NOD-SCID IL2R Research Articles

Evaluation of Four Humanized NOD-Derived Mouse Models for Dengue Virus-2 Infection.

Dengue is a significant public health problem with no specific viral treatment. One of the main challenges in studying dengue is the lack of adequate animal models recapitulating human immune responses. Most studies on humanized mice use NOD-scid IL2R gamma null (NSG) mice, which exhibit poor hematopoiesis for some cell populations. This study compares three humanized (hu) NOD-derived mouse models for dengue virus-2 (DENV-2) infection in the context of human cytokine expression. Three mouse strains (hu-NSG, hu-EXL, and hu-SGM3) received xenotransplants of human CD34+ fetal cord blood cells from a single donor, and one mouse strain received human peripheral blood mononuclear cells (hu-SGM3-PBMCs). All models exhibited infectious viruses in blood confirmed by plaque assay, but mice expressing human cytokines showed higher viremia compared to conventional NSG mice. The hu-SGM3-PBMCs model developed lethal infections, showing a significant increase in viremia and clinical signs. A detectable human cytokine response was observed in all the DENV-2-infected humanized mouse models. In conclusion, humanized NOD-derived mouse models expressing human cytokines offer a relevant platform for the study of dengue pathogenesis and antiviral therapies.

Enhancement of Subcutaneous Islet Transplant Performance by Collagen 1 Gel.

Human islets can be transplanted into the portal vein for T1 diabetes, and a similar procedure is being used in a clinical trial for stem cell-derived beta-like cells. Efforts have been underway to find an alternative transplant site that will foster better islet cell survival and function. Although conceptually attractive, the subcutaneous (SC) site has yielded disappointing results, in spite of some improvements resulting from more attention paid to vascularization and differentiation factors, including collagen. We developed a method to transplant rat islets in a disk of type 1 collagen gel and found improved efficacy of these transplants. Survival of islets following transplantation (tx) was determined by comparing insulin content of the graft to that of the pre-transplant islets from the same isolation. At 14 days after transplantation, grafts of the disks had more than double the recovered insulin than islets transplanted in ungelled collagen. SC grafts of disks had similar insulin content to grafts in a kidney site and in epididymal fat pads. In vivo disks underwent contraction to 10% of initial volume within 24 h but the islets remained healthy and well distributed. Whole mount imaging showed that residual donor vascular cells within the islets expanded and connected to ingrowing host blood vessels. Islets (400 rat islet equivalents (IEQ)) in the collagen disks transplanted into an SC site of NOD scid IL2R gammanull (NSG) mice reversed streptozotocin (STZ)-induced diabetes within 10 days as effectively as transplants in the kidney site. Thus, a simple change of placing islets into a gel of collagen 1 prior to transplantation allowed a prompt reversal of STZ-induced diabetes using SC site.

Rational Chemical and Genetic Modifications Enhance Avidity and Function of CD70-Directed CAR-T-Cells for Myeloid Leukemia

