bluuebunny/crossref_metadata_2025 huggingface dataset:
Terminal Deoxynucleotidyltransferase Is Required for the Establishment of Private Virus-Specific CD8+ TCR Repertoires and Facilitates Optimal CTL Responses
Authors: Kedzierska Katherine , Thomas Paul G. , Venturi Vanessa , Davenport Miles P. , Doherty Peter C. , Turner Stephen J. , La Gruta Nicole L.
Published: April 2014

Virus-immune CD8+ TCR repertoires specific for particular peptide-MHC class I complexes may be substantially shared between (public), or unique to, individuals (private). Because public TCRs can show reduced TdT-mediated N-region additions, we analyzed how TdT shapes the heavily public (to DbNP366) and essentially private (to DbPA224) CTL repertoires generated following influenza A virus infection of C57BL/6 (B6, H2b) mice. The DbNP366-specific CTL response was virtually clonal in TdT−/− B6 animals, with one of the three public clonotypes prominent in the wild-type (wt) response consistently dominating the TdT−/− set. Furthermore, this massive narrowing of TCR selection for DbNP366 reduced the magnitude of DbNP366-specific CTL response in the virus-infected lung. Conversely, the DbPA224-specific responses remained comparable in both magnitude and TCR diversity within individual TdT−/− and wt mice. However, the extent of TCR diversity across the total population was significantly reduced, with the consequence that the normally private wt DbPA224-specific repertoire was now substantially public across the TdT−/− mouse population. The key finding is thus that the role of TdT in ensuring enhanced diversity and the selection of private TCR repertoires promotes optimal CD8+ T cell immunity, both within individuals and across the species as a whole.
Stable CD8+ T Cell Memory during Persistent Trypanosoma cruzi Infection
Authors: Bixby Lisa M. , Tarleton Rick L.
Published: April 2014

CD8+ T cell responses to persistent infections caused by intracellular pathogens are dominated by resting T effectors and T effector memory cells, with little evidence suggesting that a T central memory (TCM) population is generated. Using a model of Trypanosoma cruzi infection, we demonstrate that in contrast to the T effector/T effector memory phenotype of the majority of T. cruzi-specific CD8+ T cells, a population of cells displaying hallmark characteristics of TCM cells is also present during long-term persistent infection. This population expressed the TCM marker CD127 and a subset expressed one or more of three other TCM markers: CD62L, CCR7, and CD122. Additionally, the majority of CD127high cells were KLRG1low, indicating that they have not been repetitively activated through TCR stimulation. These CD127high cells were better maintained than their CD127low counterparts following transfer into naive mice, consistent with their observed surface expression of CD127 and CD122, which confer the ability to self-renew in response to IL-7 and IL-15. CD127high cells were capable of IFN-γ production upon peptide restimulation and expanded in response to challenge infection, indicating that these cells are functionally responsive upon Ag re-encounter. These results are in contrast to what is typically observed during many persistent infections and indicate that a stable population of parasite-specific CD8+ T cells capable of Ag-independent survival is maintained in mice despite the presence of persistent Ag.
NF-κB Activation Controls Phagolysosome Fusion-Mediated Killing of Mycobacteria by Macrophages
Authors: Gutierrez Maximiliano Gabriel , Mishra Bibhuti B. , Jordao Luisa , Elliott Edith , Anes Elsa , Griffiths Gareth
Published: April 2014

Macrophages can potentially kill all mycobacteria by poorly understood mechanisms. In this study, we explore the role of NF-κB in the innate immune response of macrophages against Mycobacterium smegmatis, a nonpathogenic mycobacterium efficiently killed by macrophages, and Mycobacterium avium which survives within macrophages. We show that infection of macrophages with M. smegmatis induces an activation of NF-κB that is essential for maturation of mycobacterial phagosomes and bacterial killing. In contrast, the pathogenic M. avium partially represses NF-κB activation. Using microarray analysis, we identified many lysosomal enzymes and membrane-trafficking regulators, including cathepsins, LAMP-2 and Rab34, were regulated by NF-κB during infection. Our results argue that NF-κB activation increases the synthesis of membrane trafficking molecules, which may be rate limiting for regulating phagolysosome fusion during infection. The direct consequence of NF-κB inhibition is the impaired delivery of lysosomal enzymes to M. smegmatis phagosomes and reduced killing. Thus, the established role of NF-κB in the innate immune response can now be expanded to include regulation of membrane trafficking during infection.
Ly-6G+CCR2− Myeloid Cells Rather Than Ly-6ChighCCR2+ Monocytes Are Required for the Control of Bacterial Infection in the Central Nervous System
Authors: Mildner Alexander , Djukic Marija , Garbe David , Wellmer Andreas , Kuziel William A. , Mack Matthias , Nau Roland , Prinz Marco
Published: April 2014

Myeloid cell recruitment is a characteristic feature of bacterial meningitis. However, the cellular mechanisms important for the control of Streptococcus pneumoniae infection remain largely undefined. Previous pharmacological or genetic studies broadly depleted many myeloid cell types within the meninges, which did not allow defining the function of specific myeloid subsets. Herein we show that besides CD11b+Ly-6G+CCR2− granulocytes, also CD11b+Ly-6ChighCCR2+ but not Ly-6ClowCCR2− monocytes were recruited in high numbers to the brain as early as 12 h after bacterial challenge. Surprisingly, CD11b+Ly-6ChighCCR2+ inflammatory monocytes modulated local CXCL2 and IL-1β production within the meninges but did not provide protection against bacterial infection. Consistent with these results, CCR2 deficiency strongly impaired monocyte recruitment to the infected brains but was redundant for disease pathogenesis. In contrast, specific depletion of polymorphonuclear granulocytes caused elevated local bacterial titer within the brains, led to an aggravated clinical course, and enhanced mortality. These findings demonstrate that Ly-6ChighCCR2+ inflammatory monocytes play a redundant role for the host defense during bacterial meningitis and that predominantly CD11b+Ly-6G+CCR2− myeloid cells are involved in the restriction of the extracellular bacteria.
TLR Expression on Neutrophils at the Pulmonary Site of Infection: TLR1/TLR2-Mediated Up-Regulation of TLR5 Expression in Cystic Fibrosis Lung Disease
Authors: Koller Barbara , Kappler Matthias , Latzin Phillip , Gaggar Amit , Schreiner Marcus , Takyar Sherkin , Kormann Michael , Kabesch Michael , Roos Dirk , Griese Matthias , Hartl Dominik
Published: April 2014

