Type I interferon induces necroptosis in macrophages during infection with Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a virulent pathogen that induces rapid host death. Here we observed that host survival after infection with S. Typhimurium was enhanced in the absence of type I interferon signaling, with improved survival of mice deficient in the receptor for type I interferons (Ifnar1(-/-) mice) that was attributed to macrophages. Although there was no impairment in cytokine expression or inflammasome activation in Ifnar1(-/-) macrophages, they were highly resistant to S. Typhimurium-induced cell death. Specific inhibition of the kinase RIP1 or knockdown of the gene encoding the kinase RIP3 prevented the death of wild-type macrophages, which indicated that necroptosis was a mechanism of cell death. Finally, RIP3-deficient macrophages, which cannot undergo necroptosis, had similarly less death and enhanced control of S. Typhimurium in vivo. Thus, we propose that S. Typhimurium induces the production of type I interferon, which drives necroptosis of macrophages and allows them to evade the immune response.

Differentiated macrophages acquire a pro-inflammatory and cell death-resistant phenotype due to increasing XIAP and p38-mediated inhibition of RipK1.

Monocytes differentiate into macrophages, which deactivate invading pathogens. Macrophages can be resistant to cell death mechanisms in some situations, and the mechanisms involved are not clear. Here, using mouse immune cells, we investigated whether the differentiation of macrophages affects their susceptibility to cell death by the ripoptosome/necrosome pathways. We show that treatment of macrophages with a mimetic of second mitochondrial activator of caspases (SMAC) resulted in ripoptosome-driven cell death that specifically depended on tumor necrosis factor α (TNFα) expression and the receptor-interacting serine/threonine protein kinase 1 (RipK1)-RipK3-caspase-8 interaction in activated and cycling macrophages. Differentiation of macrophages increased the expression of pro-inflammatory cytokines but reduced RipK1-dependent cell death and the RipK3-caspase-8 interaction. The expression of the anti-apoptotic mediators, X-linked inhibitor of apoptosis protein (XIAP) and caspase-like apoptosis regulatory protein (cFLIPL), also increased in differentiated macrophages, which inhibited caspase activation. The resistance to cell death was abrogated in XIAP-deficient macrophages. However, even in the presence of increased XIAP expression, inhibition of the mitogen-activated protein kinase (MAPK) p38 and MAPK-activated protein kinase 2 (MK2) made differentiated macrophages susceptible to cell death. These results suggest that the p38/MK2 pathway overrides apoptosis inhibition by XIAP and that acquisition of resistance to cell death by increased expression of XIAP and cFLIPL may allow inflammatory macrophages to participate in pathogen control for a longer duration.

Type-I interferon signaling through ISGF3 complex is required for sustained Rip3 activation and necroptosis in macrophages.

Myeloid cells play a critical role in perpetuating inflammation during various chronic diseases. Recently the death of macrophages through programmed necrosis (necroptosis) has emerged as an important mechanism in inflammation and pathology. We evaluated the mechanisms that lead to the induction of necrotic cell death in macrophages. Our results indicate that type I IFN (IFN-I) signaling is a predominant mechanism of necroptosis, because macrophages deficient in IFN-α receptor type I (IFNAR1) are highly resistant to necroptosis after stimulation with LPS, polyinosinic-polycytidylic acid, TNF-α, or IFN-ß in the presence of caspase inhibitors. IFN-I-induced necroptosis occurred through both mechanisms dependent on and independent of Toll/IL-1 receptor domain-containing adaptor inducing IFN-ß (TRIF) and led to persistent phosphorylation of receptor-interacting protein 3 (Rip3) kinase, which resulted in potent necroptosis. Although various IFN-regulatory factors (IRFs) facilitated the induction of necroptosis in response to IFN-ß, IRF-9-STAT1- or -STAT2-deficient macrophages were highly resistant to necroptosis. Our results indicate that IFN-ß-induced necroptosis of macrophages proceeds through tonic IFN-stimulated gene factor 3 (ISGF3) signaling, which leads to persistent expression of STAT1, STAT2, and IRF9. Induction of IFNAR1/Rip3-dependent necroptosis also resulted in potent inflammatory pathology in vivo. These results reveal how IFN-I mediates acute inflammation through macrophage necroptosis.

