Jianhong Chu

CS1-specific chimeric antigen receptor (CAR)-engineered natural killer cells enhance in vitro and in vivo antitumor activity against human multiple myeloma

Multiple myeloma (MM) is an incurable hematological malignancy. Chimeric antigen receptor (CAR)-expressing T cells have been demonstrated successfully in the clinic to treat B-lymphoid malignancies. However, the potential utility of antigen-specific CAR-engineered natural-killer (NK) cells to treat MM has not been explored. In this study, we determined whether CS1, a surface protein that is highly expressed on MM cells, can be targeted by CAR NK cells to treat MM. We successfully generated a viral construct of a CS1-specific CAR and expressed it in human NK cells. In vitro, CS1-CAR NK cells displayed enhanced MM cytolysis and interferon-γ (IFN-γ) production, and showed a specific CS1-dependent recognition of MM cells. Ex vivo, CS1-CAR NK cells also showed similarly enhanced activities when responding to primary MM tumor cells. More importantly, in an aggressive orthotopic MM xenograft mouse model, adoptive transfer of NK-92 cells expressing CS1-CAR efficiently suppressed the growth of human IM9 MM cells and also significantly prolonged mouse survival. Thus, CS1 represents a viable target for CAR-expressing immune cells, and autologous or allogeneic transplantation of CS1-specific CAR NK cells may be a promising strategy to treat MM.

CAR-Engineered NK Cells Targeting Wild-Type EGFR and EGFRvIII Enhance Killing of Glioblastoma and Patient-Derived Glioblastoma Stem Cells

Glioblastoma (GB) remains the most aggressive primary brain malignancy. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered natural killer (NK) cells to treat GB has not been explored. Tumors from approximately 50% of GB patients express wild-type EGFR (wtEGFR) and in fewer cases express both wtEGFR and the mutant form EGFRvIII; however, previously reported CAR T cell studies only focus on targeting EGFRvIII. Here we explore whether both wtEGFR and EGFRvIII can be effectively targeted by CAR-redirected NK cells to treat GB. We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells. EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-γ production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner. In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival. These findings support intracranial administration of NK-92-EGFR-CAR cells represents a promising clinical strategy to treat GB.

Transcription factor foxo1 is a negative regulator of natural killer cell maturation and function

Little is known about the role of negative regulators in controlling natural killer (NK) cell development and effector functions. Foxo1 is a multifunctional transcription factor of the forkhead family. Using a mouse model of conditional deletion in NK cells, we found that Foxo1 negatively controlled NK cell differentiation and function. Immature NK cells expressed abundant Foxo1 and little Tbx21 relative to mature NK cells, but these two transcription factors reversed their expression as NK cells proceeded through development. Foxo1 promoted NK cell homing to lymph nodes by upregulating CD62L expression and inhibited late-stage maturation and effector functions by repressing Tbx21 expression. Loss of Foxo1 rescued the defect in late-stage NK cell maturation in heterozygous Tbx21(+/-) mice. Collectively, our data reveal a regulatory pathway by which the negative regulator Foxo1 and the positive regulator Tbx21 play opposing roles in controlling NK cell development and effector functions.

Genetic Modification of T Cells Redirected toward CS1 Enhances Eradication of Myeloma Cells

Purpose: Our goal is to test whether CS1 could be targeted by chimeric antigen receptor (CAR) T cells to treat multiple myeloma (MM). Experimental Design: We generated a retroviral construct of a CS1-specific CAR and engineered primary human T cells expressing the CAR. We then tested the capacity of CS1–CAR T cells to eradicate human MM tumor cells in vitro, ex vivo, and in vivo using orthotopic MM xenograft mouse models. Results: In vitro, compared with mock-transduced T cells, upon recognizing CS1-positive MM cells, CS1–CAR-transduced T cells secreted more IFN-γ as well as interleukin (IL)-2, expressed higher levels of the activation marker CD69, showed higher capacity for degranulation, and displayed enhanced cytotoxicity. Ectopically forced expression of CS1 in MM cells with low CS1 expression enhanced recognition and killing by CAR T cells. Ex vivo, CS1–CAR T cells also showed similarly enhanced activities when responding to primary MM cells. More importantly, in orthotopic MM xenograft mouse models, adoptive transfer of human primary T cells expressing CS1–CAR efficiently suppressed the growth of human MM.1S and IM9 myeloma cells and significantly prolonged mouse survival. Conclusions: CS1 is a promising antigen that can be targeted by CAR-expressing T cells for treatment of MM. Clin Cancer Res; 20(15); 3989–4000. ©2014 AACR.

