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PDF file - 713K, Figure S1. Kinetics of EL4 killing. Figure S2. iNKT cells kill several CD1d-expressing targets. Figure S3. EL4s express low levels of NKG2D ligands while iNKT and NK cells express NKG2D. Figure S4. Thymic but not splenic NKT cells kill EL4s in a GAg-dependent manner. Figure S5. DN3A4-1.2 NKT hybridoma cells kill GAg-loaded tumors. Figure S6. CD4+ and CD4- 8- liver iNKT cells possess cytotoxic capacity. Figure S7. iNKT cell reconstitution of NSG mice is not associated with in vivo accumulation of contaminating NK cells.

Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we found that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially induced by iNKT cell agonists of varying T-cell receptor (TCR) affinities, such as OCH, α-galactosyl ceramide, and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of TCR signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell–deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T lymphoma. Cancer Immunol Res; 2(1); 59–69. ©2013 AACR.

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BACKGROUND: Pro-inflammatory fibroblasts are critical for pathogenesis in rheumatoid arthritis, inflammatory bowel disease, interstitial lung disease, and Sjögren’s syndrome and represent a novel therapeutic target for chronic inflammatory disease. However, the heterogeneity of fibroblast phenotypes, exacerbated by the lack of a common cross-tissue taxonomy, has limited our understanding of which pathways are shared by multiple diseases. METHODS: We profiled fibroblasts derived from inflamed and non-inflamed synovium, intestine, lungs, and salivary glands from affected individuals with single-cell RNA sequencing. We integrated all fibroblasts into a multi-tissue atlas to characterize shared and tissue-specific phenotypes. FINDINGS: Two shared clusters, CXCL10(+)CCL19(+) immune-interacting and SPARC(+)COL3A1(+) vascular-interacting fibroblasts, were expanded in all inflamed tissues and mapped to dermal analogs in a public atopic dermatitis atlas. We confirmed these human pro-inflammatory fibroblasts in animal models of lung, joint, and intestinal inflammation. CONCLUSIONS: This work represents a thorough investigation into fibroblasts across organ systems, individual donors, and disease states that reveals shared pathogenic activation states across four chronic inflammatory diseases. FUNDING: Grant from F. Hoffmann-La Roche (Roche) AG.

Signaling pathways that sense amino acid abundance are integral to tissue homeostasis and cellular defense. Our laboratory has previously shown that halofuginone (HF) inhibits the prolyl-tRNA synthetase catalytic activity of glutamyl-prolyl-tRNA synthetase (EPRS), thereby activating the amino acid response (AAR). We now show that HF treatment selectively inhibits inflammatory responses in diverse cell types and that these therapeutic benefits occur in cells that lack GCN2, the signature effector of the AAR. Depletion of arginine, histidine, or lysine from cultured fibroblast-like synoviocytes recapitulates key aspects of HF treatment, without utilizing GCN2 or mammalian target of rapamycin complex 1 pathway signaling. Like HF, the threonyl-tRNA synthetase inhibitor borrelidin suppresses the induction of tissue remodeling and inflammatory mediators in cytokine-stimulated fibroblast-like synoviocytes without GCN2, but both aminoacyl-tRNA synthetase (aaRS) inhibitors are sensitive to the removal of GCN1. GCN1, an upstream component of the AAR pathway, binds to ribosomes and is required for GCN2 activation. These observations indicate that aaRS inhibitors, like HF, can modulate inflammatory response without the AAR/GCN2 signaling cassette, and that GCN1 has a role that is distinct from its activation of GCN2. We propose that GCN1 participates in a previously unrecognized amino acid sensor pathway that branches from the canonical AAR.

The role of stromal fibroblasts in chronic inflammation is unfolding. In rheumatoid arthritis, leukocyte-derived cytokines TNF and IL-17A work together, activating fibroblasts to become a dominant source of the hallmark cytokine IL-6. However, IL-17A alone has minimal effect on fibroblasts. To identify key mediators of the synergistic response to TNF and IL-17A in human synovial fibroblasts, we performed time series, dose–response, and gene-silencing transcriptomics experiments. Here we show that in combination with TNF, IL-17A selectively induces a specific set of genes mediated by factors including cut-like homeobox 1 (CUX1) and IκBζ (NFKBIZ). In the promoters of CXCL1 , CXCL2 , and CXCL3 , we found a putative CUX1–NF-κB binding motif not found elsewhere in the genome. CUX1 and NF-κB p65 mediate transcription of these genes independent of LIFR, STAT3, STAT4, and ELF3. Transcription of NFKBIZ , encoding the atypical IκB factor IκBζ, is IL-17A dose-dependent, and IκBζ only mediates the transcriptional response to TNF and IL-17A, but not to TNF alone. In fibroblasts, IL-17A response depends on CUX1 and IκBζ to engage the NF-κB complex to produce chemoattractants for neutrophil and monocyte recruitment.

Genetic studies have revealed that autoimmune susceptibility variants are over-represented in memory CD4+ T cell regulatory elements1–3. Understanding how genetic variation affects gene expression in different T cell physiological states is essential for deciphering genetic mechanisms of autoimmunity4,5. Here, we characterized the dynamics of genetic regulatory effects at eight time points during memory CD4+ T cell activation with high-depth RNA-seq in healthy individuals. We discovered widespread, dynamic allele-specific expression across the genome, where the balance of alleles changes over time. These genes were enriched fourfold within autoimmune loci. We found pervasive dynamic regulatory effects within six HLA genes. HLA-DQB1 alleles had one of three distinct transcriptional regulatory programs. Using CRISPR–Cas9 genomic editing we demonstrated that a promoter variant is causal for T cell–specific control of HLA-DQB1 expression. Our study shows that genetic variation in cis-regulatory elements affects gene expression in a manner dependent on lymphocyte activation status, contributing to the interindividual complexity of immune responses.

