ICOS protects against mortality from acute lung injury through activation of IL-5+ ILC2s.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease causing irreversible lung scarring and loss of pulmonary function. IPF Patients suffer from a high rate of pulmonary infections and acute exacerbations of disease that further contribute to pulmonary decline. Low expression of the inducible T-cell costimulatory molecule (ICOS) in peripheral blood mononuclear cells predicts decreased survival of IPF patients, but the mechanisms by which ICOS protects are unclear. Using a model of bleomycin-induced lung injury and fibrosis, we now demonstrate that ICOS expression enhances survival from lung injury rather than regulating fibrogenesis. Of ICOS-expressing cells, type 2 innate lymphocytes (ILC2s) are the first to respond to bleomycin-induced injury, and this expansion is ICOS dependent. Interestingly, a similar decrease in ICOS+ ILCs was found in lung tissue from IPF patients. Interleukin (IL)-5, produced primarily by ILC2s, was significantly reduced after lung injury in ICOS-/- mice, and strikingly, treatment with IL-5 protected both ICOS-/- and wild-type mice from mortality. These results imply that low ICOS expression and decreased lung ILC2s in IPF patients may contribute to poor recovery from infections and acute exacerbation and that IL-5 treatment may be a novel therapeutic strategy to overcome these defects and protect against lung injury.

CD43 interaction with ezrin-radixin-moesin (ERM) proteins regulates T-cell trafficking and CD43 phosphorylation.

Cell polarization is a key feature of cell motility, driving cell migration to tissues. CD43 is an abundantly expressed molecule on the T-cell surface that shows distinct localization to the migrating T-cell uropod and the distal pole complex (DPC) opposite the immunological synapse via association with the ezrin-radixin-moesin (ERM) family of actin regulatory proteins. CD43 regulates multiple T-cell functions, including T-cell activation, proliferation, apoptosis, and migration. We recently demonstrated that CD43 regulates T-cell trafficking through a phosphorylation site at Ser-76 (S76) within its cytoplasmic tail. Using a phosphorylation-specific antibody, we now find that CD43 phosphorylation at S76 is enhanced by migration signals. We further show that CD43 phosphorylation and normal T-cell trafficking depend on CD43 association with ERM proteins. Interestingly, mutation of S76 to mimic phosphorylation enhances T-cell migration and CD43 movement to the DPC while blocking ERM association, showing that CD43 movement can occur in the absence of ERM binding. We also find that protein kinase CΘ can phosphorylate CD43. These results show that while CD43 binding to ERM proteins is crucial for S76 phosphorylation, CD43 movement and regulation of T-cell migration can occur through an ERM-independent, phosphorylation-dependent mechanism.

ERM-dependent movement of CD43 defines a novel protein complex distal to the immunological synapse.

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.

Inducible costimulator regulates Th2-mediated inflammation, but not Th2 differentiation, in a model of allergic airway disease.

A novel costimulatory molecule expressed on activated T cells, inducible costimulator (ICOS), and its ligand, B7-related protein-1 (B7RP-1), were recently identified. ICOS costimulation leads to the induction of Th2 cytokines without augmentation of IL-2 production, suggesting a role for ICOS in Th2 cell differentiation and expansion. In the present study, a soluble form of murine ICOS, ICOS-Ig, was used to block ICOS/B7RP-1 interactions in a Th2 model of allergic airway disease. In this model, mice are sensitized with inactivated Schistosoma mansoni eggs and are subsequently challenged with soluble S. mansoni egg Ag directly in the airways. Treatment of C57BL/6 mice with ICOS-Ig during sensitization and challenge attenuated airway inflammation, as demonstrated by a decrease in cellular infiltration into the lung tissue and airways, as well as by a decrease in local IL-5 production. These inhibitory effects were not due to a lack of T cell priming nor to a defect in Th2 differentiation. In addition, blockade of ICOS/B7RP-1 interactions during ex vivo restimulation of lung Th2 effector cells prevented cytokine production. Thus, blockade of ICOS signaling can significantly reduce airway inflammation without affecting Th2 differentiation in this model of allergic airway disease.

ICOS costimulation: It's not just for TH2 cells anymore.

A current paradigm has ICOS participating in TH2 costimulation. New data indicates ICOS regulates not only TH2 cells, but also TH1s.

Characterization of monoclonal antibodies specific for 14-kDa human group V secretory phospholipase A2 (hVPLA2).

