Preclinical Evaluation of Off-The-Shelf PD-L1+ Human Natural Killer Cells Secreting IL15 to Treat Non-Small Cell Lung Cancer.

We described previously a human natural killer (NK) cell population that upregulates PD-L1 expression upon recognizing and reacting to tumor cells or exposure to a combination of IL12, IL18 and IL15. Here, to investigate the safety and efficacy of tumor-reactive and cytokine-activated (TRACK) NK cells, human NK cells from umbilical cord blood were expanded, transduced with a retroviral vector encoding soluble (s) IL15, and further cytokine activated to induce PD-L1 expression. Our results show cryopreserved and thawed sIL15_TRACK NK cells had significantly improved cytotoxicity against non-small cell lung cancer (NSCLC) in vitro when compared to non-transduced (NT) NK cells, PD-L1+ NK cells lacking sIL15 expression (NT_TRACK NK), or NK cells expressing sIL15 without further cytokine activation (sIL15 NK cells). Intravenous injection of sIL15_TRACK NK cells into immunodeficient mice with NSCLC significantly slowed tumor growth and improved survival when compared to NT NK and sIL15 NK cells. The addition of the anti-PD-L1 atezolizumab further improved control of NSCLC growth by sIL15_TRACK NK cells in vivo. Moreover, a dose-dependent efficacy was assessed for sIL15_TRACK NK cells without observed toxicity. These experiments indicate that the administration of frozen, off-the-shelf allogeneic sIL15_TRACK NK cells is safe in preclinical models of human NSCLC and has potent antitumor activity without and with the administration of atezolizumab. A Phase I clinical trial modeled after this preclinical study using sIL15_TRACK NK cells alone or with atezolizumab for relapsed or refractory NSCLC is currently underway (NCT05334329).

Off-the-shelf CAR-engineered natural killer cells targeting FLT3 enhance killing of acute myeloid leukemia.

The majority of patients with acute myeloid leukemia (AML) succumb to the disease or its complications, especially among older patients. Natural killer (NK) cells have been shown to have antileukemic activity in patients with AML; however, to our knowledge, primary NK cells armed with a chimeric antigen receptor (CAR) targeting antigens associated with AML as an "off-the-shelf" product for disease control have not been explored. We developed frozen, off-the-shelf allogeneic human NK cells engineered with a CAR recognizing FLT3 and secreting soluble interleukin-15 (IL-15) (FLT3 CAR_sIL-15 NK) to improve in vivo NK cell persistence and T-cell activation. FLT3 CAR_sIL-15 NK cells had higher cytotoxicity and interferon gamma secretion against FLT3+ AML cell lines when compared with activated NK cells lacking an FLT3 CAR or soluble IL-15. Frozen and thawed allogeneic FLT3 CAR_sIL-15 NK cells prolonged survival of both the MOLM-13 AML model as well as an orthotopic patient-derived xenograft AML model when compared with control NK cells. FLT3 CAR_sIL-15 NK cells showed no cytotoxicity against healthy blood mononuclear cells or hematopoietic stem cells. Collectively, our data suggest that FLT3 is an AML-associated antigen that can be targeted by frozen, allogeneic, off-the-shelf FLT3 CAR_sIL-15 NK cells that may provide a novel approach for the treatment of AML.

Adipocyte CD1d Gene Transfer Induces T Cell Expansion and Adipocyte Inflammation in CD1d Knockout Mice.

CD1d, a lipid Ag-presenting molecule for invariant NKT (iNKT) cells, is abundantly expressed on adipocytes and regulates adipose homeostasis through iNKT cells. CD1d gene expression was restored in visceral adipose tissue adipocytes of CD1d knockout (KO) mice to investigate the interactions between adipocytes and immune cells within adipose tissue. We developed an adipocyte-specific targeting recombinant adeno-associated viral vector, with minimal off-target transgene expression in the liver, to rescue CD1d gene expression in visceral adipose tissue adipocytes of CD1d KO mice, followed by assessment of immune cell alternations in adipose tissue and elucidation of the underlying mechanisms of alteration. We report that adeno-associated virus-mediated gene transfer of CD1d to adipocytes in CD1d KO mice fails to rescue iNKT cells but leads to massive and selective expansion of T cells within adipose tissue, particularly CD8+ T effector cells, that is associated with adipocyte NLRP3 inflammasome activation, dysregulation of adipocyte functional genes, and upregulation of apoptotic pathway proteins. An NLRP3 inhibitor has no effect on T cell phenotypes whereas depletion of CD8+ T cells significantly attenuates inflammasome activation and abolishes the dysregulation of adipocyte functional genes induced by adipocyte CD1d. In contrast, adipocyte overexpression of CD1d fails to induce T cell activation in wild-type mice or in invariant TCR α-chain Jα18 KO mice that have a normal lymphocyte repertoire except for iNKT cells. Our studies uncover an adipocyte CD1d → CD8+ T cell → adipocyte inflammasome cascade, in which CD8+ T cells function as a key mediator of adipocyte inflammation likely induced by an allogeneic response against the CD1d molecule.

