The clinical utility of a glaucoma polygenic risk score in four population-based European-ancestry cohorts.

OBJECTIVE: We used a polygenic risk score (PRS) to identify high-risk groups for primary open-angle glaucoma (POAG) within population-based cohorts. DESIGN: Secondary analysis of four prospective population-based studies. PARTICIPANTS: We included four European-ancestry cohorts: the United States (US) based Nurses' Health Study (NHS), NHS2, and the Health Professionals Follow-up Study, and the Rotterdam Study (RS) in the Netherlands. The US cohorts included female nurses and male health professionals aged 55+ years. The RS included residents aged 45 years or older living in Rotterdam, the Netherlands. METHODS: PRS weights were estimated by applying the Lassosum method on imputed genotype and phenotype data from the UK-Biobank. This resulted in 144,020 variants, single nucleotide polymorphism (SNPs) and indels, with non-zero betas that we used to calculate a PRS in the target populations. Using multivariable Cox proportional hazard models, we estimated the relationship between the standardized PRS and relative risk for POAG. Additionally, POAG prediction was tested by calculating these models' concordance (Harrell's C-statistic). Finally, we assessed the association between PRS tertiles and glaucoma-related traits. MAIN OUTCOME MEASURES: The relative risk for POAG and Harrell's C-statistic (the equivalent of an area-under-the-curve for longitudinal models). RESULTS: Among 1,046 cases and 38,809‬ controls, the relative risk (95% confidence interval) for POAG for participants in the highest PRS quintile was 3.99 (3.08, 5.18) in the US cohorts, and 4.89 (2.93, 8.17) in the Rotterdam Study, compared with participants with median genetic risk (3rd quintile). In restricted cubic spline analyses, the relation between continuous PRS and POAG risk increased exponentially in the US and Rotterdam cohorts (Pspline<0.05). Combining age, sex, intraocular pressure >25 mmHg, and family history resulted in a meta-analyzed concordance of 0.75 (0.73, 0.75). Adding the PRS to this model improved the concordance to 0.82 (0.80, 0.84). In a meta-analysis of all cohorts, cases in the highest tertile had a larger cup-disc ratio at diagnosis, by 0.11 (0.07, 0.15), and a 2.07-fold increased risk of requiring glaucoma surgery (1.19, 3.60). CONCLUSIONS: Incorporating a PRS into a POAG predictive model improves identification concordance from 0.75 up to 0.82, supporting its potential for guiding more cost-effective screening strategies.

Mast cells condition dendritic cells to mediate allograft tolerance.

Peripheral tolerance orchestrated by regulatory T cells, dendritic cells (DCs), and mast cells (MCs) has been studied in several models including skin allograft tolerance. We now define a role for MCs in controlling DC behavior ("conditioning") to facilitate tolerance. Under tolerant conditions, we show that MCs mediated a marked increase in tumor necrosis factor (TNFα)-dependent accumulation of graft-derived DCs in the dLN compared to nontolerant conditions. This increase of DCs in the dLN is due to the local production of granulocyte macrophage colony-stimulating factor (GM-CSF) by MCs that induces a survival advantage of graft-derived DCs. DCs that migrated to the dLN from the tolerant allograft were tolerogenic; i.e., they dominantly suppress T cell responses and control regional immunity. This study underscores the importance of MCs in conditioning DCs to mediate peripheral tolerance and shows a functional impact of peripherally produced TNFα and GM-CSF on the migration and function of tolerogenic DCs.

The immortality of humoral immunity.

Decades of high-titered antibody are sustained due to the persistence of memory B cells and long-lived plasma cells (PCs). The differentiation of each of these subsets is antigen- and T-cell driven and is dependent on signals acquired and integrated during the germinal center response. Inherent in the primary immune response must be the delivery of signals to B cells to create these populations, which have virtual immortality. Differences in biology and chemotactic behavior disperse memory B cells and long-lived PCs to a spectrum of anatomic sites. Each subset must rely on survival factors that can support their longevity. This review focuses on the generation of each of these subsets, their survival, and renewal, which must occur to sustain serological memory. In this context, we discuss the role of antigen, bystander inflammation, and cellular niches. The contribution of BAFF (B-cell activating factor belonging to the tumor necrosis factor family) and APRIL (a proliferation-inducing ligand) to the persistence of memory B cells and PCs are also detailed. Insights that have been provided over the past few years in the regulation of long-lived B-cell responses will have profound impact on vaccine development, the treatment of pre-sensitized patients for organ transplantation, and therapeutic interventions in both antibody- and T-cell-mediated autoimmunity.

