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Table S2. Principal component analysis loadings for plots shown in Figure 1C and Figure 1D, included as separate Excel file.

Figures S1-S5

Cancer cells can exhibit altered dependency on specific metabolic pathways and targeting these dependencies is a promising therapeutic strategy. Triple-negative breast cancer (TNBC) is an aggressive and genomically heterogeneous subset of breast cancer that is resistant to existing targeted therapies. To identify metabolic pathway dependencies in TNBC, we first conducted mass spectrometry–based metabolomics of TNBC and control cells. Relative levels of intracellular metabolites distinguished TNBC from nontransformed breast epithelia and revealed two metabolic subtypes within TNBC that correlate with markers of basal-like versus non-basal–like status. Among the distinguishing metabolites, levels of the cellular redox buffer glutathione were lower in TNBC cell lines compared to controls and markedly lower in non-basal–like TNBC. Significantly, these cell lines showed enhanced sensitivity to pharmacologic inhibition of glutathione biosynthesis that was rescued by N-acetylcysteine, demonstrating a dependence on glutathione production to suppress ROS and support tumor cell survival. Consistent with this, patients whose tumors express elevated levels of γ-glutamylcysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, had significantly poorer survival. We find, further, that agents that limit the availability of glutathione precursors enhance both glutathione depletion and TNBC cell killing by γ-glutamylcysteine ligase inhibitors in vitro. Importantly, we demonstrate the ability to this approach to suppress glutathione levels and TNBC xenograft growth in vivo. Overall, these findings support the potential of targeting the glutathione biosynthetic pathway as a therapeutic strategy in TNBC and identify the non-basal-like subset as most likely to respond. Mol Cancer Ther; 17(1); 264–75. ©2017 AACR.

Food allergies have increased at an alarming rate over the past 2 decades, indicating that environmental factors are driving disease progression. It has been postulated that sensitization to foods, in particular, peanut, occurs through impaired skin. Peanut allergens have been quantified in household dust and may be the culprit source. Indeed, TH2 cell–skewing innate cytokines can be driven by application of food antigens on both intact and impaired skin of mice, resulting in antigen-specific IgE production and anaphylaxis following allergen exposure. However, allergy induction through the skin can be prevented by induction of oral tolerance before skin exposure. These observations led to the dual allergen exposure hypothesis, according to which oral exposure to food antigens leads to tolerance and antigen exposure on impaired skin leads to allergy. Here, we propose the airway as an alternative route of sensitization in the dual allergen exposure hypothesis that leads to food allergy. Specifically, we will provide evidence from mouse models and human cell–based studies that together implicate the airway as a plausible route of sensitization.

The most clinically relevant food allergens are cow’s milk, hen’s egg, peanut, tree nuts, wheat, soy, fish, shellfish, and seeds. Heat-stable food allergens have molecular characteristics that enhance protein stability and gastrointestinal absorption and thus are more likely to cause systemic reactions on ingestion. In contrast, heat-labile food allergens lack these characteristics and do not typically elicit reactions if sufficiently altered by heat or acid. Immunologic cross-sensitization between food allergens is more common than clinical cross-reactivity. However, certain groups of food allergens, such as tree nuts, fish, and shellfish, are associated with high rates of clinical cross-reactivity. Knowing the rates of clinical cross-reactivity is important when providing guidance to patients with food allergy and families on what foods can be safely added to the diet and what foods should be avoided.

