bluuebunny/crossref_metadata_2025 huggingface dataset:
Lyme Arthritis Synovial γδ T Cells Instruct Dendritic Cells via Fas Ligand
Authors: Collins Cheryl , Wolfe Julie , Roessner Karen , Shi Cuixia , Sigal Leonard H. , Budd Ralph C.
Published: April 2014

γδ T cells participate in the innate immune response to a variety of infectious microorganisms. They also link to the adaptive immune response through their induction of maturation of dendritic cells (DC) during the early phase of an immune response when the frequency of Ag-specific T cells is very low. We observe that in the presence of Borrelia burgdorferi, synovial Vδ1 T cells from Lyme arthritis synovial fluid potently induce maturation of DC, including production of IL-12, and increased surface expression of CD40 and CD86. The activated DC are then able to stimulate the Vδ1 T cells to up-regulate CD25. Both of these processes are initiated primarily by Fas stimulation rather than CD40 activation of DC via high expression of Fas ligand by the Vδ1 T cells. DC are resistant to Fas-induced death due to expression of high levels of the Fas inhibitor c-FLIP. This effect serves to divert Fas-mediated signals from the caspase cascade to the ERK MAPK and NF-κB pathways. The findings affirm the importance of the interaction of certain T cell populations with DC during the early phases of the innate immune response. They also underscore the view that as levels of c-FLIP increase, Fas signaling can be diverted from induction of apoptosis to pathways leading to cell effector function.
Murine Plasmacytoid Dendritic Cells Produce IFN-γ upon IL-4 Stimulation
Authors: Suto Akira , Nakajima Hiroshi , Tokumasa Naoki , Takatori Hiroaki , Kagami Shin-ichiro , Suzuki Kotaro , Iwamoto Itsuo
Published: April 2014

IL-4 plays a key role in inducing IL-4 production in CD4+ T cells, functioning as an important determinant for Th2 cell differentiation. We show here that IL-4 induces IFN-γ production in B220+ plasmacytoid dendritic cells (PDCs). By searching for cell populations that produce IFN-γ upon IL-4 stimulation, we found that PDCs were a major IFN-γ-producing cell upon IL-4 stimulation in wild-type and Rag-2−/− splenocytes. Isolated PDCs, but not CD11b+ DCs or CD8+ DCs, produced IFN-γ upon IL-4 stimulation. In vivo, the depletion of PDCs by anti-Ly6G/C Ab prevented IFN-γ production induced by IL-4 administration. We also found that IL-4 induced IFN-γ production, but not IL-12 or IFN-α production, in PDCs and also strongly enhanced CpG oligodeoxynucleotide-induced IFN-γ production, but not CpG oligodeoxynucleotide-induced IL-12 or IFN-α production. However, IL-4 did not induce IFN-γ production in Stat6−/− PDCs. Moreover, IL-4 induced Stat4 expression in PDCs through a Stat6-dependent mechanism, and only the Stat4-expressing PDCs produced IFN-γ. Furthermore, IL-4 did not induce IFN-γ production in Stat4−/− PDCs. These results indicate that PDCs preferentially produce IFN-γ upon IL-4 stimulation by Stat6- and Stat4-dependent mechanisms.
TLR9- and FcεRI-Mediated Responses Oppose One Another in Plasmacytoid Dendritic Cells by Down-Regulating Receptor Expression
Authors: Schroeder John T. , Bieneman Anja P. , Xiao HuiQing , Chichester Kristin L. , Vasagar Kavitha , Saini Sarbjit , Liu Mark C.
Published: April 2014

