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The impact of exposure to air pollutants, such as fine particulate matter (PM), on the immune system and its consequences on pediatric asthma are not well understood. We investigated whether the ambient levels of fine PM with aerodynamic diameter ≤ 2.5 microns (PM ) are associated with alterations in circulating monocytes in children with or without asthma. Increased exposure to ambient PM was linked to specific monocyte subtypes, particularly in children with asthma. Mechanistically, we hypothesized that innate trained immunity is evoked by a primary exposure to fine PM and accounts for an enhanced inflammatory response after secondary stimulation in vitro. We determined that the trained immunity was induced in circulating monocytes by fine particulate pollutants, and it was characterized by upregulation of proinflammatory mediators, such as TNF, IL-6, and IL-8, upon stimulation with house dust mite or LPS. This phenotype was epigenetically controlled by enhanced H3K27ac marks in circulating monocytes. The specific alterations of monocytes after ambient pollution exposure suggest a possible prognostic immune signature for pediatric asthma, and pollution-induced trained immunity may provide a potential therapeutic target for asthmatic children living in areas with increased air pollution.

ObjectivesDrug reaction with eosinophilia and systemic symptoms (DRESS) is a severe, delayed hypersensitivity reaction (DHR). We observed DRESS to inhibitors of interleukin 1 (IL-1) or IL-6 in a small group of patients with Still’s disease with atypical lung disease. We sought to characterise features of patients with Still’s disease with DRESS compared with drug-tolerant Still’s controls. We analysed human leucocyte antigen (HLA) alleles for association to inhibitor-related DHR, including in a small Kawasaki disease (KD) cohort.MethodsIn a case/control study, we collected a multicentre series of patients with Still’s disease with features of inhibitor-related DRESS (n=66) and drug-tolerant Still’s controls (n=65). We retrospectively analysed clinical data from all Still’s subjects and typed 94/131 for HLA. European Still’s-DRESS cases were ancestry matched to International Childhood Arthritis Genetics Consortium paediatric Still’s cases (n=550) and compared for HLA allele frequencies. HLA association also was analysed using Still’s-DRESS cases (n=64) compared with drug-tolerant Still’s controls (n=30). KD subjects (n=19) were similarly studied.ResultsStill’s-DRESS features included eosinophilia (89%), AST-ALT elevation (75%) and non-evanescent rash (95%; 88% involving face). Macrophage activation syndrome during treatment was frequent in Still’s-DRESS (64%) versus drug-tolerant Still’s (3%; p=1.2×10−14). We found striking enrichment for HLA-DRB1*15 haplotypes in Still’s-DRESS cases versus INCHARGE Still’s controls (p=7.5×10-13) and versus self-identified, ancestry-matched Still’s controls (p=6.3×10−10). In the KD cohort, DRB1*15:01 was present only in those with suspected anakinra reactions.ConclusionsDRESS-type reactions occur among patients treated with IL-1/IL-6 inhibitors and strongly associate with common HLA-DRB1*15 haplotypes. Consideration of preprescription HLA typing and vigilance for serious reactions to these drugs are warranted.

Objective To assess the attitudes and common practices of adult rheumatologists in the US regarding health care transition (HCT) for young adults with rheumatic diseases. Methods An anonymous online survey was sent to US adult rheumatologist members of the American College of Rheumatology to collect demographic data and information on attitudes and common practices regarding the transition process. Results Of 4,064 contacted rheumatologists, 203 (5%) completed the survey. Almost half of respondents (45.1%) were never trained in transition practices, and 74.7% were not familiar with the American Academy of Pediatrics/American Academy of Family Physicians/American College of Physicians Consensus Statement About Transitions for Youth with Special Healthcare Needs. Only 56.2% felt comfortable caring for former pediatric patients. The vast majority of respondents (90.7%) did not have a multidisciplinary transition team, and 37% did not have a plan for transitioning pediatric patients into their practice. Most adult rheumatologists were unsatisfied with the current transition process (92.9%), due to insufficient resources, personnel (91.1%), and time in clinic (86.9%). They also were unsatisfied with referral data received concerning previous treatments (48.9%), hospitalization history (48%), disease activity index (45.1%), medical history summary (43.9%), comorbidities (36.4%), medication list (34.1%), and disease classification (32.6%). Three major barriers to HCT were lack of insurance reimbursement (33.7%), knowledge about community resources (30.8%), and lapses in care between primary provider and specialist (27.8%). Conclusion This survey identified substantial gaps in knowledge and resources regarding HCT for young adults with rheumatic diseases. These may be best addressed by further training, research, dedicated resources, adequate payment, and practice guidelines.

