Immunology

Unlike in infection and cancer, T cell exhaustion in autoimmune disease has not been clearly defined. Here we set out to understand inhibitory protein (PD-1, Tim3, CTLA4, Lag3) expression in CXCR5- and CXCR5+ CD8 and CD4 T cells in systemic lupus erythematosus. CXCR5+ CD8 and CD4 T cells express PD-1 and engage B cells in germinal center reactions, leading to autoantibody formation in autoimmunity. We hypothesized that CXCR5+ CD8 T cells develop an exhausted phenotype as SLE autoimmunity expands from initial to chronic, self-perpetuating disease due to chronic self-antigen exposure. Our results indicate that there is no exhaustion frequency differences between sexes, although disease kinetics vary by sex. CXCR5+ CD8 T cells express primarily IFN{gamma}, known to promote autoimmune disease development, whereas CXCR5-CD8 T cells express TNF and IFN{gamma} as disease progresses from 2-6 months. Tim3 is the highest expressed inhibitory marker for all CD4 and CD8 T cell populations demonstrating potential for terminally exhausted populations. CTLA4 expression on CD4 T cells suggests potential tolerance induction in these cells. We identified exhaustion phenotypes within autoimmune disease that progress with increasing lupus erythematosus severity and possibly provide a feedback mechanism for immunological tolerance. HighlightsO_LICXCR5- and CXCR5+ CD8 T cells expand with rate of disease in SLE mouse model. C_LIO_LICXCR5+ CD8 T cells are low contributors to TNF disease progression unlike CXCR5-CD8 T cells but may increase disease mechanisms through high IFN{gamma} production. C_LIO_LIInhibitory markers upregulate in frequency with the highest amounts seen in Tim3+ populations. Tim3+Lag3+ expression may be an indicator of terminal differentiation for all populations. C_LIO_LIInhibitory marker expression frequency was unrelated to sex. C_LI

IntroductionGlobal WHO data indicate that Sub-Saharan African (SSA) countries, such as Kenya, experienced reduced coronavirus disease 2019 (COVID-19) severe-morbidity and mortality burdens relative to their more affluent counterparts in Europe, Asia, and North America. MethodsWe analysed peripheral blood mononuclear cells (PBMC) samples collected from Kenya and Sweden before and during COVID-19. Pre-COVID-19 samples were available for 80 adults and 10 infants from Kenya, and 20 adults from Sweden. COVID-19 samples were available for 39 Kenyan adults. The samples were analysed for ex vivo IFN-{gamma} secretion using an Enzyme-Linked Immunosorbent (ELISpot) assay following in vitro stimulations with overlapping SARS-CoV-2 spike-protein peptides. T-cells expressing IFN-{gamma}, IL-2, TNF-, CD154, and CD107a were assessed following similar stimulations, using intracellular cytokine staining (ICS) and multiparameter flow cytometry. Results55.7% of the Kenyan pre-COVID-19 adults were classified as responders by ELISPOT responses to spike-protein peptides, compared with 28% of Swedish pre-COVID-19 adults (p = 0.04). The frequencies for SARS-CoV-2 spike-specific TNF- CD4+, TNF- CD8+ and IFN-{gamma} CD8+ T-cell responses, tended to be higher in the Kenyan adults although these differences did not reach statistical significance. ConclusionPre-COVID-19 T-cell responses could contribute to lower morbidity and mortality associated with SARS-CoV-2 infections in SSA relative to Europe, Asia, and North America. HighlightsO_LIHigher frequencies of cross-reactive IFN-{gamma}-secreting cells among Kenyan pre-COVID-19 adults compared to Sweden. C_LIO_LITrends of higher TNF- and IFN-{gamma} T-cell responses among Kenyan adults. C_LIO_LIPresence of pre-existing T-cell immunity in Sub-Saharan Africa. C_LI

