bluuebunny/crossref_metadata_2025 huggingface dataset:
Noncognate Interaction with MHC Class II Molecules Is Essential for Maintenance of T Cell Metabolism to Establish Optimal Memory CD4 T Cell Function
Authors: De Riva Alessandra , Bourgeois Christine , Kassiotis George , Stockinger Brigitta
Published: April 2014

CD4 memory T cells surviving in the absence of MHC class II contact lose their characteristic memory function. To investigate the mechanisms underlying the impaired function of memory T cells in the absence of MHC class II molecules, we analyzed gene expression profiles of resting memory T cells isolated from MHC class II-competent or -deficient hosts. The analysis focused on five transcripts related to T cell activation, metabolism, and survival that are underexpressed in resting memory T cells from MHC class II-deficient hosts compared with MHC class II-competent hosts. CD4 memory cells isolated from MHC class II-deficient hosts display alterations in their degree of differentiation as well as metabolic activity, and these changes are already manifest in the effector phase despite the presence of Ag-expressing dendritic cells. Our data suggest that the absence of interactions with noncognate MHC class II molecules compromises the progressive accumulation of signals that ensure optimal survival and fitness to sustain the metabolic activity of activated T cells and shape the functional capacity of the future memory compartment. Signals via AKT coordinate survival and metabolic pathways and may be one of the crucial events linking interaction with MHC class II molecules to the successful generation of a long-lived functional memory CD4 T cell population.
Characterizing T Cell Movement within Lymph Nodes in the Absence of Antigen
Authors: Beauchemin Catherine , Dixit Narendra M. , Perelson Alan S.
Published: April 2014

The recent application of two-photon microscopy to the visualization of T cell movement has presented trajectories of individual T cells within lymphoid organs both in the presence and in the absence of Ag-loaded dendritic cells. Remarkably, even though T cells largely move along conduits of the fibroblastic reticular cell network, they appear to execute random walks in lymphoid organs rather than chemotaxis. In this study, we analyze experimental trajectories of T cells using computer simulations of idealized random walks. Comparisons of simulations with experimental data provide estimates of key parameters that characterize T cell motion in vivo. For example, we find that the distance moved before turning is about twice the distance between intersections in the fibroblastic reticular cell network, suggesting that at an intersection a T cell will turn onto a new fiber ∼50% of the time. Although the calibrated model appears to offer an accurate representation of T cell movement, it has also uncovered inconsistencies across different experimental data sets.
CD56 Marks an Effector T Cell Subset in the Human Intestine
Authors: Cohavy Offer , Targan Stephan R.
Published: April 2014

T cells are key mediators of intestinal immunity, and specific T cell subsets can have differing immunoregulatory roles in animal models of mucosal inflammation. In this study, we describe human CD56+ T cells as a morphologically distinct population expressing a mature, nonproliferative phenotype that is frequent in the gut. Enhanced potential for IFN-γ and TNF synthesis suggested a proinflammatory function, and we directly demonstrate effector function mediated by direct T-T interaction with responder cells in vitro. CD56+ T cells from peripheral blood responded to the gut-related CD2 signal, and were necessary for effective CD2-mediated proliferation of peripheral blood CD56− T cells. Our findings associate CD56+ T cells with the intestinal immune compartment and suggest a putative effector function in human mucosal immunity.
Characterization of Human Lung Tumor-Associated Fibroblasts and Their Ability to Modulate the Activation of Tumor-Associated T Cells
Authors: Nazareth Michael R. , Broderick Lori , Simpson-Abelson Michelle R. , Kelleher Raymond J. , Yokota Sandra J. , Bankert Richard B.
Published: April 2014

The tumor microenvironment of human non-small cell lung cancer (NSCLC) is composed largely of stromal cells, including fibroblasts, yet these cells have been the focus of few studies. In this study, we established stromal cell cultures from primary NSCLC through isolation of adherent cells. Characterization of these cells by flow cytometry demonstrated a population which expressed a human fibroblast-specific 112-kDa surface molecule, Thy1, α-smooth muscle actin, and fibroblast activation protein, but failed to express CD45 and CD11b, a phenotype consistent with that of an activated myofibroblast. A subset of the tumor-associated fibroblasts (TAF) was found to express B7H1 (PD-L1) and B7DC (PD-L2) constitutively, and this expression was up-regulated by IFN-γ. Production of cytokines and chemokines, including IFN-γ, monokine induced by IFN-γ, IFN-γ-inducible protein-10, RANTES, and TGF-β1 was also demonstrated in these cells. Together, these characteristics provide multiple opportunities for the TAF to influence cellular interactions within the tumor microenvironment. To evaluate the ability of TAF to modulate tumor-associated T cell (TAT) activation, we conducted coculture experiments between autologous TAF and TAT. In five of eight tumors, TAF elicited a contact-dependent enhancement of TAT activation, even in the presence of a TGF-β1-mediated suppressive effect. In the three other tumors, TAF had a net suppressive effect upon TAT activation, and, in one of these cases, blockade of B7H1 or B7DC was able to completely abrogate the TAF-mediated suppression. We conclude that TAF in human NSCLC are functionally and phenotypically heterogeneous and provide multiple complex regulatory signals that have the potential to enhance or suppress TAT function in the tumor microenvironment.
Paradoxical Effect of Reduced Costimulation in T Cell-Mediated Colitis
Authors: Kim Gisen , Levin Matthew , Schoenberger Stephen P. , Sharpe Arlene , Kronenberg Mitchell
Published: April 2014

