bluuebunny/crossref_metadata_2025 huggingface dataset:
The Production of IL-10 by Human Regulatory T Cells Is Enhanced by IL-2 through a STAT5-Responsive Intronic Enhancer in the IL-10 Locus
Authors: Tsuji-Takayama Kazue , Suzuki Motoyuki , Yamamoto Mayuko , Harashima Akira , Okochi Ayumi , Otani Takeshi , Inoue Toshiya , Sugimoto Akira , Toraya Terumasa , Takeuchi Makoto , Yamasaki Fumiyuki , Nakamura Shuji , Kibata Masayoshi
Published: April 2014

STAT5 molecules are key components of the IL-2 signaling pathway, the deficiency of which often results in autoimmune pathology due to a reduced number of CD4+CD25+ naturally occurring regulatory T (Treg) cells. One of the consequences of the IL-2-STAT5 signaling axis is up-regulation of FOXP3, a master control gene for naturally occurring Treg cells. However, the roles of STAT5 in other Treg subsets have not yet been elucidated. We recently demonstrated that IL-2 enhanced IL-10 production through STAT5 activation. This occurred in two types of human Treg cells: a novel type of umbilical cord blood-derived Treg cell, termed HOZOT, and Tr1-like Treg cells, IL-10-Treg. In this study, we examined the regulatory mechanisms of IL-10 production in these Treg cells, focusing specifically on the roles of STAT5. By performing bioinformatic analysis on the IL-10 locus, we identified one STAT-responsive element within intron 4, designated I-SRE-4, as an interspecies-conserved sequence. We found that I-SRE-4 acted as an enhancer element, and clustered CpGs around the I-SRE-4 were hypomethylated in IL-10-producing Treg cells, but not in other T cells. A gel-shift analysis using a nuclear extract from IL-2-stimulated HOZOT confirmed that CpG DNA methylation around I-SRE-4 reduced STAT5 binding to the element. Chromatin immunoprecipitation analysis revealed the in situ binding of IL-2-activated STAT5 to I-SRE-4. Thus, we provide molecular evidence for the involvement of an IL-2-STAT5 signaling axis in the expression of IL-10 by human Treg cells, an axis that is regulated by the intronic enhancer, I-SRE-4, and epigenetic modification of this element.
Fibroblastic Reticular Cells Guide T Lymphocyte Entry into and Migration within the Splenic T Cell Zone
Authors: Bajénoff Marc , Glaichenhaus Nicolas , Germain Ronald N.
Published: April 2014

Although a great deal is known about T cell entry into lymph nodes, much less is understood about how T lymphocytes access the splenic white pulp (WP). We show in this study that, as recently described for lymph nodes, fibroblastic reticular cells (FRCs) form a network in the T cell zone (periarteriolar lymphoid sheath, PALS) of the WP on which T lymphocytes migrate. This network connects the PALS to the marginal zone (MZ), which is the initial site of lymphocyte entry from the blood. T cells do not enter the WP at random locations but instead traffic to that site using the FRC-rich MZ bridging channels (MZBCs). These data reveal that FRCs form a substrate for T cells in the spleen, guiding these lymphocytes from their site of entry in the MZ into the PALS, within which they continue to move on the same network.
Contrasting Responses of Lymphoid Progenitors to Canonical and Noncanonical Wnt Signals
Authors: Malhotra Sachin , Baba Yoshihiro , Garrett Karla P. , Staal Frank J. T. , Gerstein Rachel , Kincade Paul W.
Published: April 2014

The Wnt family of secreted glycoproteins has been implicated in many aspects of development, but its contribution to blood cell formation is controversial. We overexpressed Wnt3a, Wnt5a, and Dickkopf 1 in stromal cells from osteopetrotic mice and used them in coculture experiments with highly enriched stem and progenitor cells. The objective was to learn whether and how particular stages of B lymphopoiesis are responsive to these Wnt family ligands. We found that canonical Wnt signaling, through Wnt3a, inhibited B and plasmacytoid dendritic cell, but not conventional dendritic cell development. Wnt5a, which can oppose canonical signaling or act through a different pathway, increased B lymphopoiesis. Responsiveness to both Wnt ligands diminished with time in culture and stage of development. That is, only hematopoietic stem cells and very primitive progenitors were affected. Although Wnt3a promoted retention of hematopoietic stem cell markers, cell yields and dye dilution experiments indicated it was not a growth stimulus. Other results suggest that lineage instability results from canonical Wnt signaling. Lymphoid progenitors rapidly down-regulated RAG-1, and some acquired stem cell-staining characteristics as well as myeloid and erythroid potential when exposed to Wnt3a-producing stromal cells. We conclude that at least two Wnt ligands can differentially regulate early events in B lymphopoiesis, affecting entry and progression in distinct differentiation lineages.
Transfer of T Cell Surface Molecules to Dendritic Cells upon CD4+ T Cell Priming Involves Two Distinct Mechanisms
Authors: Busch Annette , Quast Thomas , Keller Sascha , Kolanus Waldemar , Knolle Percy , Altevogt Peter , Limmer Andreas
Published: April 2014

