B expression was increased at least threefold in patients with erythroleukaemia (P < 0.01 compared with controls) and megakaryocytic leukaemia (P < 0.05) as well as in their corresponding leukaemic cell lines HEL, K562, CMK and M-07e. Patients with undifferentiated or monocytic AML, ALL, MMM, MDS and CML had no significantly altered GFI1B expression, whereas GFI1B expression was decreased 10-fold in patients with SAA (P < 0.0001 compared with controls). Silencing GFI1B by transfection with small interfering RNA (siRNA) markedly reduced the proliferation rate in the leukaemic cell lines HEL, K562 and NB4 (P < 0.01). Concomitantly, we observed a two- to threefold increase in the apoptosis rate in these cells after transfection with siRNA towards GFI1B. Our data indicate that GFI1B plays a major role in AML-M6 and AML-M7 and qualifies as a target for anti-leukaemic strategies in these malignancies.

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Migration of donor-derived T cells into GVHD target organs plays a critical role in the development of GVHD and chemokines and their receptors are important molecules involved in this process. Here, we demonstrate in murine bone marrow transplantation models that the expression of the inflammatory CC chemokine receptor 2 (CCR2) on donor-derived CD8+ T cells is relevant for the control of CD8+ T-cell migration and development of GVHD. Recipients of CCR2-deficient (CCR2-/-) CD8+ T cells developed less damage of gut and liver than recipients of wild-type CD8+ T cells, which correlated with a reduction in overall GVHD morbidity and mortality. Assessment of donor CD8+ T-cell target organ infiltration revealed that CCR2-/- CD8+ T cells have an intrinsic migratory defect to the gut and liver. Other causes for the reduction in GVHD could be excluded, as alloreactive proliferation, activation, IFN-γ production and cytotoxicity of CCR2-/- CD8+ T cells were intact. Interestingly, the graft-versus-tumor effect mediated by CCR2-/- CD8+ T cells was preserved, which suggests that interference with T-cell migration by blockade of CCR2 signaling can separate GVHD from GVT activity.

Transplanting donor cells into lymph nodes regenerates liver, pancreas and thymus function in mice.

Thymopoiesis is a complex process involving crosstalk between developing thymocytes and the non-hematopoietic stromal microenvironment, which includes thymic epithelial cells (TECs), fibroblasts and endothelial cells (ECs). Despite its importance, the thymus is exquisitely sensitive to cellular insults, including cytoreductive chemo- and radiation therapy required for successful hematopoietic stem cell transplantation; therefore identification of thymic repair mechanisms will offer promising therapeutic targets for immune regeneration. Recent studies in tissues such as liver, lung and bone marrow have revealed that ECs not only passively deliver oxygen and nutrients to tissues, but also actively produce distinct paracrine factors that can orchestrate their repair. The role of thymic ECs in thymopoiesis beyond their contribution of local circulation and the importation of lymphoid progenitors has not been comprehensively studied. In order to evaluate the role of ECs in thymic regeneration, we closely examined the kinetics of thymic recovery following total body irradiation (TBI, 550cGy) in young C57BL/6 mice. Although we observed a dramatic decline in total thymic cellularity, we found no significant change in the number of thymic ECs, suggesting they are extremely radio-resistant (Fig. 1A). Interestingly, although thymic ECs appeared to be resistant to the cytoreductive effects of TBI, we revealed an increase in their proliferation as measured by expression of Ki67 shortly after injury, indicating a role in the endogenous regeneration of the thymus (Fig. 1B). Given their radioresistance and cycling after TBI, we hypothesized that thymic ECs can provide instructive signals supporting thymic regeneration. To explore potential regenerative signals stemming from ECs during thymic regeneration, we performed a transcriptome analysis of highly purified ECs of untreated mice and at days 4 and 7 following TBI. Among the 288 genes that were altered in ECs after TBI (131 upregulated and 157 downregulated), we found significant upregulation in the expression of BMP4 (which has previously been implicated in thymic regeneration) and was validated using quantitative PCR (Fig 1C). In addition, we have developed a novel light sheet fluorescence microscopy approach to visualize the thymic vasculature in 3D at high resolution (Fig. 1D). Consistent with their potential role in aiding thymic regeneration, administration of ex vivo expanded thymic ECs (ex-EC) could significantly enhance thymic regeneration when given after TBI. Intriguingly, this regenerative effect of ex-ECs was tissue specific as ex-EC derived from heart or kidney did not exhibit a similar regenerative effect (Fig. 1E). Similar to our findings in the endogenous regeneration setting, we found that thymic ex-ECs demonstrated a significantly higher BMP4 expression compared to cardiac and kidney ex-ECs (Fig. 1F). Previous reports have suggested that BMP4 signaling is capable of directly regulating the key transcription factor of TEC function, FOXN1. We found that incubation of the TEC cell line C9 with thymic ex-EC conditioned medium induced an increase in FOXN1 expression but was abrogated in the presence of Noggin, a potent BMP signaling inhibitor (Fig. 1G). These findings support the hypothesis that BMP4 mediates the regenerative effect of ECs in thymic regeneration. In summary, we found that thymic ECs are capable of mediating endogenous thymic regeneration, likely via their expression of BMP4. Adoptive transfer of ex-ECs leads to enhanced thymic regeneration after immune injury. Thus, adoptive transfer of thymic ECs represents a novel strategy to improve immune function in immunocompromised patients. Figure 1 Figure 1. Disclosures Ginsberg: Angiocrine Bioscience: Employment. Rafii:Angiocrine Biosciences: Founder Other.

