Human Hematopoietic Stem Cell Engrafted IL-15 Transgenic NSG Mice Support Robust NK Cell Responses and Sustained HIV-1 Infection.

Mice reconstituted with human immune systems are instrumental in the investigation of HIV-1 pathogenesis and therapeutics. Natural killer (NK) cells have long been recognized as a key mediator of innate anti-HIV responses. However, established humanized mouse models do not support robust human NK cell development from engrafted human hematopoietic stem cells (HSCs). A major obstacle to human NK cell reconstitution is the lack of human interleukin-15 (IL-15) signaling, as murine IL-15 is a poor stimulator of the human IL-15 receptor. Here, we demonstrate that immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice expressing a transgene encoding human IL-15 (NSG-Tg(IL-15)) have physiological levels of human IL-15 and support long-term engraftment of human NK cells when transplanted with human umbilical-cord-blood-derived HSCs. These Hu-NSG-Tg(IL-15) mice demonstrate robust and long-term reconstitution with human immune cells, but do not develop graft-versus-host disease (GVHD), allowing for long-term studies of human NK cells. Finally, we show that these HSC engrafted mice can sustain HIV-1 infection, resulting in human NK cell responses in HIV-infected mice. We conclude that Hu-NSG-Tg(IL-15) mice are a robust novel model to study NK cell responses to HIV-1.

CHMP2A regulates tumor sensitivity to natural killer cell-mediated cytotoxicity.

Natural killer (NK) cells are known to mediate killing of various cancer types, but tumor cells can develop resistance mechanisms to escape NK cell-mediated killing. Here, we use a "two cell type" whole genome CRISPR-Cas9 screening system to discover key regulators of tumor sensitivity and resistance to NK cell-mediated cytotoxicity in human glioblastoma stem cells (GSC). We identify CHMP2A as a regulator of GSC resistance to NK cell-mediated cytotoxicity and we confirm these findings in a head and neck squamous cells carcinoma (HNSCC) model. We show that deletion of CHMP2A activates NF-κB in tumor cells to mediate increased chemokine secretion that promotes NK cell migration towards tumor cells. In the HNSCC model we demonstrate that CHMP2A mediates tumor resistance to NK cells via secretion of extracellular vesicles (EVs) that express MICA/B and TRAIL. These secreted ligands induce apoptosis of NK cells to inhibit their antitumor activity. To confirm these in vitro studies, we demonstrate that deletion of CHMP2A in CAL27 HNSCC cells leads to increased NK cell-mediated killing in a xenograft immunodeficient mouse model. These findings illustrate a mechanism of tumor immune escape through EVs secretion and identify inhibition of CHMP2A and related targets as opportunities to improve NK cell-mediated immunotherapy.

Natural killer cells and cytotoxic T lymphocytes are required to clear solid tumor in a patient-derived xenograft.

Existing patient-derived xenograft (PDX) mouse models of solid tumors lack a fully tumor donor-matched, syngeneic, and functional immune system. We developed a model that overcomes these limitations by engrafting lymphopenic recipient mice with a fresh, undisrupted piece of solid tumor, whereby tumor-infiltrating lymphocytes (TILs) persisted in the recipient mice for several weeks. Successful tumor engraftment was achieved in 83% to 89% of TIL-PDX mice, and these were seen to harbor exhausted immuno-effector as well as functional immunoregulatory cells persisting for at least 6 months postengraftment. Combined treatment with interleukin-15 stimulation and immune checkpoint inhibition resulted in complete or partial tumor response in this model. Further, depletion of cytotoxic T lymphocytes and/or natural killer cells before combined immunotherapy revealed that both cell types were required for maximal tumor regression. Our TIL-PDX model provides a valuable resource for powerful mechanistic and therapeutic studies in solid tumors.

Natural Killer Cell Interactions With Myeloid Derived Suppressor Cells in the Tumor Microenvironment and Implications for Cancer Immunotherapy.

The tumor microenvironment (TME) is a complex and heterogeneous environment composed of cancer cells, tumor stroma, a mixture of tissue-resident and infiltrating immune cells, secreted factors, and extracellular matrix proteins. Natural killer (NK) cells play a vital role in fighting tumors, but chronic stimulation and immunosuppression in the TME lead to NK cell exhaustion and limited antitumor functions. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of myeloid cells with potent immunosuppressive activity that gradually accumulate in tumor tissues. MDSCs interact with innate and adaptive immune cells and play a crucial role in negatively regulating the immune response to tumors. This review discusses MDSC-mediated NK cell regulation within the TME, focusing on critical cellular and molecular interactions. We review current strategies that target MDSC-mediated immunosuppression to enhance NK cell cytotoxic antitumor activity. We also speculate on how NK cell-based antitumor immunotherapy could be improved.