Activation of CD4+ T cells by APCs occurs by multiple Ag recognition events including the exchange of costimulatory signals and cytokines. Additionally, the T cells acquire APC-derived surface molecules. Herein, we describe for the first time the transfer of human and murine T cell surface receptors to APCs after Ag-specific interaction. This transfer occurs in two qualitatively different phases. The first group of molecules (e.g., CD2) derived from the T cell surface was transferred rapidly after 2 h of interaction, was strongly bound on the DC surface (acid wash-resistant), was strictly dependent on dendritic cell-T cell contact, and transferred independently of T cell activation. The second group, including the CD3/TCR complex, CD27, and OX40, was of intracellular origin, transferred later after 10–16 h in a cell-cell contact-independent fashion, was noncovalently bound, and was strictly dependent on Ag-specific T cell activation. Functionally, murine dendritic cells that received TCR molecules from OVA-specific CD4+ T cells after Ag-specific interaction were less efficient in priming naive CD4+ T cells of the same specificity without losing their ability for CD8+ T cell stimulation, indicating that the transferred TCR molecules mask the Ag-bearing MHC II molecules, thereby reducing their accessibility to following Ag-specific CD4+ T cells. While the first group of transferred T cell surface molecules might facilitate the detachment of the CD4+ T cell from the dendritic cell during the early scanning phases, the second group could play an important immunomodulatory role in intraclonal competition of T cells for APC access, making the physical presence of CD4+ T cells unnecessary.
Involvement of LEK1 in Dendritic Cell Regulation of T Cell Immunity against Chlamydia
Authors: He Qing , Eko Francis O. , Lyn Deborah , Ananaba Godwin A. , Bandea Claudiu , Martinez Joseph , Joseph Kahaliah , Kellar Kathy , Black Carolyn M. , Igietseme Joseph U.
Published: April 2014

We investigated the hypothesis that the enhanced Ag-presenting function of IL-10-deficient dendritic cells (DCs) is related to specific immunoregulatory cytoskeletal molecules expressed when exposed to Ags. We analyzed the role of a prominent cytoskeletal protein, LEK1, in the immunoregulation of DC functions; specifically cytokine secretion, costimulatory molecule expression, and T cell activation against Chlamydia. Targeted knockdown of LEK1 expression using specific antisense oligonucleotides resulted in the rapid maturation of Chlamydia-exposed DCs as measured by FACS analysis of key activation markers (i.e., CD14, CD40, CD54, CD80, CD86, CD197, CD205, and MHC class II). The secretion of mostly Th1 cytokines and chemokines (IL-1a, IL-9, IL-12, MIP-1a, and GM-CSF but not IL-4 and IL-10) was also enhanced by blocking of LEK1. The function of LEK1 in DC regulation involves cytoskeletal changes, since the dynamics of expression of vimentin and actin, key proteins of the cellular cytoskeleton, were altered after exposure of LEK1 knockdown DCs to Chlamydia. Furthermore, targeted inhibition of LEK1 expression resulted in the enhancement of the immunostimulatory capacity of DCs for T cell activation against Chlamydia. Thus, LEK1 knockdown DCs activated immune T cells at least 10-fold over untreated DCs. These results suggest that the effect of IL-10 deficiency is mediated through LEK1-related events that lead to rapid maturation of DCs and acquisition of the capacity to activate an elevated T cell response. Targeted modulation of LEK1 expression provides a novel strategy for augmenting the immunostimulatory function of DCs for inducing an effective immunity against pathogens.
B7-H3 Contributes to the Development of Pathogenic Th2 Cells in a Murine Model of Asthma
Authors: Nagashima Osamu , Harada Norihiro , Usui Yoshihiko , Yamazaki Tomohide , Yagita Hideo , Okumura Ko , Takahashi Kazuhisa , Akiba Hisaya
Published: April 2014

B7-H3 is a new member of the B7 family. The receptor for B7-H3 has not been identified, but it seems to be expressed on activated T cells. Initial studies have shown that B7-H3 provides a stimulatory signal to T cells. However, recent studies suggest a negative regulatory role for B7-H3 in T cell responses. Thus, the immunological function of B7-H3 is controversial and unclear. In this study, we investigated the effects of neutralizing anti-B7-H3 mAb in a mouse model of allergic asthma to determine whether B7-H3 contributes to the development of pathogenic Th2 cells and pulmonary inflammation. Administration of anti-B7-H3 mAb significantly reduced airway hyperreactivity with a concomitant decrease in eosinophils in the lung as compared with control IgG-treated mice. Treatment with anti-B7-H3 mAb also resulted in decreased production of Th2 cytokines (IL-4, IL-5, and IL-13) in the draining lymph node cells. Although blockade of B7-H3 during the induction phase abrogated the development of asthmatic responses, B7-H3 blockade during the effector phase did not inhibit asthmatic responses. These results indicated an important role for B7-H3 in the development of pathogenic Th2 cells during the induction phase in a murine model of asthma.
Inside-Out Regulation of FcαRI (CD89) Depends on PP2A
Authors: Bakema Jantine E. , Bakker Annie , de Haij Simone , Honing Henk , Bracke Madelon , Koenderman Leo , Vidarsson Gestur , van de Winkel Jan G. J. , Leusen Jeanette H. W.
Published: April 2014

To achieve a correct cellular immune response toward pathogens, interaction between FcR and their ligands must be regulated. The Fc receptor for IgA, FcαRI, is pivotal for the inflammatory responses against IgA-opsonized pathogens. Cytokine-induced inside-out signaling through the intracellular FcαRI tail is important for FcαRI-IgA binding. However, the underlying molecular mechanism governing this process is not well understood. In this study, we report that PP2A can act as a molecular switch in FcαRI activation. PP2A binds to the intracellular tail of FcαRI and, upon cytokine stimulation, PP2A becomes activated. Subsequently, FcαRI is dephosphorylated on intracellular Serine 263, which we could link to receptor activation. PP2A inhibition, in contrast, decreased FcαRI ligand binding capacity in transfected cells but also in eosinophils and monocytes. Interestingly, PP2A activity was found crucial for IgA-mediated binding and phagocytosis of Neisseria meningitidis. The present findings demonstrate PP2A involvement as a molecular mechanism for FcαRI ligand binding regulation, a key step in initiating an immune response.
TH17 Cells Mediate Steroid-Resistant Airway Inflammation and Airway Hyperresponsiveness in Mice
Authors: McKinley Laura , Alcorn John F. , Peterson Alanna , DuPont Rachel B. , Kapadia Shernaaz , Logar Alison , Henry Adam , Irvin Charles G. , Piganelli Jon D. , Ray Anuradha , Kolls Jay K.
Published: April 2014

