Immunity

Secondary bacterial infections, including ventilator-associated pneumonia (VAP), lead to worse clinical outcomes and increased mortality following viral respiratory infections including in patients with coronavirus disease 2019 (COVID-19). Using a combination of tracheal aspirate bulk and single-cell RNA sequencing we assessed lower respiratory tract immune responses and microbiome dynamics in 23 COVID-19 patients, 10 of whom developed VAP, and eight critically ill uninfected controls. At a median of three days (range: 2-4 days) before VAP onset we observed a transcriptional signature of bacterial infection. At a median of 15 days prior to VAP onset (range: 8-38 days), we observed a striking impairment in immune signaling in COVID-19 patients who developed VAP. Longitudinal metatranscriptomic analysis revealed disruption of lung microbiome community composition in patients with VAP, providing a connection between dysregulated immune signaling and outgrowth of opportunistic pathogens. These findings suggest that COVID-19 patients who develop VAP have impaired antibacterial immune defense detectable weeks before secondary infection onset.

Elucidating how regulatory T (Treg) cells specialize and acquire specific suppressive capacities is key for understanding immune regulation in autoimmunity and cancer. Although IL-12 is a key driver of Th1 differentiation, its role in Treg cells remains poorly defined. Here, we investigated whether IL-12R signaling contributes to Treg cell specialization during Th1 immune responses. We show that IL-12R{beta}2 KO Treg cells display reduced type 1 specialization following LCMV infection, resulting in impaired suppressive capacity. Mechanistically, IFN-{gamma} induces a first peak of T-bet, whereas IL-12 acts downstream to sustain T-bet through a positive feedback loop. The decreased suppressive function of IL-12R{beta}2 KO Treg cells alters effector T cell responses during both acute and chronic infections. Importantly, human Treg cells also respond to IL-12, promoting enhanced type 1 specialization. Together, these findings establish IL-12 as a critical regulator of type 1 Treg specialization and highlight the therapeutic potential of targeting this pathway. TeaserIL-12R signaling in Treg cells enables sustained T-bet expression, programing type 1 Tregs for suppression of Th1 responses.

Interstitial lung diseases (ILD) are heterogeneous conditions that may lead to progressive fibrosis and death of affected individuals. Despite diversity in clinical manifestations, enlargement of lung-associated lymph nodes (LLN) in fibrotic ILD patients predicts worse survival. Herein, we revealed a common adaptive immune landscape in LLNs of all ILD patients, characterized by highly activated germinal centers and antigen-activated T cells including regulatory T cells (Tregs). In support of these findings, we identified serum reactivity to 17 candidate auto-antigens in ILD patients through a proteome-wide screening using phage immunoprecipitation sequencing. Autoantibody responses to actin binding LIM protein 1 (ABLIM1), a protein highly expressed in aberrant basaloid cells of fibrotic lungs, were correlated with LLN frequencies of T follicular helper cells and Tregs in ILD patients. Together, we demonstrate that end-stage ILD patients have converging immune mechanisms, in part driven by antigen-specific immune responses, which may contribute to disease progression.

In chronic inflammatory disorders of the central nervous system (CNS), tissue-resident self-reactive T cells perpetuate disease. The specific tissue factors governing the persistence and continuous differentiation of these cells remain undefined but could represent attractive therapeutic targets. In a model of chronic CNS autoimmunity, we find that oligodendrocyte-derived interleukin-33 (IL-33), an alarmin, is key for locally regulating the pathogenicity of self-reactive CD8+ T cells. The selective ablation of IL-33 from neo-self-antigen-expressing oligodendrocytes mitigates CNS disease. In this context, fewer self-reactive CD8+ T cells persist in the inflamed CNS, and the remaining cells are largely locked into a stem-like precursor program, failing to form TCF-1low effector progeny. Importantly, interventional IL-33 blockade by locally administered somatic gene therapy reduces T cell infiltrates and improves the disease course. Our study identifies oligodendrocyte-derived IL-33 as a druggable tissue factor regulating the differentiation and survival of self-reactive CD8+ T cell in the inflamed CNS. This finding introduces tissue factors as a novel category of immune targets for treating chronic CNS autoimmune diseases.