Innate T cells, including CD1d-restricted invariant natural killer T (iNKT) cells, are characterized by their rapid activation in response to non-peptide antigens, such as lipids. While the transcriptional profiles of naive, effector and memory adaptive T cells have been well studied, less is known about transcriptional regulation of different iNKT cell activation states. Here, using single cell RNA-sequencing, we performed longitudinal profiling of activated iNKT cells, generating a transcriptomic atlas of iNKT cell activation states. We found that transcriptional signatures of activation are highly conserved among heterogeneous iNKT cell populations, including NKT1, NKT2 and NKT17 subsets, and human iNKT cells. Strikingly, we found that regulatory iNKT cells, such as adipose iNKT cells, undergo blunted activation, and display constitutive enrichment of memory-like cMAF+ and KLRG1+ populations. Moreover, we identify a conserved cMAF-associated transcriptional network among NKT10 cells, providing novel insights into the biology of regulatory and antigen experienced iNKT cells.

Objective Delayed detection of LN associates with worse outcomes. There are conflicting recommendations regarding a threshold level of proteinuria at which biopsy will likely yield actionable management. This study addressed the association of urine protein:creatinine ratios (UPCR) with clinical characteristics and investigated the incidence of proliferative and membranous histology in patients with a UPCR between 0.5 and 1. Methods A total of 275 SLE patients (113 first biopsy, 162 repeat) were enrolled in the multicentre multi-ethnic/racial Accelerating Medicines Partnership across 15 US sites at the time of a clinically indicated renal biopsy. Patients were followed for 1 year. Results At biopsy, 54 patients had UPCR Conclusion In this prospective study, three-quarters of patients with UPCR

Immune checkpoint inhibitor (ICI) therapies that promote T cell activation have improved outcomes for advanced malignancies yet also elicit harmful autoimmune reactions. The T cell mechanisms mediating these iatrogenic autoimmune events remain unclear. Here we assayed T cells from joints of patients affected by ICI-induced inflammatory arthritis (ICI-arthritis), which can present clinically indistinguishable from rheumatoid arthritis (RA). Compared to the autoimmune arthritides RA and psoriatic arthritis (PsA), ICI-arthritis joints contained an expanded CD38hi CD127− CD8+ T cell subset that displays cytotoxic, effector, and interferon (IFN) response signatures. The abundance of CD38hi CD8 T cells in ICI-arthritis resulted from a limited number of clones that could be found proliferating in the joint. Exposure of synovial T cells to Type I IFN, more so than IFN-γ, induces the CD38hi cytotoxic phenotype. Relative to other CD8+ T cell subsets in the joints, the CD38hi population is distinct from a dysfunctional population and clonally most related to TCF7+ memory populations. Examination of synovial tissue from bilateral knee arthroplasty demonstrated considerable sharing of TCR clonotypes in the CD38hi CD8 T cell fraction from both knees. These results define a distinct CD8 T cell subset that may be directly activated by ICI therapy and mediate a tissue-specific autoimmune cellular reaction in patient joints.

Fibroblasts are important cells for the support of homeostatic tissue function. In inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease, fibroblasts take on different roles (a) as inflammatory cells themselves and (b) in recruiting leukocytes, driving angiogenesis, and enabling chronic inflammation in tissues. Recent advances in single-cell profiling techniques have transformed the ability to examine fibroblast states and populations in inflamed tissues, providing evidence of previously underappreciated heterogeneity and disease-associated fibroblast populations. These studies challenge the preconceived notion that fibroblasts are homogeneous and provide new insights into the role of fibroblasts in inflammatory pathology. In addition, new molecular insights into the mechanisms of fibroblast activation reveal powerful cell-intrinsic amplification loops that synergize with primary fibroblast stimuli to result in striking responses. In this Review, we focus on recent developments in our understanding of fibroblast heterogeneity and fibroblast pathology across tissues and diseases in rheumatoid arthritis and inflammatory bowel diseases. We highlight new approaches to, and applications of, single-cell profiling techniques and what they teach us about fibroblast biology. Finally, we address how these insights could lead to the development of novel therapeutic approaches to targeting fibroblasts in disease.

Cadherins are homophilic cell-to-cell adhesion molecules that help cells respond to environmental changes. Newly formed cadherin junctions are associated with increased cell phosphorylation, but the pathways driving this signaling response are largely unknown. Since cadherins have no intrinsic signaling activity, this phosphorylation must occur through interactions with other signaling molecules. We previously reported that cadherin-11 engagement activates joint synovial fibroblasts, promoting inflammatory and degradative pathways important in rheumatoid arthritis (RA) pathogenesis. Our objective in this study was to discover interacting partners that mediate cadherin-11 signaling. Protein array screening showed that cadherin-11 extracellular binding domains linked to an Fc domain (cad11Fc) induced platelet-derived growth factor (PDGFR)-α phosphorylation in synovial fibroblasts and glioblastoma cells. PDGFRs are growth factor receptor tyrosine kinases that promote cell proliferation, survival, and migration in mesodermally derived cells. Increased PDGFR activity is implicated in RA pathology and associates with poor prognosis in several cancers, including sarcoma and glioblastoma. PDGFRα activation by cadherin-11 signaling promoted fibroblast proliferation, a signaling pathway independent from cadherin-11-stimulated IL-6 or matrix metalloproteinase (MMP)-3 release. PDGFRα phosphorylation mediated most of the cad11Fc-induced phosphatidyl-3-kinase (PI3K)/Akt activation, but only part of the mitogen-activated protein kinase (MAPK) response. PDGFRα-dependent signaling did not require cell cadherin-11 expression. Rather, cad11Fc immunoprecipitated PDGFRα, indicating a direct interaction between cadherin-11 and PDGFRα extracellular domains. This study is the first to report an interaction between cadherin-11 and PDGFRα and adds to our growing understanding that cadherin-growth factor receptor interactions help balance the interplay between tissue growth and adhesion.