B7-1 and B7-2 play different roles in the pathogenesis of autoimmunity, but this is controversial. We analyzed colitis induced by transfer of CD45RBhighCD4+ T cells to RAG−/− recipients lacking B7-1 and/or B7-2. Surprisingly, disease was greatly accelerated in RAG−/− recipients deficient for either B7-1 or B7-2, especially in the B7-2−/− recipients. This accelerated colitis induction correlated with increased T cell division in vivo and production of Th1 cytokines. Although colitis pathogenesis following T cell transfer was inhibited in the absence of CD40L expression, CD40-CD40L interactions were not required in the B7-2−/− RAG−/− recipients. In vitro priming by APCs lacking either B7-1 or B7-2 caused decreased IL-2 production, which led to decreased CTLA-4 expression, although T cells primed in this way could respond vigorously upon restimulation by producing increased IL-2 and proinflammatory cytokines. Consistent with this mechanism, we demonstrate that blocking IL-2 early after T cell transfer accelerated colitis. Our data therefore outline a mechanism whereby synergistic costimulation by B7-1 and B7-2 molecules during priming is required for optimal IL-2 production. The consequent inhibitory effect of full CTLA-4 expression, induced by IL-2, may slow colitis, even in the absence of regulatory T cells.
Skin-Infiltrating CD8+ T Cells Initiate Atopic Dermatitis Lesions
Authors: Hennino Ana , Vocanson Marc , Toussaint Yann , Rodet Karen , Benetière Josette , Schmitt Anne-Marie , Aries Marie-Françoise , Bérard Frédéric , Rozières Aurore , Nicolas Jean-François
Published: April 2014

Skin lesions in the allergic form of atopic dermatitis (AD) are induced by allergen-specific T cells that infiltrate the skin at the site of allergen exposure. Although Th2-type CD4+ T cells appear to be crucial in AD pathophysiology, little is known about the contribution of CD8+ T cells in the development of the allergic skin inflammation. In the present study, we have analyzed the respective role of CD8+ and CD4+ T cells in the development of AD skin lesions in a mouse model of allergen-induced AD. In sensitized mice, CD8+ T cells are rapidly and transiently recruited to the allergen-exposed site and initiate the inflammatory process leading to skin infiltration with eosinophils and Th1/Th2-producing cells. CD8+ T cell-depleted mice show no inflammation, demonstrating that these cells are mandatory for the development of AD. In contrast, CD4+ T cell-depleted mice develop a severe form of eczema. Furthermore, adoptive transfer of CD8+ T cells from sensitized mice into naive recipient mice leads to skin inflammation soon after allergen exposure. These data indicate that allergen-primed CD8+ T cells are required for the development of AD-like lesions in mice.
Autoimmune Uveitis Elicited with Antigen-Pulsed Dendritic Cells Has a Distinct Clinical Signature and Is Driven by Unique Effector Mechanisms: Initial Encounter with Autoantigen Defines Disease Phenotype
Authors: Tang Jun , Zhu Wei , Silver Phyllis B. , Su Shao-Bo , Chan Chi-Chao , Caspi Rachel R.
Published: April 2014

Human autoimmune uveitis is a heterogeneous group of potentially blinding ocular diseases in which most patients who exhibit immunity recognize the same retinal Ag. It is represented by the model of experimental autoimmune uveitis (EAU) induced in mice by immunization with retinal Ag in CFA. Murine EAU is characterized by a Th1/Th17 response pattern, which may not represent all types of human uveitis. We report in this study a new model of EAU induced by injection of matured dendritic cells loaded with a uveitogenic retinal peptide. Dendritic cell-induced EAU demonstrated unique characteristics compared with traditional EAU in terms of clinical manifestations, the nature of the inflammatory infiltrating cells, the cytokine response profile, and a strict requirement for IFN-γ, whereas IL-17 appeared to play a minor role. Disease was self-limiting, but could be reinduced with the same Ag in CFA, albeit with reduced severity, suggesting postrecovery resistance. Our study demonstrates in a disease setting that the context in which the same autoantigen is initially presented to the immune system precipitates distinct forms of pathology via a distinct pathogenic pathway on the same genetic background. These findings may shed new light on the complex biology and the heterogeneous nature of human uveitis, and provide an alternative model for uveitic diseases of immune origin.
Lymphotoxin-Independent Expression of TNF-Related Activation-Induced Cytokine by Stromal Cells in Cryptopatches, Isolated Lymphoid Follicles, and Peyer’s Patches
Authors: Taylor Rebekah T. , Patel Seema R. , Lin Eugene , Butler Betsy R. , Lake Jason G. , Newberry Rodney D. , Williams Ifor R.
Published: April 2014

Stromal cells play a crucial role in the organogenesis of lymphoid tissues. We previously identified VCAM-1+ stromal cells in cryptopatches (CP) and isolated lymphoid follicles (ILF) in the small intestine of C57BL/6 mice. Nonhemopoietic stromal cell networks in CP and ILF of adult mice also expressed FDC-M1, CD157 (BP-3), and TNF-related activation-induced cytokine (TRANCE). Individual stromal cells were heterogeneous in their expression of these markers, with not all stromal cells expressing the entire set of stromal cell markers. Expression of VCAM-1, FDC-M1, and CD157 on CP stromal cells was absent in alymphoplasia mice deficient in NF-κB-inducing kinase (NIK) and NIK knockout mice. Administration of lymphotoxin β receptor (LTβR)-Ig to wild-type mice on day 13 resulted in the absence of CP on day 20; delaying administration of LTβR-Ig until day 18 resulted in an 80% decrease in the number of CP on day 22 and diminished expression of VCAM-1, FDC-M1, and CD157 on the remaining CP. In sharp contrast, TRANCE expression by stromal cells was completely independent of NIK and LTβR. In addition, expression of TRANCE in ILF was concentrated just beneath the follicle-associated epithelium, a pattern of polarization that was also observed in Peyer’s patches. These findings suggest that TRANCE on stromal cells contributes to the differentiation and maintenance of organized lymphoid aggregates in the small intestine.
A Teleost Polymeric Ig Receptor Exhibiting Two Ig-Like Domains Transports Tetrameric IgM into the Skin
Authors: Hamuro Koji , Suetake Hiroaki , Saha Nil Ratan , Kikuchi Kiyoshi , Suzuki Yuzuru
Published: April 2014

