NOD-scid IL2Rγ Research Articles

Oxazolone and ethanol induce colitis in non-obese diabetic-severe combined immunodeficiency interleukin-2Rγnull mice engrafted with human peripheral blood mononuclear cells

Oxazolone-induced colitis in mice has become a recognized model to study the efficacy of therapeutics targeting the immunological response underlying the development of inflammatory bowel disease. However, this model cannot be used when therapeutics designed to address human targets do not interact with the respective murine counterpart. In this study, we examined the induction of oxazolone mediated colitis in non-obese diabetic-severe combined immunodeficiency interleukin-2Rγ(null) (NOD-SCID IL2Rγ(null)) mice engrafted with human peripheral blood mononuclear cells (hPBMC) derived from patients suffering from ulcerative colitis (UC), atopic dermatitis (AD) and healthy volunteers. NOD-SCID IL2Rγ (null) mice were engrafted with hPBMC followed by challenge with oxazolone or ethanol vehicle. Mice developed the same symptoms as observed previously in immunocompetent mice. The clinical activity score increased and the colon architecture was characterized by the development of oedema, fibrosis, crypt loss and dense infiltration of predominantly T cells into the lamina propria. Fluorescence activated cell sorter (FACS) analysis of lymphocytes in the colon identified natural killer (NK) T cells as a major constituent. In contrast to studies with immunocompetent mice, we observed the same phenotype in the group challenged with ethanol vehicle. The phenotype was most pronounced in mice engrafted with PBMC derived from a patient suffering from UC, suggesting that the immunological history of the donors predisposes the engrafted mice to react to ethanol. The model described here has the potential to study the efficacy of therapeutics targeting human lymphocytes in a model which is more reflective of the human disease. In addition, it might be developed to elucidate molecular mechanisms underlying the disease.

Site-specific Genome Editing in PBMCs With PLGA Nanoparticle-delivered PNAs Confers HIV-1 Resistance in Humanized Mice

Biodegradable poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) encapsulating triplex-forming peptide nucleic acids (PNAs) and donor DNAs for recombination-mediated editing of the CCR5 gene were synthesized for delivery into human peripheral blood mononuclear cells (PBMCs). NPs containing the CCR5-targeting molecules efficiently entered PBMCs with low cytotoxicity. Deep sequencing revealed that a single treatment with the formulation resulted in a targeting frequency of 0.97% in the CCR5 gene and a low off-target frequency of 0.004% in the CCR2 gene, a 216-fold difference. NP-treated PBMCs efficiently engrafted immunodeficient NOD-scid IL-2rγ-/- mice, and the targeted CCR5 modification was detected in splenic lymphocytes 4 weeks posttransplantation. After infection with an R5-tropic strain of HIV-1, humanized mice with CCR5-NP–treated PBMCs displayed significantly higher levels of CD4+ T cells and significantly reduced plasma viral RNA loads compared with control mice engrafted with mock-treated PBMCs. This work demonstrates the feasibility of PLGA-NP–encapsulated PNA-based gene-editing molecules for the targeted modification of CCR5 in human PBMCs as a platform for conferring HIV-1 resistance.