Epstein-Barr virus (EBV)-associated central nervous system lymphoproliferative diseases (CNSL) are aggressive clinical conditions with poor prognosis. We have reported that durable responses in patients with primary CNS post-transplant lymphoproliferative disease (PTLD) were associated with detection of two ganciclovir (GCV)/zidovudine (AZT) viral drug target proteins, the EBV kinases BGLF4 and BXLF1 in CNSL biopsies. These are associated with lytic EBV and the mechanism for expression in latently infected EBV+ CNSL has been unknown. By carrying out RNA expression analysis in CNSL tissue biopsies (n=12), we confirmed expression of LMP1, BXLF1, and BGLF4, but not BZLF1, pointing to an incomplete lytic EBV program. Biopsies from systemic PTLD (n=24) were used for comparison and showed significantly less expression of BGLF4. By quantifying DNA methylation in EBV gene promoters we showed significantly decreased promoter methylation at BGLF4 in CNSL versus systemic PTLD (p=0.0006). Luciferase reporter analysis of the BGLF4 upstream sequence revealed 3 regions of promoter activity and 5' RACE in n=4 EBV-infected cell lines and n=5 CNSL biopsy samples identified transcription start sites at these promoters. We identified DNA methylation loss at single CpG dinucleotides which were specifically demethylated in CNSL, while surrounding EBV methylation remained high. Lastly, TET2 knockout and expression of TET1/2-suppressive mutant IDH1 in a latent HEK293 EBV model indicated that active demethylation is necessary for activity of BGLF4 promoters. We detail the epigenetic basis of BGLF4 expression in CNSL via locus-specific promoter activation that may hold value for determination of antiviral drug sensitivity. SignificanceWe show site-specific DNA methylation loss as the molecular basis of BGLF4 expression in CNSL. BGLF4 methylation holds potential for further development as a biomarker of antiviral drug response in EBV+ CNSL.

ObjectiveSystemic lupus erythematosus (SLE) is a chronic autoimmune disease causing multi-organ damage. The most specific autoantibody response in SLE, present in 20-30% of patients, targets the Sm-protein and has been shown to recognize a linear Sm-derived B cell epitope containing a post-translational modification (PTM) of arginine termed symmetrical dimethyl arginine (sDMA). As autoantibodies to other PTM-modified proteins are often promiscuous, we aimed to determine the specificity and cross-reactivity of anti-Sm antibodies. MethodsSpecificity and promiscuity/cross-reactivity of anti-Sm IgG were measured by ELISA in SLE patients and healthy donors using peptides containing either sDMA or unmodified arginine. Inhibition and cross-reactivity were determined using competitive ELISA and affinity purification. Recognition of endogenous EBNA1 by anti-Sm IgG was performed by Western blot using lysates of EBV-bearing lymphoblastoid cell lines. ResultsThe sDMA residue is recognized by anti-Sm+ SLE patients regardless of the peptide amino acid sequence, with a modest impact of amino acids flanking sDMA on recognition. Most notably, IgGs targeting sDMA comprise the overall majority ([~]90%) of the anti-Sm antibody repertoire and are highly cross-reactive between SmD3108-122 and several sDMA-containing viral-derived epitopes, including full-length EBNA1. ConclusionOur data implicate that the majority of anti-Sm IgGs target the sDMA residue irrespective of its Sm-context, thus representing a prototypic anti-PTM response. These antibodies are highly promiscuous, recognizing several sDMA-modified targets, including naturally occurring viral sDMA-expressing epitopes. These findings suggest a new mechanism by which molecular mimicry of sDMA-modified viral proteins could contribute to a breach of tolerance in anti-Sm+ SLE patients. KEY MESSAGESO_ST_ABSWhat is already known on this topicC_ST_ABSO_LISymmetrical dimethylarginine (sDMA) residues are present on RGG/RG repeat regions within the SmD1, SmD3, and SmB/B subunits of the Sm protein complex in vivo. C_LIO_LIThe introduction of sDMA is essential for recognition of minimal antigenic epitopes spanning amino acids #95-119 on the SmD1 and #108-122 on the SmD3 subunits by a specific subset of anti-Sm autoantibodies. C_LI What this study addsO_LIWe show that autoantibodies targeting sDMA residues are common in anti-Sm+ SLE patients and represent the majority of the anti-Sm autoantibody repertoire. C_LIO_LIWe demonstrate that anti-sDMA autoantibodies are highly cross-reactive and can also bind to sDMA residues on other targets, including several virus-derived epitopes, thus representing a prototypic anti-modified protein antibody (AMPA) response directed against post-translationally modified proteins through symmetrical dimethylation of arginine. C_LI How this study might affect research, practice, or policyO_LIThe high cross-reactivity of Sm antibodies to various sDMA-containing epitopes, combined with a dominant antigenic specificity, provides mechanistic insight in the potential link between viral infection and tolerance breach in anti-Sm+ SLE patients. C_LI

