Heightened TLR7 signaling primes BCR-activated B cells in chronic graft-versus-host disease for effector functions.

ABSTRACT: Chronic graft-versus-host disease (cGVHD) is a debilitating, autoimmune-like syndrome that can occur after allogeneic hematopoietic stem cell transplantation. Constitutively activated B cells contribute to ongoing alloreactivity and autoreactivity in patients with cGVHD. Excessive tissue damage that occurs after transplantation exposes B cells to nucleic acids in the extracellular environment. Recognition of endogenous nucleic acids within B cells can promote pathogenic B-cell activation. Therefore, we hypothesized that cGVHD B cells aberrantly signal through RNA and DNA sensors such as Toll-like receptor 7 (TLR7) and TLR9. We found that B cells from patients and mice with cGVHD had higher expression of TLR7 than non-cGVHD B cells. Using ex vivo assays, we found that B cells from patients with cGVHD also demonstrated increased interleukin-6 production after TLR7 stimulation with R848. Low-dose B-cell receptor (BCR) stimulation augmented B-cell responses to TLR7 activation. TLR7 hyperresponsiveness in cGVHD B cells correlated with increased expression and activation of the downstream transcription factor interferon regulatory factor 5. Because RNA-containing immune complexes can activate B cells through TLR7, we used a protein microarray to identify RNA-containing antigen targets of potential pathological relevance in cGVHD. We found that many of the unique targets of active cGVHD immunoglobulin G (IgG) were nucleic acid-binding proteins. This unbiased assay identified the autoantigen and known cGVHD target Ro-52, and we found that RNA was required for IgG binding to Ro-52. Herein, we find that BCR-activated B cells have aberrant TLR7 signaling responses that promote potential effector responses in cGVHD.

Single-cell landscape analysis unravels molecular programming of the human B cell compartment in chronic GVHD.

Alloreactivity can drive autoimmune syndromes. After allogeneic hematopoietic stem cell transplantation (allo-HCT), chronic graft-versus-host disease (cGVHD), a B cell-associated autoimmune-like syndrome, commonly occurs. Because donor-derived B cells continually develop under selective pressure from host alloantigens, aberrant B cell receptor (BCR) activation and IgG production can emerge and contribute to cGVHD pathobiology. To better understand molecular programing of B cells in allo-HCT, we performed scRNA-Seq analysis on high numbers of purified B cells from patients. An unsupervised analysis revealed 10 clusters, distinguishable by signature genes for maturation, activation, and memory. Within the memory B cell compartment, we found striking transcriptional differences in allo-HCT patients compared with healthy or infected individuals, including potentially pathogenic atypical B cells (ABCs) that were expanded in active cGVHD. To identify intrinsic alterations in potentially pathological B cells, we interrogated all clusters for differentially expressed genes (DEGs) in active cGVHD versus patients who never had signs of immune tolerance loss (no cGVHD). Active cGVHD DEGs occurred in both naive and BCR-activated B cell clusters. Remarkably, some DEGs occurred across most clusters, suggesting common molecular programs that may promote B cell plasticity. Our study of human allo-HCT and cGVHD provides understanding of altered B cell memory during chronic alloantigen stimulation.

A Phase I Trial of SYK Inhibition with Fostamatinib in the Prevention and Treatment of Chronic Graft-Versus-Host Disease.

