Explore the vast range of titles available.

Immunotherapies with chimeric antigen receptor (CAR) T cells and checkpoint inhibitors (including antibodies that antagonize programmed cell death protein 1 [PD-1]) have both opened new avenues for cancer treatment, but the clinical potential of combined disruption of inhibitory checkpoints and CAR T cell therapy remains incompletely explored. Here we show that programmed death ligand 1 (PD-L1) expression on tumor cells can render human CAR T cells (anti-CD19 4-1BBζ) hypo-functional, resulting in impaired tumor clearance in a sub-cutaneous xenograft model. To overcome this suppressed anti-tumor response, we developed a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing and lentiviral transduction to generate PD-1 deficient anti-CD19 CAR T cells. Pdcd1 (PD-1) disruption augmented CAR T cell mediated killing of tumor cells in vitro and enhanced clearance of PD-L1+ tumor xenografts in vivo . This study demonstrates improved therapeutic efficacy of Cas9-edited CAR T cells and highlights the potential of precision genome engineering to enhance next-generation cell therapies.

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4⁺ T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved inCXCR4andPD-1(PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.

Droplet single-cell RNA-seq is applied to large numbers of pooled samples from unrelated individuals. Droplet single-cell RNA-sequencing (dscRNA-seq) has enabled rapid, massively parallel profiling of transcriptomes. However, assessing differential expression across multiple individuals has been hampered by inefficient sample processing and technical batch effects. Here we describe a computational tool, demuxlet, that harnesses natural genetic variation to determine the sample identity of each droplet containing a single cell (singlet) and detect droplets containing two cells (doublets). These capabilities enable multiplexed dscRNA-seq experiments in which cells from unrelated individuals are pooled and captured at higher throughput than in standard workflows. Using simulated data, we show that 50 single-nucleotide polymorphisms (SNPs) per cell are sufficient to assign 97% of singlets and identify 92% of doublets in pools of up to 64 individuals. Given genotyping data for each of eight pooled samples, demuxlet correctly recovers the sample identity of >99% of singlets and identifies doublets at rates consistent with previous estimates. We apply demuxlet to assess cell-type-specific changes in gene expression in 8 pooled lupus patient samples treated with interferon (IFN)-β and perform eQTL analysis on 23 pooled samples.

Type 2 responses can be elicited by ILC2s and T H 2 cells. Locksley and colleagues show that the epithelium-derived cytokines IL-25, IL-33 and TSLP are required for full effector activation of poised ILC2 and primed T H 2 cell populations. Group 2 innate lymphoid cells (ILC2s) and CD4 + type 2 helper T cells (T H 2 cells) are defined by their similar effector cytokines, which together mediate the features of allergic immunity. We found that tissue ILC2s and T H 2 cells differentiated independently but shared overlapping effector function programs that were mediated by exposure to the tissue-derived cytokines interleukin 25 (IL-25), IL-33 and thymic stromal lymphopoietin (TSLP). Loss of these three tissue signals did not affect lymph node priming, but abrogated the terminal differentiation of effector T H 2 cells and adaptive lung inflammation in a T cell–intrinsic manner. Our findings suggest a mechanism by which diverse perturbations can activate type 2 immunity and reveal a shared local-tissue-elicited checkpoint that can be exploited to control both innate and adaptive allergic inflammation.

Over 90% of genetic variants associated with complex human traits map to non-coding regions, but little is understood about how they modulate gene regulation in health and disease. One possible mechanism is that genetic variants affect the activity of one or more cis-regulatory elements leading to gene expression variation in specific cell types. To identify such cases, we analyzed ATAC-seq and RNA-seq profiles from stimulated primary CD4 + T cells in up to 105 healthy donors. We found that regions of accessible chromatin (ATAC-peaks) are co-accessible at kilobase and megabase resolution, consistent with the three-dimensional chromatin organization measured by in situ Hi-C in T cells. Fifteen percent of genetic variants located within ATAC-peaks affected the accessibility of the corresponding peak (local-ATAC-QTLs). Local-ATAC-QTLs have the largest effects on co-accessible peaks, are associated with gene expression and are enriched for autoimmune disease variants. Our results provide insights into how natural genetic variants modulate cis-regulatory elements, in isolation or in concert, to influence gene expression. Analysis of ATAC-seq and RNA-seq data from stimulated T cells identifies genetic variants that disrupt transcription factor binding sites within ATAC-seq peaks. ATAC quantitative trait loci (ATAC-QTLs) are enriched for autoimmune disease-associated variants.

