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IntroductionPediatric Acute-Onset Neuropsychiatric Syndrome (PANS) is characterized by abrupt onset of obsessive-compulsive disorder or eating restriction along with the abrupt onset of other co-occurring symptoms (tics, behavioral and cognitive regression, etc.). PANS is thought to be a post-infectious immunopsychiatric disorder, although as with most post-infectious disorders, it is challenging to establish a causal relationship with proposed infectious triggers. Intravenous immunoglobulin (IVIG) can modulate inflammation and support the elimination of infection and has been used for treatment of many post-infectious inflammatory disorders and autoimmune conditions. The aim of the study is to explore the pro-inflammatory state in PANS before and after administration of IVIG.MethodsChildren with moderate-to-severe PANS received six infusions of IVIG (Octagam 5%, Octapharma) every 3 weeks with post treatment follow-up. Blood samples and psychiatric measures were obtained at Visits 1 (pre-treatment), 7 and 8 (4 and 11 weeks after last infusion, respectively). Myeloid cell activation was assessed via flow cytometry.ResultsAll ten patients included in the study were male, White, with mean age 12.4 years (range 6–16). Statistically significant improvements following IVIG treatment were demonstrated in all psychometric assessments and parent questionnaires including CY-BOCS (obsessive compulsive scale), YGTSS (tic scale) and a parent PANS rating scale (for all scales p

Still’s disease is an inflammatory syndrome linked to the development of further immune dysregulation and hypercytokinaemia termed macrophage activation syndrome. Here the authors implicate the mechanistic target of rapamycin complex 1 in murine models of Still’s disease and macrophage activation syndrome, and provide associations with clinical cases in patients

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.

T cell receptors (TCRs) recognize peptide antigens bound to major histocompatibility complex (MHC) molecules (p/MHC) that are expressed on cell surfaces; while B cell-derived antibodies (Abs) recognize soluble or cell surface native antigens of various types (proteins, carbohydrates, etc.). Immune surveillance by T and B cells thus inspects almost all formats of antigens to mount adaptive immune responses against cancer cells, infectious organisms and other foreign insults, while maintaining tolerance to self-tissues. With contributions from environmental triggers, the development of autoimmune disease is thought to be due to the expression of MHC risk alleles by antigen-presenting cells (APCs) presenting self-antigen (autoantigen), breaking through self-tolerance and activating autoreactive T cells, which orchestrate downstream pathologic events. Investigating and treating autoimmune diseases have been challenging, both because of the intrinsic complexity of these diseases and the need for tools targeting T cell epitopes (autoantigen-MHC). Naturally occurring TCRs with relatively low (micromolar) affinities to p/MHC are suboptimal for autoantigen-MHC targeting, whereas the use of engineered TCRs and their derivatives (e.g., TCR multimers and TCR-engineered T cells) are limited by unpredictable cross-reactivity. As Abs generally have nanomolar affinity, recent advances in engineering TCR-like (TCRL) Abs promise advantages over their TCR counterparts for autoantigen-MHC targeting. Here, we compare the p/MHC binding by TCRs and TCRL Abs, review the strategies for generation of TCRL Abs, highlight their application for identification of autoantigen-presenting APCs, and discuss future directions and limitations of TCRL Abs as immunotherapy for autoimmune diseases.

Summary: The non-classical Major Histocompatibility Complex class II (MHCII) protein, H2-M, edits peptides bound to conventional MHCII in favor of stable peptide/MHCII (p/MHCII) complexes. Here, we show that H2-M deficiency affects B-1 cell survival, reduces cell renewal capacity, and alters immunoglobulin repertoire, allowing for the selection of cells specific for highly abundant epitopes, but not low-frequency epitopes. H2-M-deficient B-1 cells have shorter CDR3 length, higher content of positively charged amino acids, shorter junctional regions, less mutation frequency, and a skewed clonal distribution. Mechanistically, H2-M loss reduces plasma membrane p/MHCII association with B cell receptors (BCR) on B-1 cells and diminishes integrated BCR signal strength, a key determinant of B-1 cell selection, maturation, and maintenance. Thus, H2-M:MHCII interaction serves as a cell-intrinsic regulator of BCR signaling and influences the selection of the B-1 cell clonal repertoire.

