CTLA-4 tail fusion enhances CAR-T antitumor immunity.

Chimeric antigen receptor (CAR)-T cells are powerful therapeutics; however, their efficacy is often hindered by critical hurdles. Here utilizing the endocytic feature of the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) cytoplasmic tail, we reprogram CAR function and substantially enhance CAR-T efficacy in vivo. CAR-T cells with monomeric, duplex or triplex CTLA-4 cytoplasmic tails (CCTs) fused to the C terminus of CAR exhibit a progressive increase in cytotoxicity under repeated stimulation, accompanied by reduced activation and production of proinflammatory cytokines. Further characterization reveals that CARs with increasing CCT fusion show a progressively lower surface expression, regulated by their constant endocytosis, recycling and degradation under steady state. The molecular dynamics of reengineered CAR with CCT fusion results in reduced CAR-mediated trogocytosis, loss of tumor antigen and improved CAR-T survival. CARs with either monomeric (CAR-1CCT) or duplex CCTs (CAR-2CCT) have superior antitumor efficacy in a relapsed leukemia model. Single-cell RNA sequencing and flow cytometry analysis reveal that CAR-2CCT cells retain a stronger central memory phenotype and exhibit increased persistence. These findings illuminate a unique strategy for engineering therapeutic T cells and improving CAR-T function through synthetic CCT fusion, which is orthogonal to other cell engineering techniques.

Systematic Immunotherapy Target Discovery Using Genome-Scale In Vivo CRISPR Screens in CD8 T Cells.

CD8 T cells play essential roles in anti-tumor immune responses. Here, we performed genome-scale CRISPR screens in CD8 T cells directly under cancer immunotherapy settings and identified regulators of tumor infiltration and degranulation. The in vivo screen robustly re-identified canonical immunotherapy targets such as PD-1 and Tim-3, along with genes that have not been characterized in T cells. The infiltration and degranulation screens converged on an RNA helicase Dhx37. Dhx37 knockout enhanced the efficacy of antigen-specific CD8 T cells against triple-negative breast cancer in vivo. Immunological characterization in mouse and human CD8 T cells revealed that DHX37 suppresses effector functions, cytokine production, and T cell activation. Transcriptomic profiling and biochemical interrogation revealed a role for DHX37 in modulating NF-κB. These data demonstrate high-throughput in vivo genetic screens for immunotherapy target discovery and establishes DHX37 as a functional regulator of CD8 T cells.

Multiplexed activation of endogenous genes by CRISPRa elicits potent antitumor immunity.

Immunotherapy has transformed cancer treatment. However, current immunotherapy modalities face various limitations. In the present study, we developed multiplexed activation of endogenous genes as an immunotherapy (MAEGI), a new form of immunotherapy that elicits antitumor immunity through multiplexed activation of endogenous genes in tumors. We leveraged CRISPR activation (CRISPRa) to directly augment the in situ expression of endogenous genes, and thereby the presentation of tumor antigens, leading to dramatic antitumor immune responses. Deploying this as a cell-based vaccination strategy showed efficacy in both prophylactic and therapeutic settings. Intratumoral adeno-associated virus delivery of CRISPRa libraries elicited strong antitumor immunity across multiple cancer types. Precision targeting of mutated gene sets eradicated a large fraction of established tumors at both local and distant sites. This treatment modality led to alterations in the tumor microenvironment, marked by enhanced T cell infiltration and antitumor immune signatures. Multiplexed endogenous gene activation is a versatile and highly scalable strategy to elicit potent immune responses against cancer, distinct from all existing cancer therapies.

DYRK1A promotes viral entry of highly pathogenic human coronaviruses in a kinase-independent manner.