Chimeric Antigen Receptor T cells (CAR-T) have changed the therapeutic landscape for lymphoid malignancies, but not yet in myeloid malignancies like acute myeloid leukemia (AML). The TNF-alpha family member CD70 has emerged as a promising surface target antigen in AML after high complete response rates (CR) were seen in a Phase 1 trial of the CD70 antibody cusatuzumab (Riether et al, Nature Medicine 2020). Disappointingly, phase II results with cusatuzumab found CR rates less one-half those seen in the phase 1 (Trudel ASCO 2020).Because CAR-T cells may recognize lower antigen densities than monoclonal antibodies, we sought to develop a better CAR-T strategy for targeting the CD70 antigen in AML. A recent effort to improve on the ‘first’ generation natural-ligand based CD70 CAR (full length-CD27 fused to CD3zeta) compared a variety of CARs, including the single-chain variable-fragment as the binding moiety to CD70, found that the original ‘first’ generation CAR was superior (Shaffer Blood 2011, Sauer Blood 2021). Interestingly, a second generation natural-ligand based CAR that included the 4-1BB costimulatory domain was thought to be superior to first-generation (zeta-only) CARs with the same binder (Wang Clinical Cancer Research 2016).We first confirmed that the ligand-based 4-1BB (Native) CAR, had activity against multiple AML targets in standard CAR-T assays including activation, cytolysis, and demonstrated activity in a NOD-SCID IL2R γnull (NSG) Molm13 mouse model of AML. However, these models were not curative, even in combination with azacitidine, which we confirmed mediated increase CD70 expression on the AML cells. We hypothesized that surface cleavage of CD27, which is the natural ligand of CD70, attenuated the function of the Native CARs; we confirmed this hypothesis by measuring soluble CD27 in CAR T cells co-cultured with AML targets. To abrogate surface cleavage of ligand-based CARs, we generated and tested a panel of rationally designed, novel hinge CAR variants (‘truncated’, ‘deleted’, ‘flexible’, and ‘CD8hinge&TM’, Figure 1A). We found that the CD8hinge&TM variant had improved cytolysis against AML targets in vitro as well as higher binding avidity as measured by acoustic force microscopy (Figure 1B). Furthermore, there was no detectable soluble CD27 after co-culture with AML targets, suggesting successful abrogation of hinge cleavage.When all the CAR variants were compared in vivo to the Native CAR, CD8hinge&TM CARs mediated improved tumor control, had higher CAR expansion in blood and bone marrow, a persistent central memory phenotype beyond 30 days, and mediated improved survival (Figure 1C). The effect of the CD8hinge&TM CARs was further enhanced in combination with azacitidine. We also found that tumor control (maximum flux) correlated most strongly with relative binding avidity of the hinge variants (R 2 0.906) compared to other measures of in vitro function such as IFNg production (R 2 0.548) or in vitro cytolysis (R 2 0.5982). Finally, we tested the Native and CD8hinge&TM CARs in patient-derived xenograft (PDX) models and confirmed that the Native CAR T cells did not control AML tumor and resulted in uniform lethality, whereas the CD8hinge&TM CAR T cells had superior in vivo expansion and were able to mediate AML eradication in mice. Our findings demonstrate that natural-ligand binding domains of CARs targeting CD70 in AML can be effective but require mechanisms to overcome surface cleavage. CD70-targeted CARs comprised of a fusion of truncated CD27 to a CD8 hinge and transmembrane domain have promise in patients with AML, with and without combination with azacitidine. [Display omitted] DisclosuresWehrli: Novartis: Current equity holder in publicly-traded company; Nestle: Current equity holder in publicly-traded company; CSL Behring: Patents & Royalties. Frigault: Editas: Consultancy; Takeda: Consultancy; Iovance: Consultancy; Arcellx: Consultancy; Kite: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy. Maus: Astellas: Consultancy; Arcellx: Consultancy; Agenus: Consultancy; Adaptimmune: Consultancy; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company; AstraZeneca: Consultancy; Atara: Consultancy; Bayer: Consultancy; BMS: Consultancy; Cabaletta Bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; In8bio (SAB): Consultancy; Intellia: Consultancy; GSK: Consultancy; Kite Pharma: Consultancy, Research Funding; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Novartis: Consultancy; Tmunity: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; WindMIL: Consultancy.

Identification and Development of PHE885: A Novel and Highly Potent Fully Human Anti-BCMA CAR-T Manufactured with a Novel T-Charge TM Platform for the Treatment of Multiple Myeloma

Identification and Development of PHE885: A Novel and Highly Potent Fully Human Anti-BCMA CAR-T Manufactured with a Novel T-Charge TM Platform for the Treatment of Multiple Myeloma

A humanized orthotopic tumor microenvironment alters the bone metastatic tropism of prostate cancer cells