The skin mucus IgM is an important molecule in the mucosal immune system of teleost skin. However, the transport mechanism associated with this molecule has yet to be clarified. In this study, we isolated a gene encoding a polymeric Ig receptor (pIgR) from a species of teleost fish, Takifugu rubripes (fugu). This gene is known to be an Ig transporter in the intestine of mammals. Our studies further demonstrated that fugu pIgR was expressed in the skin and that a fragment of pIgR bound to tetrameric IgM in the skin mucus. These results indicate that the skin pIgR transports tetrameric IgM into the skin mucus. The fugu pIgR exhibits a unique structure containing only two Ig-like domains corresponding to domain 1 and domain 4/5 of mammalian pIgR. This structure was sufficient for successful binding to tetrameric IgM. Teleost skin thus adopts the same Ig transport system as mammalian intestine via a unique pIgR.
TNF Trafficking to Human Mast Cell Granules: Mature Chain-Dependent Endocytosis
Authors: Olszewski Maciej B. , Groot Arjan J. , Dastych Jaroslaw , Knol Edward F.
Published: April 2014

Mast cells play a crucial role at the early stages of immune response against bacteria and parasites where their functionality is based on their capability of releasing highly bioactive compounds, among them TNF. Mast cells are considered the only cells storing preformed TNF, which allows for the immediate release of this cytokine upon contact with pathogens. We approached the question of mechanisms and amino acid motifs directing newly synthesized TNF for storage in cytoplasmic granules by analyzing the trafficking of a series of TNF-enhanced GFP fusion proteins in human mast cell lines HMC-1 and LAD2. Protein covering the full TNF sequence was successfully sorted into secretory granules in a process involving transient exposure on the outer membrane and re-endocytosis. In human cells, contrary to results previously obtained in a rodent model, TNF seems not to be glycosylated and, thus, trafficking is carbohydrate independent. In an effort to localize the amino acid motif responsible for granule targeting, we constructed additional fusion proteins and analyzed their trafficking, concluding that granule-targeting sequences are localized in the mature chain of TNF and that the cytoplasmic tail is expendable for endocytotic sorting of this cytokine, thus excluding direct interactions with intracellular adaptor proteins.
Identification of the Site of Human Mannan-Binding Lectin Involved in the Interaction with Its Partner Serine Proteases: The Essential Role of Lys55
Authors: Teillet Florence , Lacroix Monique , Thiel Steffen , Weilguny Dietmar , Agger Teit , Arlaud Gérard J. , Thielens Nicole M.
Published: April 2014

Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys55Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys55Gln and Lys55Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys55Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg47Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg47Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg47Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro53Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu49 and Leu56 were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys55, which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical.
E2A and HEB Are Required to Block Thymocyte Proliferation Prior to Pre-TCR Expression
Authors: Wojciechowski Jason , Lai Anne , Kondo Motonari , Zhuang Yuan
Published: April 2014

Thymocytes undergoing TCRβ gene rearrangements are maintained in a low or nonproliferating state during early T cell development. This block in cell cycle progression is not released until the expression of a functional pre-TCR, which is composed of a successfully rearranged TCRβ-chain and the Pre-Tα-chain. The regulatory molecules responsible for the coordination of these differentiation and proliferation events are currently unknown. E2A and HEB are structurally and functionally related basic helix-loop-helix transcription factors involved in T cell development. To reveal the function of E2A and HEB through the stage of pre-TCR expression and alleviate functional compensation between E2A and HEB, we use a double-conditional knockout model. The simultaneous deletion of E2A and HEB in developing thymocytes leads to a severe developmental block before pre-TCR expression and a dramatic reduction of Pre-Tα expression. These developmentally arrested thymocytes exhibit increased proliferation in vivo and dramatic expansion ex vivo in response to IL-7 signaling. These results suggest that E2A and HEB are not only critical for T cell differentiation but also necessary to retain developing thymocytes in cell cycle arrest before pre-TCR expression.
Human TCR-Binding Affinity is Governed by MHC Class Restriction
Authors: Cole David K. , Pumphrey Nicholas J. , Boulter Jonathan M. , Sami Malkit , Bell John I. , Gostick Emma , Price David A. , Gao George F. , Sewell Andrew K. , Jakobsen Bent K.
Published: April 2014

