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Children who grow up in traditional farm environments are protected from developing asthma and allergy. This “farm effect” can be largely explained by the child’s early life contact with farm animals, in particular cows, and their microbes. Our studies in Amish and Hutterite school children living on farms in the U.S. have further demonstrated that this protection is mediated through innate immune pathways. Although very similar with respect to ancestry and many lifestyle factors that are associated with asthma risk, Amish and Hutterites follow farming practices that are associated with profound differences in the levels of house dust endotoxin, in the prevalence of asthma and atopy among school children, and in the proportions, phenotypes, and functions of immune cells from these children. In this review, we will consider our studies in Amish and Hutterites children in the context of the many previous studies in European farm children and discuss how these studies have advanced our understanding of the asthma-protective “farm effect”.

Previous work has shown ICOS can function independently of CD28, but whether either molecule can compensate for the other in vivo is not known. Since ICOS is a potent inducer of Th2 cytokines and linked to allergy and elevated serum IgE in humans, we hypothesized that augmenting ICOS costimulation in murine allergic airway disease may overcome CD28 deficiency. While ICOS was expressed on T cells from CD28(-/-) mice, Th2-mediated airway inflammation was not induced in CD28(-/-) mice by increased ICOS costimulation. Further, we determined if augmenting CD28 costimulation could compensate for ICOS deficiency. ICOS(-/-) mice had a defect in airway eosinophilia that was not overcome by augmenting CD28 costimulation. CD28 costimulation also did not fully compensate for ICOS for antibody responses, germinal center formation or the development of follicular B helper T cells. CD28 and ICOS play complementary non-overlapping roles in the development of Th2 immunity in vivo.

The Inducible Costimulator molecule (ICOS), a member of the CD28 family of costimulatory molecules, was identified in 1999 as a molecule expressed primarily on activated human T cells. Induced upon activation, ICOS appears to be an ideal target for modifying T-cell-mediated immune responses. ICOS was also found to be highly expressed on germinal center T cells, suggesting that ICOS was involved in T:B cell interactions. While ICOS has subsequently been shown to be important for both Th1 and Th2 cell activation and effector function, a central role for ICOS in the generation and maintenance of humoral immunity is emerging. In this review, we summarize the evidence that the level of ICOS expression regulates T-cell-dependent B cell responses and propose a model for the role of ICOS in diseases characterized by dysregulated humoral immunity.

Background: Damage to the airway epithelium is one prominent feature of the damage seen in chronic asthma. Cortico-steroids induce apoptosis in inflammatory cells, which in part explains their ability to suppress airway inflammation. However, corticosteroid therapy does not necessarily reverse the epithelial damage seen in asthmatic airways. Objective: We hypothesized that corticosteroids might induce airway epithelial cell apoptosis as one potential explanation for this persistent damage. Methods: BALB/c mice were treated with 1 mg/kg dexamethasone by means of intraperitoneal injection for 3 days to 4 weeks. Additional mice were sensitized and challenged with ovalbumin and then followed for 14 days with or without dexa-methasone treatment starting on day 4 after challenge. Apoptosis was measured by using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling and immunohistochemistry for the p85 cleavage product of poly (adenosine diphosphate-ribose) polymerase. Shed epithelial cells were counted in bronchoalveolar lavage fluid. Results: In a time-dependent manner dexamethasone treatment increased epithelial cell apoptosis and shedding into the airway lumen. This was not associated with any change in the abundance of the apoptotic regulator Bcl-x L . In animals sensitized and challenged with ovalbumin, treatment with 1 mg/kg dexamethasone starting 4 days after challenge reversed the inflammatory changes but did not reverse either epithelial cell shedding or apoptosis seen after allergen challenge. Conclusion: Corticosteroids might induce apoptotic cell death of airway epithelium in vivo and fail to mitigate epithelial cell shedding and apoptosis elicited by means of allergen challenge. This raises the possibility that at least one of the major components of chronic airway damage in asthma, epithelial shedding, might in part result from a major therapy used for disease control. (J Allergy Clin Immunol 2003;111:360-6.)