Prostate cancer (PCa) is the second most commonly diagnosed cancer in men, and bone is the most frequent site of metastasis. The tumor microenvironment (TME) impacts tumor growth and metastasis, yet the role of the TME in PCa metastasis to bone is not fully understood. We used a tissue-engineered xenograft approach in NOD-scid IL2Rγnull (NSG) mice to incorporate two levels of humanization; the primary tumor and TME, and the secondary metastatic bone organ. Bioluminescent imaging, histology, and immunohistochemistry were used to study metastasis of human PC-3 and LNCaP PCa cells from the prostate to tissue-engineered bone. Here we show pre-seeding scaffolds with human osteoblasts increases the human cellular and extracellular matrix content of bone constructs, compared to unseeded scaffolds. The humanized prostate TME showed a trend to decrease metastasis of PC-3 PCa cells to the tissue-engineered bone, but did not affect the metastatic potential of PCa cells to the endogenous murine bones or organs. On the other hand, the humanized TME enhanced LNCaP tumor growth and metastasis to humanized and murine bone. Together this demonstrates the importance of the TME in PCa bone tropism, although further investigations are needed to delineate specific roles of the TME components in this context.

CBLB ablation with CRISPR/Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy

BackgroundTumors often develop resistance to surveillance by endogenous immune cells, which include natural killer (NK) cells. Ex vivo activated and/or expanded NK cells demonstrate cytotoxicity against various tumor cells and...

Tumor-infiltrating mast cells are associated with resistance to anti-PD-1 therapy

Anti-PD-1 therapy is used as a front-line treatment for many cancers, but mechanistic insight into this therapy resistance is still lacking. Here we generate a humanized (Hu)-mouse melanoma model by injecting fetal liver-derived CD34+ cells and implanting autologous thymus in immune-deficient NOD-scid IL2Rγnull (NSG) mice. Reconstituted Hu-mice are challenged with HLA-matched melanomas and treated with anti-PD-1, which results in restricted tumor growth but not complete regression. Tumor RNA-seq, multiplexed imaging and immunohistology staining show high expression of chemokines, as well as recruitment of FOXP3+ Treg and mast cells, in selective tumor regions. Reduced HLA-class I expression and CD8+/Granz B+ T cells homeostasis are observed in tumor regions where FOXP3+ Treg and mast cells co-localize, with such features associated with resistance to anti-PD-1 treatment. Combining anti-PD-1 with sunitinib or imatinib results in the depletion of mast cells and complete regression of tumors. Our results thus implicate mast cell depletion for improving the efficacy of anti-PD-1 therapy.

A novel humanized mouse model to study the function of human cutaneous memory T cells in vivo in human skin

Human skin contains a population of memory T cells that supports tissue homeostasis and provides protective immunity. The study of human memory T cells is often restricted to in vitro studies and to human PBMC serving as primary cell source. Because the tissue environment impacts the phenotype and function of memory T cells, it is crucial to study these cells within their tissue. Here we utilized immunodeficient NOD-scid IL2rγnull (NSG) mice that carried in vivo-generated engineered human skin (ES). ES was generated from human keratinocytes and fibroblasts and was initially devoid of skin-resident immune cells. Upon adoptive transfer of human PBMC, this reductionist system allowed us to study human T cell recruitment from a circulating pool of T cells into non-inflamed human skin in vivo. Circulating human memory T cells preferentially infiltrated ES and showed diverse functional profiles of T cells found in fresh human skin. The chemokine and cytokine microenvironment of ES closely resembled that of non-inflamed human skin. Upon entering the ES T cells assumed a resident memory T cell-like phenotype in the absence of infection, and a proportion of these cutaneous T cells can be locally activated upon injection of monocyte derived dendritic cells (moDCs) that presented Candida albicans. Interestingly, we found that CD69+ memory T cells produced higher levels of effector cytokines in response to Candida albicans, compared to CD69- T cells. Overall, this model has broad utility in many areas of human skin immunology research, including the study of immune-mediated skin diseases.