Despite the exciting advancement of novel therapies, chronic graft-versus-host disease (cGVHD) remains the most common cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (HCT). Frontline treatment of cGVHD involves systemic steroids, which are associated with significant morbidities. We previously found that inhibition of spleen tyrosine kinase (SYK) with fostamatinib preferentially eradicated aberrantly activated B cells in both ex vivo studies of cGVHD patient B cells, as well as in vivo mouse studies. These and other preclinical studies implicated hyper-reactive B-cell receptor signaling and increased SYK expression in the pathogenesis of cGVHD and compelled this first in-human allogeneic HCT clinical trial. We investigated the safety and efficacy of the oral SYK inhibitor, fostamatinib, for both the prevention and treatment of cGVHD. The primary objective was to evaluate the safety of fostamatinib and determine its maximum tolerated dose in the post-HCT setting. Secondary objectives included assessing the efficacy of fostamatinib in preventing and treating cGVHD, as well as examining alterations in B-cell compartments with treatment. This was a single-institution phase I clinical trial that evaluated the use of fostamatinib in allogeneic HCT patients before the development of cGVHD or at the time of steroid-refractory cGVHD (SR-cGVHD). Patients received fostamatinib at one of three dose levels using a continual reassessment algorithm to determine the maximum tolerated dose. Multiparameter flow cytometry was used to evaluate changes in B cell subpopulations over the first year of treatment with fostamatinib. Nineteen patients were enrolled in this phase I trial, with 5 in the prophylaxis arm and 14 in the therapeutic arm. One patient (5%) required discontinuation of therapy for a dose-limiting toxicity. At a median follow-up of over 3 years, no patients had cancer relapse while on fostamatinib treatment, and recurrent malignancy was observed in 1 patient 2 years after the end of therapy. In the prophylaxis arm, 1 of 5 patients (20%) developed cGVHD while on fostamatinib. In the therapeutic arm, the overall response rate was 77%, with a complete response rate of 31%. The median duration of response was 19.3 months and the 12-month failure-free survival was 69% (95% confidence interval, 48-100). Patients were able to reduce their steroid dose by a median of 80%, with 73% remaining on a lower dose at 1 year compared to baseline. There was an early reduction in the proportion of IgD-CD38hi plasmablast-like cells with fostamatinib treatment, particularly in those SR-cGVHD patients who had an eventual response. B-cell reconstitution was not significantly impacted by fostamatinib therapy after allogeneic HCT. Fostamatinib featured a favorable safety profile in the post-HCT setting. Our data suggests an early efficacy signal that was associated with effects on expected cell targets in both the prophylaxis and treatment of cGVHD, providing rationale for a phase II investigation.

A Mimic of Ankylosing Spondylitis, Ochronosis: Case Report and Review of the Literature.

PURPOSE OF REVIEW: Ochronosis and alkaptonuria are manifestations of the same condition-a rare autosomal recessive disorder resulting from a constitutional lack of homogentisate 1,2-dioxygenase (HGD) with the consequent accumulation of homogentisic acid (HGA). In ochronosis, HGA undergoes autoxidation as well as enzymatic oxidation to form an ochronotic pigment that accumulates in cartilage and connective tissues. In the beginning, there is homogentisic aciduria and pigmentation of cartilages and other connective tissues. In later years, generalized osteoarthritis of the spine and large joints, termed ochronotic arthropathy, develops. RECENT FINDINGS: The diagnosis is confirmed by quantitative measurement of HGA in urine and mutation analysis of the HGD gene. One of the differential diagnoses for the skin findings is exogenous ochronosis, a limited hyperpigmentation of skin caused by some chemicals. As for the lumbar spine findings, there can be radiographic similarities with ankylosing spondylitis (AS) including reduced intervertebral disc spaces and loss of lumbar lordosis; however, ochronosis will spare the sacroiliac joint, and the lumbar spine will show dense, wafer-like disk calcification with a vacuum disc phenomenon and broad syndesmophytes. Here, we present a case of a patient with probable ochronosis that was treated many years as ankylosing spondylitis without response, and we provide a review of the current literature on ochronosis pathogenesis, diagnosis, and treatment.

Autoimmune Theories of Chronic Spontaneous Urticaria.

Urticaria (hives) is a highly prevalent skin disorder that can occur with or without associated angioedema. Chronic spontaneous urticaria (CSU) is a condition which persists for more than 6 weeks in duration and occurs in the absence of an identifiable provoking factor. CSU results from pathogenic activation of mast cells and basophils, which gives rise to the release of proinflammatory mediators that support the generation of urticaria. Several theories have been put forth regarding the pathogenesis of CSU with much evidence pointing toward a potential autoimmune etiology in up to 50% of patients with this condition. In this review, we highlight the evidence surrounding the autoimmune pathogenesis of chronic urticaria including recent data which suggests that CSU may involve contributions from both immunoglobin G (IgG)-specific and immunoglobulin E (IgE)-specific autoantibodies against a vast array of antigens that can span beyond those found on the surface of mast cells and basophils.

Early-life antibiotics attenuate regulatory T cell generation and increase the severity of murine house dust mite-induced asthma.