Targeting Leukemia Initiating Cells by MUC1-C Subunit Inhibition

AbstractAbstract 3583 Introduction:Acute myeloid leukemia (AML) arises from a population of leukemia initiating cells (LICs) that exhibit chemotherapy resistance and serve as a reservoir for disease recurrence. We have demonstrated that the oncoprotein, MUC1, is expressed on LICs but not normal hematopoietic stem cells. We have developed an inhibitor of the MUC1-C subunit, designated GO-203, which blocks MUC1-C dimerization and its oncogenic function. In a murine NSG (NOD-scid IL2Rg) model, we examined whether MUC1high progenitors are more effective in inducing leukemic engraftment as compared to MUC1low progenitors and evaluated the capacity of GO-203 to eradicate disease. Methods and results:To confirm the hypothesis that LICs are contained within the MUChigh progenitor population, lineage−/MUC1high and lineage−/MUC1low cells were isolated from AML derived bone marrow mononuclear cells by flow cytometric sorting, and transplanted (1×106 cells/mouse) into sub-lethally irradiated NSG mice using retro orbital injections. Leukemia engraftment was observed in all recipient mice (6/6) inoculated with MUC1high cells, with no evidence of normal human engraftment as shown by immunophenotype, cell morphology, and presence of cytogenetic abnormality. In a cohort of 6 mice, treatment with GO-203 was initiated 8 weeks after challenge with MUC1high AML cells following the demonstration of circulating leukemia cells. Leukemia was eradicated in 5/6 mice treated with GO-203. In contrast, 6/6 mice inoculated with MUC1low cells demonstrated engraftment with normal human hematopoetic cells as demonstrated by immunophenotype, cell morphology and absence of cytogenetic abnormality by FISH analysis. Engraftment of normal hematopoietic cells was unaffected by treatment with GO-203 in 4/5 of mice inoculated with MUC1low cells, supporting that GO-203 does not affect non-malignant hematopoiesis. We further determined the effect of GO-203 treatment in a prevention model. NSG mice were treated with 21 days of GO-203 beginning 24 hours after inoculation with CD34+/lineage−/MUC1high cells isolated from patient derived bone marrow mononuclear cells. Treatment with GO-203 prevented leukemia engraftment in 4/5 animals with the 5th animal showing only 1.9% leukemia cells. In contrast, 4/5 animals inoculated with the CD34+/lineage-/MUC1high cells in the absence of GO-203 demonstrated evidence of extensive bone marrow infiltration with leukemia cells comprising a mean of 33% of the bone marrow mononuclear cells. Conclusions:The results demonstrate that LICs are contained within the MUC1high fraction of AML derived progenitors and that MUC1low progenitors contain normal hematopoietic stem cells capable of normal engraftment. Treatment with the MUC1-C inhibitor, GO-203, prevents engraftment of leukemia, and eradicates established disease in a murine model. Our work indicates that GO-203 is a novel therapeutic agent for AML that targets LICs and has the potential to improve disease outcomes. Disclosures:Kufe:Genus Oncology: Consultancy.

Identification of Campath-1 Antigen (CD52) As a Novel Therapeutic Target in Advanced Systemic Mastocytosis.

Abstract 2866▪▪This icon denotes a clinically relevant abstractSystemic mastocytosis (SM) is a neoplasm of mast cells (MC) and MC progenitor cells. The clinical picture in SM ranges from an indolent course to highly aggressive cases with short survival time. In a majority of all patients with SM, the KIT mutation D816V is detectable. In addition, activating RAS mutations have recently been identified in patients with advanced SM. So far, no curative therapy for advanced SM is available. To identify molecular targets in neoplastic MC, we have generated novel human MC lines by lentiviral immortalization of cord blood-derived MC progenitors with RAS G12V, SV40 large T antigen, and hTERT. These cell lines, designated MCPV1 through MCPV4, display a morphology and ultrastructure resembling immature MC progenitors. MCPV cells also express a number of MC differentiation antigens, such as KIT and tryptase, and produce an aggressive MC disease when injected into NOD-SCID IL-2RG−/− (NSG) mice. As assessed by flow cytometry, MCPV cells were found to express a number of different surface antigens, including CD13, CD30, CD33, CD44, CD52, CD54, CD63, and CD95. In consecutive analyses, we studied the cell surface target CD52, also known as CAMPATH-1 antigen. As assessed by FACS, CD52 was found to be expressed on primary neoplastic MC in patients with aggressive SM (ASM, n=3), whereas in all patients with indolent SM (ISM, n=6), neoplastic MC stained negative or only slightly positive for CD52. Normal MC stained negative for CD52 in these experiments. Expression of CD52 in neoplastic MC was also demonstrable by immunohistochemical staining of bone marrow biopsy sections. We then asked whether the putative stem cells in ISM and ASM would express CD52. Indeed, in all patients examined, including cases with ISM and ASM, the immature CD34+/CD38- cells were found to express CD52. Next, we studied the regulation of expression of CD52 in neoplastic MC. Since activating RAS mutants have been described to be expressed in neoplastic MC in patients with advanced SM, we asked whether oncogenic RAS would promote expression of CD52 in MC. Indeed, lentiviral-mediated expression of activated RAS mutants was found to upregulate mRNA expression and to promote surface expression of CD52 in the human MC line HMC-1 as well as in various other myeloid cell lines. To validate CD52 as a potential target in neoplastic MC, we applied the CD52-targeting drug alemtuzumab. In these experiments, alemtuzumab exerted only minimal direct effects on viability of MCPV cells after IgG-mediated crosslinking. However, incubation of MCPV cells with alemtuzumab induced rapid and dose-dependent cell death via complement-dependent cytotoxicity (Figure 1). Furthermore, alemtuzumab was found to induce complement-dependent cytotoxicity in CD52+ primary neoplastic MC obtained from patients with advanced SM (n=2). In conclusion, we have established a new powerful model for studying the biology of advanced SM. Moreover, our study has identified CD52 as a novel promising therapeutic target in neoplastic MC and MC progenitor cells. Whether targeting of neoplastic (stem) cells in advanced SM using alemtuzumab will improve therapy by eradicating malignant cells remains to be determined in clinical trials. [Display omitted] Disclosures:Valent:Phadia: Research Funding.

