Vγ9Vδ2 T cells recognize butyrophilin 2A1 and 3A1 heteromers.

Butyrophilin (BTN) molecules are emerging as key regulators of T cell immunity; however, how they trigger cell-mediated responses is poorly understood. Here, the crystal structure of a gamma-delta T cell antigen receptor (γδTCR) in complex with BTN2A1 revealed that BTN2A1 engages the side of the γδTCR, leaving the apical TCR surface bioavailable. We reveal that a second γδTCR ligand co-engages γδTCR via binding to this accessible apical surface in a BTN3A1-dependent manner. BTN2A1 and BTN3A1 also directly interact with each other in cis, and structural analysis revealed formation of W-shaped heteromeric multimers. This BTN2A1-BTN3A1 interaction involved the same epitopes that BTN2A1 and BTN3A1 each use to mediate the γδTCR interaction; indeed, locking BTN2A1 and BTN3A1 together abrogated their interaction with γδTCR, supporting a model wherein the two γδTCR ligand-binding sites depend on accessibility to cryptic BTN epitopes. Our findings reveal a new paradigm in immune activation, whereby γδTCRs sense dual epitopes on BTN complexes.

CD200+ fibroblasts form a pro-resolving mesenchymal network in arthritis.

Fibroblasts are important regulators of inflammation, but whether fibroblasts change phenotype during resolution of inflammation is not clear. Here we use positron emission tomography to detect fibroblast activation protein (FAP) as a means to visualize fibroblast activation in vivo during inflammation in humans. While tracer accumulation is high in active arthritis, it decreases after tumor necrosis factor and interleukin-17A inhibition. Biopsy-based single-cell RNA-sequencing analyses in experimental arthritis show that FAP signal reduction reflects a phenotypic switch from pro-inflammatory MMP3+/IL6+ fibroblasts (high FAP internalization) to pro-resolving CD200+DKK3+ fibroblasts (low FAP internalization). Spatial transcriptomics of human joints indicates that pro-resolving niches of CD200+DKK3+ fibroblasts cluster with type 2 innate lymphoid cells, whereas MMP3+/IL6+ fibroblasts colocalize with inflammatory immune cells. CD200+DKK3+ fibroblasts stabilized the type 2 innate lymphoid cell phenotype and induced resolution of arthritis via CD200-CD200R1 signaling. Taken together, these data suggest a dynamic molecular regulation of the mesenchymal compartment during resolution of inflammation.

CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification.

The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.

Characterizing Mutational Signatures in Human Cancer Cell Lines Reveals Episodic APOBEC Mutagenesis.

Multiple signatures of somatic mutations have been identified in cancer genomes. Exome sequences of 1,001 human cancer cell lines and 577 xenografts revealed most common mutational signatures, indicating past activity of the underlying processes, usually in appropriate cancer types. To investigate ongoing patterns of mutational-signature generation, cell lines were cultured for extended periods and subsequently DNA sequenced. Signatures of discontinued exposures, including tobacco smoke and ultraviolet light, were not generated in vitro. Signatures of normal and defective DNA repair and replication continued to be generated at roughly stable mutation rates. Signatures of APOBEC cytidine deaminase DNA-editing exhibited substantial fluctuations in mutation rate over time with episodic bursts of mutations. The initiating factors for the bursts are unclear, although retrotransposon mobilization may contribute. The examined cell lines constitute a resource of live experimental models of mutational processes, which potentially retain patterns of activity and regulation operative in primary human cancers.

Timing the Landmark Events in the Evolution of Clear Cell Renal Cell Cancer: TRACERx Renal.

Clear cell renal cell carcinoma (ccRCC) is characterized by near-universal loss of the short arm of chromosome 3, deleting several tumor suppressor genes. We analyzed whole genomes from 95 biopsies across 33 patients with clear cell renal cell carcinoma. We find hotspots of point mutations in the 5' UTR of TERT, targeting a MYC-MAX-MAD1 repressor associated with telomere lengthening. The most common structural abnormality generates simultaneous 3p loss and 5q gain (36% patients), typically through chromothripsis. This event occurs in childhood or adolescence, generally as the initiating event that precedes emergence of the tumor's most recent common ancestor by years to decades. Similar genomic changes drive inherited ccRCC. Modeling differences in age incidence between inherited and sporadic cancers suggests that the number of cells with 3p loss capable of initiating sporadic tumors is no more than a few hundred. Early development of ccRCC follows well-defined evolutionary trajectories, offering opportunity for early intervention.