Plasmacytoid dendritic cells (pDC) express not only TLR9 molecules through which ligation with CpG DNA favors Th1 responses but also possess IgE receptors (FcεRI) implicated in allergen presentation and induction of Th2 responses. This dichotomy prompted an investigation to determine whether TLR9- and IgE receptor-mediated responses oppose one another in pDC by affecting receptor expression and associated functional responses. Results showed that IgE cross-linking reduced TLR9 in pDC and inhibited the capacity of these cells to secrete IFN-α when stimulated with the CpG oligodeoxynucleotide (ODN)-2216. In contrast, an ∼15-fold reduction in FcεRIα mRNA and a loss in surface protein were seen in pDC first exposed to TLR9 ligation with ODN-2216. Results indicated that type I IFNs partly mediated this effect, as rIFN-α also caused a significant ∼4-fold reduction in FcεRIα mRNA. Finally, this reduction in FcεRIα mediated by ODN-2216 correlated with a selective suppression of allergen-induced CD4+ T cell proliferation, but not of responses resulting from tetanus toxoid. Overall, these results imply mechanisms by which specific innate and IgE-dependent immune responses counterregulate one another at the dendritic cell level and may have significant impact on whether an ensuing response is either of Th1 or Th2 in nature.
JNK1 Is Essential for CD8+ T Cell-Mediated Tumor Immune Surveillance
Authors: Gao Yunfei , Tao Jian , Li Ming O. , Zhang Dongqing , Chi Hongbo , Henegariu Octavian , Kaech Susan M. , Davis Roger J. , Flavell Richard A. , Yin Zhinan
Published: April 2014

JNK1 has divergent roles in regulating the effector functions of CD4+ and CD8+ T cells. However, the function of JNK1 in tumor immune surveillance is unknown. In this study, we show that similar to IFN-γ−/− mice, JNK1−/− mice are highly susceptible to tumor development after inoculation of both melanoma cell line B16 and lymphoma cell line EL-4. Using T cell depletion and reconstitution approaches, we show that CD8+ T cells, but not CD4+ T cells, from JNK1−/− mice are responsible for tumor susceptibility. JNK1−/− CD8+ T cells have an intrinsic defect in early IFN-γ gene transcription and production after activation by either anti-CD3/anti-CD28 Abs or dendritic cells loaded with specific Ag in vitro. The impaired IFN-γ production in JNK1−/− CD8+ T cells is associated with reduced expression of both T-bet and Eomesodermin, indicating that JNK1 regulates the transcription program of CD8+ T cells. Finally, JNK1−/− CD8+ T cells showed reduced perforin expression and impaired CTL function. Taken together, our results demonstrate that JNK1 plays an important role in tumor immune surveillance through regulating the effector functions of CD8+ T cells.
Recognition of Fresh Human Tumor by Human Peripheral Blood Lymphocytes Transduced with a Bicistronic Retroviral Vector Encoding a Murine Anti-p53 TCR
Authors: Cohen Cyrille J. , Zheng Zhili , Bray Regina , Zhao Yangbing , Sherman Linda A. , Rosenberg Steven A. , Morgan Richard A.
Published: April 2014

The p53 protein is markedly up-regulated in a high proportion of human malignancies. Using an HLA-A2 transgenic mouse model, it was possible to isolate high-avidity murine CTLs that recognize class I-restricted human p53 epitopes. We isolated the α- and β-chain of a TCR from a highly avid murine CTL clone that recognized the human p53264–272 epitope. These genes were cloned into a retroviral vector that mediated high efficiency gene transfer into primary human lymphocytes. Efficiencies of >90% for gene transfer into lymphocytes were obtained without selection for transduced cells. The p53 TCR-transduced lymphocytes were able to specifically recognize with high-avidity, peptide-pulsed APCs as well as HLA-A2.1+ cells transfected with either wild-type or mutant p53 protein. p53 TCR-transduced cells demonstrated recognition and killing of a broad spectrum of human tumor cell lines as well as recognition of fresh human tumor cells. Interestingly, both CD8+ and CD4+ subsets were capable of recognizing and killing target cells, stressing the potential application of such a CD8-independent TCR molecule that can mediate both helper and cytotoxic responses. These results suggest that lymphocytes genetically engineered to express anti-p53 TCR may be of value for the adoptive immunotherapy of patients with a variety of common malignancies.
IL-12 and IL-10 Expression Synergize to Induce the Immune-Mediated Eradication of Established Colon and Mammary Tumors and Lung Metastasis
Authors: Lopez M. Verónica , Adris Soraya K. , Bravo Alicia I. , Chernajovsky Yuti , Podhajcer Osvaldo L.
Published: April 2014