Molecular mechanism and function of CD40/CD40L engagement in the immune system.

SUMMARY: During the generation of a successful adaptive immune response, multiple molecular signals are required. A primary signal is the binding of cognate antigen to an antigen receptor expressed by T and B lymphocytes. Multiple secondary signals involve the engagement of costimulatory molecules expressed by T and B lymphocytes with their respective ligands. Because of its essential role in immunity, one of the best characterized of the costimulatory molecules is the receptor CD40. This receptor, a member of the tumor necrosis factor receptor family, is expressed by B cells, professional antigen-presenting cells, as well as non-immune cells and tumors. CD40 binds its ligand CD40L, which is transiently expressed on T cells and other non-immune cells under inflammatory conditions. A wide spectrum of molecular and cellular processes is regulated by CD40 engagement including the initiation and progression of cellular and humoral adaptive immunity. In this review, we describe the downstream signaling pathways initiated by CD40 and overview how CD40 engagement or antagonism modulates humoral and cellular immunity. Lastly, we discuss the role of CD40 as a target in harnessing anti-tumor immunity. This review underscores the essential role CD40 plays in adaptive immunity.

Genetic Associations Between Smoking- and Glaucoma-Related Traits.

Purpose: The purpose of this study was to describe the genetic relationship between smoking and glaucoma. Methods: We used summary-level genetic data for smoking initiation, smoking intensity (cigarettes per day [CPD]), intraocular pressure (IOP), vertical cup-disc ratio, and open-angle glaucoma (OAG) to estimate global genetic correlations (rg) and perform two-sample Mendelian randomization (MR) experiments that explored relations between traits. Finally, we examined associations between smoking genetic risk scores (GRS) and smoking traits with measured IOP and OAG in Rotterdam Study participants. Results: We identified weak inverse rg between smoking- and glaucoma-related traits that were insignificant after Bonferroni correction. However, MR analysis revealed that genetically predicted smoking initiation was associated with lower IOP (-0.18 mm Hg per SD, 95% confidence interval [CI] = -0.30 to -0.06, P = 0.003). Furthermore, genetically predicted smoking intensity was associated with decreased OAG risk (odds ratio [OR] = 0.74 per SD, 95% CI = 0.61 to 0.90, P = 0.002). In the Rotterdam Study, the smoking initiation GRS was associated with lower IOP (-0.09 mm Hg per SD, 95% CI = -0.17 to -0.01, P = 0.04) and lower odds of OAG (OR = 0.84 per SD, 95% CI = 0.73 to 0.98, P = 0.02) in multivariable-adjusted analyses. In contrast, neither smoking history nor CPD was associated with IOP (P ≥ 0.38) or OAG (P ≥ 0.54). Associations between the smoking intensity GRS and glaucoma traits were null (P ≥ 0.13). Conclusions: MR experiments and GRS generated from Rotterdam Study participants support an inverse relationship between smoking and glaucoma. Translational Relevance: Understanding the genetic drivers of the inverse relationship between smoking and glaucoma could yield new insights into glaucoma pathophysiology.

Efficacy and Safety of Micropulse Transscleral Cyclophotocoagulation.

Background: Early studies have shown that micropulse transscleral cyclophotocoagulation (MP-TSCPC) might be an effective and safe treatment option for lowering intraocular pressure (IOP). These studies were, however, somewhat limited, in particular by their retrospective nature and the length of follow-up. Therefore, we assessed the efficacy and safety of this novel treatment in a large cohort for up to 4 years. Methods: We performed a prospective cohort study, including all patients who were treated with MP-TSCPC since November 2017. The primary outcome was a reduction of IOP and the number of IOP-lowering medications. Results: The mean ± standard deviation baseline IOP and number of IOP-lowering medications were 26.6 ± 10.8 mmHg and 3.3 ± 1.3. IOP was reduced by 8.2 ± 7.9 (31.8% reduction), 6.9 ± 8.7 (28.1% reduction), and 7.1 ± 8.4 (30.2% reduction) mmHg after 6, 12, and 24 months, respectively (p < 0.001). The mean postoperative number of IOP-lowering medications was significantly reduced after 6 months by 0.6 ± 1.5 (p = 0.002) but was not significantly different after 12 or 24 months. Oral acetazolamide was significantly reduced from 28 (29%) eyes before treatment, to 9 (9%) at the last follow-up visit (p < 0.001). No major complications were observed after treatment. Conclusions: MP-TSCPC is a safe and effective treatment option for lowering IOP, but only reduced IOP-lowering medications in the first 6 months after treatment. However, MP-TSCPC is especially effective in getting patients off oral IOP-lowering drugs.

