Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy.

Manufacture of chimeric antigen receptor (CAR)-T cells usually involves the use of viral delivery systems to achieve high transgene expression. However, it can be costly and may result in random integration of the CAR into the genome, creating several disadvantages including variation in transgene expression, functional gene silencing and potential oncogenic transformation. Here, we optimized the method of nonviral, CRISPR/Cas9 genome editing using large donor DNA delivery, knocked-in an anti-tumor single chain variable fragment (scFv) into the N-terminus of CD3ε and efficiently generated fusion protein (FP) T cells. These cells displayed FP integration within the TCR/CD3 complex, lower variability in gene expression compared to CAR-T cells and good cell expansion after transfection. CD3ε FP T cells were predominantly CD8+ effector memory T cells, and exhibited anti-tumor activity in vitro and in vivo. Dual targeting FP T cells were also generated through the incorporation of scFvs into other CD3 subunits and CD28. Compared to viral-based methods, this method serves as an alternative and versatile way of generating T cells with tumor-targeting receptors for cancer immunotherapy.

CAR-T cell development for Cutaneous T cell Lymphoma: current limitations and potential treatment strategies.

Chimeric antigen receptor (CAR)-T therapy has demonstrated remarkable outcomes for B cell malignancies, however, its application for T cell lymphoma, particularly cutaneous T cell lymphoma (CTCL), has been limited. Barriers to effective CAR-T cell therapy in treating CTCL include T cell aplasia in autologous transplants, CAR-T product contamination with leukemic T cells, CAR-T fratricide (when the target antigen is present on normal T cells), and tumor heterogeneity. To address these critical challenges, innovative CAR engineering by targeting multiple antigens to strike a balance between efficacy and safety of the therapy is necessary. In this review, we discuss the current obstacles to CAR-T cell therapy and highlight potential targets in treating CTCL. Looking forward, we propose strategies to develop more powerful dual CARs that are advancing towards the clinic in CTCL therapy.

'Off-the-Shelf' Immunotherapy: Manufacture of CD8+ T Cells Derived from Hematopoietic Stem Cells.

Cellular immunotherapy is revolutionizing cancer treatment. However, autologous transplants are complex, costly, and limited by the number and quality of T cells that can be isolated from and expanded for re-infusion into each patient. This paper demonstrates a stromal support cell-free in vitro method for the differentiation of T cells from umbilical cord blood hematopoietic stem cells (HSCs). For each single HSC cell input, approximately 5 × 104 T cells were created with an initial five days of HSC expansion and subsequent T cell differentiation over 49 days. When the induced in vitro differentiated T cells were activated by cytokines and anti-CD3/CD28 beads, CD8+ T cell receptor (TCR) γδ+ T cells were preferentially generated and elicited cytotoxic function against ovarian cancer cells in vitro. This process of inducing de novo functional T cells offers a possible strategy to increase T cell yields, simplify manufacturing, and reduce costs with application potential for conversion into chimeric antigen receptor (CAR)-T cells for cancer immunotherapy and for allogeneic transplantation to restore immune competence.

Enhancing a Natural Killer: Modification of NK Cells for Cancer Immunotherapy.

Natural killer (NK) cells are potent innate immune system effector lymphocytes armed with multiple mechanisms for killing cancer cells. Given the dynamic roles of NK cells in tumor surveillance, they are fast becoming a next-generation tool for adoptive immunotherapy. Many strategies are being employed to increase their number and improve their ability to overcome cancer resistance and the immunosuppressive tumor microenvironment. These include the use of cytokines and synthetic compounds to bolster propagation and killing capacity, targeting immune-function checkpoints, addition of chimeric antigen receptors (CARs) to provide cancer specificity and genetic ablation of inhibitory molecules. The next generation of NK cell products will ideally be readily available as an "off-the-shelf" product and stem cell derived to enable potentially unlimited supply. However, several considerations regarding NK cell source, genetic modification and scale up first need addressing. Understanding NK cell biology and interaction within specific tumor contexts will help identify necessary NK cell modifications and relevant choice of NK cell source. Further enhancement of manufacturing processes will allow for off-the-shelf NK cell immunotherapies to become key components of multifaceted therapeutic strategies for cancer.

Engineered CAR-T cells targeting TAG-72 and CD47 in ovarian cancer.

