IL2Rγnull Mice Research Articles

NOD-scid IL2rγnull mice lacking TLR4 support human immune system development and the study of human-specific innate immunity.

Agents that induce inflammation have been used since the 18th century for the treatment of cancer. The inflammation induced by agents such as Toll-like receptor agonists is thought to stimulate tumor-specific immunity in patients and augment control of tumor burden. While NOD-scid IL2rγnull mice lack murine adaptive immunity (T cells and B cells), these mice maintain a residual murine innate immune system that responds to Toll-like receptor agonists. Here we describe a novel NOD-scid IL2rγnull mouse lacking murine TLR4 that fails to respond to lipopolysaccharide. NSG-Tlr4null mice support human immune system engraftment and enable the study of human-specific responses to TLR4 agonists in the absence of the confounding effects of a murine response. Our data demonstrate that specific stimulation of TLR4 activates human innate immune systems and delays the growth kinetics of a human patient-derived xenograft melanoma tumor.

Expanded natural killer cells potentiate the antimyeloma activity of daratumumab, lenalidomide, and dexamethasone in a myeloma xenograft model

The development of new treatment agents in recent decades has significantly improved the survival of patients with multiple myeloma (MM). Nonetheless, MM remains an incurable disease; therefore, novel combination therapies are required. Natural killer (NK) cells are one of the safest immunotherapeutic options. In this study, we found that the anti-myeloma activity of expanded NK cells (eNKs) was improved by daratumumab, lenalidomide, and dexamethasone (DRd) in an MM xenograft mouse model. NK cells expanded from peripheral blood mononuclear cells collected from MM patients were highly cytotoxic against DRd pretreated tumor cells in vitro. To mimic the clinical protocol, a human MM xenograft model was developed using human RPMI8226-RFP-FLuc cells in NOD/SCID IL-2Rγnull (NSG) mice. MM bearing mice were randomly divided into six groups: no treatment, eNK, Rd, Rd + eNKs, DRd, and DRd + eNKs. DRd significantly enhanced the cytotoxicity of eNKs by upregulating NK cell activation ligands and effector function. DRd in combination with eNKs significantly reduced the serum M-protein level and prolonged mouse survival. In addition, DRd significantly increased the persistence of eNK and homing to MM sites. These results show that the anti-myeloma activity of ex vivo-expanded and activated NK cells is augmented by the immunomodulatory effect of DRd in MM-bearing mice, suggesting the therapeutic potential of this combination for MM patients.

Fusion of uPA to Anti-aIIbb3 Scfv Facilitates Its Uptake By in Vitro-Generated CD34+ Megakaryocytes (MKs) to Promote Targeted Fibrinolysis

We wish to develop a clinically relevant strategy to deliver anti-thrombotic urokinase plasminogen activator (uPA)-carrying platelets (uPA-PLTs) as a targeted thromboprophylaxis that can be administered to patients with a concurrent high risk of bleeding. This intent was based on our previous data showing that transgenic mice, ectopically expressing uPA in megakaryocytes (MKs), released PLTs, containing uPA in α granules, that were effective as a thromboprophylaxis agent (PMID12689937). We faced two major challenges in further developing uPA-containing PLTs for clinical application: (1) How to get uPA into PLTs as human Plts do not take up uPA, and (2) α-granular uPA led to proteolysis of other α-granule proteins. To diminish degradation of α-granular proteins by uPA, we generated a plasmin-insensitive, but thrombin-activatable, truncated uPA mutant (uPA-T, bottom, Fig. 2). We found that both native single-chain uPA (scuPA) and uPA-T can be taken up by human MKs into the same α granules whereby they co-localize with endocytosed Factor V (FV) and/or fibrinogen (FBGN), as evidenced by confocal microscopy, suggesting that the uPA variants use the same endocytic receptor pathway, also utilized by FV and FBGN (Fig. 1). To delineate what receptor system(s) mediate uPA uptake by CD34+ MK, we found that FV (known to be partially internalized via low-density lipoprotein receptor-related protein 1 (LRP1) competed with both uPA variants for uptake, and that uptake of uPA variants and FV was partially inhibited by the LRP1 antagonist, receptor-associated protein (RAP), further supporting that LRP1 mediates internalization of the uPAs. We also found that uPA and FBGN uptake was partially inhibited by αIIbβ3 integrin antagonist ReoPro. Since we observed that LRP1 protein expression and extent of uPA uptake by MKs declines upon maturation, whereas αIIbβ3 expression does not, we tested if targeting of uPA to αIIbβ3 would facilitate its uptake by CD34+ MKs. We generated a chimeric prodrug composed of a single-chain version of the variable region (scFv) of an anti-αIIbβ3 monoclonal antibody fused to uPA-T (PLT/uPA-T, bottom Fig. 2) which binds human αIIbβ3 on both quiescent and activated PLTs, and is enzymatically activated specifically by thrombin. We have previously shown that in two murine injury models, PLT/uPA-T did not lyse preexisting clots, while it concurrently prevented the development of nascent thrombi (PMC4731161). However, direct infusion of PLT/uPA-T in mice caused significant thrombocytopenia. Now, we found that CD34+ MK internalized PLT/uPA-T into the same α granules of MKs, but with significantly higher efficiency than uPA-T protein (top Fig. 2) which will optimize loading of MKs by uPA-T for subsequent use as a source of uPA-PLTs. We found that scuPA-loaded platelets released from infused MKs prevent thrombus formation in a Rose Bengal photochemical injury model. These studies were done using immunocompromised NOD-scid IL2rγnull (NSG) mice homozygous for VWFR1326H (a VWF mutation switching binding specificity from mouse to human GPIb/IX). NSG/VWFR1326H mice had impaired clotting after vascular injury compared to NSG mice unless infused with human PLTs or MKs. We found that MKs that endocytosed uPA-T can also prevent thrombi formation in this model. Future studies will test whether PLTs released from PLT/uPA-T-loaded MKs would further enhance efficacy of this thromboprophylactic strategy, while avoiding thrombocytopenia, and can become a point-of-care thromboprophylaxis therapeutic in the clinical setting of high-risk of developing thrombi with concurrent concerns of inducing significant bleeding. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Anti-Myeloma Effect of Compound K and Rh2 in a Multiple Myeloma Xenograft Model