Islet-Specific CTL Cloned from a Type 1 Diabetes Patient Cause Beta-Cell Destruction after Engraftment into HLA-A2 Transgenic NOD/SCID/IL2RG Null Mice

Despite increasing evidence that autoreactive CD8 T-cells are involved in both the initiation of type 1 diabetes (T1D) and the destruction of beta-cells, direct evidence for their destructive role in-vivo is lacking. To address a destructive role for autoreactive CD8 T-cells in human disease, we assessed the pathogenicity of a CD8 T-cell clone derived from a T1D donor and specific for an HLA-A2-restricted epitope of islet-specific glucose-6-phosphatase catalytic-subunit related protein (IGRP). HLA-A2/IGRP tetramer staining revealed a higher frequency of IGRP-specific CD8 T-cells in the peripheral blood of recent onset human individuals than of healthy donors. IGRP265–273-specific CD8 T-cells that were cloned from the peripheral blood of a recent onset T1D individual were shown to secrete IFNγ and Granzyme B after antigen-specific activation and lyse HLA-A2-expressing murine islets in-vitro. Lytic capacity was also demonstrated in-vivo by specific killing of peptide-pulsed target cells. Using the HLA-A2 NOD-scid IL2rγnull mouse model, HLA-A2-restricted IGRP-specific CD8 T-cells induced a destructive insulitis. Together, this is the first evidence that human HLA-restricted autoreactive CD8 T-cells target HLA-expressing beta-cells in-vivo, demonstrating the translational value of humanized mice to study mechanisms of disease and therapeutic intervention strategies.

Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo

In vivo delivery is a major barrier to the use of molecular tools for gene modification. Here we demonstrate site-specific gene editing of human cells in vivo in hematopoietic stem cell-engrafted NOD.Cg-Prkdc(scid)IL2rγ(tm1Wjl) (abbreviated NOD-scid IL2rγ(null)) mice, using biodegradable nanoparticles loaded with triplex-forming peptide nucleic acids (PNAs) and single-stranded donor DNA molecules. In vitro screening showed greater efficacy of nanoparticles containing PNAs/DNAs together over PNA-alone or DNA-alone. Intravenous injection of particles containing PNAs/DNAs produced modification of the human CCR5 gene in hematolymphoid cells in the mice, with modification confirmed at the genomic DNA, mRNA and functional levels. Deep sequencing revealed in vivo modification of the CCR5 gene at frequencies of 0.43% in hematopoietic cells in the spleen and 0.05% in the bone marrow: off-target modification in the partially homologous CCR2 gene was two orders of magnitude lower. We also induced specific modification in the β-globin gene using nanoparticles carrying β-globin-targeted PNAs/DNAs, demonstrating this method's versatility. In vivo testing in an enhanced green fluorescent protein-β-globin reporter mouse showed greater activity of nanoparticles containing PNAs/DNAs together over DNA only. Direct in vivo gene modification, such as we demonstrate here, would allow for gene therapy in systemic diseases or in cells that cannot be manipulated ex vivo.

Comparison of Hematopoietic Stem Cells Derived from Fresh and Cryopreserved Whole Cord Blood in the Generation of Humanized Mice

To study the function and maturation of the human hematopoietic and immune system without endangering individuals, translational human-like animal models are needed. We compare the efficiency of CD34+ stem cells isolated from cryopreserved cord blood from a blood bank (CCB) and fresh cord blood (FCB) in generating highly engrafted humanized mice in NOD-SCID IL2Rγnull (NSG) rodents. Interestingly, the isolation of CD34+ cells from CCB results in a lower yield and purity compared to FCB. The purity of CD34+ isolation from CCB decreases with an increasing number of mononuclear cells that is not evident in FCB. Despite the lower yield and purity of CD34+ stem cell isolation from CCB compared to FCB, the overall reconstitution with human immune cells (CD45) and the differentiation of its subpopulations e.g., B cells, T cells or monocytes is comparable between both sources. In addition, independent of the cord blood origin, human B cells are able to produce high amounts of human IgM antibodies and human T cells are able to proliferate after stimulation with anti-CD3 antibodies. Nevertheless, T cells generated from FCB showed increased response to restimulation with anti-CD3. Our study reveals that the application of CCB samples for the engraftment of humanized mice does not result in less engraftment or a loss of differentiation and function of its subpopulations. Therefore, CCB is a reasonable alternative to FCB and allows the selection of specific genotypes (or any other criteria), which allows scientists to be independent from the daily changing birth rate.