Efficacy of the PRESERFLO MicroShunt and a Meta-Analysis of the Literature.

Background: Recent studies on the PRESERFLO MicroShunt suggest that it may be effective in lowering intraocular pressure (IOP); however, the number of studies on this device remains limited. Therefore, we assessed the efficacy of the PRESERFLO MicroShunt in patients with glaucoma and performed a meta-analysis of published results. Methods: Prospective study including all patients that underwent PRESERFLO MicroShunt surgery from 2018 onwards. Sub-analyses were performed for cataract-combined procedures. To compare our results, we performed a systematic review and meta-analysis. IOP, IOP-lowering medication and surgical complications reported in the retrieved studies were assessed. Results: A total of 72 eyes underwent PRESERFLO-implant surgery (59 as standalone procedure and 13 as cataract-combined procedure). No significant differences were found in IOP and IOP-lowering medication between both groups. The mean ± standard deviation IOP and IOP-lowering medications of both groups taken together declined from 21.72 ± 8.35 to 15.92 ± 8.54 mmHg (p < 0.001, 26.7% reduction) and 3.40 to 0.93 (p < 0.001, 72.6% reduction) at 1 year follow-up, respectively. Secondary surgeries were required in 19.4% of eyes, the majority (71.4%) within 6 months. The meta-analysis including 14 studies (totaling 1213 PRESERFLO MicroShunt surgeries) from the systematic review showed a mean preoperative IOP and IOP-lowering medication of 22.28 ± 5.38 and 2.97 ± 1.07, respectively. The three-years postoperative pooled mean was (weighted mean difference, 95% CI) 11.07 (10.27 [8.23−12.32], p < 0.001) mmHg and 0.91 (1.77 [1.26−2.28], p < 0.001) for IOP and IOP-lowering medication, respectively. The most common reported complication was hypotony (2−39%). Conclusion: The PRESERFLO MicroShunt is effective and safe in lowering IOP and the number of IOP-lowering medications.

Programmed death 1 ligand signaling regulates the generation of adaptive Foxp3+CD4+ regulatory T cells.

Although mature dendritic cells (DCs) are potent initiators of adaptive immune response, immature steady-state DCs contribute to immune tolerance. In this study, we show that ex vivo splenic DCs are capable of inducing conversion of naïve CD4(+) T cells to adaptive Foxp3(+)CD4(+) regulatory T cells (aTreg) in the presence of TGF-beta. In particular, when compared with splenic CD8alpha(-) DCs, the CD8alpha(+) DC subset were superior in inducing higher frequencies of conversion. This was not attributable to the difference in basal level of costimulation, because deficiency of CD40 or CD80/86 signaling did not diminish the differential induction of Foxp3. Conversion was regulated by DC maturation status. Further insights into the molecular mechanisms of conversion were gained by analyzing the contribution of several costimulatory and coinhibitory receptors. Costimulatory signals through GITR suppressed conversion, whereas coinhibitory signaling via programmed death 1 ligand (PD-L1) but not PD-L2 was required for conversion. Ex vivo PD-L1(-/-) DCs failed to support Foxp3 induction in the presence of TGF-beta. In vivo blocking PD-L1 signaling abolished conversion in a tumor-induced aTreg conversion model. Collectively, this study highlights the cellular and molecular parameters that might be exploited to control the de novo generation of aTregs and peripheral tolerance.

The effects of intravitreal injections on intraocular pressure and retinal nerve fiber layer: a systematic review and meta-analysis.