T cell recognition is initiated by the binding of TCRs to peptide-MHCs (pMHCs), the interaction being characterized by weak affinity and fast kinetics. Previously, only 16 natural TCR/pMHC interactions have been measured by surface plasmon resonance (SPR). Of these, 5 are murine class I, 5 are murine class II, and 6 are human class I-restricted responses. Therefore, a significant gap exists in our understanding of human TCR/pMHC binding due to the limited SPR data currently available for human class I responses and the absence of SPR data for human class II-restricted responses. We have produced a panel of soluble TCR molecules originating from human T cells that respond to naturally occurring disease epitopes and their cognate pMHCs. In this study, we compare the binding affinity and kinetics of eight class-I-specific TCRs (TCR-Is) to pMHC-I with six class-II-specific TCRs (TCR-IIs) to pMHC-II using SPR. Overall, there is a substantial difference in the TCR-binding equilibrium constants for pMHC-I and pMHC-II, which arises from significantly faster on-rates for TCRs binding to pMHC-I. In contrast, the off-rates for all human TCR/pMHC interactions fall within a narrow window regardless of class restriction, thereby providing experimental support for the notion that binding half-life is the principal kinetic feature controlling T cell activation.
Protein Kinase D Interaction with TLR5 Is Required for Inflammatory Signaling in Response to Bacterial Flagellin
Authors: Ivison Sabine M. , Graham Nicholas R. , Bernales Cecily Q. , Kifayet Arnawaz , Ng Natalie , Shobab Leila A. , Steiner Theodore S.
Published: April 2014

Protein kinase D (PKD), also called protein kinase C (PKC)μ, is a serine-threonine kinase that is involved in diverse areas of cellular function such as lymphocyte signaling, oxidative stress, and protein secretion. After identifying a putative PKD phosphorylation site in the Toll/IL-1R domain of TLR5, we explored the role of this kinase in the interaction between human TLR5 and enteroaggregative Escherichia coli flagellin in human epithelial cell lines. We report several lines of evidence that implicate PKD in TLR5 signaling. First, PKD phosphorylated the TLR5-derived target peptide in vitro, and phosphorylation of the putative target serine 805 in HEK 293T cell-derived TLR5 was identified by mass spectrometry. Furthermore, mutation of serine 805 to alanine abrogated responses of transfected HEK 293T cells to flagellin. Second, TLR5 interacted with PKD in coimmunoprecipitation experiments, and this association was rapidly enhanced by flagellin treatment. Third, pharmacologic inhibition of PKC or PKD with Gö6976 resulted in reduced expression and secretion of IL-8 and prevented the flagellin-induced activation of p38 MAPK, but treatment with the PKC inhibitor Gö6983 had no significant effects on these phenotypes. Finally, involvement of PKD in the p38-mediated IL-8 response to flagellin was confirmed by small hairpin RNA-mediated gene silencing. Together, these results suggest that phosphorylation of TLR5 by PKD may be one of the proximal elements in the cellular response to flagellin, and that this event contributes to p38 MAPK activation and production of inflammatory cytokines in epithelial cells.
Protection from Direct Cerebral Cryptococcus Infection by Interferon-γ-Dependent Activation of Microglial Cells
Authors: Zhou Qing , Gault Ruth A. , Kozel Thomas R. , Murphy William J.
Published: April 2014

The brain represents a significant barrier for protective immune responses in both infectious disease and cancer. We have recently demonstrated that immunotherapy with anti-CD40 and IL-2 can protect mice against disseminated Cryptococcus infection. We now applied this immunotherapy using a direct cerebral cryptococcosis model to study direct effects in the brain. Administration of anti-CD40 and IL-2 significantly prolonged the survival time of mice infected intracerebrally with Cryptococcus neoformans. The protection was correlated with activation of microglial cells indicated by the up-regulation of MHC II expression on brain CD45lowCD11b+ cells. CD4+ T cells were not required for either the microglial cell activation or anticryptococcal efficacy induced by this immunotherapy. Experiments with IFN-γ knockout mice and IFN-γR knockout mice demonstrated that IFN-γ was critical for both microglial cell activation and the anticryptococcal efficacy induced by anti-CD40/IL-2. Interestingly, while peripheral IFN-γ production and microglial cell activation were observed early after treatment, negligible IFN-γ was detected locally in the brain. These studies indicate that immunotherapy using anti-CD40 and IL-2 can augment host immunity directly in the brain against C. neoformans infection and that IFN-γ is essential for this effect.
T Cell Activation and Proliferation Characteristic for HIV-Nef Transgenic Mice Is Lymphopenia Induced
Authors: Koenen Paul G. F. , Hofhuis Frans M. , Oosterwegel Mariëtte A. , Tesselaar Kiki
Published: April 2014

The HIV-Nef protein has been implicated in generating high viral loads and T cell activation. Transgenic (tg) mice with constitutive T cell-specific Nef expression show a dramatic reduction in T cell number and highly increased T cell turnover. Previous studies in Nef tg mice attributed this T cell activation to a direct effect of Nef at the cellular level. Given the strongly reduced peripheral T cell numbers, we examined whether this enhanced T cell division might instead be lymphopenia induced. Adoptively transferred naive wild-type T cells into lymphopenic Nef tg mice showed high T cell turnover and obtained the same effector/memory phenotype as the autologous Nef tg T cells, supporting the idea that the microenvironment determines the phenotype of the T cells present. Moreover, in bone marrow chimeras from mixtures of wild-type and Nef tg bone marrow, with a full T cell compartment containing a small proportion of Nef tg T cells, Nef tg T cells kept a naive phenotype. These results demonstrate that T cell activation in the Nef tg mice is lymphopenia induced rather than due to a direct T cell-activating effect of Nef.
NK Cells Stimulate Recruitment of CXCR3+ T Cells to the Brain during Plasmodium berghei-Mediated Cerebral Malaria
Authors: Hansen Diana S. , Bernard Nicholas J. , Nie Catherine Q. , Schofield Louis
Published: April 2014