Xenogeneic Graft-versus-Host-Disease in NOD-scid IL-2Rγnull Mice Display a T-Effector Memory Phenotype

The occurrence of Graft-versus-Host Disease (GvHD) is a prevalent and potentially lethal complication that develops following hematopoietic stem cell transplantation. Humanized mouse models of xenogeneic-GvHD based upon immunodeficient strains injected with human peripheral blood mononuclear cells (PBMC; “Hu-PBMC mice”) are important tools to study human immune function in vivo. The recent introduction of targeted deletions at the interleukin-2 common gamma chain (IL-2Rγnull), notably the NOD-scid IL-2Rγnull (NSG) and BALB/c-Rag2 null IL-2Rγnull (BRG) mice, has led to improved human cell engraftment. Despite their widespread use, a comprehensive characterisation of engraftment and GvHD development in the Hu-PBMC NSG and BRG models has never been performed in parallel. We compared engrafted human lymphocyte populations in the peripheral blood, spleens, lymph nodes and bone marrow of these mice. Kinetics of engraftment differed between the two strains, in particular a significantly faster expansion of the human CD45+ compartment and higher engraftment levels of CD3+ T-cells were observed in NSG mice, which may explain the faster rate of GvHD development in this model. The pathogenesis of human GvHD involves anti-host effector cell reactivity and cutaneous tissue infiltration. Despite this, the presence of T-cell subsets and tissue homing markers has only recently been characterised in the peripheral blood of patients and has never been properly defined in Hu-PBMC models of GvHD. Engrafted human cells in NSG mice shows a prevalence of tissue homing cells with a T-effector memory (TEM) phenotype and high levels of cutaneous lymphocyte antigen (CLA) expression. Characterization of Hu-PBMC mice provides a strong preclinical platform for the application of novel immunotherapies targeting TEM-cell driven GvHD.

P059 Tumor cell derived type I IFN stimulates tumor immunosurveillance, suppressing metastasis to bone

Introduction Breast cancer is the most common type of non-skin cancer among women. Breast cancer associated morbidity is not commonly due to the primary tumor but rather the concomitant metastasis–the invasion of tumor cells to tissues such as bone. Bone is the most common site of breast cancer metastasis and once this has occurred, it is usually incurable. This severe morbidity is largely attributed to our lack of understanding of the mechanisms of bone-specific metastasis. To investigate the metastatic process we utilized the unique syngeneic 4T1.2 model of spontaneous metastasis [1] , [2] to bone and compared the transcriptional profile of tumor cells purified from primary mammary tumors and matched bone metastases. This differential transcriptome comparison revealed a substantial set of genes suppressed in metastases were highly enriched for promoter elements for the master type I IFN regulatory factor, Irf7, as well as Irf7 itself. In this study, we show for the first time that suppression of the type I IFN pathway in breast tumor cells, not immune cells as usually described, is critical to the development of bone metastases. Furthermore, we reveal the immune cell targets that are responsible for IFN-driven metastasis suppression. Methods We restored type I IFN signaling in mice bearing 4T1.2 tumors by enforced tumor cell expression of Irf7 or direct IFN administration. We then explored the consequences of altered type I IFN signaling on metastasis and immune activity in this model. In addition, we utilized type I IFN receptor null mice (Ifnar-/-), NOD SCID IL-2rγ-/- mice (lacking a functional immune system) and mice specifically depleted in certain immune cell populations, to demonstrate the critical function of the immune system in Irf7 induced metastasis suppression. Results The restoration of Irf7 gene in tumor cells significantly reduced bone metastases and prolonged metastasis free survival in the 4T1.2 murine model. Irf7-driven suppression of metastasis was reliant on host type I IFN signaling because this suppression was diminished in mice deficient in the type I IFN receptor or immune responses. In addition, systemic interferon administration greatly prolonged survival and reduced metastasis. Metastasis suppression was specifically correlated with increased CD4+, CD8+ T cells and NK cells in the peripheral blood and was reversed by depletion of these effector cell populations. Conclusion We report for the first time that tumor cell intrinsic type I IFN immune pathways stimulate host tumor immune surveillance to suppress bone metastasis. Loss of tumor cell IFN signaling predicts increased risk of development of bone metastases. Our data prompt future research into the therapeutic potential of type I IFN pathway-targeted immunotherapies in breast cancer patients. In addition, dissection of the molecular markers in primary tumors that may determine which patients are most likely to respond to such therapies and benefit from prophylactic treatment.