INTRODUCTION: Early-life exposure to antibiotics (ABX) has been linked to increases in asthma severity and prevalence in both children and laboratory animals. We explored the immunologic mechanisms behind this association using a mouse model of house dust mite (HDM)-induced asthma and early-life ABX exposure. METHODS: Mice were exposed to three short courses of ABX following weaning and experimental asthma was thereafter induced. Airway cell counts and differentials; serum immunoglobulin E (IgE); pulmonary function; lung histopathology; pulmonary regulatory T cells (Tregs); and the fecal microbiome were characterized following ABX exposure and induction of experimental asthma. RESULTS: Asthma severity was increased in mice exposed to ABX, including: airway eosinophilia, airway hyper-reactivity, serum HDM-specific IgE, and lung histopathology. ABX treatment led to sharp reduction in fecal microbiome diversity, including the loss of pro-regulatory organisms such as Lachnospira. Pulmonary Tregs were reduced with ABX treatment, and this reduction was directly proportional to diminished microbiome diversity. CONCLUSION: Intermittent exposure to ABX early in life worsened the severity of experimental asthma and reduced pulmonary Tregs; the latter change correlated with decreased microbiome diversity. These data may suggest targets for immunologic or probiotic therapy to counteract the harmful effects of childhood ABX.

Regulation of IgE activity in inhalational tolerance via formation of IgG anti-IgE/IgE immune complexes.

BACKGROUND: Allergic asthma is an inflammatory disorder of the airways that results from inappropriate production of IgE against harmless, environmental antigens. Sequestration of free IgE using humanized IgG anti-IgE is an effective therapy for asthma and other atopic disorders. However, the status of free IgE in subjects who have naturally developed immune tolerance to inhaled antigens has not been well studied. METHODS: C57BL/6 mice were sensitized and challenged with ovalbumin (OVA) for 7 days to induce allergic airway disease (AAD) or 6 weeks to induce a state of local inhalational tolerance (LIT). Serum from AAD or LIT mice, diluted to achieve equivalent levels of total OVA-specific IgE, was used to sensitize rat basophil leukemia cells for allergen-mediated degranulation. Levels of degranulation were measured in relation to serum concentrations of free IgE and IgG anti-IgE/IgE immune complexes. RESULTS: Serum from AAD animals induced a greater degree of basophil degranulation than serum from LIT animals. These results correlated with higher levels of free IgE in AAD animals, whereas LIT mice demonstrated a significant increase in IgG anti-IgE/IgE immune complexes relative to their diseased counterparts. CONCLUSIONS: Sequestration of free IgE by naturally occurring IgG anti-IgE may aid in the development of immune tolerance against inhaled allergens. The decrease in bioavailability of free IgE may, in turn, contribute to the overall reduction of asthma symptoms via a mechanism that mimics the therapeutic effects of humanized IgG anti-IgE.

Breathing Better Through Bugs: Asthma and the Microbiome.

Asthma is a highly heterogeneous disease characterized by inflammation of the airways, which invokes symptoms such as wheeze, dyspnea, and chest tightness. Asthma is the product of multiple interconnected immunological processes and represents a constellation of related, but distinct, disease phenotypes. The prevalence of asthma has more than doubled since the 1980s, and efforts to understand this increase have inspired consideration of the microbiome as a key player in the pathophysiology and regulation of this disease. While recent years have seen an explosion of new research in this area, researchers are only beginning to untangle to mechanisms by which the microbiome may influence asthma. This review will focus on the relationship between the microbiome and the immune system and how this influences development of asthma. This review will also highlight evidence that may point the way toward new therapies and potential cures for this ancient respiratory foe.

Poor Long-Term Efficacy of Prevnar-13 in Sickle Cell Disease Mice Is Associated with an Inability to Sustain Pneumococcal-Specific Antibody Titers.