NK cells are cytotoxic lymphocytes that also secrete regulatory cytokines and can therefore influence adaptive immune responses. NK cell function is largely controlled by genes present in a genomic region named the NK complex. It has been shown that the NK complex is a genetic determinant of murine cerebral malaria pathogenesis mediated by Plasmodium berghei ANKA. In this study, we show that NK cells are required for cerebral malaria disease induction and the control of parasitemia. NK cells were found infiltrating brains of cerebral malaria-affected mice. NK cell depletion resulted in inhibition of T cell recruitment to the brain of P. berghei-infected animals. NK cell-depleted mice displayed down-regulation of CXCR3 expression and a significant reduction of T cells migrating in response to IFN-γ-inducible protein 10, indicating that this chemokine pathway plays an essential role in leukocyte trafficking leading to cerebral disease and fatalities.
Antigen Presentation by Nonhemopoietic Cells Amplifies Clonal Expansion of Effector CD8 T Cells in a Pathogen-Specific Manner
Authors: Thomas Sunil , Kolumam Ganesh A. , Murali-Krishna Kaja
Published: April 2014

Professional APCs of hemopoietic-origin prime pathogen-specific naive CD8 T cells. The primed CD8 T cells can encounter Ag on infected nonhemopoietic cell types. Whether these nonhemopoietic interactions perpetuate effector T cell expansion remains unknown. We addressed this question in vivo, using four viral and bacterial pathogens, by comparing expansion of effector CD8 T cells in bone marrow chimeric mice expressing restricting MHC on all cell types vs mice that specifically lack restricting MHC on nonhemopoietic cell types or radiation-sensitive hemopoietic cell types. Absence of Ag presentation by nonhemopoietic cell types allowed priming of naive CD8 T cells in all four infection models tested, but diminished their sustained expansion by ∼10-fold during lymphocytic choriomeningitis virus and by ≤2-fold during vaccinia virus, vesicular stomatitis virus, or Listeria monocytogenes infections. Absence of Ag presentation by a majority (>99%) of hemopoietic cells surprisingly also allowed initial priming of naive CD8 T cells in all the four infection models, albeit with delayed kinetics, but the sustained expansion of these primed CD8 T cells was markedly evident only during lymphocytic choriomeningitis virus, but not during vaccinia virus, vesicular stomatitis virus, or L. monocytogenes. Thus, infected nonhemopoietic cells can amplify effector CD8 T cell expansion during infection, but the extent to which they can amplify is determined by the pathogen. Further understanding of mechanisms by which pathogens differentially affect the ability of nonhemopoietic cell types to contribute to T cell expansion, how these processes alter during acute vs chronic phase of infections, and how these processes influence the quality and quantity of memory cells will have implications for rational vaccine design.
Enrichment and Persistence of Virus-Specific CTL in the Brain of Simian Immunodeficiency Virus-Infected Monkeys Is Associated with a Unique Cytokine Environment
Authors: Marcondes Maria Cecilia G. , Burdo Tricia H. , Sopper Sieghart , Huitron-Resendiz Salvador , Lanigan Caroline , Watry Debbie , Flynn Claudia , Zandonatti Michelle , Fox Howard S.
Published: April 2014

The host reaction to infection of the brain contributes to a number of CNS pathologies including neuro-AIDS. In this study, we have identified the accumulation of SIV-specific CTL in the brains of SIV-infected animals who have neurophysiological abnormalities but are otherwise asymptomatic. SIV-specific CTL enter the brain early after viral infection and are maintained in the brain even when those reactive with an immunodominant epitope in Tat are lost from the rest of the body. The specialized CNS environment contributes to this unique outcome. Following SIV infection, brain levels of IL-15 were significantly elevated whereas IL-2 was absent, creating an environment that favors CTL persistence. Furthermore, in response to IL-15, brain-derived CD8+ T cells could expand in greater numbers than those from spleen. The accumulation, persistence, and maintenance of CTL in the brain are closely linked to the increased levels of IL-15 in the absence of IL-2 in the CNS following SIV infection.
Vesicular Stomatitis Virus Glycoprotein Displaying Retrovirus-Like Particles Induce a Type I IFN Receptor-Dependent Switch to Neutralizing IgG Antibodies
Authors: Bach Patricia , Kamphuis Elisabeth , Odermatt Bernhard , Sutter Gerd , Buchholz Christian J. , Kalinke Ulrich
Published: April 2014

Vesicular stomatitis virus (VSV) infection rapidly induces IFN-αβ that confers initial survival, whereas long-term protection is mediated by neutralizing IgG responses. Because coadministration of IFN-αβ can enhance Ab responses against soluble Ags, we addressed whether virus-induced IFN-αβ also had an impact on the induction of neutralizing Ab responses. To this end, we generated apathogenic retrovirus-like particles (VLP) displaying the VSV gp (VLP-VSV). Reminiscent of live VSV, VLP-VSV induced VSV-neutralizing IgM responses that switched to IgG in a T help-dependent manner. In type I IFN receptor-deficient (IFNAR−/−) mice, VLP-VSV injection elicited neutralizing IgM, whereas the IgG switch was absent. The lack of subclass switch was associated with a reduced germinal center reaction. Conditional knockout mice with a lymphocyte-specific IFNAR ablation showed normal Ab responses against VLP-VSV, as well as against live VSV. Thus, IFNAR triggering critically promoted the T help-dependent subclass switch of virus-neutralizing Ab responses against VLP-VSV. Interestingly, in the context of VLP-VSV as well as VSV immunization, IFNAR triggering of B lymphocytes did not play a critical role.
Neutrophil Elastase Up-Regulates Cathepsin B and Matrix Metalloprotease-2 Expression
Authors: Geraghty Patrick , Rogan Mark P. , Greene Catherine M. , Boxio Rachel M. M. , Poiriert Tiphaine , O’Mahony Michael , Belaaouaj Abderazzaq , O’Neill Shane J. , Taggart Clifford C. , McElvaney Noel G.
Published: April 2014

