A leading theory regarding the pathogenesis of biliary atresia (BA) is that bile duct injury is initiated by a virus infection, followed by an autoimmune response targeting bile ducts. In experimental models of autoimmune diseases, B cells have been shown to play an important role. The aim of this study was to determine the role of B cells in the development of biliary obstruction in the Rhesus rotavirus (RRV)-induced mouse model of BA. Wild-type (WT) and B cell-deficient (Ig-α(-/-)) mice received RRV shortly after birth. Ig-α(-/-) RRV-infected mice had significantly increased disease-free survival rate compared to WT RRV-infected BA mice (76.8% vs. 17.5%). In stark contrast to the RRV-infected BA mice, the RRV-infected Ig-α(-/-) mice did not have hyperbilirubinemia or bile duct obstruction. The RRV-infected Ig-α(-/-) mice had significantly less liver inflammation and Th1 cytokine production compared to RRV-infected WT mice. In addition, Ig-α(-/-) mice had significantly increased numbers of regulatory T cells (Tregs) at baseline and after RRV infection compared to WT mice. However, depletion of Tregs in Ig-α(-/-) mice did not induce biliary obstruction, indicating that the expanded Tregs in the Ig-α(-/-) mice were not the sole reason for protection from disease. Conclusion : B cell deficient Ig-α(-/-) mice are protected from biliary obstruction in the RRV-induced mouse model of BA, indicating a primary role of B cells in mediating disease pathology. The mechanism of protection may involve lack of B cell antigen presentation, which impairs T-cell activation and Th1 inflammation. Immune modulators that inhibit B cell function may be a new strategy for treatment of BA.

Autoreactive B cells have a major function in autoimmunity. A small subset of B cells expressing two distinct B-cell-antigen-receptors (B2R cells) is elevated in many patients with systematic lupus erythematosus (SLE) and in the MRL(/lpr) mouse model of lupus, and is often autoreactive. Here we show, using RNAseq and in vitro and in vivo analyses, signals that are required for promoting B2R cell numbers and effector function in autoimmune mice. Compared with conventional B cells, B2R cells are more responsive to Toll-like receptor 7/9 and type I/II interferon treatment, display higher levels of MHCII and co-receptors, and depend on IL-21 for their homeostasis; moreover they expand better upon T cell-dependent antigen stimulation, and mount a more robust memory response, which are characteristics essential for enhanced (auto)immune responses. Our findings thus provide insights on the stimuli for the expansion of an autoreactive B cell subset that may contribute to the etiology of SLE.

A subset of characterized HIV-1 broadly neutralizing antibodies (bnAbs) are polyreactive with additional specificities for self-antigens and it has been proposed immunological tolerance may present a barrier to their participation in protective humoral immunity. We address this hypothesis by immunizing autoimmune-prone mice with HIV-1 Envelope (Env) and characterizing the primary antibody response for HIV-1 neutralization. We find autoimmune mice generate neutralizing antibody responses to tier 2 HIV-1 strains with alum treatment alone in the absence of Env. Importantly, experimentally breaching immunological tolerance in wild-type mice also leads to the production of tier 2 HIV-1-neutralizing antibodies, which increase in breadth and potency following Env immunization. In both genetically prone and experimentally induced mouse models of autoimmunity, increased serum levels of IgM anti-histone H2A autoantibodies significantly correlated with tier 2 HIV-1 neutralization, and anti-H2A antibody clones were found to neutralize HIV-1. These data demonstrate that breaching peripheral tolerance permits a cross-reactive HIV-1 autoantibody response able to neutralize HIV-1.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited systemic treatment options. RX-5902 is a novel anti-cancer agent that inhibits phosphorylated-p68 and thus attenuates nuclear β-catenin signaling. The purpose of this study was to evaluate the ability of β-catenin signaling blockade to enhance the efficacy of anti-CTLA-4 and anti-PD-1 immune checkpoint blockade in immunocompetent, preclinical models of TNBC.