Fibrinolysis protease receptors promote activation of astrocytes to express pro-inflammatory cytokines.

BACKGROUND: Astrocytes contribute to the crosstalk that generates chronic neuro-inflammation in neurological diseases; however, compared with microglia, astrocytes respond to a more limited continuum of innate immune system stimulants. Recent studies suggest that the fibrinolysis system may regulate inflammation. The goal of this study was to test whether fibrinolysis system components activate astrocytes and if so, elucidate the responsible biochemical pathway. METHODS: Primary cultures of astrocytes and microglia were prepared from neonatal mouse brains. The ability of purified fibrinolysis system proteins to elicit a pro-inflammatory response was determined by measuring expression of the mRNAs encoding tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and chemokine (C-C motif) ligand 2 (CCL2). IκBα phosphorylation also was measured. Plasminogen activation in association with cells was detected by chromogenic substrate hydrolysis. The activity of specific receptors was tested using neutralizing antibodies and reagents. RESULTS: Astrocytes expressed pro-inflammatory cytokines when treated with plasminogen but not when treated with agonists for Toll-like Receptor-4 (TLR4), TLR2, or TLR9. Microglia also expressed pro-inflammatory cytokines in response to plasminogen; however, in these cells, the response was observed only when tissue-type plasminogen activator (tPA) was added to activate plasminogen. In astrocytes, endogenously produced urokinase-type plasminogen activator (uPA) converted plasminogen into plasmin in the absence of tPA. Plasminogen activation was dependent on the plasminogen receptor, α-enolase, and the uPA receptor, uPAR. Although uPAR is capable of directly activating cell-signaling, the receptor responsible for cytokine expression and IκBα phosphorylation response to plasmin was Protease-activated Receptor-1 (PAR-1). The pathway, by which plasminogen induced astrocyte activation, was blocked by inhibiting any one of the three receptors implicated in this pathway with reagents such as εACA, α-enolase-specific antibody, uPAR-specific antibody, the uPA amino terminal fragment, or a pharmacologic PAR-1 inhibitor. CONCLUSIONS: Plasminogen may activate astrocytes for pro-inflammatory cytokine expression through the concerted action of at least three distinct fibrinolysis protease receptors. The pathway is dependent on uPA to activate plasminogen, which is expressed endogenously by astrocytes in culture but also may be provided by other cells in the astrocytic cell microenvironment in the CNS.

Results from Phase I Clinical Trial with Intraspinal Injection of Neural Stem Cells in Amyotrophic Lateral Sclerosis: A Long-Term Outcome.

The main objective of this phase I trial was to assess the feasibility and safety of microtransplanting human neural stem cell (hNSC) lines into the spinal cord of patients with amyotrophic lateral sclerosis (ALS). Eighteen patients with a definite diagnosis of ALS received microinjections of hNSCs into the gray matter tracts of the lumbar or cervical spinal cord. Patients were monitored before and after transplantation by clinical, psychological, neuroradiological, and neurophysiological assessment. For up to 60 months after surgery, none of the patients manifested severe adverse effects or increased disease progression because of the treatment. Eleven patients died, and two underwent tracheotomy as a result of the natural history of the disease. We detected a transitory decrease in progression of ALS Functional Rating Scale Revised, starting within the first month after surgery and up to 4 months after transplantation. Our results show that transplantation of hNSC is a safe procedure that causes no major deleterious effects over the short or long term. This study is the first example of medical transplantation of a highly standardized cell drug product, which can be reproducibly and stably expanded ex vivo, comprising hNSC that are not immortalized, and are derived from the forebrain of the same two donors throughout this entire study as well as across future trials. Our experimental design provides benefits in terms of enhancing both intra- and interstudy reproducibility and homogeneity. Given the potential therapeutic effects of the hNSCs, our observations support undertaking future phase II clinical studies in which increased cell dosages are studied in larger cohorts of patients. Stem Cells Translational Medicine 2019;8:887&897.

Transplantation of clinical-grade human neural stem cells reduces neuroinflammation, prolongs survival and delays disease progression in the SOD1 rats.