The number of eye diseases treated with intravitreal injections is increasing. Obviously, an injection of fluid into the eye results in an increase of intraocular pressure (IOP), the main risk factor for glaucoma. However, the effect of these repeated IOP increases on the eye is unclear. Therefore, we performed a systematic review with meta-analyses. PubMed, Embase and Clinical Trials Registries were searched for articles investigating the relationship between intravitreal injections (anti-vascular endothelial growth factor [anti-VEGF] or steroids) and either IOP, retinal nerve fiber layer (RNFL)-thickness and glaucoma. Multiple meta-analyses were performed, combining data on intravitreal injection of anti-VEGF medication and dexamethasone implants. A total of 74 articles were eligible for meta-analyses. The short-term effect of an intravitreal injection of anti-VEGF showed a statistically significant increase in IOP. One day after injection of anti-VEGF, however, IOP was significantly lower than baseline. The long-term time-intervals showed no significant difference in IOP. After intravitreal injection of a dexamethasone implant, IOP was significantly higher than baseline 1 month post-injection. RNFL-thickness was significantly reduced 6 and 12 months post-injection of anti-VEGF, as well as at end of follow up. Caution is advised when using intravitreal medication, especially when treating patients with advanced glaucoma; in these cases, prophylactic IOP-lowering medication may be considered.

The enigmatic role of mast cells in dominant tolerance.

PURPOSE OF REVIEW: The role of regulatory T cells (Treg) in peripheral tolerance has been studied extensively in transplantation research. Recently, mast cells have been shown to play an indispensable role in allograft tolerance. The purpose of this review is to inform the reader on the current standings of the role of mast cells in dominant tolerance with an emphasis on the interaction of mast cells with Treg. RECENT FINDINGS: Mast cells are required to sustain peripheral tolerance via Treg. Treg can stabilize mast cells degranulation by contact-dependent mechanisms through the interaction of OX40 and its ligand OX40L, and by production of soluble factors, such as interleukin-10 and transforming growth factor-beta. Conversely, the activation and subsequent degranulation of mast cells break peripheral tolerance. SUMMARY: Both mast cells and Treg are needed to create a local immunosuppressive environment in the transplant. Treg are not only necessary to suppress effector T-cell responses but also to stabilize mast cells. Mast cells in return could contribute to the immunosuppressive state by release of transforming growth factor-beta, interleukin-10 and specific proteases. However, the molecular basis for mast cells control of Treg suppression in organ transplantation is still unresolved.

Induction of postsurgical tumor immunity and T-cell memory by a poorly immunogenic tumor.

The generation of protective CD8 T-cell memory against tumor-expressed self-antigens is an important but elusive goal of cancer immunotherapy. The possibility that a progressive, poorly immunogenic tumor can induce T-cell memory against self-antigens has not previously been studied. Herein, we report that growth of the poorly immunogenic B16 melanoma in the absence of regulatory T cells (T(reg)) generates CD8 T-cell responses that develop into functional memory after the tumor has been surgically excised. Tumor-primed memory T cells recognized melanocyte differentiation antigens TRP-2/DCT and gp100 and persisted for as long as 5 months following surgical tumor excision. Phenotypic analysis showed that these cells develop into both central and effector memory T-cell subsets, which produce IFN-gamma and interleukin-2 on reencounter with antigen. Most importantly, tumor-primed memory T cells mediated the rejection of intradermal and systemically disseminated challenge tumors given 30 to 60 days following surgery. Tumor-excised mice also developed autoimmune vitiligo, showing that T(reg) cells prevent tissue-specific autoimmunity in tumor-bearing hosts. This study establishes that T(reg) depletion in tumor-bearing hosts drives the natural development of protective T-cell memory. Generating such responses may aid in the clinical management of tumor recurrence and metastasis following surgery.

Studying mast cells in peripheral tolerance by using a skin transplantation model.

Mast cells (MCs) play an important role in both inflammatory and immunosuppressive responses [1]. The importance of MCs in maintaining peripheral tolerance was discovered in a FoxP3(+) regulatory T-cell (Treg)-mediated skin transplant model [2]. MCs can directly mediate tolerance by releasing anti-inflammatory mediators (reviewed in ref. 3) or by interacting with other immune cells in the graft. Here we will present protocols used to study the role of MCs in peripheral tolerance with the emphasis on how MCs can regulate T-cell functionality. First we will introduce the skin transplant model followed by reconstitution of mast cell-deficient mice (B6.Cg-Kit (W-sh) ). This includes the preparation of MCs from the bone marrow. Finally the methods used to study the influence of MCs on T-cell responses and Treg functionality will be presented by modulating the balance between tolerance and inflammation.