The authors use tiled CRISPR activation for functional enhancer discovery across two autoimmunity risk loci, CD69 and IL2RA , and identify elements with features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours a fine-mapped autoimmunity risk variant. CRISPRa mapping of enhancer functions Enhancers are gene regulatory elements that shape cell-type-specific transcriptional programs and responses to specific extracellular cues. Mapping enhancer function is challenging because of our limited understanding of the cellular context in which each enhancer contributes to gene regulation. Here, Alexander Marson and colleagues use a tiled CRISPR activation (CRISPRa) approach for functional enhancer discovery across two autoimmunity risk loci: CD69 and IL2RA. They identify several elements with features of stimulus-responsive enhancers, including an IL2RA enhancer that contains an autoimmunity risk variant. This approach should be useful for discovering functional enhancers without prior knowledge of their specific biological context. The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues 1 , 2 , 3 . Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption 4 , 5 , 6 , but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) 7 to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA . We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (T H 17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

Human regulatory T (T reg ) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains T reg cell identity, yet the complete set of key transcription factors that control T reg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human T reg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from T reg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in T reg cell function, coregulates another gene network with SATB1 and is important for T reg cell–mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human T reg cells that could be targeted for immunotherapies. T reg cells are essential for immune homeostasis, but the transcription factors controlling their cellular identity are incompletely understood. Schumann and colleagues use pooled and arrayed CRISPR screens and scRNA-seq to describe key gene networks in human T reg cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In this Letter, analysis of steady-state regulatory T (Treg) cell percentages from Il2ra enhancer deletion (EDEL) and wild-type (WT) mice revealed no differences between them (Extended Data Fig. 9d). This analysis included two mice whose genotypes were incorrectly assigned. Even after correction of the genotypes, no significant differences in Treg cell percentages were seen when data across experimental cohorts were averaged (as was done in Extended Data Fig. 9d). However, if we normalize the corrected data to account for variation among experimental cohorts, a subtle decrease in EDEL Treg cell percentages is revealed and, using the corrected and normalized data, we have redrawn Extended Data Fig. 9d in Supplementary Fig. 1. The Supplementary Information to this Amendment contains the corrected and reanalysed Extended Data Fig. 9d. The sentence “This enhancer deletion (EDEL) strain also had no obvious T cell phenotypes at steady state (Extended Data Fig. 9).” should read: “This enhancer deletion (EDEL) strain had a small decrease in the percentage of Treg cells (Extended Data Fig. 9).”. This error does not affect any of the main figures in the Letter or the data from mice with the human autoimmune-associated single nucleotide polymorphism (SNP) knocked in or with a 12-base-pair deletion at the site (12DEL). In addition, we stated in the Methods that we observed consistent immunophenotypes of EDEL mice across three founders, but in fact, we observed consistent phenotypes in mice from two founders. This does not change any of our conclusions and the original Letter has not been corrected.

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum. The ability of an organism to grow and reproduce, that is, it’s “fitness”, is determined by how its genes interact with the environment. Yeast is a model organism in which researchers can control the exact mutations present in the yeast’s genes (its genotype) and the conditions in which the yeast cells live (their environment). This allows researchers to measure how a yeast cell’s genotype and environment affect its fitness. Ubiquitin is a protein that many organisms depend on to manage cell stress by acting as a tag that targets other proteins for degradation. Essential proteins such as ubiquitin often remain unchanged by mutation over long periods of time. As a result, these proteins evolve very slowly. Like all proteins, ubiquitin is built from a chain of amino acid molecules linked together, and the ubiquitin proteins of yeast and humans are made of almost identical sequences of amino acids. Although ubiquitin has barely changed its sequence over evolution, previous studies have shown that – under normal growth conditions in the laboratory – most amino acids in ubiquitin can be mutated without any loss of cell fitness. This led Mavor et al. to hypothesize that treating the yeast cells with chemicals that cause cell stress might lead to amino acids in ubiquitin becoming more sensitive to mutation. To test this idea, a class of graduate students at the University of California, San Francisco grew yeast cells with different ubiquitin mutations together, and with different chemicals that induce cell stress, and measured their growth rates. Sequencing the ubiquitin gene in the thousands of tested yeast cells revealed that three of the chemicals cause a shared set of amino acids in ubiquitin to become more sensitive to mutation. This result suggests that these amino acids are important for the stress response, possibly by altering the ability of yeast cells to target certain proteins for degradation. Conversely, another chemical causes yeast to become more tolerant to changes in the ubiquitin sequence. The experiments also link changes in particular amino acids in ubiquitin to specific stress responses. Mavor et al. show that many of ubquitin’s amino acids are sensitive to mutation under different stress conditions, while others can be mutated to form different amino acids without effecting fitness. By testing the effects of other chemicals, future experiments could further characterize how the yeast’s genotype and environment interact.