We have developed a sensitive sandwich ELISA (sELISA) for quantitative determination of group V phospholipase A(2) (gVPLA(2)). This assay utilizes three monoclonal antibodies (mAbs) directed against human gVPLA(2) (MCL-1B7, MCL-2A5, and MCL-3G1), which recognize specifically different epitopes of gVPLA(2). A mixture of MCL-1B7 and MCL-2A5 was used as the capture mAb, and MCL-3G1 as the detector mAb; purified human gVPLA(2) was used as the standard protein. The limit of detection of the sELISA is 2 ng/ml; the intra- and inter-coefficients of variation were 4.97+/-0.81% and 8.42+/-3.4%. The validity of the sELISA was assured by the recovery of exogenous recombinant gVPLA(2), which was 99.7% to 102%, and demonstration of noninterference of the gVPLA(2) assay by a high concentrations of other protein from murine lung and heart. To assess the usefulness of this sELISA for tissue measurements, the amount of gVPLA(2) in cultured human epithelial cells and isolated human eosinophils was determined. Total gVPLA(2) mass in epithelial cells was 2.83+/-0.33 ng/10(7) cells; gVPLA(2) was not detected in eosinophils. The presence of high concentration of gVPLA(2) in epithelial cells was confirmed by immunoprecipitation/Western blot analysis and by flow cytometry. This assay allows for convenient differentiation between the highly homologous 14-kDa secretory PLA(2)s, gVPLA(2), gIIaPLA(2), gIbPLA(2) and gXPLA(2), and accurate quantitation of gVPLA(2) in biological samples.

The large mucin CD43 is actively excluded from T cell/APC interaction sites, concentrating in a membrane domain distal to the site of TCR engagement. The cytoplasmic region of CD43 was necessary and sufficient for this antipodal movement. ERM cytoskeletal adaptor proteins colocalized with CD43 in this domain. An ERM dominant-negative mutant blocked the distal accumulation of CD43 and another known ERM binding protein, Rho-GDI. Inhibition of ERM function decreased the production of IL-2 and IFNgamma, without affecting PKC(theta) focusing or CD69 upregulation. These results indicate that ERM proteins organize a complex distal to the T cell/APC interaction site and provide evidence that full T cell activation may involve removal of inhibitory proteins from the immunological synapse.

TRAF4 is one of six identified members of the family of TNFR-associated factors. While the other family members have been found to play important roles in the development and maintenance of a normal immune system, the importance of TRAF4 has remained unclear. To address this issue, we have generated TRAF4-deficient mice. Despite widespread expression of TRAF4 in the developing embryo, as well as in the adult, lack of TRAF4 expression results in a localized, developmental defect of the upper respiratory tract. TRAF4-deficient mice are born with a constricted upper trachea at the site of the tracheal junction with the larynx. This narrowing of the proximal end of the trachea results in respiratory air flow abnormalities and increases rates of pulmonary inflammation. These data demonstrate that TRAF4 is required to regulate the anastomosis of the upper and lower respiratory systems during development.