Chimeric antigen receptor (CAR) T cells have revolutionized blood cancer immunotherapy; however, their efficacy against solid tumors has been limited. A common mechanism of tumor escape from single target therapies is downregulation or mutational loss of the nominal epitope. Targeting multiple antigens may thus improve the effectiveness of CAR immunotherapies. We generated dual CAR-T cells targeting two tumor antigens: TAG-72 (tumor-associated glycoprotein 72) and CD47. TAG-72 is a pan-adenocarcinoma oncofetal antigen, highly expressed in ovarian cancers, with increased expression linked to disease progression. CD47 is ubiquitously overexpressed in multiple tumor types, including ovarian cancer; it is a macrophage "don't eat me" signal. However, CD47 is also expressed on many normal cells. To avoid this component of the dual CAR-T cells killing healthy tissue, we designed a truncated CD47 CAR devoid of intracellular signaling domains. The CD47 CAR facilitates binding to CD47+ cells, increasing the prospect of TAG-72+ cell elimination via the TAG-72 CAR. Furthermore, we could reduce the damage to normal tissue by monomerizing the CD47 CAR. Our results indicate that the co-expression of the TAG-72 CAR and the CD47-truncated monomer CAR on T cells could be an effective, dual CAR-T cell strategy for ovarian cancer, also applicable to other adenocarcinomas.

Three-Dimensional (3D) cell culture monitoring: Opportunities and challenges for impedance spectroscopy.

Three-dimensional (3D) cell culture has developed rapidly over the past 5-10 years with the goal of better replicating human physiology and tissue complexity in the laboratory. Quantifying cellular responses is fundamental in understanding how cells and tissues respond during their growth cycle and in response to external stimuli. There is a need to develop and validate tools that can give insight into cell number, viability, and distribution in real-time, nondestructively and without the use of stains or other labelling processes. Impedance spectroscopy can address all of these challenges and is currently used both commercially and in academic laboratories to measure cellular processes in 2D cell culture systems. However, its use in 3D cultures is not straight forward due to the complexity of the electrical circuit model of 3D tissues. In addition, there are challenges in the design and integration of electrodes within 3D cell culture systems. Researchers have used a range of strategies to implement impedance spectroscopy in 3D systems. This review examines electrode design, integration, and outcomes of a range of impedance spectroscopy studies and multiparametric systems relevant to 3D cell cultures. While these systems provide whole culture data, impedance tomography approaches have shown how this technique can be used to achieve spatial resolution. This review demonstrates how impedance spectroscopy and tomography can be used to provide real-time sensing in 3D cell cultures, but challenges remain in integrating electrodes without affecting cell culture functionality. If these challenges can be addressed and more realistic electrical models for 3D tissues developed, the implementation of impedance-based systems will be able to provide real-time, quantitative tracking of 3D cell culture systems.

Toward Immunocompetent 3D Skin Models.

3D human skin models provide a platform for toxicity testing, biomaterials evaluation, and investigation of fundamental biological processes. However, the majority of current in vitro models lack an inflammatory system, vasculature, and other characteristics of native skin, indicating scope for more physiologically complex models. Looking at the immune system, there are a variety of cells that could be integrated to create novel skin models, but to do this effectively it is also necessary to understand the interface between skin biology and tissue engineering as well as the different roles the immune system plays in specific health and disease states. Here, a progress report on skin immunity and current immunocompetent skin models with a focus on construction methods is presented; scaffold and cell choice as well as the requirements of physiologically relevant models are elaborated. The wide range of technological and fundamental challenges that need to be addressed to successfully generate immunocompetent skin models and the steps currently being made globally by researchers as they develop new models are explored. Induced pluripotent stem cells, microfluidic platforms to control the model environment, and new real-time monitoring techniques capable of probing biochemical processes within the models are discussed.

An antigen to remember: regulation of B cell memory in health and disease.

Vaccine success relies on the formation of immunity. Humoral immunity is critical and is mediated by long-lived antibody-secreting cells and memory B cells (MBCs). Chronic infectious diseases cause a significant global burden of disease; pathogens that evade the immune system can cause phenotypical and functional changes to immune memory populations. Thus, recent studies have focused on MBC subset function. IgM+ MBCs have emerged as important early responders in malaria. Atypical MBCs have functional qualities associated with exhaustion in chronic infectious diseases, but the requirements for their formation and where they localize remains unknown. Similarly, the T-bet-driven transcriptional program drives formation of MBCs phenotypically similar to atypical MBCs. Identifying protective or detrimental roles of MBC subsets, and their regulators, will be important for clinical intervention.

Roles of extracellular nucleotides and P2 receptors in ectodomain shedding.

Ectodomain shedding of integral membrane receptors results in the release of soluble molecules and modification of the transmembrane portions to mediate or modulate extracellular and intracellular signalling. Ectodomain shedding is stimulated by a variety of mechanisms, including the activation of P2 receptors by extracellular nucleotides. This review describes in detail the roles of extracellular nucleotides and P2 receptors in the shedding of various cell surface molecules, including amyloid precursor protein, CD23, CD62L, and members of the epidermal growth factor, immunoglobulin and tumour necrosis factor families. This review discusses the mechanisms involved in P2 receptor-mediated shedding, demonstrating central roles for the P2 receptors, P2X7 and P2Y2, and the sheddases, ADAM10 and ADAM17, in this process in a number of cell types.