Neutrophil elastase (NE) activity is increased in many diseases. Other families of proteases, including cathepsins and matrix metalloproteases (MMPs), are also present at elevated levels in similar disease conditions. We postulated that NE could induce expression of cathepsins and MMPs in human macrophages. NE exposure resulted in macrophages, producing significantly greater amounts of cathepsin B and latent and active MMP-2. Cathepsin B and MMP-2 activities were decreased in Pseudomonas-infected NE knockout mice compared with wild-type littermates. We also demonstrate that NE can activate NF-κB in macrophages, and inhibition of NF-κB resulted in a reduction of NE-induced cathepsin B and MMP-2. Also, inhibition of TLR-4 or transfection of macrophages with dominant-negative IL-1R-associated kinase-1 resulted in a reduction of NE-induced cathepsin B and MMP-2. This study describes for the first time a novel hierarchy among proteases whereby a serine protease up-regulates expression of MMPs and cathepsins. This has important implications for therapeutic intervention in protease-mediated diseases.
Neutrophil Transmigration under Shear Flow Conditions In Vitro Is Junctional Adhesion Molecule-C Independent
Authors: Sircar Monica , Bradfield Paul F. , Aurrand-Lions Michel , Fish Richard J. , Alcaide Pilar , Yang Lin , Newton Gail , Lamont Deanna , Sehrawat Seema , Mayadas Tanya , Liang Tony W. , Parkos Charles A. , Imhof Beat A. , Luscinskas Francis W.
Published: April 2014

Endothelial cell junctional adhesion molecule (JAM)-C has been proposed to regulate neutrophil migration. In the current study, we used function-blocking mAbs against human JAM-C to determine its role in human leukocyte adhesion and transendothelial cell migration under flow conditions. JAM-C surface expression in HUVEC was uniformly low, and treatment with inflammatory cytokines TNF-α, IL-1β, or LPS did not increase its surface expression as assessed by FACS analysis. By immunofluorescence microscopy, JAM-C staining showed sparse localization to cell-cell junctions on resting or cytokine-activated HUVEC. Surprisingly, staining of detergent-permeabilized HUVEC revealed a large intracellular pool of JAM-C that showed little colocalization with von Willebrand factor. Adhesion studies in an in vitro flow model showed that functional blocking JAM-C mAb alone had no inhibitory effect on polymorphonuclear leukocyte (PMN) adhesion or transmigration, whereas mAb to ICAM-1 significantly reduced transmigration. Interestingly, JAM-C-blocking mAbs synergized with a combination of PECAM-1, ICAM-1, and CD99-blocking mAbs to inhibit PMN transmigration. Overexpression of JAM-C by infection with a lentivirus JAM-C GFP fusion protein did not increase adhesion or extent of transmigration of PMN or evoke a role for JAM-C in transendothelial migration. These data suggest that JAM-C has a minimal role, if any, in PMN transmigration in this model and that ICAM-1 is the preferred endothelial-expressed ligand for PMN β2 integrins during transendothelial migration.
Dual Effect of AMD3100, a CXCR4 Antagonist, on Bleomycin-Induced Lung Inflammation
Authors: Watanabe Masaki , Matsuyama Wataru , Shirahama Yuko , Mitsuyama Hideo , Oonakahara Ken-ichi , Noma Satoshi , Higashimoto Ikkou , Osame Mitsuhiro , Arimura Kimiyoshi
Published: April 2014

The chemokine receptor CXCR4, which binds the chemokine stromal cell-derived factor 1, has been reported to be involved in the chemotaxis of inflammatory cells. In addition, AMD3100, an antagonist of CXCR4, has been reported to be an attractive drug candidate for therapeutic intervention in several disorders in which CXCR4 is critically involved. However, little is known about the therapeutic value of AMD3100 in the treatment of pulmonary fibrosis. In this study, we examined the effects of AMD3100 on a murine bleomycin-induced pulmonary fibrosis model. Concurrent administration of AMD3100 and bleomycin apparently attenuated bleomycin-induced pulmonary inflammation. In this process, an inhibition of neutrophil recruitment at early stage followed by the decrease of other inflammatory cell recruitment in the lung were observed. In addition, it also inhibited the expression of cytokines, including MCP-1, MIP-2, MIP-1α, and TGF-β. In contrast, when AMD3100 was administered following bleomycin treatment, the bleomycin-induced lung inflammation progressed and resulted in severe pulmonary fibrosis. In this process, an increase of inflammatory cell recruitment, an up-regulation of lung MCP-1 and TGF-β, and a remarkable activation of p44/42 MAPK in neutrophils were observed. U0126, an inhibitor of p44/42 MAPK, significantly abolished these effects. Thus, AMD3100 has dual effect on bleomycin-induced pulmonary fibrosis. Difference of inflammatory cell recruitment and activation might be associated with the dual effect of AMD3100 on bleomycin-induced pulmonary fibrosis.
The Phosphatidylinositol 3-Kinase Signaling Pathway Exerts Protective Effects during Sepsis by Controlling C5a-Mediated Activation of Innate Immune Functions
Authors: Wrann Christiane D. , Tabriz Navid A. , Barkhausen Tanja , Klos Andreas , van Griensven Martijn , Pape Hans C. , Kendoff Daniel O. , Guo Renfeng , Ward Peter A. , Krettek Christian , Riedemann Niels C.
Published: April 2014