Steroid-resistant asthma comprises an important source of morbidity in patient populations. TH17 cells represent a distinct population of CD4+ Th cells that mediate neutrophilic inflammation and are characterized by the production of IL-17, IL-22, and IL-6. To investigate the function of TH17 cells in the context of Ag-induced airway inflammation, we polarized naive CD4+ T cells from DO11.10 OVA-specific TCR-transgenic mice to a TH2 or TH17 phenotype by culturing in conditioned medium. In addition, we also tested the steroid responsiveness of TH2 and TH17 cells. In vitro, TH17 cytokine responses were not sensitive to dexamethasone (DEX) treatment despite immunocytochemistry confirming glucocorticoid receptor translocation to the nucleus following treatment. Transfer of TH2 cells to mice challenged with OVA protein resulted in lymphocyte and eosinophil emigration into the lung that was markedly reduced by DEX treatment, whereas TH17 transfer resulted in increased CXC chemokine secretion and neutrophil influx that was not attenuated by DEX. Transfer of TH17 or TH2 cells was sufficient to induce airway hyperresponsiveness (AHR) to methacholine. Interestingly, AHR was not attenuated by DEX in the TH17 group. These data demonstrate that polarized Ag-specific T cells result in specific lung pathologies. Both TH2 and TH17 cells are able to induce AHR, whereas TH17 cell-mediated airway inflammation and AHR are steroid resistant, indicating a potential role for TH17 cells in steroid-resistant asthma.
Impact of a Three Amino Acid Deletion in the CH2 Domain of Murine IgG1 on Fc-Associated Effector Functions
Authors: Baudino Lucie , Nimmerjahn Falk , Shinohara Yasuro , Furukawa Jun-Ichi , Petry Franz , Verbeek J. Sjef , Nishimura Shin-Ichiro , Ravetch Jeffery V. , Izui Shozo
Published: April 2014

Four murine IgG subclasses display markedly different Fc-associated effector functions because of their differential binding to three activating IgG Fc receptors (FcγRI, FcγRIII, and FcγRIV) and C1q. Previous analysis of IgG subclass switch variants of 34-3C anti-RBC monoclonal autoantibodies revealed that the IgG1 subclass, which binds only to FcγRIII and fails to activate complement, displayed the poorest pathogenic potential. This could be related to the presence of a three amino acid deletion at positions 233–235 in the CH2 domain uniquely found in this subclass. To address this question, IgG1 insertion and IgG2b deletion mutants at positions 233–235 of 34-3C anti-RBC Abs were generated, and their ability to initiate effector functions and their pathogenicity were compared with those of the respective wild-type Abs. The insertion of amino acid residues at positions 233–235 enabled the IgG1 subclass to bind FcγRIV but did not improve the binding to C1q. Accordingly, its pathogenicity was enhanced but still inferior to that of IgG2b. In contrast, the IgG2b deletion mutant lost its ability to bind to FcγRIV and activate complement. Consequently, its pathogenicity was markedly diminished to a level comparable to that of IgG1. Our results demonstrated that the initiation of FcγR- and complement-mediated effector functions of IgG2b was profoundly affected by the three amino acid deletion at positions 233–235, but that this natural three amino acid deletion could only partially explain the poor binding of IgG1 to FcγRIV and C1q. This indicates the lack in the IgG1 subclass of as yet unknown motifs promoting efficient interaction with FcγRIV and C1q.
A RAG1 Mutation Found in Omenn Syndrome Causes Coding Flank Hypersensitivity: A Novel Mechanism for Antigen Receptor Repertoire Restriction
Authors: Wong Serre-Yu , Lu Catherine P. , Roth David B.
Published: April 2014

Hypomorphic RAG mutants with severely reduced V(D)J recombination activity cause Omenn Syndrome (OS), an immunodeficiency with features of immune dysregulation and a restricted TCR repertoire. Precisely how RAG mutants produce autoimmune and allergic symptoms has been unclear. Current models posit that the severe recombination defect restricts the number of lymphocyte clones, a few of which are selected upon Ag exposure. We show that murine RAG1 R972Q, corresponding to an OS mutation, renders the recombinase hypersensitive to selected coding sequences at the hairpin formation step. Other RAG1 OS mutants tested do not manifest this sequence sensitivity. These new data support a novel mechanism for OS: by selectively impairing recombination at certain coding flanks, a RAG mutant can cause primary repertoire restriction, as opposed to a more random, limited repertoire that develops secondary to severely diminished recombination activity.
Sulfhydryl-Based Tumor Antigen-Carrier Protein Conjugates Stimulate Superior Antitumor Immunity against B Cell Lymphomas
Authors: Betting David J. , Kafi Kamran , Abdollahi-Fard Alireza , Hurvitz Sara A. , Timmerman John M.
Published: April 2014