A damaging inflammatory response is implicated in the pathogenesis of severe coronavirus disease 2019 (COVID-19), but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated immunoglobulin G (IgG) antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were associated with progression from mild to more severe COVID-19. To study the biology of afucosylated IgG immune complexes, we developed an in vivo model that revealed that human IgG–Fc-gamma receptor (FcγR) interactions could regulate inflammation in the lung. Afucosylated IgG immune complexes isolated from patients with COVID-19 induced inflammatory cytokine production and robust infiltration of the lung by immune cells. In contrast to the antibody structures that were associated with disease progression, antibodies that were elicited by messenger RNA SARS-CoV-2 vaccines were highly fucosylated and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. Vaccine-elicited IgG did not promote an inflammatory lung response. These results show that human IgG-FcγR interactions regulate inflammation in the lung and define distinct lung activities mediated by the IgG that are associated with protection against, or progression to, severe COVID-19.

Mapping MHC-II binding peptides derived from an antigenic protein for potential CD4+ T-cell epitopes has been challenging due to a lack of experimental approaches that are both quantitative and rapid. The rate-limiting steps in current approaches include the construction of single MHC allele expressing cell lines and/or the purification of the MHC-II allelic proteins for peptide elution (i.e., mass spectrometry) or in vitro peptide binding (i.e., ELISA) assays. These labor-intensive steps typically take up to 4 months or more. In this protocol, we describe a system that uses yeast cells to display "empty" (i.e., without covalently linked peptides) MHC-II heterodimers that are capable of binding exogenously added peptides of interest. This yeast-MHC-II system eliminates the time-consuming soluble MHC-II purification steps, allowing rapid identification of peptide ligands from protein antigens (RIPPA). The amount of peptide loading to MHC-II or the extent of competition between indicator and competitor peptides at the surface of yeast cells can be quantitatively determined using flow cytometric analysis. Importantly, the protocol only takes ∼1 month from the construction of plasmids and the yeast display of "empty" MHC-II to the quantitative determination of MHC-II binding peptides from a given antigen. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Yeast display of "empty" MHC-II Support Protocol: Construction of yeast shuttle vector expressing "empty" MHC-II Basic Protocol 2: Peptide competition on the surface of yeast cells Alternate Protocol: RIPPA in a 96-well format.

Major histocompatibility complex class II (MHC-II) antigen presentation underlies a wide range of immune responses in health and disease. However, how MHC-II antigen presentation is regulated by the peptide-loading catalyst HLA-DM (DM), its associated modulator, HLA-DO (DO), is incompletely understood. This is due largely to technical limitations: model antigen presenting cell (APC) systems that express these MHC-II peptidome regulators at physiologically variable levels have not been described. Likewise, computational prediction tools that account for DO and DM activities are not presently available. To address these gaps, we created a panel of single MHC-II allele, HLA-DR4-expressing APC lines that cover a wide range of DO:DM ratio states. Using a combined immunopeptidomic and proteomic discovery strategy, we measured the effects DO:DM ratios have on peptide presentation by surveying over 10,000 unique DR4-presented peptides. The resulting data provide insight into peptide characteristics that influence their presentation with increasing DO:DM ratios. These include DM-sensitivity, peptide abundance, binding affinity and motif, peptide length and register positioning on the source protein. These findings have implications for designing improved HLA-II prediction algorithms and research strategies for dissecting the variety of functions that different APCs serve in the body.IN BRIEFPeptides presented by MHC-II are critical to adaptive immune function. The non-canonical MHC molecules HLA-DM and HLA-DO cooperatively regulate MHC-II function, but how varied DO-to-DM ratios across different APCs and cellular contexts might influence their immunopeptide repertoires is unclear. We address this by measuring cell lines expressing these two proteins spanning a range of relative abundances. We found that peptides could be categorized according to how robustly they were presented at different DO:DM ratios. Importantly, this presentation was only partially linked to predicted affinity to the MHC-II molecule.HIGHLIGHTSDescribe MHC-class II peptide repertoires from a unique HLA-DR4 cell line panel with increasing DO:DM ratios.Demonstrate striking and divergent changes in MHC-II immunopeptidomes that result from the tuning function of DO:DM.These findings bridge gap in understanding and predicting MHC-II antigen presentation.GRAPHICAL ABSTRACT