Off-the-Shelf Prostate Stem Cell Antigen-Directed Chimeric Antigen Receptor Natural Killer Cell Therapy to Treat Pancreatic Cancer.

BACKGROUND & AIMS: Pancreatic cancer (PC) is the third leading cause of cancer-related death with a 5-year survival rate of approximately 10%. It typically presents as a late-stage incurable cancer and chemotherapy provides modest benefit. Here, we demonstrate the feasibility, safety, and potency of a novel human natural killer (NK) cell-based immunotherapy to treat PC. METHODS: The expression of prostate stem cell antigen (PSCA) was evaluated in primary PC at messenger RNA and protein levels. The processes of retroviral transduction, expansion, activation, and cryopreservation of primary human NK cells obtained from umbilical cord blood were optimized, allowing us to develop frozen, off-the-shelf, allogeneic PSCA chimeric antigen receptor (CAR) NK cells. The safety and efficacy of PSCA CAR NK cells also expressing soluble (s) interleukin 15 (PSCA CAR_s15 NK cells) were evaluated in vitro and in vivo. RESULTS: PSCA was elevated in primary human PC compared with the adjacent or other normal tissues. PSCA CAR_s15 NK cells displayed significant tumor-suppressive effects against PSCA(+) PC in vitro before and after 1 cycle of freeze-thaw. The viability of frozen PSCA CAR_s15 NK cells persisted more than 90 days in vivo after their last infusion and significantly prolonged the survival of mice engrafted with human PC. CONCLUSIONS: PSCA CAR_s15 NK cells showed therapeutic efficacy in human metastatic PC models without signs of systematic toxicity, providing a strong rationale to support clinical development.

Hijacking TYRO3 from Tumor Cells via Trogocytosis Enhances NK-cell Effector Functions and Proliferation.

Trogocytosis is a fast, cell-cell contact-dependent uptake of membrane patches and associated molecules by one cell from another. Here, we report our investigation of trogocytosis of TYRO3, a cell membrane protein, from tumor target cells to natural killer (NK) cells and the associated functional consequences for NK cells. We found that although NK cells did not express endogenous TYRO3 on the cell surface, activated NK cells rapidly acquired TYRO3 from tumor cells via trogocytosis in vitro and in vivo. NK cells that acquired TYRO3, which we termed TYRO3+ NK cells, had significantly enhanced cytotoxicity and IFNγ production as well as higher expression of some activated surface markers compared with TYRO3- NK cells. Furthermore, the activation status of NK cells and TYRO3 expression levels on donor cells, either endogenous or ectopic, positively correlated with trogocytosis levels. When the antigen-presenting cell (APC) K562 leukemia cell line, a feeder cell line to expand NK cells, overexpressed TYRO3, TYRO3 was transferred to NK cells via trogocytosis, which improved NK-cell proliferation ex vivo. This provides a strategy to manufacture NK cells or their engineered counterparts, such as chimeric antigen receptor NK cells, for the treatment of cancer or infectious diseases.

Environmental activation of a hypothalamic BDNF-adipocyte IL-15 axis regulates adipose-natural killer cells.

