A genome-scale screen for synthetic drivers of T cell proliferation.

The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer1. However, further improvements are needed to increase response and cure rates. CRISPR-based loss-of-function screens have been limited to negative regulators of T cell functions2-4 and raise safety concerns owing to the permanent modification of the genome. Here we identify positive regulators of T cell functions through overexpression of around 12,000 barcoded human open reading frames (ORFs). The top-ranked genes increased the proliferation and activation of primary human CD4+ and CD8+ T cells and their secretion of key cytokines such as interleukin-2 and interferon-γ. In addition, we developed the single-cell genomics method OverCITE-seq for high-throughput quantification of the transcriptome and surface antigens in ORF-engineered T cells. The top-ranked ORF-lymphotoxin-ß receptor (LTBR)-is typically expressed in myeloid cells but absent in lymphocytes. When overexpressed in T cells, LTBR induced profound transcriptional and epigenomic remodelling, leading to increased T cell effector functions and resistance to exhaustion in chronic stimulation settings through constitutive activation of the canonical NF-κB pathway. LTBR and other highly ranked genes improved the antigen-specific responses of chimeric antigen receptor T cells and γδ T cells, highlighting their potential for future cancer-agnostic therapies5. Our results provide several strategies for improving next-generation T cell therapies by the induction of synthetic cell programmes.

Pathobionts from chemically disrupted gut microbiota induce insulin-dependent diabetes in mice.

BACKGROUND: Dysbiotic gut microbiome, genetically predisposed or chemically disrupted, has been linked with insulin-dependent diabetes (IDD) including autoimmune type 1 diabetes (T1D) in both humans and animal models. However, specific IDD-inducing gut bacteria remain to be identified and their casual role in disease development demonstrated via experiments that can fulfill Koch's postulates. RESULTS: Here, we show that novel gut pathobionts in the Muribaculaceae family, enriched by a low-dose dextran sulfate sodium (DSS) treatment, translocated to the pancreas and caused local inflammation, beta cell destruction and IDD in C57BL/6 mice. Antibiotic removal and transplantation of gut microbiota showed that this low DSS disrupted gut microbiota was both necessary and sufficient to induce IDD. Reduced butyrate content in the gut and decreased gene expression levels of an antimicrobial peptide in the pancreas allowed for the enrichment of selective members in the Muribaculaceae family in the gut and their translocation to the pancreas. Pure isolate of one such members induced IDD in wildtype germ-free mice on normal diet either alone or in combination with normal gut microbiome after gavaged into stomach and translocated to pancreas. Potential human relevance of this finding was shown by the induction of pancreatic inflammation, beta cell destruction and IDD development in antibiotic-treated wildtype mice via transplantation of gut microbiome from patients with IDD including autoimmune T1D. CONCLUSION: The pathobionts that are chemically enriched in dysbiotic gut microbiota are sufficient to induce insulin-dependent diabetes after translocation to the pancreas. This indicates that IDD can be mainly a microbiome-dependent disease, inspiring the need to search for novel pathobionts for IDD development in humans. Video Abstract.

High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene Knockout and Transcriptional Modulation.

A key limitation of the widely used CRISPR enzyme S. pyogenes Cas9 is the strict requirement of an NGG protospacer-adjacent motif (PAM) at the target site. This constraint can be limiting for genome editing applications that require precise Cas9 positioning. Recently, two Cas9 variants with a relaxed PAM requirement (NG) have been developed (xCas9 and Cas9-NG), but their activity has been measured at only a small number of endogenous sites. Here, we devise a high-throughput Cas9 pooled competition screen to compare the performance of Cas9 variants at thousands of genomic loci for gene knockout, transcriptional activation, and inhibition. We show that PAM flexibility comes at a substantial cost of decreased DNA targeting and cleavage. Of the PAM-flexible variants, we find that Cas9-NG outperforms xCas9 regardless of genome engineering modality or PAM. Finally, we combine xCas9 mutations with those of Cas9-NG, creating a stronger transcriptional modulator than existing PAM-flexible Cas9 variants.

Strain-Specific Anti-inflammatory Properties of Two Akkermansia muciniphila Strains on Chronic Colitis in Mice.

Akkermansia muciniphila is potential probiotic in that its type strain ATCC BAA-835 has beneficial effects upon obesity and diabetes. However, whether A. muciniphila can improve inflammatory bowel diseases (IBD), which is a form of chronic intestinal dysbiosis, is unknown. Hence, we used an isolated murine A. muciniphila strain (designated 139) and A. muciniphila type strain ATCC, to investigate their anti-inflammatory properties in cell models and in Dextran Sulfate Sodium (DSS)-induced chronic colitis of mice. In vitro, the two A. muciniphila strains exerted similar anti-inflammatory properties as they both reduced IL-8 production by TNF-α-stimulated HT-29 cells. However, neither of the strains showed capacity to increase the differentiation of regulatory T (Treg)-cells from CD4+ T cell populations significantly. In vivo, both A. muciniphila strains exerted anti-inflammatory effects on chronic colitis as they improved clinical parameters including spleen weight, colon inflammation index, and colon histological score. They also down-regulated the expression of the pro-inflammatory cytokines including TNF-α and IFN-γ in the colon of mice. However, the anti-inflammatory effects of strain ATCC were stronger than strain 139 in that ATCC significantly reduced spleen weight, colon inflammation index, and fecal lipocalin-2 content in mice with chronic colitis, while strain 139 was not. Dysbiosis of the gut microbiota was observed in mice with chronic colitis. Both A. muciniphila strains facilitated the normalization of the gut microbiota. The specific capacity of strain ATCC to modulate the differentiation of Tregs as well as increase production of short chain fatty acids, demonstrated strain-specific characteristics for these two A. muciniphila strains. This study suggests the potential beneficial effect of A. muciniphila on IBD and the importance of the future study of the function of A. muciniphila at the strain-level.