Stem cells are emerging as a therapeutic option for incurable diseases, such as Amyotrophic Lateral Sclerosis (ALS). However, critical issues are related to their origin as well as to the need to deepen our knowledge of the therapeutic actions exerted by these cells. Here, we investigate the therapeutic potential of clinical-grade human neural stem cells (hNSCs) that have been successfully used in a recently concluded phase I clinical trial for ALS patients (NCT01640067). The hNSCs were transplanted bilaterally into the anterior horns of the lumbar spinal cord (four grafts each, segments L3-L4) of superoxide dismutase 1 G93A transgenic rats (SOD1 rats) at the symptomatic stage. Controls included untreated SOD1 rats (CTRL) and those treated with HBSS (HBSS). Motor symptoms and histological hallmarks of the disease were evaluated at three progressive time points: 15 and 40 days after transplant (DAT), and end stage. Animals were treated by transient immunosuppression (for 15 days, starting at time of transplantation). Under these conditions, hNSCs integrated extensively within the cord, differentiated into neural phenotypes and migrated rostro-caudally, up to 3.77 ± 0.63 cm from the injection site. The transplanted cells delayed decreases in body weight and deterioration of motor performance in the SOD1 rats. At 40DAT, the anterior horns at L3-L4 revealed a higher density of motoneurons and fewer activated astroglial and microglial cells. Accordingly, the overall survival of transplanted rats was significantly enhanced with no rejection of hNSCs observed. We demonstrated that the beneficial effects observed after stem cell transplantation arises from multiple events that counteract several aspects of the disease, a crucial feature for multifactorial diseases, such as ALS. The combination of therapeutic approaches that target different pathogenic mechanisms of the disorder, including pharmacology, molecular therapy and cell transplantation, will increase the chances of a clinically successful therapy for ALS.

Tissue-type plasminogen activator neutralizes LPS but not protease-activated receptor-mediated inflammatory responses to plasmin.

Tissue-type plasminogen activator (tPA) activates fibrinolysis and also suppresses innate immune system responses to LPS in bone marrow-derived macrophages (BMDMs) and in vivo in mice. The objective of this study was to assess the activity of tPA as a regulator of macrophage physiology in the presence of plasmin. Enzymatically active and enzymatically inactive (EI) tPA appeared to comprehensively block the response to LPS in BMDMs, including expression of proinflammatory cytokines such as TNF-α and IL-1ß and anti-inflammatory cytokines such as IL-10 and IL-1 receptor antagonist. The activity of EI-tPA as an LPS response modifier was conserved in the presence of plasminogen. By contrast, in BMDMs treated with tPA and plasminogen or preactivated plasmin, in the presence or absence of LPS, increased proinflammatory cytokine expression was observed and tPA failed to reverse the response. Plasmin independently activated NF-κB, ERK1/2, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase in BMDMs, which is characteristic of proinflammatory stimuli. Plasmin-induced cytokine expression was blocked by ε-aminocaproic acid, aprotinin, and inhibitors of the known plasmin substrate, Protease-activated receptor-1 (PAR-1), but not by N-methyl-d-aspartate receptor inhibitor, which blocks the effects of tPA on macrophages. Cytokine expression by BMDMs treated with the PAR-1 agonist, TFLLR, was not inhibited by EI-tPA, possibly explaining why EI-tPA does not inhibit macrophage responses to plasmin and providing evidence for specificity in the ability of tPA to oppose proinflammatory stimuli. Regulation of innate immunity by the fibrinolysis system may reflect the nature of the stimulus and a balance between the potentially opposing activities of tPA and plasmin.

Establishment of stable iPS-derived human neural stem cell lines suitable for cell therapies.

Establishing specific cell lineages from human induced pluripotent stem cells (hiPSCs) is vital for cell therapy approaches in regenerative medicine, particularly for neurodegenerative disorders. While neural precursors have been induced from hiPSCs, the establishment of hiPSC-derived human neural stem cells (hiNSCs), with characteristics that match foetal hNSCs and abide by cGMP standards, thus allowing clinical applications, has not been described. We generated hiNSCs by a virus-free technique, whose properties recapitulate those of the clinical-grade hNSCs successfully used in an Amyotrophic Lateral Sclerosis (ALS) phase I clinical trial. Ex vivo, hiNSCs critically depend on exogenous mitogens for stable self-renewal and amplification and spontaneously differentiate into astrocytes, oligodendrocytes and neurons upon their removal. In the brain of immunodeficient mice, hiNSCs engraft and differentiate into neurons and glia, without tumour formation. These findings now warrant the establishment of clinical-grade, autologous and continuous hiNSC lines for clinical trials in neurological diseases such as Huntington's, Parkinson's and Alzheimer's, among others.

Toll-like receptor 4 modulation influences human neural stem cell proliferation and differentiation.