More than 1 million apheresis platelet collections are performed annually in the United States. After 2 healthy plateletpheresis donors were incidentally found to have low CD4+ T-lymphocyte counts, we investigated whether plateletpheresis causes lymphopenia. We conducted a cross-sectional single-center study of platelet donors undergoing plateletpheresis with the Trima Accel, which removes leukocytes continuously with its leukoreduction system chamber. We recruited 3 groups of platelet donors based on the total number of plateletpheresis sessions in the prior 365 days: 1 or 2, 3 to 19, or 20 to 24. CD4+ T-lymphocyte counts were

In chronic infection, inflammation and cancer, the tissue microenvironment controls how local immune cells behave, with tissue-resident fibroblasts emerging as a key cell type in regulating activation or suppression of an immune response. Fibroblasts are heterogeneous cells, encompassing functionally distinct populations, the phenotypes of which differ according to their tissue of origin and type of inciting disease. Their immunological properties are also diverse, ranging from the maintenance of a potent inflammatory environment in chronic inflammation to promoting immunosuppression in malignancy, and encapsulating and incarcerating infectious agents within tissues. In this Review, we compare the mechanisms by which fibroblasts control local immune responses, as well as the factors regulating their inflammatory and suppressive profiles, in different tissues and pathological settings. This cross-disease perspective highlights the importance of tissue context in determining fibroblast–immune cell interactions, as well as potential therapeutic avenues to exploit this knowledge for the benefit of patients with chronic infection, inflammation and cancer. Fibroblasts are not just crucial structural cells; they exist as multiple functionally diverse populations, defined by their location and context, and regulate tissue immunity. Here, the authors review the immunological properties of fibroblasts, comprising both pro-inflammatory and immunosuppressive activities, in different tissues and disease states.

SummaryPro-inflammatory fibroblasts are critical to pathogenesis in rheumatoid arthritis, inflammatory bowel disease, interstitial lung disease, and Sjögren’s syndrome, and represent a novel therapeutic target for chronic inflammatory disease. However, the heterogeneity of fibroblast phenotypes, exacerbated by the lack of a common cross-tissue taxonomy, has limited the understanding of which pathways are shared by multiple diseases. To investigate, we profiled patient-derived fibroblasts from inflamed and non-inflamed synovium, intestine, lung, and salivary glands with single-cell RNA-sequencing. We integrated all fibroblasts into a multi-tissue atlas to characterize shared and tissue-specific phenotypes. Two shared clusters, CXCL10+CCL19+ immune-interacting and SPARC+COL3A1+ vascular-interacting fibroblasts were expanded in all inflamed tissues and additionally mapped to dermal analogues in a public atopic dermatitis atlas. We further confirmed these human pro-inflammatory fibroblasts in animal models of lung, joint, and intestinal inflammation. This work represents the first cross-tissue, single-cell fibroblast atlas revealing shared pathogenic activation states across four chronic inflammatory diseases.

Macrophages regulate protective immune responses to infectious microbes, but aberrant macrophage activation frequently drives pathological inflammation. To identify regulators of vigorous macrophage activation, we analyzed RNA-seq data from synovial macrophages and identified SLAMF7 as a receptor associated with a super-activated macrophage state in rheumatoid arthritis. We implicated IFN-γ as a key regulator of SLAMF7 expression. Engaging this receptor drove an exuberant wave of inflammatory cytokine expression, and induction of TNF-α following SLAMF7 engagement amplified inflammation through an autocrine signaling loop. We observed SLAMF7-induced gene programs not only in macrophages from rheumatoid arthritis patients, but in gut macrophages from active Crohn’s disease patients and lung macrophages from severe COVID-19 patients. This suggests a central role for SLAMF7 in macrophage super-activation with broad implications in pathology.

Patterns of innovation can be sustained (continuous) or rapid, sometimes even ‘disruptive’. A major difference is that in order to support disruptive innovation the support networks and its infrastructure often need to be changed dynamically to accommodate a rapidly evolving landscape to establish the disruptive approach. For example, introducing electric cars disrupts the support network for gasoline cars (network of gas and service stations), and at the same time requires an entirely new system of charging stations. Such disruptions occur in science, and a wonderful example was the creation of monoclonal antibody technologies. The discovery of the principle for production of monoclonal antibodies by Cesar Milstein and Georg Kohler fueled a rapid adoption of new antibody-based technologies in all areas of medicine. This transformation was strongly supported by the open workshops that catalogued antibodies from many laboratories into distinct ‘clusters of differentiation’, thereby providing a new ‘support network’ for the global use of well validated and standardised monoclonal antibodies. This new support network helped the pharmaceutical industry transition from a major focus on small chemical molecules (screened for effects in vitro) to a new targeted approach, first with recombinant proteins, later with monoclonal antibodies with the introduction of anti-tumour necrosis factor (TNF) antibodies as an example. During the last two decades, in both the USA and Europe, the need to develop public–private partnership support networks has been recognised as a means to accelerate innovation and enable translation of the rapidly expanding cellular and molecular understanding of disease pathogenesis into the development of new therapeutic agents. In Europe, the Innovative Medicine Initiative (IMI) was formed to enhance public–private partnerships, and in the USA the Accelerating Medicines Partnership (AMP).1 Identifying, validating and testing new targets based on enhanced understanding are at the core of both AMP and IMI. To facilitate continuous …

Adipose tissue invariant natural killer T (iNKT) cells are phenotypically different from other iNKT cells because they produce IL-10 and control metabolic homeostasis. Why that is the case is unclear. Here, using single-cell RNA-sequencing we found several adipose iNKT clusters, which we grouped into two functional populations based on NK1.1 expression. NK1.1(NEG) cells almost exclusively produced IL-10 and other regulatory cytokines, while NK1.1(POS) iNKT cells predominantly produced IFNγ. Mechanistically, biochemical fractionation revealed that free fatty acids drive IL-10 production primarily in NK1.1(NEG) iNKT cells via the IRE1α-XBP1s arm of the unfolded protein response. Correspondingly, adoptive transfer of adipose tissue NK1.1(NEG) iNKT cells selectively restored metabolic function in obese mice. Further, we found an unexpected role for NK1.1(POS) iNKT cells in lean adipose tissue, as IFNγ licenses natural killer cell-mediated macrophage killing to limit pathological macrophage expansion. Together, these two iNKT cell populations utilize non-redundant pathways to preserve metabolic integrity.