Enhanced humoral and HLA‐A2‐restricted dengue virus‐specific T‐cell responses in humanized BLT NSG mice

Dengue is a mosquito-borne viral disease of humans, and animal models that recapitulate human immune responses or dengue pathogenesis are needed to understand the pathogenesis of the disease. We recently described an animal model for dengue virus (DENV) infection using humanized NOD-scid IL2rγ(null) mice (NSG) engrafted with cord blood haematopoietic stem cells. We sought to further improve this model by co-transplantation of human fetal thymus and liver tissues into NSG (BLT-NSG) mice. Enhanced DENV-specific antibody titres were found in the sera of BLT-NSG mice compared with human cord blood haematopoietic stem cell-engrafted NSG mice. Furthermore, B cells generated during the acute phase and in memory from splenocytes of immunized BLT-NSG mice secreted DENV-specific IgM antibodies with neutralizing activity. Human T cells in engrafted BLT-NSG mice secreted interferon-γ in response to overlapping DENV peptide pools and HLA-A2 restricted peptides. The BLT-NSG mice will allow assessment of human immune responses to DENV vaccines and the effects of previous immunity on subsequent DENV infections.

Improved production of human beta-defensins (HBDs) in grafted site skin tissues of human IMC/mouse chimeras treated with glycyrrhizin (GL) (118.14)

Abstract Skin HBDs are very important on the elimination of pathogens at the burn wound infections. Immature myeloid cells (IMC) appearing in tissues surrounding the burn area are shown to be inhibitory on skin HBD production. In this study, the effect of GL on the impaired production of skin HBDs was studied in X-irradiated NOD-SCID IL-2rγ−/− mice (NSG mice) grafted with human IMC and their syngeneic skin tissues. NSG mice were grafted with patient skin (8 × 8 mm unburned skin removed for auto-graft surgery; skin-chimeras) and inoculated i.d. with 1 × 106 cells/mouse of CD34+CD31+ IMC isolated from the same burn patients (skin/IMC-chimeras). These chimeras were treated with 0.2 to 40 mg/kg of GL (Minophagen Pharmaceutical CO., LTD., Tokyo, Japan) 6 and 24 hrs after IMC inoculation. Two days after grafting, tissue specimens excised from grafted sites were homogenized, and the amounts of HBDs in the homogenates were measured. In the results, HBD1 and HBD2 were detected in the skin homogenates of skin-chimeras, while the peptides were not detected in the skin homogenates of skin/IMC-chimeras. However, HBDs were detected in the skin homogenates of skin/IMC-chimeras treated i.p. or i.d. with 3 to 10 mg/kg of GL. The greatest efficacy of GL on recovering HBD production was seen when skin/IMC-chimeras were treated i.d. with 10 mg/kg of GL. These results indicate that the impaired HBD production is improved in skin/IMC-chimeras treated with GL.

Abstract 5335: Identification and molecular characterization of pancreatic tumor-initiating cells

Abstract Pancreatic cancer has the worst prognosis of any major malignancy with complex etiology involving both environmental and genetic factors. Despite increasing knowledge in tumor biology, the treatment efficacy in pancreatic cancers has not improved significantly over the past decade. Thus, the identification of novel molecular pathways involved in pancreatic oncogenesis and the development of new and potent therapeutic options are highly desirable. The realization that tumors themselves function as complex organs birthed the concept that cancer cells with tumor-initiating properties of stem cells may be the key drivers of the complex machinery behind oncogenesis. Tumor-initiating cells (TICs) have been studied extensively in the hematopoietic system, however it is not known which subpopulation has the highest tumor-initiating potential in pancreatic cancer. We have identified that the CD133+/CD44+/ALDH1+ cell population derived from 31 pancreatic adenocarcinomas and 6 pancreatic cancer cell lines, has the highest tumorigenic potential, assessed by in vivo dilution tumor propagation assays in highly immunodeficient (NOD-SCID IL2Rγ-/-) mice. In addition, this population has the highest sphere-forming capacity and is highly resistant to gemcitabine treatment in comparison to the other subpopulations. RNA-sequencing analysis in CD133−/CD44−/ALDH1− and CD133+/CD44+/ALDH1+ subpopulations derived from BxPC3 pancreatic cells revealed the transcriptomic alterations between these cell types. Furthermore, gene network analysis revealed enrichment of inflammatory and lipid metabolism networks in pancreatic TICs, suggesting the potential role of inflammation and lipid metabolism in pancreatic TIC function. In addition, a gene network-based FDA-approved chemical library screen revealed that metformin perturbs the activated networks in pancreatic TICs leading to the suppression of their growth. Metformin suppressed pancreatic cancer growth, through suppression of pancreatic TICs, in different pancreatic cancer mouse models. Overall, we propose the identification of a novel pancreatic cancer subpopulation which harbors tumor-initiating properties and is regulated by inflammatory signals, contributing to our understanding of how inflammation is linked to pancreatic oncogenesis in the molecular level. Furthermore, by following a novel strategy, we have identified that metformin suppresses pancreatic tumor growth and relapse through inhibition of pancreatic TIC growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5335. doi:1538-7445.AM2012-5335

Adoptive transfer with in vitro expanded human regulatory T cells protects against porcine islet xenograft rejection via interleukin-10 in humanized mice.