Many patients with common variable immunodeficiency (CVID), including those with CTLA4 deficiency, NFKB1 variants and activated PI3K-delta syndrome (APDS), develop autoimmunity that is refractory to treatment. Despite this shared clinical phenotype, a unifying mechanism for the breakdown of B cell tolerance across monogenic forms of CVID has not been established. Here, we demonstrate that patients with loss-of-function NFKB1 variants, like those with CTLA4 variants and APDS, exhibit dysregulated CD4+ T cell expansion, accumulation of transitional B cells, and a relative lack of follicular B cells. In patients with monogenic CVID and clinical autoimmunity, we observed a relative expansion of transitional and activated naive (aN: CD21loCD11chi) B cells in peripheral blood accompanied by a marked increase in the frequency of VH4-34 expressing autoreactive 9G4+ B cells, which expanded between T2 and T3a transitional B cell stages. Single-cell transcriptomic and B cell receptor analysis further revealed a marked expansion of activated T1/2, T3 and extrafollicular activated naive and double negative (DN: IgD-CD27-) B cell subsets in APDS patients. Notably, one B cell subset appeared exclusively in the APDS disease state, characterized by high oxidative phosphorylation in transitional B cells, specifically. In APDS patients, we also observed a clonal expansion of specific extrafollicular class-switched DN B cells, which were clonally derived from activated transitional B cells. DN B cells were also identified in APDS lung tissue, consistent with the contribution of activated, extrafollicularly-derived B cells to tissue inflammation. Together, these findings suggest that in many patients with CVID and autoimmune features, premature activation of autoreactive transitional T2 and T3a B cells induces the survival and expansion, rather than the tolerization and elimination, of self-reactive B cells. This process leads to extrafollicular expansion of autoreactive B cells capable of tissue infiltration. One Sentence SummaryLoss of transitional B cell tolerance and extrafollicular expansion of autoreactive B cells drive autoimmunity in monogenic causes of CVID.

A disrupted equilibrium among gut microbiota, IL-17A-producing CD4 T-cells (Th17), and regulatory CD4 T-cells (Treg) have been linked with the pathobiology of multiple sclerosis (MS). While gut microbiota can regulate both Treg and Th17 cells, the impact of IL-17A on this gut-immune connection remains unclear. Utilizing HLA-DR3 transgenic mouse model of MS, we show that IL-17A deficiency (HLA-DR3.IL17A-/-) resulted in milder disease characterized by increased Tregs and expansion of Treg-promoting gut microbes, including Prevotella. Cohousing HLA-DR3 mice with HLA-DR3.IL17A-/- transferred the milder disease phenotype and associated microbiota changes to DR3 mice, highlighting the dominant role of gut microbiota in Treg induction and disease amelioration. DR3.IL17A-/- mice also showed a higher abundance of functional pathways linked with short-chain fatty acid synthesis and elevated IL-10 in dendritic cells. Enrichment of the Treg-promoting PPAR signaling pathway expression in the colon of HLA-DR3.IL17A-/- mice and following Prevotella administration in HLA-DR3 mice underscores the importance of gut microbiota in IL-17A-mediated immune regulation. Thus, our study uncovers a previously unappreciated role for IL-17A in shaping gut microbiota and immune regulation, with far-reaching implications for MS treatment. One-Sentence SummaryIL-17A modulates Treg and gut microbiota to control EAE

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is caused by impaired central immune tolerance due to deficiency of the Autoimmune Regulator (AIRE) and is characterized by severe, multiorgan autoimmunity. We recently identified interferon-{gamma} (IFN-{gamma}) as a dominant driver of immunopathology in APECED and showed that treatment with the JAK1/2 inhibitor ruxolitinib ameliorates disease in both AIRE-deficient mice and patients. However, broad JAK inhibition is associated with clinically relevant toxicities, raising the question of whether selective targeting of individual JAK pathways can retain efficacy while sparing nonpathogenic immune programs. Here, we systematically evaluated the effects of selective JAK1, JAK2, and JAK3 inhibition in Aire-/- mice. Selective JAK1 and JAK2 inhibition reduced autoimmune tissue injury, suppressed IFN-{gamma} signaling, and decreased accumulation of pathogenic T cells, with JAK2 inhibition providing the most robust protection, comparable to ruxolitinib. In contrast, selective JAK3 inhibition decreased T cell accumulation, but paradoxically increased the proportion of IFN-{gamma}-producing T cells and did not significantly attenuate IFN-{gamma}-driven tissue inflammation. These findings reveal an unexpected uncoupling between lymphocyte burden and pathogenic cytokine bias and identify IFN-{gamma} signaling as hierarchically dominant over {gamma}c-dependent pathways in AIRE deficiency. Together, our data indicate that effective control of APECED-associated autoimmunity requires direct suppression of the IFN-{gamma}-JAK2 axis rather than generalized lymphocyte inhibition and suggest that selective JAK2 targeting may represent a rational strategy to preserve therapeutic efficacy while minimizing disruption of JAK1-and {gamma}c-dependent immune functions.