Physical and social environments influence immune homeostasis within adipose tissue, yet the mechanisms remain poorly defined. We report that an enriched environment (EE) housing modulates the immune cell population in white adipose tissue of mice including an increase in the abundance of natural killer (NK) cells. EE upregulates the expression of IL-15 and its receptor IL-15Rα specifically within mature adipocytes. Mechanistically, we show that hypothalamic brain-derived neurotrophic factor (BDNF) upregulates IL-15 production in adipocytes via sympathetic ß-adrenergic signaling. Overexpressing BDNF mediated by recombinant adeno-associated virus (rAAV) vector in the hypothalamus expands adipose NK cells. Conversely, inhibition of hypothalamic BDNF signaling via gene transfer of a dominant negative TrkB receptor suppresses adipose NK cells. In white adipose tissue, overexpression of IL-15 using an adipocyte-specific rAAV vector stimulates adipose NK cells and inhibits the progression of subcutaneous melanoma, whereas local IL-15 knockdown blocks the EE effect. These results suggest that bio-behavioral factors regulate adipose NK cells via a hypothalamic BDNF-sympathoneural-adipocyte IL-15 axis. Targeting this pathway may have therapeutic significance for cancer.

An Oncolytic Virus Expressing IL15/IL15Rα Combined with Off-the-Shelf EGFR-CAR NK Cells Targets Glioblastoma.

IL15 is a pleiotropic cytokine with multiple roles that improve immune responses to tumor cells. Oncolytic viruses (OV) specifically lyse tumors and activate immune responses. Systemic administration of IL15 or its complex with the IL15Rα and chimeric antigen receptor (CAR) natural killer (NK) cells are currently being tested in the clinic. Here, we generated a herpes simplex 1-based OV-expressing human IL15/IL15Rα sushi domain fusion protein (named OV-IL15C), as well as off-the-shelf EGFR-CAR NK cells, and studied their monotherapy and combination efficacy in vitro and in multiple glioblastoma (GBM) mouse models. In vitro, soluble IL15/IL15Rα complex was secreted from OV-IL15C-infected GBM cells, which promoted GBM cytotoxicity and improved survival of NK and CD8+ T cells. Frozen, readily available off-the-shelf EGFR-CAR NK cells showed enhanced killing of tumor cells compared with empty vector-transduced NK cells. In vivo, OV-IL15C significantly inhibited tumor growth and prolonged survival of GBM-bearing mice in the presence of CD8+ T cells compared with parental OV. OV-IL15C plus EGFR-CAR NK cells synergistically suppressed tumor growth and significantly improved survival compared with either monotherapy, correlating with increased intracranial infiltration and activation of NK and CD8+ T cells and elevated persistence of CAR NK cells in an immunocompetent model. Collectively, OV-IL15C and off-the-shelf EGFR-CAR NK cells represent promising therapeutic strategies for GBM treatment to improve the clinical management of this devastating disease. SIGNIFICANCE: The combination of an oncolytic virus expressing the IL15/IL15Rα complex and frozen, ready-to-use EGFR-CAR NK cells elicits strong antitumor responses in glioblastoma.

Cbl-b Is Upregulated and Plays a Negative Role in Activated Human NK Cells.

The E3 ubiquitin ligase Cbl-b has been characterized as an intracellular checkpoint in T cells; however, the function of Cbl-b in primary human NK cells, an innate immune anti-tumor effector cell, is not well defined. In this study, we show that the expression of Cbl-b is significantly upregulated in primary human NK cells activated by IL-15, IL-2, and the human NK cell-sensitive tumor cell line K562 that lacks MHC class I expression. Pretreatment with JAK or AKT inhibitors prior to IL-15 stimulation reversed Cbl-b upregulation. Downregulation of Cbl-b resulted in significant increases in granzyme B and perforin expression, IFN-γ production, and cytotoxic activity against tumor cells. Collectively, we demonstrate upregulation of Cbl-b and its inhibitory effects in IL-15/IL-2/K562-activated human NK cells, suggesting that Cbl-b plays a negative feedback role in human NK cells.

CD84 is a regulator of the immunosuppressive microenvironment in multiple myeloma.