Preclinical studies demonstrated that certain cytokines are potentially useful for the induction of antitumor immune responses. However, their administration in clinical settings was only marginally useful and evoked serious toxicity. In this study, we demonstrate that the combination of autologous inactivated tumor cells expressing IL-12 and IL-10 induced tumor remission in 50–70% of mice harboring large established colon or mammary tumors and spontaneous lung metastases, with the consequent establishment of an antitumor immune memory. Mice treatment with tumor cells expressing IL-12 was only marginally effective, while expression of IL-10 was not effective at all. Administration of the combined immunotherapy stimulated the recruitment of a strong inflammatory infiltrate that correlated with local, increased expression levels of the chemokines MIP-2, MCP-1, IFN-γ-inducible protein-10, and TCA-3 and the overexpression of IFN-γ, but not IL-4. The combined immunotherapy was also therapeutically effective on established lung metastases from both colon and mammary tumors. The antitumor effect of the combined immunotherapy was mainly dependent on CD8+ cells although CD4+ T cells also played a role. The production of IFN-γ and IL-4 by spleen cells and the development of tumor-specific IgG1 and IgG2a Abs indicate that each cytokine stimulated its own Th pathway and that both arms were actively engaged in the antitumor effect. This study provides the first evidence of a synergistic antitumor effect of IL-12 and IL-10 suggesting that a Th1 and a Th2 cytokine can be effectively combined as a novel rational approach for cancer immunotherapy.
Molecular Signatures Distinguish Human Central Memory from Effector Memory CD8 T Cell Subsets
Authors: Willinger Tim , Freeman Tom , Hasegawa Hitoshi , McMichael Andrew J. , Callan Margaret F. C.
Published: April 2014

Memory T cells are heterogeneous in terms of their phenotype and functional properties. We investigated the molecular profiles of human CD8 naive central memory (TCM), effector memory (TEM), and effector memory RA (TEMRA) T cells using gene expression microarrays and phospho-protein-specific intracellular flow cytometry. We demonstrate that TCM have a gene expression and cytokine signaling signature that lies between that of naive and TEM or TEMRA cells, whereas TEM and TEMRA are closely related. Our data define the molecular basis for the different functional properties of central and effector memory subsets. We show that TEM and TEMRA cells strongly express genes with known importance in CD8 T cell effector function. In contrast, TCM are characterized by high basal and cytokine-induced STAT5 phosphorylation, reflecting their capacity for self-renewal. Altogether, our results distinguish TCM and TEM/TEMRA at the molecular level and are consistent with the concept that TCM represent memory stem cells.
Respiratory Syncytial Virus Infection of Monocyte-Derived Dendritic Cells Decreases Their Capacity to Activate CD4 T Cells
Authors: de Graaff Patricia M. A. , de Jong Esther C. , van Capel Toni M. , van Dijk Mariska E. A. , Roholl Paul J. M. , Boes Jolande , Luytjes Willem , Kimpen Jan L. L. , van Bleek Grada M.
Published: April 2014

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in children, the elderly, and immune-compromised individuals. CD4 and CD8 T cells play a crucial role in the elimination of RSV from the infected lung, but T cell memory is not sufficient to completely prevent reinfections. The nature of the adaptive immune response depends on innate immune reactions initiated after interaction of invading pathogens with host APCs. For respiratory pathogens myeloid dendritic cell (DC) precursors that are located underneath the epithelial cell layer lining the airways may play a crucial role in primary activation of T cells and regulating their functional potential. In this study, we investigated the role of human monocyte-derived DC in RSV infection. We showed that monocyte-derived DC can be productively infected, which results in maturation of the DC judged by the up-regulation of CD80, CD83, CD86, and HLA class II molecules. However, RSV infection of DC caused impaired CD4 T cell activation characterized by a lower T cell proliferation and ablation of cytokine production in activated T cells. The suppressive effect was caused by an as yet unidentified soluble factor produced by RSV-infected DC.
Signaling through a Mutant IFN-γ Receptor
Authors: Costa-Pereira Ana P. , Hermanns Heike M. , Is’harc Hayaatun , Williams Timothy M. , Watling Diane , Arulampalam Velmurugesan , Newman Sally J. , Heinrich Peter C. , Kerr Ian M.
Published: April 2014