Introduction: Several ginsenosides have demonstrated anti-cancer activity due to their ability to inhibit DNA synthesis and angiopoiesis, inducing cancer death. Rh2, a PPD-type 20(S)-ginsenoside, has been extensively studied as a major ingredient with antineoplastic properties. Moreover, the characteristics of compound K (CK), a PPD ginsenoside metabolite, resulting from the metabolic activity of enterobacteria, have been investigated. The effect of ginsenoside has been actively studied in leukemia but not in multiple myeloma (MM). Thus, the aim of study was to identify ginsenosides that have toxic effects against MM cells and to elucidate the underlying molecular mechanism. Methods: First, we screened the toxic effects of ginsenosides Rb1, Rb3, Rg1, Rg3, Rh1, Rh2, and CK at a concentration of 50 μM in several MM cell lines (ARH77, U266, and RPMI8226) after 24 hours of exposure. Furthermore, ARH77 cells were incubated with Rh2 and CK at 0, 20 μM, 50 μM, 60 μM, and 70 μM for 24 hours, and the DNA content at each stage of the cell cycle was detected. The potential cytotoxic effect of Rh2 and CK was investigated in normal hematopoietic stem cells (HSCs) isolated from healthy donors using primary peripheral blood stem cells (PBSCs). For this experiment, HSCs were isolated using a PBSC collection procedure after the recruitment of healthy donors. Briefly, granulocyte colony-stimulating factor was administered continuously until the white blood cell count exceeded the threshold of 1 × 109 cells/L. The subjects underwent leukapheresis once or twice using a blood cell separator. To quantify the production of PBSCs from each leukapheresis, flow cytometry was carried out to quantify CD34+ cells. To establish a human MM xenograft model, RPMI8226-RFP-FLuc cells (5 × 106 per mouse) were intravenously injected into 9-12-week-old male and female NOD/SCID IL-2Rγnull (NSG) mice. To assess the anti-MM effect of CK and Rh2 in the MM xenograft model, NSG mice were divided into the following treatment groups: Experiment set 1: no treatment (PBS control), CK (15 mg/kg/day), Rh2 (15 mg/kg/day), K (30 mg/kg/day), and Rh2 (30 mg/kg/day). Mice were treated with either CK or Rh2 (15 or 30 mg/kg/day) by intraperitoneal injection for 3 days in a week at a 1-day interval. Experiment set 2: no treatment (PBS control), CK (7 mg/kg/day), Rh2 (7 mg/kg/day), and Lenalidomide (1 mg/kg/day). Mice were treated with either CK or Rh2 (7 mg/kg/day) by intravenous injection for 3 days in a week at a 1-day interval. Lenalidomide (1 mg/kg/day) treatment was given by oral administration from day 14 for 5 consecutive days in a week, and it was repeated for 3 weeks at a 2-day interval in each week. Tumor growth was monitored weekly by bioluminescence imaging in the dorsal view; 10 min before imaging, mice were intraperitoneally injected with D-Luciferin (150 mg/kg/mouse). Results: The ginsenosides Rh2 and CK inhibited the cell growth, exerted cytotoxicity, and induced G1 phase cell cycle arrest in human MM cells. However, these treatments demonstrated no cytotoxic effect on the self-renewal and the differentiation capacity of normal primary PBSCs. This observation implied that the cytotoxic activity of Rh2 and CK might selectively target MM cells and that these ginsenosides are safe in other cell types. Finally, we studied the anti-MM effect of CK and Rh2 in a clinically relevant human MM xenograft model. PBS was given in the no treatment control group and all untreated tumor-bearing mice showed rapid tumor growth and severe plasmacytoma, which led to death within 7 weeks. Notably, mice treated with CK and Rh2 showed significant inhibition of tumor growth and a longer survival. Conclusions: Ginsenosides CK and Rh2 inhibited the growth of MM cells in an in vitro and in vivo model without any significant cytotoxic effect on the self-renewal and differentiation capacity of PBSCs. Thus, these ginsenosides could be used in MM treatment and can be combined with drugs without exerting any severe toxic effects. Keywords: Ginsenosides; Rh2; Compound K; Multiple myeloma Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MRSA Infection in the Thigh Muscle Leads to Systemic Disease, Strong Inflammation, and Loss of Human Monocytes in Humanized Mice.

MRSA (Methicillin-resistant Staphylococcus aureus) is the second-leading cause of deaths by antibiotic-resistant bacteria globally, with more than 100,000 attributable deaths annually. Despite the high urgency to develop a vaccine to control this pathogen, all clinical trials with pre-clinically effective candidates failed so far. The recent development of “humanized” mice might help to edge the pre-clinical evaluation closer to the clinical situation and thus close this gap. We infected humanized NSG mice (huNSG: (NOD)-scid IL2Rγ null mice engrafted with human CD34+ hematopoietic stem cells) locally with S. aureus USA300 LAC* lux into the thigh muscle in order to investigate the human immune response to acute and chronic infection. These mice proved not only to be more susceptible to MRSA infection than wild-type or “murinized” mice, but displayed furthermore inferior survival and signs of systemic infection in an otherwise localized infection model. The rate of humanization correlated directly with the severity of disease and survival of the mice. Human and murine cytokine levels in blood and at the primary site of infection were strongly elevated in huNSG mice compared to all control groups. And importantly, differences in human and murine immune cell lineages surfaced during the infection, with human monocyte and B cell numbers in blood and bone marrow being significantly reduced at the later time point of infection. Murine monocytes in contrast behaved conversely by increasing cell numbers. This study demonstrates significant differences in the in vivo behavior of human and murine cells towards S. aureus infection, which might help to sharpen the translational potential of pre-clinical models for future therapeutic approaches.