Therapeutic vaccination of B cell lymphoma patients with tumor-specific Ig (idiotype, or Id) chemically coupled to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde has shown promising results in early clinical trials, and phase III trials are underway. However, glutaraldehyde Id-KLH vaccines fail to elicit anti-Id immune and clinical responses in many patients, possibly because glutaraldehyde reacts with lysine, cysteine, tyrosine, and histidine residues, damaging critical immunogenic epitopes. A sulfhydryl-based tumor Ag-carrier protein conjugation system using maleimide chemistry was used to enhance the efficacy of Id-KLH vaccines. Maleimide Id-KLH conjugates eradicated A20 lymphoma from most tumor-bearing mice, whereas glutaraldehyde Id-KLH had little efficacy. Maleimide Id-KLH elicited tumor-specific IgG Abs and T cells, with CD8+ T cells being the major effectors of antilymphoma immunity. Maleimide Id-KLH vaccines also demonstrated superior efficacy in 38C13 and BCL-1 lymphoma models, where Abs were shown to be critical for protection. Importantly, standard glutaraldehyde Id-KLH conjugation procedures could result in “overconjugation” of the tumor Ag, leading to decreased efficacy, whereas the heterobifunctional maleimide-based conjugation yielded potent vaccine product regardless of conjugation duration. Under lysosomal processing conditions, the Id-carrier protein linkage was cleavable only after maleimide conjugation. Maleimide KLH conjugation was easily performed with human Igs analogous to those used in Id-KLH clinical trials. These data support the evaluation of sulfhydryl-based Id-KLH vaccines in lymphoma clinical trials and possibly the use of tumor Ag-carrier protein vaccines for other cancers.
Importance of TLR2 in Early Innate Immune Response to Acute Pulmonary Infection with Porphyromonas gingivalis in Mice
Authors: Hajishengallis George , Wang Min , Bagby Gregory J. , Nelson Steve
Published: April 2014

The periodontal pathogen Porphyromonas gingivalis is implicated in certain systemic diseases including atherosclerosis and aspiration pneumonia. This organism induces innate responses predominantly through TLR2, which also mediates its ability to induce experimental periodontitis and accelerate atherosclerosis. Using a validated mouse model of intratracheal challenge, we investigated the role of TLR2 in the control of P. gingivalis acute pulmonary infection. TLR2-deficient mice elicited reduced proinflammatory or antimicrobial responses (KC, MIP-1α, TNF-α, IL-6, IL-12p70, and NO) in the lung and exhibited impaired clearance of P. gingivalis compared with normal controls. However, the influx of polymorphonuclear leukocytes into the lung and the numbers of resident alveolar macrophages (AM) were comparable between the two groups. TLR2 signaling was important for in vitro killing of P. gingivalis by polymorphonuclear leukocytes or AM and, moreover, the AM bactericidal activity required NO production. Strikingly, AM were more potent than peritoneal or splenic macrophages in P. gingivalis killing, attributed to diminished AM expression of complement receptor-3 (CR3), which is exploited by P. gingivalis to promote its survival. The selective expression of CR3 by tissue macrophages and the requirement of TLR2 inside-out signaling for CR3 exploitation by P. gingivalis suggest that the role of TLR2 in host protection may be contextual. Thus, although TLR2 may mediate destructive effects, as seen in models of experimental periodontitis and atherosclerosis, we have now shown that the same receptor confers protection against P. gingivalis in acute lung infection.
Francisella tularensisLive Vaccine Strain Induces Macrophage Alternative Activation as a Survival Mechanism
Authors: Shirey Kari Ann , Cole Leah E. , Keegan Achsah D. , Vogel Stefanie N.
Published: April 2014

Francisella tularensis (Ft), the causative agent of tularemia, elicits a potent inflammatory response early in infection, yet persists within host macrophages and can be lethal if left unchecked. We report in this study that Ft live vaccine strain (LVS) infection of murine macrophages induced TLR2-dependent expression of alternative activation markers that followed the appearance of classically activated markers. Intraperitoneal infection with Ft LVS also resulted in induction of alternatively activated macrophages (AA-Mφ). Induction of AA-Mφ by treatment of cells with rIL-4 or by infection with Ft LVS promoted replication of intracellular Ftn, in contrast to classically activated (IFN-γ plus LPS) macrophages that promoted intracellular killing of Ft LVS. Ft LVS failed to induce alternative activation in IL-4Rα−/− or STAT6−/− macrophages and prolonged the classical inflammatory response in these cells, resulting in intracellular killing of Ft. Treatment of macrophages with anti-IL-4 and anti-IL-13 Ab blunted Ft-induced AA-Mφ differentiation and resulted in increased expression of IL-12 p70 and decreased bacterial replication. In vivo, Ft-infected IL-4Rα−/− mice exhibited increased survival compared with wild-type mice. Thus, redirection of macrophage differentiation by Ft LVS from a classical to an alternative activation state enables the organism to survive at the expense of the host.
A Novel Role for Non-Neutralizing Antibodies against Nucleoprotein in Facilitating Resistance to Influenza Virus
Authors: Carragher Damian M. , Kaminski Denise A. , Moquin Amy , Hartson Louise , Randall Troy D.
Published: April 2014

Current influenza vaccines elicit Abs to the hemagglutinin and neuraminidase envelope proteins. Due to antigenic drift, these vaccines must be reformulated annually to include the envelope proteins predicted to dominate in the following season. By contrast, vaccination with the conserved nucleoprotein (NP) elicits immunity against multiple serotypes (heterosubtypic immunity). NP vaccination is generally thought to convey protection primarily via CD8 effector mechanisms. However, significant titers of anti-NP Abs are also induced, yet the involvement of Abs in protection has largely been disregarded. To investigate how Ab responses might contribute to heterosubtypic immunity, we vaccinated C57BL/6 mice with soluble rNP. This approach induced high titers of NP-specific serum Ab, but only poorly detectable NP-specific T cell responses. Nevertheless, rNP immunization significantly reduced morbidity and viral titers after influenza challenge. Importantly, Ab-deficient mice were not protected by this vaccination strategy. Furthermore, rNP-immune serum could transfer protection to naive hosts in an Ab-dependent manner. Therefore, Ab to conserved, internal viral proteins, such as NP, provides an unexpected, yet important mechanism of protection against influenza. These results suggest that vaccines designed to elicit optimal heterosubtypic immunity to influenza should promote both Ab and T cell responses to conserved internal proteins.
Lipids Including Cholesteryl Linoleate and Cholesteryl Arachidonate Contribute to the Inherent Antibacterial Activity of Human Nasal Fluid
Authors: Do Thai Q. , Moshkani Safiehkhatoon , Castillo Patricia , Anunta Suda , Pogosyan Adelina , Cheung Annie , Marbois Beth , Faull Kym F. , Ernst William , Chiang Su Ming , Fujii Gary , Clarke Catherine F. , Foster Krishna , Porter Edith
Published: April 2014