The PI3K/Akt signaling pathway has been recently suggested to have controversial functions in models of acute and chronic inflammation. Our group and others have reported previously that the complement split product C5a alters neutrophil innate immunity and cell signaling during the onset of sepsis and is involved in PI3K activation. We report in this study that in vivo inhibition of the PI3K pathway resulted in increased mortality in septic mice accompanied by strongly elevated serum levels of TNF-α, IL-6, MCP-1, and IL-10 during sepsis as well as decreased oxidative burst activity in blood phagocytes. PI3K inhibition in vitro resulted in significant increases in TLR-4-mediated generation of various proinflammatory cytokines in neutrophils, whereas the opposite effect was observed in PBMC. Oxidative burst and phagocytosis activity was significantly attenuated in both neutrophils and monocytes when PI3K activation was blocked. In addition, PI3K inhibition resulted in strongly elevated TLR-4-mediated generation of IL-1β and IL-8 in neutrophils when these cells were costimulated with C5a. C5a-induced priming effects on neutrophil and monocyte oxidative burst activity as well as C5a-induced phagocytosis in neutrophils were strongly reduced when PI3K activation was blocked. Our data suggest that the PI3K/Akt signaling pathway controls various C5a-mediated effects on neutrophil and monocyte innate immunity and exerts an overall protective effect during experimental sepsis.
Systemic Inflammatory Priming in Normal Pregnancy and Preeclampsia: The Role of Circulating Syncytiotrophoblast Microparticles
Authors: Germain Sarah J. , Sacks Gavin P. , Soorana Suren R. , Sargent Ian L. , Redman Christopher W.
Published: April 2014

Systemic inflammatory responsiveness was studied in normal human pregnancy and its specific inflammatory disorder, pre-eclampsia. Compared with nonpregnancy, monocytes were primed to produce more TNF-α throughout normal pregnancy, more IL-12p70 in the first and second trimesters, and more IL-18 in the first trimester only. Intracellular cytokine measurements (TNF-α and IL12p70) showed little change by comparison. IFN-γ production was suppressed in all three trimesters. In pre-eclampsia, IL-18 secretion was increased. Secreted but not intracellular measures of TNF-α and IL-12p70 were also further enhanced compared with normal pregnancy. Inhibition of IFN-γ production was lost and involved both CD56+ NK and CD56− lymphocyte subsets. We determined whether circulating syncytiotrophoblast microparticles (STBM) could contribute to these inflammatory changes. Unbound STBM could be detected in normal pregnancy by the second trimester and increased significantly in the third. They were also bound in vivo to circulating monocytes. Women with pre-eclampsia had significantly more circulating free but not cell-bound STBMs. STBMs prepared by perfusion of normal placental lobules stimulated production of inflammatory cytokines (TNF-α, IL12p70, and IL-18 but not IFN-γ) when cultured with PBMCs from healthy nonpregnant women. Inflammatory priming of PBMCs during pregnancy is confirmed and is established by the first trimester. It is associated with early inhibition of IFN-γ production. The inflammatory response is enhanced in pre-eclampsia with loss of the IFN-γ suppression. Circulating STBMs bind to monocytes and stimulate the production of inflammatory cytokines. It is concluded that they are potential contributors to altered systemic inflammatory responsiveness in pregnancy and pre-eclampsia.
IL-22 Induces Lipopolysaccharide-Binding Protein in Hepatocytes: A Potential Systemic Role of IL-22 in Crohn’s Disease
Authors: Wolk Kerstin , Witte Ellen , Hoffmann Ute , Doecke Wolf-Dietrich , Endesfelder Stefanie , Asadullah Khusru , Sterry Wolfram , Volk Hans-Dieter , Wittig Bianca Maria , Sabat Robert
Published: April 2014

Crohn′s disease (CD) is a common, chronic, inflammatory bowel disease characterized by intestinal infiltration of activated immune cells and distortion of the intestinal architecture. In this study, we demonstrate that IL-22, a cytokine that is mainly produced by activated Th1 and Th17 cells, was present in high quantities in the blood of CD patients in contrast to IFN-γ and IL-17. In a mouse colitis model, IL-22 mRNA expression was elevated predominantly in the inflamed intestine but also in the mesenteric lymph nodes. IL-22BP, the soluble receptor for IL-22, demonstrated an affinity to IL-22 that was at least 4-fold higher than its membrane-bound receptor, and its strong constitutive expression in the intestine and lymph nodes was decreased in the inflamed intestine. To investigate the possible role of systemic IL-22 in CD, we then administered IL-22 to healthy mice and found an up-regulation of LPS-binding protein (LBP) blood levels reaching concentrations known to neutralize LPS. This systemic up-regulation was associated with increased hepatic but not renal or pulmonary LBP mRNA levels. IL-22 also enhanced the secretion of LBP in human primary hepatocytes and HepG2 hepatoma cells in vitro. This increase was mainly transcriptionally regulated and synergistic with that of other LBP inducers. Finally, elevated LBP levels were detected in CD patients and the mouse colitis model. These data suggest that systemic IL-22 may contribute to the prevention of systemic inflammation provoked by LPS present in the blood of CD patients through its induction of hepatic LBP.
Dissecting the Anti-Desmoglein Autoreactive B Cell Repertoire in Pemphigus Vulgaris Patients
Authors: Qian Ye , Diaz Luis A. , Ye Jian , Clarke Stephen H.
Published: April 2014