Activation of STAT1 and the IFN-γ response are thought to be mediated exclusively through the Y440 motif of the human IFNGR1 receptor subunit. Contrary to this accepted dogma, here it is shown that IFNGR1 with a mutant (Y440F) motif, when stably expressed in IFNGR1-negative human fibroblasts at levels similar to wild type, can sustain a substantial IFN-γ response. The mutant receptor supports selective induction of IFN-γ-inducible genes but is notably defective in the CIITA, class II HLA, suppressor of cytokine signaling and antiviral responses. Remarkably, similar selective defects are observed in human fibrosarcoma cells expressing a mutant JAK1. The phenotypes are novel and appear distinct from those observed in response to the inhibition of known additional pathways. Data from different cell types further emphasizes the importance of cellular background in determining the response.
Epigenetic Control of Highly Homologous Killer Ig-Like Receptor Gene Alleles
Authors: Chan Huei-Wei , Miller Jeffrey S. , Moore Mikel B. , Lutz Charles T.
Published: April 2014

Mature human NK lymphocytes express the highly homologous killer Ig-like receptor (KIR) genes in a stochastic fashion, and KIR transcription precisely correlates with allele-specific DNA methylation. In this study, we demonstrate that CpG methylation of a minimal KIR promoter inhibited transcription. In human peripheral blood NK cells and long-term cell lines, expressed KIR genes were associated with a moderate level of acetylated histone H3 and H4 and trimethylated histone H3 lysine 4. Histone modifications were preferentially associated with the transcribed allele in NK cell lines with monoallelic KIR expression. Although reduced, a substantial amount of histone acetylation and H3 lysine 4 trimethylation also was associated with nonexpressed KIR genes. DNA hypomethylation correlated with increased chromatin accessibility, both in vitro and in vivo. Treatment of NK cell lines and developing NK cells with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine, caused a dramatic increase in KIR RNA and protein expression, but little change in histone modification. Our findings suggest that KIR transcription is primarily controlled by DNA methylation.
The Cyclic AMP Response Element Modulator Regulates Transcription of the TCR ζ-Chain
Authors: Tenbrock Klaus , Kyttaris Vasileios C. , Ahlmann Martina , Ehrchen Jan Mauno , Tolnay Mate , Melkonyan Harutyun , Mawrin Christian , Roth Johannes , Sorg Clemens , Juang Yuang-Taung , Tsokos George C.
Published: April 2014

Systemic lupus erythematosus T cells display decreased amounts of TCR ζ mRNA that results in part from limited binding of the transcriptional enhancer Elf-1 to the TCR ζ promoter. We have identified a new cis-binding site for the cAMP response element (CRE) modulator (CREM) on the TCR ζ promoter, centered on the −390 nucleotide. Transfection of T cells with an antisense CREM α plasmid reduced the binding of CREM to the TCR ζ promoter, as shown by chromatin and reporter chromatin immunoprecipitation assays, and enhanced the production of TCR ζ mRNA and protein. Mutagenesis of the −390 CRE site prevented the binding of CREM to the TCR ζ promoter. The mechanism of CREM-mediated repression appears to be chromatin dependent, because antisense CREM promotes the acetylation of histones on the TCR ζ promoter. Finally, we established an enhanced binding of CREM to the TCR ζ-chain promoter in systemic lupus erythematosus cells compared with control T cells. Our studies demonstrate that CREM α binds to the TCR ζ promoter and repress its activity.
The High Frequency Indian Rhesus Macaque MHC Class I Molecule, Mamu-B*01, Does Not Appear to Be Involved in CD8+ T Lymphocyte Responses to SIVmac239
Authors: Loffredo John T. , Sidney John , Piaskowski Shari , Szymanski Andrew , Furlott Jessica , Rudersdorf Richard , Reed Jason , Peters Bjoern , Hickman-Miller Heather D. , Bardet Wilfried , Rehrauer William M. , O’Connor David H. , Wilson Nancy A. , Hildebrand William H. , Sette Alessandro , Watkins David I.
Published: April 2014