The distinctive germinal center phase of IgE+ B lymphocytes limits their contribution to the classical memory response.

The mechanisms involved in the maintenance of memory IgE responses are poorly understood, and the role played by germinal center (GC) IgE(+) cells in memory responses is particularly unclear. IgE(+) B cell differentiation is characterized by a transient GC phase, a bias toward the plasma cell (PC) fate, and dependence on sequential switching for the production of high-affinity IgE. We show here that IgE(+) GC B cells are unfit to undergo the conventional GC differentiation program due to impaired B cell receptor function and increased apoptosis. IgE(+) GC cells fail to populate the GC light zone and are unable to contribute to the memory and long-lived PC compartments. Furthermore, we demonstrate that direct and sequential switching are linked to distinct B cell differentiation fates: direct switching generates IgE(+) GC cells, whereas sequential switching gives rise to IgE(+) PCs. We propose a comprehensive model for the generation and memory of IgE responses.

Cross-roads in the lung: immune cells and tissue interactions as determinants of allergic asthma.

Allergic asthma is a chronic disease of the lung characterized by underlying Th2- and IgE-mediated inflammation, structural alterations of the bronchial wall, and airway hyperresponsiveness. Initial allergic sensitization and later development of chronic disease are determined by close interactions between lung structural cells and the resident and migratory immune cells in the lung. Epithelial cells play a crucial role in allergic sensitization by directly influencing dendritic cells induction of tolerant or effector T cells and production of type 2 cytokines by innate immune cells. During chronic disease, the bronchial epithelium, stroma, and smooth muscle become structurally and functionally altered, contributing to the perpetuation of tissue remodeling. Thus, targeting tissue-driven pathology in addition to inflammation may increase the effectiveness of asthma treatment.

Tryptophan hydroxylase-1 regulates immune tolerance and inflammation.

Nutrient deprivation based on the loss of essential amino acids by catabolic enzymes in the microenvironment is a critical means to control inflammatory responses and immune tolerance. Here we report the novel finding that Tph-1 (tryptophan hydroxylase-1), a synthase which catalyses the conversion of tryptophan to serotonin and exhausts tryptophan, is a potent regulator of immunity. In models of skin allograft tolerance, tumor growth, and experimental autoimmune encephalomyelitis, Tph-1 deficiency breaks allograft tolerance, induces tumor remission, and intensifies neuroinflammation, respectively. All of these effects of Tph-1 deficiency are independent of its downstream product serotonin. Because mast cells (MCs) appear to be the major source of Tph-1 and restoration of Tph-1 in the MC compartment in vivo compensates for the defect, these experiments introduce a fundamentally new mechanism of MC-mediated immune suppression that broadly impacts multiple arms of immunity.

A retinoic acid-dependent checkpoint in the development of CD4+ T cell-mediated immunity.

It is known that vitamin A and its metabolite, retinoic acid (RA), are essential for host defense. However, the mechanisms for how RA controls inflammation are incompletely understood. The findings presented in this study show that RA signaling occurs concurrent with the development of inflammation. In models of vaccination and allogeneic graft rejection, whole body imaging reveals that RA signaling is temporally and spatially restricted to the site of inflammation. Conditional ablation of RA signaling in T cells significantly interferes with CD4(+) T cell effector function, migration, and polarity. These findings provide a new perspective of the role of RA as a mediator directly controlling CD4(+) T cell differentiation and immunity.

Mast cell mediators in tolerance.

Our knowledge on the function of mast cells (MC) as part of the immune system has expanded from 'key cells in mediating allergy' to 'tunable regulators of the immune response'. Over the past years however, a large body of evidence has been presented indicating a more regulatory role for MC in the immune system by both contact dependent and independent mechanisms. Considering the vast amount of soluble mediators released by MC, it is not surprising that some are involved in the maintenance of peripheral tolerance and the control or even help to resolve ongoing inflammation. In this review we will focus on the immunosuppressive function of some of these mediators produced by MC in a wide variety of disease models.