Author Correction: γδ T cells producing interleukin-17A regulate adipose regulatory T cell homeostasis and thermogenesis.

In the version of this article initially published, three authors (Hui-Fern Kuoy, Adam P. Uldrich and Dale. I. Godfrey) and their affiliations, acknowledgments and contributions were not included. The correct information is as follows:Ayano C. Kohlgruber1,2, Shani T. Gal-Oz3, Nelson M. LaMarche1,2, Moto Shimazaki1, Danielle Duquette4, Hui-Fern Koay5,6, Hung N. Nguyen1, Amir I. Mina4, Tyler Paras1, Ali Tavakkoli7, Ulrich von Andrian2,8, Adam P. Uldrich5,6, Dale I. Godfrey5,6, Alexander S. Banks4, Tal Shay3, Michael B. Brenner1,10* and Lydia Lynch1,4,9,10*1Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, MA, USA. 2Division of Medical Sciences, Harvard Medical School, Boston, MA, USA. 3Department of Life Sciences, Ben-Gurion University of the Negev, Beersheba, Israel. 4Division of Endocrinology, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. 5Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Australia. 6ARC Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, Australia. 7Department of General and Gastrointestinal Surgery, Brigham and Women's Hospital, Boston, MA, USA. 8Department of Microbiology and Immunology, Harvard Medical School, Boston, MA, USA. 9School of Biochemistry and Immunology, Trinity College, Dublin, Ireland. 10These authors jointly supervised this work: Michael B. Brenner, Lydia Lynch. *e-mail: mbrenner@research.bwh.harvard.edu; llynch@bwh.harvard.eduAcknowledgementsWe thank A.T. Chicoine, flow cytometry core manager at the Human Immunology Center at BWH, for flow cytometry sorting. We thank D. Sant'Angelo (Rutgers Cancer Institute) for providing Zbtb16-/- mice and R. O'Brien (National Jewish Health) for providing Vg4/6-/- mice. Supported by NIH grant R01 AI11304603 (to M.B.B.), ERC Starting Grant 679173 (to L.L.), the National Health and Medical Research Council of Australia (1013667), an Australian Research Council Future Fellowship (FT140100278 for A.P.U.) and a National Health and Medical Research Council of Australia Senior Principal Research Fellowship (1117766 for D.I.G.).Author contributionsA.C.K., L.L., and M.B.B. conceived and designed the experiments, and wrote the manuscript. A.C.K., N.M.L., L.L., H.N.N., M.S., T.P., and D.D. performed the experiments. S.T.G.-O. and T.S. performed the RNA-seq analysis. A.S.B. and A.I.M. provided advice and performed the CLAMS experiments. A.T. provided human bariatric patient samples. Parabiosis experiments were performed in the laboratory of U.v.A. H.-F.K., A.P.U. and D.I.G provided critical insight into the TCR chain usage of PLZF+ γδ T cells. M.B.B., N.M.L., and L.L. critically reviewed the manuscript.The errors have been corrected in the HTML and PDF version of the article.Correction to: Nature Immunology doi:10.1038/s41590-018-0094-2 (2018), published online 18 April 2018.

An essential role for the Zn2+ transporter ZIP7 in B cell development.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.

Molecular responses to acute exercise and their relevance for adaptations in skeletal muscle to exercise training.