Structural variation in the complement 4 gene (C4) confers genetic risk for schizophrenia. The variation includes numbers of the increased C4A copy number, which predicts increased C4A mRNA expression. C4-anaphylatoxin (C4-ana) is a C4 protein fragment released upon C4 protein activation that has the potential to change the blood–brain barrier (BBB). We hypothesized that elevated plasma levels of C4-ana occur in individuals with schizophrenia (iSCZ). Blood was collected from 15 iSCZ with illness duration < 5 years and from 14 healthy controls (HC). Plasma C4-ana was measured by radioimmunoassay. Other complement activation products C3-ana, C5-ana, and terminal complement complex (TCC) were also measured. Digital-droplet PCR was used to determine C4 gene structural variation state. Recombinant C4-ana was added to primary brain endothelial cells (BEC) and permeability was measured in vitro. C4-ana concentration was elevated in plasma from iSCZ compared to HC (mean = 654 ± 16 ng/mL, 557 ± 94 respectively, p = 0.01). The patients also carried more copies of the C4AL gene and demonstrated a positive correlation between plasma C4-ana concentrations and C4A gene copy number. Furthermore, C4-ana increased the permeability of a monolayer of BEC in vitro. Our findings are consistent with a specific role for C4A protein in schizophrenia and raise the possibility that its activation product, C4-ana, increases BBB permeability. Exploratory analyses suggest the novel hypothesis that the relationship between C4-ana levels and C4A gene copy number could also be altered in iSCZ, suggesting an interaction with unknown genetic and/or environmental risk factors.

Store-operated calcium entry (SOCE) modulates cytosolic calcium in multiple cells. Endoplasmic reticulum (ER) localized STIM1 and plasma membrane (PM)-localized ORAI1 are two main components of SOCE. STIM1:ORAI1 association requires STIM1 oligomerization, its re-distribution to ER-PM junctions, and puncta formation. However, little is known about the negative regulation of these steps to prevent calcium overload. Here, we identified Tmem178 as a negative modulator of STIM1 puncta formation in myeloid cells. Using site-directed mutagenesis, co-immunoprecipitation assays and FRET imaging, we determined that Tmem178:STIM1 association occurs via their transmembrane motifs. Mutants that increase Tmem178:STIM1 association reduce STIM1 puncta formation, SOCE activation, impair inflammatory cytokine production in macrophages and osteoclastogenesis. Mutants that reduce Tmem178:STIM1 association reverse these effects. Furthermore, exposure to plasma from arthritic patients decreases Tmem178 expression, enhances SOCE activation and cytoplasmic calcium. In conclusion, Tmem178 modulates the rate-limiting step of STIM1 puncta formation and therefore controls SOCE in inflammatory conditions.

We have reported that the major histocompatibility molecule HLA-DQ2 (DQA1*05:01/DQB1*02:01) (DQ2) is relatively resistant to HLA-DM (DM), a peptide exchange catalyst for MHC class II. In this study, we analyzed the role of DQ2/DM interaction in the generation of DQ2-restricted gliadin epitopes, relevant to celiac disease, or DQ2-restricted viral epitopes, relevant to host defense. We used paired human APC, differing in DM expression (DMnull versus DMhigh) or differing by expression of wild-type DQ2, versus a DM-susceptible, DQ2 point mutant DQ2α+53G. The APC pairs were compared for their ability to stimulate human CD4+ T cell clones. Despite higher DQ2 levels, DMhigh APC attenuated T cell responses compared with DMnull APC after intracellular generation of four tested gliadin epitopes. DMhigh APC expressing the DQ2α+53G mutant further suppressed these gliadin-mediated responses. The gliadin epitopes were found to have moderate affinity for DQ2, and even lower affinity for the DQ2 mutant, consistent with DM suppression of their presentation. In contrast, DMhigh APC significantly promoted the presentation of DQ2-restricted epitopes derived intracellularly from inactivated HSV type 2, influenza hemagglutinin, and human papillomavirus E7 protein. When extracellular peptide epitopes were used as Ag, the DQ2 surface levels and peptide affinity were the major regulators of T cell responses. The differential effect of DM on stimulation of the two groups of T cell clones implies differences in DQ2 presentation pathways associated with nonpathogen- and pathogen-derived Ags in vivo.

The effector activity of IgG antibodies is regulated at several levels, including IgG subclass, modifications of the Fc glycan, and the distribution of Type I and II Fcγ receptors (FcγR) on effector cells. Here, we explore how Fc glycosylation, particularly sialylation and fucosylation, tunes cellular responses to immune complexes. We review the current understanding of the pathways and mechanisms underlying this biology, address FcγR in antigen presentation, and discuss aspects of the clinical understanding of Fc glycans in therapies and disease.