Secretory phospholipase A2 (PLA2) consists of several 14-kDa isoforms with extensive homology, which makes it difficult to identify a specific isoform. In this study, we have developed and characterized monoclonal antibodies (MAbs) directed specifically against human group V sPLA2 (hVPLA2) derived from cultured hybridomas. These hybridomas were produced from the fusion of BALB/c-derived myeloma s/p20-Ag14 and splenocytes from mice immunized with purified recombinant hVPLA2. Three hybridomas secreting MAbs, MCL-3G1, MCL-2A5, and MCL-1B7, were selected and subcloned on the basis of their specificity to recognize hVPLA2 using solid-phase enzyme-linked immunoadsorbent assay (ELISA). The purified MAbs demonstrated a common pattern of immunoreactivity to hVPLA2, but not to human group IIa isoform (hIIaPLA2). Isotype analysis indicates that these hybridomas are of the IgG1 type. Under reducing conditions, MCL-3G1 sensitively detected hVPLA2 and demonstrated no cross-reactivity to either hIIaPLA2 or group IV cytosolic PLA2. Although specific for hVPLA2, a relatively modest signal was recognized with MCL-1B7 and MCL-2A5. These newly developed MAbs allow for determination of tissue distribution and cell-specific functions of hVPLA2.

CD4+ T cell and eosinophil adhesion is mediated by specific ICAM-3 ligation and results in eosinophil activation.

T cells and eosinophils, which are found in close proximity in asthmatic lungs, express many surface receptors that are counterligands. These data suggest that direct interactions between these cell types could play an important role in regulating airway inflammation in asthma. We examined the effect of selective adhesion between counterligands on human eosinophils and CD4+ T cells to determine 1) the existence of specific adhesive interactions and 2) if augmented specific adhesion to CD4+ T cells also caused augmented secretion of leukotriene C4 (LTC4) from eosinophils. A new method for binding of human CD4+ T cells to microwell plates was developed, which allowed for specific quantitative assessment of eosinophil adhesion to individual CD4+ T cells in culture. Adhesion of CD4+ T cells to eosinophils was minimal in unstimulated cells but increased after activation of T cells by PMA. Augmented adhesion was regulated substantially through binding of ICAM-3 and only minimally by ICAM-1. We further evaluated whether this specific adhesion up-regulated stimulated secretion of LTC4 from eosinophils. Adhesion with CD4+ T cells augmented eosinophil secretion of LTC4 caused by FMLP plus cytochalasin. Blockade of ICAM-3, as well as ICAM-1, inhibited completely the augmented secretion of eosinophil LTC4. We demonstrate that eosinophils and CD4+ T cells are capable of ligand-specific adhesion that is mediated predominantly by ICAM-3 ligation and that this binding causes augmented eosinophil secretion.

RIBP, a novel Rlk/Txk- and itk-binding adaptor protein that regulates T cell activation.

A novel T cell-specific adaptor protein, RIBP, was identified based on its ability to bind Rlk/Txk in a yeast two-hybrid screen of a mouse T cell lymphoma library. RIBP was also found to interact with a related member of the Tec family of tyrosine kinases, Itk. Expression of RIBP is restricted to T and natural killer cells and is upregulated substantially after T cell activation. RIBP-disrupted knockout mice displayed apparently normal T cell development. However, proliferation of RIBP-deficient T cells in response to T cell receptor (TCR)-mediated activation was significantly impaired. Furthermore, these activated T cells were defective in the production of interleukin (IL)-2 and interferon gamma, but not IL-4. These data suggest that RIBP plays an important role in TCR-mediated signal transduction pathways and that its binding to Itk and Rlk/Txk may regulate T cell differentiation.

TCR signaling induces selective exclusion of CD43 from the T cell-antigen-presenting cell contact site.

CD43, a large highly glycosylated molecule, is arguably the most abundant molecule on the surface of T cells. Nevertheless, the function of CD43 remains unclear. Utilizing fluorescence microscopy, we find that CD43 is excluded from the T cell-APC contact site. This exclusion is Ag dependent since optimal CD43 exclusion requires Ag-pulsed APC, and since signaling through CD3, in the absence of any other receptor ligand interactions, can induce the modulation of CD43. These data suggest that CD43 may function as a barrier to nonspecific T cell-APC interactions that is removed as a result of T cell activation. Exclusion from the interaction site is a unique feature of CD43 and not universally found for all large highly glycosylated molecules since CD45 is not excluded. Thus, CD43 may represent a novel regulatory molecule on the T cell surface that can direct T cell interactions by changing its location on the cell surface.