Identifying host genes essential for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has the potential to reveal novel drug targets and further our understanding of Coronavirus Disease 2019 (COVID-19). We previously performed a genome-wide CRISPR/Cas9 screen to identify proviral host factors for highly pathogenic human coronaviruses. Few host factors were required by diverse coronaviruses across multiple cell types, but DYRK1A was one such exception. Although its role in coronavirus infection was previously undescribed, DYRK1A encodes Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A and is known to regulate cell proliferation and neuronal development. Here, we demonstrate that DYRK1A regulates ACE2 and DPP4 transcription independent of its catalytic kinase function to support SARS-CoV, SARS-CoV-2, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) entry. We show that DYRK1A promotes DNA accessibility at the ACE2 promoter and a putative distal enhancer, facilitating transcription and gene expression. Finally, we validate that the proviral activity of DYRK1A is conserved across species using cells of nonhuman primate and human origin. In summary, we report that DYRK1A is a novel regulator of ACE2 and DPP4 expression that may dictate susceptibility to multiple highly pathogenic human coronaviruses.

LRRC15 inhibits SARS-CoV-2 cellular entry in trans.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is mediated by the entry receptor angiotensin-converting enzyme 2 (ACE2). Although attachment factors and coreceptors facilitating entry are extensively studied, cellular entry factors inhibiting viral entry are largely unknown. Using a surfaceome CRISPR activation screen, we identified human LRRC15 as an inhibitory attachment factor for SARS-CoV-2 entry. LRRC15 directly binds to the receptor-binding domain (RBD) of spike protein with a moderate affinity and inhibits spike-mediated entry. Analysis of human lung single-cell RNA sequencing dataset reveals that expression of LRRC15 is primarily detected in fibroblasts and particularly enriched in pathological fibroblasts in COVID-19 patients. ACE2 and LRRC15 are not coexpressed in the same cell types in the lung. Strikingly, expression of LRRC15 in ACE2-negative cells blocks spike-mediated viral entry in ACE2+ cell in trans, suggesting a protective role of LRRC15 in a physiological context. Therefore, LRRC15 represents an inhibitory attachment factor for SARS-CoV-2 that regulates viral entry in trans.

Single-cell longitudinal analysis of SARS-CoV-2 infection in human airway epithelium identifies target cells, alterations in gene expression, and cell state changes.

There are currently limited Food and Drug Administration (FDA)-approved drugs and vaccines for the treatment or prevention of Coronavirus Disease 2019 (COVID-19). Enhanced understanding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and pathogenesis is critical for the development of therapeutics. To provide insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2, we performed single-cell (sc) RNA sequencing (RNA-seq) of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface (ALI) cultures over a time course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target at the onset of infection, which we confirmed by electron and immunofluorescence microscopy. Over the course of infection, the cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III interferons (IFNs) and interleukin (IL)-6 but not IL-1. This results in expression of interferon-stimulated genes (ISGs) in both infected and bystander cells. This provides a detailed characterization of genes, cell types, and cell state changes associated with SARS-CoV-2 infection in the human airway.

LAMP3/CD63 Expression in Early and Late Endosomes in Human Vaginal Epithelial Cells Is Associated with Enhancement of HSV-2 Infection.

Herpes simplex virus 2 (HSV-2) is a lifelong sexually transmitted virus that disproportionately infects women through heterosexual transmission in the vaginal tract. The vaginal epithelium is known to be highly susceptible to HSV-2 infection; however, the cellular mechanism of HSV-2 uptake and replication in vaginal epithelium has not been extensively studied. Previously, we observed that lysosomal-associated membrane protein-3 (LAMP3/CD63) was among the highly upregulated genes during HSV-2 infection of human vaginal epithelial cell line VK2, leading us to posit that LAMP3/CD63 may play a role in HSV-2 infection. Consequently, we generated two gene-altered VK2-derived cell lines, a LAMP3-overexpressed (OE) line and a LAMP3 knockout (KO) line. The wild-type VK2 and the LAMP3 OE and KO cell lines were grown in air-liquid interface (ALI) cultures for 7 days and infected with HSV-2. Twenty-four hours postinfection, LAMP3 OE cells produced and released significantly higher numbers of HSV-2 virions than wild-type VK2 cells, while virus production was greatly attenuated in LAMP3 KO cells, indicating a functional association between LAMP3/CD63 expression and HSV-2 replication. Fluorescence microscopy of HSV-2-infected cells revealed that HSV-2 colocalized with LAMP3 in both early endosomes and lysosomal compartments. In addition, blocking endosomal maturation or late endosomal/lysosomal fusion using specific inhibitors resulted in reduced HSV-2 replication in VK2 cells. Similarly, LAMP3 KO cells exhibited very low viral entry and association with endosomes, while LAMP3 OE cells demonstrated large amounts of virus that colocalized with LAMP3/CD63 in endosomes and lysosomes. IMPORTANCE Collectively, these results showed that HSV-2 is taken up by human vaginal epithelial cells through an endosomal-lysosomal pathway in association with LAMP3, which plays a crucial role in the enhancement of HSV-2 replication. These findings provide the basis for the future design of antiviral agents for prophylactic measures against HSV-2 infection.

