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Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract.sup.1-4. The protective effects of IL-2 involve the generation, maintenance and function of regulatory T (T.sub.reg) cells.sup.4-8, and the use of low doses of IL-2 has emerged as a potential therapeutic strategy for patients with inflammatory bowel disease.sup.9. However, the cellular and molecular pathways that control the production of IL-2 in the context of intestinal health are undefined. Here we show, in a mouse model, that IL-2 is acutely required to maintain T.sub.reg cells and immunological homeostasis throughout the gastrointestinal tract. Notably, lineage-specific deletion of IL-2 in T cells did not reduce T.sub.reg cells in the small intestine. Unbiased analyses revealed that, in the small intestine, group-3 innate lymphoid cells (ILC3s) are the dominant cellular source of IL-2, which is induced selectively by IL-1[beta]. Macrophages in the small intestine produce IL-1[beta], and activation of this pathway involves MYD88- and NOD2-dependent sensing of the microbiota. Our loss-of-function studies show that ILC3-derived IL-2 is essential for maintaining T.sub.reg cells, immunological homeostasis and oral tolerance to dietary antigens in the small intestine. Furthermore, production of IL-2 by ILC3s was significantly reduced in the small intestine of patients with Crohn's disease, and this correlated with lower frequencies of T.sub.reg cells. Our results reveal a previously unappreciated pathway in which a microbiota- and IL-1[beta]-dependent axis promotes the production of IL-2 by ILC3s to orchestrate immune regulation in the intestine. A microbiota- and IL-1[beta]-dependent axis of IL-2 production by group-3 innate lymphoid cells is shown in a mouse model to be necessary to maintain immunological homeostasis and regulatory T cells in the small intestine., Author(s): Lei Zhou [sup.1] [sup.2] [sup.3] , Coco Chu [sup.1] [sup.2] [sup.3] , Fei Teng [sup.1] [sup.2] [sup.3] , Nicholas J. Bessman [sup.1] [sup.2] [sup.3] , Jeremy Goc [sup.1] [sup.2] [...]

There is growing evidence that tumour neoantigens have important roles in generating spontaneous antitumour immune responses and predicting clinical responses to immunotherapies.sup.1,2. Despite the presence of numerous neoantigens in patients, complete tumour elimination is rare, owing to failures in mounting a sufficient and lasting antitumour immune response.sup.3,4. Here we show that durable neoantigen-specific immunity is regulated by mRNA N.sup.6-methyadenosine (m.sup.6A) methylation through the m.sup.6A-binding protein YTHDF1.sup.5. In contrast to wild-type mice, Ythdf1-deficient mice show an elevated antigen-specific CD8.sup.+ T cell antitumour response. Loss of YTHDF1 in classical dendritic cells enhanced the cross-presentation of tumour antigens and the cross-priming of CD8.sup.+ T cells in vivo. Mechanistically, transcripts encoding lysosomal proteases are marked by m.sup.6A and recognized by YTHDF1. Binding of YTHDF1 to these transcripts increases the translation of lysosomal cathepsins in dendritic cells, and inhibition of cathepsins markedly enhances cross-presentation of wild-type dendritic cells. Furthermore, the therapeutic efficacy of PD-L1 checkpoint blockade is enhanced in Ythdf1.sup.-/- mice, implicating YTHDF1 as a potential therapeutic target in anticancer immunotherapy.The m.sup.6A reader protein YTHDF1 suppresses the clearance of tumour cells by enhancing the translation of lysosomal proteases in dendritic cells and thereby suppressing tumour antigen presentation., Author(s): Dali Han [sup.1] [sup.2] [sup.3] , Jun Liu [sup.4] [sup.5] [sup.6] , Chuanyuan Chen [sup.1] [sup.2] [sup.3] , Lihui Dong [sup.7] , Yi Liu [sup.7] , Renbao Chang [sup.1] [...]