As treatment of the early, inflammatory phase of sepsis improves, post-sepsis immunosuppression and secondary infection have increased in importance. How early inflammation drives immunosuppression remains unclear. Although IFN-γ typically helps microbial clearance, we found that increased plasma IFN-γ in early clinical sepsis was associated with the later development of secondary Candida infection. Consistent with this observation, we found that exogenous IFN-γ suppressed macrophage phagocytosis of zymosan in vivo, and antibody blockade of IFN-γ after endotoxemia improved survival of secondary candidemia. Transcriptomic analysis of innate lymphocytes during endotoxemia suggested that NKT cells drove IFN-γ production by NK cells via mTORC1. Activation of invariant NKT (iNKT) cells with glycolipid antigen drove immunosuppression. Deletion of iNKT cells in Cd1d(–/–) mice or inhibition of mTOR by rapamycin reduced immunosuppression and susceptibility to secondary Candida infection. Thus, although rapamycin is typically an immunosuppressive medication, in the context of sepsis, rapamycin has the opposite effect. These results implicated an NKT cell/mTOR/IFN-γ axis in immunosuppression following endotoxemia or sepsis. In summary, in vivo iNKT cells activated mTORC1 in NK cells to produce IFN-γ, which worsened macrophage phagocytosis, clearance of secondary Candida infection, and mortality.

© The Author(s), under exclusive licence to Springer Nature America, Inc. 2021, Metabolic programming controls immune cell lineages and functions, but little is known about γδ T cell metabolism. Here, we found that γδ T cell subsets making either interferon-γ (IFN-γ) or interleukin (IL)-17 have intrinsically distinct metabolic requirements. Whereas IFN-γ+ γδ T cells were almost exclusively dependent on glycolysis, IL-17+ γδ T cells strongly engaged oxidative metabolism, with increased mitochondrial mass and activity. These distinct metabolic signatures were surprisingly imprinted early during thymic development and were stably maintained in the periphery and within tumors. Moreover, pro-tumoral IL-17+ γδ T cells selectively showed high lipid uptake and intracellular lipid storage and were expanded in obesity and in tumors of obese mice. Conversely, glucose supplementation enhanced the antitumor functions of IFN-γ+ γδ T cells and reduced tumor growth upon adoptive transfer. These findings have important implications for the differentiation of effector γδ T cells and their manipulation in cancer immunotherapy., This work was supported by the Wellcome Trust (092973/Z/10/Z to D.J.P.), Biotechnology and Biological Sciences Research Council (BBSRC) UK (BB/R017808/1 to D.J.P.), European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 646701 to B.S.-S.; StG_679173 to L.L.), Science Foundation Ireland (SFI) (16/FRL/3865 to L.L.), NIH (NS115064, HG008155, AG062377 to M.K.), NIH (R01 AI134861 and metabolic core grant S10 OD020100 to L.L.), Fundação Astrazeneca (Prémio FAZ Ciência 2019 to B.S.-S. and N.L.) and PAC-PRECISE LISBOA-01-0145-FEDER-016394, co-funded by FEDER (POR Lisboa 2020 (Programa Operacional Regional de Lisboa, do Portugal 2020)) and Fundação para a Ciência e a Tecnologia (Portugal). N.L. is supported by a postdoctoral fellowship from EMBO (ALTF 752-2018); S.M. was supported by a studentship from the Medical Research Council (MRC) UK; G.F. is supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 752932; and A.D., S.C., L.D. and H.P. are supported by Irish Research Council fellowships.

PTPRS-targeted FLS-directed therapy synergizes with TNF inhibition in therapy of arthritis., Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase–mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.

Up to 49% of certain types of cancer are attributed to obesity, and potential mechanisms include overproduction of hormones, adipokines, and insulin. Cytotoxic immune cells, including natural killer (NK) cells and CD8+ T cells, are important in tumor surveillance, but little is known about the impact of obesity on immunosurveillance. Here, we show that obesity induces robust peroxisome proliferator-activated receptor (PPAR)-driven lipid accumulation in NK cells, causing complete ‘paralysis’ of their cellular metabolism and trafficking. Fatty acid administration, and PPARα and PPARδ (PPARα/δ) agonists, mimicked obesity and inhibited mechanistic target of rapamycin (mTOR)-mediated glycolysis. This prevented trafficking of the cytotoxic machinery to the NK cell–tumor synapse. Inhibiting PPARα/δ or blocking the transport of lipids into mitochondria reversed NK cell metabolic paralysis and restored cytotoxicity. In vivo, NK cells had blunted antitumor responses and failed to reduce tumor growth in obesity. Our results demonstrate that the lipotoxic obese environment impairs immunosurveillance and suggest that metabolic reprogramming of NK cells may improve cancer outcomes in obesity.

Genome-wide association studies (GWAS) have identified many disease-associated noncoding variants, but cannot distinguish functional single-nucleotide polymorphisms (fSNPs) from others that reside incidentally within risk loci. To address this challenge, we developed an unbiased high-throughput screen that employs type IIS enzymatic restriction to identify fSNPs that allelically modulate the binding of regulatory proteins. We coupled this approach, termed SNP-seq, with flanking restriction enhanced pulldown (FREP) to identify regulation of CD40 by three disease-associated fSNPs via four regulatory proteins, RBPJ, RSRC2 and FUBP-1/TRAP150. Applying this approach across 27 loci associated with juvenile idiopathic arthritis, we identified 148 candidate fSNPs, including two that regulate STAT4 via the regulatory proteins SATB2 and H1.2. Together, these findings establish the utility of tandem SNP-seq/FREP to bridge the gap between GWAS and disease mechanism.

Mycobacterium tuberculosis is an extremely successful pathogen, and its success is widely attributed to its ability to manipulate the intracellular environment of macrophages. A central phenomenon of tuberculosis pathology enabling immune evasion is the capacity of virulent M. tuberculosis (H37Rv) to induce macrophage necrosis, which facilitates the escape of the mycobacteria from the macrophage and spread of infection. In contrast, avirulent M. tuberculosis (H37Ra) induces macrophage apoptosis, which permits Ag presentation and activation of adaptive immunity. Previously, we found that H37Rv induces plasma membrane microdisruptions, leading to necrosis in the absence of plasma membrane repair. In contrast, H37Ra permits plasma membrane repair, which changes the host cell death modality to apoptosis, suggesting that membrane repair is critical for sequestering the pathogen in apoptotic vesicles. However, mechanisms of plasma membrane repair induced in response to M. tuberculosis infection remain unknown. Plasma membrane repair is known to induce a Ca2+-mediated signaling, which recruits lysosomes to the area of damaged plasma membrane sites for its resealing. In this study, we found that the small GTPase Arl8b is required for plasma membrane repair by controlling the exocytosis of lysosomes in cell lines and in human primary macrophages. Importantly, we found that the Arl8b secretion pathway is crucial to control the type of cell death of the M. tuberculosis–infected macrophages. Indeed, Arl8b-depleted macrophages infected with avirulent H37Ra undergo necrotic instead of apoptotic cell death. These findings suggest that membrane repair mediated by Arl8b may be an important mechanism distinguishing avirulent from virulent M. tuberculosis–induced necrotic cell death.

