Enhanced anti-serpin antibody activity inhibits autoimmune inflammation in type 1 diabetes.

Intracellular (clade B) OVA-serpin protease inhibitors play an important role in tissue homeostasis by protecting cells from death in response to hypo-osmotic stress, heat shock, and other stimuli. It is not known whether these serpins influence immunological tolerance and the risk for autoimmune diseases. We found that a fraction of young autoimmune diabetes-prone NOD mice had elevated levels of autoantibodies against a member of clade B family known as serpinB13. High levels of anti-serpinB13 Abs were accompanied by low levels of anti-insulin autoantibodies, reduced numbers of islet-associated T cells, and delayed onset of diabetes. Exposure to anti-serpinB13 mAb alone also decreased islet inflammation, and coadministration of this reagent and a suboptimal dose of anti-CD3 mAb accelerated recovery from diabetes. In a fashion similar to that discovered in the NOD model, a deficiency in humoral activity against serpinB13 was associated with early onset of human type 1 diabetes. These findings suggest that, in addition to limiting exposure to proteases within the cell, clade B serpins help to maintain homeostasis by inducing protective humoral immunity.

EBI3 deficiency leads to diminished T helper type 1 and increased T helper type 2 mediated airway inflammation.

Despite extensive investigation of the signals required for development of T helper type 1 (Th1) and type 2 (Th2) immune responses, the mechanisms involved are still not well-defined. A critical role for Epstein-Barr virus-induced gene 3 (EBI3) in these responses has been proposed. EBI3, initially discovered as a transcriptionally activated gene in Epstein-Barr virus-infected B lymphocytes, codes for a subunit of the cytokine interleukin-27 (IL-27). While initial studies suggested that it had an important role in promoting Th1 responses, subsequent studies have revealed that EBI3 receptor signalling influences a variety of immune cell types and can inhibit both Th1 and Th2 responses. In the present study, we evaluated EBI3(-/-) mice for their ability to mount both Th1-mediated and Th2-mediated airway inflammatory responses. The EBI3(-/-) mice sensitized by exposure to inhaled ovalbumin plus a high dose of lipopolysaccharide, which normally results in Th1 responses in wild-type (WT) mice, instead developed Th2 type airway inflammation, with increased numbers of eosinophils. The EBI3(-/-) mice that were exposed to inhaled ovalbumin with a low dose of lipopolysaccharide, which induces Th2 responses in WT mice, showed a marked enhancement of these responses, with increased airway eosinophils, increased serum IgE levels and increased levels of Th2 cytokines (IL-4, IL-5 and IL-13) in culture supernatants of mediastinal lymph node cells. Increased production of Th2 cytokines was also seen when naive CD4(+) T cells from EBI3(-/-) mice were stimulated in vitro compared with cells from WT mice. These results provide the first evidence that EBI3 may play an inhibitory role in allergic asthma development.

Targeting Toll-like receptors on dendritic cells modifies the T(H)2 response to peanut allergens in vitro.

BACKGROUND: Delivery of allergens with bacterial adjuvants has been shown to be a successful immunotherapeutic strategy for food allergy treatment in animal models. How microbial signals, acting through the innate immune system, reshape ongoing allergic responses is poorly understood. OBJECTIVE: To investigate the contribution of Toll-like receptors (TLRs) in the response to bacterial adjuvants, we designed an in vitro system to characterize the effect of heat-killed Escherichia coli vector (HKE) on peanut-induced responses of dendritic cells (DCs) and T cells. METHODS: Wild-type or TLR signaling-deficient bone marrow-derived DCs were pulsed with crude peanut extract (CPE) alone (50 microg/mL) in the presence of HKE (10(6)/mL). DC maturation was analyzed by means of flow cytometry. Treated DCs were cocultured with carboxyfluorescein succinimidyl ester (CFSE)-labeled CD4(+) T cells from sensitized mice. Cytokine production from DCs and T cells was measured by using Bioplex assays. RESULTS: Peanut-pulsed DCs induced the production of IL-4, IL-5, and IL-13, as well as IL-17 and IFN-gamma, from primed T cells. Adding HKE to CPE-pulsed DCs resulted in a significant decrease in T(H)2 cytokine production associated with an increase in IFN-gamma levels and profound attenuation of T-cell proliferation. These effects were linked to HKE-induced TLR-dependent changes in DC reactivity to CPE, especially the production of polarizing cytokines, such as IL-12. CONCLUSIONS: TLR signals modulate peanut-induced DC maturation in vitro, leading to changes in the T-cell response to peanut. These TLR effects must be confirmed in vivo and might constitute another alternative for allergen immunotherapies.