T cell-mediated rejection remains a barrier to the clinical application of islet xenotransplantation. Regulatory T cells (Treg) regulate immune responses by suppressing effector T cells. This study aimed to determine the ability of human Treg to prevent islet xenograft rejection and the mechanism(s) involved. Neonatal porcine islet transplanted NOD-SCID IL2rγ−/− mice received human peripheral blood mononuclear cells (PBMC) with in vitro expanded autologous Treg in the absence or presence of anti-human interleukin-10 (IL-10) monoclonal antibody. In addition, human PBMC-reconstituted recipient mice received recombinant human IL-10 (rhIL-10). Adoptive transfer with expanded autologous Treg prevented islet xenograft rejection in human PBMC-reconstituted mice by inhibiting graft infiltration of effector cells and their function. Neutralization of human IL-10 shortened xenograft survival in mice receiving human PBMC and Treg. In addition, rhIL-10 treatment led to prolonged xenograft survival in human PBMC-reconstituted mice. This study demonstrates the ability of human Treg to prevent T-cell effector function and the importance of IL-10 in this response. In vitro Treg expansion was a simple and effective strategy for generating autologous Treg and highlighted a potential adoptive Treg cell therapy to suppress antigraft T-cell responses and reduce the requirement for immunosuppression in islet xenotransplantation.

The Hsp32 Inhibitors SMA-ZnPP and PEG-ZnPP Exert Major Growth-Inhibitory Effects on D34+/CD38+ and CD34+/CD38- AML Progenitor Cells

Heat shock protein 32 (Hsp32), also known as heme oxygenase 1 (HO-1), has recently been identified as a potential target in various hematologic malignancies. We provide evidence that Hsp32 is constitutively expressed in primary leukemic cells in patients with acute myeloid leukemia (AML) and in various AML cell lines (HL60, U937, KG1). Expression of Hsp32 mRNA was demonstrable by qPCR, and expression of the Hsp32 protein by immunocytochemistry and Western blotting. The stem cell-enriched CD34+/CD38+ and CD34+/CD38- fractions of AML cells were found to express Hsp32 mRNA in excess over normal CD34+ progenitor cells. Two Hsp32-targeting drugs, pegylated zinc-protoporphyrin (PEG-ZnPP) and styrene-maleic-acid-copolymer-micelle-encapsulated ZnPP (SMAZnPP), were found to inhibit cytokine-dependent and spontaneous proliferation in all 3 AML cell lines as well as in primary AML cells. Growth inhibitory effects of SMA-ZnPP and PEG-ZnPP were dose-dependent with IC50 values ranging between 1 and 20 μM, and were accompanied by apoptosis as evidenced by light- and electron microscopy, Tunel assay, and caspase-3 activation. Finally, we were able to demonstrate that SMA-ZnPP inhibits cytokine-dependent proliferation of CD34+/CD38+ and CD34+/CD38- AML progenitor cells in vitro in all patients as well as leukemiainitiation of AML stem cells in NOD-SCID IL-2Rγ(-/-) (NSG) mice in vivo. Together, our data suggest that Hsp32 plays an important role as a survival factor in leukemic stem cells and as a potential new target in AML.

The Leukemic Stem Cell (LSC) in Ph+ CML Is a CD34+/CD38−/Lin− Cell That Co-Expresses Dipeptidylpeptidase IV (CD26) and Disrupts LSC-Niche Interactions by Degrading the CXCR4 Ligand SDF-1α