BACKGROUND Asthma is a heterogenous disease that can be classified into eosinophilic (type 2-high) and noneosinophilic (type 2-low) endotypes. The type 2-low endotype of asthma can be characterized by the presence of neutrophilic airway inflammation that is poorly responsive to corticosteroids. Dysregulated innate immune responses to microbial products including Toll-like receptor (TLR) ligands have been associated with the pathogenesis of neutrophilic asthma. The key molecules that regulate inflammatory responses in individuals with neutrophilic asthma remain unclear. We previously reported that the immunoregulatory receptor neuropilin-2 (NRP2) is expressed by murine and human alveolar macrophage (AM) and suppresses lipopolysaccharide (LPS)-induced neutrophilic airway inflammation. METHODS Here, we investigated the immunoregulatory role of NRP2 in a mouse model of neutrophilic asthma. RESULTS We found that TLR ligands, but not T helper 2 (Th2)-promoting adjuvants, induced NRP2 expression by AM. Using an LPS-mediated model of neutrophilic asthma, we demonstrate that NRP2 was increased in AM and other lung antigen-presenting cells following airway challenge with antigen. Conditional deletion of NRP2 in myeloid cells exacerbated airway inflammation in a neutrophilic asthma model. In contrast, myeloid-specific ablation of NRP2 did not affect airway inflammation in a Th2-mediated eosinophilic asthma model. Myeloid-specific ablation of NRP2 did not affect Th1/Th17 responses to inhaled antigens or expression of neutrophil chemokines but rather resulted in impaired efferocytosis by AM, which is necessary for effective resolution of airway inflammation. CONCLUSION Our findings suggest that NRP2 is a negative regulator of airway inflammation associated with neutrophilic asthma.

Acute ozone (O3) exposure is associated with multiple adverse cardiorespiratory outcomes, the severity of which varies across individuals in human populations and inbred mouse strains. However, molecular determinants of response, including susceptibility biomarkers that distinguish who will develop severe injury and inflammation, are not well characterized. We and others have demonstrated that airway macrophages (AMs) are an important resident immune cell type that are functionally and transcriptionally responsive to O3 inhalation. Here, we sought to explore influences of strain, exposure, and strain-by-O3 exposure interactions on AM gene expression and identify transcriptional correlates of O3-induced inflammation and injury across six mouse strains, including five Collaborative Cross (CC) strains. We exposed adult mice of both sexes to filtered air (FA) or 2 ppm O3 for 3 h and measured inflammatory and injury parameters 21 h later. Mice exposed to O3 developed airway neutrophilia and lung injury with strain-dependent severity. In AMs, we identified a common core O3 transcriptional response signature across all strains, as well as a set of genes exhibiting strain-by-O3 exposure interactions. In particular, a prominent gene expression contrast emerged between a low- (CC017/Unc) and high-responding (CC003/Unc) strain, as reflected by cellular inflammation and injury. Further inspection indicated that differences in their baseline gene expression and chromatin accessibility profiles likely contribute to their divergent post-O3 exposure transcriptional responses. Together, these results suggest that aspects of O3-induced respiratory responses are mediated through altered AM transcriptional signatures and further confirm the importance of gene-environment interactions in mediating differential responsiveness to environmental agents.

Zeta-chain-associated protein kinase 70 (ZAP-70) plays an integral role in the T-cell antigenic receptor complex. A deficiency of this kinase leads to a phenotype of severe combined immunodeficiency, while hypomorphic mutations of the kinase lead to more mild immunodeficiency phenotypes. We present a case of a 21-year-old patient with lymphadenopathy who was found to have Epstein–Barr virus (EBV) lymphoproliferative disease (LPD) and the development of hemophagocytic lymphohistiocytosis (HLH). On further workup, the patient was ultimately found to have a homozygous intrionic mutation in ZAP-70. This is a novel ZAP-70 mutation (c.1623 + 5G > A) associated with combined immunodeficiency and an EBV-positive LPD. A primary immunodeficiency is important to consider in a young, otherwise healthy patient presenting with an EBV-positive LPD.