NF-kB plays important roles in immunity and oncogenesis, indicating that therapeutic targeting of this pathway could be beneficial in various clinical settings; however,an NF-kB-specific inhibitor does not exist in clinical practice to date. One approach toward development of such a compound is small-molecule-mediated direct inhibition of one or several members of the NF-kB family of transcription factors, a network that comprises five structurally related proteins including p50, p52, RelA, RelB and c-Rel. After screening of a library of 15,000 small molecules with a biochemical assay, we identified two scaffolds with inhibitory activity specific for the NF-kB subunit c-Rel. These scaffolds act as direct c-Rel inhibitors by modifying the conformation of the c-Rel protein, thus preventing DNA binding. We previously reported that in vitro treatment of T cells with the thiohydantoin IT-603 induces c-Rel deficiency, resulting in suppression of T cell alloactivation without compromising T cell activation triggered by recognition of tumor-associated or viral antigens (Shono et al., Cancer Discovery, 2014). Here, we for the first time demonstrate in vivo efficacy of a c-Rel inhibitor treatment regimen in mouse models of graft-versus-host disease (GVHD) and graft-versus-lymphoma (GVL), as well as xenograft models of human B cell lymphomas, revealing that inhibition of c-Rel activity allows not only for suppression of GVHD while retaining GVL activity, but it also mediates promising anti-lymphoma effects. We first show that the novel small molecule IT-901 is a more potent c-Rel inhibitor than IT-603 and has a superior pharmacokinetic profile. IT-901 displayed significantly improved in vivo efficacy, ameliorating GVHD while preserving the anti-lymphoma activity of T cells (Figure 1a,b). Recent genetic evidence has established a pathogenetic role for NF-kB signaling in lymphoid malignancies. We therefore sought to explore the potential of IT-901 for targeted therapy of human lymphomas. We analyzed six representative diffuse large B cell lymphoma (DLBCL) cell lines including activated B-like (ABC; HBL1, TMD8, U2932) and germinal center B-like (GCB; Ly19, SU-DHL4, SU-DHL8) cell lines for nuclear translocation of c-Rel and found that c-Rel was constitutively active in all cell lines. To examine if c-Rel inhibition with IT-901 alters cytokine production by DLBCL cells, we analyzed cytokine levels in the supernatant after in vitro incubation with IT-901. IT-901 treatment resulted in decreased levels of a wide range of cytokines in TMD8 cells, with the notable exceptions of interleukin 8 (IL-8), tumor necrosis factor (TNF)-α, and TNF-β (P We next analyzed primary lymphoma cells and found that the c-Rel gene is widely expressed in human B cell malignancies and frequently amplified in DLBCL and EBV-transformed B cells. Importantly, intranuclear analysis of the c-Rel protein demonstrated that this transcription factor can be constitutively active in a wide range of human lymphomas. IT-901 efficiently inhibited growth of EBV-transformed B cells in vitro, and mediated significant anti-lymphoma activity in a xenograft model of EBV-induced lymphoma (P In summary, our findings underscore multiple therapeutic benefits and great potential for clinical translation of a novel c-Rel inhibitor. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Abstract 454 Allogeneic hematopoietic stem cell transplantation (HSCT) was initially developed as a rescue from therapy-related bone marrow failure following high dose chemo/radiation therapy to rescue from therapy-related bone marrow failure, the emphasis has shifted towards allogeneic HSCT as a strategy to facilitate graft-versus-tumor (GVT) activity. Strategies to suppress graft-versus-host diesase (GVHD) are often associated with broader suppression of the immune system leading to immune deficiency and compromised GVT activity. The aim of this study was to modulate T cell responses in the context of GVHD by targeting the NF-kappaB family member c-Rel, a transcription factor that upon antigen receptor triggering regulates lymphocyte survival and proliferation. In T cells, the main target gene of c-Rel is interleukin 2 (IL-2), a cytokine required for normal T cell proliferative and differentiative responses. The effect of c-Rel deficiency on T cell differentiation and function has been investigated previously demonstrating that c-Rel deficient T cells show reduced Th1, Treg and Th17 but normal Th2 responses. Inhibition of c-Rel is therefore expected to impair T cell activation, differentiation, and function. Using methods to inhibit the c-Rel pathway we developed a novel strategy to minimize the severity of GVHD while leaving GVT activity intact. We investigated the role of c-Rel during GVHD in MHC mismatched as well as MHC matched, minor antigen mismatched (more clinically relevant) murine HSCT models. c-Rel−/− T cells caused significantly less GVHD than normal T cells, as determined by survival (p We next evaluated if c-Rel−/− T cells were able to mediate antitumor activity. We challenged allogeneic HSCT recipients with either a liquid tumor (A20 lymphoma) or a solid tumor (RENCA-renal cell carcinoma) and found that GVT activity in c-Rel−/− T cells recipients was intact in the absence of GVHD, resulting in significantly improved survival compared to recipients of wildtype T cells (p The potential for clinical translation of our approach is highlighted by a series of experiments testing the efficacy of a recently developed Pyrimidinetrione-based small molecule c-Rel inhibitor compound. We found that incubation of T cells with this compound resulted in efficient downregulation of c-Rel expression as well as IL-2 expression in vitro. When we administered T cells that were pre-incubated with this c-Rel antagonizing small molecule, we were able to reproduce our above described findings regarding the effect of c-Rel deficient T cells on GVHD (Figure 1). Importantly, normal T cells pre-incubated with a c-Rel antagonist mediated intact GVT activity, indicating that they are viable and functional T cells. This strongly suggests that the diminished capacity of these cells to induce GVHD is due to the c-Rel inhibitory mechanism of the small molecule compound and not due to a non-specific cytotoxic effect of the in vitro manipulation procedure. Taken together, our findings identify c-Rel as a promising target for the development of a clinical strategy to prevent or ameliorate GVHD while preserving GVT activity. Disclosures: No relevant conflicts of interest to declare.