Because Tissue-Resident Memory T (TRM) cells contribute critically to body-surface immunoprotection and/or immunopathology in multiple settings, their regulation is biologically and clinically important. Interestingly, TRM commonly develop in epithelia part-shaped by innate-like lymphocytes that become tissue-intrinsic during development. Here we show that polyclonal TRM cells induced by allergic contact dermatitis (ACD) interact with signature intraepidermal {gamma}{delta} T cells, facilitating a feedback-loop wherein TRM-derived IFN{gamma} upregulates PD-L1 on {gamma}{delta} cells that can thereupon regulate PD1+ TRM. Thus, TRM induced by ACD in mice lacking either local {gamma}{delta} cells, or lacking a single gene (IFN{gamma}R) expressed by local {gamma}{delta} cells, displayed enhanced proliferative and effector potentials. Those phenotypes were associated with strikingly limited motility, reduced TRM quality. and an impaired capacity to restrain melanoma. Thus, inter-individual and tissue-specific variation in how tissue-intrinsic lymphocytes integrate with TRM may sit upstream of variation in responses to cancer, allergens and other challenges, and may likewise underpin inflammatory pathologies repeatedly observed in {gamma}{delta}-deficient animals.

Multisystem inflammatory syndrome in children (MIS-C) is a severe, post-infectious sequela of SARS-CoV-2 infection, yet the pathophysiological mechanism connecting the infection to the broad inflammatory syndrome remains unknown. Here we leveraged a large set of MIS-C patient samples (n=199) to identify a distinct set of host proteins that are differentially targeted by patient autoantibodies relative to matched controls. We identified an autoreactive epitope within SNX8, a protein expressed primarily in immune cells which regulates an antiviral pathway associated with MIS-C pathogenesis. In parallel, we also probed the SARS-CoV-2 proteome-wide MIS-C patient antibody response and found it to be differentially reactive to a distinct domain of the SARS-CoV-2 nucleocapsid (N) protein relative to controls. This viral N region and the mapped SNX8 epitope bear remarkable biochemical similarity. Furthermore, we find that many children with anti-SNX8 autoantibodies also have T-cells cross-reactive to both SNX8 and this distinct domain of the SARS-CoV-2 N protein. Together, these findings suggest that MIS-C patients develop a distinct immune response against the SARS-CoV-2 N protein that is associated with cross reactivity to the self-protein SNX8, demonstrating a link from the infection to the inflammatory syndrome.

Breakdown of B cell tolerance is a central feature of autoimmune diseases, yet the molecular mechanisms underlying the female predominance of these diseases remain unclear. Here, we identified miR-130b as an essential component of a miRNA network, defined by the GUGCA seed motif, that regulates central B cell tolerance. Elevated miR-130b levels impaired tolerance by promoting the survival of immature B cells upon B cell antigen receptor engagement. This network converged on the estrogen receptor pathway through downregulation of Esr1 and Pten. Genetic ablation of Esr1 in immature B cells was sufficient to compromise central B cell tolerance in females, but not in males, revealing an unrecognized role for ER in this process. In males, Esr1 deficiency reduced bone marrow B cell numbers, whereas in females, B cell numbers were preserved, with a greater proportion of cells displaying lower surface CD19 levels. Transcriptomic analysis revealed sex-specific genetic programs characterized by defective B cell development and skewed immunoglobulin gene usage in males, and increased PI3K-AKT signaling in females. In females, this molecular rewiring compensated for developmental defects, but also attenuated central tolerance mechanisms, allowing the escape of autoreactive B cells to the periphery. In patients with multiple sclerosis, elevated levels of miR-130b in circulating vesicles correlated with more severe disease, including increased formation of new demyelinating lesions, cognitive decline, and neurodegeneration. Together, our findings identify a sex-specific ER-dependent checkpoint in B cell tolerance controlled by a seed-driven miRNA network, providing a mechanistic framework for female predisposition to autoimmunity.

