Multimodal single-cell assays provide high-resolution snapshots of complex cell populations, but are mostly limited to transcriptome plus an additional modality. Here, we describe expanded ...CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell. We demonstrate application of ECCITE-seq to multimodal CRISPR screens with robust direct single-guide RNA capture and to clonotype-aware multimodal phenotyping of cancer samples.
Antibodies' protective, pathological, and therapeutic properties result from their considerable diversity. This diversity is almost limitless in potential, but actual diversity is still poorly ...understood. Here we use deep sequencing to characterize the diversity of the heavy-chain CDR3 region, the most important contributor to antibody binding specificity, and the constituent V, D, and J segments that comprise it. We find that, during the stepwise D-J and then V-DJ recombination events, the choice of D and J segments exert some bias on each other; however, we find the choice of the V segment is essentially independent of both. V, D, and J segments are utilized with different frequencies, resulting in a highly skewed representation of VDJ combinations in the repertoire. Nevertheless, the pattern of segment usage was almost identical between two different individuals. The pattern of V, D, and J segment usage and recombination was insufficient to explain overlap that was observed between the two individuals' CDR3 repertoires. Finally, we find that while there are a near-infinite number of heavy-chain CDR3s in principle, there are about 3-9 million in the blood of an adult human being.
Pathogenic Th17 cells play important roles in many autoimmune and inflammatory diseases. Their function depends on T cell receptor (TCR) signaling and cytokines that activate signal transducer and ...activator of transcription 3 (STAT3). TCR engagement activates stromal interaction molecule 1 (STIM1) and calcium (Ca2+) influx through Ca2+-release-activated Ca2+ (CRAC) channels. Here, we show that abolishing STIM1 and Ca2+ influx in T cells expressing a hyperactive form of STAT3 (STAT3C) attenuates pathogenic Th17 cell function and inflammation associated with STAT3C expression. Deletion of STIM1 in pathogenic Th17 cells reduces the expression of genes required for mitochondrial function and oxidative phosphorylation (OXPHOS) but enhances reactive oxygen species (ROS) production. STIM1 deletion or inhibition of OXPHOS is associated with a non-pathogenic Th17 gene expression signature and impaired pathogenic Th17 cell function. Our findings establish Ca2+ influx as a critical regulator of mitochondrial function and oxidative stress in pathogenic Th17 cell-mediated multiorgan inflammation.
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•STIM1-mediated calcium flux controls ETC expression in pathogenic Th17 cells•STIM1 deficiency impairs OXPHOS and increases mROS in Th17 cells•Loss of STIM1 and OXPHOS inhibition results in a non-pathogenic Th17 gene signature•Lack of STIM1 protects from Th17 cell-mediated lung, intestinal, and skin inflammation
Kaufmann et al. show an essential role of the store-operated calcium entry (SOCE) pathway in pathogenic Th17 cell function by regulating mitochondrial function and OXPHOS, and preventing oxidative stress. SOCE inhibition could be a potential therapeutic avenue in Th17 inflammatory diseases such as MS, colitis, psoriasis, or steroid-resistant asthma.
Rewiring of metabolic pathways is a hallmark of tumorigenesis as cancer cells acquire novel nutrient dependencies to support oncogenic growth. A major genetic subtype of lung adenocarcinoma with ...KEAP1/NRF2 mutations, which activates the endogenous oxidative stress response, undergoes significant metabolic rewiring to support enhanced antioxidant production. We demonstrate that cancers with high antioxidant capacity exhibit a general dependency on exogenous non-essential amino acids (NEAAs) that is driven by the Nrf2-dependent secretion of glutamate through system xc− (XCT), which limits intracellular glutamate pools that are required for NEAA synthesis. This dependency can be therapeutically targeted by dietary restriction or enzymatic depletion of individual NEAAs. Importantly, limiting endogenous glutamate levels by glutaminase inhibition can sensitize tumors without alterations in the Keap1/Nrf2 pathway to dietary restriction of NEAAs. Our findings identify a metabolic strategy to therapeutically target cancers with genetic or pharmacologic activation of the Nrf2 antioxidant response pathway by restricting exogenous sources of NEAAs.
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•Keap1 mutations drive non-essential amino acid (NEAA) dependency in cancer•Intracellular glutamate levels dictate cellular ability to survive NEAA deprivation•Restriction of NEAA can suppress Keap1 mutant tumor growth in vivo•Limiting glutamate by glutaminase inhibition enhances response to NEAA deprivation
LeBoeuf et al. describe a general mechanism through which cancer cells depend on non-essential amino acids. Tumor cells with intrinsically low intracellular glutamate require amino acids to be supplied from the extracellular environment. By reducing the amount of circulating amino acids, the authors reduce tumor growth in vivo.