Cathepsins limit macrophage necroptosis through cleavage of Rip1 kinase.

It has recently been shown that programmed necrosis, necroptosis, may play a key role in the development of inflammation. Deciphering the regulation of this pathway within immune cells may therefore have implications in pathology associated with inflammatory diseases. We show that treatment of macrophages with the pan caspase inhibitor (zVAD-FMK) results in both increased phosphorylation and decreased cleavage of receptor interacting protein kinase-1 (Rip1), leading to necroptosis that is dependent on autocrine TNF signaling. Stimulation of cells with TLR agonists such as LPS in the presence of zVAD-FMK also induced Rip1-phosphorylation via a TNFR-independent mechanism. Further examination of Rip1 expression under these stimulatory conditions revealed a regulatory cleavage of Rip1 in macrophages that is not apparently attributable to caspase-8. Instead, we provide novel evidence that cysteine family cathepsins, which are highly abundant in myeloid cells, can also cleave Rip1 kinase. Using small interfering RNA knockdown, specific cathepsin inhibitors, and cell-free cleavage assays, we demonstrate that cysteine cathepsins B and S can directly cleave Rip1. Finally, we demonstrate that only through combined inhibition of cathepsins and caspase-8 could a potent induction of macrophage necroptosis be achieved. These data reveal a novel mechanism of regulation of necroptosis by cathepsins within macrophage cells.

A Flow Cytometry-Based Method for Assessing CAR Cell Binding Kinetics Using Stable CAR Jurkat Cells.

Chimeric antigen receptors (CARs) are synthetic fusion proteins that can reprogram immune cells to target specific antigens. CAR-expressing T cells have emerged as an effective treatment method for hematological cancers; despite this success, the mechanisms and structural properties that govern CAR responses are not fully understood. Here, we provide a simple assay to assess cellular avidity using a standard flow cytometer. This assay measures the interaction kinetics of CAR-expressing T cells and targets antigen-expressing target cells. By co-culturing stably transfected CAR Jurkat cells with target positive and negative cells for short periods of time in a varying effector-target gradient, we were able to observe the formation of CAR-target cell doublets, providing a readout of actively bound cells. When using the optimized protocol reported here, we observed unique cellular binding curves that varied between CAR constructs with differing antigen binding domains. The cellular binding kinetics of unique CARs remained consistent, were dependent on specific target antigen expression, and required active biological signaling. While existing literature is not clear at this time whether higher or lower CAR cell binding is beneficial to CAR therapeutic activity, the application of this simplified protocol for assessing CAR binding could lead to a better understanding of the proximal signaling events that regulate CAR functionality. Key features • Determines CAR receptor cellular interaction kinetics using a Jurkat cell model. • Can be used for a wide variety of CAR target antigens, including both hematological and solid tumor targets. • Experiments can be performed in under two hours with no staining using a standard flow cytometer. • Requires stable CAR Jurkat cells and target cells with stable fluorescent marker expression for optimal results.

Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic.

Simultaneous engineering of natural killer cells for CAR transgenesis and CRISPR-Cas9 knockout using retroviral particles.

Natural killer (NK) cells are potent cytotoxic innate lymphocytes that can be used for cancer immunotherapy. Since the balance of signals from activating and inhibitory receptors determines the activity of NK cells, their anti-tumor activity can be potentiated by overexpressing activating receptors or knocking out inhibitory receptors via genome engineering, such as chimeric antigen receptor (CAR) transgenesis and CRISPR-Cas9-mediated gene editing, respectively. Here, we report the development of a one-step strategy for CRISPR-Cas9-mediated gene knockout and CAR transgenesis in NK cells using retroviral particles. We generated NK cells expressing anti-epidermal growth factor receptor (EGFR)-CAR with simultaneous TIGIT gene knockout using single transduction and evaluated the consequence of the genetic modifications in vitro and in vivo. Taken together, our results demonstrate that retroviral particle-mediated engineering provides a strategy readily applicable to simultaneous genetic modifications of NK cells for efficient immunotherapy.