FC041: Regeneration of Nephrons and Renal Stroma using the Renal Developmental Environment of Animal Fetuses

Abstract BACKGROUND AND AIMS The kidney is derived from three types of progenitor cells: nephron progenitor cells (NPCs), stromal progenitor cells (SPCs) and ureteric buds (UBs). Extensive research in regenerative medicine has made it possible to induce pluripotent stem cells (PSCs) to develop into NPCs and UBs in vitro and generate the organoids of nephrons and collecting ducts. Renal stroma, including mesangial cells and erythropoietin- and renin-producing cells, is essential for maintaining biological functions. However, to our knowledge, the induction of SPC differentiation from PSCs has not been established yet. Moreover, the renal stroma cannot mature in the in vitro environment, making it difficult to generate the whole kidney, including the renal stroma. In this study, we attempted to regenerate the renal stroma in the in vivo environment by transplanting rat SPCs into the nephrogenic zone of mouse fetuses to provide rat SPCs with the renal developmental mechanism of heterologous mouse fetuses. Furthermore, we developed an interspecies dual replacement of progenitor (i-DROP) system wherein two progenitor cell lines, NPCs and SPCs, were genetically modified and replaced with transplanted progenitor cells and examined the feasibility of the regeneration of kidneys, including nephrons and renal stroma. METHOD We crossed Foxd1GC/+ mice with R26RDTR/DTR mice to generate Foxd1GC/+; R26RDTR/+ mice (Foxd–DTR mice) can eliminate Foxd1-positive SPCs. A group of SPC-rich cells extracted from green fluorescent protein (GFP) rat metanephros targeting platelet-derived growth factor receptor alpha (PDGFRa) was transplanted into the kidneys of Foxd1–DTR mouse fetuses. Subsequently, the organ-cultured metanephros or the transplanted metanephros in immunodeficient NOD/Shi- scid IL2rγnull (NOG) mice or Sprague–Dawley (SD) rats were evaluated using immunostaining. Next, we assessed whether the i-DROP system could regenerate interspecies nephrons and stroma. We crossed Six2TGC/+; Foxd1GC/+ mice obtained from Six2TGC/+ mice and Foxd1GC/+ mice with R26RDTR/DTR mice to generate Six2TGC/+; Foxd1GC/+; and R26RDTR/+ mice (Six2/Foxd1–DTR mice) that can simultaneously eliminate Six2-positive NPCs and Foxd1-positive SPCs. The SPCs targeted to PDGFRa+ and NPCs targeted to PDGFRa−/integrin alpha 8 (Itga8)+ from GFP rat metanephros were simultaneously transferred to Six2/Foxd1–DTR mouse metanephros and evaluated similarly. RESULTS In the organ culture of Foxd1–DTR mouse metanephros, SPCs around the cap mesenchyme replaced from mouse to GFP rat. We achieved extensive rat stromal cell regeneration in mouse kidneys, including endocrine functions such as erythropoietin production in the rat in response to anemia in the host mouse. Next, in the organ culture of the hind kidney of Six2/Foxd1–DTR mice equipped with the i-DROP system, the NPCs and SPCs of GFP rats aggregated around the mouse UB and the cap mesenchyme reconstituted. In vivo, rat nephrons and renal stroma were regenerated simultaneously in the mouse kidneys. The newly generated glomeruli were composed of GFP-positive rat cells, both podocytes and mesangial cells of different lineages (Figure 1). Intravenous injection of fluorescently labeled dextran into host mice revealed that the regenerated nephrons had glomerular filtration and tubular reabsorption capabilities. Furthermore, taking the xenotransplantation of mouse metanephros into SD rats as a control, the transplantation of regenerated rat kidneys into SD rats using our method resulted in reduced xenogenic immune rejection. CONCLUSION The newly developed i-DROP system regenerated multifunctional kidneys, composed of nephrons and renal stroma originating from different species, with urine production and endocrine functions. Functional human kidneys could be regenerated in the future using the renal developmental mechanism of large heterologous animals, such as pigs.

Development and characterization of a humanized mouse model of osteoarthritis

In light of the role of immune cells in OA pathogenesis, the development of sophisticated animal models closely mimicking the immune dysregulation during the disease development and progression could be instrumental for the preclinical evaluation of novel treatments. Among these models, immunologically humanized mice may represent a relevant system, particularly for testing immune-interacting DMOADs or cell therapies before their transfer to the clinic. Our objective, therefore, was to develop an experimental model of OA by destabilization of the medial meniscus (DMM) in humanized mice. Irradiated 5-week-old NOD/LtSz-scid IL2Rγnull (NSG) mice were humanized by intravenous injection of CD34+ human hematopoietic stem cells. The engraftment efficiency was evaluated by flow cytometry 17 weeks after the humanization procedure. Humanized and non-humanized NSG mice underwent DMM or sham surgery and OA development was assessed 1, 6, and 12 weeks after the surgery. 120 days after the humanization, human T and B lymphocytes, macrophages and NK cells, were present in the blood and spleen of the humanized NSG mice. The DMM surgery induced articular cartilage and meniscal alterations associated with an increase in OA and the meniscal score. Moreover, the surgery triggered an inflammatory response that was sustained at a low grade in the DMM group. Our study shows for the first time the feasibility of inducing OA by DMM in humanized mice. This novel OA model could constitute a useful tool to bridge the gap between the preclinical and clinical evaluation of immune interacting DMOADs and cell-based therapies.