Liposomal delivery of gene therapy for ovarian cancer: a systematic review.

OBJECTIVE: To systematically identify and narratively synthesize the evidence surrounding liposomal delivery of gene therapy and the outcome for ovarian cancer. METHODS: An electronic database search of the Embase, MEDLINE and Web of Science from inception until July 7, 2023, was conducted to identify primary studies that investigated the effect of liposomal delivery of gene therapy on ovarian cancer outcomes. Retrieved studies were assessed against the eligibility criteria for inclusion. RESULTS: The search yielded 564 studies, of which 75 met the inclusion criteria. Four major types of liposomes were identified: cationic, neutral, polymer-coated, and ligand-targeted liposomes. The liposome with the most evidence involved cationic liposomes which are characterized by their positively charged phospholipids (n = 37, 49.3%). Similarly, those with neutrally charged phospholipids, such as 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, were highly researched as well (n = 25, 33.3%). Eight areas of gene therapy research were identified, evaluating either target proteins/transcripts or molecular pathways: microRNAs, ephrin type-A receptor 2 (EphA2), interleukins, mitogen-activated protein kinase (MAPK), human-telomerase reverse transcriptase/E1A (hTERT/EA1), suicide gene, p53, and multidrug resistance mutation 1 (MDR1). CONCLUSION: Liposomal delivery of gene therapy for ovarian cancer shows promise in many in vivo studies. Emerging polymer-coated and ligand-targeted liposomes have been gaining interest as they have been shown to have more stability and specificity. We found that gene therapy involving microRNAs was the most frequently studied. Overall, liposomal genetic therapy has been shown to reduce tumor size and weight and improve survivability. More research involving the delivery and targets of gene therapy for ovarian cancer may be a promising avenue to improve patient outcomes.

Epithelial cell plasticity: breaking boundaries and changing landscapes.

Epithelial tissues respond to a wide variety of environmental and genotoxic stresses. As an adaptive mechanism, cells can deviate from their natural paths to acquire new identities, both within and across lineages. Under extreme conditions, epithelial tissues can utilize "shape-shifting" mechanisms whereby they alter their form and function at a tissue-wide scale. Mounting evidence suggests that in order to acquire these alternate tissue identities, cells follow a core set of "tissue logic" principles based on developmental paradigms. Here, we review the terminology and the concepts that have been put forward to describe cell plasticity. We also provide insights into various cell intrinsic and extrinsic factors, including genetic mutations, inflammation, microbiota, and therapeutic agents that contribute to cell plasticity. Additionally, we discuss recent studies that have sought to decode the "syntax" of plasticity-i.e., the cellular and molecular principles through which cells acquire new identities in both homeostatic and malignant epithelial tissues-and how these processes can be manipulated for developing novel cancer therapeutics.

Chronic Lymphocytic Leukemia Presenting as Bilateral Periorbital Edema Treated With Low-dose Radiation Therapy.