Summary
DNA methylation has pivotal regulatory roles in mammalian development, retrotransposon silencing, genomic imprinting, and X‐chromosome inactivation. Cancer cells display highly dysregulated ...DNA methylation profiles characterized by global hypomethylation in conjunction with hypermethylation of promoter CpG islands that presumably lead to genome instability and aberrant expression of tumor suppressor genes or oncogenes. The recent discovery of ten‐eleven‐translocation (TET) family dioxygenases that oxidize 5mC to 5‐hydroxymethylcytosine (5hmC), 5‐formylcytosine (5fC), and 5‐carboxylcytosine (5caC) in DNA has led to profound progress in understanding the mechanism underlying DNA demethylation. Among the three TET genes, TET2 recurrently undergoes inactivating mutations in a wide range of myeloid and lymphoid malignancies. TET2 functions as a bona fide tumor suppressor particularly in the pathogenesis of myeloid malignancies resembling chronic myelomonocytic leukemia (CMML) and myelodysplastic syndromes (MDS) in human. Here we review diverse functions of TET proteins and the novel epigenetic marks that they generate in DNA methylation/demethylation dynamics and normal and malignant hematopoietic differentiation. The impact of TET2 inactivation in hematopoiesis and various mechanisms modulating the expression or activity of TET proteins are also discussed. Furthermore, we also present evidence that TET2 and TET3 collaborate to suppress aberrant hematopoiesis and hematopoietic transformation. A detailed understanding of the normal and pathological functions of TET proteins may provide new avenues to develop novel epigenetic therapies for treating hematological malignancies.
A salient feature of pancreatic ductal adenocarcinoma (PDAC) is an abundant fibroinflammatory response characterized by the recruitment of immune and mesenchymal cells and the consequent ...establishment of a protumorigenic microenvironment. Here, we report the prominent presence of B cells in human pancreatic intraepithelial neoplasia and PDAC lesions as well as in oncogenic Kras-driven pancreatic neoplasms in the mouse. The growth of orthotopic pancreatic neoplasms harboring oncogenic Kras was significantly compromised in B-cell-deficient mice (μMT), and this growth deficiency could be rescued by the reconstitution of a CD1d(hi)CD5(+) B-cell subset. The protumorigenic effect of B cells was mediated by their expression of IL35 through a mechanism involving IL35-mediated stimulation of tumor cell proliferation. Our results identify a previously unrecognized role for IL35-producing CD1d(hi)CD5(+) B cells in the pathogenesis of pancreatic cancer and underscore the potential significance of a B-cell/IL35 axis as a therapeutic target.
This study identifies a B-cell subpopulation that accumulates in the pancreatic parenchyma during early neoplasia and is required to support tumor cell growth. Our findings provide a rationale for exploring B-cell-based targeting approaches for the treatment of pancreatic cancer.
Understanding metabolic pathways that regulate Th17 development is important to broaden therapeutic options for Th17-mediated autoimmunity. Here, we report a pivotal role of mitochondrial oxidative ...phosphorylation (OXPHOS) for lineage specification toward pathogenic Th17 differentiation. Th17 cells rapidly increase mitochondrial respiration during development, and this is necessary for metabolic reprogramming following T cell activation. Surprisingly, specific inhibition of mitochondrial ATP synthase ablates Th17 pathogenicity in a mouse model of autoimmunity by preventing Th17 pathogenic signature gene expression. Notably, cells activated under OXPHOS-inhibited Th17 conditions preferentially express Foxp3, rather than Th17 genes, and become suppressive Treg cells. Mechanistically, OXPHOS promotes the Th17 pioneer transcription factor, BATF, and facilitates T cell receptor (TCR) and mTOR signaling. Correspondingly, overexpression of BATF rescues Th17 development when ATP synthase activity is restricted. Together, our data reveal a regulatory role of mitochondrial OXPHOS in dictating the fate decision between Th17 and Treg cells by supporting early molecular events necessary for Th17 commitment.
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•CD4 T cells rapidly increase mitochondrial respiration during Th17 differentiation•OXPHOS is essential for Th17 cell pathogenicity in a mouse model of MS•Mitochondrial respiration shapes the Th17 and Treg cell fate decision•OXPHOS facilitates TCR and mTOR signaling, which in turn support BATF induction
Shin et al. report that ATP-linked mitochondrial respiration controls the Th17 and Treg cell fate decision by supporting TCR signaling and Th17-associated molecular events. Inhibition of mitochondrial OXPHOS ablates Th17 pathogenicity in a mouse model of MS and results in generation of functionally suppressive Treg cells under Th17 conditions.
The Ten-Eleven-Translocation 2 (TET2) gene encodes a member of TET family enzymes that alters the epigenetic status of DNA by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Somatic ...loss-of-function mutations of TET2 are frequently observed in patients with diverse myeloid malignancies, including myelodysplastic syndromes, myeloproliferative neoplasms, and chronic myelomonocytic leukemia. By analyzing mice with targeted disruption of the Tet2 catalytic domain, we show here that Tet2 is a critical regulator of self-renewal and differentiation of hematopoietic stem cells (HSCs). Tet2 deficiency led to decreased genomic levels of 5hmC and augmented the size of the hematopoietic stem/progenitor cell pool in a cell-autonomous manner. In competitive transplantation assays, Tet2-deficient HSCs were capable of multilineage reconstitution and possessed a competitive advantage over wild-type HSCs, resulting in enhanced hematopoiesis into both lymphoid and myeloid lineages. In vitro, Tet2 deficiency delayed HSC differentiation and skewed development toward the monocyte/macrophage lineage. Our data indicate that Tet2 has a critical role in regulating the expansion and function of HSCs, presumably by controlling 5hmC levels at genes important for the self-renewal, proliferation, and differentiation of HSCs.
B lymphocytes are essential for an efficient immune response against a variety of pathogens. A large fraction of hematologic malignancies is of B cell origin suggesting that development and ...activation of B cells need to be tightly regulated. In recent years, increasing evidence has emerged demonstrating that microRNAs (miRNAs) − a class of non-coding RNAs that control gene expression − are involved in the regulation of B cell development and function. Here, we provide an overview of the current knowledge on the role of miRNAs and their relevant targets in B cell development, B cell activation and B cell malignant transformation.
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