An instructive role for Interleukin-7 receptor \u03b1 in the development of human B-cell precursor leukemia

Kinase signaling fuels growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Yet its role in leukemia initiation is unclear and has not been shown in primary human hematopoietic cells. We previously described activating mutations in interleukin-7 receptor alpha (IL7RA) in poor-prognosis “ph-like” BCP-ALL. Here we show that expression of activated mutant IL7RA in human CD34+ hematopoietic stem and progenitor cells induces a preleukemic state in transplanted immunodeficient NOD/LtSz-scid IL2Rγnull mice, characterized by persistence of self-renewing Pro-B cells with non-productive V(D)J gene rearrangements. Preleukemic CD34+CD10highCD19+ cells evolve into BCP-ALL with spontaneously acquired Cyclin Dependent Kinase Inhibitor 2 A (CDKN2A) deletions, as commonly observed in primary human BCP-ALL. CRISPR mediated gene silencing of CDKN2A in primary human CD34+ cells transduced with activated IL7RA results in robust development of BCP-ALLs in-vivo. Thus, we demonstrate that constitutive activation of IL7RA can initiate preleukemia in primary human hematopoietic progenitors and cooperates with CDKN2A silencing in progression into BCP-ALL.

Insights into Endogenous Vs Exogenous Cargo-Containing Platelet Alpha-Granules

Alpha granules in megakaryocytes contain a mixture of endogenously expressed proteins as well as proteins taken up from the intramedullar fluid. Both pools are thought to be found in all alpha granules in the megakaryocytes and released platelets. We have been studying the ectopic expression of urokinase (uPA) in platelets as a targeting strategy for fibrinolysis of nascent thrombi without causing fibrinolysis of established thrombi. These studies also demonstrated that there are two distinct pools of alpha granules, an endogenous cargo pool of granules and an exogenous uptake cargo pool of granules.Using in vitro grown megakaryocytes from two sources (1) CD34+-hematopoietic progenitor cells and (2) induced-pluripotent stem cell derived line imMKCL kindly provided by Dr. Koji Eto at Kyoto University, we demonstrated that urokinase can be localized within alpha granules in the megakaryocytes by either adding urokinase to the media or by ectopically expressing the protein using a lentiviral strategy. We observed that both a human single-chain uPA (scuPA) or a plasmin-insensitive but thrombin-activatable truncated human uPA mutant (uPA-T) in the media were internalized into granules distinct from granules containing ectopically expressed mouse scuPA following lentiviral transduction. Endocytosed uPA showed no co-localization with endogenous von Willebrand Factor (vWF), but significant colocalization with endocytosed Factor V or plasminogen (PLG) on confocal immunofluorescent microscopy. Further, Factor V competed with both uPA variants for uptake from the media. Uptake of these proteins was inhibited by the LRP1 antagonist receptor-associated protein (RAP) and by anti-LRP1 antibodies. This suggests that both proteins use the same endocytic receptor pathway and share this pathway with other proteins taken up from the media, including Factor V.We found that in vitro-generated CD34+ megakaryocytes pre-loaded with exogenously added PLG and co-incubated thereafter with recombinant scuPA and FV significantly degraded FV; however, no vWF degradation was observed in CD34+-derived megakaryocytes that had endocytosed or ectopically expressed scuPA with exogenously added PLG, suggesting that only the proteins which are endocytosed by in vitro-generated megakaryocytes are degraded by uPA-generated plasmin, whereas endogenous alpha-granular proteins remain intact.We then asked whether uPA localized in these two distinct pools can be released at sites of nascent thrombus formation and be effective in preventing nascent thrombus growth. We infused CD34+-derived MKs into NOD-scid IL2rγnull (NSG) mice homozygous for VWF R1326H (a mutation switching binding VWF specificity from mouse to human GPIb/IX). NSG/VWF R1326H mice have impaired clotting after vascular injury compared to NSG mice unless infused with human platelets or MKs . Significantly less post-injury clotting was seen upon infusion of either endogenous or exogenous scuPA-containing MK infusion. Further studies to define relative efficacy at the same levels of scuPA are being pursued.These studies show that there are two sets of alpha granules that remain separate during megakaryopoiesis in vitro: granules with endogenously expressed cargo and granules with endocytosed cargo with limited mixing between the two pools by confocal microscopy studies and following PLG uptake studies. The extent of mixing that occurs subsequently in released platelets was not studied nor has these finding been done with primary MKs not grown in culture; however, we believe that these studies extend our understanding of the nature of alpha granules and offer new insights into how to manipulate their cargo. DisclosuresCines: Dova: Consultancy; Rigel: Consultancy; Treeline: Consultancy; Arch Oncol: Consultancy; Jannsen: Consultancy; Taventa: Consultancy; Principia: Other: Data Safety Monitoring Board.

Human stem cell-derived thymic epithelial cells enhance human T-cell development in a xenogeneic thymus

Generation of thymic tissue from pluripotent stem cells would provide therapies for acquired and congenital thymic insufficiency states. This study aimed to generate human thymic epithelial progenitors from human embryonic stem cells (hES-TEPs) and to assess their thymopoietic function invivo. This study differentiated hES-TEPs by mimicking developmental queues with FGF8, retinoic acid, SHH, Noggin, and BMP4. Their function was assessed in reaggregate cellular grafts under the kidney capsule and in hybrid thymi by incorporating them into swine thymus (SwTHY) grafts implanted under the kidney capsules of immunodeficient mice that received human hematopoietic stem and progenitor cells (hHSPCs) intravenously. Cultured hES-TEPs expressed FOXN1 and formed colonies expressing EPCAM and both cortical and medullary thymic epithelial cell markers. In thymectomized immunodeficient mice receiving hHSPCs, hES-TEPs mixed with human thymic mesenchymal cells supported human T-cell development. Hypothesizing that support from non-epithelial thymic cells might allow long-term function of hES-TEPs, the investigators injected them into SwTHY tissue, which supports human thymopoiesis in NOD severe combined immunodeficiency IL2Rγnull mice receiving hHSPCs. hES-TEPs integrated into SwTHY grafts, enhanced human thymopoiesis, and increased peripheral CD4+ naive T-cell reconstitution. This study has developed and demonstrated invivo thymopoietic function of hES-TEPs generated with a novel differentiation protocol. The SwTHY hybrid thymus model demonstrates beneficial effects on human thymocyte development of hES-TEPs maturing in the context of a supportive thymic structure.