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis, affecting up to 40% of patients with psoriasis. Constitutive expression by CD4+ T cells of an active form of STAT3, a signal transducer and transcription factor, has been shown to induce many of the major features of PsA in an animal model. We used high dimensional mass cytometry (CyTOF) to probe ex-vivo levels of phosphorylated STAT3 (pSTAT3) in circulating immune cell subpopulations from PsA patients during active and inactive states. We evaluated the frequency of 16 immune cell populations and the levels of the activated forms of STAT3 (pSTAT3) and, for comparison, STAT1 (pSTAT1) and Src (pSrc) in whole blood fixed shortly after collection. In addition to PsA patients, we studied active rheumatoid arthritis (RA) patients. Increased levels of pSTAT3 were found in all the CD4+ T cell subsets analyzed, specifically, Th1, Th2, Th17, T follicular helper (Tfh) and T regulatory (Treg) as well as in CD14+CD16- (classical) monocytes from active PsA patients compared to inactive patients. After correcting for body mass index (BMI), smoking and conventional disease modifying antirheumatic drugs (c-DMARDs), levels of pSTAT3 levels remained increased in Th1 and Tfh CD4+ T cells, and in CD14+CD16- monocytes from active patients compared to inactive patients. No differences between the patient groups were observed for pSTAT1 or pSrc. No differences were found between the active PsA and active RA groups after correction for multiple testing. During active PsA, circulating Th1 and Tfh CD4+ T cells, and CD14+CD16- monocytes expressing high levels of pSTAT3 may play a role in PsA pathophysiology, perhaps by migration to inflamed sites.

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.

Monocytes are crucial regulators of inflammation, and are characterized by three distinct subsets in humans, of which classical and non-classical are the most abundant. Different subsets carry out different functions and have been previously associated with multiple inflammatory conditions. Dissecting the contribution of different monocyte subsets to disease is currently limited by samples and cohorts, often resulting in underpowered studies and poor reproducibility. Publicly available transcriptome profiles 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 cell levels. 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 maintain their accuracy regardless of disease state in an independent cohort profiled by RNA-sequencing and are specific to their respective subset when compared to other immune cells from both myeloid and lymphoid lineages profiled across 6160 transcriptome profiles. Consequently, we show that these signatures can be used to quantify changes in monocyte subsets levels in expression profiles from patients in clinical trials. Finally, we show that proteins encoded by our signature genes can be used in cytometry-based assays to specifically sort monocyte subsets. Our results demonstrate the robustness, versatility, and utility of our computational approach and provide a framework for the discovery of new cellular markers.

The role of intestinal epithelial cells (IECs) in mucosal tolerance and immunity remains poorly understood. We present a method for inducing MHC class II (MHC-II) in human enteroids, “mini-guts” derived from small intestinal crypt stem cells, and show that the intracellular MHC-II peptide-pathway is intact and functional in IECs. Our approach enables human enteroids to be used for novel in vitro studies into IEC MHC-II regulation and function during health and disease.

Two types of single-walled carbon nanotubes (SWCNTs), HiPco- and carboxyl-SWCNT, are evaluated as drug carriers for the traditional anti-inflammatory drug methotrexate (MTX) and a small interfering RNA (siRNA) targeting NOTCH1 gene. The nanotubes are solubilized by PEGylation and covalently loaded with MTX. The coupling efficiency (CE%) of MTX is 77–79% for HiPco-SWCNT and 71–83% for carboxyl-SWCNT. siRNA is noncovalently attached to the nanotubes with efficiency of 90–97% for HiPco-SWCNT and 87–98% for carboxyl-SWCNT. Through whole body imaging in the second near-infrared window (NIR-II window, 1000–1700 nm), SWCNTs were found to be selectively accumulated in inflamed joints in a serum transfer mouse model. We further investigated the interactions of the siRNA/MTX loaded nanotubes with human blood and mice bone marrow cells. In human blood, both types of unloaded SWCNTs were associated with B cells, monocytes and neutrophils. Interestingly, loading with MTX suppressed SWCNTs targeting specificity to immune cells, especially B cells; in contrast, loading siRNA alone enhanced the targeting specificity. Loading both MTX and siRNA to carboxyl-SWCNT enhanced targeting specificity to neutrophils and monocytes but not B cells. The targeting specificity of SWCNTs can potentially be adjusted by altering the ratio of MTX and siRNA loaded. The combined results show that carbon nanotubes have the potential for delivery of cargo drugs specifically to immune cells involved in rheumatoid arthritis.