Cystic fibrosis (CF) lung disease is characterized by infection with Pseudomonas aeruginosa and a sustained accumulation of neutrophils. In this study, we analyzed 1) the expression of MyD88-dependent TLRs on circulating and airway neutrophils in P. aeruginosa-infected CF patients, P. aeruginosa-infected non-CF bronchiectasis patients, and noninfected healthy control subjects and 2) studied the regulation of TLR expression and functionality on neutrophils in vitro. TLR2, TLR4, TLR5, and TLR9 expression was increased on airway neutrophils compared with circulating neutrophils in CF and bronchiectasis patients. On airway neutrophils, TLR5 was the only TLR that was significantly higher expressed in CF patients compared with bronchiectasis patients and healthy controls. Studies using confocal microscopy and flow cytometry revealed that TLR5 was stored intracellularly in neutrophils and was mobilized to the cell surface in a protein synthesis-independent manner through protein kinase C activation or after stimulation with TLR ligands and cytokines characteristic of the CF airway microenvironment. The most potent stimulator of TLR5 expression was the bacterial lipoprotein Pam3CSK4. Ab-blocking experiments revealed that the effect of Pam3CSK4 was mediated through cooperation of TLR1 and TLR2 signaling. TLR5 activation enhanced the phagocytic capacity and the respiratory burst activity of neutrophils, which was mediated, at least partially, via a stimulation of IL-8 production and CXCR1 signaling. This study demonstrates a novel mechanism of TLR regulation in neutrophils and suggests a critical role for TLR5 in neutrophil-P. aeruginosa interactions in CF lung disease.
Lymphocytes Support Oval Cell-Dependent Liver Regeneration
Authors: Strick-Marchand Hélène , Masse Guillemette X. , Weiss Mary C. , Di Santo James P.
Published: April 2014

In case of hepatic damage, the liver uses a unique regeneration mechanism through proliferation of hepatocytes. If this process is inhibited, bipotent oval stem cells proliferate and differentiate to hepatocytes and bile ducts, thus restoring liver mass. Although oval cell accumulation in the liver is often associated with inflammatory processes, the role of lymphocytes in oval cell-mediated hepatic regeneration is poorly understood. We treated wild-type and immunodeficient mice with an oval cell-inducing diet: in the absence of T cells (CD3ε−/− and Rag2−/−) there were fewer oval cells, whereas in alymphoid mice (Rag2−/−γc−/−) a strongly reduced oval cell response and higher mortality, due to liver failure, was observed. Adoptive transfer of T cells into alymphoid mice protected them from liver failure, but was insufficient to restore the oval cell response. Treatment of Rag2−/− mice with an NK cell-depleting Ab resulted in a significantly diminished oval cell response. These genetic experiments point to a major role for NK and T cells in oval cell expansion. In wild-type mice, oval cell proliferation is accompanied by an intrahepatic inflammatory response, characterized by the recruitment of Kupffer, NK, NKT, and T cells. Under these conditions, lymphocytes produce TH1 proinflammatory cytokines (IFN-γ and TNF-α) that are mitogenic for oval cells. Our data suggest that T and NK lymphocytes stimulate oval cell expansion by local cytokine secretion. This beneficial cross-talk between the immune system and liver stem cells operates under noninfectious conditions and could promote tissue regeneration following acute liver damage.
Impaired Restoration of Plasmacytoid Dendritic Cells in HIV-1-Infected Patients with Poor CD4 T Cell Reconstitution Is Associated with Decrease in Capacity to Produce IFN-α but Not Proinflammatory Cytokines
Authors: Sachdeva Naresh , Asthana Vishwaratn , Brewer Toye H. , Garcia Deborah , Asthana Deshratn
Published: April 2014

We analyzed reconstitution characteristics of plasmacytoid dendritic cells (PDCs) and myeloid DCs-1 in 38 HIV-1-infected patients with impaired restoration of CD4 T cell counts despite prolonged suppression of plasma viremia (discordant) and compared them with 42 patients showing good immunological and virological responses following highly active antiretroviral therapy (HAART). While myeloid DCs showed spontaneous recovery following HAART in both the groups, the discordant patients demonstrated poor peripheral reconstitution of PDCs as compared with concordant patients. The ability of PDCs to produce IFN-α following stimulation with TLR7 ligand imiquimod and TLR9 ligand CpG ODN-2216 was also impaired in discordant patients even after 2 years following initiation of HAART. Lower IFN-α expression in the PDCs following TLR stimulation was further associated with lower expression of transcription factor, IFN regulatory factor-7. In contrast, production of TNF-α and IL-6 following TLR stimulation was comparable in both groups of patients, indicating that impaired reconstitution characteristics do not affect the capacity of PDCs to produce proinflammatory cytokines. The discordant patients had significantly lower baseline CD4 T cell counts and higher baseline viral load at the initiation of HAART implying that lower baseline CD4 T cell counts and higher plasma viral load are associated with impaired restoration of CD4 T cells and PDCs, thus, increasing the susceptibility of discordant patients toward opportunistic infections despite virological control.
Tumor-Educated Tolerogenic Dendritic Cells Induce CD3ε Down-Regulation and Apoptosis of T Cells through Oxygen-Dependent Pathways
Authors: Kuang Dong-Ming , Zhao Qiyi , Xu Jing , Yun Jing-Ping , Wu Changyou , Zheng Limin
Published: April 2014