Serologic markers that predict severe COVID-19 disease trajectories could enable early medical interventions and reduce morbidity and mortality. We found that distinct features of IgG Fab and Fc domain structures were present within three days of a positive test that predicted two separate disease trajectories in a prospective cohort of patients with COVID-19. One trajectory was defined by early production of neutralizing antibodies and led to mild disease. A distinct trajectory, characterized by an initial period of mild symptoms followed by rapid progression to more severe outcomes, was predicted by the absence of early neutralizing antibody responses with concomitant production of afucosylated IgGs. Elevated frequencies of monocytes expressing the receptor for afucosylated IgGs, FcγRIIIa (CD16a), were an additional antecedent in patients with the more severe outcomes. In mechanistic studies, afucosylated immune complexes in the lung triggered an inflammatory infiltrate and cytokine production that was dependent on CD16a. Finally, in healthy subjects, mRNA SARS-CoV-2 vaccination elicited neutralizing antibodies that were enriched for Fc fucosylation and sialylation and distinct from both infection-induced trajectories. These data show the importance of combined Fab and Fc domain functions in the antiviral response, define an early antibody signature in people who progressed to more severe COVID-19 outcomes and have implications for novel therapeutic strategies targeting FcγRIIIa pathways.

In addition to their role as antibody producing cells, B cells make a critical contribution to adaptive immune responses by functioning as professional antigen-presenting cells (APC). Distinctive features of B cells as APC include the expression of the B cell receptor (BCR) for antigen and regulated expression of HLA-DO. Here, we discuss recent progress in investigation of B cells as APC. We start with an update on the canonical MHC class II antigen presentation pathway in B cells and alternative pathways, including generation of extracellular vesicles. Turning to APC function, we highlight the roles of B cells as thymic APC, as APC for T follicular helper (TFH), as APC for CD4 memory T cells and as presenters of idiotypic BCR determinants. We also note recent examples that link B cell Ag-presentation to disease. Emerging evidence indicates that, in addition to unique features of B cells compared to other professional APC, there is appreciable heterogeneity among B cells, arising from, for example, B cell activation state or the microenvironment.

Juvenile idiopathic arthritis (JIA) is a chronic inflammatory disease affecting the joints and other organs that occurs in 1 in 1,000 children in the United States. Given the various categories of JIA, interpretation of the literature can be difficult. In this review, new developments in understanding non-systemic JIA and its treatment will be covered. Recent advances in the journey toward personalized treatment in JIA will be highlighted, including a review of currently available biologic modifiers. Uveitis and the temporomandibular joint will be discussed as particularly challenging treatment issues. Recent guideline updates and literature-guided treatment decisions will be reviewed.

Monocytes and monocyte-derived cells play important roles in the regulation of inflammation, both as precursors as well as effector cells. Monocytes are heterogeneous and characterized by three distinct subsets in humans. Classical and non-classical monocytes represent the most abundant subsets, each carrying out distinct biological functions. Consequently, altered frequencies of different subsets have been associated with inflammatory conditions, such as infections and autoimmune disorders including lupus, rheumatoid arthritis, inflammatory bowel disease, and, more recently, COVID-19. Dissecting the contribution of different monocyte subsets to disease is currently limited by samples and cohorts, often resulting in underpowered studies and, consequently, poor reproducibility. Public transcriptomes provide an alternative source of data characterized by high statistical power and real world heterogeneity. However, most transcriptome datasets profile bulk blood or tissue samples, requiring the use of in silico approaches to quantify changes in the levels of specific cell types.Here, we integrated 853 publicly available microarray expression profiles of sorted human monocyte subsets from 45 independent studies to identify robust and parsimonious gene expression signatures, consisting of 10 genes specific to each subset. These signatures, although derived using only datasets profiling healthy individuals, maintain their accuracy independent of the disease state in an independent cohort profiled by RNA-sequencing (AUC = 1.0). Furthermore, we demonstrate that our signatures are specific to monocyte subsets compared to other immune cells such as B, T, dendritic cells (DCs) and natural killer (NK) cells (AUC = 0.87~0.88, p