As the primary barrier between an organism and its environment, epithelial cells are well-positioned to regulate tolerance while preserving immunity against pathogens. Class II major histocompatibility complex molecules (MHC class II) are highly expressed on the surface of epithelial cells (ECs) in both the lung and intestine, although the functional consequences of this expression are not fully understood. Here, we summarize current information regarding the interactions that regulate the expression of EC MHC class II in health and disease. We then evaluate the potential role of EC as non-professional antigen presenting cells. Finally, we explore future areas of study and the potential contribution of epithelial surfaces to gut-lung crosstalk.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Gene therapy mediated by bone marrow-derived hematopoietic stem cells (BM-HSC) has been widely used in treating genetic deficiencies in both pre-clinical and clinical settings. Using mitotically inactive cell-targeting lentivirus with separate promoters for our gene of interest (the murine MHC class II (MHCII) chaperone, invariant chain (Ii)) and a GFP reporter, we monitored the expression and function of introduced Ii in various types of professional antigen presenting cells (B cells, macrophages and DC) from different organs (spleen, pancreatic lymph nodes (PLN), BM and blood). Ii and GFP were detected. Ii levels correlated with GFP levels only in macrophages and monocytes from spleen, monocytes from PLN and macrophage precursors from blood. By cell type, Ii levels in PLN cells were more similar to those in spleen cells than to those in blood or BM cells. Functionally, Ii expressed in PLN or spleen had more effect on MHCII abundance than Ii expressed in BM or blood. The results have implications for analysis of the outcomes of gene therapy when both therapeutic and reporter genes are introduced. The findings also have implications for understanding the development of immune molecule function.

Autoantibodies against antigens expressed by insulin-producing β cells are circulating in both healthy individuals and patients at risk of developing Type 1 diabetes. Recent studies suggest that another set of antibodies (anti-idiotypic antibodies) exists in this antibody/antigen interacting network to regulate auto-reactive responses. Anti-idiotypic antibodies may block the antigen-binding site of autoantibodies or inhibit autoantibody expression and secretion. The equilibrium between autoantibodies and anti-idiotypic antibodies plays a critical role in mediating or preventing autoimmunity. In order to investigate the molecular mechanisms underlying such a network in autoimmunity and potentially develop neutralizing reagents to prevent or treat Type 1 diabetes, we need to produce autoantibodies and autoantigens with high quality and purity. Herein, using GAD65/anti-GAD65 autoantibodies as a model system, we aimed to establish reliable approaches for the preparation of highly pure autoantibodies suitable for downstream investigation.

Regulatory T cells have an important role in limiting immune reactions and are essential regulators of selftolerance. Among them, CD4+CD25high regulatory T cells are the best-described subset. In this article, we summarize current knowledge on the phenotype, function, and development of CD4+CD25high regulatory T cells. We also review the literature on the role of these T cells in rheumatic diseases and discuss the potential for their use in immunotherapy

Autoantibodies against antigens expressed by insulin-producing β cells are circulating in both healthy individuals and patients at risk of developing Type 1 diabetes. Recent studies suggest that another set of antibodies (anti-idiotypic antibodies) exists in this antibody/antigen interacting network to regulate auto-reactive responses. Anti-idiotypic antibodies may block the antigen-binding site of autoantibodies or inhibit autoantibody expression and secretion. The equilibrium between autoantibodies and anti-idiotypic antibodies plays a critical role in mediating or preventing autoimmunity. Herein, using GAD65/anti-GAD65 autoantibodies as a model system, we aimed at establishing reliable approaches for purification of highly pure autoantibodies for the downstream investigation of molecular mechanisms underlying such a network.