Pemphigus vulgaris (PV) encompasses two clinical phenotypes, one producing mucosal blisters and the other mucosal and skin lesions (mcPV). The mucosal blister-producing PV variant is characterized by autoantibodies against desmoglein (Dsg)3, whereas mucosal and skin lesion-producing PV is characterized by autoantibodies to Dsg3 and Dsg1. The present study was aimed at disclosing the diversity and clonality of the anti-Dsg3 response, as well as whether anti-Dsg3 B cells are Ag selected. Human-mouse heterohybridomas were generated by fusion of EBV-transformed or freshly isolated PBLs from six PV patients with mouse myeloma cells. A total of 73 anti-Dsg hybridomas (47 IgM and 26 IgG) were isolated. Over 90% are specific for both Dsg1 and Dsg3 indicating extensive cross-reactivity between these responses. VH gene segment use by IgM hybridomas is diverse, but is restricted among IgG hybridomas, where the majority uses one of two VH genes. VL gene segment use was diverse even among IgG hybridomas suggesting that the VL is less critical to defining desmoglein specificity. Additionally, the IgG hybridomas were extensively mutated and the distribution and nature of the mutations suggested that they had been Ag selected. We conclude that the potentially pathogenic IgG anti-Dsg response is restricted in VH use, is somatically mutated, and is Ag selected.
Down-Regulation of Human Complement Factor H Sensitizes Non-Small Cell Lung Cancer Cells to Complement Attack and Reduces In Vivo Tumor Growth
Authors: Ajona Daniel , Hsu Yi-Fan , Corrales Leticia , Montuenga Luis M. , Pio Ruben
Published: April 2014

Malignant cells are often resistant to complement activation through the enhanced expression of complement inhibitors. In this work, we examined the protective role of factor H, CD46, CD55, and CD59 in two non-small cell lung cancer cell lines, H1264 and A549, upon activation of the classical pathway of complement. Complement was activated with polyclonal Abs raised against each cell line. After blocking factor H activity with a neutralizing Ab, C3 deposition and C5a release were more efficient. Besides, a combined inhibition of factor H and CD59 significantly increased complement-mediated lysis. CD46 and CD55 did not show any effect in the control of complement activation. Factor H expression was knockdown on A549 cells using small interfering RNA. In vivo growth of factor H-deficient cells in athymic mice was significantly reduced. C3 immunocytochemistry on explanted xenografts showed an enhanced activation of complement in these cells. Besides, when mice were depleted of complement with cobra venom factor, growth was recovered, providing further evidence that complement was important in the reduction of in vivo growth. In conclusion, we show that expression of the complement inhibitor factor H by lung cancer cells can prevent complement activation and improve tumor development in vivo. This may have important consequences in the efficiency of complement-mediated immunotherapies.
Selective Role of NFATc3 in Positive Selection of Thymocytes
Authors: Canté-Barrett Kirsten , Winslow Monte M. , Crabtree Gerald R.
Published: April 2014

The four Ca2+-dependent NFATc proteins are both signal transducers and transcription factors that reside in the cytoplasm until dephosphorylation by calcineurin. Dephosphorylation exposes nuclear import sequences and sends NFATc proteins into the nucleus where they assemble with nuclear partners into NFAT transcription complexes. Recent genetic studies have indicated that calcineurin-NFAT signaling is a major determinant of vertebrate morphogenesis and development. Mice lacking calcineurin activity show a complete block in positive selection of CD4 and CD8 double-positive thymocytes, yet the role of the NFATc proteins in T cell development has been controversial. In this study, we address the requirement for NFATc3 in T cell development by generating NFATc3 conditional knockout mice. We show that specific deletion of NFATc3 in thymocytes causes a partial block at the double-negative stage 3 and also a partial block in positive selection. Furthermore, the defect does not become more pronounced when NFATc2 is also absent, consistent with the fact that NFATc2-null mice do not have a T cell developmental defect. Expression of a nuclear (and constitutively active) NFATc1 even at subphysiological levels can rescue the transition of double-negative to double-positive thymocytes in RAG-null mice, but is unable to rescue development of CD4 and CD8 single-positive cells. In addition to NFATc3, this suggests a role for NFATc1 in T cell development. Our studies indicate that the signals that direct positive selection likely use both NFATc1 and NFATc3 downstream of calcineurin.
IL-15 Is Required for Sustained Lymphopenia-Driven Proliferation and Accumulation of CD8 T Cells
Authors: Sandau Michelle M. , Winstead Colleen J. , Jameson Stephen C.
Published: April 2014

Naive T cells undergo slow homeostatic proliferation in response to T cell lymphopenia, which is also called lymphopenia-induced proliferation (LIP). IL-7 is critically required for this process, but previous studies suggested IL-15 was expendable for LIP of naive CD8 T cells. In contrast, we show that IL-15 is important for sustained CD8 T cell proliferation and accumulation in a lymphopenic setting, as revealed by truncated LIP in IL-15−/− hosts. At the same time, we find that IL-12 enhances LIP by acting directly on the CD8 T cells and independently of IL-15, suggesting distinct pathways by which cytokines can regulate homeostatic proliferation. Interestingly, the memory-phenotype CD8 T cell generated by LIP in IL-15−/− hosts are phenotypically distinct from the rare endogenous memory-phenotype cells found in IL-15−/− animals, suggesting these cells are generated by different means. These findings demonstrate that cytokine requirements for LIP change during the process itself, illustrating the need to identify factors that regulate successive stages of lymphopenia-driven proliferation.