Background There is growing evidence that environmental peanut exposure through non-oral routes, including the skin and respiratory tract, can result in peanut sensitization. Environmental adjuvants in indoor dust can promote sensitization to inhaled antigens, but whether they contribute to peanut allergy development is unclear. Objective We investigated whether indoor dust promotes airway sensitization to peanut and peanut allergy development in mice. Methods Female and male C57BL/6J mice were exposed via the airways to peanut, indoor dust extract, or both for 2 weeks. Mice were then challenged with peanut and assessed for anaphylaxis. Peanut-specific immunoglobulins, peanut uptake by lung conventional dendritic cells (cDCs), lung innate cytokines, and T cell differentiation in lung-draining lymph nodes were quantified. Innate cytokine production by primary human bronchial epithelial cells exposed to indoor dust was also determined. Results Inhalational exposure to low levels of peanut in combination with indoor dust, but neither alone, resulted in production of peanut-specific IgE and development of anaphylaxis upon peanut challenge. Indoor dust triggered production of innate cytokines in murine lungs and in primary human bronchial epithelial cells. Additionally, inhaled indoor dust stimulated maturation and migration of peanut-laden lung type 1 cDCs to draining lymph nodes. Inhalational exposure to peanut and indoor dust induced peanut-specific T helper 2 cell differentiation and accumulation of T follicular helper cells in draining lymph nodes, which were associated with increased B cell numbers and peanut-specific immunoglobulin production. Conclusions & clinical relevance Indoor dust promotes airway sensitization to peanut and development of peanut allergy in mice. Our findings suggest that environmental adjuvants in indoor dust may be determinants of peanut allergy development in children.

Background The National Academy of Medicine has identified stigma surrounding substance use disorders (SUDs) and infectious diseases (ID) as a key barrier to integration of opioid use disorder (OUD) and ID services. Prior literature on stigmatizing language in OUD clinical care focuses on surveys and theoretical scenarios rather than real-world data. As part of a larger study of patients admitted for infectious complications of injection drug use (CHOICE), we sought to determine how inpatient physicians describe persons with OUD, as well as associations of this language with outcomes along the OUD continuum of care. Methods CHOICE is a retrospective review of adults hospitalized with an infectious complication of OUD and IDU at four academic medical centers. Included patients were hospitalized between 1/1/2018-12/31/2018, had ICD9/10 diagnosis codes consistent with OUD and acute bacterial/fungal infection, and chart review verification of active infection associated with OUD. Data was abstracted regarding demographics, inpatient interventions, transitions of care, and outcomes 1 year after admission. Potentially stigmatizing language was identified based on the discharge summary. “Abuse” and “misuse” were considered potentially stigmatizing; “use disorder” was considered best practice. Associations of language and outcomes were analyzed via logistic generalized estimating equations. Results A total of 287 subjects met inclusion criteria; 119 (42%) were female and the median age was 40 years (IQR: 32 – 52). The most common terms used to describe OUD were “abuse” (190, 66%) and “IVDU” (119, 42%). “Use disorder” was noted in only 72 (25%) charts. In a regression analysis, any mention of “use disorder” was associated with not leaving against medical advice (OR 2.48, 95% CI 1.24 – 4.95), a plan for ongoing OUD treatment (OR 5.17, 95% CI 2.05-13.0), and addiction-specific follow up (OR 2.94, 95% CI 2.34-3.68). Conclusion In this multicenter retrospective study, inpatient physicians commonly referred to patients with OUD using stigmatizing language. When NIH-preferred language was used this was associated with improved outcomes along the OUD continuum of care, possibly reflecting increased awareness of best practices for treating patients with OUD. Disclosures Ellen Eaton, MD , Gilead (Grant/Research Support) Ellen Eaton, MD , Gilead (Individual(s) Involved: Self): Research Grant or Support Greer A. Burkholder, MD, MSPH, Eli Lilly (Grant/Research Support) Sarah Kattakuzhy, MD, Gilead Sciences (Scientific Research Study Investigator, Research Grant or Support) Elana S. Rosenthal, MD, Gilead Sciences (Research Grant or Support)Merck (Research Grant or Support)