To explore the cytokine responses associated with T cell epitopes from human cartilage glycoprotein 39 (HC gp-39) and the potential for modifying cytokine secretion using altered peptide ligands (APLs). Draining lymph node cells were harvested from HLA–DR*0401 transgenic mice that had been immunized with HC gp-39. Cytokine responses to 5 previously identified HLA–DR*0401–restricted HC gp-39 T cell epitopes were studied in vitro. The anchor and T cell receptor (TCR) contact residues of peptide 322–337 were identified, and this information was used to design alanine-substituted APLs. T cells were primed in vivo with wild-type peptide 322–337, restimulated with wild-type peptide or APLs, and the cytokine profiles were compared. Restimulation with individual peptides elicited distinct cytokine profiles. HC gp-39 (peptide 322–337) elicited a dominant interferon-γ (IFNγ) response. Residues within the core (positions P1–P9) 322–337 peptide sequence were critical for T cell recognition. Surprisingly, the N-terminal flanking region was also important for recognition by 6 of 10 specific T cell hybridomas. Substitutions of charged TCR contact residues in the 322–337 core epitope (E332A and K335A) were associated with a significant reduction in the IFNγ and interleukin-10 (IL-10) stimulation indices. Restimulation with peptides W325A and V326A was also associated with a trend toward reduced IFNγ and IL-10 secretion. In contrast, restimulation with peptide D330N elicited cytokine profiles more comparable with those resulting from restimulation with wild-type peptide. This study indicates that APLs of a proinflammatory HC gp-39 T cell epitope may be used to alter the cytokine response from a memory T cell population. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) act as a first-line recognition system for invading pathogens, such as influenza A. The interaction of DC with influenza A virus results in DC activation via endosomal Toll-like receptors and also leads to presentation of viral peptides on MHC class II molecules. Prior work demonstrated that influenza A virus (A/HK×31; H3N2) infection of BALB/c mice activates lung DCs for antigen presentation, and that the enhanced function of these cells persists long after viral clearance and resolution of the virus-induced inflammatory response. Whether influenza A virus has acute or longer-lasting effects on the endo/lysosomal antigen-processing machinery of DCs has not been studied. Here, we show that antigen presentation from intact protein antigen, but not peptide presentation, results in increased T cell stimulation by influenza-exposed lung DCs, suggesting increased antigen processing/loading in these DCs. We find that cathepsin (Cat) B levels and activity are substantially up-regulated in murine lung DCs, harvested 30 days after A/HK×31 infection. CatB levels and activity are also increased in murine splenic and bone marrow-derived DCs, following short-term in vitro exposure to UV-inactivated influenza A virus. Modest effects on CatX are also seen during in vivo and in vitro exposure to influenza A virus. Using a cell permeable Cat inhibitor, we show Cats in influenza-exposed DCs to be functional and required for generation of a T cell epitope from intact ovalbumin. Our findings indicate that influenza A virus affects the MHC class II antigen-processing pathway, an essential pathway for CD4+ T cell activation. [ABSTRACT FROM AUTHOR]

In this study, we developed a series of synthetic oligonucleotides that allowed us to investigate the details on the antigen recognition by autoimmune antibodies in localized scleroderma subjects. Besides dramatically improved analytical specificity of the assay, our data suggests a potential linking for antibodies to DNA to the biological status of disease state in localized scleroderma. Moreover, introducing chemical modifications into short synthetic deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules completely changed the binding titers of corresponding antibodies and their clinical relevance. The strongest observed effect was registered for the localized scleroderma skin damage index (LoSDI) on the IgG antibodies to TC dinucleotide-rich double-stranded antigen (p < 0.001). In addition to providing valuable tools for diagnosis of clinically relevant biomarkers, we believe that this work opens up new opportunities for research on antibodies to nucleic acids in localized scleroderma and other autoimmune diseases.

Monocytes play a critical role in the innate and adaptive immune systems, performing phagocytosis, presenting antigen, and producing cytokines. They are a heterogeneous population that has been divided in humans into classical, intermediate, and non-classical subsets, but the roles of these subsets are incompletely understood. In this study, we investigated the expression patterns of MHC class II (MHCII) and associated molecules and find that the intermediate monocytes express the highest levels of the MHC molecules, HLA-DR (tested in n = 30 samples), HLA-DP (n = 30), and HLA-DQ (n = 10). HLA-DM (n = 30), which catalyzes the peptide exchange on the MHC molecules, is also expressed at the highest levels in intermediate monocytes. To measure HLA-DM function, we measured levels of MHCII-bound CLIP (class II invariant chain peptide, n = 23), which is exchanged for other peptides by HLA-DM. We calculated CLIP:MHCII ratios to normalize CLIP levels to MHCII levels, and found that intermediate monocytes have the lowest CLIP:MHCII ratio. We isolated the different monocyte subsets (in a total of 7 samples) and analyzed their responses to selected cytokines as model of monocyte activation: two M1-polarizing cytokines (IFNγ, GM-CSF), an M2-polarizing cytokine (IL-4) and IL-10. Classical monocytes exhibit the largest increases in class II pathway expression in response to stimulatory cytokines (IFNγ, GM-CSF, IL-4). All three subsets decrease HLA-DR levels after IL-10 exposure. Our findings argue that intermediate monocytes are the most efficient constitutive antigen presenting subset, that classical monocytes are recruited into an antigen presentation role during inflammatory responses and that IL-10 negatively regulates this function across all subsets.