Abstract 961In Philadelphia-positive (Ph+) chronic myeloid leukemia (CML), leukemic stem cells (LSC) supposedly reside in a CD34+/CD38−/Lin− fraction of the leukemic clone. However, little is known about phenotypic properties of LSC in CML. We screened for novel LSC markers and targets in CML by gene chip studies and extensive flow cytometry analyses using monoclonal antibodies against various surface antigens (n=50). A total number of 240 bone marrow or peripheral blood samples (CML, n=95; AML, n=103; CMML, n=10, control marrow, n=32) were examined. In common with normal SC, CD34+/CD38− CML LSC were found to co-express the homing-receptor CD44, G-CSF-R (CD114), KIT (CD117), FLT3 (CD135), and CXCR4 (CD184). Similar to LSC in AML and CMML, CML LSC were found to display higher levels of Siglec-3 (CD33) and IL-3RA (CD123). Most significantly, however, we found that in contrast to normal CD34+/CD38− stem cells, CD34+/CD38− CML LSC aberrantly express IL-2RA (CD25), dipeptidylpeptidase IV (DPPIV=CD26), and IL-1RAP. In other myeloid leukemias (AML, CMML), CD34+/CD38− LSC also co-expressed CD25, but usually did not express CD26 or IL-1RAP. Whereas CD26 was expressed almost invariably on CD34+/CD38− cells in all CML patients tested, the surface enzyme was neither detectable in more mature CD34+/CD38+ progenitor cells nor on CD34+/CD38− stem cells in reactive bone marrow or healthy controls. During successful treatment with imatinib or nilotinib (patients examined at CCyR and/or MMR), CD34+/CD38− stem cells invariably showed a ‘normal’ phenotype (CD25−, CD26−, IL-1RAP−), whereas in relapsing CML, CD34+/CD38− cells were again found to co-express CD25 and CD26. Sorted Lin−/CD26− stem cells obtained from CML patients (at diagnosis) engrafted irradiated NOD-SCID IL-2Rγ−/− (NSG) mice with normal multilineage BCR/ABL1− hematopoiesis, whereas Lin−/CD26+ stem cells were found to engraft NSG mice with BCR/ABL+ cells. We next examined the regulation of expression of CD25 and CD26 on CML LSC. Whereas expression of CD25 was found to depend on BCR/ABL1 and STAT5-activity, CD26 expression was found to be expressed independent of BCR/ABL1 and independent of STAT5-signaling. In a next step, we examined the potential function of CD26 on CML LSC. In these studies, CD26 was identified as a target-enzyme disrupting the niche-related SDF-1α/CXCR4 axis by degrading SDF-1α. Correspondingly, CD26-targeting gliptins (sitagliptin, 1 μM; vildagliptin, 1 μM) were found to revert recombinant DPPIV/CD26-induced or cellular CD26-induced inhibition of SDF-1α-mediated in vitro migration of CD26+ leukemic cells. Finally, we found that in a CML patient treated with nilotinib, in whom uncontrolled diabetes mellitus required therapy with saxagliptin, BCR/ABL1 levels (in percent of ABL according to IS) that were found to increase before the start of saxagliptin (IS before saxagliptin: 1.6 [-4 months], 2.3 [-3 months], and 2.4 [at therapy-start]), decreased over time during saxagliptin-therapy (IS: 1.0 [+1 month], 1.0 [+3 months], 0.8 [+5 months]). Together, the CML-initiating LSC is a CD34+/CD38− cell that exhibits aberrant expression of IL-1RAP, CD25, and DPPIV/CD26. All three markers may be useful for purification of CML LSC. DPPIV/CD26 appears to be a functionally and pathogenetically relevant antigen that may facilitate niche-independent uncontrolled redistribution and thus extramedullary spread of LSC and LSC-derived progenitor cells in CML. Whether CD26 can be developed as a novel therapeutic target in CML is currently under investigation. Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding.

Pre‐differentiated human committed T‐lymphoid progenitors promote peripheral T‐cell re‐constitution after stem cell transplantation in immunodeficient mice

T-cell re-constitution after allogeneic stem cell transplantation (alloSCT) is often dampened by the slow differentiation of human peripheral blood CD34(+) (huCD34(+) ) hematopoietic stem cells (HSCs) into mature T cells. This process may be accelerated by the co-transfer of in vitro-pre-differentiated committed T/NK-lymphoid progenitors (CTLPs). Here, we analysed the developmental potential of huCD34(+) HSCs compared with CTLPs from a third-party donor in a murine NOD-scid IL2Rγ(null) model of humanised chimeric haematopoiesis. CTLPs (CD34(+) lin(-) CD45RA(+) CD7(+) ) could be generated in vitro within 10 days upon co-culture of huCD34(+) or cord blood CD34(+) (CB-CD34) HSCs on murine OP9/N-DLL-1 stroma cells but not in a novel 3-D cell-culture matrix with DLL-1(low) human stroma cells. In both in vitro systems, huCD34(+) and CB-CD34(+) HSCs did not give rise to mature T cells. Upon transfer into 6-wk-old immune-deficient mice, CTLPs alone did not engraft. However, transplantation of CTLPs together with huCD34(+) HSCs resulted in rapid T-cell engraftment in spleen, bone marrow and thymus at day 28. Strikingly, at this early time point mature T cells originated exclusively from CTLPs, whereas descendants of huCD34(+) HSCs still expressed a T-cell-precursor phenotype (CD7(+) CD5(+) CD1a(+/-) ). This strategy to enhance early T-cell re-constitution with ex vivo-pre-differentiated T-lymphoid progenitors could bridge the gap until full T-cell recovery in severely immunocompromised patients after allogeneic stem cell transplantation.