The innate immune system is equipped with multiple receptors to detect microbial nucleic acids and induce type I interferon (IFN) to restrict viral replication. When dysregulated these receptor pathways induce inflammation in response to host nucleic acids and promote development and persistence of autoimmune diseases like Systemic Lupus Erythematosus (SLE). IFN production is regulated by the Interferon Regulatory Factor (IRF) transcription factor family of proteins that function downstream of several innate immune receptors such as Toll-like receptors (TLRs) and Stimulator of Interferon Genes (STING). Although both TLRs and STING activate the same downstream molecules, the pathway by which TLRs and STING activate IFN response are thought to be independent. Here we show that STING plays a previously undescribed role in human TLR8 signaling. Stimulation with the TLR8 ligands induced IFN secretion in primary human monocytes, and inhibition of STING reduced IFN secretion from primary monocytes from 8 healthy donors. We demonstrate that TLR8-induced IRF activity was reduced by STING inhibitors. Moreover, TLR8-induced IRF activity was blocked by inhibition or loss of IKK{varepsilon}, but not TBK1. Bulk RNA transcriptomic analysis supported a model where TLR8 induces transcriptional responses associated with SLE that can be downregulated by inhibition of STING. These data demonstrate that STING is required for full TLR8-to-IRF signaling and provide evidence for a new framework of crosstalk between cytosolic and endosomal innate immune receptors, which could be leveraged to treat IFN driven autoimmune diseases. BackgroundHigh levels of type I interferon (IFN) is characteristic of multiple autoimmune diseases, and while TLR8 is associated with autoimmune disease and IFN production, the mechanisms of TLR8-induced IFN production are not fully understood. ResultsSTING is phosphorylated following TLR8 signaling, which is selectively required for the IRF arm of TLR8 signaling and for TLR8-induced IFN production in primary human monocytes. ConclusionSTING plays a previously unappreciated role in TLR8-induced IFN production SignificanceNucleic acid-sensing TLRs contribute to development and progression of autoimmune disease including interferonopathies, and we show a novel role for STING in TLR-induced IFN production that could be a therapeutic target.

BackgroundAtopic dermatitis (AD) is a chronic skin disease that typically occurs in early childhood. In this cross-sectional case-control study, our objective was to employ machine learning approaches to identify novel clusters of protective or susceptibility features associated with AD. Methods and FindingsWe utilised an integrated dataset comprising previously established environmental, cytokine, antibody, and gene expression data from AmaXhosa children, both healthy and with AD, living in either rural or urban settings of South Africa, aged 12-36 months. The applied machine learning methods included the GeneSelectR workflow to identify a subset of relevant genes, the calculation of SHAP values to explain the machine learning output, and the use of DIABLO to integrate the datasets for a comprehensive analysis. Key findings included the identification of a protective cluster of environmental features primarily found in the rural setting, which were correlated with plasma cytokine levels and with expression of autophagy-related genes. Additionally, we identified AD susceptibility clusters where levels of allergen-specific and total IgE antibodies correlated with the cytokines MCP-4 and TARC. Lastly, we identified an RNA-Seq feature signature specific to the disease endotype. ConclusionsThe application of various machine learning methods enabled the identification of significant factors associated with AD in a complex, multi-modular dataset, making the output explainable and potentially informing targeted interventions and improved diagnostic criteria.

While naive CD4+ T cells have historically been considered a homogenous population, recent studies have provided evidence that functional heterogeneity exists within this population. Using single cell RNA sequencing (scRNAseq), we identify five transcriptionally distinct naive CD4+ T cell subsets that emerge within the single positive stage in the thymus: a quiescence cluster (TQ), a memory-like cluster (TMEM), a TCR reactive cluster (TTCR), an IFN responsive cluster (TIFN), and an undifferentiated cluster (TUND). Elevated expression of transcription factors KLF2, Mx1, and Nur77 within the TQ, TIFN, and TMEM clusters, respectively, allowed enrichment of these subsets for further analyses. Functional studies using sorted cells revealed that naive T cell subsets have distinctive functional biases upon stimulation. Furthermore, treatment of mice with inflammatory stimuli imparted a state of reduced responsiveness on naive T cells, evidenced by a reduction in cytokine production ex vivo. In human lupus patients, naive CD4+ T cell cluster frequencies were distorted, with the TIFN cluster expanding proportionately with disease score. Our data show that naive T cells are influenced by host environment, with functional consequences manifesting upon activation. These findings highlight a need to explore how naive T cells can become distorted in cancer, autoimmunity, and infectious diseases. SummaryThis study describes the transcriptional heterogeneity of murine and human naive CD4+ T cells as comprising of multiple discrete clusters that impact CD4+ T cell fate and trajectories. Naive CD4+ T cells experiencing inflammatory environments exhibit an altered transcriptional state that influences their functional trajectory.