Brief ex vivo Fas-ligand incubation attenuates GvHD without compromising stem cell graft performance

Graft versus host disease (GvHD) remains a limiting factor for successful hematopoietic stem cell transplantation (HSCT). T cells and antigen-presenting cells (APCs) are major components of the hematopoietic G-CSF mobilized peripheral blood cell (MPBC) graft. Here we show that a short incubation (2 h) of MPBCs with hexameric Fas ligand (FasL) selectively induces apoptosis of specific donor T cell subsets and APCs but not of CD34+ cells. FasL treatment preferentially induces apoptosis in mature T cell subsets which express high levels of Fas (CD95), such as T stem cell memory, T central memory, and T effector memory cells, as well as TH1 and TH17 cells. Anti-CD3/CD28 stimulated T cells derived from FasL-treated-MPBCs express lower levels of CD25 and secrete lower levels of IFN-γ as compared to control cells not treated with FasL. FasL treatment also induces apoptosis of transitional, naïve, memory and plasmablastoid B cells leading to a reduction in their numbers in the graft and following engraftment in transplanted mice. Most importantly, ex vivo treatment of MPBCs with FasL prior to transplant in conditioned NOD-scid IL2Rγnull (NSG) mice prevented GvHD while preserving graft versus leukemia (GvL) effects, and leading to robust stem cell engraftment.

The Impact of Smoking on Survival in Patients (Pts) with Newly Diagnosed Philadelphia Chromosome Positive (Ph+) Acute Lymphoblastic Leukemia (ALL) Treated with the Combination of Intensive Therapy with Tyrosine Kinase Inhibitor (TKI)

The Impact of Smoking on Survival in Patients (Pts) with Newly Diagnosed Philadelphia Chromosome Positive (Ph+) Acute Lymphoblastic Leukemia (ALL) Treated with the Combination of Intensive Therapy with Tyrosine Kinase Inhibitor (TKI)

Genetically engineered CAR NK cells display selective cytotoxicity against FLT3-positive B-ALL and inhibit in vivo leukemia growth.

Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells represent a promising effector cell type for adoptive cancer immunotherapy. Both, genetically modified donor-derived NK cells as well as continuously expanding NK-92 cells are currently under clinical development. To enhance their therapeutic utility for the treatment of pre-B-cell acute lymphoblastic leukemia (B-ALL), we engineered NK-92 cells by lentiviral gene transfer to express a FMS-like tyrosine kinase 3 (FLT3)-specific CAR that contains a composite CD28-CD3ζ signaling domain. FLT3 has primarily been described as a therapeutic target for acute myeloid leukemia, but overexpression of FLT3 has also been reported in B-ALL. Exposure of FLT3-positive targets to CAR NK-92 cells resulted in conjugate formation between NK and leukemia cells, NK-cell degranulation and selective cytotoxicity toward established B-ALL cell lines and primary blasts that were resistant to parental NK-92. In a SEM B-ALL xenograft model in NOD-SCID IL2R γnull mice, treatment with CAR NK-92 but not parental NK-92 cells markedly inhibited disease progression, demonstrating high antileukemic activity in vivo. As FLT3 is known to be also expressed on precursor cells, we assessed the feasibility of incorporating an inducible caspase-9 (iCasp9) suicide switch to enhance safety of our approach. Upon addition of the chemical dimerizer AP20187 to NK-92 cells coexpressing the FLT3-specific CAR and iCasp9, rapid iCasp9 activation was observed, precluding further CAR-mediated cytotoxicity. Our data demonstrate that B-ALL can be effectively targeted by FLT3-specific CAR NK cells which may complement CD19-directed immunotherapies, particularly in cases of inherent or acquired resistance to the latter.