Lung vascular endothelial growth factor expression induces local myeloid dendritic cell activation.

We previously demonstrated that vascular endothelial growth factor (VEGF) expression in the murine lung increases local CD11c+MHCII+ DC number and activation. In this study, employing a multicolor flow cytometry, we report increases in both myeloid (mDC) and plasmacytoid (pDC) DC in the lungs of VEGF transgenic (tg) compared to WT mice. Lung pDC from VEGF tg mice exhibited higher levels of activation with increased expression of MHCII and costimulatory molecules. As VEGF tg mice display an asthma-like phenotype and lung mDC play a critical role in asthmatic setting, studies were undertaken to further characterize murine lung mDC. Evaluations of sorted mDC from VEGF tg lungs demonstrated a selective upregulation of cathepsin K, MMP-8, -9, -12, and -14, and chemokine receptors as compared to those obtained from WT control mice. They also had increased VEGFR2 but downregulated VEGFR1 expression. Analysis of chemokine and regulatory cytokine expression in these cells showed an upregulation of macrophage chemotactic protein-3 (MCP-3), thymus-expressed chemokine (TECK), secondary lymphoid organ chemokine (SLC), macrophage-derived chemokine (MDC), IL-1beta, IL-6, IL-12 and IL-13. The antigen (Ag) OVA-FITC uptake by lung DC and the migration of Ag-loaded DC to local lymph nodes were significantly increased in VEGF tg mice compared to WT mice. Thus, VEGF may predispose the lung to inflammation and/or repair by activating local DC. It regulates lung mDC expression of innate immunity effector molecules. The data presented here demonstrate how lung VEGF expression functionally affects local mDC for the transition from the innate response to a Th2-type inflammatory response.

p21 Ras/impedes mitogenic signal propagation regulates cytokine production and migration in CD4 T cells.

The propensity of T cells to generate coordinated cytokine responses is critical for the host to develop resistance to pathogens while maintaining the state of immunotolerance to self-antigens. The exact mechanisms responsible for preventing the overproduction of proinflammatory cytokines including interferon (IFN)-gamma are not fully understood, however. In this study, we examined the role of a recently described Ras GTPase effector and repressor of the Raf/MEK/ERK cascade called impedes mitogenic signal propagation (Imp) in limiting the induction of T-cell cytokines. We found that stimulation of the T cell receptor complex leads to the rapid development of a physical association between Ras and Imp. Consistent with the hypothesis that Imp inhibits signal transduction, we also found that disengagement of this molecule by the Ras(V12G37) effector loop mutant or RNA interference markedly enhances the activation of the NFAT transcription factor and IFN-gamma secretion. A strong output of IFN-gamma is responsible for the distinct lymphocyte traffic pattern observed in vivo because the transgenic or retroviral expression of Ras(V12G37) caused T cells to accumulate preferentially in the lymph nodes and delayed their escape from the lymphoid tissue, respectively. Together, our results describe a hitherto unrecognized negative regulatory role for Imp in the production of IFN-gamma in T cells and point to Ras-Imp binding as an attractive target for therapeutic interventions in conditions involving the production of this inflammatory cytokine.

Assessment of myosin II, Va, VI and VIIa loss of function on endocytosis and endocytic vesicle motility in bone marrow-derived dendritic cells.