Mucosal surfaces provide first-line defense against microbial invasion through their complex secretions. The antimicrobial activities of proteins in these secretions have been well delineated, but the contributions of lipids to mucosal defense have not been defined. We found that normal human nasal fluid contains all major lipid classes (in micrograms per milliliter), as well as lipoproteins and apolipoprotein A-I. The predominant less polar lipids were myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid, cholesterol, and cholesteryl palmitate, cholesteryl linoleate, and cholesteryl arachidonate. Normal human bronchioepithelial cell secretions exhibited a similar lipid composition. Removal of less-polar lipids significantly decreased the inherent antibacterial activity of nasal fluid against Pseudomonas aeruginosa, which was in part restored after replenishing the lipids. Furthermore, lipids extracted from nasal fluid exerted direct antibacterial activity in synergism with the antimicrobial human neutrophil peptide HNP-2 and liposomal formulations of cholesteryl linoleate and cholesteryl arachidonate were active against P. aeruginosa at physiological concentrations as found in nasal fluid and exerted inhibitory activity against other Gram-negative and Gram-positive bacteria. These data suggest that host-derived lipids contribute to mucosal defense. The emerging concept of host-derived antimicrobial lipids unveils novel roads to a better understanding of the immunology of infectious diseases.
Trafficking of Antigen-Specific CD8+ T Lymphocytes to Mucosal Surfaces following Intramuscular Vaccination
Authors: Kaufman David R. , Liu Jinyan , Carville Angela , Mansfield Keith G. , Havenga Menzo J. E. , Goudsmit Jaap , Barouch Dan H.
Published: April 2014

A critical goal of vaccine development for a wide variety of pathogens is the induction of potent and durable mucosal immunity. However, it has been assumed that this goal would be difficult to achieve by systemic vaccination due to the anatomic and functional distinctness of the systemic and mucosal immune systems and the resultant compartmentalization of immune responses. In this study, we show that Ag-specific CD8+ T lymphocytes traffic efficiently to mucosal surfaces following systemic vaccination. Intramuscular immunization with recombinant adenovirus (rAd) vector-based vaccines expressing SIV Gag resulted in potent, durable, and functional CD8+ T lymphocyte responses at multiple mucosal effector sites in both mice and rhesus monkeys. In adoptive transfer studies in mice, vaccine-elicited systemic CD8+ T lymphocytes exhibited phenotypic plasticity, up-regulated mucosal homing integrins and chemokine receptors, and trafficked rapidly to mucosal surfaces. Moreover, the migration of systemic CD8+ T lymphocytes to mucosal compartments accounted for the vast majority of Ag-specific mucosal CD8+ T lymphocytes induced by systemic vaccination. Thus, i.m. vaccination can overcome immune compartmentalization and generate robust mucosal CD8+ T lymphocyte memory. These data demonstrate that the systemic and mucosal immune systems are highly coordinated following vaccination.
Elevated Numbers of FcγRIIIA+ (CD16+) Effector CD8 T Cells with NK Cell-Like Function in Chronic Hepatitis C Virus Infection
Authors: Björkström Niklas K. , Gonzalez Veronica D. , Malmberg Karl-Johan , Falconer Karolin , Alaeus Annette , Nowak Greg , Jorns Carl , Ericzon Bo-Göran , Weiland Ola , Sandberg Johan K. , Ljunggren Hans-Gustaf
Published: April 2014

CTL are crucial in the defense against viral infections. In the course of investigating peripheral blood and intrahepatic CD8 T cells in patients with chronic hepatitis C virus (HCV) infection, we observed a significant population of CD8 T cells expressing the FcγRIIIA (CD16) receptor. This observation led us to characterize these cells with respect to their phenotype and function in a cohort of patients with chronic HCV infection as well as in healthy blood donors. On average, 10% of peripheral blood CD8 T cells from HCV-infected patients expressed CD16 compared with only a few percent in healthy donors. CD16+ CD8 T cells displayed a late-stage effector phenotype with high levels of perforin. These cells exhibited a restricted TCR profile suggesting underlying clonal expansion. Stimulation of CD16 on CD8 T cells evoked a vigorous response similar to that of CD16 stimulation in NK cells. Our data suggest that CD8 T cells, during chronic HCV infection in humans, continue to differentiate beyond defined stages of terminal effector cells, acquiring CD16 and NK cell-like functional properties.
PIR-B-Deficient Mice Are Susceptible to Salmonella Infection
Authors: Torii Ikuko , Oka Satoshi , Hotomi Muneki , Benjamin William H. , Takai Toshiyuki , Kearney John F. , Briles David E. , Kubagawa Hiromi
Published: April 2014