Advancements in CAR-NK therapy: lessons to be learned from CAR-T therapy.

Advancements in chimeric antigen receptor engineered T-cell (CAR-T) therapy have revolutionized treatment for several cancer types over the past decade. Despite this success, obstacles including the high price tag, manufacturing complexity, and treatment-associated toxicities have limited the broad application of this therapy. Chimeric antigen receptor engineered natural killer cell (CAR-NK) therapy offers a potential opportunity for a simpler and more affordable "off-the-shelf" treatment, likely with fewer toxicities. Unlike CAR-T, CAR-NK therapies are still in early development, with few clinical trials yet reported. Given the challenges experienced through the development of CAR-T therapies, this review explores what lessons we can apply to build better CAR-NK therapies. In particular, we explore the importance of optimizing the immunochemical properties of the CAR construct, understanding factors leading to cell product persistence, enhancing trafficking of transferred cells to the tumor, ensuring the metabolic fitness of the transferred product, and strategies to avoid tumor escape through antigen loss. We also review trogocytosis, an important emerging challenge that likely equally applies to CAR-T and CAR-NK cells. Finally, we discuss how these limitations are already being addressed in CAR-NK therapies, and what future directions may be possible.

A simplified function-first method for the discovery and optimization of bispecific immune engaging antibodies.

Bi-specific T-cell engager antibodies (BiTEs) are synthetic fusion molecules that combine multiple antibody-binding domains to induce active contact between T-cells and antigen expressing cells in the body. Blinatumomab, a CD19-CD3 BiTE is now a widely used therapy for relapsed B-cell malignancies, and similar BiTE therapeutics have shown promise for treating various other forms of cancer. The current process for new BiTE development is time consuming and costly, requiring characterization of the individual antigen binding domains, followed by bi-specific design, protein production, purification, and eventually functional screening. Here, we sought to establish a more cost-efficient approach for generating novel BiTE sequences and assessing bioactivity through a function first approach without purification. We generate a plasmid with a bi-modular structure to allow high-throughput exchange of either binding arm, enabling rapid screening of novel tumour-targeting single chain variable (scFv) domains in combination with the well-characterized OKT3 scFv CD3-targeting domain. We also demonstrate two systems for high throughput functional screening of BiTE proteins based on Jurkat T cells (referred to as BiTE-J). Using BiTE-J we evaluate four EGFRvIII-scFv sequenced in BiTE format, identifying two constructs with superior activity for redirecting T-cells against the EGFRvIII-tumour specific antigen. We also confirm activity in primary T cells, where novel EGFRvIII-BiTEs induced T cell activation and antigen selective tumor killing. We finally demonstrate similar exchange the CD3-interacting element of our bi-modular plasmid. By testing several novel CD3-targeting scFv elements for activity in EGFRvIII-targeted BiTEs, we were able to identify highly active BiTE molecules with desirable functional activity for downstream development. In summary, BiTE-J presents a low cost, high-throughput method for the rapid assessment of novel BiTE molecules without the need for purification and quantification.

Packaging cells for lentiviral vectors generated using the cumate and coumermycin gene induction systems and nanowell single-cell cloning.

Lentiviral vectors (LVs) are important for cell therapy because of their capacity to stably modify the genome after integration. This study describes a novel and relatively simple approach to generate packaging cells and producer clones for self-inactivating (SIN) LVs pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). A novel gene regulation system, based on the combination of the cumate and coumermycin induction systems, was developed to ensure tight control for the expression of cytotoxic packaging elements. To accelerate clone isolation and ensure monoclonality, the packaging genes were transfected simultaneously into human embryonic kidney cells (293SF-3F6) previously engineered with the induction system, and clones were isolated after limiting dilution into nanowell arrays using a robotic cell picking instrument with scanning capability. The method's effectiveness to isolate colonies derived from single cells was demonstrated using mixed populations of cells labeled with two different fluorescent markers. Because the recipient cell line grew in suspension culture, and all the procedures were performed without serum, the resulting clones were readily adaptable to serum-free suspension culture. The best producer clone produced LVs expressing GFP at a titer of 2.3 × 108 transduction units (TU)/mL in the culture medium under batch mode without concentration.