Targeting ulcerative colitis by suppressing glucose uptake with ritonavir

ABSTRACTGlucose is the preferred source of energy in activated inflammatory cells. Glucose uptake into the cell is ensured by a family of glucose uptake transporters (GLUTs), which have been identified as off-target molecules of the HIV protease inhibitor ritonavir. In this study, we examined the effect of ritonavir on inflammation in vitro and in vivo. Peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3 in the presence or absence of ritonavir and analyzed by flow cytometric analysis. Frequencies of CD4+ cells were significantly affected by ritonavir (CD69+ P=3E-05; CD134 P=4E-06; CD25+ P=E-07; central memory P=0.02; effector P=6E-03; effector memory P=6E-05). To corroborate that inflammation has a metabolic effect in vivo, a mouse model was used that is based on immunocompromised NOD-scid IL-2Rγ null mice reconstituted with PBMCs from patients with ulcerative colitis (UC). Inflammation had a significant effect on amino acid (AA) levels (Glu P=1E-07, Asp P=1E-04). Principal component analysis (PCA) discriminated between unchallenged and challenged groups. Finally, the efficacy of ritonavir was tested in the same mouse model. Dependent variables were clinical and histological scores, frequencies of human leukocytes isolated from spleen and colon, and levels of AA in sera of mice. Mice benefited from treatment with ritonavir as indicated by significantly decreased colon (P=7E-04) and histological (P=1E-04) scores, frequencies of M2 monocytes (CD14+ CD163; P=0.02), and Glu levels (P=2E-05). PCA discriminated between control and challenged groups (P=0.026). Thus, inhibition of glucose uptake might be a promising therapeutic intervention point for active UC.

The Suppressive Function of the Human Regulatory Macrophages In-Vitro have more Potency but not In-Vivo

Centre for Transplant & Renal Research. Introduction Human regulatory macrophages (Mregs) have been shown to suppress mitogen-driven T cell proliferation in vitro and are under evaluation as a cellular therapy in renal transplantation in THE ONE STUDY clinical trial. In this study, we investigated whether human Mregs were capable of suppressing the allogeneic response in vitro and protecting islet xeno-grafts in humanizing mice in vivo. Methods Human CD14+ cells were isolated from healthy donor peripheral blood monocytes (PBMCs) using CD14 magnetic beads. Mregs induction was performed by culturing CD14+ cells with M-CSF for 6 days and be stimulated with IFN-γ for a further 24 hr. The phenotype of Mregs was characterized by flow cytometry, and their suppressive activity was tested by mixed-lymphocyte reaction (MLR) using human PBMCs as responder cells, autologous Mregs as suppressor cells and irradiated allogeneic PBMCs as stimulator cells. In a separate transwell MLR setting, responder and allogeneic stimulator cells were cultured in the upper chamber and autologous Mregs were seeded in the lower chamber. Supernatants collected from MLR cultures were used for cytokine detection. Isolated the neonatal porcine islet cell clusters (NICCs) and cultured for 6 days. In vivo, every 5,000 IEQ NICCs were transplanted into NOD-SCID IL2R gamma -/- mice under the two sides of renal capsules. Adoptive transfer of human PBMCs and Mregs were suspended in PBS and injected into mice at doses of 10×106 and 2×106 by tail vein within 7 days after transplant. Graft rejection was defined as little or no intact graft with a heavy lymphocytic infiltrate on histological examination, survival was analyzed by H&E staining and endocrine secreting cells was analyzed by insulin staining. Results The resulting induced human Mregs demonstrated a typical Mregs phenotype, CD14lowCD80lowCD86+HLA-DR+CD83-/lowCD16-, and as a result, potent suppression of proliferating alloreactive PBMC even at Mregs: responder cells ratios >1:16. Separation of Mregs from responder and allogeneic stimulators in a transwell MLR system had the mild impact on their suppressive potency in the allogeneic response at all ratios of Mregs: responder cells tested. Upregulated levels of Mregs function related molecules TGF-b and IDO were detected in the supernatants collected from both non-transwell and transwell MLR cultures containing Mregs. In treated mice, the human PBMC were identified in the porcine-NICC-xenografts, as well as systemically in the spleens, blood, and kidneys. However, injected Mregs cannot be detected by flow cytometry, and there were no intact xenografts were found with mid-term. Conclusions In vitro induced human Mregs showed the capacity to suppress the allogeneic response in vitro and that was not cell-cell contact dependent. The porcine-NICC-xenografts cannot be protected by Human-Mregs in vivo as Mregs cannot proliferation. The suppressive function of the Mregs have more potency in vitro, but not in vivo.