Multiple myeloma (MM) is characterized by an accumulation of malignant plasma cells (PCs) within the BM. The BM microenvironment supports survival of the malignant cells and is composed of cellular fractions that foster myeloma development and progression by suppression of the immune response. Despite major progress in understanding the biology and pathophysiology of MM, this disease is still incurable and requires aggressive treatment with significant side effects. CD84 is a self-binding immunoreceptor belonging to the signaling lymphocyte activation molecule (SLAM) family. Previously, we showed that CD84 bridges between chronic lymphocytic leukemia cells and their microenvironment, and it regulates T cell function. In the current study, we investigated the role of CD84 in MM. Our results show that MM cells express low levels of CD84. However, these cells secrete the cytokine macrophage migration inhibitory factor (MIF), which induces CD84 expression on cells in their microenvironment. Its activation leads to an elevation of expression of genes regulating differentiation to monocytic/granulocytic-myeloid-derived suppressor cells (M-MDSCs and G-MDSCs, respectively) and upregulation of PD-L1 expression on MDSCs, which together suppress T cell function. Downregulation of CD84 or its blocking reduce MDSC accumulation, resulting in elevated T cell activity and reduced tumor load. Our data suggest that CD84 might serve as a novel therapeutic target in MM.

Enriched environment enhances NK cell maturation through hypothalamic BDNF in male mice.

Macroenvironmental factors, including a patient's physical and social environment, play a role in cancer risk and progression. Our previous preclinical studies have shown that the enriched environment (EE) confers anti-obesity and anti-cancer phenotypes that are associated with enhanced adaptive immunity and are mediated by brain-derived neurotrophic factor (BDNF). Natural killer (NK) cells have anti-cancer and anti-viral properties, and their absence or depletion is associated with inferior clinical outcomes. In this study, we investigated the effects of EE on NK cell maturation following their depletion. Mice living in EE displayed a higher proportion of NK cells in the spleen, bone marrow, and blood, compared to those living in the standard environment (SE). EE enhanced NK cell maturation in the spleen and was associated with upregulation of BDNF expression in the hypothalamus. Hypothalamic BDNF overexpression reproduced the EE effects on NK cell maturation in secondary lymphoid tissues. Conversely, hypothalamic BDNF knockdown blocked the EE modulation on NK cell maturation. Our results demonstrate that a bio-behavior intervention enhanced NK cell maturation and was mediated at least in part by hypothalamic BDNF.

CSF1R inhibitor PLX5622 and environmental enrichment additively improve metabolic outcomes in middle-aged female mice.

As the elderly population grows, chronic metabolic dysfunction including obesity and diabetes are becoming increasingly common comorbidities. Hypothalamic inflammation through CNS resident microglia serves as a common pathway between developing obesity and developing systemic aging pathologies. Despite understanding aging as a life-long process involving interactions between individuals and their environment, limited studies address the dynamics of environment interactions with aging or aging therapeutics. We previously demonstrated environmental enrichment (EE) is an effective model for studying improved metabolic health and overall healthspan in mice, which acts through a brain-fat axis. Here we investigated the CSF1R inhibitor PLX5622 (PLX), which depletes microglia, and its effects on metabolic decline in aging in interaction with EE. PLX in combination with EE substantially improved metabolic outcomes in middle-aged female mice over PLX or EE alone. Chronic PLX treatment depleted 75% of microglia from the hypothalamus and reduced markers of inflammation without affecting brain-derived neurotrophic factor levels induced by EE. Adipose tissue remodeling and adipose tissue macrophage modulation were observed in response to CSF1R inhibition, which may contribute to the combined benefits seen in EE with PLX. Our study suggests benefits exist from combined drug and lifestyle interventions in aged animals.

Adipocytes: A Novel Target for IL-15/IL-15Rα Cancer Gene Therapy.

IL-15 is a proinflammatory cytokine that plays an essential role in the development and activation of natural killer (NK) cells. Adipose tissue acts as an endocrine organ that secretes cytokines and is an important reservoir for lymphocytes. We hypothesized that activation of the IL-15 signaling in adipose tissue will activate and expand the NK cell population and control tumor growth. We recently developed an adipocyte-targeting recombinant adeno-associated viral (rAAV) vector with minimal off-target transgene expression in the liver. Here, we used this rAAV system to deliver an IL-15/IL-15Rα complex to the abdominal fat by intraperitoneal (i.p.) injection. Adipose IL-15/IL-15Rα complex gene transfer led to the expansion of NK cells in the adipose tissue and spleen in normal mice without notable side effects. The i.p. injection of rAAV-IL-15/IL-15Rα complex significantly suppressed the growth of Lewis lung carcinoma implanted subcutaneously and exerted a significant survival advantage in a B16-F10 melanoma metastasis model. The antitumor effects were associated with the expansion of the NK cells in the blood, spleen, abdominal fat, and tumor, as well as the enhancement of NK cell maturity. Our proof-of-concept preclinical studies demonstrate the safety and efficacy of the adipocyte-specific IL-15/IL-15Rα complex vector as a novel cancer immune gene therapy.

