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

Background: Tumors often develop resistance to surveillance by endogenous immune cells, which include natural killer (NK) cells. Ex vivo activated and/or expanded NK cells demonstrate cytotoxicity against various tumor cells and are promising therapeutics for adoptive cancer immunotherapy. Genetic modification can further enhance NK effector cell activity or activation sensitization.

Expression of carcinoma, apoptosis, and cell-death-related genes are determinants for sensitivity of pediatric cancer cell lines to lysis by natural killer cells.

Natural killer (NK) cells have potential utility in pediatric cancer immunotherapy for their ability to lyse diverse tumor targets, lack of dependence on mutation-associated tumor antigens, and for their relative safety demonstrated so far in clinical trials. Here, we evaluate the cytotoxic potential of expanded NK cells against a well-characterized panel of pediatric cancer cell lines representing Ewing sarcoma, rhabdomyosarcoma, neuroblastoma, lymphoma, leukemia, and brain tumors. We correlate their sensitivity NK cell lysis with tumor phenotypic, transcriptomic, and genetic determinants, and correlate known immunogenetic determinants with donor NK cell potency.

Correction to: Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

There was a typo in author Andrew Wahba's name in the initial online publication. The original article has been corrected.

Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

Purpose: Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent studies have shown the ability of natural killer (NK) cells to lyse MB cell lines in vitro, but in vivo successes remain elusive and the efficacy and fate of NK cells in vivo remain unknown.

Methods: To address these questions, we injected MB cells into the cerebellum of immunodeficient mice and examined tumor growth at various days after tumor establishment via bioluminescence imaging.

NCR1 Expression Identifies Canine Natural Killer Cell Subsets with Phenotypic Similarity to Human Natural Killer Cells.

Canines spontaneously develop many cancers similar to humans - including osteosarcoma, leukemia, and lymphoma - offering the opportunity to study immune therapies in a genetically heterogeneous and immunocompetent environment. However, a lack of antibodies recognizing canine NK cell markers has resulted in suboptimal characterization and unknown purity of NK cell products, hindering the development of canine models of NK cell adoptive immunotherapy. To this end, we generated a novel antibody to canine NCR1 (NKp46), the putative species-wide marker of NK cells, enabling purification of NK cells for further characterization.

Electroporation of siRNA to Silence Gene Expression in Primary NK Cells.

Gene silencing through siRNA is an effective experimental tool to unravel molecular mechanisms involved in cellular processes. Here we describe a method to silence gene expression in primary human natural killer (NK) cells by transfecting ON-TARGETplus SMART pool siRNA using an electroporation-based method called Nucleofection(®). The technique yields effective silencing of the target gene without any off-target effects.

Engineering Receptor Expression on Natural Killer Cells Through Trogocytosis.

Trogocytosis is a rapid contact-dependent process by which lymphocytes acquire membrane patches from the target cells ('donor' cells) with which they interact and this phenomenon has been shown to occur in various immune cells. The surface molecules acquired through trogocytosis are functionally incorporated in the 'acceptor' cells transiently. We had previously demonstrated that trogocytosis can be utilized in place of gene transfer to engineer surface receptor expression on NK cells for adoptive immunotherapy applications.

Ex Vivo Expansion of Human NK Cells Using K562 Engineered to Express Membrane Bound IL21.

Natural killer (NK) cells have gained significant attention for adoptive immunotherapy of cancer due to their well-documented antitumor function. In order to evaluate the therapeutic efficacy of NK cell adoptive immunotherapy in preclinical models with a potential for clinical translation, there is a need for a reliable platform for ex vivo expansion of NK cells. Numerous methods are reported in literature using cytokines and feeder cells to activate and expand human NK cells, and many of these methods are limited by low-fold expansion, cytokine dependency of expanded NK cells or expansion-related senescence.

Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry Method.

Although natural killer (NK) cells produce various cytokines that regulate other lymphocytes of the immune system, the primary effector function of NK cells is the direct cytolysis of their targets. Hence analyzing the cytotoxic potential of these lymphocytes is fundamental to understanding their biology and their clinical impact. We have previously shown that release-based cytotoxicity assays, such as calcein release assay, could potentially underestimate percent specific lysis if the entrapped reporter is not completely released and demonstrated that an Image cytometry method can overcome this caveat.

