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.

Human gene age dating reveals an early and rapid evolutionary construction of the adaptive immune system.

T cells are a type of white blood cell that play a critical role in the immune response against foreign pathogens through a process called T Cell Adaptive Immunity (TCAI). However, the evolution of the genes and nucleotide sequences involved in TCAI is not well understood. To investigate this, we performed comparative studies of gene annotations and genome assemblies of 28 vertebrate species and identified sets of human genes that are involved in TCAI, carcinogenesis, and ageing.

Cas12a/Cpf1 knock-in mice enable efficient multiplexed immune cell engineering.

Cas9 transgenic animals have drastically accelerated the discovery of novel immune modulators. But due to its inability to process its own CRISPR RNAs (crRNAs), simultaneous multiplexed gene perturbations using Cas9 remains limited, especially by pseudoviral vectors. Cas12a/Cpf1, however, can process concatenated crRNA arrays for this purpose.

CTLA-4 tail fusion enhances CAR-T anti-tumor 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 (CT), we reprogram CAR function and substantially enhance CAR-T efficacy . CAR-T cells with monomeric, duplex, or triplex CTLA-4 CTs (CCTs) fused to the C-terminus of CAR exhibit a progressive increase in cytotoxicity under repeated stimulation, accompanied by reduced activation and production of pro-inflammatory cytokines.

Multiplexed LNP-mRNA vaccination against pathogenic coronavirus species.

Although COVID-19 vaccines have been developed, multiple pathogenic coronavirus species exist, urging on development of multispecies coronavirus vaccines. Here we develop prototype lipid nanoparticle (LNP)-mRNA vaccine candidates against SARS-CoV-2 Delta, SARS-CoV, and MERS-CoV, and we test how multiplexing LNP-mRNAs can induce effective immune responses in animal models. Triplex and duplex LNP-mRNA vaccinations induce antigen-specific antibody responses against SARS-CoV-2, SARS-CoV, and MERS-CoV.

Variant-specific vaccination induces systems immune responses and potent in vivo protection against SARS-CoV-2.

Lipid nanoparticle (LNP)-mRNA vaccines offer protection against COVID-19; however, multiple variant lineages caused widespread breakthrough infections. Here, we generate LNP-mRNAs specifically encoding wild-type (WT), B.1.

Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617.

COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical.

A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-T therapy.

Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and immunological diseases has made great strides, but it still faces multiple hurdles. Finding the right molecular targets to engineer T cells toward a desired function has broad implications for the armamentarium of T cell-centered therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation screen in primary CD8 T cells and identified gain-of-function (GOF) targets for CAR-T engineering.

Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617.

COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical.

High-content CRISPR screening.

CRISPR screens are a powerful source of biological discovery, enabling the unbiased interrogation of gene function in a wide range of applications and species. In pooled CRISPR screens, various genetically encoded perturbations are introduced into pools of cells. The targeted cells proliferate under a biological challenge such as cell competition, drug treatment or viral infection.

Tumor immunology CRISPR screening: present, past, and future.

Recent advances in immunotherapy have fundamentally changed the landscape of cancer treatment by leveraging the specificity and selectivity of the adaptive immune system to kill cancer cells. These successes have ushered in a new wave of research aimed at understanding immune recognition with the hope of developing newer immunotherapies. The advent of clustered regularly interspaced short palindromic repeats (CRISPR) technologies and advancement of multiomics modalities have greatly accelerated the discovery process.

Nonstructural Protein 1 of SARS-CoV-2 Is a Potent Pathogenicity Factor Redirecting Host Protein Synthesis Machinery toward Viral RNA.

The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome.

CRISPR-GEMM Pooled Mutagenic Screening Identifies KMT2D as a Major Modulator of Immune Checkpoint Blockade.

Immune checkpoint blockade (ICB) has shown remarkable clinical efficacy in several cancer types. However, only a fraction of patients will respond to ICB. Here, we performed pooled mutagenic screening with CRISPR-mediated genetically engineered mouse models (CRISPR-GEMM) in ICB settings, and identified KMT2D as a major modulator of ICB response across multiple cancer types.

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.

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.

Ketorolac Use Shortens Hospital Length of Stay After Bariatric Surgery: a Single-Center 5-Year Experience.