Group A Streptococcus Modulates Host Inflammation by Manipulating Polymorphonuclear Leukocyte Cell Death Responses.

Polymorphonuclear leukocyte (PMN) cell death strongly influences the resolution of inflammatory episodes, and may exacerbate adverse pathologies in response to infection. We investigated PMN cell death mechanisms following infection by virulent group A Streptococcus (GAS). Human PMNs were infected in vitro with a clinical, virulent GAS isolate and an avirulent derivative strain, and compared for phagocytosis, the production of reactive oxygen species (ROS), mitochondrial membrane depolarization and apoptotic markers. C57BL/6J mice were then infected, in order to observe the effects on murine PMNs in vivo. Human PMNs phagocytosed virulent GAS less efficiently, produced less ROS and underwent reduced mitochondrial membrane depolarization compared with phagocytosis of avirulent GAS. Morphological and biochemical analyses revealed that PMNs infected with avirulent GAS exhibited nuclear fragmentation and caspase-3 activation consistent with an anti-inflammatory apoptotic phenotype. Conversely, virulent GAS induced PMN vacuolization and plasma membrane permeabilization, leading to a necrotic form of cell death. Infection of the mice with virulent GAS engendered significantly higher systemic pro-inflammatory cytokine release and localized infiltration of murine PMNs, with cells associated with virulent GAS infection exhibiting reduced apoptotic potential. Avirulent GAS infection was associated with lower levels of proinflammatory cytokines and tissue PMN apoptosis. We propose that the differences in PMN cell death mechanisms influence the inflammatory responses to infection by GAS.

Activation of the P2X7 receptor induces the rapid shedding of CD23 from human and murine B cells.

Activation of the P2X7 receptor by the extracellular damage-associated molecular pattern, adenosine 5'-triphosphate (ATP), induces the shedding of cell surface molecules including the low-affinity IgE receptor, CD23, from human leukocytes. A disintegrin and metalloprotease (ADAM) 10 mediates P2X7-induced shedding of CD23 from multiple myeloma RPMI 8226 B cells; however, whether this process occurs in primary B cells is unknown. The aim of the current study was to determine whether P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells. Flow cytometric and ELISA measurements showed that ATP treatment of human and murine B cells induced the rapid shedding of CD23. Treatment of cells with the specific P2X7 antagonist, AZ10606120, near-completely impaired ATP-induced CD23 shedding from both human and murine B cells. ATP-induced CD23 shedding was also impaired in B cells from P2X7 knockout mice. The absence of full-length, functional P2X7 in the P2X7 knockout mice was confirmed by immunoblotting of splenic cells, and by flow cytometric measurements of ATP-induced YO-PRO-1(2+) uptake into splenic B and T cells. The broad-spectrum metalloprotease antagonist, BB-94, and the ADAM10 antagonist, GI254023X, impaired P2X7-induced CD23 shedding from both human and murine B cells. These data indicate that P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells and that this process may be mediated by ADAM10.

Human P2X7 receptor activation induces the rapid shedding of CXCL16.

Activation of the purinergic P2X7 receptor by extracellular ATP induces the shedding of cell-surface molecules including the low-affinity IgE receptor, CD23 from leukocytes. CD23 is a known substrate of a disintegrin and metalloprotease (ADAM)10. The aim of the current study was to determine if P2X7 activation induced the shedding of the chemokine CXCL16, an ADAM10 substrate. Using immunolabelling and flow cytometry we demonstrate that human RPMI 8226 multiple myeloma B cells, which have been previously shown to express P2X7, also express CXCL16. Flow cytometric and ELISA measurements of ATP-induced loss of cell-surface CXCL16 showed that ATP treatment of RPMI 8226 cells induced the rapid shedding of CXCL16. Treatment of RPMI 8226 cells with the specific P2X7 antagonists, AZ10606120 and KN-62 impaired ATP-induced CXCL16 shedding by ~86% and ~90% respectively. RT-PCR demonstrated that ADAM10 is expressed in these cells and treatment of cells with the ADAM10 inhibitor, GI254023X, impaired ATP-induced CXCL16 shedding by ~87%. GI254023X also impaired P2X7-induced CD23 shedding by ∼57%. This data indicates that human P2X7 activation induces the rapid shedding of CXCL16 and that this process involves ADAM10.

P2X7 receptor activation mediates organic cation uptake into human myeloid leukaemic KG-1 cells.