CD4+ T cells orchestrate adaptive immune responses via binding of antigens to their receptors through specific peptide/MHC-II complexes. To study these responses, it is essential to identify protein-derived MHC-II peptide ligands that constitute epitopes for T cell recognition. However, generating cells expressing single MHC-II alleles and isolating these proteins for use in peptide elution or binding studies is time consuming. Here, we express human MHC alleles (HLA-DR4 and HLA-DQ6) as native, noncovalent αβ dimers on yeast cells for direct flow cytometry-based screening of peptide ligands from selected antigens. We demonstrate rapid, accurate identification of DQ6 ligands from pre-pro-hypocretin, a narcolepsy-related immunogenic target. We also identify 20 DR4-binding SARS-CoV-2 spike peptides homologous to SARS-CoV-1 epitopes, and one spike peptide overlapping with the reported SARS-CoV-2 epitope recognized by CD4+ T cells from unexposed individuals carrying DR4 subtypes. Our method is optimized for immediate application upon the emergence of novel pathogens.

Major histocompatibility complex class II (MHCII) molecules display peptides on antigen-presenting cells (APCs) for inspection by CD4+ T cells. MHCII surface levels and life span are regulated post-translationally by peptide loading and ubiquitin-dependent lysosomal targeting, but proteases responsible for MHCII protein degradation remain unidentified. Here, we examined the role of aspartyl proteases in MHCII protein degradation and characterised the form of MHCII that is degraded. Exposure of immature monocyte-derived dendritic cells (MoDCs) and KG-1 acute myeloid leukaemia cells to the aspartyl protease inhibitor, pepstatin A (PepA), caused accumulation of human leukocyte antigen (HLA)-DR molecules in intracellular vesicles. Statistically significant PepA effects on MHCII protein expression were also observed in murine APCs. In KG-1 cells, cathepsin D (CatD) was the sole expressed aspartyl protease, and shRNA-mediated knockdown ablated the PepA effect, providing formal proof of CatD involvement. In vitro, CatD initiated specific cleavage of recombinant DR at αF54, a site flanking the peptide-binding groove. Immunochemical characteristics of PepA-rescued DR molecules in KG-1 cells were consistent with selective CatD attack on HLA-DR molecules that lack association with the MHCII chaperone, invariant chain, or with stably bound peptide. We propose that CatD has a critical role in the selective lysosomal disposal of mature HLA-DR molecules that have lost, or never acquired, bound peptide, explaining how MHCII protein life span is coupled to peptide loading.

Approximately 5% of children with juvenile idiopathic arthritis (JIA) are diagnosed with the psoriatic form of the disease. In recent years, there has been substantial scholarship demonstrating both heterogeneity within the disease as well as similarities with other forms of JIA, culminating in a recent proposal for the categorization of JIA that excluded the psoriatic form altogether. The purpose of the review is to summarize the clinical, epidemiologic, and genetic features of psoriatic JIA (PsJIA), comparing it with other categories of JIA including spondyloarthritis. We conclude that there are sufficient unique clinical and genetic features within PsJIA as well as similarities with its adult counterpart that warrant including it within the JIA paradigm.

We were very interested to read the correspondence from Dr Bader-Meunier and colleagues, who report a case of systemic juvenile idiopathic arthritis (sJIA) with associated lung disease.1 This case indeed matches the description of and provides further evidence for the striking and unusual clinical characteristics we recently detailed in an international case series of such patients.2 We thank the authors for this communication. One aspect of particular importance in the reported case is the development of drug reaction with eosinophilia and systemic symptoms (DReSS), a delayed hypersensitivity reaction, to canakinumab (monoclonal antibody to interleukin (IL)-1β). As we noted, DReSS is a possibly under-recognised event preceding the clinical recognition or development of this lung disorder in at least a subset of patients. …

Juvenile psoriatic arthritis (JPsA), a subtype of juvenile idiopathic arthritis (JIA), constitutes 5% of JIA. The literature is inconsistent regarding features of JPsA, and physicians debate whether it is a distinct entity within JIA. A biphasic age of onset distribution has been noted. Early-onset disease is characterized by female predominance, small joint involvement, dactylitis, and positive antinuclear antibodies. Late-onset JPsA resembles adult-onset psoriatic arthritis (PsA), with male predominance, psoriasis, enthesitis, and axial disease. Recent studies report improved outcomes, likely due to the widespread use of traditional and biologic disease-modifying antirheumatic drugs. Conflicting HLA associations have been reported in JPsA, but notably both HLA class I and II allele associations are suggested. Similar to PsA cohorts, subjects with JPsA have a lower frequency of a protective interleukin 23R allele than controls or other JIA subtypes. Data in the Childhood Arthritis and Rheumatology Research Alliance (CARRA) patient registry suggest the aggressive characteristics of JPsA: 24.6% of children have joint damage 4.6 years after symptom onset. Pediatric and adult PsA classification criteria define different JPsA cohorts within the registry and support a previous suggestion that the International League of Associations for Rheumatology criteria for JPsA may be overly stringent. Increased collaboration between pediatric and adult physicians and comparative research on these clinically related conditions are warranted.