In principle, humans can produce an antibody response to any non-self-antigen molecule in the appropriate context. This flexibility is achieved by the presence of a large repertoire of naive antibodies, the diversity of which is expanded by somatic hypermutation following antigen exposure.sup.1. The diversity of the naive antibody repertoire in humans is estimated to be at least 10.sup.12 unique antibodies.sup.2. Because the number of peripheral blood B cells in a healthy adult human is on the order of 5 × 10.sup.9, the circulating B cell population samples only a small fraction of this diversity. Full-scale analyses of human antibody repertoires have been prohibitively difficult, primarily owing to their massive size. The amount of information encoded by all of the rearranged antibody and T cell receptor genes in one person--the 'genome' of the adaptive immune system--exceeds the size of the human genome by more than four orders of magnitude. Furthermore, because much of the B lymphocyte population is localized in organs or tissues that cannot be comprehensively sampled from living subjects, human repertoire studies have focused on circulating B cells.sup.3. Here we examine the circulating B cell populations of ten human subjects and present what is, to our knowledge, the largest single collection of adaptive immune receptor sequences described to date, comprising almost 3 billion antibody heavy-chain sequences. This dataset enables genetic study of the baseline human antibody repertoire at an unprecedented depth and granularity, which reveals largely unique repertoires for each individual studied, a subpopulation of universally shared antibody clonotypes, and an exceptional overall diversity of the antibody repertoire. A genetic study of the baseline human antibody repertoire, based on the circulating B cell populations of ten subjects, reveals universally shared antibody clonotypes within repertoires that are largely unique to the individual., Author(s): Bryan Briney [sup.1] [sup.2] [sup.3] [sup.4] [sup.5] , Anne Inderbitzin [sup.1] [sup.6] , Collin Joyce [sup.1] [sup.2] [sup.3] [sup.4] , Dennis R. Burton [sup.1] [sup.2] [sup.4] [sup.5] [sup.7] Author [...]

Most patients with cancer either do not respond to immune checkpoint blockade or develop resistance to it, often because of acquired mutations that impair antigen presentation. Here we show that loss of function of the RNA-editing enzyme ADAR1 in tumour cells profoundly sensitizes tumours to immunotherapy and overcomes resistance to checkpoint blockade. In the absence of ADAR1, A-to-I editing of interferon-inducible RNA species is reduced, leading to double-stranded RNA ligand sensing by PKR and MDA5; this results in growth inhibition and tumour inflammation, respectively. Loss of ADAR1 overcomes resistance to PD-1 checkpoint blockade caused by inactivation of antigen presentation by tumour cells. Thus, effective anti-tumour immunity is constrained by inhibitory checkpoints such as ADAR1 that limit the sensing of innate ligands. The induction of sufficient inflammation in tumours that are sensitized to interferon can bypass the therapeutic requirement for CD8.sup.+ T cell recognition of cancer cells and may provide a general strategy to overcome immunotherapy resistance.Deletion of the A-to-I double-stranded RNA-editing enzyme ADAR1 sensitizes tumour cells to immunotherapy., Author(s): Jeffrey J. Ishizuka [sup.1] [sup.2] [sup.3] , Robert T. Manguso [sup.1] [sup.3] , Collins K. Cheruiyot [sup.1] [sup.3] , Kevin Bi [sup.1] [sup.3] , Arpit Panda [sup.1] [sup.3] [sup.4] [...]

The placenta is the extraembryonic organ that supports the fetus during intrauterine life. Although placental dysfunction results in major disorders of pregnancy with immediate and lifelong consequences for the mother and child, our knowledge of the human placenta is limited owing to a lack of functional experimental models.sup.1. After implantation, the trophectoderm of the blastocyst rapidly proliferates and generates the trophoblast, the unique cell type of the placenta. In vivo, proliferative villous cytotrophoblast cells differentiate into two main sub-populations: syncytiotrophoblast, the multinucleated epithelium of the villi responsible for nutrient exchange and hormone production, and extravillous trophoblast cells, which anchor the placenta to the maternal decidua and transform the maternal spiral arteries.sup.2. Here we describe the generation of long-term, genetically stable organoid cultures of trophoblast that can differentiate into both syncytiotrophoblast and extravillous trophoblast. We used human leukocyte antigen (HLA) typing to confirm that the organoids were derived from the fetus, and verified their identities against four trophoblast-specific criteria.sup.3. The cultures organize into villous-like structures, and we detected the secretion of placental-specific peptides and hormones, including human chorionic gonadotropin (hCG), growth differentiation factor 15 (GDF15) and pregnancy-specific glycoprotein (PSG) by mass spectrometry. The organoids also differentiate into HLA-G.sup.+ extravillous trophoblast cells, which vigorously invade in three-dimensional cultures. Analysis of the methylome reveals that the organoids closely resemble normal first trimester placentas. This organoid model will be transformative for studying human placental development and for investigating trophoblast interactions with the local and systemic maternal environment.An in vitro system that generates three-dimensional cultures of extraembryonic fetal trophoblast cells that differentiate into the two main types of trophoblast can be used to study human placental development., Author(s): Margherita Y. Turco [sup.1] [sup.2] [sup.3] , Lucy Gardner [sup.1] [sup.3] , Richard G. Kay [sup.4] , Russell S. Hamilton [sup.2] [sup.3] , Malwina Prater [sup.2] [sup.3] , Michael [...]

Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of difficult-to-treat, often fatal infections in humans.sup.1,2. Most humans have antibodies against S. aureus, but these are highly variable and often not protective in immunocompromised patients.sup.3. Previous vaccine development programs have not been successful.sup.4. A large percentage of human antibodies against S. aureus target wall teichoic acid (WTA), a ribitol-phosphate (RboP) surface polymer modified with N-acetylglucosamine (GlcNAc).sup.5,6. It is currently unknown whether the immune evasion capacities of MRSA are due to variation of dominant surface epitopes such as those associated with WTA. Here we show that a considerable proportion of the prominent healthcare-associated and livestock-associated MRSA clones CC5 and CC398, respectively, contain prophages that encode an alternative WTA glycosyltransferase. This enzyme, TarP, transfers GlcNAc to a different hydroxyl group of the WTA RboP than the standard enzyme TarS.sup.7, with important consequences for immune recognition. TarP-glycosylated WTA elicits 7.5-40-fold lower levels of immunoglobulin G in mice than TarS-modified WTA. Consistent with this, human sera contained only low levels of antibodies against TarP-modified WTA. Notably, mice immunized with TarS-modified WTA were not protected against infection with tarP-expressing MRSA, indicating that TarP is crucial for the capacity of S. aureus to evade host defences. High-resolution structural analyses of TarP bound to WTA components and uridine diphosphate GlcNAc (UDP-GlcNAc) explain the mechanism of altered RboP glycosylation and form a template for targeted inhibition of TarP. Our study reveals an immune evasion strategy of S. aureus based on averting the immunogenicity of its dominant glycoantigen WTA. These results will help with the identification of invariant S. aureus vaccine antigens and may enable the development of TarP inhibitors as a new strategy for rendering MRSA susceptible to human host defences.Strains of methicillin-resistant Staphylococcus aureus use a prophage-encoded glycosyltransferase to alter the glycosylation of their wall teichoic acid and thereby evade antibody-mediated immune responses., Author(s): David Gerlach [sup.1] [sup.2] , Yinglan Guo [sup.3] , Cristina De Castro [sup.4] , Sun-Hwa Kim [sup.5] , Katja Schlatterer [sup.1] [sup.2] , Fei-Fei Xu [sup.6] , Claney Pereira [...]

Mixed phenotype acute leukaemia (MPAL) is a high-risk subtype of leukaemia with myeloid and lymphoid features, limited genetic characterization, and a lack of consensus regarding appropriate therapy. Here we show that the two principal subtypes of MPAL, T/myeloid (T/M) and B/myeloid (B/M), are genetically distinct. Rearrangement of ZNF384 is common in B/M MPAL, and biallelic WT1 alterations are common in T/M MPAL, which shares genomic features with early T-cell precursor acute lymphoblastic leukaemia. We show that the intratumoral immunophenotypic heterogeneity characteristic of MPAL is independent of somatic genetic variation, that founding lesions arise in primitive haematopoietic progenitors, and that individual phenotypic subpopulations can reconstitute the immunophenotypic diversity in vivo. These findings indicate that the cell of origin and founding lesions, rather than an accumulation of distinct genomic alterations, prime tumour cells for lineage promiscuity. Moreover, these findings position MPAL in the spectrum of immature leukaemias and provide a genetically informed framework for future clinical trials of potential treatments for MPAL.A large-scale genomics study shows that the cell of origin and founding mutations determine disease subtype and lead to the expression of multiple haematopoietic lineage-defining antigens in mixed phenotype acute leukaemia., Author(s): Thomas B. Alexander [sup.1] [sup.2] , Zhaohui Gu [sup.3] , Ilaria Iacobucci [sup.3] , Kirsten Dickerson [sup.3] , John K. Choi [sup.3] , Beisi Xu [sup.4] , Debbie Payne-Turner [...]

The UK Biobank project is a prospective cohort study with deep genetic and phenotypic data collected on approximately 500,000 individuals from across the United Kingdom, aged between 40 and 69 at recruitment. The open resource is unique in its size and scope. A rich variety of phenotypic and health-related information is available on each participant, including biological measurements, lifestyle indicators, biomarkers in blood and urine, and imaging of the body and brain. Follow-up information is provided by linking health and medical records. Genome-wide genotype data have been collected on all participants, providing many opportunities for the discovery of new genetic associations and the genetic bases of complex traits. Here we describe the centralized analysis of the genetic data, including genotype quality, properties of population structure and relatedness of the genetic data, and efficient phasing and genotype imputation that increases the number of testable variants to around 96 million. Classical allelic variation at 11 human leukocyte antigen genes was imputed, resulting in the recovery of signals with known associations between human leukocyte antigen alleles and many diseases.Deep phenotype and genome-wide genetic data from 500,000 individuals from the UK Biobank, describing population structure and relatedness in the cohort, and imputation to increase the number of testable variants to 96 million., Author(s): Clare Bycroft [sup.1] , Colin Freeman [sup.1] , Desislava Petkova [sup.1] [sup.12] , Gavin Band [sup.1] , Lloyd T. Elliott [sup.2] , Kevin Sharp [sup.2] , Allan Motyer [sup.3] [...]