Bat-mouse bone marrow chimera: a novel animal model for dissecting the uniqueness of the bat immune system

Bats are an important animal model with long lifespans, low incidences of tumorigenesis and an ability to asymptomatically harbour pathogens. Currently, in vivo studies of bats are hampered due to their low reproduction rates. To overcome this, we transplanted bat cells from bone marrow (BM) and spleen into an immunodeficient mouse strain NOD-scid IL-2R−/− (NSG), and have successfully established stable, long-term reconstitution of bat immune cells in mice (bat-mice). Immune functionality of our bat-mouse model was demonstrated through generation of antigen-specific antibody response by bat cells following immunization. Post-engraftment of total bat BM cells and splenocytes, bat immune cells survived, expanded and repopulated the mouse without any observable clinical abnormalities. Utilizing bat’s remarkable immunological functions, this novel model has a potential to be transformed into a powerful platform for basic and translational research.

Design and validation of a disease network of inflammatory processes in the NSG-UC mouse model

BackgroundUlcerative colitis (UC) is a highly progressive inflammatory disease that requires the interaction of epithelial, immune, endothelial and muscle cells and fibroblasts. Previous studies suggested two inflammatory conditions in UC-patients: ‘acute’ and ‘remodeling’ and that the design of a disease network might improve the understanding of the inflammatory processes. The objective of the study was to design and validate a disease network in the NOD-SCID IL2rγnull (NSG)-UC mouse model to get a better understanding of the inflammatory processes.MethodsLeukocytes were isolated from the spleen of NSG-UC mice and subjected to flow cytometric analysis. RT-PCR and RNAseq analysis were performed from distal parts of the colon. Based on these analyses and the effects of interleukins, chemokines and growth factors described in the literature, a disease network was designed. To validate the disease network the effect of infliximab and pitrakinra was tested in the NSG-UC model. A clinical- and histological score, frequencies of human leukocytes isolated from spleen and mRNA expression levels from distal parts of the colon were determined.ResultsAnalysis of leukocytes isolated from the spleen of challenged NSG-UC mice corroborated CD64, CD163 and CD1a expressing CD14+ monocytes, CD1a expressing CD11b+ macrophages and HGF, TARC, IFNγ and TGFß1 mRNA as inflammatory markers. The disease network suggested that a proinflammatory condition elicited by IL-17c and lipids and relayed by cytotoxic T-cells, Th17 cells and CD1a expressing macrophages and monocytes. Conversely, the remodeling condition was evoked by IL-34 and TARC and promoted by Th2 cells and M2 monocytes. Mice benefitted from treatment with infliximab as indicated by the histological- and clinical score. As predicted by the disease network infliximab reduced the proinflammatory response by suppressing M1 monocytes and CD1a expressing monocytes and macrophages and decreased levels of IFNγ, TARC and HGF mRNA. As predicted by the disease network inflammation aggravated in the presence of pitrakinra as indicated by the clinical and histological score, elevated frequencies of CD1a expressing macrophages and TNFα and IFNγ mRNA levels.ConclusionsThe combination of the disease network and the NSG-UC animal model might be developed into a powerful tool to predict efficacy or in-efficacy and potential mechanistic side effects.