An essential feature of dendritic cell immune surveillance is endocytic sampling of the environment for non-self antigens primarily via macropinocytosis and phagocytosis. The role of several members of the myosin family of actin based molecular motors in dendritic cell endocytosis and endocytic vesicle movement was assessed through analysis of dendritic cells derived from mice with functionally null myosin mutations. These include the dilute (myosin Va), Snell's waltzer (myosin VI) and shaker-1 (myosin VIIa) mouse lines. Non muscle myosin II function was assessed by treatment with the inhibitor, blebbistatin. Flow cytometric analysis of dextran uptake by dendritic cells revealed that macropinocytosis was enhanced in Snell's waltzer dendritic cells while shaker-1 and blebbistatin-treated cells were comparable to controls. Comparison of fluid phase uptake using pH insensitive versus pH sensitive fluorescent dextrans revealed that in dilute cells rates of uptake were normal but endosomal acidification was accelerated. Phagocytosis, as quantified by uptake of E. coli, was normal in dilute while dendritic cells from Snell's waltzer, shaker-1 and blebbistatin treated cells exhibited decreased uptake. Microtubule mediated movements of dextran-or transferrin-tagged endocytic vesicles were significantly faster in dendritic cells lacking myosin Va. Loss of myosin II, VI or VIIa function had no significant effects on rates of endocytic vesicle movement.

Innate immune control of pulmonary dendritic cell trafficking.

Dendritic cells (DC) are potent antigen-presenting cells that are essential for initiating adaptive immune responses. Residing within the airway mucosa, pulmonary DC continually sample the antigenic content of inhaled air and migrate to draining lymph nodes, where they present these antigens to naive T cells. The migratory patterns of pulmonary DC are highly dependent upon inflammatory conditions in the lung. Under steady-state, or non-inflammatory, conditions, pulmonary DC undergo slow but constitutive migration to draining lymph nodes, where they remain for several days and confer antigen-specific tolerance. With the onset of pulmonary inflammation, airway DC trafficking increases dramatically, and these cells rapidly accumulate within draining lymph nodes. However, within a few days, the number of airway-derived DC in lymph nodes stabilizes or declines, even in the face of ongoing pulmonary inflammation. Here, we summarize current understanding of the molecular and cellular mechanisms underlying pulmonary DC trafficking to the lymph node and the recruitment of DC precurors to the lung. It is hoped that an improved understanding of these mechanisms will lead to novel DC-mediated therapeutic strategies to treat immune-related pulmonary disease.

CTLA-4 differentially regulates the immunological synapse in CD4 T cell subsets.

Primary murine Th1 and Th2 cells differ in the organization of the immunological synapse, with Th1 cells, but not Th2 cells, clustering signaling molecules at the T cell/B cell synapse site. We sought to determine whether differential costimulatory signals could account for the differences observed. We found that Th2 cells express higher levels of CTLA-4 than Th1 cells, and demonstrated that Th2 cells lacking CTLA-4 are now able to cluster the TCR with the same frequency as Th1 cells. Furthermore, reconstitution of CTLA-4 into CTLA-4-deficient Th2 cells, or into Th1 cells, inhibits the clustering of the TCR. We have also shown that Th2 cells, but not Th1 cells, show variations in the organization of the immunological synapse depending on levels of expression of CD80/CD86 on the APC. These studies demonstrate a unique role for CTLA-4 as a critical regulator of Th2 cells and the immunological synapse.

MyD88-dependent induction of allergic Th2 responses to intranasal antigen.

MyD88 is a common Toll-like receptor (TLR) adaptor molecule found to be essential for induction of adaptive Th1 immunity. Conversely, innate control of adaptive Th2 immunity has been shown to occur in a MyD88-independent manner. In this study, we show that MyD88 is an essential innate component in the induction of TLR4-dependent Th2 responses to intranasal antigen; thus we demonstrate what we believe to be a novel role for MyD88 in pulmonary Th2 immunity. Induction of the MyD88-independent type I IFN response to LPS is defective in the pulmonary environment. Moreover, in the absence of MyD88, LPS-induced upregulation of costimulatory molecule expression on pulmonary DCs is defective, in contrast to what has been observed with bone marrow-derived DCs (BMDCs). Reconstitution of Th2 responses occurs upon adoptive pulmonary transfer of activated BMDCs to MyD88-deficient recipients. Furthermore, the dependence of Th2 responses on MyD88 is governed by the initial route of antigen exposure; this demonstrates what we believe are novel site-specific innate mechanisms for control of adaptive Th2 immunity.

Regulated recruitment of MHC class II and costimulatory molecules to lipid rafts in dendritic cells.