Timing of Calorie Restriction in Mice Impacts Host Metabolic Phenotype with Correlative Changes in Gut Microbiota.

Calorie restriction (CR) is accompanied by self-imposed daily restriction of food intake and an extended fasting period between meals. The impact of restricting feeding to the dark or light phase on the effects of CR remains elusive. Here, light-fed CR mice showed physiological changes, such as muscle loss, concomitant with changes in the gut microbiota structure and composition. After switching to ad libitum access to food, light-fed mice had a period of food-craving behavior and short-lived physiological changes, while dark-fed mice displayed lasting changes in fat accumulation, glucose metabolism, intestinal barrier function, and systemic inflammatory markers. Moreover, the gut microbiota was modulated by when the food was consumed, and the most abundant Lactobacillus operational taxonomic unit (OTU) promoted by CR was enhanced in dark-fed mice. After switching to ad libitum feeding, the gut microbiota of dark-fed mice returned to the state resembling that of mice fed normal chow ad libitum, but that of light-fed mice was still significantly different from the other two groups. Together, these data indicate that for CR, restricting food consumption to the active phase brought better metabolic phenotype associated with potentially beneficial structural shifts in the gut microbiota.IMPORTANCE Aberrant feeding patterns whereby people eat more frequently throughout the day and with a bias toward late-night eating are prevalent in society today. However, whether restriction of food to daytime in comparison to nighttime, coupled with restricted calorie intake, can influence gut microbiota, metabolism, and overall health requires further investigation. We surveyed the effects of the shift in feeding time on gut microbiota and metabolic phenotype in calorie-restricted mice and found that avoiding eating during the rest period may generate more beneficial effects in mice. This work strengthens the evidence for using "when to eat" as an intervention to improve health during calorie restriction.

Genetically Obese Human Gut Microbiota Induces Liver Steatosis in Germ-Free Mice Fed on Normal Diet.

Dysbiotic gut microbiota contributes to genetically obese phenotype in human. However, the effect of genetic obesity-associated gut microbiota on host hepatic metabolic deteriorations remains largely unknown. Gut microbiota from a genetically obese human donor before and after a dietary weight loss program was transplanted into germ-free C57BL/6J male mice, grouped as PreM and PostM groups, respectively. The gut microbiome, liver pathology and transcriptome response in the gnotobiotic mice were evaluated. After being fed on normal chow diet for 4 weeks, PreM group developed liver macrovesicular steatosis accompanied with higher concentrations of hepatic triglyceride and cholesterol, while PostM group exhibited normal hepatic physiology. The gut microbiota in PreM and PostM groups was significantly different from each other and was more resembling with their respective donor. RNA-sequencing revealed that, in comparison with PostM group, PreM group showed a foregoing pro-steatotic transcriptional response in liver featuring by the repression of lipid beta-oxidation and the activation of lipid absorption and cholesterol uptake before the pathology of liver steatosis. Moreover, peroxisome proliferator-activated receptor alpha (PPARα), which was repressed in PreM group, may act as crucial regulator of the hepatic transcriptional profile of lipid metabolism between two groups. Our results show that gut microbiota from a genetically obese human promotes the onset of liver steatosis by impacting hepatic transcriptional profile of lipid metabolism in mice. This adds new evidence that gut microbiota may play a causative role in the development of non-alcoholic fatty liver disease.

Gut bacteria selectively promoted by dietary fibers alleviate type 2 diabetes.

The gut microbiota benefits humans via short-chain fatty acid (SCFA) production from carbohydrate fermentation, and deficiency in SCFA production is associated with type 2 diabetes mellitus (T2DM). We conducted a randomized clinical study of specifically designed isoenergetic diets, together with fecal shotgun metagenomics, to show that a select group of SCFA-producing strains was promoted by dietary fibers and that most other potential producers were either diminished or unchanged in patients with T2DM. When the fiber-promoted SCFA producers were present in greater diversity and abundance, participants had better improvement in hemoglobin A1c levels, partly via increased glucagon-like peptide-1 production. Promotion of these positive responders diminished producers of metabolically detrimental compounds such as indole and hydrogen sulfide. Targeted restoration of these SCFA producers may present a novel ecological approach for managing T2DM.