IntroductionPlasmacytoid dendritic cells (pDCs) are unique antigen presenting cells that may be implicated in allergic disease because they bind IgE on their surface and modulate important Th1/Th2 cytokine responses. While conducting in vitro experiments using excess omalizumab to capture IgE, we discovered that this immunoglobulin is not readily removed from pDC to the same extent observed for basophils suggesting that a portion of the IgE on pDC is membrane bound. MethodsBasophils and pDC were prepared from leukopacks using established protocols. In order to isolate PBMCs, blood was also drawn from consenting donors with a wide range in total serum IgE levels. B cells and pDCs were identified by flow cytometry by gating on CD19/CD27 and BDCA2/CD123, respectively. Quilizumab, a mouse anti-human monoclonal IgG1 antibody with specificity only for membrane-bound IgE, was used to detect this molecule in both cell types. ResultsWhen used in vitro at 1.5 mg/ml, omalizumab removed [~]80-90% of the IgE expressed by basophils. In contrast, IgE expression decreased only 30-40% on pDC treated likewise. Upon analyzing pDC for membrane-bound IgE, there was no significant difference in number of target-bound cells and mean fluorescence between the mouse isotype control and quilizumab among pDCs (P = 0.125, 0.165). However, the ratio of the proportion of target-bound CD19+27+ B cells compared to other cells was 32:1 for isotype (P < 0.001) and 54:1 for quilizumab (P = 0.015). ConclusionOverall, this study demonstrates that while pDCs express significant levels of Fc{varepsilon}RI that binds IgE to the surface, there is no appreciable amount of membrane-bound IgE noted. The reason for omalizumabs poor ability to remove IgE from pDCs remains unknown.

Systemic Lupus Erythematous (SLE) is a chronic autoimmune disorder, broadly characterized by systemic inflammation along with heterogeneous clinical manifestations, severe morbidity, moribund organ failure and eventual mortality. In our study, SLE patients displayed higher percentage of activated, inflamed and hyper polarized CD8+ T cells, dysregulated CD8+ T cell differentiation, significantly elevated serum inflammatory cytokines, higher accumulation of cellular ROS when compared to healthy controls. Importantly, these hyper inflammatory/hyper polarized CD8+ T cells responded better to an anti-oxidant than to an oxidant. Terminally differentiated Tc1 cells also showed plasticity upon Oxidant/antioxidant treatment but was in contrast to the SLE CD8+ T cell response. Our studies suggest that the differential phenotype and redox response of SLE CD8+ T cells and Tc1 cells could be attributed to their cytokine environs during their respective differentiation and eventual activation environs. Polarisation of Tc1 cells with IL-21 drove hyper cytoxicity without hyper polarisation suggesting that SLE inflammatory environment could drive the extreme aberrancy in SLE CD8+ T cell.

BackgroundDendritic cells are potently activated by the synergistic action of CD40 stimulation in conjunction with signaling through toll like receptors, subsequently activating antigen specific T cells. Cancer vaccines targeting the activation of dendritic cells in this manner show promise in murine models and are being developed for human cancer patients. While vaccine efficacy has been established, further investigation is needed to understand the mechanism of tumor control and how vaccination alters tumor infiltrating immune cells. MethodsMice bearing established murine melanoma tumors were vaccinated with agonist anti-CD40, polyI:C, and tumor antigen. Intratumoral T cell numbers, differentiation state, proliferation, and survival were assessed by flow cytometry. T cell effector function was measured both within the tumor and ex vivo by flow cytometry. T cell trafficking was blocked to examine changes to intratumoral T cells present at the time of vaccination. ResultsVaccination led to increased intratumoral T cell numbers and delayed tumor growth. Expansion of T cells and tumor control did not require trafficking of T cells from the periphery. The increase in intratumoral T cells was associated with an acute burst in proliferation but not changes in viability. Intratumoral T cells had lower PD-1 and Eomes expression but were less functional after vaccination on a per cell basis. However, the increased intratumoral T cell numbers yielded increased effector T cells per tumor. ConclusionsPre-infiltrated CD8 T cells are responsive to CD40/TLR-mediated vaccination and sufficient for vaccination to delay tumor growth when additional T cell trafficking is blocked. This indicates that the existing T cell response and intratumoral DC could be critical for vaccination efficacy. This also suggests that circulating T cells may not be an appropriate biomarker for vaccination efficacy.