Alveolar macrophages (AM) play a central role in initiation and resolution of lung inflammation, but the integration of these opposing core functions is poorly understood. AM expression of cholesterol 25-hydroxylase (CH25H), the primary biosynthetic enzyme for 25-hydroxycholesterol (25HC), far exceeds the expression of macrophages in other tissues, but no role for CH25H has been defined in lung biology. As 25HC is an agonist for the antiinflammatory nuclear receptor, liver X receptor (LXR), we speculated that CH25H might regulate inflammatory homeostasis in the lung. Here, we show that, of natural oxysterols or sterols, 25HC is induced in the inflamed lung of mice and humans. Ch25h-/- mice fail to induce 25HC and LXR target genes in the lung after LPS inhalation and exhibit delayed resolution of airway neutrophilia, which can be rescued by systemic treatment with either 25HC or synthetic LXR agonists. LXR-null mice also display delayed resolution, suggesting that native oxysterols promote resolution. During resolution, Ch25h is induced in macrophages upon their encounter with apoptotic cells and is required for LXR-dependent prevention of AM lipid overload, induction of Mertk, efferocytic resolution of airway neutrophilia, and induction of TGF-β. CH25H/25HC/LXR is, thus, an inducible metabolic axis that programs AMs for efferocytic resolution of inflammation.

Over the past decade, immunotherapies have revolutionized the treatment of cancer. Although the success of immunotherapy is remarkable, it is still limited to a subset of patients. More than 1500 clinical trials are currently ongoing with a goal of improving the efficacy of immunotherapy through co-administration of other agents. Preclinical, small-animal models are strongly desired to increase the pace of scientific discovery, while reducing the cost of combination drug testing in humans. Human immune system (HIS) mice are highly immune-deficient mouse recipients rtpeconstituted with human hematopoietic stem cells. These HIS-mice are capable of growing human tumor cell lines and patient-derived tumor xenografts. This model allows rapid testing of multiple, immune-related therapeutics for tumors originating from unique clinical samples. Using a cord blood-derived HIS-BALB/c-Rag2nullIl2rγnullSIRPαNOD (BRGS) mouse model, we summarize our experiments testing immune checkpoint blockade combinations in these mice bearing a variety of human tumors, including breast, colorectal, pancreatic, lung, adrenocortical, melanoma and hematological malignancies. We present in-depth characterization of the kinetics and subsets of the HIS in lymph and non-lymph organs and relate these to protocol development and immune-related treatment responses. Furthermore, we compare the phenotype of the HIS in lymph tissues and tumors. We show that the immunotype and amount of tumor infiltrating leukocytes are widely-variable and that this phenotype is tumor-dependent in the HIS-BRGS model. We further present flow cytometric analyses of immune cell subsets, activation state, cytokine production and inhibitory receptor expression in peripheral lymph organs and tumors. We show that responding tumors bear human infiltrating T cells with a more inflammatory signature compared to non-responding tumors, similar to reports of "responding" patients in human immunotherapy clinical trials. Collectively these data support the use of HIS mice as a preclinical model to test combination immunotherapies for human cancers, if careful attention is taken to both protocol details and data analysis.

About 5% of B cells in healthy mice and humans are allelically or isotypically included and hence co-express two different antibodies. In mice, dual antibody B cells (B2R) expand with systemic autoimmunity, co-express autoreactive and non-autoreactive antibodies, and participate in immune responses, but this phenomenon is strain dependent. This study was developed with two goals: 1) to establish the contribution of TLR and IFN receptor signaling to the development of germinal center B cells that express two antibodies in MRL/lpr mice; and 2) to determine whether B2R B cells are increased and particularly activated in a subset of adult patients diagnosed with systemic lupus erythematosus (SLE). Results from the MRL/lpr studies indicate that the enhanced differentiation of dual-κ B cells into germinal center B cells is due to a heightened response to TLR7 and TLR9 signaling, further fueled by an increased response to type II IFN. To understand the clinical and translational implications of our observations in mouse B2R B cells, cohorts of SLE patients and healthy controls were recruited and evaluated for expression of dual BCRs. Results from flow cytometry and microscopy revealed supraphysiological frequencies of κ+λ+ B2R cells in one fourth of the SLE patients. Abnormal numbers of κ+λ+ B cells correlated with higher frequencies of activated naïve B cells and age-associated B cells, and a lower proportion of "B cells that are naïve IgD+" (BND). However, results from single cell V(D)J sequencing demonstrated that these high κ+λ+ SLE patients harbored normal frequencies of κ+λ+ and other B2R B cells. and we further show that their B cells were instead decorated by κ and λ VH4-34 autoantibodies. Thus, our findings indicate that elevated flow cytometric detection of isotypically-included B cells can identify patients with high titers of B cell-reactive VH4-34 autoantibodies and abnormal distribution of B cell subsets relevant to autoimmunity.