T cell activation has long been associated with the partitioning of Ag receptors and associated molecules to lipid microdomains. We now show that dendritic cells (DCs) also accomplish the selective recruitment to lipid rafts of molecules critical for Ag presentation. Using mouse bone marrow-derived DCs, we demonstrate that MHC class II molecules become substantially localized to rafts upon DC maturation. Even more striking is the fact that CD86 is recruited to rafts upon T cell-DC interaction. Recruitment is Ag dependent and requires CD28 on T cells. Despite the regulated recruitment of MHC class II and CD86 to rafts, unlike the counter-receptors in T cells, DCs do not polarize these molecules to sites of DC-T cell contact. This difference may reflect the necessity for DCs to interact with multiple T cells simultaneously and emphasizes that the biochemical and morphological correlates of lipid rafts are not necessarily equivalent.

Understanding asthma pathogenesis: linking innate and adaptive immunity.

PURPOSE OF REVIEW: Treatment and even prevention of allergic asthma will require a detailed understanding of disease pathogenesis and in particular identification of factors that govern T-helper type 2 (Th2) immunity. This review defines the priming and differentiation steps necessary to develop antiallergen Th2 immunity and highlights recently identified stimuli that satisfy these requirements. RECENT FINDINGS: Striking discoveries in innate immunity have advanced our understanding of how adaptive immune responses are initiated, yet only recently have these principles been applied to allergic disease. Signaling through certain innate immune receptors, the toll-like receptors (TLR) have been shown to modulate Th2-mediated disease in animal models. The dendritic cell has emerged as the central player in the intricate interplay between the adaptive and innate systems of immunity. Recent studies have also uncovered alternative pathways of initiating allergen sensitization that depend entirely on adaptive, rather than innate immune, triggers. SUMMARY: The adaptive immune system cannot initiate a response without the "permission" of the innate immune system, and this holds true for Th2 responses to aeroallergens, although induction of Th2 immunity in response to TLR signaling varies with the type and dose of TLR ligand. However, under conditions of ongoing Th2 inflammation, the adaptive immune system can act as its own adjuvant and provide the necessary activating signals to initiate an immune response to foreign protein antigens. This may be the mechanism underlying the clinically observed phenomenon of polysensitization in atopic patients and provides another therapeutic target in asthma.

CD4 raft association and signaling regulate molecular clustering at the immunological synapse site.

T cell activation is associated with the partitioning of TCRs and other signaling proteins, forming an immunological synapse. This study demonstrates a novel function for the CD4 coreceptor in regulating molecular clustering at the immunological synapse site. We show using transgenic mouse and retroviral reconstitution studies that CD4 is required for TCR/protein kinase C (PKC) theta clustering. Specifically, we demonstrate that CD4 palmitoylation sequences are required for TCR/PKCtheta raft association and subsequent clustering, indicating a particular role for raft-associated CD4 molecules in regulating immune synapse organization. Although raft association of CD4 is necessary, it is not sufficient to mediate clustering, as cytoplasmic tail deletion mutants are able to localize to rafts, but are unable to mediate TCR/PKCtheta clustering, indicating an additional requirement for CD4 signaling. These studies suggest that CD4 coreceptor function is regulated not only through its known signaling function, but also by posttranslational lipid modifications which regulate localization of CD4 in lipid rafts.

IL-4-dependent Th2 collateral priming to inhaled antigens independent of Toll-like receptor 4 and myeloid differentiation factor 88.

Allergic asthma is an inflammatory lung disease thought to be initiated and directed by type 2 helper T cells responding to environmental Ags. The mechanisms by which allergens induce Th2-adaptive immune responses are not well understood, although it is now clear that innate immune signals are required to promote DC activation and Th2 sensitization to inhaled proteins. However, the effect of ongoing Th2 inflammation, as seen in chronic asthma, on naive lymphocyte activation has not been explored. It has been noted that patients with atopic disorders demonstrate an increased risk of developing sensitivities to new allergens. This suggests that signals from an adaptive immune response may facilitate sensitization to new Ags. We used a Th2-adoptive transfer murine model of asthma to identify a novel mechanism, termed "collateral priming," in which naive CD4(+) T cells are activated by adaptive rather than innate immune signals. Th2 priming to newly encountered Ags was dependent on the production of IL-4 by the transferred Th2 population but was independent of Toll-like receptor 4 signaling and the myeloid differentiation factor 88 Toll-like receptor signaling pathway. These results identify a novel mechanism of T cell priming in which an Ag-specific adaptive immune response initiates distinct Ag-specific T cell responses in the absence of classical innate immune system triggering signals.