BackgroundAntinuclear antibodies (ANA) and extractable nuclear antigens (ENA) are crucial biomarkers for the diagnosis of autoimmune diseases (AID) such as systemic lupus erythematosus (SLE), Sjogrens syndrome, systemic sclerosis, and polymyositis. In the present study, we assessed the most frequent ANA patterns associated with the most detectable ENA antigen (Ag) that could be used as a diagnostic and efficient prognostic marker of AID. Materials and MethodsThe primary objective of this study was to investigate the association between immunofluorescence (IF) ANA and ENA in patients with AIDs. This was a retrospective cross-sectional study. The study was performed at the Immunology Department of the Armed Forces Institute of Pathology, Rawalpindi. Retrospective data from 76 patients were tested for ANA and ENA from June 2020 to Nov 2020. ResultsA total of 76 patients comprising 14 (18.4%) males and 62(81.6%) females were tested for AIDs. The most frequent pattern among AID patients was coarse speckled, followed by the peripheral ANA pattern. The most frequent ENA Ags were Sjogrens syndrome A (SSA) and B (SSB). SSA was significantly associated with coarse speckled and peripheral ANA patterns, whereas SSB was associated with coarse speckled ANA patterns. These associations are relevant for accurate diagnosis of autoimmune diseases. ConclusionSSA was associated with coarse speckled and peripheral ANA patterns, whereas SSB was associated with coarse speckled ANA patterns. The ANA patterns were significantly associated with ENA antigens.

BackgroundHLA-A29-positive birdshot chorioretinitis (BCR) is an inflammatory eye disorder that is generally assumed to be caused by an autoimmune response to HLA-A29-presented peptides from retinal arrestin (SAG), yet the epitopes recognized by CD8+ T cells from patients remain to be identified. ObjectivesThe identification of natural ligands of SAG presented by HLA-A29. To quantify CD8+ T cells reactive to antigenic SAG peptides presented by HLA-A29 in patients and controls. MethodsWe performed mass-spectrometry based immunopeptidomics of HLA-A29 of antigen-presenting cell lines from patients engineered to express SAG. MHC-I Dextramer technology was utilised to identify antigen-specific CD8+ T cells reactive to SAG peptides in complex with HLA-A29 in a cohort of BCR patients, HLA-A29-positive controls, and HLA-A29-negative controls. ResultsWe report on the naturally presented antigenic SAG peptides identified by sequencing the HLA-A29 immunopeptidome of antigen-presenting cells of patients. We show that the N-terminally extended SAG peptide precursors can be trimmed in vitro by the antigen-processing aminopeptidases ERAP1 and ERAP2. Unexpectedly, no antigen engagement by CD8+ T cells upon stimulation with SAG peptides was observed in patients or HLA-A29-positive controls. Multiplexed HLA-A29-peptide dextramer profiling of a case-control cohort revealed that CD8+ T cells specific for these SAG peptides were neither detectable in peripheral blood nor in eye biopsies of patients. ConclusionsCollectively, these findings demonstrate that SAG is not a CD8+ T cell autoantigen and sharply contrast the paradigm in the pathogenesis of BCR. Therefore, the mechanism by which HLA-A29 is associated with BCR does not involve SAG.