Coagulopathy in severe COVID-19 is common but poorly understood. The purpose of this study was to determine how SARS-CoV-2 infection impacts histone levels, fibrin structure, and endogenous thrombin potential in the presence and absence of endothelial cells. We studied individuals with SARS-CoV-2 infection and acute respiratory distress syndrome at the time of initiation of mechanical ventilation compared to healthy controls. Circulating histone-DNA complexes were elevated in the plasma of COVID-19 patients relative to healthy controls (n=6, each group). Using calibrated automated thrombography, thrombin generation was altered in COVID-19 patient plasma samples. Despite having increased endogenous thrombin potential, patient plasma samples exhibited prolonged lag times and times to peak thrombin in the presence of added tissue factor and PCPS. Strikingly different results were observed when endothelial cells were used in place of tissue factor and PCPS. While healthy control plasma samples did not generate measurable thrombin after 60 min, plasma samples from COVID-19+ patients formed thrombin (mean lag time ~20 min). Consistent with the observed alterations in thrombin generation, clots from COVID-19 subjects exhibited a denser fibrin network, thinner fibers and lower fibrin resolvability. Elevated histones, aberrant fibrin formation, and increased endothelial-dependent thrombin generation may contribute to coagulopathy in COVID-19.

Although increased neuropeptides are often detected in lungs that exhibit respiratory distress, whether they contribute to the condition is unknown. Here, we show in a mouse model of neuroendocrine cell hyperplasia of infancy, a pediatric disease with increased pulmonary neuroendocrine cells (PNECs), excess PNEC-derived neuropeptides are responsible for pulmonary manifestations including hypoxemia. In mouse postnatal lung, prolonged signaling from elevated neuropeptides such as calcitonin gene-related peptide (CGRP) activate receptors enriched on endothelial cells, leading to reduced cellular junction gene expression, increased endothelium permeability, excess lung fluid, and hypoxemia. Excess fluid and hypoxemia were effectively attenuated by either prevention of PNEC formation, inactivation of CGRP gene, endothelium-specific inactivation of CGRP receptor gene, or treatment with CGRP receptor antagonist. Neuropeptides were increased in human lung diseases with excess fluid such as acute respiratory distress syndrome. Our findings suggest that restricting neuropeptide function may limit fluid and improve gas exchange in these conditions.

Phagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue homeostasis and drives anti-inflammatory programming in engulfing macrophages. Here, we assess metabolites in naive and inflammatory macrophages following engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to increases in the arginine-derived polyamines, spermidine and spermine, in vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does not arise from retention of apoptotic cell metabolites or de novo synthesis but from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase. Blocking polyamine import prevents efferocytosis from suppressing macrophage interleukin (IL)-1β or IL-6. This identifies efferocytosis as a trigger for polyamine import and accumulation, and imported polyamines as mediators of efferocytosis-induced immune reprogramming.

Airway mucus is essential for lung defense, but excessive mucus in asthma obstructs airflow, leading to severe and potentially fatal outcomes. Current asthma treatments have minimal effects on mucus, and the lack of therapeutic options stems from a poor understanding of mucus function and dysfunction at a molecular level and in vivo. Biophysical properties of mucus are controlled by mucin glycoproteins that polymerize covalently via disulfide bonds. Once secreted, mucin glycopolymers can aggregate, form plugs, and block airflow. Here we show that reducing mucin disulfide bonds disrupts mucus in human asthmatics and reverses pathological effects of mucus hypersecretion in a mouse allergic asthma model. In mice, inhaled mucolytic treatment loosens mucus mesh, enhances mucociliary clearance, and abolishes airway hyperreactivity (AHR) to the bronchoprovocative agent methacholine. AHR reversal is directly related to reduced mucus plugging. These findings establish grounds for developing treatments to inhibit effects of mucus hypersecretion in asthma.

Alveolar macrophages serve as central orchestrators of inflammatory responses in the lungs, both initiating their onset and promoting their resolution. However, the mechanisms that program macrophages for these dynamic responses are not fully understood. Over 95% of all mammalian genes undergo alternative pre-mRNA splicing. While alternative splicing has been shown to regulate inflammatory responses in macrophages in vitro, it has not been investigated on a genome-wide scale in vivo Here we used RNAseq to investigate alternative pre-mRNA splicing in alveolar macrophages isolated from lipopolysaccharide (LPS)-treated mice during the peak of inflammation and during its resolution. We found that lung inflammation induced substantial alternative pre-mRNA splicing in alveolar macrophages. The number of changes in isoform usage was greatest at the peak of inflammation and involved multiple classes of alternative pre-mRNA splicing events. Comparative pathway analysis of inflammation-induced changes in alternative pre-mRNA splicing and differential gene expression revealed overlap of pathways enriched for immune responses such as chemokine signaling and cellular metabolism. Moreover, alternative pre-mRNA splicing of genes in metabolic pathways differed in tissue resident vs. recruited (blood monocyte-derived) alveolar macrophages and corresponded to changes in core metabolism, including a switch to Warburg-like metabolism in recruited macrophages with increased glycolysis and decreased flux through the tricarboxylic acid cycle.