Expression of CD47 in de novo acute myelogenous leukemia with normal karyotype and its clinical significance

Objective To investigate the CD47 expression in de novo acute myelogenous leukemia (AML) patients with normal karyotype and its clinical significance. Methods One hundred thirty-seven cases of de novo AML with normal karyotype and 3 healthy volunteers were selected. Relative CD47 expressions in normal bone marrow hematopoietic stem cells (HSC) and multipotent progenitor (MPP) from healthy volunteers, as well as bone marrow mononuclear cells (MNC) and leukemia stem cells (LSC, Lin- CD34+ CD38- CD90-) from AML patients were determined by flow cytometry. CD47 expression on the Lin- CD34+ CD38- LSC-enriched fraction of specimen was determined by flow cytometry. The FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) was detected by using the Genome Analyzer platform. CD34+ CD38- CD47hi and CD34+ CD38- CD47lo expressing cells were identified and purified using FACS. Two groups of cells were inoculated with MethoCult H4445 medium on agarose-containing methylcellulose plates. After 12 days, MPP colony forming units (CFU) were counted, and 1×105 CD34+ CD38- CD47lo and CD34+ CD38- CD47hi cells were transplanted into NSG (NOD-SCID IL-2R γ null) mice irradiated by 280 cGy, and mice were sacrificed after 8 weeks. The ratio of human CD45+ cells was detected by flow cytometry. Results The expression of CD47 in AML patients was higher than that in the healthy control. CD47 was expressed in all FAB (French-American-British) subtypes of AML. No significant difference in CD47 expression among different FAB subtypes was found (F= 0.545, P > 0.05). Among the 37 patients with CD34+ CD38- CD47hi, 17 (46%) were FLT3-ITD negative, and 20 (54%) were FLT3-ITD positive. Among the 100 patients with CD34+ CD38- CD47hi, 63 (63%) cases were FLT3-ITD negative, 37 (37%) cases were FLT3-ITD positive. The rate of FLT3-ITD positive in patients with CD34+ CD38- CD47lo had no statistical difference compared with patients with CD34+ CD38- CD47hi (χ 2= 3.79, P > 3.79). The CD34+ CD38- CD47lo or CD34+ CD38- CD47hi which was selected by FACS, was inoculated with the methylcellulose plate containing agarose for 12 days, and CD34+ CD38- CD47lo cells could form CFU. The NSG mouse transplantation experiment showed that CD34+ CD38- CD47lo cells could be reconstructed hematopoiesis, and CD34+ CD38- CD47hi implantation failed. Conclusion CD34+ CD38- CD47hi could enrich LSC, which may be a potential marker to detect minimal residual disease. Key words: Leukemia, myelogenous, acute; Antigens, CD47; Colony-forming units assay

Intrathymic injection of hematopoietic progenitor cells establishes functional T cell development in a mouse model of severe combined immunodeficiency

BackgroundEven 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.MethodsHematopoietic 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.ResultsDespite 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.ConclusionsOur 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.

Continuously expanding CAR NK-92 cells display selective cytotoxicity against B-cell leukemia and lymphoma

Background aimsNatural killer (NK) cells can rapidly respond to transformed and stressed cells and represent an important effector cell type for adoptive immunotherapy. In addition to donor-derived primary NK cells, continuously expanding cytotoxic cell lines such as NK-92 are being developed for clinical applications. MethodsTo enhance their therapeutic utility for the treatment of B-cell malignancies, we engineered NK-92 cells by lentiviral gene transfer to express chimeric antigen receptors (CARs) that target CD19 and contain human CD3ζ (CAR 63.z), composite CD28-CD3ζ or CD137-CD3ζ signaling domains (CARs 63.28.z and 63.137.z). ResultsExposure of CD19-positive targets to CAR NK-92 cells resulted in formation of conjugates between NK and cancer cells, NK-cell degranulation and selective cytotoxicity toward established B-cell leukemia and lymphoma cells. Likewise, the CAR NK cells displayed targeted cell killing of primary pre-B-ALL blasts that were resistant to parental NK-92. Although all three CAR NK-92 cell variants were functionally active, NK-92/63.137.z cells were less effective than NK-92/63.z and NK-92/63.28.z in cell killing and cytokine production, pointing to differential effects of the costimulatory CD28 and CD137 domains. In a Raji B-cell lymphoma model in NOD-SCID IL2R γnull mice, treatment with NK-92/63.z cells, but not parental NK-92 cells, inhibited disease progression, indicating that selective cytotoxicity was retained in vivo. ConclusionsOur data demonstrate that it is feasible to generate CAR-engineered NK-92 cells with potent and selective antitumor activity. These cells may become clinically useful as a continuously expandable off-the-shelf cell therapeutic agent.