Introduction: There has been a recent increased interest in the use of non-steroidal anti-inflammatory drugs (NSAIDs) such as ketorolac for post-operative pain management to minimize opioid use and decrease hospital length of stay (LOS). Although NSAID use has been controversial following bariatric surgery due to anecdotal concerns for increased gastric bleeding, the impact of ketorolac as an adjunct to opioids needs further investigation on LOS and post-operative complications like bleeding.

Objective: This study aims to evaluate the impact of post-operative ketorolac use on opioid consumption, LOS, and bleeding risk after bariatric surgery.

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.

Convergent Identification and Interrogation of Tumor-Intrinsic Factors that Modulate Cancer Immunity In Vivo.

The genetic makeup of cancer cells directs oncogenesis and influences the tumor microenvironment. In this study, we massively profiled genes that functionally drive tumorigenesis using genome-scale in vivo CRISPR screens in hosts with different levels of immunocompetence. As a convergent hit from these screens, Prkar1a mutant cells are able to robustly outgrow as tumors in fully immunocompetent hosts.

Is bariatric surgery effective for co-morbidity resolution in the super-obese patients?

Background: Type 2 diabetes (T2D), obstructive sleep apnea (OSA), hypertension (HTN), and hyperlipidemia (HLD) are common co-morbidities that are strongly associated with obesity.

Objective: The purpose of this study was to compare the rate of obesity-related co-morbidity remission and percent total body weight loss of super-obese patients with a body mass index (BMI) ≥50 kg/m with bariatric patients who have a BMI of 30 to 49.9 kg/m.

Mapping a functional cancer genome atlas of tumor suppressors in mouse liver using AAV-CRISPR-mediated direct in vivo screening.

Cancer genomics consortia have charted the landscapes of numerous human cancers. Whereas some mutations were found in classical oncogenes and tumor suppressors, others have not yet been functionally studied in vivo. To date, a comprehensive assessment of how these genes influence oncogenesis is lacking.

AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma.

A causative understanding of genetic factors that regulate glioblastoma pathogenesis is of central importance. Here we developed an adeno-associated virus-mediated, autochthonous genetic CRISPR screen in glioblastoma. Stereotaxic delivery of a virus library targeting genes commonly mutated in human cancers into the brains of conditional-Cas9 mice resulted in tumors that recapitulate human glioblastoma.

Determinants of Divergent Adaptive Immune Responses after Airway Sensitization with Ligands of Toll-Like Receptor 5 or Toll-Like Receptor 9.

Excessive type 2 helper T cell responses to environmental antigens can cause immunopathology such as asthma and allergy, but how such immune responses are induced remains unclear. We studied this process in the airways by immunizing mice intranasally with the antigen ovalbumin together with either of two Toll-like receptor (TLR) ligands. We found the TLR5 ligand flagellin promoted a type 2 helper T cell response, whereas, a TLR9 ligand CpG oligodeoxyribonucleotide (ODN) promoted a type 1 helper T cell response.

Choleduodenal fistula with gastric outlet obstruction.

Bouveret's syndrome, cholecystoduodenal fistula with gastric outlet obstruction secondary to an impacted gallstone, is a rare but serious complication of cholelithiasis. We report the case of a 69-year-old woman who presented with epigastric pain and vomiting in whom cross-sectional imaging revealed a duodenal mass with cholecystoduodenal fistula and pneumobilia. Endoscopic evaluation identified a large gallstone obstructing the pyloric channel.

Despite Increased Type 1 IFN, Autoimmune Nonobese Diabetic Mice Display Impaired Dendritic Cell Response to CpG and Decreased Nuclear Localization of IFN-Activated STAT1.

Innate immune signals help break self-tolerance to initiate autoimmune diseases such as type 1 diabetes, but innate contributions to subsequent regulation of disease progression are less clear. Most studies have measured in vitro innate responses of GM-CSF dendritic cells (DCs) that are functionally distinct from conventional DCs (cDCs) and do not reflect in vivo DC subsets. To determine whether autoimmune NOD mice have alterations in type 1 IFN innate responsiveness, we compared cDCs from prediabetic NOD and control C57BL/6 (B6) mice stimulated in vivo with the TLR9 ligand CpG, a strong type 1 IFN inducer.