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 (T(reg)) 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(+) T(reg) 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.

Glycolipid antigens recognized by αβ T-cell receptors (TCRs) drive the activation of invariant natural killer T (iNKT) cells, a specialized subset of innate T lymphocytes. Glycolipids with α-linked anomeric carbohydrates have been identified as potent microbial lipid antigens for iNKT cells, and their unusual α-anomeric linkage has been thought to define a "foreign" lipid antigen motif. However, mammals use endogenous lipids to select iNKT cells, and there is compelling evidence for iNKT cell responses in various types of sterile inflammation. The nature of endogenous or environmental lipid antigens encountered by iNKT cells is not well defined. Here, we sought to identify lipid antigens in cow's milk, a prominent part of the human diet. We developed a method to directly capture lipid antigens within CD1d-lipid-TCR complexes, while excluding CD1d bound to nonantigenic lipids, followed by direct biochemical analysis of the lipid antigens trapped at the TCR-CD1d interface. The specific antigens captured by this "TCR trap" method were identified as α-linked monohexosylceramides by mass spectrometry fragmentation patterns that distinguished α- from β-anomeric monohexosylceramides. These data provide direct biochemical evidence for α-linked lipid antigens from a common dietary source.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell–B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4(+) T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1(hi)CXCR5(–)CD4(+) T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1(hi)CXCR5(–)CD4(+) T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5(–) T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c(+) B cells in SLE patients. PD-1(hi)CD4(+) T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.

Current prevailing knowledge on EMT (epithelial mesenchymal transition) deems epithelial cells acquire the characters of mesenchymal cells to be capable of invading and metastasizing on their own. One of the signature events of EMT is called “cadherin switch”, e.g. the epithelial E-cadherin switching to the mesenchymal Cadherin-11. Here, we report the critical events after EMT that cancer cells utilize cadherin-11 to hijack the endogenous cadherin-11 positive fibroblasts. Numerous 3-D cell invasion assays with high-content live cell imaging methods reveal that cadherin-11 positive cancer cells adhere to and migrate back and forth dynamically on the cell bodies of fibroblasts. By adhering to fibroblasts for co-invasion through 3-D matrices, cancer cells acquire higher invasion speed and velocity, as well as significantly elevated invasion persistence, which are exclusive characteristics of fibroblast invasion. Silencing cadherin-11 in cancer cells or in fibroblasts, or in both, significantly decouples such physical co-invasion. Additional bioinformatics studies and PDX (patient derived xenograft) studies link such cadherin-11 mediated cancer hijacking fibroblasts to the clinical cancer progression in human such as triple-negative breast cancer patients. Further animal studies confirm cadherin-11 mediates cancer hijacking fibroblasts in vivo and promotes significant solid tumor progression and distant metastasis. Moreover, overexpression of cadherin-11 strikingly protects 4T1-luc cells from implant rejection against firefly luciferase in immunocompetent mice. Overall, our findings report and characterize the critical post-EMT event of cancer hijacking fibroblasts in cancer progression and suggest cadherin-11 can be a therapeutic target for solid tumors with stroma. Our studies hence provide significant updates on the “EMT” theory that EMT cancer cells can hijack fibroblasts to achieve full mesenchymal behaviors in vivo for efficient homing, growth, metastasis and evasion of immune surveillance. Our studies also reveal that cadherin-11 is the key molecule that helps link cancer cells to stromal fibroblasts in the “Seed & Soil” theory.Graphical Abstract

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.

Background: African-American ethnicity is associated with a 3-fold higher risk of developing systemic lupus erythematosus (SLE). In addition, there is an increased risk of lupus nephritis (2-fold), high-risk histological features, and resistance to treatment. This may account for the increased mortality rate compared to Caucasian patients, especially in women. Objectives: In Phase One of the Accelerating Medicines Partnership (AMP) study, we used single-cell RNA sequencing on kidney biopsies from patients with active lupus nephritis to identify pathways that were differentially expressed in African-American patients. Methods: Single cell RNA sequencing was performed on renal biopsies obtained for clinical purpose for active nephritis using CEL-Seq2. Cell clusters with similar expression profile were identified using t-distributed stochastic neighbor embedding (t-SNE). First, the relative abundance of a cluster in AAs compared to Caucasian was determined using a logistic mixed model. Second, the differential expression profile was determined for each cell cluster and we applied Ingenuity Pathway Analysis (IPA) (QIAGEN Bioinformatics) to identify pathways of interest. Results: Samples from 13 AA and 7 Caucasian patients were obtained. Of the 3097 sequenced cell libraries, we used 2354 which passed our quality filter for a total of 30155 unique molecular identifiers. We identified 16 cell clusters including CD4, CD8, B and plasma cells, NK, myeloid cells, and tubular cells. We identified 2 cell clusters unique to African-American patients, a T and a B cell population with high expression of interferon inducible genes. We also identified that same cell populations may have differential gene expression profiles across ethnicity. For example, CD4 T cells in African-Americans have a higher expression of type 1 and type 2 interferon pathways. In contrast, myeloid cells have several upregulated pathways in Caucasians, including ERK/MAPK signaling. Conclusion: African-American lupus nephritis patients may have a stronger interferon pathway activation in infiltrating immune cells. Several other pathways, including ERK/MAPK, are differentially expressed in infiltrating cells based on ethnicity. These results suggest that ethnicity might predict a response to both current and upcoming treatments, paving the way for a more personalized approach to treatment in lupus nephritis. Further work in Phase 2 of AMP will confirm and extend these findings. Disclosure of Interests: Andrea Fava: None declared, Yuji Zhang: None declared, Nir Hacohen: None declared, Arnon Arazi: None declared, Celine Berthier: None declared, Deepak Rao: None declared, Michael Brenner Grant/research support from: Roche: sponsored research agreement on stromal cells (but has nothing to do with checkpoint related disease), Consultant for: GSK: consultant. (I am part of their immunology network, a group of about 8 immunologists who advise them regularly and broadly in the areas of inflammation and infection)., David Wofsy Consultant for: GlaxoSmithKline – Member, data safety monitoring board Novartis – Member, data safety monitoring board Celgene – member, scientific advisory board, Anne Davidson: None declared, Matthias Kretzler: None declared, David Hildeman: None declared, E. Steve Woodle: None declared, Betty Diamond: None declared, Michelle A Petri Shareholder of: Pfizer, Merck, Grant/research support from: AstraZeneca, Exagen, Consultant for: Eli Lilly, GSK, Merck EMD Serono, Janssen, Amgen, Novartis, Quintiles, Exagen, Inova Diagnostics, AstraZeneca, Blackrock, Glenmark, UCB, and the Annenberg Center for Health Sciences