Identifying effect modifiers of CAR-T cell therapeutic efficacy: a systematic review and individual patient data meta-analysis protocol.

BACKGROUND: Chimeric antigen receptor T cell therapy (CAR-T) represents a promising and exciting new therapy for hematologic malignancies, where prognosis for relapsed/refractory patients remains poor. Encouraging results from clinical trials have often been tempered by heterogeneity in response to treatment among patients, as well as safety concerns including cytokine release syndrome. The identification of specific patient or treatment-specific factors underlying this heterogeneity may provide the key to the long-term sustainability of this complex and expensive therapy. An individual patient data meta-analysis (IPMDA) may provide potential explanations for the high degree of heterogeneity. Therefore, our objective is to perform a systematic review and IPDMA of CAR-T cell therapy in patients with hematologic malignancies to explore potential effect modifiers of CAR-T cell therapy. METHODS AND ANALYSIS: We will search MEDLINE, Embase, and the Cochrane Central Register of Controlled Clinical Trials. Studies will be screened in duplicate at the abstract level, then at the full-text level by two independent reviewers. We will include any prospective clinical trial of CAR-T cell therapy in patients with hematologic malignancies. Our primary outcome is complete response, while secondary outcomes of interest include overall response, progression-free survival, overall survival, and safety. IPD will be collected from each included trial and, in the case of missing data, corresponding authors/study sponsors will be contacted. Standard aggregate meta-analyses will be performed, followed by the IPD meta-analysis using a one-stage approach. A modified Institute of Health Economics tool will be used to evaluate the risk of bias of included studies. ETHICS AND DISSEMINATION: Identifying characteristics that may act as modifiers of CAR-T cell efficacy is of paramount importance and can help shape future clinical trials in the field. Results from this study will be submitted for publication in a peer-reviewed scientific journal, presented at relevant conferences and shared with relevant stakeholders.

Application of blood brain barrier models in pre-clinical assessment of glioblastoma-targeting CAR-T based immunotherapies.

Human blood brain barrier (BBB) models derived from induced pluripotent stem cells (iPSCs) have become an important tool for the discovery and preclinical evaluation of central nervous system (CNS) targeting cell and gene-based therapies. Chimeric antigen receptor (CAR)-T cell therapy is a revolutionary form of gene-modified cell-based immunotherapy with potential for targeting solid tumors, such as glioblastomas. Crossing the BBB is an important step in the systemic application of CAR-T therapy for the treatment of glioblastomas and other CNS malignancies. In addition, even CAR-T therapies targeting non-CNS antigens, such as the well-known CD19-CAR-T therapies, are known to trigger CNS side-effects including brain swelling due to BBB disruption. In this study, we used iPSC-derived brain endothelial-like cell (iBEC) transwell co-culture model to assess BBB extravasation of CAR-T based immunotherapies targeting U87MG human glioblastoma (GBM) cells overexpressing the tumor-specific mutated protein EGFRvIII (U87vIII). Two types of anti-EGFRvIII targeting CAR-T cells, with varying tonic signaling profiles (CAR-F263 and CAR-F269), and control Mock T cells were applied on the luminal side of BBB model in vitro. CAR-F263 and CAR-F269 T cells triggered a decrease in transendothelial electrical resistance (TEER) and an increase in BBB permeability. CAR-T cell extravasation and U87vIII cytotoxicity were assessed from the abluminal compartment using flow cytometry and Incucyte real-time viability imaging, respectively. A significant decrease in U87vIII cell viability was observed over 48 h, with the most robust cytotoxicity response observed for the constitutively activated CAR-F263. CAR-F269 T cells showed a similar cytotoxic profile but were approximately four fold less efficient at killing the U87vIII cells compared to CAR-F263, despite similar transmigration rates. Visualization of CAR-T cell extravasation across the BBB was further confirmed using BBTB-on-CHIP models. The described BBB assay was able to discriminate the cytotoxic efficacies of different EGFRvIII-CARs and provide a measure of potential alterations to BBB integrity. Collectively, we illustrate how BBB models in vitro can be a valuable tool in deciphering the mechanisms of CAR-T-induced BBB disruption, accompanying toxicity and effector function on post-barrier target cells.