MicroRNAs activate natural killer cells through Toll-like receptor signaling.

MicroRNAs (miRNAs) bind to complementary sequences of target mRNAs, resulting in translational repression or target degradation and thus gene silencing. miRNAs are abundant in circulating blood, yet it is not known whether, as a class of regulatory molecules, they interact with human natural killer (NK) cells. Here we found that the treatment of human NK cells with several mature miRNAs in the presence of a low concentration of interleukin-12 induced CD69 expression, interferon-γ production, and degranulation marker CD107a expression. In vivo, infusion of several miRNAs alone in murine peripheral blood also resulted in comparable NK-cell activation, but not T-cell activation. Furthermore, miRNA administration significantly protected mice from tumor development in an NK cell-dependent manner. Mechanistically, we found that miRNA stimulation led to downstream activation of nuclear factor κB (NF-κB), an effect that was blunted by a block in Toll-like receptor 1(TLR1) signaling and attenuated in lymphoma patients. Knockdown of TLR1 resulted in less activation by miRNAs. Collectively, we show that miRNAs have a capacity to selectively activate innate immune effector cells that is, at least in part, via the TLR1-NF-κB signaling pathway. This may be important in the normal host defense against infection and/or malignant transformation.

A combinational therapy of EGFR-CAR NK cells and oncolytic herpes simplex virus 1 for breast cancer brain metastases

Breast cancer brain metastases (BCBMs) are common in patients with metastatic breast cancer and indicate a poor prognosis. These tumors are especially resistant to currently available treatments due to multiple factors. However, the combination of chimeric antigen receptor (CAR)-modified immune cells and oncolytic herpes simplex virus (oHSV) has not yet been explored in this context. In this study, NK-92 cells and primary NK cells were engineered to express the second generation of EGFR-CAR. The efficacies of anti-BCBMs of EGFR-CAR NK cells, oHSV-1, and their combination were tested in vitro and in a breast cancer intracranial mouse model. In vitro, compared with mock-transduced NK-92 cells or primary NK cells, EGFR-CAR-engineered NK-92 cells and primary NK cells displayed enhanced cytotoxicity and IFN-γ production when co-cultured with breast cancer cell lines MDA-MB-231, MDA-MB-468, and MCF-7. oHSV-1 alone was also capable of lysing and destroying these cells. However, a higher cytolytic effect of EGFR-CAR NK-92 cells was observed when combined with oHSV-1 compared to the monotherapies. In the mice intracranially pre-inoculated with EGFR-expressing MDA-MB-231 cells, intratumoral administration of either EGFR-CAR-transduced NK-92 cells or oHSV-1 mitigated tumor growth. Notably, the combination of EGFR-CAR NK-92 cells with oHSV-1 resulted in more efficient killing of MDA-MB-231 tumor cells and significantly longer survival of tumor-bearing mice when compared to monotherapies. These results demonstrate that regional administration of EGFR-CAR NK-92 cells combined with oHSV-1 therapy is a potentially promising strategy to treat BCBMs.

TGFβ Treatment Enhances Glioblastoma Virotherapy by Inhibiting the Innate Immune Response

Oncolytic viruses, including oncolytic herpes simplex virus (oHSV), have produced provocative therapeutic responses in patients with glioblastoma, the most aggressive brain tumor. Paradoxically, innate immune responses mediated by natural killer (NK) cells and macrophages/microglia appear to limit oHSV efficacy. Therefore, we investigated whether pretreatment with an immunosuppressive cytokine, TGFβ, might reverse these effects and thereby potentiate oHSV efficacy. TGFβ treatment of NK cells rendered them less cytolytic against oHSV-infected glioblastoma cells and stem-like cells in vitro. Furthermore, TGFβ treatment of NK cells, macrophages, or microglia increased viral titers of oHSV in cocultures with glioblastoma cells. In a syngeneic mouse model of glioblastoma, administering TGFβ prior to oHSV injection inhibited intracranial infiltration and activation of NK cells and macrophages. Notably, a single administration of TGFβ prior to oHSV therapy was sufficient to phenocopy NK-cell depletion and suppress tumor growth and prolong survival in both xenograft and syngeneic models of glioblastoma. Collectively, our findings show how administering a single dose of TGFβ prior to oncolytic virus treatment of glioblastoma can transiently inhibit innate immune cells that limit efficacy, thereby improving therapeutic responses and survival outcomes.