Background Immune checkpoint inhibitors (CI) have revolutionized cancer management, but can also cause immune-related adverse events. Five percent of CI-treated patients develop inflammatory arthritis, but it is poorly defined phenotypically and immunologically. Objectives To characterize phenotypes of CI-associated arthritis, and compare cytokine levels in these patients to rheumatoid arthritis (RA) and osteoarthritis (OA) controls. Methods Patients referred for CI-associated arthralgia or arthritis were prospectively enrolled in an institutional registry. Serum was collected when patients underwent phlebotomy for a clinical indication. We used a Luminex Human Magnetic Assay to measure levels of IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12, IL-17, IL-22, TNF, IFN-γ, PD-L1, CCL2, CXCL2, CXCL13, OSM, CCL20, GM-CSF, CXCL-11 in CI-treated patients, and in stored serum from 7 RA patients (matched for medication, age, sex, CDAI) and 4 OA patients (age, sex matched). All comparisons were planned a priori. Results Thirty-six patients were enrolled 5/1/18-1/25/19. Median [IQR] age was 67[58-78], 18(50%) were female, 14(39%) were smokers and 13(36%) had melanoma. Twenty-two (61%) were on anti-PD1 or PD-L1 monotherapy, and the remainder were on combination CI. Phenotypes included 1. Small joint involvement in 17(47%), 2. Exclusively large joint involvement in 6(17%), 3. Arthralgia without arthritis in 9(25%), and 4. Polymyalgia rheumatica in 4(11%). In all, 7(19%) had concomitant tenosynovitis or enthesitis, mostly accompanying large joint arthritis or arthralgia (6/7). Median CDAI at entry was 11[7-23] and median ESR 29[18-44]. The majority (58%) of patients with the small joint phenotype were RF and/or CCP positive and one had erosive disease, compared to none with the large joint phenotype. ANA positivity was common (74%) and did not vary across phenotypes. Median time of symptom onset was 4[0.8-12] months after CI initiation. Median follow up was 7[3-22] months but only 5(14%) had resolution of arthritis off medication during that period. 22(61%) required CI discontinuation, 5(14%) due to arthritis. 29(81%) required steroids including 15(42%) who required >20mg prednisone. 19(53%) required a synthetic DMARD and 5(14%) required a biologic DMARD. Of 33 patients with known cancer status, 15(45%) had a complete response, 5(15%) a partial response, 5(15%) “stable” disease, 6(18%) progression, and 2(6%) died. Cytokines were measured in 22 patients, who were similar to the cohort as a whole except for more CCP positivity (p=0.02). CI patients with the small joint phenotype had higher levels of IL-6 than RA controls (p=0.04) (Figure 1) and IL-6 levels trended higher in the small joint vs. exclusively large joint phenotype (Figure 2). The B cell chemoattractant CXCL13, a factor produced by T peripheral helper cells in RA synovium1, trended higher in the serum of CI patients (Figure 3). Other cytokine levels did not differ significantly in patients with the small joint vs. exclusively large joint phenotype, or in CI-arthritis patients vs. controls. Conclusion Half of patients with CI-arthritis present with an RA-like phenotype with small joint involvement and frequent seropositivity, and have elevated levels of IL-6 compared to RA patients. This small joint phenotype may represent an accelerated form of RA. References [1] Rao DA, et al. Nature 2017: 542(7639):110-114 Disclosure of Interests Karmela Kim Chan Grant/research support from: Roche: sponsored research agreement on stromal cells, Consultant for: GSK: consultant. (I am part of their immunology network, a group of about 8 immunologists who advise them regularly and broadly in the areas of inflammation and infection)., Gregory Vitone: None declared, Sara Shanaj: None declared, Aidan Tirpack: None declared, Jackie Finik: None declared, Laura Donlin: None declared, Deepak Rao Grant/research support from: Merck - Sponsored research project funding support, Consultant for: Janssen - Consultant Scipher Medicine - Consultant Amgen - Scientific advisory board Patent submitted on Tph cells, Caroline Benson: None declared, Vivian Bykerk Grant/research support from: Mallinckrodt, BMS, Crescendo Biosciences, Sanofi/Regeneron., Consultant for: Amgen, Pfizer, UCB, Scipher, Sanofi/Genzyme/Regeneron, Dana Orange Consultant for: Astra Zeneca and Pfizer., Michael Brenner Grant/research support from: Roche: sponsored research agreement on stromal cells (but has nothing to do with checkpoint related disease), Consultant for: GSK: consultant. (I am part of their immunology network, a group of about 8 immunologists who advise them regularly and broadly in the areas of inflammation and infection)., Susan Goodman Grant/research support from: Novartis: research support, Consultant for: Novartis, UCB, Pfizer: consulting, Anne Bass: None declared