T cell deficiencies can occur in many settings, including aging, autoimmune and genetic disorders, hematological malignancies, infectious diseases, and exposure to cytotoxic/cytostatics agents. Notch-based culture systems can be utilized for ex vivo generation of large numbers of T lineage committed lymphoid precursor cells, and we recently reported that the adoptive transfer of T cell precursors significantly enhances T cell reconstitution and function after allogeneic T cell depleted HSCT. The aim of this study was to evaluate if allogeneic T cell precursors are safe and effective when used for adoptive transfer across MHC barriers in the absence of allogeneic HSCs to overcome radiation injury, enhance T cell function and improve anti-tumor activity in immunosuppressed recipients. We found that positive selection of adoptively transferred allogeneic (C57BL/6) precursor cells ± syngeneic (BALB/c) HSCs in irradiated hosts (BALB/c) is dependent on MHC molecules on non-hematopoietic host cells, resulting in the in vivo development of host-MHC restricted allogeneic T cells. We demonstrate that negative selection of these cells is mediated by donor-derived antigen presenting cells (APCs), resulting in host-tolerance. Adoptively transferred allogeneic T cell precursors significantly improved survival of BALB/c mice after irradiation (675 cGy) and enhanced anti-tumor activity against liquid (A20 lymphoma) and solid (renal cell carcinoma) tumors in syngeneic HSCT recipients. Furthermore, we demonstrate the feasibility of genetic engineering of antigen-specific T cell precursors, by transducing them to express a chimeric antigen receptor (CAR) targeting human CD19. Transduction efficiencies were routinely in the range of 50%–70%. Adoptive transfer of CAR-expressing T cell precursors resulted in the in vivo generation of high numbers of appropriately selected CAR-expressing T cells with significantly enhanced anti-tumor activity (compared with CAR-negative T cell precursors) against a CAR-sensitive tumor, but without any undesirable auto/alloreactivity. We conclude that adoptively transferred allogeneic T cell precursors develop into host-MHC restricted and host-tolerant T cells characterized by selection of a functional TCR repertoire even in a fully mismatched thymic epithelial MHC environment. This strategy overcomes important limitations of conventional adoptive T cell therapies: rejection, alloreactivity and impaired antigen recognition due to restriction to MHC disparate from the one expressed on APCs. The use of allogeneic instead of autologous cells eliminates the risk of contamination with residual malignant patient cells and allows the generation and storage of virtually unlimited quantities of precursor cells for ‘off-the-shelf’ immunotherapy. This procedure has not only substantial logistic advantages, but it facilitates ex vivo manipulation, in particular genetic engineering, to generate antigen-specific or otherwise enhanced designer cells. Adoptive transfer of MHC mismatched and genetically enhanced T cell precursors therefore represents a promising novel strategy for targeted ‘off-the-shelf’ immunotherapy in immunosuppressed patients.