Respiratory viral pandemics result in large numbers of cases of acute respiratory failure arising from a single etiology, thus reducing the heterogeneity of precepting insult and allowing improved insight into the variation of host responses. In 2009-2011, an influenza pandemic occurred, with pH1N1 infecting millions of people worldwide. Here, we have used novel bioinformatic methods to combine clinical, protein biomarker, and genomic data from patients with influenza-associated acute respiratory failure to identify three mechanistically discrete sub-types with significantly different clinical outcomes. The three endotypes identified can be described as "neutrophil-driven" (16.3%), "adaptive" (51.9%), and "endothelial leak" (31.7%). The neutrophil driven patients display evidence of innate immune activation with associated multi-organ dysfunction and reduced 30-day survival. These patients could be differentiated from the adaptive endotype by an alteration in the GAIT-mechanism, a late transcriptional regulatory response to IFN-{gamma} that acts to suppress innate immunity by reducing caeruloplasmin mRNA translation. Patients with the neutrophil-driven endotype had significantly increased IFN-{gamma} levels but appeared unable to suppress their innate immune response. The endothelial leak endotype could be distinguished from both the neutrophil driven and adaptive endotypes by alterations in Slit-Robo signalling, a pathway important in the maintenance of endothelial barrier integrity; Although patients with this endotype required mechanical ventilation, they did not develop multi-organ failure in the manner of the neutrophil-driven endotype patients, and had significantly better clinical outcomes. Importantly, the endotypes identified were stable over 48 hours opening up the possibility of stratified interventional clinical trials in the future. One Sentence SummaryWe have identified three new mechanistically distinct subtypes of influenza associated acute respiratory failure, with differential clinical outcomes.

Glycoprotein 90K, encoded by the interferon-stimulated gene LGALS3BP, displays broad antiviral activity. It reduces HIV-1 infectivity by interfering with Env maturation and virion incorporation, and increases survival of Influenza A virus-infected mice via antiviral innate immune signaling. Here, we analyzed the expression of 90K/LGALS3BP in 44 hospitalized COVID-19 patients. 90K protein serum levels were significantly elevated in COVID-19 patients compared to uninfected sex- and age-matched controls. Furthermore, PBMC-associated concentrations of 90K protein were overall reduced by SARS-CoV-2 infection in vivo, suggesting enhanced secretion into the extracellular space. Mining of published PBMC scRNA-seq datasets uncovered monocyte-specific induction of LGALS3BP mRNA expression in COVID-19 patients. In functional assays, neither 90K overexpression in susceptible cell lines nor exogenous addition of purified 90K consistently inhibited SARS-CoV-2 infection. Our data suggests that 90K/LGALS3BP contributes to the global type I IFN response during SARS-CoV-2 infection in vivo without displaying detectable antiviral properties.

Th17 cells are crucial mediators of protective immunity against extracellular pathogens but also promote inflammatory responses in pathological settings such as MS. Distinct transcriptional programs can generate Th17 populations termed pathogenic or non-pathogenic. Understanding the transcriptional regulation of these T cell states is emerging and presents opportunities to modulate pathogenic T cell responses. In this study, we demonstrate that pro-inflammatory Th17 cells in MS and EAE express high levels of the transcription factor RUNX2. T cell specific deletion of RUNX2 lowers the threshold for induction of CNS autoimmunity in mice by increasing generation of pathogenic GM-CSF-expressing Th17 cells. RUNX2-dependent inhibition of pathogenic Th17 priming relied on the extent of neutrophil-driven pathogenic Th17 priming in vivo. EAE-induced immature neutrophils elicit more pathogenic Th17 cells in the absence of RUNX2 in T cells, an effect reliant on neutrophil-derived extracellular traps. These data uncover a role for RUNX2 in suppressing NET-driven Th17 pathogenicity and suggest that increasing RUNX2 activity in T cells may be a strategy to constrain pathological inflammatory responses where Th17 cells and inflammatory neutrophils interplay.