Narcolepsy is a chronic sleep disorder caused by the loss of neurons that produce hypocretin. The close association with HLA-DQB1*06:02, evidence for immune dysregulation and increased incidence upon influenza vaccination together suggest that this disorder has an autoimmune origin. However, there is little evidence of autoreactive lymphocytes in patients with narcolepsy. Here we used sensitive cellular screens and detected hypocretin-specific CD4.sup.+ T cells in all 19 patients that we tested; T cells specific for tribbles homologue 2--another self-antigen of hypocretin neurons--were found in 8 out of 13 patients. Autoreactive CD4.sup.+ T cells were polyclonal, targeted multiple epitopes, were restricted primarily by HLA-DR and did not cross-react with influenza antigens. Hypocretin-specific CD8.sup.+ T cells were also detected in the blood and cerebrospinal fluid of several patients with narcolepsy. Autoreactive clonotypes were serially detected in the blood of the same--and even of different--patients, but not in healthy control individuals. These findings solidify the autoimmune aetiology of narcolepsy and provide a basis for rapid diagnosis and treatment of this disease.The detection of hypocretin-specific autoreactive CD4.sup.+ and CD8.sup.+ T cells in patients with narcolepsy reveals the autoimmune aetiology of this disorder., Author(s): Daniela Latorre [sup.1] [sup.2] , Ulf Kallweit [sup.3] [sup.4] , Eric Armentani [sup.1] , Mathilde Foglierini [sup.1] [sup.5] , Federico Mele [sup.1] , Antonino Cassotta [sup.1] [sup.2] , Sandra [...]

Plasmodium vivax is the most widely distributed malaria parasite that infects humans.sup.1. P. vivax invades reticulocytes exclusively, and successful entry depends on specific interactions between the P. vivax reticulocyte-binding protein 2b (PvRBP2b) and transferrin receptor 1 (TfR1).sup.2. TfR1-deficient erythroid cells are refractory to invasion by P. vivax, and anti-PvRBP2b monoclonal antibodies inhibit reticulocyte binding and block P. vivax invasion in field isolates.sup.2. Here we report a high-resolution cryo-electron microscopy structure of a ternary complex of PvRBP2b bound to human TfR1 and transferrin, at 3.7 Å resolution. Mutational analyses show that PvRBP2b residues involved in complex formation are conserved; this suggests that antigens could be designed that act across P. vivax strains. Functional analyses of TfR1 highlight how P. vivax hijacks TfR1, an essential housekeeping protein, by binding to sites that govern host specificity, without affecting its cellular function of transporting iron. Crystal and solution structures of PvRBP2b in complex with antibody fragments characterize the inhibitory epitopes. Our results establish a structural framework for understanding how P. vivax reticulocyte-binding protein engages its receptor and the molecular mechanism of inhibitory monoclonal antibodies, providing important information for the design of novel vaccine candidates. Structural studies show that conserved residues in Plasmodium vivax reticulocyte-binding protein 2b determine interactions with transferrin receptor 1 that are essential for host invasion, suggesting avenues for designing vaccines that work across P. vivax strains., Author(s): Jakub Gruszczyk [sup.1] , Rick K. Huang [sup.2] , Li-Jin Chan [sup.1] [sup.3] , Sébastien Menant [sup.1] , Chuan Hong [sup.2] , James M. Murphy [sup.1] [sup.3] , Yee-Foong [...]

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes.sup.1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites.sup.3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair.sup.5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.A non-viral strategy to introduce large DNA sequences into T cells enables the correction of a pathogenic mutation that causes autoimmunity, and the replacement of an endogenous T-cell receptor with an engineered receptor that can recognize cancer antigens., Author(s): Theodore L. Roth [sup.1] [sup.2] [sup.3] [sup.4] [sup.5] , Cristina Puig-Saus [sup.6] , Ruby Yu [sup.3] [sup.4] [sup.5] , Eric Shifrut [sup.3] [sup.4] [sup.5] , Julia Carnevale [sup.7] , [...]

