In vitro modeling of CD8+ T cell exhaustion enables CRISPR screening to reveal a role for BHLHE40.

Identifying molecular mechanisms of exhausted CD8 T cells (Tex) is a key goal of improving immunotherapy of cancer and other diseases. However, high-throughput interrogation of in vivo Tex can be costly and inefficient. In vitro models of Tex are easily customizable and quickly generate high cellular yield, enabling CRISPR screening and other high-throughput assays. We established an in vitro model of chronic stimulation and benchmarked key phenotypic, functional, transcriptional, and epigenetic features against bona fide in vivo Tex. We leveraged this model of in vitro chronic stimulation in combination with CRISPR screening to identify transcriptional regulators of T cell exhaustion. This approach identified several transcription factors, including BHLHE40. In vitro and in vivo validation defined a role for BHLHE40 in regulating a key differentiation checkpoint between progenitor and intermediate Tex subsets. By developing and benchmarking an in vitro model of Tex, then applying high-throughput CRISPR screening, we demonstrate the utility of mechanistically annotated in vitro models of Tex.

In Vitro Modeling of CD8 T Cell Exhaustion Enables CRISPR Screening to Reveal a Role for BHLHE40.

Identifying novel molecular mechanisms of exhausted CD8 T cells (T ex ) is a key goal of improving immunotherapy of cancer and other diseases. However, high-throughput interrogation of in vivo T ex can be costly and inefficient. In vitro models of T ex are easily customizable and quickly generate high cellular yield, offering an opportunity to perform CRISPR screening and other high-throughput assays. We established an in vitro model of chronic stimulation and benchmarked key phenotypic, functional, transcriptional, and epigenetic features against bona fide in vivo T ex . We leveraged this model of in vitro chronic stimulation in combination with pooled CRISPR screening to uncover transcriptional regulators of T cell exhaustion. This approach identified several transcription factors, including BHLHE40. In vitro and in vivo validation defined a role for BHLHE40 in regulating a key differentiation checkpoint between progenitor and intermediate subsets of T ex . By developing and benchmarking an in vitro model of T ex , we demonstrate the utility of mechanistically annotated in vitro models of T ex , in combination with high-throughput approaches, as a discovery pipeline to uncover novel T ex biology.

Multiscale 3D genome organization underlies ILC2 ontogenesis and allergic airway inflammation.

Innate lymphoid cells (ILCs) are well-characterized immune cells that play key roles in host defense and tissue homeostasis. Yet, how the three-dimensional (3D) genome organization underlies the development and functions of ILCs is unknown. Herein, we carried out an integrative analysis of the 3D genome structure, chromatin accessibility and gene expression in mature ILCs. Our results revealed that the local 3D configuration of the genome is rewired specifically at loci associated with ILC biology to promote their development and functional differentiation. Importantly, we demonstrated that the ontogenesis of ILC2s and the progression of allergic airway inflammation are determined by a unique local 3D configuration of the region containing the ILC-lineage-defining factor Id2, which is characterized by multiple interactions between the Id2 promoter and distal regulatory elements bound by the transcription factors GATA-3 and RORα, unveiling the mechanism whereby the Id2 expression is specifically controlled in group 2 ILCs.

The miR-181 family regulates colonic inflammation through its activity in the intestinal epithelium.

The intestinal epithelium is a key physical interface that integrates dietary and microbial signals to regulate nutrient uptake and mucosal immune cell function. The transcriptional programs that regulate intestinal epithelial cell (IEC) quiescence, proliferation, and differentiation have been well characterized. However, how gene expression networks critical for IECs are posttranscriptionally regulated during homeostasis or inflammatory disease remains poorly understood. Herein, we show that a conserved family of microRNAs, miR-181, is significantly downregulated in IECs from patients with inflammatory bowel disease and mice with chemical-induced colitis. Strikingly, we showed that miR-181 expression within IECs, but not the hematopoietic system, is required for protection against severe colonic inflammation in response to epithelial injury in mice. Mechanistically, we showed that miR-181 expression increases the proliferative capacity of IECs, likely through the regulation of Wnt signaling, independently of the gut microbiota composition. As epithelial reconstitution is crucial to restore intestinal homeostasis after injury, the miR-181 family represents a potential therapeutic target against severe intestinal inflammation.

Single-cell lineage tracing of metastatic cancer reveals selection of hybrid EMT states.