P-006: Expanded natural killer cells with daratumumab, lenalidomide and dexamethasone combination potentiates antimyeloma activity in a myeloma xenograft model

<h3>Background</h3> Advances in the treatment of multiple myeloma (MM) significantly improved overall survival of patients. Nonetheless, MM still remains an incurable disease. This emphasizes urgent need for novel combination therapies for the treatment of MM. Recently, natural killer (NK) cells are becoming an attractive and safe tool in the field of immunotherapy. <h3>Methods</h3> Here, we investigated how daratumumab, lenalidomide, and dexamethasone (DRd) treatment potentiate antitumor effects of NK cells in a MM xenograft mouse model. NK cells were expanded and activated using K562-OX40 ligand and membrane-bound IL-18 and IL-21 in the presence of IL-2 and IL-15 from peripheral blood mononuclear cells of MM patients. A human MM xenograft model was established using human RPMI8226-RFP-FLuc cells in NOD/SCID IL-2Rγnull (NSG) mice. Tumor bearing mice were divided into six treatment groups: no treatment, expanded NK cells (eNK), Rd, Rd + eNK, DRd, and DRd + eNK. DRd treatment strongly enhanced the cytotoxicity of eNK by upregulating NK cell's activation ligands and effector function. <h3>Results</h3> DRd combination with eNK significantly prolonged mouse survival and reduced MM progression and serum M-protein level. Moreover, DRd treatment significantly increased eNK persistence and homing into MM sites. <h3>Conclusions</h3> Our findings suggest that DRd treatment augments the anti-myeloma effects of ex vivo expanded and activated NK cells by modulating immune responses in MM-bearing mice.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34&lt;sup&gt;+&lt;/sup&gt; Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2Rγnull (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2Rγnull (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Gene therapy in a murine model of chronic eosinophilic leukemia-not otherwise specified (CEL-NOS).

Chronic eosinophilic leukemia-not otherwise specified (CEL-NOS) is a rare, aggressive, fatal disease characterized by blood eosinophilia and dysfunction of organs infiltrated with eosinophils. Clinically, the disease manifests with weight loss, cough, weakness, diarrhea, and multi-organ dysfunction that is unresponsive to therapy. We developed a one-time gene therapy for CEL-NOS using an adeno-associated virus (AAV) expressing an anti-eosinophil monoclonal antibody (AAVrh.10mAnti-Eos) to provide sustained suppression of eosinophil numbers in blood, thus reducing eosinophil tissue invasion and organ dysfunction. A novel CEL-NOS model was developed in NOD-scid IL2rγnull (NSG) mice by administration of AAV expressing the cytokine IL5 (AAVrh.10mIL5), resulting in marked peripheral and tissue eosinophilia of the heart, lung, liver, and spleen, and eventually death. Mice were administered AAVrh.10mAnti-Eos (1011 genome copies) 4 wk after administration of AAVrh.10mIL5 and evaluated for anti-eosinophil antibody expression, blood eosinophil counts, organ eosinophil invasion, and survival. AAVrh.10mAnti-Eos expressed persistent levels of the anti-eosinophil antibody for >24 wk. Strikingly, CEL-NOS treated mice had markedly lower blood eosinophil levels and reduced mortality when compared with control treated mice. These results suggest that a single treatment with AAVrh.10mAnti-Eos has the potential to provide substantial therapeutic benefit to patients with CEL-NOS, a fatal malignant disorder.

PD44-05 N-CADHERIN ANTAGONISTS N-Ac-CHAVC-NH2 RESCUE IMMUNOSUPPRESS CAUSED BY PD- L1/IDO-1 IN PROSTATE

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II (PD44)1 Sep 2021PD44-05 N-CADHERIN ANTAGONISTS N-Ac-CHAVC-NH2 RESCUE IMMUNOSUPPRESS CAUSED BY PD- L1/IDO-1 IN PROSTATE Yi Sun, Jiaoti Huang, Qiang Wei, and Lu Yang Yi SunYi Sun More articles by this author , Jiaoti HuangJiaoti Huang More articles by this author , Qiang WeiQiang Wei More articles by this author , and Lu YangLu Yang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002058.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Few prostate cancer patients benefit from current immunotherapies. We therefore aimed to explore new strategies to change this paradigm. METHODS: Human tissues, cell lines and in vivo experiments were used to determine whether and how N-cadherin impacts the production of PD-L1 and IDO-1. Then, we used PC3 bearing humanized nonobese diabetic/severe combined immunodeficiency IL2Rγnull (hNSG) mice with an intravenous injection of human CD34+ haematopoietic stem cells into the tail vein to test if the N-cadherin antagonists N-Ac-CHAVC-NH2 (designated ADH-1) could improve the therapeutic effect of tumour-infiltrating lymphocyte (TIL)-related treatment. RESULTS: N-cadherin dramatically upregulated the expression of PD-L1 and IDO-1 through IFN-g signalling, which could be reversed by N-cadherin knockout. In preclinical experiments, immune reconstitution obtained by TILs slowed tumour growth and extended survival time; however, this effect disappeared after immune system suppression by PD-L1 and IDO-1. Furthermore, ADH-1 effectively reduced the immunosuppression and enforced TIL-related therapy. CONCLUSIONS: These data show that the N-cadherin antagonist ADH-1 promotes TIL antitumour responses. This important hurdle must be overcome for tumours to generate a response to immunotherapy. Source of Funding: This study was supported by the National Natural Science Foundation of China (Grant No. 81370855, 81300627, 81702536, 81770756 and 81200551) for L.Y. and Q.W., This study was supported by the National key research and development program of China (Grant No. SQ2017YFSF090096) Q.W., the Prostate Cancer Foundation Young Investigator Award 2013, Foundation of Science &Technology Department of Sichuan Province (Grant No. 2015SZ0230, 2013SZ0006 and 2013SZ0093) for Q.W., Programs from Science and Technology Department of Sichuan Province (Grant No. 2018JY0089 and 2017HH0063) for Q.W., Young Investigator Award of Sichuan University 2017, and the Scientific Research Project of Health Department of Sichuan Province (No. 120203) for L.Y. Guangdong Basic and Applied Basic Research Foundation (2019A1515110125) for J.J. The National Natural Science Foundation of China (grant no. 81860454) for J.G. © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e738-e739 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yi Sun More articles by this author Jiaoti Huang More articles by this author Qiang Wei More articles by this author Lu Yang More articles by this author Expand All Advertisement Loading ...