Defects in the CD3/TCR complex and impairment of T cell function are necessary for tumor evasion, but the underlying mechanisms are incompletely understood. We found that culture supernatants from several types of solid tumor cell lines drove human monocytes to become tolerogenic semimature dendritic cells (TDCs). Upon encountering T cells, the TDCs triggered rapid down-regulation of CD3ε and TCR-α/β and subsequent apoptosis in autologous T cells. Consistent with these results, accumulation of immunosuppressive DCs coincided with CD3ε down-regulation and T cell deletion in cancer nests of human tumors. The impaired T cell function was mediated by factor(s) released by live TDCs after direct interaction with lymphocytes. Also, the TDC-induced effect on T cells was markedly reduced by blocking of NADPH oxidase but not by inhibition of arginase, inducible NO synthase (iNOS), IDO, or IFN-γ. Moreover, we found that hyaluronan fragments constituted a common factor produced by a variety of human tumor cell lines to induce formation of TDCs. These observations indicate that tumor microenvironments, including hyaluronan fragments derived from cancer cells, educate DCs to adopt a semimature phenotype, which in turn aids tumor immune escape by causing defects in the CD3/TCR complex and deletion of T cells.
Radiation-Induced CXCL16 Release by Breast Cancer Cells Attracts Effector T Cells
Authors: Matsumura Satoko , Wang Baomei , Kawashima Noriko , Braunstein Steve , Badura Michelle , Cameron Thomas O. , Babb James S. , Schneider Robert J. , Formenti Silvia C. , Dustin Michael L. , Demaria Sandra
Published: April 2014

Recruitment of effector T cells to inflamed peripheral tissues is regulated by chemokines and their receptors, but the factors regulating recruitment to tumors remain largely undefined. Ionizing radiation (IR) therapy is a common treatment modality for breast and other cancers. Used as a cytocidal agent for proliferating cancer cells, IR in combination with immunotherapy has been shown to promote immune-mediated tumor destruction in preclinical studies. In this study we demonstrate that IR markedly enhanced the secretion by mouse and human breast cancer cells of CXCL16, a chemokine that binds to CXCR6 on Th1 and activated CD8 effector T cells, and plays an important role in their recruitment to sites of inflammation. Using a poorly immunogenic mouse model of breast cancer, we found that irradiation increased the migration of CD8+CXCR6+ activated T cells to tumors in vitro and in vivo. CXCR6-deficient mice showed reduced infiltration of tumors by activated CD8 T cells and impaired tumor regression following treatment with local IR to the tumor and Abs blocking the negative regulator of T cell activation, CTLA-4. These results provide the first evidence that IR can induce the secretion by cancer cells of proinflammatory chemotactic factors that recruit antitumor effector T cells. The ability of IR to convert tumors into “inflamed” peripheral tissues could be exploited to overcome obstacles at the effector phase of the antitumor immune response and improve the therapeutic efficacy of immunotherapy.
Sodium-Dependent Glucose Transporter-1 as a Novel Immunological Player in the Intestinal Mucosa
Authors: Palazzo Marco , Gariboldi Silvia , Zanobbio Laura , Selleri Silvia , Dusio Giuseppina F. , Mauro Valentina , Rossini Anna , Balsari Andrea , Rumio Cristiano
Published: April 2014

In this study, we demonstrate the protective effect of the activation of sodium-dependent glucose transporter-1 (SGLT-1) on damages induced by TLR ligands, in intestinal epithelial cells and in a murine model of septic shock. In intestinal epithelial cell lines, glucose inhibited the IL-8/keratinocyte-derived chemokine production and the activation of the TLR-related transcription factor NF-κB stimulated by LPS or CpG-oligodeoxynucleotide. Oral ingestion of glucose was found to protect 100% of mice from lethal endotoxic shock induced by i.p. LPS administration; protection was only observed when glucose was administered orally, not by i.p. route, suggesting the important role of intestinal epithelial cells in this protection. In addition, we observed that the in vivo protection depends on an increase of anti-inflammatory cytokine IL-10. The cornerstone of the observed immunomodulatory and life-saving effects resides in activation of SGLT-1; in fact, the glucose analog 3-O-methyl-d-gluco-pyranose, which induces the transporter activity, but is not metabolized, exerted the same inhibitory effects as glucose both in vitro and in vivo. Thus, we propose that activated SGLT-1, apart from its classical metabolic function, may be a promising target for inhibition of bacteria-induced inflammatory processes and life-saving treatments, assuming a novel role as an immunological player.
Reprogrammed FoxP3+ T Regulatory Cells Become IL-17+ Antigen-Specific Autoimmune Effectors In Vitro and In Vivo
Authors: Radhakrishnan Suresh , Cabrera Rosalyn , Schenk Erin L. , Nava-Parada Pilar , Bell Michael P. , Van Keulen Virginia P. , Marler Ronald J. , Felts Sara J. , Pease Larry R.
Published: April 2014