Dendritic cells orchestrate the crosstalk between innate and adaptive immunity. CD8[alpha].sup.+ dendritic cells present antigens to CD8.sup.+ T cells and elicit cytotoxic T cell responses to viruses, bacteria and tumours.sup.1. Although lineage-specific transcriptional regulators of CD8[alpha].sup.+ dendritic cell development have been identified.sup.2, the molecular pathways that selectively orchestrate CD8[alpha].sup.+ dendritic cell function remain elusive. Moreover, metabolic reprogramming is important for dendritic cell development and activation.sup.3,4, but metabolic dependence and regulation of dendritic cell subsets are largely uncharacterized. Here we use a data-driven systems biology algorithm (NetBID) to identify a role of the Hippo pathway kinases Mst1 and Mst2 (Mst1/2) in selectively programming CD8[alpha].sup.+ dendritic cell function and metabolism. Our NetBID analysis reveals a marked enrichment of the activities of Hippo pathway kinases in CD8[alpha].sup.+ dendritic cells relative to CD8[alpha].sup.- dendritic cells. Dendritic cell-specific deletion of Mst1/2--but not Lats1 and Lats2 (Lats1/2) or Yap and Taz (Yap/Taz), which mediate canonical Hippo signalling--disrupts homeostasis and function of CD8.sup.+ T cells and anti-tumour immunity. Mst1/2-deficient CD8[alpha].sup.+ dendritic cells are impaired in presentation of extracellular proteins and cognate peptides to prime CD8.sup.+ T cells, while CD8[alpha].sup.- dendritic cells that lack Mst1/2 have largely normal function. Mechanistically, compared to CD8[alpha].sup.- dendritic cells, CD8[alpha].sup.+ dendritic cells exhibit much stronger oxidative metabolism and critically depend on Mst1/2 signalling to maintain bioenergetic activities and mitochondrial dynamics for their functional capacities. Further, selective expression of IL-12 by CD8[alpha].sup.+ dendritic cells depends on Mst1/2 and the crosstalk with non-canonical NF-[kappa]B signalling. Our findings identify Mst1/2 as selective drivers of CD8[alpha].sup.+ dendritic cell function by integrating metabolic activity and cytokine signalling, and highlight that the interplay between immune signalling and metabolic reprogramming underlies the unique functions of dendritic cell subsets.A data-driven analysis helps to identify specific roles of the Hippo signalling kinases Mst1 and Mst2 in integrating metabolic activity and cytokine signalling in dendritic cells, and thereby orchestrating immune cell function., Author(s): Xingrong Du [sup.1] , Jing Wen [sup.1] , Yanyan Wang [sup.1] , Peer W. F. Karmaus [sup.1] , Alireza Khatamian [sup.2] , Haiyan Tan [sup.3] [sup.4] , Yuxin Li [...]

Various forms of immunotherapy, such as checkpoint blockade immunotherapy, are proving to be effective at restoring T cell-mediated immune responses that can lead to marked and sustained clinical responses, but only in some patients and cancer types.sup.1-4. Patients and tumours may respond unpredictably to immunotherapy partly owing to heterogeneity of the immune composition and phenotypic profiles of tumour-infiltrating lymphocytes (TILs) within individual tumours and between patients.sup.5,6. Although there is evidence that tumour-mutation-derived neoantigen-specific T cells play a role in tumour control.sup.2,4,7-10, in most cases the antigen specificities of phenotypically diverse tumour-infiltrating T cells are largely unknown. Here we show that human lung and colorectal cancer CD8.sup.+ TILs can not only be specific for tumour antigens (for example, neoantigens), but also recognize a wide range of epitopes unrelated to cancer (such as those from Epstein-Barr virus, human cytomegalovirus or influenza virus). We found that these bystander CD8.sup.+ TILs have diverse phenotypes that overlap with tumour-specific cells, but lack CD39 expression. In colorectal and lung tumours, the absence of CD39 in CD8.sup.+ TILs defines populations that lack hallmarks of chronic antigen stimulation at the tumour site, supporting their classification as bystanders. Expression of CD39 varied markedly between patients, with some patients having predominantly CD39.sup.- CD8.sup.+ TILs. Furthermore, frequencies of CD39 expression among CD8.sup.+ TILs correlated with several important clinical parameters, such as the mutation status of lung tumour epidermal growth factor receptors. Our results demonstrate that not all tumour-infiltrating T cells are specific for tumour antigens, and suggest that measuring CD39 expression could be a straightforward way to quantify or isolate bystander T cells.Human lung and colorectal tumours contain a population of tumour-infiltrating lymphocytes that are specific for tumour-unrelated antigens and, unlike tumour-antigen-specific tumour-infiltrating lymphocytes, do not express CD39., Author(s): Yannick Simoni [sup.1] , Etienne Becht [sup.1] , Michael Fehlings [sup.1] [sup.2] , Chiew Yee Loh [sup.1] , Si-Lin Koo [sup.3] , Karen Wei Weng Teng [sup.1] , Joe [...]