Efficacy of a combination therapy targeting CDK4/6 and autophagy in a mouse xenograft model of t(8;21) acute myeloid leukemia

One of the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML) is t(8;21). Although patients with t(8;21) AML have a more favorable prognosis than other cytogenetic subgroups, relapse is still common and novel therapeutic approaches are needed. A recent study showed that t(8;21) AML is characterized by CCND2 deregulation and that co-inhibition of CDK4/6 and autophagy induces apoptosis in t(8;21) AML cells. In this study, we examined the in vivo effects of co-inhibiting CDK4/6 and autophagy. We used a mouse model in which t(8;21)-positive Kasumi-1 cells were subcutaneously inoculated into NOD/Shi-scid IL2Rgnull mice. The mice were treated with the autophagy inhibitor chloroquine (CQ), a CDK4/6 inhibitor (either abemaciclib or palbociclib), or a CDK4/6 inhibitor plus CQ. After 20 days of treatment, tumor volume was measured, and immunostaining and transmission electron microscopy observations were performed. There was no change in tumor growth in CQ-treated mice. However, mice treated with a CDK4/6 inhibitor plus CQ had significantly less tumor growth than mice treated with a CDK4/6 inhibitor alone. CDK4/6 inhibitor treatment increased the formation of autophagosomes. The number of single-strand DNA-positive (apoptotic) cells was significantly higher in the tumors of mice treated with a CDK4/6 inhibitor plus CQ than in mice treated with either CQ or a CDK4/6 inhibitor. These results show that CDK4/6 inhibition induces autophagy, and that co-inhibition of CDK4/6 and autophagy induces apoptosis in t(8;21) AML cells in vivo. The results suggest that inhibiting CDK4/6 and autophagy could be a novel and promising therapeutic strategy in t(8;21) AML.

Abstract 85: Metastasizing melanoma cells exhibit increased dependence on the pentose phosphate pathway to manage oxidative stress

Abstract To study distant metastasis we developed a patient-derived xenograft (PDX) assay in which melanoma cells from patients spontaneously metastasize in NOD/SCID IL2Rγnull (NSG) mice in a manner that correlates with metastatic behavior in patients (Sci Transl Med 4:159). Using this assay, we discovered that distant melanoma metastasis is limited by oxidative stress (Nature 527:186). Reactive Oxygen Species (ROS) increase dramatically in melanoma cells as they metastasize through the blood. The rare cells that survive undergo reversible metabolic changes that confer oxidative stress resistance. We found that metastatic melanomas had significant increases in metabolites from the pentose phosphate pathway as compared to primary cutaneous melanomas. Metabolites in the oxidative branch of the pentose phosphate pathway, downstream of the NADPH-regenerating enzyme Glucose-6-Phosphate Dehydrogenase (G6PD), were the most dramatically increased in metastatic nodules. We hypothesized that metastasizing melanoma cells increase their dependence on the pentose phosphate pathway in order to manage oxidative stress. We used CRISPR gene editing to create loss-of-function mutations in G6PD in patient-derived melanomas. When transplanted into NSG mice, the tumors formed by G6PD mutant melanomas showed significantly reduced oxidative pentose phosphate pathway activity, increased ROS levels, decreased NADPH/NADP ratios, and reduced metastatic potential as compared to parental control melanomas. Our results demonstrate that metastasizing melanoma cells exhibit increased dependence upon the pentose phosphate pathway as compared to primary cutaneous tumors. Untargeted metabolomics revealed that G6PD mutant melanomas exhibit increased glutamine utilization, perhaps to compensate for increased oxidative stress. Citation Format: Arin B. Aurora, VIshal Khivansara, Ashley Leach, Misty Martin-Sandoval, Thomas Mathews, Sean J. Morrison. Metastasizing melanoma cells exhibit increased dependence on the pentose phosphate pathway to manage oxidative stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 85.