Objective.Adult patients with psoriatic arthritis are at increased risk for obesity and metabolic syndrome, but data regarding adiposity in children with juvenile psoriatic arthritis (JPsA) are limited. Our study assessed adiposity in children with JPsA in the Childhood Arthritis and Rheumatology Research Alliance (CARRA) registry.Methods.Patients with JPsA in the CARRA registry were divided into nonoverweight and overweight groups using recommendations from the US Centers for Disease Control, and differences in demographic and clinical characteristics between groups at baseline and after 1-year followup were assessed using chi-square test, Fisher’s exact test, T test, or Mann-Whitney U test, as appropriate. The prevalence of overweight status in the JPsA registry patients was compared to rheumatoid factor–positive and −negative polyarticular juvenile idiopathic arthritis (RF+polyJIA; RF−polyJIA) registry cohorts and the US pediatric population, using a chi-square goodness-of-fit test.Results.Overweight children represented 36.3% of this JPsA cohort (n = 320). Compared to nonoverweight children, they were significantly older at symptom onset and rheumatologist’s first assessment, and scored significantly worse on patient/physician outcome measures. At 1-year followup, changes in body mass index were not associated with changes in clinical features or outcome measures. The prevalence of overweight and obesity in patients with JPsA was significantly higher than in RF+polyJIA patients, RF−polyJIA patients, and the US pediatric population.Conclusion.In this registry, almost 1 in 5 patients with JPsA were obese and more than one-third were overweight. This is significantly more than expected compared to the US pediatric population, and appropriate longterm followup of this JPsA subgroup is warranted.

Individuals with narcolepsy suffer from abnormal sleep patterns due to loss of neurons that uniquely supply hypocretin (HCRT). Previous studies found associations of narcolepsy with the human leukocyte antigen (HLA)-DQ6 allele and T-cell receptor α (TRA) J24 gene segment and also suggested that in vitro-stimulated T cells can target HCRT. Here, we present evidence of in vivo expansion of DQ6-HCRT tetramer

Chromatin undergoes extensive reprogramming during immune cell differentiation. Here we report the repression of controlled histone H3 amino terminus proteolytic cleavage (H3ΔN) during monocyte-to-macrophage development. This abundant histone mark in human peripheral blood monocytes is catalyzed by neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase and proteinase 3. NSPs are repressed as monocytes mature into macrophages. Integrative epigenomic analysis reveals widespread H3ΔN distribution across the genome in a monocytic cell line and primary monocytes, which becomes largely undetectable in fully differentiated macrophages. H3ΔN is enriched at permissive chromatin and actively transcribed genes. Simultaneous NSP depletion in monocytic cells results in H3ΔN loss and further increase in chromatin accessibility, which likely primes the chromatin for gene expression reprogramming. Importantly, H3ΔN is reduced in monocytes from patients with systemic juvenile idiopathic arthritis, an autoinflammatory disease with prominent macrophage involvement. Overall, we uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development. Chromatin undergoes extensive reprogramming during immune cell differentiation. Here Kuo and colleagues uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development.

Dysregulation of monocyte and macrophage responses are often observed in children with systemic juvenile idiopathic arthritis (sJIA) and cytokine storm syndrome (CSS), a potentially fatal complication of chronic rheumatic diseases. Both conditions are associated with activation of TLR signaling in monocyte and macrophage lineage cells, leading to overwhelming inflammatory responses. Despite the importance of TLR engagement in activating proinflammatory macrophages, relatively little is known about activation of intrinsic negative regulatory pathways to attenuate excessive inflammatory responses. In this study, we demonstrate that loss of diacylglycerol (DAG) kinase (Dgk) ζ, an enzyme which converts DAG into phosphatidic acid, limits inflammatory cytokine production in an arthritic mouse model dependent on TLR2 signaling and in a CSS mouse model dependent on TLR9 signaling. In vitro, Dgkζ deficiency results in reduced production of TNF-α, IL-6, and IL-1β and in limited M1 macrophage polarization. Mechanistically, Dgkζ deficiency decreases STAT1 and STAT3 phosphorylation. Moreover, Dgkζ levels are increased in macrophages derived from mice with CSS or exposed to plasma from sJIA patients with active disease. Our data suggest that Dgkζ induction in arthritic conditions perpetuates systemic inflammatory responses mediated by macrophages and highlight a potential role of Dgkζ–DAG/phosphatidic acid axis as a modulator of inflammatory cytokine production in sJIA and CSS.