Lymphocyte differentiation from naive CD4+ T cells into mature Th1, Th2, Th17, or T regulatory cell (Treg) phenotypes has been considered end stage in character. In this study, we demonstrate that dendritic cells (DCs) activated with a novel immune modulator B7-DC XAb (DCXAb) can reprogram Tregs into T effector cells. Down-regulation of FoxP3 expression after either in vitro or in vivo Treg-DCXAb interaction is Ag-specific, IL-6-dependent, and results in the functional reprogramming of the mature T cell phenotype. The reprogrammed Tregs cease to express IL-10 and TGFβ, fail to suppress T cell responses, and gain the ability to produce IFN-γ, IL-17, and TNF-α. The ability of IL-6+ DCXAb and the inability of IL-6−/− DCXAb vaccines to protect animals from lethal melanoma suggest that exogenously modulated DC can reprogram host Tregs. In support of this hypothesis and as a test for Ag specificity, transfer of DCXAb into RIP-OVA mice causes a break in immune tolerance, inducing diabetes. Conversely, adoptive transfer of reprogrammed Tregs but not similarly treated CD25− T cells into naive RIP-OVA mice is also sufficient to cause autoimmune diabetes. Yet, treatment of normal mice with B7-DC XAb fails to elicit generalized autoimmunity. The finding that mature Tregs can be reprogrammed into competent effector cells provides new insights into the plasticity of T cell lineage, underscores the importance of DC-T cell interaction in balancing immunity with tolerance, points to Tregs as a reservoir of autoimmune effectors, and defines a new approach for breaking tolerance to self Ags as a strategy for cancer immunotherapy.
Expression and Functional Significance of SOCS-1 and SOCS-3 in Astrocytes
Authors: Qin Hongwei , Niyongere Sandrine A. , Lee Sun Jung , Baker Brandi J. , Benveniste Etty N.
Published: April 2014

Astrocytes play a number of important physiological roles in CNS homeostasis. Inflammation stimulates astrocytes to secrete cytokines and chemokines that guide macrophages/microglia and T cells to sites of injury/inflammation. Herein, we describe how these processes are controlled by the suppressor of cytokine signaling (SOCS) proteins, a family of proteins that negatively regulate adaptive and innate immune responses. In this study, we describe that the immunomodulatory cytokine IFN-β induces SOCS-1 and SOCS-3 expression in primary astrocytes at the transcriptional level. SOCS-1 and SOCS-3 transcriptional activity is induced by IFN-β through IFN-γ activation site (GAS) elements within their promoters. Studies in STAT-1α-deficient astrocytes indicate that STAT-1α is required for IFN-β-induced SOCS-1 expression, while STAT-3 small interfering RNA studies demonstrate that IFN-β-induced SOCS-3 expression relies on STAT-3 activation. Specific small interfering RNA inhibition of IFN-β-inducible SOCS-1 and SOCS-3 in astrocytes enhances their proinflammatory responses to IFN-β stimulation, such as heightened expression of the chemokines CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), and CXCL10 (IP-10), and promoting chemotaxis of macrophages and CD4+ T cells. These results indicate that IFN-β induces SOCS-1 and SOCS-3 in primary astrocytes to attenuate its own chemokine-related inflammation in the CNS.
Human Anti-IgG1 Hinge Autoantibodies Reconstitute the Effector Functions of Proteolytically Inactivated IgGs
Authors: Brezski Randall J. , Luongo Jennifer L. , Petrone Diane , Ryan Mary H. , Zhong Degang , Tam Susan H. , Schmidt Albert P. , Kruszynski Marian , Whitaker Brian P. , Knight David M. , Jordan Robert E.
Published: April 2014

A number of proteases of potential importance to human physiology possess the ability to selectively degrade and inactivate Igs. Proteolytic cleavage within and near the hinge domain of human IgG1 yielded products including Fab and F(ab′)2 possessing full Ag binding capability but absent several functions needed for immune destruction of cellular pathogens. In parallel experiments, we showed that the same proteolytically generated Fabs and F(ab′)2s become self-Ags that were widely recognized by autoantibodies in the human population. Binding analyses using various Fab and F(ab′)2, as well as single-chain peptide analogues, indicated that the autoantibodies targeted the newly exposed sequences where proteases cleave the hinge. The point of cleavage may be less of a determinant for autoantibody binding than the exposure of an otherwise cryptic stretch of hinge sequence. It was noted that the autoantibodies possessed an unusually high proportion of the IgG3 isotype in contrast to Abs induced against foreign immunogens in the same human subjects. In light of the recognized potency of IgG3 effector mechanisms, we adopted a functional approach to determine whether human anti-hinge (HAH) autoantibodies could reconstitute the (missing) Fc region effector functions to Fab and F(ab′)2. Indeed, in in vitro cellular assays, purified HAH autoantibodies restored effector functions to F(ab′)2 in both Ab-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays. The results indicate that HAH autoantibodies selectively bind to proteolytically cleaved IgGs and can thereby provide a surrogate Fc domain to reconstitute cell lytic functions.
Colocalization of Antigen-Specific B and T Cells within Ectopic Lymphoid Tissue following Immunization with Exogenous Antigen
Authors: Weinstein Jason S. , Nacionales Dina C. , Lee Pui Y. , Kelly-Scumpia Kindra M. , Yan Xiao-Jie , Scumpia Philip O. , Vale-Cruz Dustin S. , Sobel Eric , Satoh Minoru , Chiorazzi Nicholas , Reeves Westley H.
Published: April 2014