Ocular manifestations in chronic lymphocytic leukemia (CLL) have been reported in 30% to 40% of patients and may be a result of direct tissue infiltration, concomitant blood dyscrasias, or a result of therapeutic intervention. Leukemia cutis, defined as infiltration of the epidermis or dermis by neoplastic lymphocytes, is rare. Herein, we present a case report of a patient with leukemia who presented with periorbital edema and ecchymosis. This is the first known case to date of periorbital CLL successfully treated with low-dose radiation therapy (4 Gy in 2 fractions). Clinicians should be aware of the possibility of ocular involvement from CLL, given the importance of prompt diagnosis and treatment.

In vivo profiling of metastatic double knockouts through CRISPR-Cpf1 screens.

Systematic investigation of the genetic interactions that influence metastatic potential has been challenging. Here we developed massively parallel CRISPR-Cpf1/Cas12a crRNA array profiling (MCAP), an approach for combinatorial interrogation of double knockouts in vivo. We designed an MCAP library of 11,934 arrays targeting 325 pairwise combinations of genes implicated in metastasis. By assessing the metastatic potential of the double knockouts in mice, we unveiled a quantitative landscape of genetic interactions that drive metastasis.

Non-steroidal anti-inflammatory drugs dampen the cytokine and antibody response to SARS-CoV-2 infection.

Identifying drugs that regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing area of investigation during the coronavirus disease 2019 (COVID-19) pandemic. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are frequently used for the relief of pain and inflammation, could modulate both SARS-CoV-2 infection and the host response to the virus. NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which mediate the production of prostaglandins (PGs). As PGs play diverse biological roles in homeostasis and inflammatory responses, inhibiting PG production with NSAIDs could affect COVID-19 pathogenesis in multiple ways, including: (1) altering susceptibility to infection by modifying expression of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2; (2) regulating replication of SARS-CoV-2 in host cells; and (3) modulating the immune response to SARS-CoV-2. Here, we investigate these potential roles. We demonstrate that SARS-CoV-2 infection upregulates COX-2 in diverse human cell culture and mouse systems. However, suppression of COX-2 by two commonly used NSAIDs, ibuprofen and meloxicam, had no effect on ACE2 expression, viral entry, or viral replication. In contrast, in a mouse model of SARS-CoV-2 infection, NSAID treatment reduced production of pro-inflammatory cytokines and impaired the humoral immune response to SARS-CoV-2 as demonstrated by reduced neutralizing antibody titers. Our findings indicate that NSAID treatment may influence COVID-19 outcomes by dampening the inflammatory response and production of protective antibodies rather than modifying susceptibility to infection or viral replication.ImportancePublic health officials have raised concerns about the use of nonsteroidal anti-inflammatory drugs (NSAIDs) for treating symptoms of coronavirus disease 2019 (COVID-19). NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which are critical for the generation of prostaglandins - lipid molecules with diverse roles in homeostasis and inflammation. Inhibition of prostaglandin production by NSAIDs could therefore have multiple effects on COVID-19 pathogenesis. Here, we demonstrate that NSAID treatment reduced both the antibody and pro-inflammatory cytokine response to SARS-CoV-2 infection. The ability of NSAIDs to modulate the immune response to SARS-CoV-2 infection has important implications for COVID-19 pathogenesis in patients. Whether this occurs in humans and whether it is beneficial or detrimental to the host remains an important area of future investigation. This also raises the possibility that NSAIDs may alter the immune response to SARS-CoV-2 vaccination.

Parent stem cells can serve as niches for their daughter cells.