Graft-versus-host-disease (GVHD) is a serious complication of allogeneic bone marrow transplantation (allo-BMT). The physiology of GVHD is dominated by alloactivated donor T cells, yet current treatments are often nonspecific and offer limited efficacy with relatively high toxicities. We screened for novel drug targets in alloactivated T cells in a murine allo-BMT model using a flow cytometric technique for the in vivo analysis of intracellular signaling. We defined the signaling profile of normal T cells and alloreactive T cells during GVHD, focusing on pathways involved in T cell receptor (TCR), costimulatory, and cytokine signaling. This analysis revealed that although proteins in multiple pathways (MAP kinases, PI3K, Jak/STAT signaling) were all heavily phosphorylated in alloactivated T cells, phosphorylation of STAT-3 and ERK1/2 were particularly prominently increased in donor alloactivated CD4 T cells. We further analyzed the importance of STAT-3 and ERK1/2 signaling in alloactivated T cells via the use of small-molecule inhibitors of STAT-3 (curcurbitacin E/I) and ERK1/2 phosphorylation (SL327). Treatment with these inhibitors attenuated T cell proliferation in response to anti-CD3+CD28 stimulation and in mixed leukocyte reactions in vitro in a dose-dependent fashion (figure 1). Figure 1 Figure 1. Pre-incubation of donor splenocytes with cucurbitacin E significanly reduced T cell activation (CD25, CD69) at 24 hours in adoptive transfer experiments in vivo (p Figure 2 Figure 2. We conclude that flow cytometric analysis of signaling pathways in single cells represents a novel methodology to assess the in vivo signaling profiles of specific cell populations in order to select drug targets for further study. STAT-3 and ERK1/2 phosphorylation may also represent potential targets to selectively inhibit donor T cell alloactivation and proliferation in GVHD.

Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with a varying period of immunoincompetence that particularly affects he T cell lineage resulting in significant morbidity and mortality from opportunistic infections. Recent studies have shown that murine T cells and their precursors can be generated from hematopoietic stem cells (HSC) in vitro using a OP9-DL1 coculture system consisting of OP9 bone marrow stromal cells expressing the Notch 1 ligand Delta-like 1 and growth factors (interleukin 7 and fms-like tyrosine kinase-3 ligand). In this study we determined the effects of adoptively transferred in vitro generated T cell precursors on T cell reconstitution after allogeneic HSCT. We selected HSC (Lin- Sca-1hi c-kithi) from bone marrow (BM) of C57BL/6 mice and cultured these cells on a monolayer of OP9-DL1 cells in the presence of growth factors. These HSC expanded 2,000–5,000-fold within 3–4 weeks and consisted of >95% CD4-CD8-double negative (DN) T cell precursors after 16–28 days of culture. We infused these cells (8x106) with T cell depleted (TCD) BM (5x106) or purified HSC into allogeneic recipients using minor antigen mismatched and MHC class I/II mismatched transplant models. Control mice received TCD BM or purified HSC only. Progeny of OP9-DL1 derived T cell precursors were found in thymus and spleen increasing thymic cellularity and significantly improving thymic and splenic donor T cell chimerism. This effect was even more pronounced when purified HSC instead of whole BM were used as allograft. T cell receptor repertoire and proliferative response to foreign antigen (determined by third party MLR) of in vivo differentiated OP9-DL1 derived mature T cells were intact. Administration of in vitro generated T cell precursors did not induce graft-versus-host disease (GVHD) but mediated significant graft-versus-tumor (GVT) activity (determined by in vivo bioluminescence imaging) resulting in a subsequent significant survival benefit. This advantage was associated with better cytokine responses (IL-2, INF-g, TNF-a) in T cells originating from OP9-DL1 derived T cell precursors compared to BM donor derived T cells. We conclude that the adoptive transfer of OP9-DL1 derived T cell precursors significantly enhances post-transplant T cell reconstitution and GVT activity in the absence GVHD.