Although the SIV-infected Indian rhesus macaque (Macaca mulatta) is the animal model most widely used for studying HIV infection, our current understanding of the functional macaque MHC class I molecules is limited. To date, SIV-derived CD8+ T lymphocyte epitopes from only three high frequency macaque MHC class I molecules have been extensively characterized. In this study, we defined the peptide-binding properties of the high frequency Indian rhesus macaque class I molecule, Mamu-B*01 (∼26%). We first identified a preliminary binding motif by eluting and sequencing endogenously bound Mamu-B*01 ligands. We further characterized the peptide-binding characteristics using panels of single amino acid substitution analogs. Using this detailed motif, 507 peptides derived from SIVmac239 were identified and tested for their Mamu-B*01 binding capacity. Surprisingly, only 11 (2.2%) of these motif-containing peptides bound with IC50 values ≤500 nM. We assessed the immunogenicity of these peptides using freshly isolated PBMC from ten Mamu-B*01+ SIV-infected rhesus macaques in IFN-γ ELISPOT and IFN-γ/TNF-α intracellular cytokine staining assays. Lymphocytes from these SIV-infected macaques responded to none of these peptides. Furthermore, there was no sequence variation indicative of escape in the regions of the virus that encoded these peptides. Additionally, we could not confirm previous reports of SIV-derived Mamu-B*01-restricted epitopes in the Env and Gag proteins. Our results suggest that the high frequency MHC class I molecule, Mamu-B*01, is not involved in SIV-specific CD8+ T lymphocyte responses.
TNF Receptor-Associated Factor 6-Dependent CD40 Signaling Primes Macrophages to Acquire Antimicrobial Activity in Response to TNF-α
Authors: Andrade Rosa M. , Wessendarp Matthew , Portillo Jose-Andres C. , Yang Jun-Qi , Gomez Francisco J. , Durbin Joan E. , Bishop Gail A. , Subauste Carlos S.
Published: April 2014

IFN-γ is considered an essential stimulus that allows macrophages to acquire activity against intracellular pathogens in response to a second signal such as TNF-α. However, protection against important pathogens can take place in the absence of IFN-γ through mechanisms that are still dependent on TNF-α. Engagement of CD40 modulates antimicrobial activity in macrophages. However, it is not known whether CD40 can replace IFN-γ as priming signal for induction of this response. We show that CD40 primes mouse macrophages to acquire antimicrobial activity in response to TNF-α. The effect of CD40 was not caused by modulation of IL-10 and TGF-β production or TNFR expression and did not require IFN-αβ signaling. Induction of antimicrobial activity required cooperation between TNFR-associated factor 6-dependent CD40 signaling and TNFR2. These results support a paradigm where TNFR-associated factor 6 signaling downstream of CD40 alters the pattern of response of macrophages to TNF-α leading to induction of antimicrobial activity.
Differential TLR Recognition of Leptospiral Lipid A and Lipopolysaccharide in Murine and Human Cells
Authors: Nahori Marie-Anne , Fournié-Amazouz Edith , Que-Gewirth Nanette S. , Balloy Viviane , Chignard Michel , Raetz Christian R. H. , Saint Girons Isabelle , Werts Catherine
Published: April 2014

Leptospira interrogans is a spirochete that is responsible for leptospirosis, a zoonotic disease. This bacterium possesses an unusual LPS that has been shown to use TLR2 instead of TLR4 for signaling in human cells. The structure of its lipid A was recently deciphered. Although its overall hexa-acylated disaccharide backbone is a classical feature of all lipid A forms, the lipid A of L. interrogans is peculiar. In this article, the functional characterization of this lipid A was studied in comparison to whole parental leptospiral LPS in terms of cell activation and use of TLR in murine and human cells. Lipid A from L. interrogans did not coagulate the Limulus hemolymph. Although leptospiral lipid A activated strongly murine RAW cells, it did not activate human monocytic cells. Results obtained from stimulation of peritoneal-elicited macrophages from genetically deficient mice for TLR2 or TLR4 clearly showed that lipid A stimulated the cells through TLR4 recognition, whereas highly purified leptospiral LPS utilized TLR2 as well as TLR4. In vitro experiments with transfected human HEK293 cells confirmed that activation by lipid A occurred only through murine TLR4-MD2 but not through human TLR4-MD2, nor murine or human TLR2. Similar studies with parental leptospiral LPS showed that TLR2/TLR1 were the predominant receptors in human cells, whereas TLR2 but also TLR4 contributed to activation in murine cells. Altogether these results highlight important differences between human and mouse specificity in terms of TLR4-MD2 recognition that may have important consequences for leptospiral LPS sensing and subsequent susceptibility to leptospirosis.
Nef Is Physically Recruited into the Immunological Synapse and Potentiates T Cell Activation Early after TCR Engagement
Authors: Fenard David , Yonemoto Wes , de Noronha Carlos , Cavrois Marielle , Williams Samuel A. , Greene Warner C.
Published: April 2014