Background Treatment selection of biologic therapy for patients with rheumatoid arthritis (RA) is currently a trial and error process, with approximately 40% failing to respond well to the first biologic. The lack of biomarkers to predict treatment response leads to further pain, joint damage, patient anxiety and is cost ineffective for those who are non-responders. Objectives We aim to identify immune signatures from a pre-treatment blood test to inform the choice of treatment strategies for stratified medicine. Methods RA patients with active disease (DAS28 > 5.1) who failed to respond to conventional DMARDs and were due to commence biologic treatment were included in the BRAGGSS cohort. Peripheral blood mononuclear cells (PBMCs) taken before the initiation of biologic treatment were available for 300 patients (good (60%), moderate (25%), and non-responders (15%) to various biologics (anti-TNFs (68%), Rituximab (16%), Tocilizumab (13%), Abatacept (3%)). PBMCs were left unstimulated or stimulated (anti-CD3/CD28 beads or 10 ng/mL LPS) and stained with three flow cytometry panels including specific surface, intracellular and intranuclear markers to deeply characterise the function of several subsets of monocytes, NK cells, T cells and B cells (e.g. disease-associated cell populations such as PD-1hiCXCR5- peripheral T helpers (Rao DA Nature 2017), CD27-HLA-DR+ effector CD4+ T cells (Fonseka CY Sci Transl Med 2018) and cytotoxic PD-1+CXCR5- CD8+ T cell subsets, Tbet+CD11c+ autoimmune-associated B cells, CD14+IL1β+ pro-inflammatory monocytes (Zhang F BioRxiv 2018)). Hypothesis-free clustering algorithms were also used for data analysis to identify unreported cell populations. Statistical association testing was preformed using mixed effects ordinal (EULAR response) or linear (DAS28 or its components) regression models. Results Preliminary interim analysis identified associations between pan-biologic non-response and spontaneous expression of pro-inflammatory cytokines (Th1, Th17 lineage) by CD4+ T cells (p Conclusion Ongoing work involves the replication of these results. The identification of immune cell types associated with response to a particular class of biologic drugs (e.g. anti-TNFs, but not other classes) might inform the choice of first line biologic drugs based on individual patients’ immune profiles (stratification to treatment response categories). Cells or cytokines generally associated with non-response to all biologics could represent new therapeutic targets, at least in some patients groups. Acknowledgement AMP RA/SLE Disclosure of Interests Gemma Radley: None declared, Ben Mulhearn: None declared, Laura Donlin: None declared, Jennifer H. Anolik: None declared, Michael Brenner Grant/research support from: Roche: sponsored research agreement on stromal cells (but has nothing to do with checkpoint related disease), Consultant for: GSK: consultant. (I am part of their immunology network, a group of about 8 immunologists who advise them regularly and broadly in the areas of inflammation and infection)., Soumya Raychaudhuri: None declared, Kimme Hyrich Grant/research support from: Grants to institution: BMS, Pfizer, UCB, Ann Morgan: None declared, Gerry Wilson: None declared, John Isaacs Grant/research support from: Pfizer, Grant/research support from: Pfizer, Consultant for: Abbvie, Pfizer, Roche, Galvani, Merck, Gilead, Eli Lilly, Amgen, Janssen, Celltrion, NAPP, Consultant for: Abbvie, Pfizer, Roche, Galvani, Merck, Gilead, Eli Lilly, Amgen, Janssen, Celltrion, NAPP, Speakers bureau: Abbvie, Pfizer, Eli Lilly, Speakers bureau: Abbvie, Pfizer, Eli Lilly, Tracy Hussell: None declared, Anne Barton: None declared, Sebastien Viatte: None declared

The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune-mediated inflammatory diseases (IMIDs)1,2. However, it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue-driven processes observed in IMIDs, such as inflammation and damage3-5. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of fibroblast activation protein-α (FAPα)+ fibroblasts suppressed both inflammation and bone erosions in mouse models of resolving and persistent arthritis. Single-cell transcriptional analysis identified two distinct fibroblast subsets within the FAPα+ population: FAPα+THY1+ immune effector fibroblasts located in the synovial sub-lining, and FAPα+THY1- destructive fibroblasts restricted to the synovial lining layer. When adoptively transferred into the joint, FAPα+THY1- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation, whereas transfer of FAPα+ THY1+ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell-based therapies aimed at modulating inflammation and tissue damage.

Background African-American ethnicity is associated with a 3-fold higher risk of developing systemic lupus erythematosus (SLE). In addition, there is an increased risk of lupus nephritis (2-fold), high-risk histological features, and resistance to treatment. This may account for the increased mortality rate compared to Caucasian patients, especially in women. In Phase One of the Accelerating Medicines Partnership (AMP) study, we used single-cell RNA sequencing on kidney biopsies from patients with active lupus nephritis to identify pathways that were differentially expressed in African-American patients. Methods Single cell RNA sequencing was performed on renal biopsies obtained for clinical purpose for active nephritis using CEL-Seq2. Cell clusters with similar expression profile were identified using t-distributed stochastic neighbor embedding (t-SNE). First, the relative abundance of a cluster in AAs compared to Caucasian was determined using a logistic mixed model. Second, the differential expression profile was determined for each cell cluster and we applied Ingenuity Pathway Analysis (IPA) (QIAGEN Bioinformatics) to identify pathways of interest. Results Samples from 13 AA and 7 Caucasian patients were obtained. Of the 3097 sequenced cell libraries, we used 2354 which passed our quality filter for a total of 30 155 unique molecular identifiers. We identified 16 cell clusters including CD4, CD8, B and plasma cells, NK, myeloid cells, and tubular cells. We identified 2 cell clusters unique to African-American patients, a T and a B cell population with high expression of interferon inducible genes. We also identified that same cell populations may have differential gene expression profiles across ethnicity. For example, CD4 T cells in African-Americans have a higher expression of type 1 and type 2 interferon pathways. In contrast, myeloid cells have several upregulated pathways in Caucasians, including ERK/MAPK signaling. Conclusions African-American lupus nephritis patients may have a stronger interferon pathway activation in infiltrating immune cells. Several other pathways, including ERK/MAPK, are differentially expressed in infiltrating cells based on ethnicity. These results suggest that ethnicity might predict a response to both current and upcoming treatments, paving the way for a more personalized approach to treatment in lupus nephritis. Further work in Phase 2 of AMP will confirm and extend these findings. Funding Source(s): NIH foundation partnership with AbbVie, Biogen, Bristol-Myers Squibb, Celgene, GlaxoSmithKline, Johnson and Johnson, Lilly, Merck, Pfizer, Sanofi, Takeda, and Verily.