Paired Ig-like receptors of activating (PIR-A) and inhibitory (PIR-B) isoforms are expressed by many hematopoietic cells, including B lymphocytes and myeloid cells. To determine the functional roles of PIR-A and PIR-B in primary bacterial infection, PIR-B-deficient (PIR-B−/−) and wild-type (WT) control mice were injected i.v. with an attenuated strain of Salmonella enterica Typhimurium (WB335). PIR-B−/− mice were found to be more susceptible to Salmonella infection than WT mice, as evidenced by high mortality rate, high bacterial loads in the liver and spleen, and a failure to clear bacteria from the circulation. Although blood levels of major cytokines and Salmonella-specific Abs were mostly comparable in the two groups of mice, distinct patterns of inflammatory lesions were found in their livers at 7–14 days postinfection: diffuse spreading along the sinusoids in PIR-B−/− mice vs nodular restricted localization in WT mice. PIR-B−/− mice have more inflammatory cells in the liver but fewer B cells and CD8+ T cells in the spleen than WT mice at 14 days postinfection. PIR-B−/− bone marrow-derived macrophages (BMMφ) failed to control intracellular replication of Salmonella in vitro, in part due to inefficient phagosomal oxidant production, when compared with WT BMMφ. PIR-B−/− BMMφ also produced more nitrite and TNF-α upon exposure to Salmonella than WT BMMφ did. These findings suggest that the disruption of PIR-A and PIR-B balance affects their regulatory roles in host defense to bacterial infection.
Identification of Functional Roles for Both IL-17RB and IL-17RA in Mediating IL-25-Induced Activities
Authors: Rickel Erika A. , Siegel Lori A. , Yoon Bo-Rin Park , Rottman James B. , Kugler David G. , Swart David A. , Anders Penny M. , Tocker Joel E. , Comeau Michael R. , Budelsky Alison L.
Published: April 2014

IL-25 (IL-17E) is a unique IL-17 family ligand that promotes Th2-skewed inflammatory responses. Intranasal administration of IL-25 into naive mice induces pulmonary inflammation similar to that seen in patients with allergic asthma, including increases in bronchoalveolar lavage fluid eosinophils, bronchoalveolar lavage fluid IL-5 and IL-13 concentrations, goblet cell hyperplasia, and increased airway hyperresponsiveness. IL-25 has been reported to bind and signal through IL-17RB (IL-17BR, IL-17Rh1). It has been demonstrated recently that IL-17A signals through a heteromeric receptor composed of IL-17RA and IL-17RC. We sought to determine whether other IL-17 family ligands also utilize heteromeric receptor complexes. The required receptor subunits for IL-25 biological activities were investigated in vitro and in vivo using a combination of knockout (KO) mice and antagonistic Abs. Unlike wild-type mice, cultured splenocytes from either IL-17RB KO or IL-17RA KO mice did not produce IL-5 or IL-13 in response to IL-25 stimulation, and both IL-17RB KO and IL-17RA KO mice did not respond to intranasal administration of IL-25. Furthermore, treatment with antagonistic mAbs to either IL-17RB or IL-17RA completely blocked IL-25-induced pulmonary inflammation and airway hyperresponsiveness in naive BALB/c mice, similar to the effects of an antagonistic Ab to IL-25. Finally, a blocking Ab to human IL-17RA prevented IL-25 activity in a primary human cell-based assay. These data demonstrate for the first time that IL-25-mediated activities require both IL-17RB and IL-17RA and provide another example of an IL-17 family ligand that utilizes a heteromeric receptor complex.
Serum Amyloid A Induces CCL2 Production via Formyl Peptide Receptor-Like 1-Mediated Signaling in Human Monocytes
Authors: Lee Ha Young , Kim Sang Doo , Shim Jae Woong , Lee Sun Young , Lee Hwahyung , Cho Kyung-Hyun , Yun Jeanho , Bae Yoe-Sik
Published: April 2014

Although the presence of an elevated level of serum amyloid A (SAA) has been regarded as a cardiovascular risk factor, the role of SAA on the progress of atherosclerosis has not been fully elucidated. In the present study, we investigated the effect of SAA on the production of CCL2, an important mediator of monocyte recruitment, and the mechanism underlying the action of SAA in human monocytes. The stimulation of human monocytes with SAA elicited CCL2 production in a concentration-dependent manner. The production of CCL2 by SAA was found to be mediated by the activation of NF-κB. Moreover, the signaling events induced by SAA included the activation of ERK and the induction of cyclooxygenase-2, which were required for the production of CCL2. Moreover, SAA-induced CCL2 induction was inhibited by a formyl peptide receptor-like 1 (FPRL1) antagonist. We also found that the stimulation of FPRL1-expressing RBL-2H3 cells induced CCL2 mRNA accumulation, but the vector-expressing RBL-2H3 cells combined with SAA did not. Taken together, our findings suggest that SAA stimulates CCL2 production and, thus, contributes to atherosclerosis. Moreover, FPRL1 was found to be engaged in SAA-induced CCL2 induction, and cyclooxygenase-2 induction was found to be essential for SAA-induced CCL2 expression. These results suggest that SAA and FPRL1 offer a developmental starting point for the treatment of atherosclerosis.
Potent Tumor-Specific Protection Ignited by Adoptively Transferred CD4+ T Cells
Authors: Liu Zuqiang , Noh Hae S. , Chen Janet , Kim Jin H. , Falo Louis D. , You Zhaoyang
Published: April 2014

Administration of anti-CD25 mAb before an aggressive murine breast tumor inoculation provoked effective antitumor immunity. Compared with CD4+ T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4+ T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by dendritic cells (DCs) produced more IFN-γ and IL-2, and less IL-17 in vitro, and ignited protective antitumor immunity in vivo in an adoptive transfer model. Tumor Ag-loaded DCs activated naive CD8+ T cells in the presence of these CD4+ T cells in vitro. Tumor Ag and adoptively transferred CD4+ T cells were both required for inducing a long-term tumor-specific IFN-γ-producing cellular response and potent protective antitumor activity. Although adoptively transferred CD4+ T cells ignited effective tumor-specific antitumor immunity in wild-type mice, they failed to do so in endogenous NK cell-depleted, Gr-1+ cell-depleted, CD40−/−, CD11c+ DC-depleted, B cell−/−, CD8+ T cell-depleted, or IFN-γ−/− mice. Collectively, the data suggest that adoptively transferred CD4+ T cells orchestrate both endogenous innate and adaptive immunity to generate effective tumor-specific long-term protective antitumor immunity. The data also demonstrate the pivotal role of endogenous DCs in the tumor-specific protection ignited by adoptively transferred CD4+ T cells. Thus, these findings highlight the importance of adoptively transferred CD4+ T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity.
Rapid Deletional Peripheral CD8 T Cell Tolerance Induced by Allogeneic Bone Marrow: Role of Donor Class II MHC and B Cells
Authors: Fehr Thomas , Wang Sophia , Haspot Fabienne , Kurtz Josef , Blaha Peter , Hogan Timothy , Chittenden Meredith , Wekerle Thomas , Sykes Megan
Published: April 2014