The intestinal immune system has the challenging task of tolerating foreign nutrients and the commensal microbiome, while excluding or eliminating ingested pathogens. Failure of this balance leads to conditions such as inflammatory bowel diseases, food allergies and invasive gastrointestinal infections.sup.1. Multiple immune mechanisms are therefore in place to maintain tissue integrity, including balanced generation of effector T (T.sub.H) cells and FOXP3.sup.+ regulatory T (pT.sub.reg) cells, which mediate resistance to pathogens and regulate excessive immune activation, respectively.sup.1-4. The gut-draining lymph nodes (gLNs) are key sites for orchestrating adaptive immunity to luminal perturbations.sup.5-7. However, it is unclear how they simultaneously support tolerogenic and inflammatory reactions. Here we show that gLNs are immunologically specific to the functional gut segment that they drain. Stromal and dendritic cell gene signatures and polarization of T cells against the same luminal antigen differ between gLNs, with the proximal small intestine-draining gLNs preferentially giving rise to tolerogenic responses and the distal gLNs to pro-inflammatory T cell responses. This segregation permitted the targeting of distal gLNs for vaccination and the maintenance of duodenal pT.sub.reg cell induction during colonic infection. Conversely, the compartmentalized dichotomy was perturbed by surgical removal of select distal gLNs and duodenal infection, with effects on both lymphoid organ and tissue immune responses. Our findings reveal that the conflict between tolerogenic and inflammatory intestinal responses is in part resolved by discrete gLN drainage, and encourage antigen targeting to specific gut segments for therapeutic immune modulation. Immune responses in the gut and associated draining lymph nodes differ between tolerogenic and inflammatory depending on their anatomical location., Author(s): Daria Esterházy [sup.1] [sup.4] , Maria C. C. Canesso [sup.1] [sup.2] , Luka Mesin [sup.3] , Paul A. Muller [sup.1] , Tiago B. R. de Castro [sup.1] [sup.2] , [...]

The L-type amino acid transporter 1 (LAT1; also known as SLC7A5) catalyses the cross-membrane flux of large neutral amino acids in a sodium- and pH-independent manner.sup.1-3. LAT1, an antiporter of the amino acid-polyamine-organocation superfamily, also catalyses the permeation of thyroid hormones, pharmaceutical drugs, and hormone precursors such as l-3,4-dihydroxyphenylalanine across membranes.sup.2-6. Overexpression of LAT1 has been observed in a wide range of tumour cells, and it is thus a potential target for anti-cancer drugs.sup.7-11. LAT1 forms a heteromeric amino acid transporter complex with 4F2 cell-surface antigen heavy chain (4F2hc; also known as SLC3A2)--a type II membrane glycoprotein that is essential for the stability of LAT1 and for its localization to the plasma membrane.sup.8,9. Despite extensive cell-based characterization of the LAT1-4F2hc complex and structural determination of its homologues in bacteria, the interactions between LAT1 and 4F2hc and the working mechanism of the complex remain largely unknown.sup.12-19. Here we report the cryo-electron microscopy structures of human LAT1-4F2hc alone and in complex with the inhibitor 2-amino-2-norbornanecarboxylic acid at resolutions of 3.3 Å and 3.5 Å, respectively. LAT1 exhibits an inward open conformation. Besides a disulfide bond association, LAT1 also interacts extensively with 4F2hc on the extracellular side, within the membrane, and on the intracellular side. Biochemical analysis reveals that 4F2hc is essential for the transport activity of the complex. Together, our characterizations shed light on the architecture of the LAT1-4F2hc complex, and provide insights into its function and the mechanisms through which it might be associated with disease. The cryo-EM structure of the LAT1-4F2hc complex, a heteromeric amino acid transporter, is characterized, providing insights into its function and the mechanisms through which mutations of this complex could cause disease., Author(s): Renhong Yan [sup.1] , Xin Zhao [sup.1] , Jianlin Lei [sup.2] , Qiang Zhou [sup.3] Author Affiliations: (1) Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for [...]

The cytokine interferon-[gamma] (IFN[gamma]) is a central coordinator of innate and adaptive immunity, but its highly pleiotropic actions have diminished its prospects for use as an immunotherapeutic agent. Here, we took a structure-based approach to decoupling IFN[gamma] pleiotropy. We engineered an affinity-enhanced variant of the ligand-binding chain of the IFN[gamma] receptor IFN[gamma]R1, which enabled us to determine the crystal structure of the complete hexameric (2:2:2) IFN[gamma]-IFN[gamma]R1-IFN[gamma]R2 signalling complex at 3.25 Å resolution. The structure reveals the mechanism underlying deficits in IFN[gamma] responsiveness in mycobacterial disease syndrome resulting from a T168N mutation in IFN[gamma]R2, which impairs assembly of the full signalling complex. The topology of the hexameric complex offers a blueprint for engineering IFN[gamma] variants to tune IFN[gamma] receptor signalling output. Unexpectedly, we found that several partial IFN[gamma] agonists exhibited biased gene-expression profiles. These biased agonists retained the ability to induce upregulation of major histocompatibility complex class I antigen expression, but exhibited impaired induction of programmed death-ligand 1 expression in a wide range of human cancer cell lines, offering a route to decoupling immunostimulatory and immunosuppressive functions of IFN[gamma] for therapeutic applications. The X-ray structure of the hexameric complex of interferon-[gamma] bound to its receptors is solved at 3.25 Å resolution, providing a basis for engineering variants of interferon-[gamma] that enable decoupling of its immunomodulatory functions., Author(s): Juan L. Mendoza [sup.1] [sup.2] [sup.7] , Nichole K. Escalante [sup.3] [sup.4] , Kevin M. Jude [sup.1] [sup.2] , Junel Sotolongo Bellon [sup.5] , Leon Su [sup.1] [sup.2] , [...]

Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats.sup.1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan.sup.1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR [alpha]-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism. The DR isotype of the human leukocyte antigen of the MHC class II--or its homologues in bats, pigs, mice and chickens--is an essential cell entry determinant for bat influenza A viruses., Author(s): Umut Karakus [sup.1] , Thiprampai Thamamongood [sup.2] [sup.3] [sup.4] [sup.5] , Kevin Ciminski [sup.2] [sup.3] , Wei Ran [sup.2] [sup.3] , Sira C. Günther [sup.1] , Marie O. Pohl [...]

The human genome contains approximately 20 thousand protein-coding genes.sup.1, but the size of the collection of antigen receptors of the adaptive immune system that is generated by the recombination of gene segments with non-templated junctional additions (on B cells) is unknown--although it is certainly orders of magnitude larger. It has not been established whether individuals possess unique (or private) repertoires or substantial components of shared (or public) repertoires. Here we sequence recombined and expressed B cell receptor genes in several individuals to determine the size of their B cell receptor repertoires, and the extent to which these are shared between individuals. Our experiments revealed that the circulating repertoire of each individual contained between 9 and 17 million B cell clonotypes. The three individuals that we studied shared many clonotypes, including between 1 and 6% of B cell heavy-chain clonotypes shared between two subjects (0.3% of clonotypes shared by all three) and 20 to 34% of [lambda] or [kappa] light chains shared between two subjects (16 or 22% of [lambda] or [kappa] light chains, respectively, were shared by all three). Some of the B cell clonotypes had thousands of clones, or somatic variants, within the clonotype lineage. Although some of these shared lineages might be driven by exposure to common antigens, previous exposure to foreign antigens was not the only force that shaped the shared repertoires, as we also identified shared clonotypes in umbilical cord blood samples and all adult repertoires. The unexpectedly high prevalence of shared clonotypes in B cell repertoires, and identification of the sequences of these shared clonotypes, should enable better understanding of the role of B cell immune repertoires in health and disease. Many clonotypes in human B cell repertoires are shared, including between adult and umbilical cord blood repertoires, which indicates that this similarity is not driven only by exposure to common antigens., Author(s): Cinque Soto [sup.1] [sup.2] , Robin G. Bombardi [sup.1] , Andre Branchizio [sup.1] , Nurgun Kose [sup.1] , Pranathi Matta [sup.1] , Alexander M. Sevy [sup.3] , Robert S. [...]

Understanding the determinants of broadly neutralizing antibody (bNAb) evolution is crucial for the development of bNAb-based HIV vaccines.sup.1. Despite emerging information on cofactors that promote bNAb evolution in natural HIV-1 infections, in which the induction of bNAbs is genuinely rare.sup.2, information on the impact of the infecting virus strain on determining the breadth and specificity of the antibody responses to HIV-1 is lacking. Here we analyse the influence of viral antigens in shaping antibody responses in humans. We call the ability of a virus strain to induce similar antibody responses across different hosts its antibody-imprinting capacity, which from an evolutionary biology perspective corresponds to the viral heritability of the antibody responses. Analysis of 53 measured parameters of HIV-1-binding and neutralizing antibody responses in a cohort of 303 HIV-1 transmission pairs (individuals who harboured highly related HIV-1 strains and were putative direct transmission partners or members of an HIV-1 transmission chain) revealed that the effect of the infecting virus on the outcome of the bNAb response is moderate in magnitude but highly significant. We introduce the concept of bNAb-imprinting viruses and provide evidence for the existence of such viruses in a systematic screening of our cohort. The bNAb-imprinting capacity can be substantial, as indicated by a transmission pair with highly similar HIV-1 antibody responses and strong bNAb activity. Identification of viruses that have bNAb-imprinting capacities and their characterization may thus provide the potential to develop lead immunogens.Similarity of antibody responses in HIV-1 transmission pairs reveals a significant impact of the virus genome on imprinting antibody responses., Author(s): Roger D. Kouyos [sup.1] [sup.2] , Peter Rusert [sup.1] , Claus Kadelka [sup.1] [sup.2] , Michael Huber [sup.1] , Alex Marzel [sup.1] [sup.2] , Hanna Ebner [sup.1] , Merle [...]