The synovium is a mesenchymal tissue composed mainly of fibroblasts, with a lining and sublining that surround the joints. In rheumatoid arthritis the synovial tissue undergoes marked hyperplasia, becomes inflamed and invasive, and destroys the joint1,2. It has recently been shown that a subset of fibroblasts in the sublining undergoes a major expansion in rheumatoid arthritis that is linked to disease activity3-5; however, the molecular mechanism by which these fibroblasts differentiate and expand is unknown. Here we identify a critical role for NOTCH3 signalling in the differentiation of perivascular and sublining fibroblasts that express CD90 (encoded by THY1). Using single-cell RNA sequencing and synovial tissue organoids, we found that NOTCH3 signalling drives both transcriptional and spatial gradients-emanating from vascular endothelial cells outwards-in fibroblasts. In active rheumatoid arthritis, NOTCH3 and Notch target genes are markedly upregulated in synovial fibroblasts. In mice, the genetic deletion of Notch3 or the blockade of NOTCH3 signalling attenuates inflammation and prevents joint damage in inflammatory arthritis. Our results indicate that synovial fibroblasts exhibit a positional identity that is regulated by endothelium-derived Notch signalling, and that this stromal crosstalk pathway underlies inflammation and pathology in inflammatory arthritis.

How innate T cells (ITC), including invariant natural killer T (iNKT) cells, mucosal-associated invariant T (MAIT) cells, and γδ T cells, maintain a poised effector state has been unclear. Here we address this question using low-input and single-cell RNA-seq of human lymphocyte populations. Unbiased transcriptomic analyses uncover a continuous ‘innateness gradient’, with adaptive T cells at one end, followed by MAIT, iNKT, γδ T and natural killer cells at the other end. Single-cell RNA-seq reveals four broad states of innateness, and heterogeneity within canonical innate and adaptive populations. Transcriptional and functional data show that innateness is characterized by pre-formed mRNA encoding effector functions, but impaired proliferation marked by decreased baseline expression of ribosomal genes. Together, our data shed new light on the poised state of ITC, in which innateness is defined by a transcriptionally-orchestrated trade-off between rapid cell growth and rapid effector function., Innate T cells (ITC) contain many subsets and are poised to promptly respond to antigens and pathogens, but how this poised state is maintained is still unclear. Here the authors perform single-cell RNA-seq to align the various ITC subsets along an ‘innateness gradient’ that is associated with changes in proliferation and effector functions.

Macrophages tailor their function to the signals found in tissue microenvironments, taking on a wide spectrum of phenotypes. In human tissues, a detailed understanding of macrophage phenotypes is limited. Using single-cell RNA-sequencing, we define distinct macrophage subsets in the joints of patients with the autoimmune disease rheumatoid arthritis (RA), which affects ~1% of the population. The subset we refer to as HBEGF+inflammatory macrophages is enriched in RA tissues and shaped by resident fibroblasts and the cytokine TNF. These macrophages promote fibroblast invasiveness in an EGF receptor dependent manner, indicating that inflammatory intercellular crosstalk reshapes both cell types and contributes to fibroblast-mediated joint destruction. In anex vivotissue assay, the HBEGF+inflammatory macrophage is targeted by several anti-inflammatory RA medications, however, COX inhibition redirects it towards a different inflammatory phenotype that is also expected to perpetuate pathology. These data highlight advances in understanding the pathophysiology and drug mechanisms in chronic inflammatory disorders can be achieved by focusing on macrophage phenotypes in the context of complex interactions in human tissues.One Sentence SummaryA newly identified human macrophage phenotype from patients with the autoimmune condition RA is found to promote joint tissue invasiveness and demonstrates variable sensitivities to anti-inflammatory medications used to treat the disease.

Adipose-resident invariant natural killer T (iNKT) cells are key players in metabolic regulation. iNKT cells are innate lipid sensors, and their activation, using their prototypic ligand α-galactosylceramide (αGalCer), induces weight loss and restores glycemic control in obesity. Here, iNKT activation induced fibroblast growth factor 21 (FGF21) production and thermogenic browning of white fat. Complete metabolic analysis revealed that iNKT cell activation induced increased body temperature, V02, VC02, and fatty acid oxidation, without affecting food intake or activity. FGF21 induction played a major role in iNKT cell-induced weight loss, as FGF21 null mice lost significantly less weight after αGalCer treatment. The glucagon-like peptide 1 (GLP-1) receptor agonist, liraglutide, also activated iNKT cells in humans and mice. In iNKT-deficient mice, liraglutide promoted satiety but failed to induce FGF21, resulting in less weight loss. These findings reveal an iNKT cell-FGF21 axis that defines a new immune-mediated pathway that could be targeted for glycemic control and weight regulation.

Invariant natural killer T (iNKT) cells are a specialized T cell subset that plays an important role in host defense, orchestrating both innate and adaptive immune effector responses against a variety of microbes. Specific microbial lipids and mammalian self lipids displayed by the antigen-presenting molecule CD1d can activate iNKT cells through their semi-invariant αβ T cell receptors (TCRs). iNKT cells also constitutively express receptors for inflammatory cytokines typically secreted by antigen-presenting cells (APCs) after recognition of pathogen-associated molecular patterns (PAMPs), and they can be activated through these cytokine receptors either in combination with TCR signals, or in some cases even in the absence of TCR signaling. During infection, experimental evidence suggests that both TCR-driven and cytokine-driven mechanisms contribute to iNKT cell activation. While the relative contributions of these two signaling mechanisms can vary widely depending on the infectious context, both lipid antigens and PAMPs mediate reciprocal activation of iNKT cells and APCs, leading to downstream activation of multiple other immune cell types to promote pathogen clearance. In this review, we discuss the mechanisms involved in iNKT cell activation during infection, focusing on the central contributions of both lipid antigens and PAMP-induced inflammatory cytokines, and highlight in vivo examples of activation during bacterial, viral, and fungal infections.