Stem cells integrate inputs from multiple sources. Stem cell niches provide signals that promote stem cell maintenance, while differentiated daughter cells are known to provide feedback signals to regulate stem cell replication and differentiation. Recently, stem cells have been shown to regulate themselves using an autocrine mechanism. The existence of a 'stem cell niche' was first postulated by Schofield in 1978 to define local environments necessary for the maintenance of haematopoietic stem cells. Since then, an increasing body of work has focused on defining stem cell niches. Yet little is known about how progenitor cell and differentiated cell numbers and proportions are maintained. In the airway epithelium, basal cells function as stem/progenitor cells that can both self-renew and produce differentiated secretory cells and ciliated cells. Secretory cells also act as transit-amplifying cells that eventually differentiate into post-mitotic ciliated cells . Here we describe a mode of cell regulation in which adult mammalian stem/progenitor cells relay a forward signal to their own progeny. Surprisingly, this forward signal is shown to be necessary for daughter cell maintenance. Using a combination of cell ablation, lineage tracing and signalling pathway modulation, we show that airway basal stem/progenitor cells continuously supply a Notch ligand to their daughter secretory cells. Without these forward signals, the secretory progenitor cell pool fails to be maintained and secretory cells execute a terminal differentiation program and convert into ciliated cells. Thus, a parent stem/progenitor cell can serve as a functional daughter cell niche.

Appraisal of systematic reviews on interventions for postpartum depression: systematic review.

BACKGROUND: Postpartum depression (PPD) is a highly prevalent mental health problem that affects parental health with implications for child health in infancy, childhood, adolescence and beyond. The primary aim of this study was to critically appraise available systematic reviews describing interventions for PPD. The secondary aim was to evaluate the methodological quality of the included systematic reviews and their conclusions. METHODS: An electronic database search of MEDLINE, Embase, and the Cochrane Library from 2000 to 2020 was conducted to identify systematic reviews that examined an intervention for PPD. A Measurement Tool to Assess Systematic Reviews was utilized to independently score each included systematic review which was then critically appraised to better define the most effective therapeutic options for PPD. RESULTS: Of the 842 studies identified, 83 met the a priori criteria for inclusion. Based on the systematic reviews with the highest methodological quality, we found that use of antidepressants and telemedicine were the most effective treatments for PPD. Symptoms of PPD were also improved by traditional herbal medicine and aromatherapy. Current evidence for physical exercise and cognitive behavioural therapy in treating PPD remains equivocal. A significant, but weak relationship between AMSTAR score and journal impact factor was observed (p = 0.03, r = 0.24; 95% CI, 0.02 to 0.43) whilst no relationship was found between the number of total citations (p = 0.27, r = 0.12; 95% CI, - 0.09 to 0.34), or source of funding (p = 0.19). CONCLUSION: Overall the systematic reviews on interventions for PPD are of low-moderate quality and are not improving over time. Antidepressants and telemedicine were the most effective therapeutic interventions for PPD treatment.

Quality appraisal of systematic reviews on methods of labour induction: a systematic review.

PURPOSE: Induction of labour has become more common over the last decade, together with an increase in the number of systematic reviews of the subject. However, with multiple systematic reviews it is necessary to evaluate the methodological rigor to ensure the reliability of conclusions and recommendations for clinical practice. Therefore, the aim of this study was to appraise the quality of systematic reviews that examined the efficacy and/or safety of labour induction methods. METHODS: An electronic search of MEDLINE, Embase, and the Cochrane Library from 2000 to 2020 was conducted. Study selection, data extraction and quality assessment were conducted using A Measurement Tool to Assess Systematic Reviews (AMSTAR) by two independent reviewers, in duplicate. RESULTS: The search identified 387 publications, of which 48 studies (13%) met the a priori inclusion criteria. No significant relationships were found between study quality and number of citations, journal impact factor, or publication year. CONCLUSION: Methodological quality for systematic reviews on the induction of labour were ranked as moderate with no significant changes in quality over the past 2 decades. Publication characteristics are not significantly associated with methodological quality, indicating that healthcare professionals should critically appraise studies before applying them to practice.

Novel frequency analysis of signal-averaged electrocardiograms is predictive of adverse outcomes in implantable cardioverter defibrillator patients.