Mixed chimerism and donor-specific tolerance are achieved in mice receiving 3 Gy of total body irradiation and anti-CD154 mAb followed by allogeneic bone marrow (BM) transplantation. In this model, recipient CD4 cells are critically important for CD8 tolerance. To evaluate the role of CD4 cells recognizing donor MHC class II directly, we used class II-deficient donor marrow and were not able to achieve chimerism unless recipient CD8 cells were depleted, indicating that directly alloreactive CD4 cells were necessary for CD8 tolerance. To identify the MHC class II+ donor cells promoting this tolerance, we used donor BM lacking certain cell populations or used positively selected cell populations. Neither donor CD11c+ dendritic cells, B cells, T cells, nor donor-derived IL-10 were critical for chimerism induction. Purified donor B cells induced early chimerism and donor-specific cell-mediated lympholysis tolerance in both strain combinations tested. In contrast, positively selected CD11b+ monocytes/myeloid cells did not induce early chimerism in either strain combination. Donor cell preparations containing B cells were able to induce early deletion of donor-reactive TCR-transgenic 2C CD8 T cells, whereas those devoid of B cells had reduced activity. Thus, induction of stable mixed chimerism depends on the expression of MHC class II on the donor marrow, but no requisite donor cell lineage was identified. Donor BM-derived B cells induced early chimerism, donor-specific cell-mediated lympholysis tolerance, and deletion of donor-reactive CD8 T cells, whereas CD11b+ cells did not. Thus, BM-derived B cells are potent tolerogenic APCs for alloreactive CD8 cells.
CD8+ T Cells Targeting a Single Immunodominant Epitope are Sufficient for Elimination of Established SV40 T Antigen-Induced Brain Tumors
Authors: Tatum Angela M. , Mylin Lawrence M. , Bender Susan J. , Fischer Matthew A. , Vigliotti Beth A. , Tevethia M. Judith , Tevethia Satvir S. , Schell Todd D.
Published: April 2014

Immunotherapy of established solid tumors is rarely achieved, and the mechanisms leading to success remain to be elucidated. We previously showed that extended control of advanced-stage autochthonous brain tumors is achieved following adoptive transfer of naive C57BL/6 splenocytes into sublethally irradiated line SV11 mice expressing the SV40 T Ag (T Ag) oncoprotein, and was associated with in vivo priming of CD8+ T cells (TCD8) specific for the dominant epitope IV (T Ag residues 404–411). Using donor lymphocytes derived from mice that are tolerant to epitope IV or a newly characterized transgenic mouse line expressing an epitope IV-specific TCR, we show that epitope IV-specific TCD8 are a necessary component of the donor pool and that purified naive epitope IV-specific TCD8 are sufficient to promote complete and rapid regression of established tumors. While transfer of naive TCR-IV cells alone induced some initial tumor regression, increased survival of tumor-bearing mice required prior conditioning of the host with a sublethal dose of gamma irradiation and was associated with complete tumor eradication. Regression of established tumors was associated with rapid accumulation of TCR-IV T cells within the brain following initial priming against the endogenous T Ag in the peripheral lymphoid organs. Additionally, persistence of functional TCR-IV cells in both the brain and peripheral lymphoid organs was associated with long-term tumor-free survival. Finally, we show that production of IFN-γ, but not perforin or TNF-α, by the donor lymphocytes is critical for control of autochthonous brain tumors.
Elevated Maternal IL-16 Levels, Enhanced IL-16 Expressions in Endothelium and Leukocytes, and Increased IL-16 Production by Placental Trophoblasts in Women with Preeclampsia
Authors: Gu Yang , Lewis David F. , Deere Kelli , Groome Lynn J. , Wang Yuping
Published: April 2014

Cytokine IL-16 plays an important role in innate immune responses. However, little information is available about IL-16 function in human pregnancy. In this study, we collected maternal blood samples from 125 pregnant women between 26 and 41 wk of gestation, 63 from normal pregnant women and 62 from women with preeclampsia (PE). Serum IL-16C levels were measured by ELISA. We also examined IL-16C and IL-16N immunostaining in maternal vessels and protein expression in leukocytes from normal and PE pregnant women. In addition, IL-16C production by placental trophoblasts was also determined. Our results showed that IL-16C levels were significantly higher in severe PE than in mild PE and normal pregnant controls, 515 ± 58 vs 287 ± 46 (p < 0.05) and 163 ± 9 pg/ml (p < 0.01), respectively, indicating that increased IL-16 levels in PE is associated with the severity of the disease. There was no difference for the IL-16C levels in normal pregnant women throughout the third trimester. The correlation of maternal IL-16C levels with labor and body mass index was also analyzed. IL-16C levels were neither associated with labor nor associated with body mass index. Moreover, increased IL-16C immunostaining in maternal vessel endothelium and enhanced IL-16C protein expression in leukocytes were observed in PE. We also found that IL-16C production was increased by trophoblasts from PE placentas. Our study demonstrated up-regulation of the IL-16 profile in both the maternal and the placental systems in PE, suggesting that IL-16 could be an important cytokine engaged in the altered immune system and exaggerated inflammatory response in PE syndrome.
Transcriptional Outcome of Wnt-Frizzled Signal Transduction in Inflammation: Evolving Concepts
Authors: Sen Malini , Ghosh Gourisankar
Published: April 2014