Background The transcription factor promyelocytic leukemia zinc finger (PLZF) is transiently expressed during development of type 2 innate lymphoid cells (ILC2s) but is not present at the mature stage. We hypothesized that PLZF-deficient ILC2s have functional defects in the innate allergic response and represent a tool for studying innate immunity in a mouse with a functional adaptive immune response. Objective We determined the consequences of PLZF deficiency on ILC2 function in response to innate and adaptive immune stimuli by using PLZF −/− mice and mixed wild-type:PLZF −/− bone marrow chimeras. Methods PLZF −/− mice, wild-type littermates, or mixed bone marrow chimeras were treated with the protease allergen papain or the cytokines IL-25 and IL-33 or infected with the helminth Nippostrongylus brasiliensis to induce innate type 2 allergic responses. Mice were sensitized with intraperitoneal ovalbumin-alum, followed by intranasal challenge with ovalbumin alone, to induce adaptive T H 2 responses. Lungs were analyzed for immune cell subsets, and alveolar lavage fluid was analyzed for ILC2-derived cytokines. In addition, ILC2s were stimulated ex vivo for their capacity to release type 2 cytokines. Results PLZF-deficient lung ILC2s exhibit a cell-intrinsic defect in the secretion of IL-5 and IL-13 in response to innate stimuli, resulting in defective recruitment of eosinophils and goblet cell hyperplasia. In contrast, the adaptive allergic inflammatory response to ovalbumin and alum was unimpaired. Conclusions PLZF expression at the innate lymphoid cell precursor stage has a long-range effect on the functional properties of mature ILC2s and highlights the importance of these cells for innate allergic responses in otherwise immunocompetent mice.

SummaryIt is well understood that antigen-presenting cells (APCs) within tumors typically do not maintain cytotoxic T cell (CTL) function, despite engaging them. Across multiple mouse tumor models and human tumor biopsies, we have delineated the intratumoral dendritic cell (DC) populations as distinct from macrophage populations. Within these, CD103+ DCs are extremely sparse and yet remarkably capable CTL stimulators. These are uniquely dependent on IRF8, Zbtb46, and Batf3 transcription factors and are generated by GM-CSF and FLT3L cytokines. Regressing tumors have higher proportions of these cells, T-cell-dependent immune clearance relies on them, and abundance of their transcripts in human tumors correlates with clinical outcome. This cell type presents opportunities for prognostic and therapeutic approaches across multiple cancer types.

Purpose Bronchiolitis Obliterans Syndrome (BOS) is the major limitation to long-term survival after lung transplantation. Its pathogenesis, however, is poorly understood and no effective predictive biomarkers have been identified. We hypothesized that peripheral blood mononuclear cell gene expression profiles could identify patients at risk of BOS. Methods and Materials Gene expression profiling was performed in PBMCs collected from 26 lung transplant recipients at 3 and 9 months after transplantation and prior to development of BOS. Differential gene expression was tested over time using a linear regression mixed effect model. Results A substantial proportion of genes (~1800, false discovery rate (FDR) Conclusions We postulate that differential patterns of gene expression associated with dysregulated biological pathways after lung transplantation predispose to, and may predict, BOS development.

Purpose The development of Bronchiolitis Obliterans Syndrome (BOS) has been previously linked to microbial pathogens within the transplanted lung. With advanced molecular technology, we are now able to further define microbial communities within the transplanted lung. We hypothesized that these microbial communities will be distinct in lung transplant recipients who develop BOS compared to those who remain stable. Methods and Materials Eighteen lung transplant recipients were included in this analysis. Nine patients developed BOS within 2 years. Bronchoscopy with bronchoalveolar lavage (BAL) was performed at 1, 3, 6, and 12 months post transplant. Bacterial 16S rDNA extracted from the BAL fluid was amplified using barcoded primers and PCR products were pooled and sequenced using 454 pyrosequencing. All the sequence reads were denoised and clustered into similar operational taxonomic units. Taxonomy annotation was assigned according to Ribosomal Database Project. Results The bacterial community of transplanted lungs was taxonomically diversified and consisted of micro-organisms belonging to 14 phyla. Inter-individual variation of the microbial community was greater than intra-individual variation for all patients. There was a distinct difference in the microbial community at the phylum and class taxonomic level between BOS and non-BOS patients (p Conclusions The pulmonary microbiota in lung transplant recipients is taxonomically diverse and dynamic over time and is distinct in patients who develop early BOS compared to those who remain stable. Our findings provide novel approaches to address the relationship between microbial communities and BOS development after lung transplantation.