A new humanized in vivo model of KIT D816V+ advanced systemic mastocytosis monitored using a secreted luciferase.

Systemic mastocytosis are rare neoplasms characterized by accumulation of mast cells in at least one internal organ. The majority of systemic mastocytosis patients carry KIT D816V mutation, which activates constitutively the KIT receptor. Patient with advanced forms of systemic mastocytosis, such as aggressive systemic mastocytosis or mast cell leukemia, are poorly treated to date. Unfortunately, the lack of in vivo models reflecting KIT D816V+ advanced disease hampers pathophysiological studies and preclinical development of new therapies for such patients. Here, we describe a new in vivo model of KIT D816V+ advanced systemic mastocytosis developed by transplantation of the human ROSAKIT D816V-Gluc mast cell line in NOD-SCID IL-2R g−/− mice, using Gaussia princeps luciferase as a reporter. Intravenous injection of ROSAKIT D816V-Gluc cells led, in 4 weeks, to engraftment in all injected primary recipient mice. Engrafted cells were found at high levels in bone marrow, and at lower levels in spleen, liver and peripheral blood. Disease progression was easily monitored by repeated quantification of Gaussia princeps luciferase activity in peripheral blood. This quantification evidenced a linear relationship between the number of cells injected and the neoplastic mast cell burden in mice. Interestingly, the secondary transplantation of ROSAKIT D816V-Gluc cells increased their engraftment capability. To conclude, this new in vivo model mimics at the best the features of human KIT D816V+ advanced systemic mastocytosis. In addition, it is a unique and convenient tool to study the kinetics of the disease and the potential in vivo activity of new drugs targeting neoplastic mast cells.

Targeting the entry region of Hsp90's ATP binding pocket with a novel 6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl amide

The molecular chaperone Hsp90 plays an important role in cancer cell survival and proliferation by regulating the maturation and stabilization of numerous oncogenic proteins. Due to its potential to simultaneously disable multiple signaling pathways, Hsp90 has emerged as an attractive therapeutic target for cancer treatment. In this study, the design, synthesis, and biological evaluation of a series of Hsp90 inhibitors are described. Among the synthetic compounds, 6,7-dihydrothieno [3,2-c]pyridin-5(4H)-yl amide 19 exhibits a remarkable binding affinity to the N-terminus of Hsp90 in a fluorescence polarization (FP) binding assay (IC50 = 50.3 nM). Furthermore, it effectively inhibits the proliferation of H1975 non-small cell lung cancer (NSCLC) and Skbr3 breast cancer cell lines with GI50 values of 0.31 μM and 0.11 μM, respectively. Compound 19 induces the degradation of the Hsp90 client proteins including EGFR, Her2, Met, c-Raf, and Akt, and consequently promotes apoptotic cancer cell death. Compound 19 also inhibits the growth of H1975 xenografts in NOD-scid IL2R gammanull mice without any apparent body-weight loss. The immunohistologic evaluation indicates that compound 19 decreases the expression of Akt in xenograft tumor tissue via an inhibition of the Hsp90 chaperon function. Additionally, the cytochrome P450 assay indicates that compound 19 has no effect on the activities of five major P450 isoforms (IC50 > 50 μM for 1A2, 2C9, 2C19, 2D6, and 3A), suggesting that clinical interactions between compound 19 and the substrate drugs of the five major P450 isoforms are not expected. Overall, compound 19 represents a new class of Hsp90 inhibitor with its 6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl-amide structure, and it has the therapeutic potential to overcome drug resistance in cancer chemotherapy.