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.

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. Our cryo-EM structure of the Nsp1-40S ribosome complex shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the structure of the 48S preinitiation complex formed by Nsp1, 40S, and the cricket paralysis virus internal ribosome entry site (IRES) RNA, which shows that it is nonfunctional because of the incorrect position of the mRNA 3' region. Our results elucidate the mechanism of host translation inhibition by SARS-CoV-2 and advance understanding of the impacts from a major pathogenicity factor of SARS-CoV-2.

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.

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. In response to CpG, NOD mice produce more type 1 IFN and express higher levels of CD40, and NOD monocyte DCs make more TNF. However, the overall CpG-induced transcriptional response is muted in NOD cDCs. Of relevance the costimulatory proteins CD80/CD86, signals needed for regulatory T cell homeostasis, are upregulated less on NOD cDCs. Interestingly, NOD Rag1(-/-) mice also display a defect in CpG-induced CD86 upregulation compared with B6 Rag1(-/-), indicating this particular innate alteration precedes adaptive autoimmunity. The impaired response in NOD DCs is likely downstream of the IFN-α/ß receptor because DCs from NOD and B6 mice show similar CpG-induced CD86 levels when anti-IFN-α/ß receptor Ab is added. IFN-α-induced nuclear localization of activated STAT1 is markedly reduced in NOD CD11c(+) cells, consistent with lower type 1 IFN responsiveness. In conclusion, NOD DCs display altered innate responses characterized by enhanced type 1 IFN and activation of monocyte-derived DCs but diminished cDC type 1 IFN response.

Interleukin-2-mediated inhibition of dendritic cell development correlates with decreased CD135 expression and increased monocyte/macrophage precursors.

We have previously shown that interleukin-2 (IL-2) inhibits dendritic cell (DC) development from mouse bone marrow (BM) precursors stimulated with the ligand for FMS-like tyrosine kinase 3 receptor (Flt3L), and have provided evidence that this inhibition occurs at the monocyte DC precursor stage of DC development. Here, we explored the mechanism of IL-2-mediated inhibition of DC development. First, we showed that these in vitro cultures accurately model DCs that develop in vivo by comparing gene and protein expression of the three main Flt3L-induced DC subsets from the BM, CD11b(+) and CD24(+) conventional DCs (cDCs) and plasmacytoid DCs (pDCs) with their respective ex vivo spleen DC subsets (CD11b(+), CD8(+) and pDCs). Next, gene expression changes were quantified in Flt3L DC subsets that developed in the presence of IL-2. These changes included increased expression of Bcl2l11, which encodes the apoptosis-inducing protein Bim, and decreased expression of Flt3 (CD135), the receptor that initiates DC development. Interleukin-2 also significantly reduced Flt3 protein expression on all three Flt3L DC subsets, and attenuated Flt3L-induced STAT3 phosphorylation in DCs. Based on these data, we hypothesized that decreased Flt3 signalling may divert BM precursors down monocyte and macrophage lineages. Indeed, addition of IL-2 led to increases in Flt3(-) cells, including cKit(+) Ly6C(+) CD11b(-) populations consistent with the recently identified committed monocyte/macrophage progenitor. Therefore, IL-2 can inhibit DC development via decreased signalling through Flt3 and increased monocyte/macrophage development.

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. We found that these gene sets share interaction pathways which may have contributed to the evolution of longevity in the vertebrate lineage leading to humans. Our human gene age dating analyses revealed that there was rapid origination of genes with TCAI-related functions prior to the Cretaceous eutherian radiation and these new genes mainly encode negative regulators. We identified no new TCAI-related genes after the divergence of placental mammals, but we did detect an extensive number of amino acid substitutions under strong positive selection in recently evolved human immunity genes suggesting they are co-evolving with adaptive immunity. More specifically, we observed that antigen processing and presentation and checkpoint genes are significantly enriched among new genes evolving under positive selection. These observations reveal an evolutionary process of T Cell Adaptive Immunity that were associated with rapid gene duplication in the early stages of vertebrates and subsequent sequence changes in TCAI-related genes. These processes together suggest an early genetic construction of the vertebrate immune system and subsequent molecular adaptation to diverse antigens.

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 in vivo . 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. 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 anti-tumor 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.

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. Here, we created conditional and constitutive LbCas12a knock-in transgenic mice. With these mice, we demonstrated efficient multiplexed gene editing and surface protein knockdown within individual primary immune cells. We showed genome editing across multiple types of primary immune cells including CD4 and CD8 T cells, B cells, and bone-marrow derived dendritic cells. These transgenic animals, along with the accompanying viral vectors, together provide a versatile toolkit for a broad range of ex vivo and in vivo gene editing applications, including fundamental immunological discovery and immune gene engineering.

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. Here, we review the current literature surrounding CRISPR screens within the context of tumor immunology, provide essential components needed to conduct immune-specific CRISPR screens, and present avenues for future research.

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.1.351, and B.1.617 SARS-CoV-2 spikes, and systematically study their immune responses. All three LNP-mRNAs induced potent antibody and T cell responses in animal models; however, differences in neutralization activity have been observed between variants. All three vaccines offer potent protection against in vivo challenges of authentic viruses of WA-1, Beta, and Delta variants. Single-cell transcriptomics of WT- and variant-specific LNP-mRNA-vaccinated animals reveal a systematic landscape of immune cell populations and global gene expression. Variant-specific vaccination induces a systemic increase of reactive CD8 T cells and altered gene expression programs in B and T lymphocytes. BCR-seq and TCR-seq unveil repertoire diversity and clonal expansions in vaccinated animals. These data provide assessment of efficacy and direct systems immune profiling of variant-specific LNP-mRNA vaccination in vivo.

