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.

Applications of CRISPR technology in cellular immunotherapy.

CRISPR technology has transformed multiple fields, including cancer and immunology. CRISPR-based gene editing and screening empowers direct genomic manipulation of immune cells, opening doors to unbiased functional genetic screens. These screens aid in the discovery of novel factors that regulate and reprogram immune responses, offering novel drug targets. The engineering of immune cells using CRISPR has sparked a transformation in the cellular immunotherapy field, resulting in a multitude of ongoing clinical trials. In this review, we discuss the development and applications of CRISPR and related gene editing technologies in immune cells, focusing on functional genomics screening, gene editing-based cell therapies, as well as future directions in this rapidly advancing field.

Hypomethylation of STAT1 and HLA-DRB1 is associated with type-I interferon-dependent HLA-DRB1 expression in lupus CD8+ T cells.

OBJECTIVE: We examined genome-wide DNA methylation changes in CD8+ T cells from patients with lupus and controls and investigated the functional relevance of some of these changes in lupus. METHODS: Genome-wide DNA methylation of lupus and age, sex and ethnicity-matched control CD8+ T cells was measured using the Infinium MethylationEPIC arrays. Measurement of relevant cell subsets was performed via flow cytometry. Gene expression was quantified by qPCR. Inhibiting STAT1 and CIITA was performed using fludarabine and CIITA siRNA, respectively. RESULTS: Lupus CD8+ T cells had 188 hypomethylated CpG sites compared with healthy matched controls. Among the most hypomethylated were sites associated with HLA-DRB1. Genes involved in the type-I interferon response, including STAT1, were also found to be hypomethylated. IFNα upregulated HLA-DRB1 expression on lupus but not control CD8+ T cells. Lupus and control CD8+ T cells significantly increased STAT1 mRNA levels after treatment with IFNα. The expression of CIITA, a key interferon/STAT1 dependent MHC-class II regulator, is induced by IFNα in lupus CD8+ T cells, but not healthy controls. CIITA knockdown and STAT1 inhibition experiments revealed that HLA-DRB1 expression in lupus CD8+ T cells is dependent on CIITA and STAT1 signalling. Coincubation of naïve CD4+ T cells with IFNα-treated CD8+ T cells led to CD4+ T cell activation, determined by increased expression of CD69 and cytokine production, in patients with lupus but not in healthy controls. This can be blocked by neutralising antibodies targeting HLA-DR. CONCLUSIONS: Lupus CD8+ T cells are epigenetically primed to respond to type-I interferon. We describe an HLA-DRB1+ CD8+ T cell subset that can be induced by IFNα in patients with lupus. A possible pathogenic role for CD8+ T cells in lupus that is dependent on a high type-I interferon environment and epigenetic priming warrants further characterisation.

CD4+CD28+KIR+CD11ahi T cells correlate with disease activity and are characterized by a pro-inflammatory epigenetic and transcriptional profile in lupus patients.

OBJECTIVE: The goal of this study was to comprehensively characterize CD4+CD28+ T cells overexpressing CD11a and KIR genes, and examine the relationship between this T cell subset, genetic risk, and disease activity in lupus. METHODS: The size of the CD4+CD28+KIR+CD11ahi T cell subset was determined by flow cytometry, and total genetic risk for lupus was calculated in 105 female patients using 43 confirmed genetic susceptibility loci. Primary CD4+CD28+KIR+CD11ahi T cells were isolated from lupus patients or were induced from healthy individuals using 5-azacytidine. Genome-wide DNA methylation was analyzed using an array-based approach, and the transcriptome was assessed by RNA sequencing. Transcripts in the CDR3 region were used to assess the TCR repertoire. Chromatin accessibility was determined using ATAC-seq. RESULTS: A total of 31,019 differentially methylated sites were identified in induced KIR+CD11ahi T cells with >99% being hypomethylated. RNA sequencing revealed a clear pro-inflammatory transcriptional profile. TCR repertoire analysis suggests less clonotype diversity in KIR+CD11ahi compared to autologous KIR-CD11alow T cells. Similarly, primary KIR+CD11ahi T cells isolated from lupus patients were hypomethylated and characterized by a pro-inflammatory chromatin structure. We show that the genetic risk for lupus was significantly higher in African-American compared to European-American lupus patients. The demethylated CD4+CD28+KIR+CD11ahi T cell subset size was a better predictor of disease activity in young (age ≤ 40) European-American patients independent of genetic risk. CONCLUSION: CD4+CD28+KIR+CD11ahi T cells are demethylated and characterized by pro-inflammatory epigenetic and transcriptional profiles in lupus. Eliminating these cells or blocking their pro-inflammatory characteristics might present a novel therapeutic approach for lupus.