Expression of Fas (CD95) and FasL (CD95L) in human airway epithelium.

The cell surface molecule Fas (CD95) is a member of the tumor necrosis factor receptor family. Ligation of the Fas receptor can lead to induction of apoptosis in inflammatory cells. It has been suggested that expression of the Fas receptor and its ligand (FasL) in airway epithelium may modulate the inflammatory response commonly found in asthmatic lungs. We examined Fas and FasL expression on primary human tissues, on bronchial epithelial cells in primary culture, and on the immortalized human airway epithelial cell line, 1HAEo-. Receptor and ligand expression were demonstrated using multiple antibodies and multiple techniques, including immunohistochemistry, flow cytometry, Western blots, and reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical staining demonstrated that both columnar and basal cells of intact human lung tissues expressed cell surface Fas and FasL. In addition, both primary cultured and immortalized 1HAEo- cells expressed cell surface Fas and FasL, as demonstrated by flow cytometry; expression of Fas and FasL was confirmed at the transcription level using RT-PCR and, for additional confirmation of FasL, using Western blots. We demonstrate that both Fas and FasL are expressed by human airway epithelial cell subtypes. Expression of these molecules may play an important role in regulation of the inflammatory response.

CTLA4Ig inhibits airway eosinophilia and hyperresponsiveness by regulating the development of Th1/Th2 subsets in a murine model of asthma.

Complete T-cell activation requires two distinct signals, one delivered via the T-cell receptor, and the second "co-stimulatory" signal through CD28/B7 ligation. Previous studies showed that the blockade of CD28/B7 ligation alters differentiation of Th1/Th2 lymphocyte subsets in vitro and in vivo. The present study was designed to determine the effect of a CD28/B7 antagonist (CTLA4Ig) on Th1/Th2 development in Schistosoma mansoni-sensitized and airway-challenged mice. Treatment of mice with CTLA4Ig beginning 1 wk after sensitization abolished airway responsiveness to intravenous methacholine determined 96 h following antigen challenge. We also found a significant reduction in bronchoalveolar lavage (BAL) eosinophilia, and reduced peribronchial eosinophilic infiltration and mucoid-cell hyperplasia. Furthermore, CTLA4Ig treatment significantly decreased interleukin (IL)-4 and IL-5 content in BAL fluid in vivo, and the production of IL-5 by lung lymphocytes stimulated with soluble egg antigen (SEA) in vitro. In contrast, the content of interferon-gamma in BAL fluid and supernatant from SEA-stimulated lung lymphocytes from CTLA4Ig-treated mice was increased significantly compared with untreated animals. Thus, CTLA4Ig inhibits eosinophilic airway inflammation and airway hyperresponsiveness in S. mansoni-sensitized and airway-challenged mice, most likely due to attenuated secretion of Th2-type cytokines and increased secretion of Th1-type cytokines.

Selective expansion of Vgamma2-Vdelta7 TCR gammadelta cells in C57BL/6 mice is postnatal and extrathymic.

Previous studies have shown that TCR-gammadelta cells expressing Vgamma2 region elements are selectively expanded in vivo in C57BL/6 (B6), but not DBA/2, mice. Genetic analysis demonstrated that the expansion of Vgamma2+ was linked to the TCR alphadelta loci, suggesting that a particular Vgamma-Vdelta pair may be necessary for the expansion. In the studies presented here, we find that the expanding TCR gammadelta cells in B6 mice express a Vgamma2+/Vdelta7+ TCR. The Vgamma2-Jgamma and Vdelta7-Ddelta-Jdelta junctional amino acid sequences of these cells display wide variation in length, suggesting that expansion is based on variable region usage and not junctional diversity. The kinetics and dynamics of Vgamma2+/Vdelta7+ T cell expression were studied to determine the biological basis of clonal expansion. Although expression of the Vgamma2+ cells in B6 and DBA/2 neonates was similar, Vgamma2+ cells in the B6 mice expanded fourfold by 4 wk of age, while the expression in DBA/2 mice remained constant. In addition, expansion of the Vgamma2+ cells occurred in athymic nude mice, suggesting that expansion was driven by extrathymic stimuli. Finally, B6 mice housed under germfree conditions expressed expanded levels of Vgamma2+ gammadelta T cells similar to their normally housed counterparts. Thus, expansion and diversification of Vgamma2+/Vdelta7+ T cells are postnatal extrathymic events that do not require microbial antigenic exposure.