Reliable measurement of clinically relevant autoimmune antibodies toward phospholipid-protein conjugates is highly desirable in research and clinical assays. To date, the development in this field has been limited to the use of natural heterogeneous antigens. However, this approach does not take structural features of biologically active antigens into account and leads to low reliability and poor scientific test value. Here we describe novel phospholipid-protein conjugates for specific detection of human autoimmune antibodies. Our synthetic approach includes mild oxidation of synthetic phospholipid cardiolipin, and as the last step, coupling of the product with azide-containing linker and copper-catalyzed click chemistry with β2-glycoprotein I and prothrombin. To prove utility of the product antigens, we used enzyme-linked immunosorbent assay and three cohorts of samples obtained from patients in Denmark (n = 34) and the USA (n = 27 and n = 14). Afterwards we analyzed correlation of the obtained autoantibody titers with clinical parameters for each patient. Our results prove that using novel antigens clinically relevant autoantibodies can be detected with high repeatability, sensitivity and specificity. Unlike previously used antigens the obtained autoantibody titers strongly correlate with high disease activity and in particular, with arthritis, renal involvement, anti-Smith antibodies and high lymphocyte count. Importantly, chemical composition of antigens has a strong influence on the correlation of detected autoantibodies with disease activity and manifestations. This confirms the crucial importance of antigens' composition on research and diagnostic assays, and opens up exciting perspectives for synthetic antigens in future studies of autoimmunity.

Type I diabetes (T1D), mediated by autoreactive T cell destruction of insulin-producing islet beta cells, has been treated with bone marrow-derived hematopoietic stem cell (BM-HSC) transplantation. Older non-obese diabetic (NOD) mice recipients (3m, at disease-onset stage) receiving syngeneic BM-HSC progressed more rapidly to end-stage diabetes post-transplantation than younger recipients (4-6w, at disease-initiation stage). FACS analyses showed a higher percentage and absolute number of regulatory T cells (Treg) and lower proportion of proliferating T conventional cells (Tcon) in pancreatic lymph nodes from the resistant mice among the younger recipients compared to the rapid progressors among the older recipients. Treg distribution in spleen, mesenteric lymph nodes (MLN), blood and thymus between the two groups was similar. However, the percentage of thymic Tcon and the proliferation of Tcon in MLN and blood were lower in the young resistants. These results suggest recipient age and associated disease stage as a variable to consider in BM-HSC transplantation for treating T1D.

Epstein-Barr Virus (EBV) is an enveloped double-stranded DNA virus of the gammaherpesvirinae sub-family that predominantly infects humans through epithelial cells and B cells. Three EBV glycoproteins, gH, gL and gp42, form a complex that targets EBV infection of B cells. Human leukocyte antigen (HLA) class II molecules expressed on B cells serve as the receptor for gp42, triggering membrane fusion and virus entry. The mechanistic role of gHgL in herpesvirus entry has been largely unresolved, but it is thought to regulate the activation of the virally-encoded gB protein, which acts as the primary fusogen. Here we study the assembly and function of the reconstituted B cell entry complex comprised of gHgL, gp42 and HLA class II. The structure from negative-stain electron microscopy provides a detailed snapshot of an intermediate state in EBV entry and highlights the potential for the triggering complex to bring the two membrane bilayers into proximity. Furthermore, gHgL interacts with a previously identified, functionally important hydrophobic pocket on gp42, defining the overall architecture of the complex and playing a critical role in membrane fusion activation. We propose a macroscopic model of the initiating events in EBV B cell fusion centered on the formation of the triggering complex in the context of both viral and host membranes. This model suggests how the triggering complex may bridge the two membrane bilayers, orienting critical regions of the N- and C- terminal ends of gHgL to promote the activation of gB and efficient membrane fusion.