Acute ozone (O3) exposure is associated with multiple adverse cardiorespiratory outcomes, the severity of which varies across human populations and rodent models from diverse genetic backgrounds. However, molecular determinants of response, including biomarkers that distinguish which individuals will develop more severe injury and inflammation (i.e., high responders), are poorly characterized. Here, we exposed adult, female and male mice from 6 strains, including 5 Collaborative Cross (CC) strains, to filtered air (FA) or 2 ppm O3for 3 hours, and measured several inflammatory and injury parameters 21 hours later. Additionally, we collected airway macrophages and performed RNA-seq analysis to investigate influences of strain, treatment, and strain-by-treatment interactions on gene expression as well as transcriptional correlates of lung phenotypes. Animals exposed to O3developed airway neutrophilia and lung injury, with varying degrees of severity. We identified many genes that were altered by O3exposure across all strains, and examination of genes whose expression was influenced by strain-by-treatment interactions revealed prominent differences in response between the CC017/Unc and CC003/Unc strains, which were low- and high-responders, respectively (as measured by cellular inflammation and injury). Further investigation of this contrast indicated that baseline gene expression differences likely contribute to their divergent post-O3exposure transcriptional responses. We also observed alterations in chromatin accessibility that differed by strain and with strain-by-treatment interactions, lending further plausibility that baseline differences can modulate post-exposure responses. Together, these results suggest that aspects of the respiratory response to O3exposure may be mediated through altered airway macrophage transcriptional signatures, and further confirms the importance of gene-by-environment interactions in mediating differential responsiveness to environmental agents.

PURPOSE OF REVIEW: The recent increase in childhood food allergy prevalence strongly suggests that environmental exposures are contributing to food allergy development. This review summarizes current knowledge about the role of the external exposome in food allergy. RECENT FINDINGS: There is growing evidence that environmental exposure to food antigens in house dust through non-oral routes contributes to food sensitization and allergy. Co-exposure to environmental adjuvants in house dust, such as microbial products and fungal allergens, may also facilitate allergic sensitization. While a high-microbe environment is associated with decreased atopy, studies are mixed on whether endotoxin exposure protects against food sensitization. Several chemicals and air pollutants have been associated with food sensitization, but their role in food allergy remains understudied. SUMMARY: Children are exposed to numerous environmental agents that can influence food allergy risk. Further studies are needed to identify the key early-life exposures that promote or inhibit food allergy development.

Neuropilins are multifunctional receptors that play important roles in immune regulation. Neuropilin-2 (NRP2) is expressed in the lungs, but whether it regulates airway immune responses is unknown. Here, we report that Nrp2 is weakly expressed by alveolar macrophages (AMs) in the steady state but is dramatically upregulated following in vivo lipopolysaccharide (LPS) inhalation. Ex vivo treatment of human AMs with LPS also increased NRP2 mRNA expression and cell-surface display of NRP2 protein. LPS-induced Nrp2 expression in AMs was dependent upon the myeloid differentiation primary response 88 signaling pathway and the transcription factor NF-κB. In addition to upregulating display of NRP2 on the cell membrane, inhaled LPS also triggered AMs to release soluble NRP2 into the airways. Finally, myeloid-specific ablation of NRP2 resulted in increased expression of the chemokine (C-C motif) ligand 2 ( Ccl2) in the lungs and prolonged leukocyte infiltration in the airways following LPS inhalation. These findings suggest that NRP2 expression by AMs regulates LPS-induced inflammatory cell recruitment to the airways and reveal a novel role for NRP2 during innate immune responses in the lungs.