Chronic inflammation promotes the formation of ectopic lymphoid tissue morphologically resembling secondary lymphoid tissues, though it is unclear whether this is a location where Ag-specific immune responses develop or merely a site of lymphocyte accumulation. Ectopic lymphoid tissue formation is associated with many humoral autoimmune diseases, including lupus induced by tetramethylpecadentane in mice. We examined whether an immune response to 4-hydroxy-3-nitrophenyl acetyl-keyhole limpet hemocyanin (NP-KLH) and NP-OVA develops within ectopic lymphoid tissue (“lipogranulomas”) induced by tetramethylpecadentane in C57BL/6 mice. Following primary immunization, NP-specific B cells bearing V186.2 and related heavy chains as well as λ-light chains accumulated within ectopic lymphoid tissue. The number of anti-NP-secreting B cells in the ectopic lymphoid tissue was greatly enhanced by immunization with NP-KLH. Remarkably, the H chain sequences isolated from individual lipogranulomas from these mice were diverse before immunization, whereas individual lipogranulomas from single immunized mice had unique oligo- or monoclonal populations of presumptive NP-specific B cells. H chain CDR sequences bore numerous replacement mutations, consistent with an Ag-driven and T cell-mediated response. In mice adoptively transferred with OT-II or DO11 T cells, there was a striking accumulation of OVA-specific T cells in lipogranulomas after s.c. immunization with NP-OVA. The selective colocalization of proliferating, Ag-specific T and B lymphocytes in lipogranulomas from tetramethylpecadentane-treated mice undergoing primary immunization implicates ectopic lymphoid tissue as a site where Ag-specific humoral immune responses can develop. This has implications for understanding the strong association of humoral autoimmunity with lymphoid neogenesis, which may be associated with deficient censoring of autoreactive cells.
IL-2 Producing Memory CD4+ T Lymphocytes Are Closely Associated with the Generation of IgG-Secreting Plasma Cells
Authors: Litjens Nicolle H. R. , Huisman Martin , Hijdra Daniëlle , Lambrecht Bart M. N. , Stittelaar Koert J. , Betjes Michiel G. H.
Published: April 2014

The role of specific CD4+ T cell subsets in the induction of humoral immune responses in humans is largely unknown. In this study, the generation of hepatitis B surface Ag-specific CD4+ T lymphocytes following vaccination was closely monitored and characterized at the single-cell level. The appearance and absolute numbers of hepatitis B surface Ag-specific IL-2 producing effector memory CD4+ T lymphocytes was solely and tightly related to Ab titers reached. This relation remained present many years after vaccination. Subsequently, a relation was found between Ab titers and number of IL-2 producing memory CD4+ T lymphocytes for various other Ags. These observations matched the findings of an in vitro assay, using different T cell subsets to induce B cell differentiation into IgG-producing plasma cells. By depleting for IL-2 producing memory T cells, we demonstrated that these cells are important for B cell differentiation into IgG-producing plasma cells. Finally, blocking the action of IL-2 with an IL-2R-α Ab inhibited the differentiation of B lymphocytes into IgG-producing plasma cells. Based on these findings, we conclude that the development of Ag-specific IL-2-producing memory T cells appears to be essential for the development of IgG-secreting plasma cells in humans.
Increased Regulatory versus Effector T Cell Development Is Associated with Thymus Atrophy in Mouse Models of Multiple Myeloma
Authors: Laronne-Bar-On Ayelet , Zipori Dov , Haran-Ghera Nechama
Published: April 2014

CD4+CD25+Foxp3+ regulatory T cells (Tregs) play a central role in cancer tolerance. However, mechanisms leading to their accumulation in cancer remain unknown. Although the thymus is the main site of Treg development, thymic contribution to Treg expansion in cancer has not been directly examined. Herein, we used two murine models of multiple myeloma (MM), 5T2 MM and 5T33 MM, to examine Treg accumulation in peripheral lymphoid organs, including spleen, lymph nodes, bone marrow, and blood, and to explore thymic Treg development during malignancy. We found that peripheral ratios of suppressive-functional Tregs increased in both models of MM-inflicted mice. We found that thymic ratios of Treg development in MM increased, in strong association with thymus atrophy and altered developmental processes in the thymus. The CD4+CD8+ double-positive population, normally the largest thymocyte subset, is significantly decreased, whereas the CD4−CD8− double-negative population is increased. Administration of thymocytes from MM-inflicted mice compared with control thymocytes resulted in increased progression of the disease, and this effect was shown to be mediated by Tregs in the thymus of MM-inflicted mice. Our data suggest that increased ratios of Treg development in the thymus may contribute to disease progression in MM-inflicted mice.
Cutting Edge: Alum Adjuvant Stimulates Inflammatory Dendritic Cells through Activation of the NALP3 Inflammasome
Authors: Kool Mirjam , Pétrilli Virginie , De Smedt Thibaut , Rolaz Aline , Hammad Hamida , van Nimwegen Menno , Bergen Ingrid M. , Castillo Rosa , Lambrecht Bart N. , Tschopp Jürg
Published: April 2014

Adjuvants are vaccine additives that stimulate the immune system without having any specific antigenic effect of itself. In this study we show that alum adjuvant induces the release of IL-1β from macrophages and dendritic cells and that this is abrogated in cells lacking various NALP3 inflammasome components. The NALP3 inflammasome is also required in vivo for the innate immune response to OVA in alum. The early production of IL-1β and the influx of inflammatory cells into the peritoneal cavity is strongly reduced in NALP3-deficient mice. The activation of adaptive cellular immunity to OVA-alum is initiated by monocytic dendritic cell precursors that induce the expansion of Ag-specific T cells in a NALP3-dependent way. We propose that, in addition to TLR stimulators, agonists of the NALP3 inflammasome should also be considered as vaccine adjuvants.
Role of β-Catenin in B Cell Development and Function
Authors: Yu Qing , Quinn William J. , Salay Theresa , Crowley Jenni E. , Cancro Michael P. , Sen Jyoti Misra
Published: April 2014