IFI44L promoter methylation as a blood biomarker for systemic lupus erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease with limited reliable diagnostic biomarkers. We investigated whether gene methylation could meet sensitivity and specificity criteria for a robust biomarker. METHODS: IFI44L promoter methylation was examined using DNA samples from a discovery set including 377 patients with SLE, 358 healthy controls (HCs) and 353 patients with rheumatoid arthritis (RA). Two independent sets including 1144 patients with SLE, 1350 HCs, 429 patients with RA and 199 patients with primary Sjögren's syndrome (pSS) were used for validation. RESULTS: Significant hypomethylation of two CpG sites within IFI44L promoter, Site1 (Chr1: 79 085 222) and Site2 (Chr1: 79 085 250; cg06872964), was identified in patients with SLE compared with HCs, patients with RA and patients with pSS. In a comparison between patients with SLE and HCs included in the first validation cohort, Site1 methylation had a sensitivity of 93.6% and a specificity of 96.8% at a cut-off methylation level of 75.5% and Site2 methylation had a sensitivity of 94.1% and a specificity of 98.2% at a cut-off methylation level of 25.5%. The IFI44L promoter methylation marker was also validated in an European-derived cohort. In addition, the methylation levels of Site1 and Site2 within IFI44L promoter were significantly lower in patients with SLE with renal damage than those without renal damage. Patients with SLE showed significantly increased methylation levels of Site1 and Site2 during remission compared with active stage. CONCLUSIONS: The methylation level of IFI44L promoter can distinguish patients with SLE from healthy persons and other autoimmune diseases, and is a highly sensitive and specific diagnostic marker for SLE.

Scleroderma dermal microvascular endothelial cells exhibit defective response to pro-angiogenic chemokines.

OBJECTIVES: Angiogenesis plays a critical role in SSc (scleroderma). The aim of this study was to examine the expression of growth-regulated protein-γ (Gro-γ/CXCL3), granulocyte chemotactic protein 2 (GCP-2/CXCL6) and their receptor CXCR2 in endothelial cells (ECs) isolated from SSc skin and determine whether these cells mount an angiogenic response towards pro-angiogenic chemokines. The downstream signalling pathways as well as the pro-angiogenic transcription factor inhibitor of DNA-binding protein 1 (Id-1) were also examined. METHODS: Skin biopsies were obtained from patients with dcSSc. ECs were isolated via magnetic positive selection. Angiogenesis was measured by EC chemotaxis assay. RESULTS: Gro-γ/CXCL3 and GCP-2/CXCL6 were minimally expressed in both skin types but elevated in SSc serum. Pro-angiogenic chemokine mRNA was greater in SSc ECs than in normal ECs. SSc ECs did not migrate to vascular endothelial growth factor (VEGF), Gro-γ/CXCL3, GCP-2/CXCL6 or CXCL16. The signalling pathways stimulated by these chemokines were also dysregulated. Id-1 mRNA in SSc ECs was lower compared with normal ECs, and overexpression of Id-1 in SSc ECs increased their ability to migrate towards VEGF and CXCL16. CONCLUSION: Our results show that SSc ECs are unable to respond to pro-angiogenic chemokines despite their increased expression in serum and ECs. This might be due to the differences in the signalling pathways activated by these chemokines in normal vs SSc ECs. In addition, the lower expression of Id-1 also decreases the angiogenic response. The inability of pro-angiogenic chemokines to promote EC migration provides an additional mechanism for the impaired angiogenesis that characterizes SSc.

Saccharomyces cerevisiae lysophospholipid acyltransferase, Lpt1, requires Asp146 and Glu297 for catalysis.