AimsType I interferons (IFNs) are linked to an increased risk of cardiovascular disease and are chronically elevated in systemic autoimmune diseases such as systemic lupus erythematosus (SLE). We evaluated the effect of chronic IFNa exposure on cardiac and cardiomyocyte function in order to better understand IFN-driven cardiac disease from the perspective of SLE. Methods and ResultsAdministration of the TLR7/8 agonist, resiquimod, to C57BL6 mice, drove chronic IFN induction and reduced ejection fraction and fractional shortening compared to mice treated with vehicle control. Multiomic analysis demonstrated increased IFN signature in resiquimod treated hearts (transcriptomic and proteomic) and a decrease in genes and proteins representing electron transport chain (ETC) and cytochrome complex assembly. Integration of metabolomics with transcriptomics revealed pathways representing chemokine signaling, Toll-like receptor signaling, HIF-1 signaling and Dilated cardiomyopathy. As our in vivo model of IFN-driven SLE-like heart disease showed both proteomic and transcriptomic changes that reflected changes in mitochondrial and potentially cardiac function, we conducted an in vitro analysis of the effects of acute and chronic IFN on cardiomyocytes, the most energetically demanding cell type in the heart, and the cells most susceptible to stress and inflammation. As with our in vivo findings, proteomic and transcriptomic analysis of AC16 cardiomyocytes exposed to chronic IFN indicated perturbation of mitochondrial pathways. Extracellular flux analysis of chronically exposed AC16 cells showed impaired basal respiration, maximal respiration, ATP production and non-glycolytic acidification, glycolysis and glycolytic capacity, indicating increased mitochondrial stress in response to chronic exposure to IFNa. MitoTracker green staining showed increased fragmentation and perinuclear localization in AC16 chronically exposed to IFN and an increase in mtDNA release and expression of proteins known to contribute to mtDNA release and detection. ConclusionsOur results directly support a role for chronic IFN in driving cardiac dysfunction in both a mouse model of IFN-driven disease that mimics SLE and in cardiomyocytes, though enhanced mitochondrial stress, mtDNA release and potentially exacerbation of cGAS-STING-IFN axis. Translational PerspectivesMany immune features of SLE such as elevated type I interferons, chronic inflammation, and persistent autoantibody production are strongly correlated with increased cardiovascular risk, but it remains difficult to prove direct biological causation. This study demonstrates that chronic IFN exposure induces mitochondrial dysfunction and mtDNA release in cardiomyocytes, directly complementing an in vivo model of SLE-cardiac disease. It suggests that targeting IFNs may reduce CVD risk in IFN-driven autoimmunity.

BackgroundWe have previously isolated a highly mutated VH1-02 antibody termed group C 76-Q13-6F5 (6F5) that targets a conformational epitope on gp41. 6F5 has the capacity to mediate Ab dependent cell cytotoxicity (ADCC). When the VH1-02 group C 76 antibodies variable chain sequence was reverted to germline (76Canc), this still retained ADCC activity. Due to this ability for the 76Canc germline antibody to functionally target this epitope, we sought to identify a protein target for vaccine development. MethodsInitially, we interrogated peptide targeting by screening a microarray containing 29,127 linear peptides. Western blot and ELISAs were used to confirm binding and explore human serum targeting. Autoimmune targeting was further interrogated on a yeast-displayed human protein microarray. Results76Canc specifically recognized a number of acidic peptides. Meme analysis identified a peptide sequence similar to a non-structural protein of Hepacivirus previously implicated in Kawasaki disease (KD). Binding was confirmed to top peptides, including the Hepacivirus-related and KD-related peptide. On serum competitions studies using samples from children with KD compared to controls, targeting of this epitope showed no specific correlation to having KD. Human protein autoantigen screening was also reassuring. ConclusionsThis study identifies a peptide that can mimic the gp41 epitope targeted by 76C group antibodies (i.e. a mimotope). We show little risk of autoimmune targeting including any inflammation similar to KD, implying non-specific targeting of this peptide during KD. Development of such peptides as the basis for vaccination should proceed cautiously.

ObjectivePhenotypic diversity of autoimmune diseases presents an ongoing diagnostic and drug development challenge for clinicians and scientists. Recent discovery of mutations in RELA (encoding RELA) in patients with different diagnoses has highlighted that different pathogenic molecular mechanisms are at play and may explain the observed phenotypic diversity. We identified seven novel/rare RELA variants in patients with autoimmune diseases and examine the functional consequences on immune signalling. MethodsWild type and mutant RELA proteins were ectopically expressed in HEK293 cells. Western blot and NF-{kappa}B/IFN{beta} luciferase reporter assays were used to determine RELA expression and transcriptional activity, respectively. In patients (n=3), B and T cell populations were examined via flow cytometry and NF-{kappa}B and interferon stimulated genes in PBMCs were assessed via qPCR following toll-like receptor activation. ResultsRELAI250V, RELAR295H and RELAE3* displayed a loss in NF-{kappa}B transcriptional activity. Comparative to RELAWT, RELAI250V protein expression was reduced. Two variants, RELAI250V and RELAR295H, induced hyperactivation of the IFN{beta} promoter. An elevated IFN gene signature was not detected in patient PBMCs following toll-like receptor activation, however the patient heterozygous for I250V had elevated IFN{beta} transcripts at baseline and after TLR7/8 activation. A reduction in transitional, unswitched memory and memory B cell and cTfh (CCR6-CXCR3-) T cell subsets was shared by the patient group. ConclusionWe expand upon the clinical syndromes linked to RELA dysfunction and uncover rare and novel variants that have distinct functional effects on gene transcription downstream of NF-{kappa}B and IFN{beta} promoter elements. These findings reinforce an important role for RELA in a range of autoimmune and autoinflammatory diseases.