The underpinnings of cancer metastasis remain poorly understood, in part due to a lack of tools for probing their emergence at high resolution. Here we present macsGESTALT, an inducible CRISPR-Cas9-based lineage recorder with highly efficient single-cell capture of both transcriptional and phylogenetic information. Applying macsGESTALT to a mouse model of metastatic pancreatic cancer, we recover ∼380,000 CRISPR target sites and reconstruct dissemination of ∼28,000 single cells across multiple metastatic sites. We find that cells occupy a continuum of epithelial-to-mesenchymal transition (EMT) states. Metastatic potential peaks in rare, late-hybrid EMT states, which are aggressively selected from a predominately epithelial ancestral pool. The gene signatures of these late-hybrid EMT states are predictive of reduced survival in both human pancreatic and lung cancer patients, highlighting their relevance to clinical disease progression. Finally, we observe evidence for in vivo propagation of S100 family gene expression across clonally distinct metastatic subpopulations.

The gut microbiota regulates white adipose tissue inflammation and obesity via a family of microRNAs.

The gut microbiota is a key environmental determinant of mammalian metabolism. Regulation of white adipose tissue (WAT) by the gut microbiota is a process critical to maintaining metabolic fitness, and gut dysbiosis can contribute to the development of obesity and insulin resistance (IR). However, how the gut microbiota regulates WAT function remains largely unknown. Here, we show that tryptophan-derived metabolites produced by the gut microbiota controlled the expression of the miR-181 family in white adipocytes in mice to regulate energy expenditure and insulin sensitivity. Moreover, dysregulation of the gut microbiota-miR-181 axis was required for the development of obesity, IR, and WAT inflammation in mice. Our results indicate that regulation of miR-181 in WAT by gut microbiota-derived metabolites is a central mechanism by which host metabolism is tuned in response to dietary and environmental changes. As we also found that MIR-181 expression in WAT and the plasma abundance of tryptophan-derived metabolites were dysregulated in a cohort of obese human children, the MIR-181 family may represent a potential therapeutic target to modulate WAT function in the context of obesity.

The long noncoding RNA Morrbid regulates CD8 T cells in response to viral infection.

The transcriptional programs that regulate CD8 T-cell differentiation and function in the context of viral infections or tumor immune surveillance have been extensively studied; yet how long noncoding RNAs (lncRNAs) and the loci that transcribe them contribute to the regulation of CD8 T cells during viral infections remains largely unexplored. Here, we report that transcription of the lncRNA Morrbid is specifically induced by T-cell receptor (TCR) and type I IFN stimulation during the early stages of acute and chronic lymphocytic choriomeningitis virus (LCMV) infection. In response to type I IFN, the Morrbid RNA and its locus control CD8 T cell expansion, survival, and effector function by regulating the expression of the proapoptotic factor, Bcl2l11, and by modulating the strength of the PI3K-AKT signaling pathway. Thus, our results demonstrate that inflammatory cue-responsive lncRNA loci represent fundamental mechanisms by which CD8 T cells are regulated in response to pathogens and potentially cancer.

CD8+ T cell cytotoxicity mediates pathology in the skin by inflammasome activation and IL-1ß production.

Deregulated CD8+ T cell cytotoxicity plays a central role in enhancing disease severity in several conditions. However, we have little understanding of the mechanisms by which immunopathology develops as a consequence of cytotoxicity. Using murine models of inflammation induced by the protozoan parasite leishmania, and data obtained from patients with cutaneous leishmaniasis, we uncovered a previously unrecognized role for NLRP3 inflammasome activation and IL-1ß release as a detrimental consequence of CD8+ T cell-mediated cytotoxicity, ultimately resulting in chronic inflammation. Critically, pharmacological blockade of NLRP3 or IL-1ß significantly ameliorated the CD8+ T cell-driven immunopathology in leishmania-infected mice. Confirming the relevance of these findings to human leishmaniasis, blockade of the NLRP3 inflammasome in skin biopsies from leishmania-infected patients prevented IL-1ß release. Thus, these studies link CD8+ T cell cytotoxicity with inflammasome activation and reveal novel avenues of treatment for cutaneous leishmaniasis, as well as other of diseases where CD8+ T cell-mediated cytotoxicity induces pathology.

Continuity and Change in Cognition and Autism Severity from Toddlerhood to School Age.

This paper charted the cognitive and behavioural profiles from toddlerhood to middle childhood in 48 children diagnosed with ASD at 24-months. The Mullen Scales of Early Learning (MSEL) was administered at 24- and 48-months and the Wechsler Abbreviated Scale of Intelligence (WASI) at school age. Autism severity was derived using The Autism Diagnostic Observation Schedule (ADOS) Results: Developmental Disability/Intellectual Disability (DD/ID; Developmental Quotient <70) reduced from 64% at 24-months to 8% at outcome. Seventy-three percent of children continued to meet ADOS cut-off at school age. CONCLUSION: Diagnoses at 24-months, appear to be reliable and stable. Further research is needed to investigate whether early identification, which provides more opportunity to access early intervention, may in turn facilitate cognitive development over time.