The P2X7 purinergic receptor is an ATP-gated cation channel with an emerging role in neoplasia. In this study we demonstrate that the human KG-1 cell line, a model of acute myelogenous leukaemia, expresses functional P2X7. RT-PCR and immunochemical techniques demonstrated the presence of P2X7 mRNA and protein respectively in KG-l cells, as well as in positive control multiple myeloma RPMI 8226 cells. Flow cytometric measurements demonstrated that ATP induced ethidium(+) uptake into KG-l cells suspended in sucrose medium (EC(50) of ≈ 3 µM), but not into cells in NaCl medium. In contrast, ATP induced ethidium(+) uptake into RPMI 8226 cells suspended in either sucrose or NaCl medium (EC(50) of ≈ 3 or ≈ 99 µM, respectively), as well as into RPMI 8226 cells in KCl medium (EC(50) of ≈ 18 µM). BzATP and to a lesser extent ATPγS and αß-methylene ATP, but not ADP or UTP, also induced ethidium(+) uptake into KG-1 cells. ATP-induced ethidium(+) uptake was completely impaired by the P2X7 antagonists, AZ10606120 and A-438079. ATP-induced ethidium(+) uptake was also impaired by probenecid but not by carbenoxolone, both pannexin-1 antagonists. ATP induced YO-PRO-1(2+) and propidium(2+) uptake into KG-1 cells. Finally, sequencing of full-length P2X7 cDNA identified several single nucleotide polymorphisms (SNPs) in KG-1 cells including H155Y, A348T, T357S and Q460R. RPMI 8226 cells contained A348T, A433V and H521Q SNPs. In conclusion, the KG-1 cell line expresses functional P2X7. This cell line may help elucidate the signalling pathways involved in P2X7-induced survival and invasiveness of myeloid leukaemic cells.

P2X7 receptor activation induces cell death and CD23 shedding in human RPMI 8226 multiple myeloma cells.

BACKGROUND: The extracellular ATP-gated cation channel, P2X7 receptor, has an emerging role in neoplasia, however progress in the field is limited by a lack of malignant cell lines expressing this receptor. METHODS: Immunofluorescence labelling and a fixed-time ATP-induced ethidium+ uptake assay were used to screen a panel of human malignant cell lines for the presence of functional P2X7. The presence of P2X7 was confirmed by RT-PCR, immunoblotting and pharmacological approaches. ATP-induced cell death was measured by colourimetric tetrazolium-based and cytofluorometric assays. ATP-induced CD23 shedding was measured by immunofluorescence labelling and ELISA. RESULTS: RPMI 8226 multiple myeloma cells expressed P2X7 mRNA and protein, as well as P2X1, P2X4 and P2X5 mRNA. ATP induced ethidium+ uptake into these cells with an EC50 of ~116 µM, and this uptake was reduced in the presence of extracellular Ca²+ and Mg²+. The P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not UTP, induced ethidium+ uptake. ATP-induced ethidium+ uptake was impaired by the P2X7 antagonists, KN-62 and A-438079. ATP induced death and CD23 shedding in RPMI 8226 cells, and both processes were impaired by P2X7 antagonists. The metalloprotease antagonists, BB-94 and GM6001, impaired ATP-induced CD23 shedding but not ethidium+ uptake. CONCLUSIONS: P2X7 receptor activation induces cell death and CD23 shedding in RPMI 8226 cells. GENERAL SIGNIFICANCE: RPMI 8226 cells may be useful to study the role of P2X7 in multiple myeloma and B-lymphocytes.

Murine epidermal Langerhans cells and keratinocytes express functional P2X7 receptors.

Extracellular ATP via the activation of purinergic P2 receptors has an emerging role in cutaneous biology; however, the distribution of these receptors in mouse skin is poorly defined. This study investigated whether murine epidermal cell subpopulations express functional purinergic P2X(7) receptors. P2X(7) expression was examined by immunoblotting and immunofluorescence staining of epidermal cells from C57Bl/6 mice. P2X(7) function was evaluated by nucleotide-induced ethidium(+) uptake measurements in epidermal cells from C57Bl/6 mice, and from P2X(7) deficient mice and wild-type littermate controls. P2X(7) was detected in whole epidermal cell preparations, and specifically on Langerhans cells (LCs) and keratinocytes (KCs). ATP induced ethidium(+) uptake into LCs and KCs, with EC(50) values of 503 and 482 microm, respectively. BzATP, and to a lesser extent ATPgammaS and ADP, also induced ethidium(+) uptake; while UTP, alphabeta-meth-ATP and NAD were ineffective. ATP-induced ethidium(+) uptake was impaired by Na(+) and Mg(2+), and the P2X(7) antagonist, A-438079 and was absent in LCs and KCs from P2X(7) deficient mice. These results demonstrate that murine LCs and KCs express functional P2X(7), and support a role for this receptor in cutaneous biology.