BACKGROUND: One of the most common causes of morbidity and mortality in children with sickle cell disease (SCD) is infection with the pneumococcal bacterium (Streptococcus pneumoniae). Unfortunately, the polysaccharide-conjugate vaccine appears to be less effective in individuals with SCD when compared to the general population. We sought to better understand the relative efficacy of pneumococcal vaccination in a SCD mouse challenge model. METHODS: Transgenic control and SCD mice were monitored for mortality after intranasal pneumococcal infection or pneumococcal vaccination with Prevnar-13 and type-matched challenge. Anti-pneumococcal antibody titers were measured by ELISA and opsonophagocytosis was measured in vitro. RESULTS: Mortality after pneumococcal infection was similar between control and SCD mice. However, after three intramuscular polysaccharide-conjugate vaccinations, all control mice were protected following high-dose intranasal infection, whereas 60% of SCD mice died. Anti-pneumococcal antibody titers showed initial IgG and IgM responses in both groups, but waning titers were observed in the SCD group, even after boosting. When functionally assayed in vitro, serum from SCD mice 13 weeks after a second booster shot maintained little to no ability to opsonize pneumococci, while serum from control mice sustained a significantly higher capacity opsonization. Thus, it appears that SCD mice do not maintain antibody responses to pneumococcal polysaccharides after Prevnar-13 vaccination, thereby leaving them susceptible to mortality after type-matched infection. CONCLUSION: Our results emphasize the need to better understand the correlates of immune protection in SCD so that pneumococcal vaccines can be improved and mortality reduced in this susceptible population.

Long-Term Exposure to House Dust Mite Leads to the Suppression of Allergic Airway Disease Despite Persistent Lung Inflammation.

BACKGROUND: Allergic asthma is a major cause of worldwide morbidity and results from inadequate immune regulation in response to innocuous, environmental antigens. The need exists to understand the mechanisms that promote nonreactivity to human-relevant allergens such as house dust mite (HDM) in order to develop curative therapies for asthma. The aim of our study was to compare the effects of short-, intermediate- and long-term HDM administration in a murine asthma model and determine the ability of long-term HDM exposure to suppress allergic inflammation. METHODS: C57BL/6 mice were intranasally instilled with HDM for short-term (2 weeks), intermediate-term (5 weeks) and long-term (11 weeks) periods to induce allergic airway disease (AAD). The severity of AAD was compared across all stages of the model via both immunological and pulmonary parameters. RESULTS: Short- and intermediate-term HDM exposure stimulated the development of AAD that included eosinophilia in the bronchoalveolar lavage fluid (BALF), pronounced airway hyperreactivity (AHR) and evidence of lung inflammation. Long-term HDM exposure promoted the suppression of AAD, with a loss of BALF eosinophilia and AHR despite persistent mononuclear inflammation in the lungs. Suppression of AAD with long-term HDM exposure was associated with an increase in both Foxp3+ regulatory T cells and IL-10-positive alveolar macrophages at the site of inflammation. CONCLUSIONS: This model recapitulates the key features of human asthma and may facilitate investigation into the mechanisms that promote immunological tolerance against clinically relevant aeroallergens.

Transgenic sickle cell disease mice have high mortality and dysregulated immune responses after vaccination.

BACKGROUND: Children with sickle cell disease (SCD) are susceptible to recurrent infections, which are often life threatening and necessitate frequent vaccinations. Given the altered baseline immunity and proinflammatory state associated with SCD, we sought to determine the relative safety and efficacy of vaccination in transgenic SCD mice. METHODS: Eight-week-old SCD mice were vaccinated with ovalbumin and aluminum hydroxide weekly for 3 wk by the intraperitoneal or intramuscular route. One week after the third vaccination, serum cytokines/chemokines, immunoglobulins, and bronchoalveolar lavage fluid cytokines were measured. RESULTS: Only SCD mice were prone to mortality associated with vaccination, as 40% of the animals died after the intraperitoneal vaccinations and 50% died after the intramuscular vaccinations. Serum IgG2b and IgM were significantly lower in SCD mice than in C57BL/6 mice after vaccination, but ovalbumin-specific IgE was significantly higher. Serum interleukin (IL)-1α, IL-2, IL-5, macrophage inflammatory protein 1α, and granulocyte macrophage-colony stimulating factor were significantly lower in SCD mice than in C57BL/6 mice after vaccination, whereas bronchoalveolar lavage fluid IL-1ß and IL-6 were increased. CONCLUSION: Mice with SCD appear to have a dysregulated immune response to vaccination. Thus, the relative safety and immunogenicity of vaccination should be studied in greater detail in the context of SCD.