The HIV-1 protein Nef enhances viral pathogenicity and accelerates disease progression in vivo. Nef potentiates T cell activation by an unknown mechanism, probably by optimizing the intracellular environment for HIV replication. Using a new T cell reporter system, we have found that Nef more than doubles the number of cells expressing the transcription factors NF-κB and NFAT after TCR stimulation. This Nef-induced priming of TCR signaling pathways occurred independently of calcium signaling and involved a very proximal step before protein kinase C activation. Engagement of the TCR by MHC-bound Ag triggers the formation of the immunological synapse by recruiting detergent-resistant membrane microdomains, termed lipid rafts. Approximately 5–10% of the total cellular pool of Nef is localized within lipid rafts. Using confocal and real-time microscopy, we found that Nef in lipid rafts was recruited into the immunological synapse within minutes after Ab engagement of the TCR/CD3 and CD28 receptors. This recruitment was dependent on the N-terminal domain of Nef encompassing its myristoylation. Nef did not increase the number of cell surface lipid rafts or immunological synapses. Recently, studies have shown a specific interaction of Nef with an active subpopulation of p21-activated kinase-2 found only in the lipid rafts. Thus, the corecruitment of Nef and key cellular partners (e.g., activated p21-activated kinase-2) into the immunological synapse may underlie the increased frequency of cells expressing transcriptionally active forms of NF-κB and NFAT and the resultant changes in T cell activation.
CD80/CD86 Costimulation Regulates Acute Vascular Rejection
Authors: Hosiawa Karoline A. , Wang Hao , DeVries Mark E. , Garcia Bertha , Liu Weihua , Zhou Dejun , Akram Ali , Jiang Jifu , Sun Hongtao , Cameron Mark J. , Zhong Robert , Kelvin David J.
Published: April 2014

Xenotransplantation may provide the only solution to the shortage of human donor organs. Although hyperacute rejection associated with xenotransplantation can now be overcome, acute vascular rejection (AVR) remains a primary barrier to xenotransplantation. To date, standard immunosuppressive agents fail to block AVR or prolong xenograft survival. The present study was undertaken to determine the role of CD80/CD86 costimulatory molecules in regulating AVR. Lewis rat hearts were transplanted heterotopically into wild-type or IL-12, CD80- or CD86-deficient C57BL/6 mice. Wild-type recipients were treated with CD80 or CD86 neutralizing Ab with and without daily cyclosporin A (CsA, 15 mg/kg). Transplanted hearts in untreated wild-type recipients were rejected on postoperative days (POD) 17–21 and showed cell-mediated rejection (CMR) and AVR pathologies. In contrast, transplanted hearts in IL-12 and CD80 recipients or wild-type recipients treated with CD80 neutralizing Ab were rapidly rejected on POD 5 and 6 with AVR pathology. Interestingly, hearts transplanted into CD86 knockout recipients or wild-type recipients treated with CD86 neutralizing Ab underwent CMR on POD 17. Finally, blockade of CD86 but not CD80 rendered xenograft recipients sensitive to daily CsA therapy, leading to indefinite xenograft survival. To conclude, we demonstrate that AVR can be overcome by blocking the CD86 costimulatory pathway. Furthermore, we demonstrate that CD80 and CD86 have opposing roles in regulation of xenotransplantation rejection, where CD80 drives CMR and attenuates AVR while CD86 drives AVR. Most strikingly, indefinite xenograft survival can be achieved by suppressing AVR with CD86 neutralization in combination of CsA therapy, which inhibits CMR.
Protection against Osteoporosis by Active Immunization with TRANCE/RANKL Displayed on Virus-Like Particles
Authors: Spohn Gunther , Schwarz Katrin , Maurer Patrik , Illges Harald , Rajasekaran Narendiran , Choi Yongwon , Jennings Gary T. , Bachmann Martin F.
Published: April 2014