β-Catenin is a central mediator of Wnt signaling pathway, components of which have been implicated in B cell development and function. B cell progenitors and bone marrow stromal cells express Wnt ligands, Frizzled receptors and Wnt antagonists, suggesting fine tuned regulation of this pathway in B cell development. In particular, deletion of Frizzled 9 gene results in developmental defects at the pre-B stage of development and an accumulation of plasma cells. Furthermore, Wnt signals regulate B cell proliferation through lymphocyte enhancer-binding factor-1. However, it is not known whether Wnt signaling in B cell development is mediated by β-catenin and whether β-catenin plays a role in mature B cell function. In this report, we show that mice bearing B cell-specific deletion of β-catenin have normal B cell development in bone marrow and periphery. A modest defect in plasma cell generation in vitro was documented, which correlated with a defective expression of IRF-4 and Blimp-1. However, B cell response to T-dependent and T-independent Ags in vivo was found to be normal. Thus, β-catenin expression was found to be dispensable for normal B cell development and function.
Cathelicidin Antimicrobial Peptides Inhibit Hyaluronan-Induced Cytokine Release and Modulate Chronic Allergic Dermatitis
Authors: Morioka Yasuhide , Yamasaki Kenshi , Leung Donald , Gallo Richard L.
Published: April 2014

Antimicrobial peptides such as cathelicidins can modulate inflammation by interfering with TLR function. Small fragment hyaluronan (HA) is released following injury, and is an endogenous ligand for TLR4 as well as CD44. In this study, we examined the interactions of cathelicidin with HA. Cathelicidin inhibited HA induced MIP-2 release from mouse bone marrow derived macrophages in a CD44 dependent manner but did not inhibit MALP2-induced MIP-2 release. This inhibitory activity was more potent than that of a peptide inhibitor of HA binding (Pep-1) and independent of Gi protein coupled or EGF-R signaling, both targets of cathelicidin inhibited HA-induced MIP-2 release. In assay of cell binding to HA, cathelicidins also significantly inhibited this process, suggesting that this antimicrobial peptide can interfere in other membrane binding events mediated by HA. The significance of this inhibition was demonstrated in a skin inflammation model induced by repeated application of 2,4-dinitrofluorobenzene. This induced an increase in HA at the site of application and was partially CD44 dependent. Camp−/− mice lacking cathelcidin demonstrated a large increase in ear swelling, cell infiltration, and MIP-2 expression compared with wild type mice. These results suggest that cathelicidin has anti-inflammatory activity in skin that may be mediated in part by inhibition of HA-mediated processes.
Functionally Competent Eosinophils Differentiated Ex Vivo in High Purity from Normal Mouse Bone Marrow
Authors: Dyer Kimberly D. , Moser Jennifer M. , Czapiga Meggan , Siegel Steven J. , Percopo Caroline M. , Rosenberg Helene F.
Published: April 2014

We have devised an ex vivo culture system which generates large numbers of eosinophils at high purity (>90%) from unselected mouse bone marrow progenitors. In response to 4 days of culture with recombinant mouse FLT3-L and recombinant mouse stem cell factor followed by recombinant mouse IL-5 alone thereafter, the resulting bone marrow-derived eosinophils (bmEos) express immunoreactive major basic protein, Siglec F, IL-5R α-chain, and transcripts encoding mouse eosinophil peroxidase, CCR3, the IL-3/IL-5/GM-CSF receptor common β-chain, and the transcription factor GATA-1. BmEos are functionally competent: they undergo chemotaxis toward mouse eotaxin-1 and produce characteristic cytokines, including IFN-γ, IL-4, MIP-1α, and IL-6. The rodent pathogen pneumonia virus of mice replicates in bmEos and elevated levels of IL-6 are detected in supernatants of bmEos cultures in response to active infection. Finally, differentiating bmEos are readily transfected with lentiviral vectors, suggesting a means for rapid production of genetically manipulated cells.
A Human Dendritic Cell Subset Receptive to the Venezuelan Equine Encephalitis Virus-Derived Replicon Particle Constitutively Expresses IL-32
Authors: Nishimoto Kevin P. , Laust Amanda K. , Nelson Edward L.
Published: April 2014

Dendritic cells (DCs) are a diverse population with the capacity to respond to a variety of pathogens. Because of their critical role in pathogenesis and Ag-specific adaptive immune responses, DCs are the focus of extensive study and incorporation into a variety of immunotherapeutic strategies. The diversity of DC subsets imposes a substantial challenge to the successful development of DC-based therapies, requiring identification of the involved subset(s) and the potential roles each contributes to the immunologic responses. The recently developed and promising Venezuelan equine encephalitis replicon particle (VRP) vector system has conserved tropism for a subset of myeloid DCs. This immunotherapeutic vector permits in situ targeting of DCs; however, it targets a restricted subset of DCs, which are heretofore uncharacterized. Using a novel technique, we isolated VRP-receptive and -nonreceptive populations from human monocyte-derived DCs. Comparative gene expression analysis revealed significant differential gene expression, supporting the existence of two distinct DC populations. Further analysis identified constitutive expression of the proinflammatory cytokine IL-32 as a distinguishing characteristic of VRP-receptive DCs. IL-32 transcript was exclusively expressed (>50 fold) in the VRP-receptive DC population relative to the background level of expression in the nonreceptive population. The presence of IL-32 transcript was accompanied by protein expression. These data are the first to identify a subset of immature monocyte-derived DCs constitutively expressing IL-32 and they provide insights into both DC biology and potential mechanisms employed by this potent vector system.
Cholera Toxin B Accelerates Disease Progression in Lupus-Prone Mice by Promoting Lipid Raft Aggregation
Authors: Deng Guo-Min , Tsokos George C.
Published: April 2014