The esterification of lysophospholipids contributes to phospholipid synthesis, remodeling, and scavenging. Acyl-CoA-dependent lysophospholipid acyltransferase activity with broad substrate use is mediated by Saccharomyces cerevisiae Lpt1p. We sought to identify Lpt1p active site amino acids besides the histidine conserved among homologs and repeatedly found to be required for catalysis. In vitro Lpt1p assays with amino acid modifying agents implicated aspartate, glutamate, and lysine as active site residues. Threonine and tyrosine were not ruled out. Aligning the primary structures of functionally characterized LPT1 homologs from fungi, plants, and animals identified 11 conserved aspartate, glutamate, lysine, threonine, and tyrosine residues. Site-directed mutagenesis of the respective codons showed that changing D146 and E297 abolished activity without abolishing protein expression. The mechanism of Lpt1p was further analyzed using monounsaturated acyl-CoA species with different double bond positions. Delta 6 species showed the highest catalytic efficiency. We propose that D146 and E297 act in conjunction with H382 as nucleophiles that attack the hydroxyl group in lysophospholipids in a general acid/base mechanism. This sequential mechanism provides a precedent for other members of the membrane bound O-acyltransferase family. Also, Lpt1p optimally orients acyl-CoA substrates with 7.5 Å between a double bond and the thioester bond.

The DNA methylation signature of human TCRαß+CD4-CD8- double negative T cells reveals CG demethylation and a unique epigenetic architecture permissive to a broad stimulatory immune response.

T cell receptor (TCR) αß+CD4-CD8- double negative T cells represent a rare T cell subset implicated in the pathogenesis of several autoimmune diseases. We investigated the DNA methylation signature of double negative T cells to gain insight into the epigenetic architecture of peripheral blood primary human double negative T cells compared to autologous CD4+ and CD8+ T cells. We identified 2984 CG sites across the genome with unique loss of DNA methylation in double negative T cells, and showed significant reduction in mRNA expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B. DNA methylation was increased in CD8A/CD8B and CD4 consistent with epigenetic repression of both the CD8 and CD4 genetic loci in double negative T cells. We show a consistent increase in non-CG methylation in double negative T cells, a finding suggestive of pluripotency. Network analyses indicate a strong relationship between double negative T cells and functions related to cell-cell interaction, cell adhesion, and cell activation pathways. Our data also suggest a robust pro-inflammatory epigenetic signature in double negative T cells, consistent with a transcriptional permissiveness in key inflammatory cytokines including IFNγ, IL-17F, IL-12B, IL-5, IL-18, TNFSF11 (RANKL), and TNFSF13B (BLYS or BAFF). These findings highlight an epigenetic basis for a role of double negative T cells in autoimmunity.

Renal involvement in lupus is characterized by unique DNA methylation changes in naïve CD4+ T cells.

Systemic lupus erythematosus is a multi-system disease characterized by wide-spread DNA methylation changes. To identify epigenetic susceptibility loci for lupus nephritis, genome-wide DNA methylation changes in naïve CD4+ T cells were compared between two sets of lupus patients with and without a history of renal involvement. A total of 56 lupus patients (28 with renal involvement and 28 without renal involvement), and 56 age-, sex-, and ethnicity-matched healthy controls were included in our study. We identified 191 CG sites and 121 genes that were only differentially methylated in lupus patients with but not without a history of renal involvement. The tyrosine kinase gene TNK2 involved in cell trafficking and tissue invasion, and the phosphatase gene DUSP5 which dephosphorylates and inhibits the ERK signaling pathway, were among the most hypomethylated. Independent of disease activity, renal involvement is characterized by more robust demethylation in interferon regulated genes differentially methylated in both sets of lupus patients with and without renal involvement (fold change 1.4, P = 0.0014). The type-I interferon master regulator gene IRF7 is only hypomethylated in lupus patients with renal involvement. IRF-7 is an upstream transcription factor that regulates several loci demethylated only with renal involvement such as CD80, HERC5, IFI44, IRF7, ISG15, ISG20, ITGAX, and PARP12 (P = 1.78 × 10(-6)). Among the CG sites only hypomethylated with renal involvement, CG10152449 in CHST12 has a sensitivity of 85.7% and a specificity of 64.3% for stratifying lupus patients for a history of renal involvement (P = 0.0029). Our data identified novel epigenetic susceptibility loci that are differentially methylated with renal involvement in lupus. These loci will help better understand lupus nephritis, and provide a proof of principle for the potential applicability of specific methylation changes as predictors for specific organ involvement in lupus.

Genome scale CRISPR screens identify actin capping proteins as key modulators of therapeutic responses to radiation and immunotherapy.