Maternal Immune Cell and Cytokine Profiles to Predict Cardiovascular Risk Six Months after Preeclampsia.

Women who develop preeclampsia (PE) are at high risk for cardiovascular disease (CVD). Early identification of women with PE who may benefit the most from early cardiovascular risk screening and interventions remains challenging. Our objective was to assess whether cytokine and immune cell profiles after PE are helpful in distinguishing women at low and high CVD risk at 6-months postpartum. Individuals who developed PE were followed for immune cell phenotyping and plasma cytokine quantification at delivery, at 3-months, and at 6-months postpartum. Lifetime CVD risk was assessed at 6-months postpartum, and the immune cell and cytokine profiles were compared between risk groups at each time point. Among 31 participants, 18 (58.1%) exhibited high CVD-risk profiles at 6-months postpartum. The proportion of circulating NK-cells was significantly lower in high-risk participants at delivery (p = 0.04). At 3-months postpartum, high-risk participants exhibited a lower proportion of FoxP3+ regulatory T-cells (p = 0.01), a greater proportion of CD8+ T cells (p = 0.02) and a lower CD4+:CD8+ ratio (p = 0.02). There were no differences in immune cell populations at 6-months postpartum. There were no differences in plasma cytokines levels between risk groups at any time point. Subtle differences in immune cell profiles may help distinguish individuals at low and high CVD risk in the early postpartum period and warrants further investigation.

A New Functional Screening Platform Identifies Colistin Sulfate as an Enhancer of Natural Killer Cell Cytotoxicity.

Due to their crucial role in tumor immunity, NK cells have quickly became a prime target for immunotherapies, with the adoptive transfer of NK cells and the use of NK cell engagers quickly moving to the clinical stage. On the other hand, only a few studies have focused on small molecule drugs capable of unleashing NK cells against cancer. In this context, repurposing small molecules is an attractive strategy to identify new immunotherapies from already approved drugs. Here, we developed a new platform to screen small molecule compounds based on a high-throughput luciferase-release cytotoxicity assay. We tested 1200 FDA approved drugs from the Prestwick Chemical Library, to identify compounds that increase NK cells' cytotoxic potential. We found that the antibiotic colistin sulfate increased the cytotoxicity of human NK cells towards cancer cells. The effect of colistin was short lived and was not observed when NK cells were pretreated with the drug, showing how NK cell activity was potentiated only when the compound was present at the time of recognition of cancer cells. Further studies are needed to uncover the mechanism of action and the pre-clinical efficacy of colistin sulfate in mouse cancer models.

Programmable Attenuation of Antigenic Sensitivity for a Nanobody-Based EGFR Chimeric Antigen Receptor Through Hinge Domain Truncation.

Epidermal growth factor family receptor (EGFR) is commonly overexpressed in many solid tumors and an attractive target for chimeric antigen receptor (CAR)-T therapy, but as EGFR is also expressed at lower levels in healthy tissues a therapeutic strategy must balance antigenic responsiveness against the risk of on-target off-tumor toxicity. Herein, we identify several camelid single-domain antibodies (also known as nanobodies) that are effective EGFR targeting moieties for CARs (EGFR-sdCARs) with very strong reactivity to EGFR-high and EGFR-low target cells. As a strategy to attenuate their potent antigenic sensitivity, we performed progressive truncation of the human CD8 hinge commonly used as a spacer domain in many CAR constructs. Single amino acid hinge-domain truncation progressively decreased both EGFR-sdCAR-Jurkat cell binding to EGFR-expressing targets and expression of the CD69 activation marker. Attenuated signaling in hinge-truncated EGFR-sdCAR constructs increased selectivity for antigen-dense EGFR-overexpressing cells over an EGFR-low tumor cell line or healthy donor derived EGFR-positive fibroblasts. We also provide evidence that epitope location is critical for determining hinge-domain requirement for CARs, as hinge truncation similarly decreased antigenic sensitivity of a membrane-proximal epitope targeting HER2-CAR but not a membrane-distal EGFRvIII-specific CAR. Hinge-modified EGFR-sdCAR cells showed clear functional attenuation in Jurkat-CAR-T cells and primary human CAR-T cells from multiple donors in vitro and in vivo. Overall, these results indicate that hinge length tuning provides a programmable strategy for throttling antigenic sensitivity in CARs targeting membrane-proximal epitopes, and could be employed for CAR-optimization and improved tumor selectivity.