Trastuzumab upregulates expression of HLA-ABC and T cell costimulatory molecules through engagement of natural killer cells and stimulation of IFNγ secretion.

It is increasingly recognized that trastuzumab, an antibody approved for treating human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer, exerts some of its antitumor effects through enhanced T cell responses. Full activation of CD8 T cells requires both expression of major histocompatibility complex class I molecules (HLA-ABC) and expression of the T cell costimulatory molecule CD80 or CD86; however, the impact of trastuzumab treatment on the expression of HLA-ABC and CD80 and CD86 has not been investigated in HER2-overexpressing breast cancer cells. In this study, we found that, while there is no direct correlation between the expression of HER2 and HLA-ABC in breast cancer, knockdown of HER2 or inhibition of HER2 kinase by lapatinib downregulated HLA-ABC expression.

Natural killer cells stimulated with PM21 particles expand and biodistribute in vivo: Clinical implications for cancer treatment.

Background Aims: Natural killer (NK) cell immunotherapy for treatment of cancer is promising, but requires methods that expand cytotoxic NK cells that persist in circulation and home to disease site.

Methods: We developed a particle-based method that is simple, effective and specifically expands cytotoxic NK cells from peripheral blood mononuclear cells (PBMCs) both ex vivo and in vivo. This method uses particles prepared from plasma membranes of K562-mb21-41BBL cells, expressing 41BBL and membrane bound interleukin-21 (PM21 particles).

A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry.

Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The 51Chromium release assay, is the "gold standard" for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods.

Transcription of the activating receptor NKG2D in natural killer cells is regulated by STAT3 tyrosine phosphorylation.

Signal transducer and activator of transcription 3 (STAT3) is considered a negative regulator of inflammation, as inhibition of STAT3 signaling enhances antitumor immunity. However, STAT3 activation is a key oncogenic pathway in natural killer (NK)-lineage large granular lymphomas, and we recently reported enhanced proliferation and function of human NK cells activated with IL-21, which signals primarily through STAT3. These IL-21-expanded NK cells also have increased NKG2D expression, which led us to focus our investigation on whether STAT3 regulates NKG2D.

Universal artificial antigen presenting cells to selectively propagate T cells expressing chimeric antigen receptor independent of specificity.

T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to nonviral gene transfer in T cells uses ex vivo numeric expansion of CAR T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA.

Growth and activation of natural killer cells ex vivo from children with neuroblastoma for adoptive cell therapy.

Purpose: Adoptive transfer of natural killer (NK) cells combined with tumor-specific monoclonal antibodies (mAb) has therapeutic potential for malignancies. We determined if large numbers of activated NK (aNK) cells can be grown ex vivo from peripheral blood mononuclear cells (PBMC) of children with high-risk neuroblastoma using artificial antigen-presenting cells (aAPC).

Experimental Design: Irradiated K562-derived Clone 9.

Decitabine has a biphasic effect on natural killer cell viability, phenotype, and function under proliferative conditions.

DNA hypermethylation resulting in aberrant epigenetic silencing plays an important role in the oncogenesis of many cancer types, including acute myelogenous leukemia (AML).(4) The modulation of NK cell receptors and their cognate ligands is a known mechanism of immune escape in AML, and some membrane proteins, such as killer immunoglobulin-like receptors (KIR), are known to be transcriptionally regulated by DNA methylation of their promoter regions. Thus, restoring proper expression of immunoreceptors or their ligands with immunosensitizing drugs is an attractive approach to improving cancer immunotherapy.

Fenretinide sensitizes multidrug-resistant human neuroblastoma cells to antibody-independent and ch14.18-mediated NK cell cytotoxicity.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Combining passive immunotherapy with an antibody to the disialoganglioside GD2 (ch14.18/SP2/0) and cytokines with 13-cis-retinoic acid for post-myeloablative maintenance therapy increased survival in high-risk NB, but the overall prognosis for these children is still in need of improvement.

Engineering lymph node homing of ex vivo-expanded human natural killer cells via trogocytosis of the chemokine receptor CCR7.

Natural killer (NK) cells have gained significant attention in adoptive immunotherapy for cancer. Consequently, novel methods of clinical-grade expansion of NK cells have emerged. Subsets of NK cells express a variety of chemokine receptors.

Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells.

NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening.

Expansion, purification, and functional assessment of human peripheral blood NK cells.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).