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. Single-cell RNA sequencing profiles the global systemic immune repertoires and respective transcriptome signatures of vaccinated animals, revealing a systemic increase in activated B cells and differential gene expression across major adaptive immune cells. Sequential vaccination shows potent antibody responses against all three species, significantly stronger than simultaneous vaccination in mixture. These data demonstrate the feasibility, antibody responses, and single-cell immune profiles of multispecies coronavirus vaccination. The direct comparison between simultaneous and sequential vaccination offers insights into optimization of vaccination schedules to provide broad and potent antibody immunity against three major pathogenic coronavirus species.

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. Targeted knockin or overexpression of a lead target, PRODH2, enhanced CAR-T-based killing and in vivo efficacy in multiple cancer models. Transcriptomics and metabolomics in CAR-T cells revealed that augmenting PRODH2 expression reshaped broad and distinct gene expression and metabolic programs. Mitochondrial, metabolic, and immunological analyses showed that PRODH2 engineering enhances the metabolic and immune functions of CAR-T cells against cancer. Together, these findings provide a system for identification of GOF immune boosters and demonstrate PRODH2 as a target to enhance CAR-T efficacy.

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. Subsequently, the perturbation-induced effects are evaluated by sequencing-based counting of the guide RNAs that specify each perturbation. The typical results of such screens are ranked lists of genes that confer sensitivity or resistance to the biological challenge of interest. Contributing to the broad utility of CRISPR screens, adaptations of the core CRISPR technology make it possible to activate, silence or otherwise manipulate the target genes. Moreover, high-content read-outs such as single-cell RNA sequencing and spatial imaging help characterize screened cells with unprecedented detail. Dedicated software tools facilitate bioinformatic analysis and enhance reproducibility. CRISPR screening has unravelled various molecular mechanisms in basic biology, medical genetics, cancer research, immunology, infectious diseases, microbiology and other fields. This Primer describes the basic and advanced concepts of CRISPR screening and its application as a flexible and reliable method for biological discovery, biomedical research and drug development - with a special emphasis on high-content methods that make it possible to obtain detailed biological insights directly as part of the screen.

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. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identify two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generate a bispecific antibody. Lead antibodies show strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solve several cryo-EM structures at ~3 Å resolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and reveal distinct epitopes, binding patterns, and conformations. The lead clones also show potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generate and characterize a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants.

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. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identified two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generated a bispecific antibody. Lead antibodies showed strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solved several cryo-EM structures at ∼3 Šresolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and revealed distinct epitopes, binding patterns, and conformations. The lead clones also showed potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generated and characterized a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants.

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. KMT2D encodes a histone H3K4 methyltransferase and is among the most frequently mutated genes in patients with cancer. Kmt2d loss led to increased DNA damage and mutation burden, chromatin remodeling, intron retention, and activation of transposable elements. In addition, Kmt2d-mutant cells exhibited increased protein turnover and IFNγ-stimulated antigen presentation. In turn, Kmt2d-mutant tumors in both mouse and human were characterized by increased immune infiltration. These data demonstrate that Kmt2d deficiency sensitizes tumors to ICB by augmenting tumor immunogenicity, and also highlight the power of CRISPR-GEMMs for interrogating complex molecular landscapes in immunotherapeutic contexts that preserve the native tumor microenvironment. SIGNIFICANCE: ICB is ineffective in the majority of patients. Through direct in vivo CRISPR mutagenesis screening in GEMMs of cancer, we find Kmt2d deficiency sensitizes tumors to ICB. Considering the prevalence of KMT2D mutations, this finding potentially has broad implications for patient stratification and clinical decision-making.This article is highlighted in the In This Issue feature, p. 1775.

In vivo CRISPR screening in CD8 T cells with AAV-Sleeping Beauty hybrid vectors identifies membrane targets for improving immunotherapy for glioblastoma.

Targeting membrane proteins could improve the efficacy of T cell-based immunotherapies. To facilitate the identification of T cell targets, we developed a hybrid genetic screening system where the Sleeping Beauty (SB) transposon and single guide RNA cassette are nested in an adeno-associated virus (AAV). SB-mediated genomic integration of the single guide RNA cassette enables efficient gene editing in primary murine T cells as well as a screen readout. We performed in vivo AAV-SB-CRISPR screens for membrane protein targets in CD8+ T cells in mouse models of glioblastoma (GBM). We validated screen hits by demonstrating that adoptive transfer of CD8+ T cells with Pdia3, Mgat5, Emp1 or Lag3 gene editing enhances the survival of GBM-bearing mice in both syngeneic and T-cell receptor transgenic models. Transcriptome profiling, single cell sequencing, cytokine assays and T cell signaling analysis showed that Pdia3 editing in T cells enhances effector functions. Engineered PDIA3 mutant EGFRvIII chimeric antigen T cells are more potent in antigen-specific killing of human GBM cells.

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. Functional interrogation showed that Prkar1a loss greatly altered the transcriptome and proteome involved in inflammatory and immune responses as well as extracellular protein production. Single-cell transcriptomic profiling and flow cytometry analysis mapped the tumor microenvironment of Prkar1a mutant tumors and revealed the transcriptomic alterations in host myeloid cells. Taken together, our data suggest that tumor-intrinsic mutations in Prkar1a lead to drastic alterations in the genetic program of cancer cells, thereby remodeling the tumor microenvironment.