Targeting Tregs is a potential strategy to improve cancer therapies. However, which Tregs accumulate in response to tumoral processes, and how tumors affect their phenotype, is poorly understood. Here we show that tumor Tregs are equivalent to effector tissue Tregs in steady state organs. We used a mouse model of intestinal neoplasia to demonstrate that one early event in carcinogenesis is sufficient to induce local accumulation of Tregs resembling human tumor Tregs. Treg accumulation was driven by TCR-dependent oligoclonal expansion of tissue Tregs with an effector Treg phenotype. Treg expansion was independent of CCR8, IL33R and CD137, which were previously linked to tumor Treg. In contrast, GATA3 was required for effector tissue Tregs and for their expansion in response to neoplasia. Our findings identify GATA3-dependent clonal expansion of effector tissue Tregs as a key event in promoting tumor growth. HighlightsO_LIAn early tumorigenic event alone drives accumulation of effector tissue Tregs C_LIO_LITregs in tumors are phenotypically akin to effector tissue Tregs C_LIO_LIThe accumulation of Tregs is driven by TCR-dependent oligoclonal expansion C_LIO_LIGATA3 controls tumor-promoting effector tissue Tregs C_LI

Extraintestinal autoimmune diseases are multifactorial with translocating gut pathobionts implicated as instigators and perpetuators in mice. However, the microbial contributions to autoimmunity in humans remain largely unclear, including whether specific pathological human adaptive immune responses are triggered by such pathobionts. We show here that the translocating pathobiont Enterococcus gallinarum induces human IFN{gamma}+ Th17 differentiation and IgG3 subclass switch of anti-E. gallinarum RNA and correlating anti-human RNA autoantibody responses in patients with systemic lupus erythematosus and autoimmune hepatitis. Human Th17 induction by E. gallinarum is cell-contact dependent and involves TLR8-mediated human monocyte activation. In murine gnotobiotic lupus models, E. gallinarum translocation triggers IgG3 anti-RNA autoantibody titers that correlate with renal autoimmune pathophysiology and with disease activity in patients. Overall, we define cellular mechanisms of how a translocating pathobiont induces human T- and B-cell-dependent autoimmune responses, providing a framework for developing host- and microbiota-derived biomarkers and targeted therapies in extraintestinal autoimmune diseases. One Sentence SummaryTranslocating pathobiont Enterococcus gallinarum promotes human Th17 and IgG3 autoantibody responses linked to disease activity in autoimmune patients.

Epithelial injury calls for a regenerative response from a coordinated network of epithelial stem cells and immune cells. Defining this network is key to preserving the repair process for acute resolution, but also for preventing a remodeling process with chronic dysfunction. We recently identified an immune niche for basal-epithelial stem cells using mouse models of injury after respiratory viral infection. Niche function depended on an early sentinel population of monocyte-derived dendritic cells (moDCs) that provided ligand GPNMB to basal-ESC receptor CD44 for reprogramming towards chronic lung disease. These same cell and molecular control points worked directly in mouse and human basal-ESC organoids, but the findings were not yet validated in vivo in human disease. Further, persistence of GPNMB expression in moDCs and M2-macrophages in mouse models suggested utility as a long-term disease biomarker in humans. Here we show increased expression of GPNMB localized to moDC-macrophage populations in lung tissue samples from long-term Covid, asthma, and COPD. The findings thereby provide initial evidence of a persistent and correctable pathway from acute injury to chronic disease with implications for cellular reprogramming and inflammatory memory. New and noteworthyRecent work indicates that a sentinel immune niche provides GPNMB to epithelial stem cells to drive structural remodeling and disease as exemplified by the response to respiratory viral injury. The present study provides initial evidence that this niche can be detected in humans in the context of comparable diseases (long-term Covid, asthma, and COPD) also linked to viral infection. The results support a persistent mechanism for inflammatory disease that might be correctable with GPNMB blockade directly or indirectly through related signaling pathways.