The mononuclear phagocyte (MP) system consists of macrophages, monocytes, and dendritic cells (DCs). MP subtypes play distinct functional roles in steady-state and inflammatory conditions. Although murine MPs are well characterized, their pulmonary and lymph node (LN) human homologs remain poorly understood. To address this gap, we have created a gene expression compendium across 24 distinct human and murine lung and LN MPs, along with human blood and murine spleen MPs, to serve as validation datasets. In-depth RNA sequencing identifies corresponding human-mouse MP subtypes and determines marker genes shared and divergent across species. Unexpectedly, only 13%-23% of the top 1,000 marker genes (i.e., genes not shared across species-specific MP subtypes) overlap in corresponding human-mouse MP counterparts. Lastly, CD88 in both species helps distinguish monocytes/macrophages from DCs. Our cross-species expression compendium serves as a resource for future translational studies to investigate beforehand whether pursuing specific MP subtypes or genes will prove fruitful.

B cell receptors (BCRs) generate tonic signals critical for B cell survival and early B cell development. To determine whether these signals also mediate the development of transitional and mature B cells, we examined B cell development using a mouse strain in which nonautoreactive immunoglobulin heavy and light chain-targeted B cells express low surface BCR levels. We found that reduced BCR expression translated into diminished tonic BCR signals that strongly impaired the development of transitional and mature B cells. Constitutive expression of Bcl-2 did not rescue the differentiation of BCR-low B cells, suggesting that this defect was not related to decreased cell survival. In contrast, activation of the Ras pathway rescued the differentiation of BCR-low immature B cells both in vitro and in vivo, whereas extracellular signal-regulated kinase (Erk) inhibition impaired the differentiation of normal immature B cells. These results strongly suggest that tonic BCR signaling mediates the differentiation of immature into transitional and mature B cells via activation of Erk, likely through a pathway requiring Ras.

Humoral immunity to viruses and encapsulated bacteria is comprised of T cell-independent type 2 (TI-2) antibody responses that are characterized by rapid antibody production by marginal zone and B1 B cells. We demonstrate that toll-like receptor (TLR) ligands influence the TI-2 antibody response not only by enhancing the overall magnitude but also by skewing this response to one that is dominated by IgG isotypes. Importantly, TLR ligands facilitate this response by inducing type I interferon (IFN), which in turn elicits rapid and significant amounts of antigen-specific IgG2c predominantly from FO (follicular) B cells. Furthermore, we show that although the IgG2c antibody response requires B cell-autonomous IFN-alpha receptor signaling, it is independent of B cell-intrinsic TLR signaling. Thus, innate signals have the capacity to enhance TI-2 antibody responses by promoting participation of FO B cells, which then elaborate effective IgG anti-pathogen antibodies.

Rare dual-reactive B cells expressing two types of Ig light or heavy chains have been shown to participate in immune responses and differentiate into IgG(+) cells in healthy mice. These cells are generated more often in autoreactive mice, leading us to hypothesize they might be relevant in autoimmunity. Using mice bearing Igk allotypic markers and a wild-type Ig repertoire, we demonstrate that the generation of dual-κ B cells increases with age and disease progression in autoimmune-prone MRL and MRL/lpr mice. These dual-reactive cells express markers of activation and are more frequently autoreactive than single-reactive B cells. Moreover, dual-κ B cells represent up to half of plasmablasts and memory B cells in autoimmune mice, whereas they remain infrequent in healthy mice. Differentiation of dual-κ B cells into plasmablasts is driven by MRL genes, whereas the maintenance of IgG(+) cells is partly dependent on Fas inactivation. Furthermore, dual-κ B cells that differentiate into plasmablasts retain the capacity to secrete autoantibodies. Overall, our study indicates that dual-reactive B cells significantly contribute to the plasmablast and memory B cell populations of autoimmune-prone mice suggesting a role in autoimmunity.