CD28 costimulation promotes the production of Th2 cytokines.

CD28 ligation augments TCR-mediated proliferation, IL-2 production, and T cell survival. However, the role of CD28 costimulation in T cell differentiation remains controversial. To address this issue, CD28+ and CD28-deficient TCR alphabeta transgenic (Tg) mice were used to examine cytokine production by T cells following antigenic stimulation. Increasing CD28 ligation resulted in increased production of IL-4 and IL-5, consistent with differentiation toward a Th2 phenotype, in both CD4+ TCR Tg T cells and CD8+ TCR Tg T cells. The same result was obtained with CD4+ TCR Tg mice bred to RAG2-deficient mice, indicating that the Th2 differentiation observed with increased CD28 ligation was not due to the presence of memory T cells. Although CD28 costimulation is an essential factor regulating IL-2 synthesis, differentiation toward a Th2-like phenotype by CD28 ligation was not an indirect effect of enhanced IL-2 production. In contrast, blockade of IL-4 during the primary cultures of the T cells resulted in a profound inability to produce Th2-type cytokines upon restimulation. The critical role of IL-4 was confirmed by the finding that CD28-deficient TCR alphabeta Tg+ T cells cultured with rIL-4 differentiated into Th2-like T cells. Therefore, CD28 ligation promotes the production of Th2-type cytokines by naive murine T cells via an IL-4-dependent mechanism.

CD28/B7 interactions deliver a unique signal to naive T cells that regulates cell survival but not early proliferation.

CD28/B7 ligation provides costimulatory signals important for the development of T cell responses. In the present study, we examined whether CD28/B7 interactions have a specialized role in the regulation of cell cycle progression and sustained T cell proliferative responses in naive T cell populations using TCR transgenic mice. CD28-mediated signaling was shown to be uniquely capable of regulating cell survival compared with TCR-mediated signaling. Increasing the strength of the TCR-mediated signal 1 increased early proliferative responses, but had no effect on sustained cell survival. In contrast, CD28 ligation, signal 2, was not required for early proliferative responses, but dramatically influenced long term T cell survival. The increased cell survival after CD28 ligation was not due to increased IL-2 production, but was linked to up-regulation of Bcl-xL. The Bcl-xL protein could not be induced following increased TCR cross-linking in the absence of CD28 signaling. In addition, survival of T cells from Bcl-xL transgenic mice was not inhibited by blocking CD28 ligation, suggesting that CD28-induced T cell survival is regulated by Bcl-xL expression. Together, these results suggest that the unique role of CD28 signaling is not to costimulate the initial activation of naive T cells, but is, in fact, to sustain the late proliferative response and enhance long term cell survival.

TCR-gamma delta cells in CD3 zeta-deficient mice contain Fc epsilon RI gamma in the receptor complex but are specifically unresponsive to antigen.

Unlike TCR-alpha beta cells, TCR-gamma delta cells express a distinct member of the zeta family, the gamma-chain of Fc epsilon RI (Fc epsilon RI gamma) within the TCR complex. To study the role of the Fc epsilon RI gamma-chain in TCR-gamma delta cells, a TCR-gamma delta transgenic mouse (G8) has been crossed with CD3 zeta-chain-deficient mice (G8.zeta-/-). Thy-1+ spleen and lymph node cells of these animals expressed low levels of CD3/TCR. These results suggested that the zeta-chain is required for effective TCR transport to the cell surface. In contrast, intraepithelial TCR-gamma delta cells of G8.zeta-/- mice expressed high levels of TCR. Immunoprecipitation with anti-CD3 showed that Fc epsilon RI gamma-chains were associated with the TCR complex in T cells isolated from zeta-deficient mice. Although the Fc epsilon RI gamma-expressing T cells proliferated in response to stimulation by TCR-specific Abs including anti-CD3 epsilon, anti-pan gamma delta, and anti-V gamma 2 mAb, the G8.zeta-/- T cells did not respond to the G8-specific Ag (T10b), anti-Thy-1 mAb, or Con A. The unresponsiveness to the Ag was not due to the reduced TCR expression, because intraepithelial TCR-gamma delta cells from the zeta-deficient mice did not respond to Ag. The inability of the G8.zeta-/- T cells to respond to Ag could not be overcome by providing an anti-CD28 costimulatory signal or by adding exogenous rIL-2. Taken together, our data suggest that the Fc epsilon RI gamma-chain associates with the TCR-gamma delta complex in the absence of the zeta-chain, but it is not able to substitute for the zeta-chain for effective transport of TCR to the cell surface or functional responses to Ag.