Wnt-Frizzled signaling was first identified as a key event in Drosophila development. Over the years, ample evidence has accumulated regarding the multiple roles of Wnt-Frizzled signaling in mammalian cell differentiation and tissue/organ morphogenesis. It is thus not surprising that variations in the regulatory network of the Wnt signaling scheme would lead to alterations in cellular organization and cell activation and to the development of pathogenic conditions. Several reports have accordingly implied the involvement of Wnt-Frizzled signaling in the activation of proinflammatory mediators in inflammatory disorders. We will discuss how Wnt-Frizzled signaling may initiate/augment inflammation, focusing on its transcriptional outcome.
Only a Subset of Phosphoantigen-Responsive γ9δ2 T Cells Mediate Protective Tuberculosis Immunity
Authors: Spencer Charles T. , Abate Getahun , Blazevic Azra , Hoft Daniel F.
Published: April 2014

Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin (BCG) induce potent expansions of human memory Vγ9+Vδ2+ T cells capable of IFN-γ production, cytolytic activity, and mycobacterial growth inhibition. Certain phosphoantigens expressed by mycobacteria can stimulate γ9δ2 T cell expansions, suggesting that purified or synthetic forms of these phosphoantigens may be useful alone or as components of new vaccines or immunotherapeutics. However, we show that while mycobacteria-activated γ9δ2 T cells potently inhibit intracellular mycobacterial growth, phosphoantigen-activated γ9δ2 T cells fail to inhibit mycobacteria, although both develop similar effector cytokine and cytolytic functional capacities. γ9δ2 T cells receiving TLR-mediated costimulation during phosphoantigen activation also failed to inhibit mycobacterial growth. We hypothesized that mycobacteria express Ags, other than the previously identified phosphoantigens, that induce protective subsets of γ9δ2 T cells. Testing this hypothesis, we compared the TCR sequence diversity of γ9δ2 T cells expanded with BCG-infected vs phosphoantigen-treated dendritic cells. BCG-stimulated γ9δ2 T cells displayed a more restricted TCR diversity than phosphoantigen-activated γ9δ2 T cells. In addition, only a subset of phosphoantigen-activated γ9δ2 T cells functionally responded to mycobacteria-infected dendritic cells. Furthermore, differential inhibitory functions of BCG- and phosphoantigen-activated γ9δ2 T cells were confirmed at the clonal level and were not due to differences in TCR avidity. Our results demonstrate that BCG infection can activate and expand protective subsets of phosphoantigen-responsive γ9δ2 T cells, and provide the first indication that γ9δ2 T cells can develop pathogen specificity similar to αβ T cells. Specific targeting of protective γ9δ2 T cell subsets will be important for future tuberculosis vaccines.
Hepatitis C Virus-Specific Th17 Cells Are Suppressed by Virus-Induced TGF-β
Authors: Rowan Aileen G. , Fletcher Jean M. , Ryan Elizabeth J. , Moran Barry , Hegarty John E. , O'Farrelly Cliona , Mills Kingston H. G.
Published: April 2014

IL-17-secreting T (Th17) cells play a protective role in certain bacterial infections, but they are major mediators of inflammation and are pathogenic in organ-specific autoimmune diseases. However, human Th17 cells appear to be resistant to suppression by CD4+CD25+FoxP3+ regulatory T cells, suggesting that they may be regulated by alternative mechanisms. Herein we show that IL-10 and TGF-β suppressed IL-17 production by anti-CD3-stimulated PBMC from normal individuals. TGF-β also suppressed IL-17 production by purified CD4+ T cells, whereas the inhibitory effect of IL-10 on IL-17 production appears to be mediated predominantly by its effect on APC. An examination of patients infected with hepatitis C virus (HCV) demonstrated that Ag-specific Th17 cells are induced during infection and that these cells are regulated by IL-10 and TGF-β. PBMC from HCV Ab-positive donors secreted IL-17, IFN-γ, IL-10, and TGF-β in response to stimulation with the HCV nonstructural protein 4 (NS4). Furthermore, NS4 induced innate TGF-β and IL-10 expression by monocytes from normal donors and at higher levels from HCV-infected patients. Neutralization of TGF-β, and to a lesser extent IL-10, significantly enhanced NS4-specific IL-17 and IFN-γ production by T cells from HCV-infected donors. Our findings suggest that both HCV-specific Th1 and Th17 cells are suppressed by NS4-induced production of the innate anti-inflammatory cytokines IL-10 and TGF-β. This may represent a novel immune subversion mechanism by the virus to evade host-protective immune responses. Our findings also suggest that TGF-β and IL-10 play important roles in constraining the function of Th17 cells in general.
Lentiviral-Mediated Transcriptional Targeting of Dendritic Cells for Induction of T Cell Tolerance In Vivo
Authors: Dresch Christiane , Edelmann Stephanie L. , Marconi Peggy , Brocker Thomas
Published: April 2014

Dendritic cells (DCs) are important APCs able to induce both tolerance and immunity. Therefore, DCs are attractive targets for immune intervention. However, the ex vivo generation and manipulation of DCs at sufficient numbers and without changing their original phenotypic and functional characteristics are major obstacles. To manipulate DCs in vivo, we developed a novel DC-specific self-inactivating lentiviral vector system using the 5′ untranslated region from the DC-STAMP gene as a putative promoter region. We show that a gene therapy approach with these DC-STAMP-lentiviral vectors yields long-term and cell-selective transgene expression in vivo. Furthermore, transcriptionally targeted DCs induced functional, Ag-specific CD4 and CD8 T cell tolerance in vivo, which could not be broken by viral immunization. Tolerized CTL were unable to induce autoimmune diabetes in a murine autoimmune model system. Therefore, delivering transgenes specifically to DCs by using viral vectors might be a promising tool in gene therapy.