Cancer cells can exhibit altered dependency on specific metabolic pathways and targeting these dependencies is a promising therapeutic strategy. Triple-negative breast cancer (TNBC) is an aggressive and genomically heterogeneous subset of breast cancer that is resistant to existing targeted therapies. To identify metabolic pathway dependencies in TNBC, we first conducted mass spectrometry–based metabolomics of TNBC and control cells. Relative levels of intracellular metabolites distinguished TNBC from nontransformed breast epithelia and revealed two metabolic subtypes within TNBC that correlate with markers of basal-like versus non-basal–like status. Among the distinguishing metabolites, levels of the cellular redox buffer glutathione were lower in TNBC cell lines compared to controls and markedly lower in non-basal–like TNBC. Significantly, these cell lines showed enhanced sensitivity to pharmacologic inhibition of glutathione biosynthesis that was rescued by N-acetylcysteine, demonstrating a dependence on glutathione production to suppress ROS and support tumor cell survival. Consistent with this, patients whose tumors express elevated levels of γ-glutamylcysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, had significantly poorer survival. We find, further, that agents that limit the availability of glutathione precursors enhance both glutathione depletion and TNBC cell killing by γ-glutamylcysteine ligase inhibitors in vitro. Importantly, we demonstrate the ability to this approach to suppress glutathione levels and TNBC xenograft growth in vivo. Overall, these findings support the potential of targeting the glutathione biosynthetic pathway as a therapeutic strategy in TNBC and identify the non-basal-like subset as most likely to respond. Mol Cancer Ther; 17(1); 264–75. ©2017 AACR.

Toll-like receptor ligands in indoor dust act as environmental adjuvants to promote sensitization to inhaled peanut in mice. The timing of exposure to environmental adjuvants relative to peanut is a determinant of peanut allergy development.

The induction of allergen-specific T helper 2 (Th2) cells by lung dendritic cells (DCs) is a critical step in allergic asthma development. Airway delivery of purified allergens or microbial products can promote Th2 priming by lung DCs, but how environmentally relevant quantities and combinations of these factors affect lung DC function is unclear. Here, we investigated the ability of house dust extract (HDE), which contains a mixture of environmental adjuvants, to prime Th2 responses against an innocuous inhaled antigen. Inhalational exposure to HDE conditioned lung conventional DCs, but not monocyte-derived DCs, to induce antigen-specific Th2 differentiation. Conditioning of DCs by HDE was independent of Toll-like receptor 4 signaling, indicating that environmental endotoxin is dispensable for programming DCs to induce Th2 responses. DCs directly treated with HDE underwent maturation but were poor stimulators of Th2 differentiation. In contrast, DCs treated with bronchoalveolar lavage fluid (BALF) from HDE-exposed mice induced robust Th2 differentiation. DC conditioning by BALF was independent of the proallergic cytokines IL-25, IL-33, and thymic stromal lymphopoietin. BALF treatment of DCs resulted in upregulation of CD80 but low expression of CD40, CD86, and IL-12p40, which was associated with Th2 induction. These findings support a model whereby environmental adjuvants in house dust indirectly program DCs to prime Th2 responses by triggering the release of endogenous soluble factor(s) by airway cells. Identifying these factors could lead to novel therapeutic targets for allergic asthma.

Differential display of the integrins CD103 and CD11b are widely used to distinguish two major dendritic cell (DC) subsets in nonlymphoid tissues. CD103+ DCs arise from FLT3-dependent DC precursors (preDCs), whereas CD11bhi DCs can arise either from preDCs or FLT3-independent monocytes. Functional characterization of these two lineages of CD11bhi DCs has been hindered by the lack of a widely applicable method to distinguish between them. We performed gene expression analysis of fractionated lung DCs from C57BL/6 mice and found that monocyte-derived DCs (moDCs), including CD11bhiLy-6Clo tissue-resident and CD11bhiLy-6Chi inflammatory moDCs, express the complement 5a receptor 1/CD88, whereas preDC-derived conventional DCs (cDCs), including CD103+ and CD11bhi cDCs, express dipeptidyl peptidase-4/CD26. Flow cytometric analysis of multiple organs, including the kidney, liver, lung, lymph nodes, small intestine, and spleen, confirmed that reciprocal display of CD88 and CD26 can reliably distinguish FLT3-independent moDCs from FLT3-dependent cDCs in C57BL/6 mice. Similar results were obtained when DCs from BALB/c mice were analyzed. Using this novel approach to study DCs in mediastinal lymph nodes, we observed that most blood-derived lymph node–resident DCs, as well as tissue-derived migratory DCs, are cDCs. Furthermore, cDCs, but not moDCs, stimulated naive T cell proliferation. We anticipate that the use of Abs against CD88 and CD26 to distinguish moDCs and cDCs in multiple organs and mouse strains will facilitate studies aimed at assigning specific functions to distinct DC lineages in immune responses.