CD28 expression is not essential for positive and negative selection of thymocytes or peripheral T cell tolerance.

Thymocyte development requires positive selection of clones that can recognize Ag presented by MHC molecules and negative selection of clones that are self-reactive. However, the costimulatory signals required for negative selection and cell death, or positive selection and the transition to the peripheral lymphoid system, are not well understood. Many molecular interactions that are important for T cell activation have also been found to play a role in thymocyte development. The importance of the CD28/B7 interaction in the activation of mature T cells and recent observations that CD28 may play a role negative selection of developing CD4+CD8+ thymocytes suggest that CD28 may also be involved in development and maintenance of T cell tolerance. CD28-deficient mice were crossed to alpha beta and gamma delta TCR transgenic as well as H-2k Mlsc-bearing animals and were used to address the role of CD28 in positive and negative selection of developing T cells. The CD28-deficient animals demonstrated no obvious deficiency in either positive or negative selection of developing thymocytes. However, when mixed bone marrow chimeras were created with cells derived from both CD28-deficient and wild-type mice, the CD28+ T cells had a selective advantage over the CD28-deficient T cells. Therefore, it appears that CD28, although not essential for the selection of T cells during development, may allow for additional signals that increase the efficiency of selection and/or expansion of peripheral T cell populations.

Two waves of gamma delta T cells expressing different V delta genes are recruited into schistosome-induced liver granulomas.

Isolated granulomas provide a unique model to study T cells in the site of inflammation. Schistosoma mansoni-infected mice develop liver granulomas in response to schistosome egg deposition and the granulomas contain gamma delta T cells that appear to be activated (Pgp-1high and L-selectin low). Analysis of kinetics and TCR gene usage of granuloma gamma delta T cells revealed a limited TCR repertoire restricted to V gamma 1.1, V delta 4, and V delta 6 genes, suggesting that the occurrence of gamma delta T cells in the granuloma is influenced by TCR V gene usage. The V delta 4+, but not the V delta 6+, gamma delta T cells expressed CD69, a marker of recent activation. To determine if there was a preferred order of accumulation of the gamma delta T cells in granulomas, s.c. sponge grafts were implanted into schistosome-infected mice, schistosome eggs were injected into the grafts, and accumulated T cells were sequentially analyzed. The earliest gamma delta T cell immigrants expressed V delta 6 and later immigrants expressed V delta 4 V genes. Additional evidence for a role of TCR specificity in the accumulation of gamma delta T cells in granulomas is their absence from schistosome granulomas in TCR transgenic mice that express only a single MHC-specific gamma delta TCR. Finally, gamma delta T cell recruitment into the granulomas did not require beta 2-microglobulin, since gamma delta T cells were present in liver granulomas of beta 2-microglobulin gene-disrupted mice. The analysis of the influx of gamma delta T cells into schistosome-induced, liver granulomas and schistosome egg-containing sponges provides a model system to investigate the role, if any, of gamma delta T cells in schistosome infections.

CD43 is a murine T cell costimulatory receptor that functions independently of CD28.

Costimulation mediated by the CD28 receptor has been shown to play an important role in the development of a vigorous T cell immune response. Nevertheless, CD28-deficient mice can mount effective T cell-dependent immune responses. These data suggest that other costimulatory molecules may play a role in T cell activation. In a search for other costimulatory receptors on T cells, we have characterized a monoclonal antibody (mAb) that can costimulate T cells in the absence of accessory cells. Similar to CD28 antibodies, this mAb, R2/60, was found to synergize with T cell receptor engagement in inducing proliferation. Independent ligation of CD3 and the ligand recognized by R2/60 results in T cell proliferation, suggesting that the two molecules do not have to colocalize to activate the R2/60 costimulatory pathway. R2/60 does not react with CD28, and furthermore, R2/60 costimulates in a CD28-independent fashion since the mAb costimulates T cells from the CD28-deficient mice as well as wild-type mice. Expression cloning of the R2/60 antigen identified the ligand as murine CD43. Together, these data demonstrate that CD43 can serve as a receptor on T cells that can provide CD28-independent costimulation.