Radiotherapy (RT), is a fundamental treatment for malignant tumors and is used in over half of cancer patients. As radiation can promote anti-tumor immune effects, a promising therapeutic strategy is to combine radiation with immune checkpoint inhibitors (ICIs). However, the genetic determinants that impact therapeutic response in the context of combination therapy with radiation and ICI have not been systematically investigated. To unbiasedly identify the tumor intrinsic genetic factors governing such responses, we perform a set of genome-scale CRISPR screens in melanoma cells for cancer survival in response to low-dose genotoxic radiation treatment, in the context of CD8 T cell co-culture and with anti-PD1 checkpoint blockade antibody. Two actin capping proteins, Capza3 and Capg, emerge as top hits that upon inactivation promote the survival of melanoma cells in such settings. Capza3 and Capg knockouts (KOs) in mouse and human cancer cells display persistent DNA damage due to impaired homology directed repair (HDR); along with increased radiation, chemotherapy, and DNA repair inhibitor sensitivity. However, when cancer cells with these genes inactivated were exposed to sublethal radiation, inactivation of such actin capping protein promotes activation of the STING pathway, induction of inhibitory CEACAM1 ligand expression and resistance to CD8 T cell killing. Patient cancer genomics analysis reveals an increased mutational burden in patients with inactivating mutations in CAPG and/or CAPZA3, at levels comparable to other HDR associated genes. There is also a positive correlation between CAPG expression and activation of immune related pathways and CD8 T cell tumor infiltration. Our results unveil the critical roles of actin binding proteins for efficient HDR within cancer cells and demonstrate a previously unrecognized regulatory mechanism of therapeutic response to radiation and immunotherapy.

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.

RAMIHM generates fully human monoclonal antibodies by rapid mRNA immunization of humanized mice and BCR-seq.

As a clinical vaccine, lipid nanoparticle (LNP) mRNA has demonstrated potent and broad antibody responses, leading to speculation about its potential for antibody discovery. Here, we developed RAMIHM, a highly efficient strategy for developing fully human monoclonal antibodies that employs rapid mRNA immunization of humanized mice followed by single B cell sequencing (scBCR-seq). We immunized humanized transgenic mice with RAMIHM and generated 15 top-ranked clones from peripheral blood, plasma B, and memory B cell populations, demonstrating a high rate of antigen-specificity (93.3%). Two Omicron-specific neutralizing antibodies with high potency and one broad-spectrum neutralizing antibody were discovered. Furthermore, we extended the application of RAMIHM to cancer immunotherapy targets, including a single transmembrane protein CD22 and a multi-transmembrane G protein-coupled receptor target, GPRC5D, which is difficult for traditional protein immunization methods. RAMIHM-scBCR-seq is a broadly applicable platform for the rapid and efficient development of fully human monoclonal antibodies against an assortment of targets.

Immunogenetic Metabolomics Reveals Key Enzymes That Modulate CAR T-cell Metabolism and Function.

Immune evasion is a critical step of cancer progression that remains a major obstacle for current T cell-based immunotherapies. Hence, we investigated whether it is possible to genetically reprogram T cells to exploit a common tumor-intrinsic evasion mechanism whereby cancer cells suppress T-cell function by generating a metabolically unfavorable tumor microenvironment (TME). In an in silico screen, we identified ADA and PDK1 as metabolic regulators. We then showed that overexpression (OE) of these genes enhanced the cytolysis of CD19-specific chimeric antigen receptor (CAR) T cells against cognate leukemia cells, and conversely, ADA or PDK1 deficiency dampened this effect. ADA-OE in CAR T cells improved cancer cytolysis under high concentrations of adenosine, the ADA substrate, and an immunosuppressive metabolite in the TME. High-throughput transcriptomics and metabolomics analysis of these CAR T cells revealed alterations of global gene expression and metabolic signatures in both ADA- and PDK1-engineered CAR T cells. Functional and immunologic analyses demonstrated that ADA-OE increased proliferation and decreased exhaustion in CD19-specific and HER2-specific CAR T cells. ADA-OE improved tumor infiltration and clearance by HER2-specific CAR T cells in an in vivo colorectal cancer model. Collectively, these data unveil systematic knowledge of metabolic reprogramming directly in CAR T cells and reveal potential targets for improving CAR T-cell therapy.

Perturbomics of tumor-infiltrating NK cells.