Toll-like receptor 4 (TLR4) activation is pivotal to innate immunity and has been shown to regulate proliferation and differentiation of human neural stem cells (hNSCs) in vivo. Here we study the role of TLR4 in regulating hNSC derived from the human telencephalic-diencephalic area of the fetal brain and cultured in vitro as neurospheres in compliance with Good Manifacture Procedures (GMP) guidelines. Similar batches have been used in recent clinical trials in ALS patients. We found that TLR2 and 4 are expressed in hNSCs as well as CD14 and MD-2 co-receptors, and TLR4 expression is downregulated upon differentiation. Activation of TLR4 signaling by lipopolysaccharide (LPS) has a positive effect on proliferation and/or survival while the inverse is observed with TLR4 inhibition by a synthetic antagonist. TLR4 activation promotes neuronal and oligodendrocyte differentiation and/or survival while TLR4 inhibition leads to increased apoptosis. Consistently, endogenous expression of TLR4 is retained by hNSC surviving after transplantation in ALS rats or immunocompromised mice, thus irrespectively of the neuroinflammatory environment. The characterization of downstream signaling of TLR4 in hNSCs has suggested some activation of the inflammasome pathway. This study suggests TLR4 signaling as essential for hNSC self-renewal and as a novel target for the study of neurogenetic mechanisms.

The Urokinase Receptor Induces a Mesenchymal Gene Expression Signature in Glioblastoma Cells and Promotes Tumor Cell Survival in Neurospheres.

PLAUR encodes the urokinase receptor (uPAR), which promotes cell survival, migration, and resistance to targeted cancer therapeutics in glioblastoma cells in culture and in mouse model systems. Herein, we show that patient survival correlates inversely with PLAUR mRNA expression in gliomas of all grades, in glioblastomas, and in the subset of glioblastomas that demonstrate the mesenchymal gene expression signature. PLAUR clusters with genes that define the more aggressive mesenchymal subtype in transcriptome profiles of glioblastoma tissue and glioblastoma cells in neurospheres, which are enriched for multipotent cells with stem cell-like qualities. When PLAUR was over-expressed or silenced in glioblastoma cells, neurosphere growth and expression of mesenchymal subtype biomarkers correlated with uPAR abundance. uPAR also promoted glioblastoma cell survival in neurospheres. Constitutively-active EGF Receptor (EGFRvIII) promoted neurosphere growth; however, unlike uPAR, EGFRvIII did not induce the mesenchymal gene expression signature. Immunohistochemical analysis of human glioblastomas showed that uPAR is typically expressed by a small sub-population of the cancer cells; it is thus reasonable to conclude that this subpopulation of cells is responsible for the effects of PLAUR on patient survival. We propose that uPAR-expressing glioblastoma cells demonstrate a mesenchymal gene signature, an increased capacity for cell survival, and stem cell-like properties.

Neural Stem Cells and Human Induced Pluripotent Stem Cells to Model Rare CNS Diseases.

BACKGROUND & OBJECTIVE: Despite the great effort spent over recent decades to unravel the pathological mechanisms underpinning the development of central nervous system disorders, most of them still remain unclear. In particular, the study of rare CNS diseases is hampered by the lack of postmortem samples and of reliable epidemiological studies, thus the setting of in vitro modeling systems appears essential to dissect the puzzle of genetic and environmental alterations affecting neural cells viability and functionality. The isolation and expansion in vitro of embryonic (ESC) and fetal neural stem cells (NSC) from human tissue have allowed the modeling of several neurological diseases "in a dish" and have also provided a novel platform to test potential therapeutic strategies in a pre-clinical setting. In recent years, the development of induced pluripotent stem cell (iPS) technology has added enormous value to the aforementioned approach, thanks to their capability for generating diseaserelevant cell phenotypes in vitro and to their perspective use in autologous transplantation. However, while the potentiality of ESC, NSC and iPS has been widely sponsored, the pitfalls related to the available protocols for differentiation and the heterogeneity of lines deriving from different individuals have been poorly discussed. Here we present pro and contra of using ESC, NSC or iPS for modeling rare diseases like Lysosomal Storage disorders and Motor Neuron Diseases. CONCLUSION: In this view, the advent of gene editing technologies is a unique opportunity to standardize the data analysis in preclinical studies and to tailor clinical protocols for stem cell-mediated therapy.

Glial degeneration with oxidative damage drives neuronal demise in MPSII disease.