Infectious agents, including bacteria and viruses, are thought to provide triggers for the development or exacerbation of autoimmune diseases such as systemic lupus erythematosus in the genetically predisposed individual. Molecular mimicry and engagement of TLRs have been assigned limited roles that link infection to autoimmunity, but additional mechanisms are suspected to be involved. In this study we show that T cells from lupus-prone mice display aggregated lipid rafts that harbor signaling, costimulatory, inflammatory, adhesion, and TLR molecules. The percentage of T cells with clustered lipid rafts increases with age and peaks before the development of lupus pathology. We show that cholera toxin B, a component of Vibrio cholerae, promotes autoantibody production and glomerulonephritis in lupus-prone mice by enhancing lipid raft aggregation in T cells. In contrast, disruption of lipid raft aggregation results in delay of disease pathology. Our results demonstrate that lipid rafts contribute significantly to the pathogenesis of lupus and provide a novel mechanism whereby aggregated lipid rafts represent a potential link between infection and autoimmunity.
Linked Suppression across an MHC-Mismatched Barrier in a Miniature Swine Kidney Transplantation Model
Authors: Griesemer Adam D. , LaMattina John C. , Okumi Masayoshi , Etter Justin D. , Shimizu Akira , Sachs David H. , Yamada Kazuhiko
Published: April 2014

We have demonstrated previously that a 12-day course of FK506 permits the induction of tolerance to fully MHC-mismatched renal transplants in miniature swine. In the present study, we examined the mechanism of this tolerance by assessing the possibility that the survival of one-haplotype mismatched third-party kidneys might be prolonged via linked suppression. Ten SLAd/d miniature swine received fully MHC-mismatched renal allografts from SLAc/c donors with 12 days of FK506. Six animals received second SLAc/c kidneys without immunosuppression to confirm tolerance. Regulatory mechanisms were assessed by mixed lymphocyte reaction (MLR) and cell-mediated lympholysis coculture assays and ELISA for regulatory cytokines. Linked suppression was investigated by transplanting SLAa/c or SLAa/d allografts into long-term tolerant recipients without immunosuppression. All recipients showed donor-specific unresponsiveness in standard cell-mediated lympholysis and MLR assays. Tolerant cells prestimulated with donor Ag and then cocultured with naive recipient MHC-matched cells inhibited antidonor responses, confirming the presence of regulatory cells. ELISA and MLR assays showed that TGF-β2 was involved in mediating the suppression in vitro. SLAa/d renal allografts transplanted into tolerant recipients were rejected by postoperative day 8 (median, 7 days; range, 6–8). In contrast, SLAa/c allografts showed markedly prolonged survival (median, 52 days; range, 28–78; p = 0.0246), suggesting linked suppression. Animals not challenged with a second donor-matched graft did not manifest linked suppression consistent with in vitro data showing that re-exposure to tolerated Ags is important for generation of regulatory cells. To our knowledge, these data represent the first evidence of linked suppression across fully MHC-mismatched barriers in a large animal model.
α4β7/MAdCAM-1 Interactions Play an Essential Role in Transitioning Cryptopatches into Isolated Lymphoid Follicles and a Nonessential Role in Cryptopatch Formation
Authors: Wang Caihong , McDonough Jacquelyn S. , McDonald Keely G. , Huang Conway , Newberry Rodney D.
Published: April 2014

The α4 integrins α4β7 and α4β1, and their ligands mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) and VCAM-1, have diverse functions, including roles in the formation of secondary lymphoid tissues at early time points during the colonization and clustering of the fetal lymphoid tissue inducer (LTi) cells and at later time points during the recruitment of lymphocytes. In this study, we evaluated the role of α4 integrins in the development of a recently appreciated class of intestinal lymphoid tissues, isolated lymphoid follicles (ILFs). We observed that diverse ILF cellular populations express α4β7 and α4β1, including the LTi-like cells and lymphocytes, while ILF stromal cells and vessels within ILFs express VCAM-1 and MAdCAM-1, respectively. Evaluation of adult and neonatal β7−/− mice and adult and neonatal mice given blocking Abs to α4β7, MAdCAM-1, or VCAM-1 did not identify a role for α4 integrins in cryptopatch (CP) development; however, these studies demonstrated that α4β7 and MAdCAM-1 are required for the transitioning of CP into lymphoid tissues containing lymphocytes or ILFs. Competitive bone marrow transfers demonstrated that β7−/− LTi-like cells had a reduced but not significantly impaired ability to localize to CP. Bone marrow transfers and adoptive transfers of B lymphocytes revealed that β7 expression by B lymphocytes was essential for their entry into the developing ILFs. These findings demonstrate an essential role for α4β7/MAdCAM-1 in ILF development corresponding to the influx of β7-expressing lymphocytes and a nonessential role for β7-localizing LTi-like cells to the small intestine.
Multiple Levels of Selection Responsive to Immunoglobulin Light Chain and Heavy Chain Structures Impede the Development of Dμ-Expressing B Cells
Authors: Guloglu F. Betul , Smith Brendan P. , Roman Christopher A. J.
Published: April 2014