Asthma is associated with exposure to a wide variety of allergens and adjuvants. The extent to which overlap exists between the cellular and molecular mechanisms triggered by these various agents is poorly understood, but it might explain the differential responsiveness of patients to specific therapies. In particular, it is unclear why some, but not all, patients benefit from blockade of TNF. Here, we characterized signaling pathways triggered by distinct types of adjuvants during allergic sensitization. Mice sensitized to an innocuous protein using TLR ligands or house dust extracts as adjuvants developed mixed eosinophilic and neutrophilic airway inflammation and airway hyperresponsiveness (AHR) following allergen challenge, whereas mice sensitized using proteases as adjuvants developed predominantly eosinophilic inflammation and AHR. TLR ligands, but not proteases, induced TNF during allergic sensitization. TNF signaled through airway epithelial cells to reprogram them and promote Th2, but not Th17, development in lymph nodes. TNF was also required during the allergen challenge phase for neutrophilic and eosinophilic inflammation. In contrast, TNF was dispensable for allergic airway disease in a protease-mediated model of asthma. These findings might help to explain why TNF blockade improves lung function in only some patients with asthma.

Graft-versus-host disease (GVHD) is a systemic inflammatory response due to the recognition of major histocompatibility complex disparity between donor and recipient after hematopoietic stem cell transplantation (HSCT). T-cell activation is critical to the induction of GVHD, and data from our group and others have shown that regulatory T cells (Tregs) prevent GVHD when given at the time of HSCT. Using multiphoton laser scanning microscopy, we examined the single cell dynamics of donor T cells and dendritic cells (DCs) with or without Tregs postallogeneic transplantation. We found that donor conventional T cells (Tcons) spent very little time screening host DCs. Tcons formed stable contacts with DCs very early after transplantation and only increased velocity in the lymph node at 20 hours after transplant. We also observed that Tregs reduced the interaction time between Tcons and DCs, which was dependent on the generation of interleukin 10 by Tregs. Imaging using inducible Tregs showed similar disruption of Tcon-DC contact. Additionally, we found that donor Tregs induce host DC death and down-regulate surface proteins required for donor T-cell activation. These data indicate that Tregs use multiple mechanisms that affect host DC numbers and function to mitigate acute GVHD.

Background Environmental factors, as well as genetic predisposition, are known to be critical for the development of autoimmunity. However, the environmental agents that trigger autoimmune responses have remained elusive. One possible explanation is the "hit-and-run" mechanism in which the inciting antigens that initiate autoimmune responses are not present at the time of overt autoimmune disease. Objective After our previous findings that some allergens can incite autoimmune responses, we investigated the potential role of environmental allergens in triggering autoantibody development in patients with an autoimmune skin disease, pemphigus vulgaris (PV). Methods Revertant/germline mAbs (with mutations on variable regions of heavy and light chains reverted to germline forms) of 8 anti–desmoglein (Dsg) 3 pathogenic mAbs from patients with PV were tested for reactivity against a panel of possible allergens, including insects, pollens, epithelia, fungi, and food antigens. Results All the PV germline mAbs were reactive to antigens from walnut, including the well-known allergen Jug r 2 and an uncharacterized 85-kDa protein component. Sera from patients with PV contained significantly greater levels of anti-Dsg3 autoantibodies than walnut-specific antibodies, suggesting that the autoreactive B-cell response in patients with PV might be initially triggered by walnut antigens but is subsequently driven by Dsg3. Conclusion Our findings suggest that walnut antigens/allergens can initiate autoantibody development in patients with PV through a "hit-and-run" mechanism. The revertant/germline mAb approach might provide a paradigm for the etiological study of other allergic and autoimmune diseases.