Repeated, episodic bouts of skeletal muscle contraction undertaken frequently as structured exercise training are a potent stimulus for physiological adaptation in many organs. Specifically, in skeletal muscle, remarkable plasticity is demonstrated by the remodeling of muscle structure and function in terms of muscular size, force, endurance, and contractile velocity as a result of the functional demands induced by various types of exercise training. This plasticity, and the mechanistic basis for adaptations to skeletal muscle in response to exercise training, are underpinned by activation and/or repression of molecular pathways and processes in response to each individual acute exercise session. These pathways include the transduction of signals arising from neuronal, mechanical, metabolic, and hormonal stimuli through complex signal transduction networks, which are linked to a myriad of effector proteins involved in the regulation of pre- and posttranscriptional processes, and protein translation and degradation processes. This review therefore describes acute exercise-induced signal transduction and the molecular responses to acute exercise in skeletal muscle including emerging concepts such as epigenetic pre- and posttranscriptional regulation and the regulation of protein translation and degradation. A critical appraisal of methodological approaches and the current state of knowledge informs a series of recommendations offered as future directions in the field.

γδ T cells producing interleukin-17A regulate adipose regulatory T cell homeostasis and thermogenesis.

γδ T cells are situated at barrier sites and guard the body from infection and damage. However, little is known about their roles outside of host defense in nonbarrier tissues. Here, we characterize a highly enriched tissue-resident population of γδ T cells in adipose tissue that regulate age-dependent regulatory T cell (Treg) expansion and control core body temperature in response to environmental fluctuations. Mechanistically, innate PLZF+ γδ T cells produced tumor necrosis factor and interleukin (IL) 17 A and determined PDGFRα+ and Pdpn+ stromal-cell production of IL-33 in adipose tissue. Mice lacking γδ T cells or IL-17A exhibited decreases in both ST2+ Treg cells and IL-33 abundance in visceral adipose tissue. Remarkably, these mice also lacked the ability to regulate core body temperature at thermoneutrality and after cold challenge. Together, these findings uncover important physiological roles for resident γδ T cells in adipose tissue immune homeostasis and body-temperature control.

T cell autoreactivity directed toward CD1c itself rather than toward carried self lipids.

The hallmark function of αß T cell antigen receptors (TCRs) involves the highly specific co-recognition of a major histocompatibility complex molecule and its carried peptide. However, the molecular basis of the interactions of TCRs with the lipid antigen-presenting molecule CD1c is unknown. We identified frequent staining of human T cells with CD1c tetramers across numerous subjects. Whereas TCRs typically show high specificity for antigen, both tetramer binding and autoreactivity occurred with CD1c in complex with numerous, chemically diverse self lipids. Such extreme polyspecificity was attributable to binding of the TCR over the closed surface of CD1c, with the TCR covering the portal where lipids normally protrude. The TCR essentially failed to contact lipids because they were fully seated within CD1c. These data demonstrate the sequestration of lipids within CD1c as a mechanism of autoreactivity and point to small lipid size as a determinant of autoreactive T cell responses.

The complement system drives local inflammatory tissue priming by metabolic reprogramming of synovial fibroblasts.

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.

Interspecies systems biology uncovers metabolites affecting C. elegans gene expression and life history traits.

Diet greatly influences gene expression and physiology. In mammals, elucidating the effects and mechanisms of individual nutrients is challenging due to the complexity of both the animal and its diet. Here, we used an interspecies systems biology approach with Caenorhabditis elegans and two of its bacterial diets, Escherichia coli and Comamonas aquatica, to identify metabolites that affect the animal's gene expression and physiology. We identify vitamin B12 as the major dilutable metabolite provided by Comamonas aq. that regulates gene expression, accelerates development, and reduces fertility but does not affect lifespan. We find that vitamin B12 has a dual role in the animal: it affects development and fertility via the methionine/S-Adenosylmethionine (SAM) cycle and breaks down the short-chain fatty acid propionic acid, preventing its toxic buildup. Our interspecies systems biology approach provides a paradigm for understanding complex interactions between diet and physiology.

A three-stage intrathymic development pathway for the mucosal-associated invariant T cell lineage.