PurposeSystemic lupus erythematosus is an autoimmune disease hallmarked by a plethora of autoantibodies, interferon-signature, auto-immune complexes, dysregulation of soluble factors in circulation, and abnormal neutrophils. We hypothesized that lupus humoral "environment" could be responsible, at least in part, for these neutrophil abnormalities. We characterized the neutrophil phenotype in a cohort of lupus patients and assessed the impact of the "lupus environment". MethodsBlood samples were analyzed for neutrophil viability and expression of surface markers, by flow cytometry. Neutrophil-enriched suspensions were stimulated with serum samples from lupus patients, and apoptosis was monitored in real-time. Plasma samples were subjected to multiplex analysis. Whole blood and neutrophil-enriched suspensions were stimulated with lupus-relevant soluble factors or with heat-aggregated IgGs, which mimic the engagement of Fc gamma (Fc{gamma}) receptors by immune complexes, for viability and activation analysis. ResultsLupus neutrophils had reduced viability and increased apoptosis. They also displayed significant alterations in the expression of surface markers of adhesion, complement regulation, degranulation, and immune complex response. Stimulation of healthy neutrophils with lupus serum increased apoptosis. Lupus-relevant soluble factors accelerated neutrophil apoptosis. Heat-aggregated IgGs mirrored most of the key alterations in viability and surface marker expression observed in lupus neutrophils. ConclusionThis study demonstrates that Fc{gamma} receptors engagement is a major driver of the phenotype observed in lupus neutrophils, including increased apoptosis and dysregulated surface marker expression. These findings suggest that lupus neutrophils may not be inherently defective but rather educated by lupus-associated environmental cues.

PurposeNeutrophils express Fc receptors on their surface to trap immune complexes. While the implication of Fc{gamma}RIIa and Fc{gamma}RIIIb have extensively been studied in that context, that of Fc{gamma}RI remains elusive. Recently, aggregated IgGs have been shown to induce rapid Fc{gamma}RI upregulation and reactive oxygen species (ROS) generation, but the biological relevance of this process is still unclear. MethodsBlood samples were obtained from healthy volunteers and patients with lupus. Aggregated IgGs were prepared as a model of immune complex. Fc{gamma}Rs surface expression on circulating leukocytes and on freshly isolated neutrophils was measured by flow cytometry. ROS production was monitored with luminol-based chemiluminescence. ResultsIncubation of blood samples from healthy volunteers with aggregated IgGs rapidly upregulated the surface expression of Fc{gamma}RI, predominantly on neutrophils. Stimulation of isolated neutrophils from healthy donors with aggregated IgGs resulted in the production of ROS in an Fc{gamma}RI-dependent fashion. Cytochalasin B potentiated Fc{gamma}RI expression and ROS production. Positive correlations between neutrophil Fc{gamma}RI and ROS production were observed in resting blood from both healthy volunteers and lupus patients. In the lupus cohort, monocyte Fc{gamma}RI also correlated with ROS production. ConclusionThis study unveils a previously underappreciated role for neutrophil Fc{gamma}RI in ROS production in both healthy individuals and patients with lupus, and identifies Fc{gamma}RI as a potential biomarker of oxidative response.

BackgroundCurrently the role of dengue virus (DENV) specific T cell responses in disease pathogenesis and protection are not well understood, including potential differences in those who have obesity. We sought to investigate the functionality and phenotype of T cells in patients with acute dengue fever (DF) or dengue haemorrhagic fever (DHF). MethodsT cell function was assessed in patients with DF (n=50) and DHF (n=12), recruited within [&le;]4 days of illness and again on day 5 to 7, using 109 peptides representing CD8{square} epitopes and 90 peptides targeting CD4+ T cell epitopes. Phenotypic analysis was in DF (n=21) and DHF patients (n=21), recruited between days 6-8 since onset of illness, by multicolor flow cytometry. ResultsThe frequency of ex vivo IFN{gamma} ELISpot responses to both the CD4+ and CD8+ peptides pools significantly increased from the first to second time point in patients with DF (p<0.0001) but not with DHF. The frequency of ex vivo IFN{gamma} ELISpot responses to CD4+ (p=0.001) and CD8+ peptides pools (p=0.0002) also significantly increased from the first to second time point in lean patients compared obese patients. Cutaneous lymphocyte associated antigen (CLA) expression was significantly higher in the CD8+ T cell subset in patients with DF and DHF compared to HC and these differences were most significant in CD8+CD45RA- T cells. CD8+CD45RA-CLA+ T cells consisted of >50% of the T cells in 9/21 patients with DHF, with 92.7% expressing CD38. CLA expression was highest in the CD8+CD45RA- of obese individuals, which was significantly higher compared to lean individuals (p=0.01). CD27 and CD127 were both significantly downregulated in patients with DHF compared to DF, with ICOS expression being significantly higher in CD8+ T cells in DHF. DiscussionPatients with DHF and obese individuals had impaired T cell functionality. Activated and skin homing CD8+ T cells were associated with DHF, with downregulation of CD27 and CD127. Therefore, the role of skin homing T cells, which have impaired functionality in disease pathogenesis, should be further investigated.