Oncolytic virus driven T-cell-based combination immunotherapy platform for colorectal cancer.

Colorectal cancer is the third most diagnosed cancer and the second leading cause of cancer mortality worldwide, highlighting an urgent need for new therapeutic options and combination strategies for patients. The orchestration of potent T cell responses against human cancers is necessary for effective antitumour immunity. However, regression of a limited number of cancers has been induced by immune checkpoint inhibitors, T cell engagers (TCEs) and/or oncolytic viruses. Although one TCE has been FDA-approved for the treatment of hematological malignancies, many challenges exist for the treatment of solid cancers. Here, we show that TCEs targeting CEACAM5 and CD3 stimulate robust activation of CD4 and CD8-positive T cells in in vitro co-culture models with colorectal cancer cells, but in vivo efficacy is hindered by a lack of TCE retention in the tumour microenvironment and short TCE half-life, as demonstrated by HiBiT bioluminescent TCE-tagging technology. To overcome these limitations, we engineered Bispecific Engager Viruses, or BEVirs, a novel tumour-targeted vaccinia virus platform for intra-tumour delivery of these immunomodulatory molecules. We characterized virus-mediated TCE-secretion, TCE specificity and functionality from infected colorectal cancer cells and patient tumour samples, as well as TCE cytotoxicity in spheroid models, in the presence and absence of T cells. Importantly, we show regression of colorectal tumours in both syngeneic and xenograft mouse models. Our data suggest that a different profile of cytokines may contribute to the pro-inflammatory and immune effects driven by T cells in the tumour microenvironment to provide long-lasting immunity and abscopal effects. We establish combination regimens with immune checkpoint inhibitors for aggressive colorectal peritoneal metastases. We also observe a significant reduction in lung metastases of colorectal tumours through intravenous delivery of our oncolytic virus driven T-cell based combination immunotherapy to target colorectal tumours and FAP-positive stromal cells or CTLA4-positive Treg cells in the tumour microenvironment. In summary, we devised a novel combination strategy for the treatment of colorectal cancers using oncolytic vaccinia virus to enhance immune-payload delivery and boost T cell responses within tumours.

Identification of transcriptional subtypes in lung adenocarcinoma and squamous cell carcinoma through integrative analysis of microarray and RNA sequencing data.

Classification of tumors into subtypes can inform personalized approaches to treatment including the choice of targeted therapies. The two most common lung cancer histological subtypes, lung adenocarcinoma and lung squamous cell carcinoma, have been previously divided into transcriptional subtypes using microarray data, and corresponding signatures were subsequently used to classify RNA-seq data. Cross-platform unsupervised classification facilitates the identification of robust transcriptional subtypes by combining vast amounts of publicly available microarray and RNA-seq data. However, cross-platform classification is challenging because of intrinsic differences in data generated using the two gene expression profiling technologies. In this report, we show that robust gene expression subtypes can be identified in integrated data representing over 3500 normal and tumor lung samples profiled using two widely used platforms, Affymetrix HG-U133 Plus 2.0 Array and Illumina HiSeq RNA sequencing. We tested and analyzed consensus clustering for 384 combinations of data processing methods. The agreement between subtypes identified in single-platform and cross-platform normalized data was then evaluated using a variety of statistics. Results show that unsupervised learning can be achieved with combined microarray and RNA-seq data using selected preprocessing, cross-platform normalization, and unsupervised feature selection methods. Our analysis confirmed three lung adenocarcinoma transcriptional subtypes, but only two consistent subtypes in squamous cell carcinoma, as opposed to four subtypes previously identified. Further analysis showed that tumor subtypes were associated with distinct patterns of genomic alterations in genes coding for therapeutic targets. Importantly, by integrating quantitative proteomics data, we were able to identify tumor subtype biomarkers that effectively classify samples on the basis of both gene and protein expression. This study provides the basis for further integrative data analysis across gene and protein expression profiling platforms.