Food allergies are increasing in prevalence and present an emerging epidemic for westernized countries. Strict dietary avoidance is the only approved management for food allergy, but accidental exposures regularly occur, leading to significant patient anxiety and decreased quality of life. Over the past decade, oral and sublingual immunotherapies have emerged as potential treatments for food allergy. While several small clinical trials have demonstrated that immunotherapy can desensitize food-allergic individuals, strategies for further enhancing safety and definitively establishing long-term efficacy are needed. This review presents an overview of recent oral and sublingual immunotherapy trials, and provides a glimpse into what the next generation of food immunotherapy may entail.

There is a growing evidence that allergen immunotherapy (AIT) can provide significant and long-lasting clinical benefit for a number of allergic individuals. However, it is less clear if AIT results in clinical tolerance, which is characterized by a persistent state of clinical non-reactivity to allergens after therapy is finished. Addressing this knowledge gap is particularly relevant for patients undergoing AIT for food allergies, as anything less than complete tolerance could have potentially devastating consequences. An increasing number of studies, in particular those involving oral immunotherapy, are attempting to assess tolerance induction following AIT. Clinical tolerance does appear to be achievable in a subset of patients undergoing AIT, but whether this is equivalent to the type of tolerance observed in nonallergic individuals remains unknown. Developing established criteria for assessing tolerance induction, as well as the use of consistent terminology when describing clinical tolerance, will be important for determining the disease-modifying potential of AIT.

OBJECTIVES/SPECIFIC AIMS: Allergic asthma is a chronic lung disease driven by inappropriate inflammatory responses against inhaled allergens. Neuropilin-2 (NRP2) is a pleiotropic transmembrane receptor expressed in the lung, but its role in allergic airway inflammation is unknown. Here, we characterized NRP2 expression in lung immune cells and investigated the effects of NRP2 deficiency on airway inflammation. METHODS/STUDY POPULATION: NRP2 expression by lung immune cells from NRP2 reporter mice was determined by flow cytometry. NRP2 expression by human alveolar macrophages (AM) from healthy individuals was determined by mRNA analysis and flow cytometry. Airway inflammation in NRP2-deficient mice was assessed by bronchoalveolar lavage (BAL) cytology and inflammatory gene expression in lung tissue. RESULTS/ANTICIPATED RESULTS: NRP2 expression in lung immune cells was negligible under steady-state conditions. In contrast, inhalational exposure to lipopolysaccharide (LPS) adjuvant dramatically induced NRP2 expression in AM, as 63.3% of AM from LPS-treated mice were NRP2+ compared with 1.5% of AM from control mice. Ex vivo treatment of human AM with LPS resulted in a 1.5-fold and 2.6-fold increase in NRP2 mRNA and surface protein expression, respectively. Compared to littermate controls, NRP2-deficient mice had greater numbers of BAL leukocytes and increased lung expression of the T helper type 2 cytokines IL-4 and IL-5. Furthermore, NRP2 deficiency resulted in stochastic development of allergic airway inflammation, as spontaneous airway eosinophilia was detected in 25% (2/8) of NRP2-deficient mice compared with 0% (0/8) of littermate controls. DISCUSSION/SIGNIFICANCE OF IMPACT: NRP2 is expressed by activated human and murine AM and suppresses the spontaneous development of allergic airway inflammation. These findings suggest that NRP2 may play a key role in allergic asthma pathogenesis, and could prove to be an important therapeutic target in patients with asthma and other allergic diseases.