The PD-L1/2 - PD-1 immune checkpoint is essential for the proper induction of peripheral tolerance and limits autoimmunity, whereas tumor cells exploit their expression to promote immune evasion. Many different cell types express PD-L1/2, either constitutively or upon stimulation, but the factors driving this expression are often not defined. Here, using genome-wide CRISPR-activation screening, we identified three factors that upregulate PD-L1 expression; GATA2, MBD6, and VGLL3. GATA2 and VGLL3 act as transcriptional regulators and their expression induced PD-L1 in many different cell types. Conversely, loss of VGLL3 impaired IFN{gamma}-induced PD-L1/2 expression in keratinocytes. Mechanistically, by performing a second screen to identify proteins acting together with VGLL3, we found that VGLL3 forms a complex with TEAD1 and RUNX1/3 to drive expression of PD-L1/2. Collectively, our work identified a new transcriptional network controlling PD-L1/2 expression and suggests that VGLL3, in addition to its known role in the expression of pro-inflammatory genes, can balance inflammation by upregulating the anti-inflammatory factors PD-L1 and PD-L2.

Immune responses to pathogens and effective vaccines elicit germinal center (GC) responses wherein B cells undergo affinity maturation and develop into plasma cells (PCs) and memory B cells (MBCs). The GC reaction is initially seeded by a limited group of founder B cells, and subsequently further diversified by continual entry of naive B cells that compete with GC founder cells for antigen and T cell help. Whether these later-arriving invaders contribute to the development of PCs or MBCs is not known. To investigate the fate of GC invaders we developed a dual-recombinase reporter approach that enables pre- and post-GC B cell lineage tracing and used it to examine immune responses to vaccination and influenza infection. Notably, fate-mapped invaders preferentially give rise to MBCs as opposed to PCs. Moreover, antibodies expressed by invader-derived MBCs harbor fewer somatic mutations, exhibit lower affinity, and their antibodies bind to subdominant antigenic epitopes relative to founder MBCs. Our findings indicate that invader GC B cells are an important source of humoral immune memory diversification after infection or vaccination.

Sjogrens disease (SjD) causes localised and systemic inflammation due to autoantibody production against intracellular proteins, such as TRIM21/Ro52. TRIM21 is an E3 ubiquitin ligase which binds antibody Fc domains on opsonised pathogens, which have escaped extracellular immunity and entered cytosols; TRIM21 ubiquitinates these, driving their proteasomal degradation. How and why TRIM21 becomes an autoantigen remains unclear. We show that TRIM21 is released upon lytic cell death (pyroptosis/necroptosis) but not apoptosis. Released TRIM21 binds circulating antibody Fc domains, and forms large immune complexes (ICs). These are further enhanced with TRIM21/Ro52 seropositive SjD plasma antibodies, where interactions are mediated via both Fc and F(ab)2 domains. TRIM21-ICs are taken up by macrophages, which in high interferon environments drive pro-inflammatory responses, antigen presentation, and inflammatory and metabolic transcriptional changes. Whilst many cytosolic proteins are released by dead cells, due to its high affinity for antibodies, TRIM21 can generate large ICs. This may perpetuate inflammation and antigen presentation, causing TRIM21 to be highly autoimmunogenic. One Sentence SummaryHow the intracellular protein TRIM21 becomes an autoantigen.

While age-associated B cells (ABCs) are known to expand and persist following viral infection and during autoimmunity, their interactions are yet to be studied together in these contexts. Epstein-Barr virus (EBV) infection has long been implicated in multiple sclerosis (MS), and it is not known whether ABCs could play a role in mediating viral contribution to autoimmunity. Here, we show that the circulating ABC population is expanded in people with MS and that EBV infection and MS status differentially impact the circulating ABC phenotype. We then directly compared ABCs during viral infection and autoimmunity using mouse models of EBV, gammaherpesvirus 68 ({gamma}HV68), and MS, experimental autoimmune encephalomyelitis (EAE). We observed that splenic ABCs are expanded in a sex-biased manner during both latent virus infection and EAE, and each event drives the ABC population to opposing phenotypes. We have previously shown that latent{gamma} HV68 infection exacerbates EAE and here we show that mice lacking ABCs fail to display{gamma} HV68-enhanced disease. Collectively, these findings indicate that latent viral infection and central nervous system autoimmunity differentially impact the ABC population and suggests that viral infections such as EBV prime ABCs to contribute pathogenically in MS.