Self-Cutting and Integrating CRISPR Plasmids Enable Targeted Genomic Integration of Genetic Payloads for Rapid Cell Engineering.

Since observations that CRISPR nucleases function in mammalian cells, many strategies have been devised to adapt them for genetic engineering. Here, we investigated self-cutting and integrating CRISPR-Cas9 plasmids (SCIPs) as easy-to-use gene editing tools that insert themselves at CRISPR-guided locations. SCIPs demonstrated similar expression kinetics and gene disruption efficiency in mouse (EL4) and human (Jurkat) cells, with stable integration in 3-6% of transfected cells. Clonal sequencing analysis indicated that integrants showed bi- or mono-allelic integration of entire CRISPR plasmids in predictable orientations and with limited insertion or deletion formation. Interestingly, including longer homology arms (HAs; 500 bp) in varying orientations only modestly increased knock-in efficiency (by around twofold). Using a SCIP-payload design (SCIPpay) that liberates a promoter-less sequence flanked by HAs thereby requiring perfect homology-directed repair for transgene expression, longer HAs resulted in higher integration efficiency and precision of the payload but did not affect integration of the remaining plasmid sequence. As proofs of concept, we used SCIPpay to insert (1) a gene fragment encoding tdTomato into the CD69 locus of Jurkat cells, thereby creating a cell line that reports T-cell activation, and (2) a chimeric antigen receptor gene into the TRAC locus. Here, we demonstrate that SCIPs function as simple, efficient, and programmable tools useful for generating gene knock-out/knock-in cell lines, and we suggest future utility in knock-in site screening/optimization, unbiased off-target site identification, and multiplexed, iterative, and/or library-scale automated genome engineering.

Current Advances and Hurdles in Chimeric Antigen Receptor Technology.

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TNFR2 is required for RIP1-dependent cell death in human leukemia.

Despite major advances in the treatment of patients with acute lymphoblastic leukemia in the last decades, refractory and/or relapsed disease remains a clinical challenge, and relapsed leukemia patients have an exceedingly dismal prognosis. Dysregulation of apoptotic cell death pathways is a leading cause of drug resistance; thus, alternative cell death mechanisms, such as necroptosis, represent an appealing target for the treatment of high-risk malignancies. We and other investigators have shown that activation of receptor interacting protein kinase 1 (RIP1)-dependent apoptosis and necroptosis by second mitochondria derived activator of caspase mimetics (SMs) is an attractive antileukemic strategy not currently exploited by standard chemotherapy. However, the underlying molecular mechanisms that determine sensitivity to SMs have remained elusive. We show that tumor necrosis factor receptor 2 (TNFR2) messenger RNA expression correlates with sensitivity to SMs in primary human leukemia. Functional genetic experiments using clustered regularly interspaced short palindromic repeats/Cas9 demonstrate that TNFR2 and TNFR1, but not the ligand TNF-α, are essential for the response to SMs, revealing a ligand-independent interplay between TNFR1 and TNFR2 in the induction of RIP1-dependent cell death. Further potential TNFR ligands, such as lymphotoxins, were not required for SM sensitivity. Instead, TNFR2 promotes the formation of a RIP1/TNFR1-containing death signaling complex that induces RIP1 phosphorylation and RIP1-dependent apoptosis and necroptosis. Our data reveal an alternative paradigm for TNFR2 function in cell death signaling and provide a rationale to develop strategies for the identification of leukemias with vulnerability to RIP1-dependent cell death for tailored therapeutic interventions.