Individuals with narcolepsy suffer from abnormal sleep patterns due to loss of neurons that uniquely supply hypocretin (HCRT). Previous studies found associations of narcolepsy with the human leukocyte antigen (HLA)-DQ6 allele and T-cell receptor α (TRA) J24 gene segment and also suggested that in vitro-stimulated T cells can target HCRT. Here, we present evidence of in vivo expansion of DQ6-HCRT tetramer+/TRAJ24+/CD4+ T cells in DQ6+ individuals with and without narcolepsy. We identify related TRAJ24+ TCRαβ clonotypes encoded by identical α/β gene regions from two patients and two controls. TRAJ24-G allele+ clonotypes only expand in the two patients, whereas a TRAJ24-C allele+ clonotype expands in a control. A representative tetramer+/G-allele+ TCR shows signaling reactivity to the epitope HCRT87–97. Clonally expanded G-allele+ T cells exhibit an unconventional effector phenotype. Our analysis of in vivo expansion of HCRT-reactive TRAJ24+ cells opens an avenue for further investigation of the autoimmune contribution to narcolepsy development., T cells from narcolepsy patients were recently reported to recognize hypocretin, a wakefulness-promoting neurohormone, suggesting autoimmune origin of the disease. Here the authors show that hypocretin-specific T cells expand both in healthy controls and in narcolepsy patients, and identify preliminary features that may distinguish them.

The complicated clinical picture and biomolecular pattern of human autoimmune diseases (ADs) make knowledge on their etiology still fragmentary. The diagnostic approaches for ADs require improvement both for clinical and research effort to progress. Synthetic biomolecular antigens find growing applications for diagnosis and investigation of ADs. The main goal of this work is to detect interaction between synthetic antigens and autoantibodies in systemic lupus erythematosus within a combined, high-throughput assay. A panel of synthetic antigens has been prepared from DNA, RNA, locked nucleic acids and apolipoprotein H. The binding of synthetic antigens to autoantibodies has been confirmed in sera samples from those with active systemic lupus erythematosus (SLE) by indirect enzyme linked immunosorbent assay. Our study provides an efficient methodology for combined autoantibody profiling in SLE.

The complicated clinical picture and biomolecular pattern of human autoimmune diseases (ADs) make knowledge on their etiology still fragmentary. The diagnostic approaches for ADs require improvement both for clinical and research effort to progress. Synthetic biomolecular antigens find growing applications for diagnosis and investigation of ADs. The main goal of this work is to detect interaction between synthetic antigens and autoantibodies in systemic lupus erythematosus within a combined, high-throughput assay. A panel of synthetic antigens has been prepared from DNA, RNA, locked nucleic acids and apolipoprotein H. The binding of synthetic antigens to autoantibodies has been confirmed in sera samples from those with active systemic lupus erythematosus (SLE) by indirect enzyme linked immunosorbent assay. Our study provides an efficient methodology for combined autoantibody profiling in SLE.

B cell receptors and surface-displayed peptide/MHCII complexes constitute two key components of the B-cell machinery to sense signals and communicate with other cell types during antigen-triggered activation. However, critical pathways synergizing antigen-BCR interaction and antigenic peptide-MHCII presentation remain elusive. Here, we report the discovery of factors involved in establishing such synergy. We applied a single-cell measure coupled with super-resolution microscopy to investigate the integrated function of two lysosomal regulators for peptide loading, HLA-DM and HLA-DO. In model cell lines and human tonsillar B cells, we found that tunable DM/DO stoichiometry governs DMfree activity for exchange of placeholder CLIP peptides with high affinity MHCII ligands. Compared to their naïve counterparts, memory B cells with less DMfree concentrate a higher proportion of CLIP/MHCII in lysosomal compartments. Upon activation mediated by high affinity BCR, DO tuning is synchronized with antigen internalization and rapidly potentiates DMfree activity to optimize antigen presentation for T-cell recruitment.