Vitamin A (VitA) and its derivative retinoic acid (RA) are essential for immunological responses. In VitA deficient (VAD) mice, acquisition of effector responses is impeded, but little is known about maintenance and expression of previously acquired effector function under the VAD conditions. We examined the impact of VAD on progression of autoimmune diseases using two models of uveitis, experimental autoimmune uveitis (EAU) induced by active immunization and spontaneous uveitis in retina-specific T cell receptor transgenic (R161H) mice, and in the model of experimental autoimmune encephalomyelitis (EAE). VAD was induced by dietary lack of VitA from before birth, or by daily injections of a pan-RA receptor inhibitor BMS493 in adult mice fed with the standard diet. VAD mice were essentially resistant to induction of EAU or EAE and displayed impaired effector T cell responses. Defective priming/acquisition of effector function by VAD T cells was also evident. By contrast, spontaneously uveitic R161H mice fed with VAD diet, in which priming of pathogenic T cells occurs before onset of full VAD, only moderately attenuated uveitis compared to VitA sufficient R161H mice. To reconcile somewhat different results between induced model and spontaneous model of uveitis, we examined EAU in partial VAD mice or adoptive transfer into VAD hosts. The results supported that effector T cells primed in VitA-sufficient environment were able to function in VAD environment and induced EAU. We conclude that although priming of naive T cells in the VAD environment is defective, effector function acquired under VitA sufficient conditions is maintained and can be expressed under VAD conditions. Because dietary lack of VitA is rarely profound and may be seasonal, our findings may shed light on immunity and autoimmunity in geographical regions where dietary VitA is limiting.

BackgroundFood allergen immunotherapy can induce remission of food allergies in certain individuals, but the mechanisms underlying this remission are largely unknown. Prior work has identified differences in immunomodulatory metabolites between older children who develop remission versus non-remission on oral immunotherapy (OIT). Here we aim to characterize metabolomic changes during OIT in young children to and validate patterns of immunomodulatory metabolites previously observed. MethodsUntargeted plasma metabolomic profiling was performed on samples from the DEVIL peanut OIT trial (n=41, ages 9-36 months). Remission status was determined by oral food challenges conducted at the end-of-therapy and the end of a 1-month avoidance period. Linear and logistic regression models were used to detect differences in individual metabolites over time on OIT and by remission status. Pathway analyses were used to determine enrichment of chemical subclasses and biological pathways. These pathways were then compared to prior findings generated from similar profiling performed from the PNOIT peanut OIT trial (n=20, ages 7-13). ResultsDuring OIT, glycerophospholipid metabolites (q=3.8x10-5) increased over time and most amino acid metabolites (q=6.1x10-45) decreased over time. Participants who went on to develop remission, had higher levels of amino acids (q=4.3x10-8) and bile acids (q=0.00014), whereas children who developed non-remission had higher levels of glycerophospholipids (q=4.3x10-10). Comparison of these findings with our second cohort of OIT in older children, showed replication of the enrichment of glycerophosphocholines (q=1.0x10-13) and amino acids (q=2.1x10-5) among metabolites that changed over time on OIT and replication of glycerophospholipids(q=5.7x10-16), amino acids (PNOIT q=7.2x10- 7), and bile acids (PNOIT q=3.8x10-8) among metabolites that varied by remission status. ConclusionsMetabolomic profiles on OIT differed both over time on OIT and by OIT remission status in young children. Between two independent OIT cohorts of different ages we observed replication of significantly enriched chemical subclasses of glycerophospholipids, amino acids, and bile acids. Given the potentially immunomodulatory roles of some of these metabolites, our results suggest that glycerophospholipids, amino acids, and bile acids may be involved in the mechanisms of remission on OIT.