Analysis of ACE2 in polarized epithelial cells: surface expression and function as receptor for severe acute respiratory syndrome-associated coronavirus.

The primary target of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is epithelial cells in the respiratory and intestinal tract. The cellular receptor for SARS-CoV, angiotensin-converting enzyme 2 (ACE2), has been shown to be localized on the apical plasma membrane of polarized respiratory epithelial cells and to mediate infection from the apical side of these cells. Here, these results were confirmed and extended by including a colon carcinoma cell line (Caco-2), a lung carcinoma cell line (Calu-3) and Vero E6 cells in our analysis. All three cell types expressed human ACE2 on the apical membrane domain and were infected via this route, as determined with vesicular stomatitis virus pseudotypes containing the S protein of SARS-CoV. In a histological analysis of the respiratory tract, ACE2 was detected in the trachea, main bronchus and alveoli, and occasionally also in the small bronchi. These data will help us to understand the pathogenesis of SARS-CoV infection.

Airway eosinophils accumulate in the mediastinal lymph nodes but lack antigen-presenting potential for naive T cells.

Asthma is characterized by infiltration of the airway wall with eosinophils. Although eosinophils are considered to be effector cells, recent studies have reported their ability to activate primed Th2 cells. In this study, we investigated whether eosinophils are capable of presenting Ag to unprimed T cells in draining lymph nodes (DLN) of the lung and compared this capacity with professional dendritic cells (DC). During development of eosinophilic airway inflammation in OVA-sensitized and challenged mice, CCR3(+) eosinophils accumulated in the DLN. To study their function, eosinophils were isolated from the bronchoalveolar lavage fluid of mice by sorting on CCR3(+)B220(-)CD3(-)CD11c(dim) low autofluorescent cells, avoiding contamination with other APCs, and were intratracheally injected into mice that previously received CFSE-labeled OVA TCR-transgenic T cells. Eosinophils did not induce divisions of T cells in the DLN, whereas DC induced on average 3.7 divisions in 45.7% of T cells. To circumvent the need for Ag processing or migration in vivo, eosinophils were pulsed with OVA peptide and were still not able to induce T cell priming in vitro, whereas DC induced vigorous proliferation. This lack of Ag-presenting ability was explained by the very weak expression of MHC class II on fresh eosinophils, despite expression of the costimulatory molecules CD80 and ICAM-1. This investigation does not support any role for airway eosinophils as APCs to naive T cells, despite their migration to the DLN at times of allergen exposure. DC are clearly superior in activating T cells in the DLN of the lung.

Lipopolysaccharide-induced suppression of airway Th2 responses does not require IL-12 production by dendritic cells.

The prevalence of atopic asthma, a Th2-dependent disease, is reaching epidemic proportions partly due to improved hygiene in industrialized countries. There is an inverse correlation between the level of environmental endotoxin exposure and the prevalence of atopic sensitization. As dendritic cells (DC) have been implicated in causing sensitization to inhaled Ag, we studied the effect of endotoxin on Th2 development induced by bone marrow DC in vitro and by intratracheal injection in vivo, with particular emphasis on the role played by the polarizing cytokine IL-12. Bone marrow-derived DC stimulated with Escherichia coli O26:B6 LPS produced IL-12p70 for a limited period of time, after which production became refractory to further stimulation with CD40 ligand, a phenomenon previously called "exhaustion." The level of IL-12 production of DC did not correlate with Th1 development, as exhausted OVA-pulsed DC were still capable of shifting the cytokine pattern of responding OVA-specific Th cells toward Th1 in vitro and in vivo. When mice were first immunized by intratracheal injection of OVA-DC and subsequently challenged with OVA aerosol, prior in vitro stimulation of DC with LPS reduced the development of airway eosinophilia and Th2 cytokine production. Most surprisingly, the capacity of LPS to reduce Th2-dependent eosinophilic airway inflammation was IL-12-independent altogether, as IL-12p40 knockout DC had a similar reduced capacity to prime for Th2 responses. These results suggest that LPS reduces sensitization to inhaled Ag by reducing DC-driven Th2 development, but that IL-12 is not necessary for this effect.