The truncated/VH-less mouse H chain Dμ forms precursor B cell receptors with the surrogate L chain complex that promotes allelic exclusion but not other aspects of pre-B cell development, causing most progenitor B cells expressing this H chain to be eliminated at the pre-B cell checkpoint. However, there is evidence that Dμ-λ1 complexes can be made and are positively selected during fetal life but cannot sustain adult B lymphopoiesis. How surrogate and conventional L chains interpret Dμ’s unusual structure and how that affects signaling outcome are unclear. Using nonlymphoid and primary mouse B cells, we show that secretion-competent λ1 L chains could associate with both full-length H chains and Dμ, whereas secretion-incompetent λ1 L chains could only do so with full-length H chains. In contrast, Dμ could not form receptors with a panel of κ L chains irrespective of their secretion properties. This was due to an incompatibility of Dμ with the κ-joining and constant regions. Finally, the Dμ-λ1 receptor was less active than the full-length mouse μ-λ1 receptor in promoting growth under conditions of limiting IL-7. Thus, multiple receptor-dependent mechanisms operating at all stages of B cell development limit the contribution of B cells with Dμ H chain alleles to the repertoire.
Acid Sphingomyelinase Amplifies Redox Signaling in Pseudomonas aeruginosa-Induced Macrophage Apoptosis
Authors: Zhang Yang , Li Xiang , Carpinteiro Alexander , Gulbins Erich
Published: April 2014

Recent studies indicate that distinct membrane microdomains, also named lipid rafts, and ceramide play an important role in infectious biology. Ceramide forms larger ceramide-enriched membrane platforms that are required for diverse signal transduction. In this study, we demonstrate that ceramide-enriched membrane platforms are critically involved in redox signaling that regulates alveolar macrophage apoptosis upon infection with Pseudomonas aeruginosa. In freshly isolated alveolar macrophages, P. aeruginosa infection results in rapid activation of acid sphingomyelinase (Asm), release of ceramide, and formation of ceramide-enriched membrane platforms, which are required for P. aeruginosa-induced activation of NADPH oxidase and production of reactive oxygen species (ROS). Inhibition of NADPH oxidase or removal of intracellular ROS reduced P. aeruginosa-induced activation of the Asm and formation of ceramide-enriched membrane platforms, suggesting that NADPH oxidase-derived ROS regulate Asm-initiated redox signaling in a positive feedback manner. Furthermore, stimulation of JNK and induction of apoptosis upon P. aeruginosa infections are dependent on NADPH oxidase-derived ROS. These findings indicate that ceramide-enriched membrane platforms are essential for amplification of Asm-mediated redox signaling, which mediates JNK activation and thereby apoptosis of alveolar macrophages upon P. aeruginosa infection.
RAGE Ligation Affects T Cell Activation and Controls T Cell Differentiation
Authors: Chen Yali , Akirav Eitan M. , Chen Wei , Henegariu Octavian , Moser Bernhard , Desai Dharmesh , Shen Jane M. , Webster Jeffery C. , Andrews Robert C. , Mjalli Adnan M. , Rothlein Robert , Schmidt Ann Marie , Clynes Raphael , Herold Kevan C.
Published: April 2014

The pattern recognition receptor, RAGE, has been shown to be involved in adaptive immune responses but its role on the components of these responses is not well understood. We have studied the effects of a small molecule inhibitor of RAGE and the deletion of the receptor (RAGE−/− mice) on T cell responses involved in autoimmunity and allograft rejection. Syngeneic islet graft and islet allograft rejection was reduced in NOD and B6 mice treated with TTP488, a small molecule RAGE inhibitor (p < 0.001). RAGE−/− mice with streptozotocin-induced diabetes showed delayed rejection of islet allografts compared with wild type (WT) mice (p < 0.02). This response in vivo correlated with reduced proliferative responses of RAGE−/− T cells in MLRs and in WT T cells cultured with TTP488. Overall T cell proliferation following activation with anti-CD3 and anti-CD28 mAbs were similar in RAGE−/− and WT cells, but RAGE−/− T cells did not respond to costimulation with anti-CD28 mAb. Furthermore, culture supernatants from cultures with anti-CD3 and anti-CD28 mAbs showed higher levels of IL-10, IL-5, and TNF-α with RAGE−/− compared with WT T cells, and WT T cells showed reduced production of IFN-γ in the presence of TTP488, suggesting that RAGE may be important in the differentiation of T cell subjects. Indeed, by real-time PCR, we found higher levels of RAGE mRNA expression on clonal T cells activated under Th1 differentiating conditions. We conclude that activation of RAGE on T cells is involved in early events that lead to differentiation of Th1+ T cells.
Intradermal Administration of Thymic Stromal Lymphopoietin Induces a T Cell- and Eosinophil-Dependent Systemic Th2 Inflammatory Response
Authors: Jessup Heidi K. , Brewer Avery W. , Omori Miyuki , Rickel Erika A. , Budelsky Alison L. , Yoon Bo-Rin Park , Ziegler Steven F. , Comeau Michael R.
Published: April 2014

The epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) is sufficient to induce asthma or atopic dermatitis-like phenotypes when selectively overexpressed in transgenic mice, or when driven by topical application of vitamin D3 or low-calcemic analogues. Although T and B cells have been reported to be dispensable for the TSLP-induced inflammation in these models, little is known about the downstream pathways or additional cell types involved in the inflammatory response driven by TSLP. To characterize the downstream effects of TSLP in vivo, we examined the effects of exogenous administration of TSLP protein to wild-type and genetically deficient mice. TSLP induced a systemic Th2 inflammatory response characterized by increased circulating IgE and IgG1 as well as increased draining lymph node size and cellularity, Th2 cytokine production in draining lymph node cultures, inflammatory cell infiltrates, epithelial hyperplasia, subcuticular fibrosis, and up-regulated Th2 cytokine and chemokine messages in the skin. Responses to TSLP in various genetically deficient mice demonstrated T cells and eosinophils were required, whereas mast cells and TNF-α were dispensable. TSLP-induced responses were significantly, but not completely reduced in IL-4- and IL-13-deficient mice. These results shed light on the pathways and cell types involved in TSLP-induced inflammation.