Leukemogenic Pathway of MLL-AF4 Fusion-Positive Acute Lymphoblastic Leukemia

Background Many hematologic malignancies, including leukemia, are caused by genetic abnormalities such as chromosomal translocation. These gene defects usually perturb differentiation and proliferation of hematopoietic stem/progenitor cells. As observed in most cancers, multiple gene defects are required to develop overt leukemia, and multi-step tumorigenesis is also the rule in leukemogenesis. Leukemia-specific fusion genes are usually generated early in leukemogenesis as essential genetic defects (1st hit) producing pre-leukemic stem cells. Additional genetic defect(s) occur in pre-leukemic stem cells, finally generating leukemic stem cells that expand to present overt leukemia. Infant acute lymphoblastic leukemia (ALL) are frequently characterized by chromosome translocations involving 11q23, resulting in the rearrangement of the mixed-lineage leukemia (MLL) gene and subsequent generation of MLL fusion genes. Amongst the > 50 genes that have been identified as fusion partners of MLL, fusion with AF4 is characteristically and commonly observed in infant ALL, representing a hallmark of poor prognosis. In most cases of ALL with MLL-AF4, the fusion gene originates in utero as a primary event. Although mutations in RAS and FLT3 are frequently observed in MLL-AF4 -positive ALL, recent genetic data showed significantly lower number of accompanying mutations, which suggested that 2nd hit mutations were not necessary for developing MLL fusion-positive leukemia. The necessity of 2nd hit mutations in leukemogenesis with MLL-AF4 is still under debate. Materials and Methods Several previous studies have modeled leukemic transformation by introducing fusion genes into hematopoietic stem/progenitor cells; however, reproduction of certain forms of ALL, such as MLL-AF4 -positive infant ALL, has been challenging. We used the immature hematopoietic cells derived from mouse embryonic stem (ES) cells as targets for MLL-AF4 -mediated transformation. The MLL-AF4 fusion gene was introduced into mouse ES cells to obtain ES cell clones with stable expression of MLL-AF4 . These ES cells were conditioned to generate embryoid bodies (EBs) and subsequently differentiated into hematopoietic lineages. Tie2-positive cells, which contain immature hematopoietic stem cells, were separated and transplanted into immunodeficient non-obese diabetic/Shi-scid, IL-2Rγ null (NOG) mice to investigate leukemia development. Retroviral insertional mutagenesis was used to introduce additional genetic defect(s) as candidates for tumor-promoting 2nd hit mutations. Purified Tie2-positive cells were transduced using the murine stem cell virus (MSCV) and transplanted into NOG mice. Results Mouse ES cells with stable expression of the MLL-AF4 fusion were viable and could proliferate and differentiate to produce EBs. However, EBs derived from MLL-AF4 -positive ES cells differentiated via the mesenchymal pathway rather than into the Tie2-positive hematopoietic lineage. Mice transplanted with Tie2-positive cells with MLL-AF4 expression did not develop leukemia within the observed period. However, addition of an additional genetic change by retroviral insertional mutagenesis accelerated tumorigenesis in the recipient mice. The MLL-AF4 -expressing Tie2-positive cells with insertional mutagenesis showed clonal expansion and formed mostly extra-medullary tumors with B220 expression, but did not fully develop into tumors of hematopoietic organs. Viral integration sites of these tumors were analyzed to determine additional genetic events inducing tumor formation. In the MLL-AF4 model, viral integration often caused overexpression of gene(s) located near the integration site. Conclusion Considering the in utero formation of MLL-AF4 harboring pre-leukemic stem cells in ALL, hematopoietic progenitor cells or immature fetal cells may be potential targets of leukemic transformation. Immature hematopoietic cells such as the Tie2-positive cells derived from ES cells could be suitable candidates for transformation with MLL-AF4 . Clonal expansion of MLL-AF4 -positive cells after introduction of retroviral insertional mutagenesis suggests the necessity of additional genetic change(s) that activate certain target gene(s) in leukemogenesis with MLL-AF4 . We are currently analyzing the roles of these genes in the leukemic transformation of MLL-AF4 -positive hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

CRISPR/Cas9 Gene-Edited Hematopoietic Stem Cell Therapy for Sickle Cell Disease

Sickle cell disease (SCD) is a life-threatening hereditary disorder that affects thirty million individuals worldwide. The disease results from a single amino acid change in the β-globin gene, which causes polymerization of hemoglobin and deformation of red blood cells, leading to vaso-occlusion, severe pain crisis, multi-organ dysfunction, and ultimately reduction of lifespan in affected individuals. Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative therapy for SCD, yet its success is limited by donor availability and high risk of graft-versus-host disease. Increasing expression of fetal γ-globin in adults is an accepted therapeutic strategy for treating SCD. Supporting this approach are GWAS studies that have identified genetic loci, including within the BCL11A gene, that are associated with elevated fetal hemoglobin (HbF) and mitigated disease severity. Leveraging CRISPR/Cas9 genome editing technology, we aim to develop a safe and curative therapy by autologous, genome-modified HSCT leading to sustained generation of HbF-containing, functional red blood cells. Guide RNAs (gRNAs) targeting the erythroid-specific enhancer region of the BCL11A gene were assayed in CD34+ hematopoietic stem and progenitor cells (HSPCs), and one gRNA was selected for in vivo evaluation based on editing efficiency and functional potency. We achieved approximately 80% target site editing in human CD34+ HSPCs and 65% in CD34+CD38-CD90+CD45RA-CD49f+ defined LT-HSCs. Potential off-target editing sites were identified by two approaches: in silico prediction and unbiased oligo insertion method. These sites were then directly interrogated in HSPCs by targeted PCR amplification and NGS analysis, and to date our selected gRNA had zero validated off-target sites. In addition, HSPC composition was not altered following electroporation of ribonucleoprotein complex, and cells were capable of in vitro expansion and multi-lineage differentiation as measured by CFU assays. Upon in vitro differentiation into the erythroid lineage, edited cells demonstrated ~40% reduction in BCL11A mRNA with a corresponding two-fold increase in γ-globin transcript, translating to a 30-40% increase in HbF positive cells. Most importantly, when CD34+ cells derived from SCD patients were gene-edited and differentiated into erythroid cells, a similar editing efficiency and increase in HbF positive cells was observed. To evaluate whether gene editing alters the self-renewal, engraftment or lineage differentiation capacity of HSCs, gene-edited HSPCs were transplanted into NOD- scid IL2rγnull (NSG) mice to assess reconstitution of the hematopoietic system. Mice displayed 30-40% human CD45+ cell engraftment and multi-lineage reconstitution at 16 weeks following transplantation. When long-term engrafted bone marrow cells were harvested from the recipients and differentiated into erythroid cells in vitro, gene-edited bone marrow cells produced significantly higher numbers of HbF positive cells compared to mock-edited bone marrow cells. These data support the development of autologous genome-modified HSPC transplantation as a novel cell therapy for SCD. DisclosuresYu:Novartis Institutes for BioMedical Research: Employment. Liu:Novartis Institutes for BioMedical Research: Employment. Curran:Novartis Institutes for BioMedical Research: Employment. Zhang:Novartis: Employment. Snead:Novartis: Employment. Schmedt:Novartis: Employment. Yang:Novartis Institutes for BioMedical Research: Employment. Lin:Novartis: Employment. Tschantz:Novartis Institutes for BioMedical Research: Employment. Quinn:Novartis Institutes for BioMedical Research: Employment. Russ:Novartis: Employment. Clarkson:Novartis: Employment. Janiak:Novartis: Employment. Stewart:Intellia Therapeutics: Employment. Mulumba:Intellia Therapeutics: Employment. Lescarbeau:Intellia Therapeutics: Employment. Murray:Intellia Therapeutics: Employment. Seitzer:Intellia Therapeutics: Employment. Strapps:Intellia Therapeutics: Employment. Huang:Intellia Therapeutics: Employment. Sloan:Intellia Therapeutics: Employment. Mickanin:Novartis: Employment. Klickstein:Novartis: Employment. Stevenson:Novartis: Employment.