Background & Aims Intestinal microfold (M) cells are a unique subset of intestinal epithelial cells in the Peyer's patches that regulate mucosal immunity, serving as portals for sampling and uptake of luminal antigens. The inability to efficiently develop human M cells in cell culture has impeded studies of the intestinal immune system. We aimed to identify signaling pathways required for differentiation of human M cells and establish a robust culture system using human ileum enteroids. Methods We analyzed transcriptome data from mouse Peyer's patches to identify cell populations in close proximity to M cells. We used the human enteroid system to determine which cytokines were required to induce M-cell differentiation. We performed transcriptome, immunofluorescence, scanning electron microscope, and transcytosis experiments to validate the development of phenotypic and functional human M cells. Results A combination of retinoic acid and lymphotoxin induced differentiation of glycoprotein 2-positive human M cells, which lack apical microvilli structure. Upregulated expression of innate immune-related genes within M cells correlated with a lack of viral antigens after rotavirus infection. Human M cells, developed in the enteroid system, internalized and transported enteric viruses, such as rotavirus and reovirus, across the intestinal epithelium barrier in the enteroids. Conclusions We identified signaling pathways required for differentiation of intestinal M cells, and used this information to create a robust culture method to develop human M cells with capacity for internalization and transport of viruses. Studies of this model might increase our understanding of antigen presentation and the systemic entry of enteric pathogens in the human intestine.

Pulse-chase analysis is a commonly used technique for studying the synthesis, processing, and transport of proteins. Cultured cells expressing proteins of interest are allowed to take up radioactively labeled amino acids for a brief interval ("pulse"), during which all newly synthesized proteins incorporate the label. The cells are then returned to nonradioactive culture medium for various times ("chase"), during which proteins may undergo conformational changes, trafficking, or degradation. Proteins of interest are isolated (usually by immunoprecipitation) and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the fate of radiolabeled molecules is examined by autoradiography. This chapter describes a pulse-chase protocol suitable for studies of major histocompatibility complex (MHC) class II biosynthesis and maturation. We discuss how results are affected by the recognition by certain anti-class II antibodies of distinct class II conformations associated with particular biosynthetic states. Our protocol can be adapted to follow the fate of many other endogenously synthesized proteins, including viral or transfected gene products, in cultured cells.

Our understanding of the mechanisms underlying HLA associations with inflammatory arthritis continues to evolve. Disease associations have been refined, and interactions of HLA genotype with other genes and environmental risk factors in determining disease risk have been identified. This Review provides basic information on the genetics and molecular function of HLA molecules, as well as general features of HLA associations with disease. Evidence is discussed regarding the various peptide-dependent and peptide-independent mechanisms by which HLA alleles might contribute to the pathogenesis of three types of inflammatory arthritis: rheumatoid arthritis, spondyloarthritis and systemic juvenile idiopathic arthritis. Also discussed are HLA allelic associations that shed light on the genetic heterogeneity of inflammatory arthritides and on the relationships between adult and paediatric forms of arthritis. Clinical implications range from improved diagnosis and outcome prediction to the possibility of using HLA associations in developing personalized strategies for the treatment and prevention of these diseases.

Cytokine storm syndrome (CSS) is a life-threatening condition characterized by excessive activation of T cells and uncontrolled inflammation, mostly described in patients with familial hemophagocytic lymphohistiocytosis and certain systemic auto-inflammatory diseases, such as systemic juvenile idiopathic arthritis (sJIA). Defects in T cell cytotoxicity as a mechanism for uncontrolled inflammation following viral infections fail to represent the whole spectrum of CSS. Evidence implicates dysregulated innate immune responses, especially activation of monocytes and macrophages, in patients with CSS. However, the direct contribution of monocytes/macrophages to CSS development and the signaling pathways involved in their activation have not been formally investigated. We find that depletion of monocytes/macrophages during early stages of CSS development, by clodronate-liposomes or neutralizing anti-CSF1 antibody, reduces mortality and inflammatory cytokine levels in two CSS mouse models, one dependent on T cells and the second induced by repeated TLR9 stimulation. We further demonstrate that activation of Plcγ2 in myeloid cells controls CSS development by driving macrophage pro-inflammatory responses. Intriguingly, the Plcγ2 downstream effector Tmem178, a negative modulator of calcium levels, acts in a negative feedback loop to restrain inflammatory cytokine production. Genetic deletion of Tmem178 leads to pro-inflammatory macrophage polarization in vitro and more severe CSS in vivo. Importantly, Tmem178 levels are reduced in macrophages from mice with CSS and after exposure to plasma from sJIA patients with active disease. Our data identify a novel Plcγ2/Tmem178 axis as a modulator of inflammatory cytokine production by monocytes/macrophages. We also find that loss of Tmem178 accentuates the pro-inflammatory responses in CSS.