Tissue-specific autoimmunity occurs when selected antigens presented by susceptible alleles of the major histocompatibility complex are recognized by T cells. However, the reason why certain specific self-antigens dominate the response and are indispensable for triggering autoreactivity is unclear. Spontaneous presentation of insulin is essential for initiating autoimmune type 1 diabetes in non-obese diabetic mice.sup.1,2. A major set of pathogenic CD4 T cells specifically recognizes the 12-20 segment of the insulin B-chain (B:12-20), an epitope that is generated from direct presentation of insulin peptides by antigen-presenting cells.sup.3,4. These T cells do not respond to antigen-presenting cells that have taken up insulin that, after processing, leads to presentation of a different segment representing a one-residue shift, B:13-21.sup.4. CD4 T cells that recognize B:12-20 escape negative selection in the thymus and cause diabetes, whereas those that recognize B:13-21 have only a minor role in autoimmunity.sup.3-5. Although presentation of B:12-20 is evident in the islets.sup.3,6, insulin-specific germinal centres can be formed in various lymphoid tissues, suggesting that insulin presentation is widespread.sup.7,8. Here we use live imaging to document the distribution of insulin recognition by CD4 T cells throughout various lymph nodes. Furthermore, we identify catabolized insulin peptide fragments containing defined pathogenic epitopes in [beta]-cell granules from mice and humans. Upon glucose challenge, these fragments are released into the circulation and are recognized by CD4 T cells, leading to an activation state that results in transcriptional reprogramming and enhanced diabetogenicity. Therefore, a tissue such as pancreatic islets, by releasing catabolized products, imposes a constant threat to self-tolerance. These findings reveal a self-recognition pathway underlying a primary autoantigen and provide a foundation for assessing antigenic targets that precipitate pathogenic outcomes by systemically sensitizing lymphoid tissues.A sensitive T cell tracking assay reveals immunogenic activity of specific catabolized peptide fragments of insulin and their effects on T cell activity in lymph nodes, highlighting communication between pancreatic islets and lymphoid tissue., Author(s): Xiaoxiao Wan [sup.1] , Bernd H. Zinselmeyer [sup.1] , Pavel N. Zakharov [sup.1] , Anthony N. Vomund [sup.1] , Ruth Taniguchi [sup.2] , Laura Santambrogio [sup.3] , Mark S. [...]

The thymus is responsible for generating a diverse yet self-tolerant pool of T cells.sup.1. Although the thymic medulla consists mostly of developing and mature AIRE.sup.+ epithelial cells, recent evidence has suggested that there is far greater heterogeneity among medullary thymic epithelial cells than was previously thought.sup.2. Here we describe in detail an epithelial subset that is remarkably similar to peripheral tuft cells that are found at mucosal barriers.sup.3. Similar to the periphery, thymic tuft cells express the canonical taste transduction pathway and IL-25. However, they are unique in their spatial association with cornified aggregates, ability to present antigens and expression of a broad diversity of taste receptors. Some thymic tuft cells pass through an Aire-expressing stage and depend on a known AIRE-binding partner, HIPK2, for their development. Notably, the taste chemosensory protein TRPM5 is required for their thymic function through which they support the development and polarization of thymic invariant natural killer T cells and act to establish a medullary microenvironment that is enriched in the type 2 cytokine, IL-4. These findings indicate that there is a compartmentalized medullary environment in which differentiation of a minor and highly specialized epithelial subset has a non-redundant role in shaping thymic function.A comprehensive analysis of the thymic medulla identifies a tuft-cell-like thymic epithelial cell population that is necessary for shaping thymic function., Author(s): Corey N. Miller [sup.1] [sup.2] , Irina Proekt [sup.1] [sup.2] , Jakob von Moltke [sup.2] [sup.3] [sup.4] [sup.9] , Kristen L. Wells [sup.5] , Aparna R. Rajpurkar [sup.5] , [...]

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies.sup.1-3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells.sup.4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.Genetically engineered T cells that induced remission in a patient with chronic lymphocytic leukaemia were found to have disruption of the TET2 gene, which caused T cell changes that potentiated their anti-tumour effects., Author(s): Joseph A. Fraietta [sup.1] [sup.2] [sup.3] [sup.4] , Christopher L. Nobles [sup.5] , Morgan A. Sammons [sup.6] [sup.10] , Stefan Lundh [sup.1] [sup.2] , Shannon A. Carty [sup.2] [sup.11] [...]