The master regulators of allergic inflammation: dendritic cells in Th2 sensitization.

The development of Th2 responses to inhaled proteins represents a malfunction of the adaptive immune system in that protein antigens are not microbial in nature and should not elicit an adaptive immune reaction. This derailing of the immune system may result from false alarms generated by the innate immune system, resulting in unexpected dendritic cell (DC) maturation after exposure to allergens. Conditions in the local microenvironment during DC maturation may also result in the preferential induction of Th2 responses. Recent progress has been made in our understanding of the role of DCs in both Th2 sensitization to aeroallergens and the regulation of Th2 and Th1 immunity.

Cytolytic CD4(+)-T-cell clones reactive to EBNA1 inhibit Epstein-Barr virus-induced B-cell proliferation.

In the absence of immune surveillance, Epstein-Barr virus (EBV)-infected B cells generate neoplasms in vivo and transformed cell lines in vitro. In an in vitro system which modeled the first steps of in vivo immune control over posttransplant lymphoproliferative disease and lymphomas, our investigators previously demonstrated that memory CD4(+) T cells reactive to EBV were necessary and sufficient to prevent proliferation of B cells newly infected by EBV (S. Nikiforow et al., J. Virol. 75:3740-3752, 2001). Here, we show that three CD4(+)-T-cell clones reactive to the latent EBV antigen EBNA1 also prevent the proliferation of newly infected B cells from major histocompatibility complex (MHC) class II-matched donors, a crucial first step in the transformation process. EBNA1-reactive T-cell clones recognized B cells as early as 4 days after EBV infection through an HLA-DR-restricted interaction. They secreted Th1-type and Th2-type cytokines and lysed EBV-transformed established lymphoblastoid cell lines via a Fas/Fas ligand-dependent mechanism. Once specifically activated, they also caused bystander regression and bystander killing of non-MHC-matched EBV-infected B cells. Since EBNA1 is recognized by CD4(+) T cells from nearly all EBV-seropositive individuals and evades detection by CD8(+) T cells, EBNA1-reactive CD4(+) T cells may control de novo expansion of B cells following EBV infection in vivo. Thus, EBNA1-reactive CD4(+)-T-cell clones may find use as adoptive immunotherapy against EBV-related lymphoproliferative disease and many other EBV-associated tumors.

Human gamma/delta T-cell proliferation and IFN-gamma production induced by hexamethylene diisocyanate.

BACKGROUND: The human immune response to isocyanate, a leading cause of occupational asthma, remains incompletely characterized, including the cell types involved and the form of the chemical that acts as an antigen. OBJECTIVE: The purpose of this investigation was to characterize human T cells that respond to hexamethylene diisocyanate (HDI), an aliphatic isocyanate routinely used in the automobile body industry. METHODS: Human T-cell lines were generated and characterized from peripheral blood of HDI-exposed and HDI-unexposed subjects, using two different HDI antigens, HDI-conjugated albumin and HDI-exposed human airway epithelial cells (NCI-H292). Flow cytometry was used to characterize the phenotype of HDI-responsive T cells. ELISA and intracellular staining techniques were used to evaluate HDI-induced cytokine production. DNA sequence analysis of T-cell receptors was used to further define clonal populations of HDI-responsive T cells. RESULTS: HDI antigen preparations but not "mock exposed" control antigens lead to increased proliferation of specific cell types, CD3+CD4-CD8(dim) and/or CD3+CD4-CD8- cells, from HDI-exposed but not from HDI-unexposed subjects. These HDI-responsive T cells expressed unique oligoclonal gamma/delta rather than alpha/beta T-cell receptors, with characteristics suggestive of antigen-mediated selection and specificity. The HDI-stimulated gamma/delta T cells were associated with T(H)1-like cytokines and produce IFN-gamma but not IL-5 or IL-13. CONCLUSIONS: These data are the first to demonstrate that HDI can selectively stimulate gamma/delta T cells with the potential to modulate the human immune response to exposure.