Detection of Recurrent Genetic Mutations in Intravascular Large B-Cell Lymphoma By Comprehensive Genetic Analyses Using Peripheral Blood Circulating Cell-Free DNA

Abstract Background: Intravascular large B-cell lymphoma (IVLBCL) is a subtype of diffuse large B-cell lymphoma (DLBCL) characterized by the selective growth of tumor cells in the lumina of small vessels. The characteristics of the disease, such as the lack of remarkable lymphadenopathy and the presence of non-specific clinical symptoms, including documented fever of unknown origin, make precise diagnosis difficult; random organ biopsies from bone marrow, skin, and lung are still performed to detect tumor cells. Recently, many recurrent genetic mutations have been detected, especially in DLBCL, but the genetic background in IVLBCL has not been fully elucidated because of the difficulty in obtaining sufficient live tumor cells from diagnostic specimens for subsequent genetic analyses. Previously, we demonstrated that peripheral blood circulating cell-free DNA (cfDNA) in DLBCL is an alternative tumor DNA (tDNA) source for comprehensive genetic analyses (Suzuki Y, et al. ASH 2014 #1658). In this report, we demonstrate that the concentration of cfDNA is significantly higher in IVLBCL than in DLBCL, and that efficiently accumulating the tDNA in peripheral blood is useful for comprehensive genetic analyses to detect recurrent genetic mutations in IVLBCL. Aims: Confirmation of recurrent genetic mutations in IVLBCL using plasma cfDNA. Methods: The study population included 54 B-cell lymphoma patients (IVLBCL, N=21; DLBCL, N=21; follicular lymphoma, N=12) and healthy volunteer donors (N=14). In IVLBCL patients, primary bone marrow (BM) cells containing tumor cells were transplanted into NOD/Shi- scid IL2Rγnull mice to generate a xenograft model (Shimada K, et al. Leukemia 2016), and the tumor cells from tumors formed in the spleen of the mouse model were purified using anti-CD19 antibody-conjugated magnetic beads to obtain tDNA. For genetic analyses using cfDNA and DNAs from purified tumor cells, whole-exome sequencing (WES) using the HiSeq 2000 (Illumina) and Sanger sequencing for reconfirmation of those mutations were performed. Results: The cfDNA concentrations were significantly higher in DLBCL and IVLBCL than in normal donors (p=0.037 and 0.036, respectively), and the concentration was significantly higher in IVLBCL than in DLBCL (p Discussion: The concentration of cfDNA is significantly higher in IVLBCL than in DLBCL, and it appeared to be sufficient to carry out comprehensive genetic analyses to accumulate tDNA. Moreover, the WES analysis using cfDNA could detect genetic mutations more efficiently than that using corresponding BM-DNA. The profiling of recurrent genetic mutations in IVLBCL is partly similar to that in activated B-cell type DLBCL with mutations in NF-κB pathway-related factors, and, intriguingly, they resemble those in B-cell lymphomas that infiltrate into immunologically sanctuary organs, e.g., primary CNS, testis, and breast lymphomas. The present data suggest that genetic analysis using cfDNA in addition to random organ biopsies may become a powerful, safer, and painless strategy to predict the presence of IVLBCL in the clinical setting. Disclosures Shimada: Kyowa Hakko Kirin Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Eisai Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria. Kusumoto: Chugai: Honoraria, Other: GALLIUM and GOYA are sponsored by F. Hoffmann-La Roche Ltd. Third-party medical writing support, under the direction of Shigeru Kusumoto, was provided by Cheryl Wright of Gardiner-Caldwell Communications, and was funded by F. Hoffmann-La Roche Ltd, Research Funding. Masaki: MSD: Research Funding; Eisai: Research Funding; Astellas Pharma: Research Funding; Kyowa Hakko Kirin: Research Funding; Daiichi Sankyo: Research Funding; Taisho Toyama Pharmaceuticals: Research Funding; Taiho Pharmaceutical: Research Funding; Takeda Pharmaceuticals: Research Funding; Mitsubishi Tanabe Pharmaceutical: Research Funding; Chugai Pharmaceutical: Research Funding; Teijin Pharmaceutical: Research Funding; Japan Blood Products Organization: Research Funding; Pfizer: Research Funding; Asahi Kasei Pharmaceutical: Research Funding; Ono Pharmaceutical: Research Funding; Shinogi Pharmaceutical: Research Funding; Meiji Seika Pharma: Research Funding; Mochida Pharmaceutical: Research Funding. Kiyoi: Meiji Seika Pharma Co.,Ltd.: Research Funding; Phizer Japan Inc.: Honoraria, Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; MSD K.K.: Research Funding; JCR Pharmaceuticals Co.,Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; ONO Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Celgene Corporation: Consultancy, Research Funding; Novartis Pharma K.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.