SR Wilson, L The, LM Batia. Cell. 2013;155(2):285–295 Atopic dermatitis (AD) is a cutaneous disorder characterized by inflamed and pruritic (itchy) skin. The proallergic cytokine thymic stromal lymphopoietin (TSLP) is produced by keratinocytes and plays a central role in the pathogenesis of AD. Whether TSLP is directly responsible for the severe itching associated with AD is unclear. Studies were performed with mice and human cells. TSLP-mediated neuronal signaling was assessed by using calcium imaging and electrophysiology. TSLP-inducing signaling pathways …

GVHD is a syndrome that results from minor and major histocompatibility complex incompatibilities between the donor and recipient. More than 50 years after its initial description, the pathophysiology of GVHD remains poorly understood. Nonetheless, donor T cells have been shown to be critical to the pathophysiology of acute and chronic GVHD, yet precisely how they function remains unclear. The effector mechanisms by which donor T cells mediate tissue inflammation is even less well understood. Identification of several new lineages of CD4+ T cells made in the past decade and their roles in the pathophysiology of T cell–mediated diseases has shed new light on these effector mechanisms. In this review, we summarize the recent descriptions of these T-cell lineages and the current data supporting their role in acute and to a lesser extent chronic GVHD. Investigations into the activity of these new T-cell lineages may provide more rationale approaches to the treatment or prevention of GVHD.

A novel genetic vaccine that is based on a Venezuelan equine encephalitis virus (VEE) replicon launched from plasmid DNA is described. The plasmid encodes a VEE replicon under the transcriptional control of the cytomegalovirus immediate-early promoter (VEE DNA). The VEE DNA consistently expressed 3- to 15-fold more green fluorescent protein in vitro than did a conventional DNA vaccine. Furthermore, transfection with the DNA-launched VEE replicon induced apoptosis and type I interferon production. Inoculation of mice with VEE DNA encoding human immunodeficiency virus type 1 gp160 significantly increased humoral responses by several orders of magnitude compared to an equal dose of a conventional DNA vaccine. These increases were also observed at 10- and 100-fold-lower doses of the VEE DNA. Cellular immune responses measured by gamma interferon and interleukin 2 enzyme-linked immunospot assay were significantly higher in mice immunized with the VEE DNA at decreased doses. The immune responses induced by the VEE DNA-encoded antigen, however, were independent of an intact type I interferon signaling pathway. Moreover, the DNA-launched VEE replicon induced an efficient prime to a VEE replicon particle (VRP) boost, increasing humoral and cellular immunity by at least 1 order of magnitude compared to VEE DNA only. Importantly, immunization with VEE DNA, as opposed to VRP, did not induce any anti-VRP neutralizing antibodies. Increased potency of DNA vaccines and reduced vector immunity may ultimately have an impact on the design of vaccination strategies in humans.

While dendritic cell (DC) vaccines can protect hosts from tumor challenge, their ability to effectively inhibit the growth of established tumors remains indeterminate. Previously, we have shown that human DCs transduced with Venezuelan equine encephalitis virus replicon particles (VRPs) were potent stimulators of antigen-specific T cells in vitro. Therefore, we investigated the ability of VRP-transduced DCs (VRP-DCs) to induce therapeutic immunity in vivo against tumors overexpressing the neu oncoprotein. Transduction of murine DCs with VRPs resulted in high-level transgene expression, DC maturation and secretion of proinflammatory cytokines. Vaccination with VRP-transduced DCs (VRP-DCs) expressing a truncated neu oncoprotein induced robust neu-specific CD8+ T cell and anti-neu IgG responses. Furthermore, a single vaccination with VRP-DCs induced the regression of large established tumors in wild-type mice. Interestingly, depletion of CD4+, but not CD8+, T cells completely abrogated inhibition of tumor growth following vaccination. Taken together, our results demonstrate that VRP-DC vaccines induce potent immunity against established tumors, and emphasize the importance of the generation of both CD4+ T cell and B cell responses for efficient tumor inhibition. These findings provide the rationale for future evaluation VRP-DC vaccines in the clinical setting.