Humanized Mouse Model of Ovarian Cancer Recapitulates Patient Solid Tumor Progression, Ascites Formation, and Metastasis

Ovarian cancer is the most common cause of death from gynecological cancer. Understanding the biology of this disease, particularly how tumor-associated lymphocytes and fibroblasts contribute to the progression and metastasis of the tumor, has been impeded by the lack of a suitable tumor xenograft model. We report a simple and reproducible system in which the tumor and tumor stroma are successfully engrafted into NOD-scid IL2Rγnull (NSG) mice. This is achieved by injecting tumor cell aggregates derived from fresh ovarian tumor biopsy tissues (including tumor cells, and tumor-associated lymphocytes and fibroblasts) i.p. into NSG mice. Tumor progression in these mice closely parallels many of the events that are observed in ovarian cancer patients. Tumors establish in the omentum, ovaries, liver, spleen, uterus, and pancreas. Tumor growth is initially very slow and progressive within the peritoneal cavity with an ultimate development of tumor ascites, spontaneous metastasis to the lung, increasing serum and ascites levels of CA125, and the retention of tumor-associated human fibroblasts and lymphocytes that remain functional and responsive to cytokines for prolonged periods. With this model one will be able to determine how fibroblasts and lymphocytes within the tumor microenvironment may contribute to tumor growth and metastasis, and will make it possible to evaluate the efficacy of therapies that are designed to target these cells in the tumor stroma.

Ex Vivo Expanded Hematopoietic Stem Cells Overcome the MHC Barrier in Allogeneic Transplantation

The lack of understanding of the interplay between hematopoietic stem cells (HSCs) and the immune system has severely hampered the stem cell research and practice of transplantation. Major problems for allogeneic transplantation include low levels of donor engraftment and high risks of graft-versus-host disease (GVHD). Transplantation of purified allogeneic HSCs diminishes the risk of GVHD but results in decreased engraftment. Here we show that exvivo expanded mouse HSCs efficiently overcame the major histocompatibility complex barrier and repopulated allogeneic-recipient mice. An 8-day expansion culture led to a 40-fold increase of the allograft ability of HSCs. Both increased numbers of HSCs and culture-induced elevation of expression of the immune inhibitor CD274 (B7-H1 or PD-L1) on the surface of HSCs contributed to the enhancement. Our study indicates the great potential of utilizing exvivo expanded HSCs for allogeneic transplantation and suggests that the immune privilege of HSCs can be modulated.

Humanized tumor mice—A new model to study and manipulate the immune response in advanced cancer therapy

The immunological impact on antibody-based anticancer therapies remains incompletely understood due to the lack of appropriate animal models for in vivo analysis. Here, we present a novel humanized tumor mouse (HTM) model, generated by concurrent transplantation of human hematopoietic stem cells (HSCs) and human breast cancer cells in neonatal NOD-scid IL2Rγ(null) mice. Five weeks after intrahepatic transplantation, a functional human immune system was developed in all organs, and, in addition, tumor cells were detectable in lung and bone marrow (early dissemination). After 3 months posttransplant, tumor-cell effusions and macroscopic tumors associated with liver or spleen were found. Furthermore, disseminated cells in different lymphoid and nonlymphoid organs were measurable. Tumor growth was accompanied by specific T-cell maturation and tumor cell-specific T-cell activation. In addition, Natural-Killer cell accumulation and activation were observed in HTM, which was further enhanced upon IL-15 treatment facilitating the possibility of immune cell modulation in, e.g., antibody-dependent cellular cytotoxicity-based immunotherapeutic approaches. This novel mouse model makes it possible to combine transfer of MHC mismatched tumor cells together with human HSCs resulting in a solid coexistence and interaction without evidence for rejection. Overall, humanized tumor mice represent a powerful in vivo model that for the first time permits the investigation of human immune system-related target cancer therapy and resistance.

Human CD34+ CD133+ Hematopoietic Stem Cells Cultured with Growth Factors Including Angptl5 Efficiently Engraft Adult NOD-SCID Il2rγ−/− (NSG) Mice

Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34+ CD133+ cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg−/− (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34+ CD133+ fraction of expanded cells and that CD34+ CD133+ cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.