BACKGROUND: Current noninvasive risk stratification methods offer limited prediction of arrhythmic events when selecting patients for ICD implantation. Our laboratory has recently developed a signal processing metric called Layered Symbolic Decomposition frequency (LSDf) that quantifies the percentage of hidden QRS wave frequency components in signal-averaged ECG (SAECG) recordings. The purpose of this pilot study was to determine whether LSDf can be predictive of ventricular arrhythmia or death in an ICD patient cohort. METHODS AND RESULTS: Fifty-two ICD patients were recruited from 2008 to 2009. These were followed for a mean of 8.5 ± 0.4 years for the primary outcome of first appropriately treated ventricular arrhythmia (VT/VF) or death. Thirty-four subjects met the primary outcome. LSDf was significantly lower, and 12-lead QRS duration was significantly greater in patients meeting the primary outcome (12.14 ± 3.97% vs. 16.45 ± 3.73%; p = 0.001) and (111.59 ± 14.96 ms vs. 97.69 ± 13.51 ms; p = 0.012) respectively. A 13.25% LSDf threshold (0.74 sensitivity and 0.85 specificity) was selected based on an ROC curve. Kaplan-Meier survival analysis was conducted; patients above the 13.25% threshold demonstrated significantly better survival outcomes (log-rank p < 0.001). In Cox multivariate regression analysis, the LSDf threshold (13.25%) was compared to LVEF (28.5%), 12-lead QRSd (100 ms), age, % male sex, NYHA classification, and antiarrhythmic usage. LSDf was a predictor of the primary outcome (p = 0.005) and an independent predictor for solely ventricular arrhythmia (p = 0.002). CONCLUSION: Layered Symbolic Decomposition frequency analysis in SAECG recordings may be a viable predictor of negative ICD survival outcomes.

Real-world evaluation of deep learning algorithms to classify functional pathogenic germline variants.

Deep learning models for variant pathogenicity prediction can recapitulate expert-curated annotations, but their performance remains unexplored on actual disease phenotypes in a real-world setting. Here, we apply three state-of-the-art pathogenicity prediction models to classify hereditary breast cancer gene variants in the UK Biobank. Predicted pathogenic variants in BRCA1, BRCA2 and PALB2, but not ATM and CHEK2, were associated with increased breast cancer risk. We explored gene-specific score thresholds for variant pathogenicity, finding that they could improve model performance. However, when specifically tasked with classifying variants of uncertain significance, the deep learning models were generally of limited clinical utility.

In vivo AAV-SB-CRISPR screens of tumor-infiltrating primary NK cells identify genetic checkpoints of CAR-NK therapy.

Natural killer (NK) cells have clinical potential against cancer; however, multiple limitations hinder the success of NK cell therapy. Here, we performed unbiased functional mapping of tumor-infiltrating NK (TINK) cells using in vivo adeno-associated virus (AAV)-SB (Sleeping Beauty)-CRISPR (clustered regularly interspaced short palindromic repeats) screens in four solid tumor mouse models. In parallel, we characterized single-cell transcriptomic landscapes of TINK cells, which identified previously unexplored subpopulations of NK cells and differentially expressed TINK genes. As a convergent hit, CALHM2-knockout (KO) NK cells showed enhanced cytotoxicity and tumor infiltration in mouse primary NK cells and human chimeric antigen receptor (CAR)-NK cells. CALHM2 mRNA reversed the CALHM2-KO phenotype. CALHM2 KO in human primary NK cells enhanced their cytotoxicity, degranulation and cytokine production. Transcriptomics profiling revealed CALHM2-KO-altered genes and pathways in both baseline and stimulated conditions. In a solid tumor model resistant to unmodified CAR-NK cells, CALHM2-KO CAR-NK cells showed potent in vivo antitumor efficacy. These data identify endogenous genetic checkpoints that naturally limit NK cell function and demonstrate the use of CALHM2 KO for engineering enhanced NK cell-based immunotherapies.

Mutations in IFN-γ signaling genes sensitize tumors to immune checkpoint blockade.

Song and Chow demonstrate that while tumor-intrinsic mutations in the IFN-γ signaling pathway confer immune resistance across in vitro co-culture systems, such alterations associate with enhanced anti-tumor immunity in vivo and improved responsiveness to immune checkpoint blockade therapy in patients with cancer.