Enriched environment regulates thymocyte development and alleviates experimental autoimmune encephalomyelitis in mice.

Environmental and social factors have profound impacts on immune homeostasis. Our work on environmental enrichment (EE) has revealed a novel anti-obesity and anticancer phenotype associated with enhanced activity of CD8+ cytotoxic T lymphocytes in secondary lymphoid tissues. Here we investigated how an EE modulated thymus and thymocyte development. EE decreased thymus mass and cellularity, decreased the double positive thymocyte population, increased the proportion of CD8+ T cells, reduced the CD4:CD8 ratio, and downregulated CD69 expression in T cells. In a model of multiple sclerosis: experimental autoimmune encephalomyelitis (EAE), EE alleviated symptoms, inhibited spinal cord inflammation through regulation of type 1 T-helper cells mediated by glucocorticoid receptor signaling, and prevented EAE-induced thymic disturbance. Our mechanistic studies demonstrated that hypothalamic BDNF activated a hypothalamic-pituitary-adrenal axis mediating the EE's thymic effects. Our results indicate that a lifestyle intervention links the nervous, endocrine, and adaptive immune system, allowing the body to adapt to internal and external environments.

CD56 Expression Marks Human Group 2 Innate Lymphoid Cell Divergence from a Shared NK Cell and Group 3 Innate Lymphoid Cell Developmental Pathway.

According to the established model of murine innate lymphoid cell (ILC) development, helper ILCs develop separately from natural killer (NK) cells. However, it is unclear how helper ILCs and NK cells develop in humans. Here we elucidated key steps of NK cell, ILC2, and ILC3 development within human tonsils using ex vivo molecular and functional profiling and lineage differentiation assays. We demonstrated that while tonsillar NK cells, ILC2s, and ILC3s originated from a common CD34-CD117+ ILC precursor pool, final steps of ILC2 development deviated independently and became mutually exclusive from those of NK cells and ILC3s, whose developmental pathways overlapped. Moreover, we identified a CD34-CD117+ ILC precursor population that expressed CD56 and gave rise to NK cells and ILC3s but not to ILC2s. These data support a model of human ILC development distinct from the mouse, whereby human NK cells and ILC3s share a common developmental pathway separate from ILC2s.

An optimized xylene-free protein extraction method adapted to formalin-fixed paraffin embedded tissue sections for western blot analysis.

Deparaffinization of formalin-fixed paraffin embedded (FFPE) tissues with xylene currently remains a major challenge to the biomedical community. We developed an efficient xylene-free protocol to isolate proteins from archived FFPE human tissue sections. A total of 79 different types of FFPE tissue sections of 8 µm thickness were obtained from various archived FFPE specimens. Deparaffinization was conducted by gently washing each section with around 1 ml of hot distilled water (≈80°C). The deparaffinized tissues were homogenized in lysis buffer, and the isolated proteins were quantified and efficiently resolved using western blot analysis for the presence of Protein kinase B (PKB/AKT) and ß-actin. Moreover, a significant amount of proteins was successfully isolated with an average of 2.31 µg/µl. The migration pattern of AKT and ß-actin obtained from the specimens was similar to the positive control obtained from protein lysates prepared from in vitro cultured MDA231 cancer cell lines. AKT was successfully identified in all specimens, and ß-actin protein was resolved with an efficiency higher than 80%. The entire extraction procedure requires only 20 minutes. This newly developed technique is an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissue sections adequate for molecular analysis.

Transfusion Therapy in Children With Sickle Cell Disease.

Hydroxyurea, blood transfusions, and hematopoietic stem cell transplantation represent the 3 disease-modifying therapies in children with sickle cell disease (SCD). Blood transfusions play an increasingly important role in both prevention and management of SCD complications in this age group. This review will focus on the indications of blood transfusion in children with SCD and modalities of its administration. It will also highlight the complications of this life-saving therapy and ways of optimizing transfusion to minimize its associated risks.