The Natural Product Phyllanthusmin C Enhances IFN-γ Production by Human NK Cells through Upregulation of TLR-Mediated NF-κB Signaling

Natural products are a major source for cancer drug development. NK cells are a critical component of innate immunity with the capacity to destroy cancer cells, cancer-initiating cells, and clear viral infections. However, few reports describe a natural product that stimulates NK cell IFN-γ production and unravel a mechanism of action. In this study, through screening, we found that a natural product, phyllanthusmin C (PL-C), alone enhanced IFN-γ production by human NK cells. PL-C also synergized with IL-12, even at the low cytokine concentration of 0.1 ng/ml, and stimulated IFN-γ production in both human CD56bright and CD56dim NK cell subsets. Mechanistically, TLR1 and/or TLR6 mediated PL-C’s activation of the NF-κB p65 subunit that in turn bound to the proximal promoter of IFNG and subsequently resulted in increased IFN-γ production in NK cells. However, IL-12 and IL-15Rs and their related STAT signaling pathways were not responsible for the enhanced IFN-γ secretion by PL-C. PL-C induced little or no T cell IFN-γ production or NK cell cytotoxicity. Collectively, we identify a natural product with the capacity to selectively enhance human NK cell IFN-γ production. Given the role of IFN-γ in immune surveillance, additional studies to understand the role of this natural product in prevention of cancer or infection in select populations are warranted.

Prenatal inflammation-induced NF-κB dyshomeostasis contributes to renin-angiotensin system over-activity resulting in prenatally programmed hypertension in offspring

Studies involving the use of prenatally programmed hypertension have been shown to potentially contribute to prevention of essential hypertension (EH). Our previous research has demonstrated that prenatal inflammatory stimulation leads to offspring’s aortic dysfunction and hypertension in pregnant Sprague-Dawley rats challenged with lipopolysaccharide (LPS). The present study found that prenatal LPS exposure led to NF-κB dyshomeostasis from fetus to adult, which was characterized by PI3K-Akt activation mediated degradation of IκBα protein and impaired NF-κB self-negative feedback loop mediated less newly synthesis of IκBα mRNA in thoracic aortas (gestational day 20, postnatal week 7 and 16). Prenatal or postnatal exposure of the IκBα degradation inhibitor, pyrollidine dithiocarbamate, effectively blocked NF-κB activation, endothelium dysfunction, and renin-angiotensin system (RAS) over-activity in thoracic aortas, resulting in reduced blood pressure in offspring that received prenatal exposure to LPS. Surprisingly, NF-κB dyshomeostasis and RAS over-activity were only found in thoracic aortas but not in superior mesenteric arteries. Collectively, our data demonstrate that the early life NF-κB dyshomeostasis induced by prenatal inflammatory exposure plays an essential role in the development of EH through triggering RAS over-activity. We conclude that early life NF-κB dyshomeostasis is a key predictor of EH, and thus, NF-κB inhibition represents an effective interventional strategy for EH prevention.

Cellular and molecular mechanisms in graft‐versus‐host disease

Graft‐versus‐host disease is a complication in patients undergoing hematopoietic stem cell transplantation. Graft‐versus‐host disease includes acute graft‐versus‐host disease and chronic graft‐versus‐host disease. Host APCs (e.g., dendritic cells and macrophages), effector T cells (e.g., Th1, Th17, and abnormal Th17:regulatory T cell ratio), B cells, and NK cells are implicated in graft‐versus‐host disease physiopathology. Proinflammation cytokines (e.g., IL‐17, IL‐1β, and TNF‐α) are increased in graft‐versus‐host disease. Costimulatory molecules play an important role in inducing graft‐versus‐host disease. Pattern‐recognition receptors, such as TLRs and nucleotide‐binding oligomerization domain‐like receptors, are critically involved in the pathogenesis of graft‐versus‐host disease. Complement system C3 mediates Th1/Th17 polarization in human T cell activation and skin graft‐versus‐host disease. Accumulation of CD26 T cells in graft‐versus‐host disease target organs was found. As a therapeutic target, soluble CD83 molecules or antibodies have been demonstrated to have therapeutic effects against graft‐versus‐host disease, and signaling molecules promote the inflammatory and immune process of graft‐versus‐host disease. These immune cells and molecules could be the predictors of graft‐versus‐host disease development and the drug targets of the treatments for graft‐versus‐host disease. This article focuses on major advances on cellular and molecular mechanisms in graft‐versus‐host disease.