Differential roles for CD4 and CD8 T cells after diisocyanate sensitization: genetic control of TH2-induced lung inflammation.

BACKGROUND: Exposure to diisocyanates is a major cause of occupational asthma. We previously developed a novel mouse model of diisocyanate-induced asthma involving epicutaneous sensitization to hexamethylene diisocyanate (HDI) that demonstrates many features of the human disease, including airway eosinophilia and mucus hypersecretion. OBJECTIVE: To determine what factors are critical for the development of HDI-induced airway inflammation, we investigated the strain distribution of this response and the roles of CD4(+) and CD8(+) T cells. METHODS: Mice were epicutaneously exposed to HDI and then challenged with HDI, either by means of inhalation to induce airway inflammation or on the ear to induce contact hypersensitivity (CHS). Lymph node cytokine production and serum antibodies were also measured. RESULTS: Induction of airway eosinophilia was highly dependent on the mouse strain used, with C57BL/6, A/J, CBA, C3H, and C57BL/10 mice all having significantly fewer eosinophils than BALB/c mice. HDI-specific antibodies and lymph node IL-5 and IL-13 production were also diminished in non-BALB/c strains. In contrast, CHS to HDI developed in all strains tested. Studies in mice deficient in either CD4(+) or CD8(+) T cells revealed that CD4(+) T cells were critical for HDI-induced airway eosinophilia, whereas CD8(+) T cells were the major effector cells in CHS. CONCLUSION: The data suggest that, in contrast to CHS, induction of T(H)2 responses after epicutaneous exposure to diisocyanates is strongly genetically influenced. Furthermore, the lung inflammatory response to inhaled HDI appears to depend primarily on effective generation of these CD4(+) T(H)2 responses, as is the case in atopic asthma.

To respond or not to respond: T cells in allergic asthma.

The incidence of allergic asthma has almost doubled in the past two decades. Numerous epidemiological studies have linked the recent surge in atopic disease with decreased exposure to infections in early childhood as a result of a more westernized lifestyle. However, a clear mechanistic explanation for how this might occur is still lacking. An answer might lie in the presently unfolding story of various regulatory T-cell populations that can limit adaptive immune responses, including T helper 2 (T(H)2)-cell-mediated allergic airway disease.

Activation of CD4 T cells by Raf-independent effectors of Ras.

Small GTPase Ras is capable of mediating activation in T lymphocytes by using Raf kinase-dependent signaling pathway. Other effectors of Ras exist, however, suggesting that targets of Ras alternative to Raf may also contribute to T cell functions. Here we demonstrate that Ras(V12G37) mutant that fails to bind Raf, potently increases intracellular calcium concentration and cytokine production in primary antigen-stimulated T cells. From three known effectors which retain the ability to interact with Ras(V12G37), overexpression of phospholipase C epsilon but not that of RIN1 or Ral guanine nucleotide exchange factors enhanced cytokine and nuclear factor-activated T cell reporter T cell responses. Hence T cell activation can be critically regulated by the Ras effector pathway independent from Raf that can be mimicked by phospholipase C epsilon.

P27kip1 regulates the cell cycle arrest and survival of activated T lymphocytes in response to interleukin-2 withdrawal.

The majority of activated T lymphocytes undergo cell death at the end of a primary immune response, while a minority survive as memory cells. The mechanisms that control the decision between these two fates are unknown. In the present study we examined the response of activated T cells to interleukin-2 (IL-2) withdrawal. Within hours, the percentage of T lymphocytes in cell cycle showed a steady decrease, while the percentage arrested in G1 increased proportionally. Deprivation of IL-2 resulted in upregulation of the cell cycle inhibitor p27kip1. Comparison with resting T-cell populations revealed that the highest expression of p27kip1 occurs in activated T cells undergoing cell cycle arrest following IL-2 withdrawal. T cells deficient in p27kip1 expression showed an impaired ability to undergo cell cycle arrest in response to IL-2 deprivation. Moreover, T cells deficient in p27kip1 showed significantly more apoptosis after IL-2 withdrawal. Collectively, this study demonstrates that p27kip1 regulates both the cell cycle arrest and the apoptosis of antigen-specific T lymphocytes.