TNF-related activation-induced cytokine (TRANCE), also known as receptor activator of NF-κB ligand (RANKL), is the key molecule responsible for the bone loss observed in osteoporosis. Passive administration of osteoprotegerin, the soluble decoy receptor of TRANCE/RANKL, is efficient in blocking disease progression, but may not find widespread clinical use due to patient compliance problems and the expected high costs. In this study, we describe an efficient, safe, and potentially cost-effective active immunization strategy against TRANCE/RANKL. We show in mice that immunization with TRANCE/RANKL covalently linked to virus-like particles can overcome the natural tolerance of the immune system toward self proteins and produce high levels of specific Abs without the addition of any adjuvant. Serum Abs of immunized mice neutralized TRANCE/RANKL activity in vitro and were highly active in preventing bone loss in a mouse model of osteoporosis. Active immunization against TRANCE/RANKL was essentially reversible and did not produce any measurable immunosuppressive side effects, underscoring its potential as a new therapeutic approach to the treatment of human bone-degenerative disorders.
TCR trans-Rearrangements: Biological Significance in Antigen Recognition vs the Role as Lymphoma Biomarker
Authors: Allam Atef , Kabelitz Dieter
Published: April 2014

V(D)J rearrangements occur within loci of TCR and BCR genes, thus generating the diversity of the AgR repertoire. In addition, interlocus V(D)J rearrangements occur, giving rise to so-called “trans-rearrangements.” Such trans-rearrangements increase the diversity of the immune receptor repertoire and can be expressed as functional chimeric TCR proteins on the surface of T cells. Although chimeric receptors are not pathogenic per se, the frequency of AgR trans-rearrangements correlates with the level of genetic instability and thus could be used as a predictive biomarker for lymphoma risk.
Cutting Edge: Constitutive B Cell Receptor Signaling Is Critical for Basal Growth of B Lymphoma
Authors: Gururajan Murali , Jennings C. Darrell , Bondada Subbarao
Published: April 2014

B lymphomas account for the majority of the lymphoma cases. BCR expression appears to be important for B lymphoma because most oncogenes are translocated to nonrearranged Ig loci and because all of the variants that arise in anti-idiotypic Ab-treated lymphoma patients remain BCR positive. Based on this and the fact that BCR is required for mature B cell survival, we tested the requirement for continued expression of BCR for the growth and survival of B lymphoma cells. Using Igα or Igβ-specific small interfering RNA (siRNA) to inhibit BCR expression, we demonstrate for the first time that constitutive signaling by BCR is critical for survival and proliferation of both murine and human B lymphoma cells. The BCR signals in lymphoma appear to be mediated by Syk, as it is constitutively active in a variety of B lymphoma cells. Blocking Syk activity by selective inhibitors suppresses growth of several murine and human B lymphomas.
Cutting Edge: Nonproliferating Mature Immune Cells Form a Novel Type of Organized Lymphoid Structure in Idiopathic Pulmonary Fibrosis
Authors: Marchal-Sommé Joëlle , Uzunhan Yurdagul , Marchand-Adam Sylvain , Valeyre Dominique , Soumelis Vassili , Crestani Bruno , Soler Paul
Published: April 2014

Ectopic formation of secondary lymphoid tissue is initiated by the local attraction of naive T and B cells. In this study, we describe a novel type of organized lymphoid structure in the lung of human idiopathic pulmonary fibrosis, with key features of lymphoid neogenesis, including: 1) recently activated CD40 ligand (CD40L)+ T cells; 2) variable numbers of activated CD40+/CD40L+ B cells, sometimes organized in follicles; 3) fully mature dendritic cells (DC) expressing CD40, CD83, CD86, and DC-lysosome-associated membrane protein; 4) the expression of the chemokine CCL21; 5) the presence of vessels with characteristics of high endothelial venules; and 6) a dense network of follicular DC. Surprisingly, these structures are devoid of CCR7+ naive T cells, proliferating lymphocytes, and germinal centers, suggesting that newly recruited activated DC and Ag-experienced lymphocytes can drive lymphoid neogenesis and that factors present within the lymphoid aggregates, such as CD40L, are essential to induce DC maturation.
CD25+Foxp3+ Regulatory T Cells Facilitate CD4+ T Cell Clonal Anergy Induction during the Recovery from Lymphopenia
Authors: Vanasek Tracy L. , Nandiwada Sarada L. , Jenkins Marc K. , Mueller Daniel L.
Published: April 2014