Mucopolysaccharidosis type II (MPSII) is a lysosomal storage disorder due to the deficit of the iduronate 2-sulfatase (IDS) enzyme, causing progressive neurodegeneration in patients. Neural stem cells (NSCs) derived from the IDS-ko mouse can recapitulate MPSII pathogenesis in vitro. In differentiating IDS-ko NSCs and in the aging IDS-ko mouse brain, glial degeneration precedes neuronal degeneration. Here we show that pure IDS-ko NSC-derived astrocytes are selectively able to drive neuronal degeneration when cocultured with healthy neurons. This phenotype suggests concurrent oxidative damage with metabolic dysfunction. Similar patterns were observed in murine IDS-ko animals and in human MPSII brains. Most importantly, the mutant phenotype of IDS-ko astrocytes was reversed by low oxygen conditions and treatment with vitamin E, which also reversed the toxic effect on cocultured neurons. Moreover, at very early stages of disease we detected in vivo the development of a neuroinflammatory background that precedes astroglial degeneration, thus suggesting a novel model of MPSII pathogenesis, with neuroinflammation preceding glial degeneration, which is finally followed by neuronal death. This hypothesis is also consistent with the progression of white matter abnormalities in MPSII patients. Our study represents a novel breakthrough in the elucidation of MPSII brain pathogenesis and suggests the antioxidant molecules as potential therapeutic tools to delay MPSII onset and progression.

Human neural stem cell transplantation in ALS: initial results from a phase I trial.

BACKGROUND: We report the initial results from a phase I clinical trial for ALS. We transplanted GMP-grade, fetal human neural stem cells from natural in utero death (hNSCs) into the anterior horns of the spinal cord to test for the safety of both cells and neurosurgical procedures in these patients. The trial was approved by the Istituto Superiore di Sanità and the competent Ethics Committees and was monitored by an external Safety Board. METHODS: Six non-ambulatory patients were treated. Three of them received 3 unilateral hNSCs microinjections into the lumbar cord tract, while the remaining ones received bilateral (n = 3 + 3) microinjections. None manifested severe adverse events related to the treatment, even though nearly 5 times more cells were injected in the patients receiving bilateral implants and a much milder immune-suppression regimen was used as compared to previous trials. RESULTS: No increase of disease progression due to the treatment was observed for up to18 months after surgery. Rather, two patients showed a transitory improvement of the subscore ambulation on the ALS-FRS-R scale (from 1 to 2). A third patient showed improvement of the MRC score for tibialis anterior, which persisted for as long as 7 months. The latter and two additional patients refused PEG and invasive ventilation and died 8 months after surgery due to the progression of respiratory failure. The autopsies confirmed that this was related to the evolution of the disease. CONCLUSIONS: We describe a safe cell therapy approach that will allow for the treatment of larger pools of patients for later-phase ALS clinical trials, while warranting good reproducibility. These can now be carried out under more standardized conditions, based on a more homogenous repertoire of clinical grade hNSCs. The use of brain tissue from natural miscarriages eliminates the ethical concerns that may arise from the use of fetal material. TRIAL REGISTRATION: EudraCT:2009-014484-39 .

Differential pathotropism of non-immortalized and immortalized human neural stem cell lines in a focal demyelination model.

Cell therapy is reaching the stage of phase I clinical trials for post-traumatic, post-ischemic, or neurodegenerative disorders, and the selection of the appropriate cell source is essential. In order to assess the capacity of different human neural stem cell lines (hNSC) to contribute to neural tissue regeneration and to reduce the local inflammation after an acute injury, we transplanted GMP-grade non-immortalized hNSCs and v-myc (v-IhNSC), c-myc T58A (T-IhNSC) immortalized cells into the corpus callosum of adult rats after 5 days from focal demyelination induced by lysophosphatidylcholine. At 15 days from transplantation, hNSC and T-IhNSC migrated to the lesioned area where they promoted endogenous remyelination and differentiated into mature oligodendrocytes, while the all three cell lines were able to integrate in the SVZ. Moreover, where demyelination was accompanied by an inflammatory reaction, a significant reduction of microglial cells' activation was observed. This effect correlated with a differential migratory pattern of transplanted hNSC and IhNSC, significantly enhanced in the former, thus suggesting a specific NSC-mediated immunomodulatory effect on the local inflammation. We provide evidence that, in the subacute phase of a demyelination injury, different human immortalized and non-immortalized NSC lines, all sharing homing to the stem niche, display a differential pathotropism, both through cell-autonomous and non-cell autonomous effects. Overall, these findings promote IhNSC as an inexhaustible cell source for large-scale preclinical studies and non-immortalized GMP grade hNSC lines as an efficacious, safe, and reliable therapeutic tool for future clinical applications.