Natural killer (NK) cells are an innate immune cell type that serves at the first level of defense against pathogens and cancer. NK cells have clinical potential, however, multiple current limitations exist that naturally hinder the successful implementation of NK cell therapy against cancer, including their effector function, persistence, and tumor infiltration. To unbiasedly reveal the functional genetic landscape underlying critical NK cell characteristics against cancer, we perform perturbomics mapping of tumor infiltrating NK cells by joint in vivo AAV-CRISPR screens and single cell sequencing. We establish a strategy with AAV-SleepingBeauty(SB)- CRISPR screening leveraging a custom high-density sgRNA library targeting cell surface genes, and perform four independent in vivo tumor infiltration screens in mouse models of melanoma, breast cancer, pancreatic cancer, and glioblastoma. In parallel, we characterize single-cell transcriptomic landscapes of tumor-infiltrating NK cells, which identifies previously unexplored sub-populations of NK cells with distinct expression profiles, a shift from immature to mature NK (mNK) cells in the tumor microenvironment (TME), and decreased expression of mature marker genes in mNK cells. CALHM2, a calcium homeostasis modulator that emerges from both screen and single cell analyses, shows both in vitro and in vivo efficacy enhancement when perturbed in chimeric antigen receptor (CAR)-NK cells. Differential gene expression analysis reveals that CALHM2 knockout reshapes cytokine production, cell adhesion, and signaling pathways in CAR- NKs. These data directly and systematically map out endogenous factors that naturally limit NK cell function in the TME to offer a broad range of cellular genetic checkpoints as candidates for future engineering to enhance NK cell-based immunotherapies.

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.

Immunogenetic metabolomics revealed key enzymes that modulate CAR-T metabolism and function.

Immune evasion is a critical step of cancer progression that remains a major obstacle for current T cell-based immunotherapies. Hence, we seek to genetically reprogram T cells to exploit a common tumor-intrinsic evasion mechanism, whereby cancer cells suppress T cell function by generating a metabolically unfavorable tumor microenvironment (TME). Specifically, we use an in silico screen to identify ADA and PDK1 as metabolic regulators, in which gene overexpression (OE) enhances the cytolysis of CD19-specific CD8 CAR-T cells against cognate leukemia cells, and conversely, ADA or PDK1 deficiency dampens such effect. ADA -OE in CAR-T cells improves cancer cytolysis under high concentrations of adenosine, the ADA substrate and an immunosuppressive metabolite in the TME. High-throughput transcriptomics and metabolomics in these CAR-Ts reveal alterations of global gene expression and metabolic signatures in both ADA- and PDK1- engineered CAR-T cells. Functional and immunological analyses demonstrate that ADA -OE increases proliferation and decreases exhaustion in α-CD19 and α-HER2 CAR-T cells. ADA-OE improves tumor infiltration and clearance by α-HER2 CAR-T cells in an in vivo colorectal cancer model. Collectively, these data unveil systematic knowledge of metabolic reprogramming directly in CAR-T cells, and reveal potential targets for improving CAR-T based cell therapy. Synopsis: The authors identify the adenosine deaminase gene (ADA) as a regulatory gene that reprograms T cell metabolism. ADA-overexpression (OE) in α-CD19 and α-HER2 CAR-T cells increases proliferation, cytotoxicity, memory, and decreases exhaustion, and ADA-OE α-HER2 CAR-T cells have enhanced clearance of HT29 human colorectal cancer tumors in vivo .

Massively parallel knock-in engineering of human T cells.

The efficiency of targeted knock-in for cell therapeutic applications is generally low, and the scale is limited. In this study, we developed CLASH, a system that enables high-efficiency, high-throughput knock-in engineering. In CLASH, Cas12a/Cpf1 mRNA combined with pooled adeno-associated viruses mediate simultaneous gene editing and precise transgene knock-in using massively parallel homology-directed repair, thereby producing a pool of stably integrated mutant variants each with targeted gene editing. We applied this technology in primary human T cells and performed time-coursed CLASH experiments in blood cancer and solid tumor models using CD3, CD8 and CD4 T cells, enabling pooled generation and unbiased selection of favorable CAR-T variants. Emerging from CLASH experiments, a unique CRISPR RNA (crRNA) generates an exon3 skip mutant of PRDM1 in CAR-Ts, which leads to increased proliferation, stem-like properties, central memory and longevity in these cells, resulting in higher efficacy in vivo across multiple cancer models, including a solid tumor model. The versatility of CLASH makes it broadly applicable to diverse cellular and therapeutic engineering applications.

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.