Mucosal-associated invariant T cells (MAIT cells) detect microbial vitamin B2 derivatives presented by the antigen-presenting molecule MR1. Here we defined three developmental stages and checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and stage 2 MAIT cells predominated in thymus, while stage 3 cells progressively increased in abundance extrathymically. Transition through each checkpoint was regulated by MR1, whereas the final checkpoint that generated mature functional MAIT cells was controlled by multiple factors, including the transcription factor PLZF and microbial colonization. Furthermore, stage 3 MAIT cell populations were expanded in mice deficient in the antigen-presenting molecule CD1d, suggestive of a niche shared by MAIT cells and natural killer T cells (NKT cells). Accordingly, this study maps the developmental pathway and checkpoints that control the generation of functional MAIT cells.

Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.

Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.

A multi-epigenomic map of endurance exercise training.

The Molecular Transducers of Physical Activity Consortium (MoTrPAC) aims to comprehensively map molecular alterations in response to acute exercise and chronic training. In one of a recent series of papers from MoTrPAC, Nair et al. provide the first multi-epigenomic and transcriptomic integration across eight tissues in both sexes following adaptation to endurance exercise training (EET).

Correcting PCR amplification errors in unique molecular identifiers to generate accurate numbers of sequencing molecules.

Unique molecular identifiers are random oligonucleotide sequences that remove PCR amplification biases. However, the impact that PCR associated sequencing errors have on the accuracy of generating absolute counts of RNA molecules is underappreciated. We show that PCR errors are a source of inaccuracy in both bulk and single-cell sequencing data, and synthesizing unique molecular identifiers using homotrimeric nucleotide blocks provides an error-correcting solution that allows absolute counting of sequenced molecules.

The burgeoning family of unconventional T cells.

While most studies of T lymphocytes have focused on T cells reactive to complexes of peptide and major histocompatibility complex (MHC) proteins, many other types of T cells do not fit this paradigm. These include CD1-restricted T cells, MR1-restricted mucosal associated invariant T cells (MAIT cells), MHC class Ib-reactive T cells, and γδ T cells. Collectively, these T cells are considered 'unconventional', in part because they can recognize lipids, small-molecule metabolites and specially modified peptides. Unlike MHC-reactive T cells, these apparently disparate T cell types generally show simplified patterns of T cell antigen receptor (TCR) expression, rapid effector responses and 'public' antigen specificities. Here we review evidence showing that unconventional T cells are an abundant component of the human immune system and discuss the immunotherapeutic potential of these cells and their antigenic targets.

αß T cell antigen receptor recognition of CD1a presenting self lipid ligands.

A central paradigm in αß T cell-mediated immunity is the simultaneous co-recognition of antigens and antigen-presenting molecules by the αß T cell antigen receptor (TCR). CD1a presents a broad repertoire of lipid-based antigens. We found that a prototypical autoreactive TCR bound CD1a when it was presenting a series of permissive endogenous ligands, while other lipid ligands were nonpermissive to TCR binding. The structures of two TCR-CD1a-lipid complexes showed that the TCR docked over the A' roof of CD1a in a manner that precluded direct contact with permissive ligands. Nonpermissive ligands indirectly inhibited TCR binding by disrupting the TCR-CD1a contact zone. The exclusive recognition of CD1a by the TCR represents a previously unknown mechanism whereby αß T cells indirectly sense self antigens that are bound to an antigen-presenting molecule.

T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex.

Central to adaptive immunity is the interaction between the αß T cell receptor (TCR) and peptide presented by the major histocompatibility complex (MHC) molecule. Presumably reflecting TCR-MHC bias and T cell signaling constraints, the TCR universally adopts a canonical polarity atop the MHC. We report the structures of two TCRs, derived from human induced T regulatory (iT(reg)) cells, complexed to an MHC class II molecule presenting a proinsulin-derived peptide. The ternary complexes revealed a 180° polarity reversal compared to all other TCR-peptide-MHC complex structures. Namely, the iT(reg) TCR α-chain and ß-chain are overlaid with the α-chain and ß-chain of MHC class II, respectively. Nevertheless, this TCR interaction elicited a peptide-reactive, MHC-restricted T cell signal. Thus TCRs are not 'hardwired' to interact with MHC molecules in a stereotypic manner to elicit a T cell signal, a finding that fundamentally challenges our understanding of TCR recognition.