The mechanisms by which B cells undergo tolerance, such as receptor editing, clonal deletion, and anergy, have been established in mice. However, corroborating these mechanisms in humans remains challenging. To study how autoreactive human B cells undergo tolerance, we developed a novel humanized mouse model. Mice expressing an anti-human Igκ membrane protein to serve as a ubiquitous neo self-antigen (Ag) were transplanted with a human immune system. By following the fate of self-reactive human κ(+) B cells relative to nonautoreactive λ(+) cells, we show that tolerance of human B cells occurs at the first site of self-Ag encounter, the bone marrow, via a combination of receptor editing and clonal deletion. Moreover, the amount of available self-Ag and the genetics of the cord blood donor dictate the levels of central tolerance and autoreactive B cells in the periphery. Thus, this model can be useful for studying specific mechanisms of human B cell tolerance and to reveal differences in the extent of this process among human populations.

Dendritic cells (DCs) are required for the induction of cytotoxic T cells (CTL). In most tissues, including the lung, the resident DCs fall into two types expressing the integrin markers CD103 and CD11b. The current supposition is that DC function is predetermined by lineage, designating the CD103(+) DC as the major cross-presenting DC able to induce CTL. Here we show that Poly I:C (TLR3 agonist) or R848 (TLR7 agonist) do not activate all endogenous DCs. CD11b(+) DCs can orchestrate a CTL response in vivo in the presence of a TLR7 agonist but not a TLR3 agonist, whereas CD103(+) DCs require ligation of TLR3 for this purpose. This selectivity does not extend to antigen cross-presentation for T-cell proliferation but is required for induction of cytotoxicity. Thus, we demonstrate that the ability of DCs to induce functional CTLs is specific to the nature of the pathogen-associated molecular pattern (PAMP) encountered by endogenous DC.

Double negative (DN) B cells (CD27-IgD-) comprise a heterogenous population of DN1, DN2, and the recently described DN3 and DN4 subsets. In autoimmune disease, DN2 cells are reported to be precursors to autoreactive antibody secreting cells and expansion of DN2 cells is linked to elevated interferon levels. Severe SARS-CoV-2 infection is characterized by elevated systemic levels of pro-inflammatory cytokines and serum autoantibodies and expansion of the DN2 subset in severe SARS-CoV-2 infection has been reported. However, the activation status, functional capacity and contribution to virally-induced autoantibody production by DN subsets is not established. Here, we validate the finding that severe SARS-CoV-2 infection is associated with a reduction in the frequency of DN1 cells coinciding with an increase in the frequency of DN2 and DN3 cells. We further demonstrate that with severe viral infection DN subsets are at a heightened level of activation, display changes in immunoglobulin class isotype frequency and have functional BCR signaling. Increases in overall systemic inflammation (CRP), as well as specific pro-inflammatory cytokines (TNFα, IL-6, IFNγ, IL-1β), significantly correlate with the skewing of DN1, DN2 and DN3 subsets during severe SARS-CoV-2 infection. Importantly, the reduction in DN1 cell frequency and expansion of the DN3 population during severe infection significantly correlates with increased levels of serum autoantibodies. Thus, systemic inflammation during SARS-CoV-2 infection drives changes in Double Negative subset frequency, likely impacting their contribution to generation of autoreactive antibodies.

Newly generated bone marrow B cells are positively selected into the peripheral lymphoid tissue only when they express a B cell receptor (BCR) that is nonautoreactive or one that binds self-antigen with only minimal avidity. This positive selection process, moreover, is critically contingent on the ligand-independent tonic signals transduced by the BCR. We have previously shown that when autoreactive B cells express an active form of the rat sarcoma (RAS) oncogene, they upregulate the receptor for the B cell activating factor (BAFFR) and undergo differentiation in vitro and positive selection into the spleen in vivo, overcoming central tolerance. Based on the in vitro use of pharmacologic inhibitors, we further showed that this cell differentiation process is critically dependent on the activation of the mitogen-activated protein kinase kinase pathway MEK (MAPKK)-extracellular signal-regulated kinase (ERK), which is downstream of RAS. Here, we next investigated if activation of ERK is not only necessary but also sufficient to break central B cell tolerance and induce differentiation of autoreactive B cells in vitro and in vivo. Our results demonstrate that activation of ERK is critical for upregulating BAFFR and overcoming suboptimal levels of tonic BCR signals or low amounts of antigen-induced BCR signals during in vitro B cell differentiation. However, direct activation of ERK does not lead high avidity autoreactive B cells to increase BAFFR levels and undergo positive selection and differentiation in vivo. B cell-specific MEK-ERK activation in mice is also unable to lead to autoantibody secretion, and this in spite of a general increase of serum immunoglobulin levels. These findings indicate that additional pathways downstream of RAS are required for high avidity autoreactive B cells to break central and/or peripheral tolerance.