T cell clonal anergy induction in lymphopenic nu/nu mice was found to be ineffective. Exposure to a tolerizing peptide Ag regimen instead induced aggressive CD4+ cell cycle progression and increased Ag responsiveness (priming). Reconstitution of T cell-deficient mice by an adoptive transfer of mature peripheral lymphocytes was accompanied by the development of a CD25+Foxp3+CTLA-4+CD4+ regulatory T cell population that acted to dampen Ag-driven cell cycle progression and facilitate the induction of clonal anergy in nearby responder CD25−CD4+ T cells. Thus, an early recovery of CD25+ regulatory T cells following a lymphopenic event can prevent exuberant Ag-stimulated CD4+ cell cycle progression and promote the development of clonal anergy.
Inducible Costimulator: A Modulator of IFN-γ Production in Human Tuberculosis
Authors: Quiroga María F. , Pasquinelli Virginia , Martínez Gustavo J. , Jurado Javier O. , Zorrilla Liliana Castro , Musella Rosa M. , Abbate Eduardo , Sieling Peter A. , García Verónica E.
Published: April 2014

Effective host defense against Mycobacterium tuberculosis requires the induction of Th1 cytokine responses. We investigated the regulated expression and functional role of the inducible costimulator (ICOS), a receptor known to regulate Th cytokine production, in the context of human tuberculosis. Patients with active disease, classified as high responder (HR) or low responder (LR) patients according to their in vitro T cell responses against the Ag, were evaluated for T cell expression of ICOS after M. tuberculosis-stimulation. We found that ICOS expression significantly correlated with IFN-γ production by tuberculosis patients. ICOS expression levels were regulated in HR patients by Th cytokines: Th1 cytokines increased ICOS levels, whereas Th2-polarizing conditions down-regulated ICOS in these individuals. Besides, in human polarized Th cells, engagement of ICOS increased M. tuberculosis IFN-γ production with a magnitude proportional to ICOS levels on those cells. Moreover, ICOS ligation augmented Ag-specific secretion of the Th1 cytokine IFN-γ from responsive individuals. In contrast, neither Th1 nor Th2 cytokines dramatically affected ICOS levels on Ag-stimulated T cells from LR patients, and ICOS activation did not enhance IFN-γ production. However, simultaneous activation of ICOS and CD3 slightly augmented IFN-γ secretion by LR patients. Together, our data suggest that the regulation of ICOS expression depends primarily on the response of T cells from tuberculosis patients to the specific Ag. IFN-γ released by M. tuberculosis-specific T cells modulates ICOS levels, and accordingly, ICOS ligation induces IFN-γ secretion. Thus, ICOS activation may promote the induction of protective Th1 cytokine responses to intracellular bacterial pathogens.
Dual Effects of Sprouty1 on TCR Signaling Depending on the Differentiation State of the T Cell
Authors: Choi Heonsik , Cho Sung-Yup , Schwartz Ronald H. , Choi Kyungho
Published: April 2014

Sprouty (Spry) is known to be a negative feedback inhibitor of growth factor receptor signaling through inhibition of the Ras/MAPK pathway. Several groups, however, have reported a positive role for Spry involving sequestration of the inhibitory protein c-Cbl. Thus, Spry may have various functions in the regulation of receptor-mediated signaling depending on the context. In the immune system, the function of Spry is unknown. In this study, we investigated the role of Spry1 in T cell activation. Spry1, among the four mammalian homologs, was specifically induced by TCR signaling of CD4+ murine T cells. In fully differentiated Th1 clones, overexpressed Spry1 inhibited TCR signaling and decreased IL-2 production while reducing expression with specific siRNA transfection had the opposite effect, increasing IL-2 production. In contrast, in naive T cells, Spry1 overexpression enhanced TCR signaling, and increased proliferation and IL-2 production, while siRNA transfection again had the opposite effect, reducing IL-2 production following activation. The enhancing effect in naive cells was abrogated by preactivation of the T cells with Ag and APC, indicating that the history of exposure to Ag is correlated with a hierarchy of T cell responsiveness to Spry1. Furthermore, both the NF-AT and MAPK pathways were influenced by Spry1, implying a different molecular mechanism from that for growth factor receptor signaling. Thus, Spry1 uses a novel mechanism to bring about differential effects on TCR signaling through the same receptor, depending on the differentiation state of the T cell.
Monoclonal Antibodies for Bacillus anthracis Spore Detection and Functional Analyses of Spore Germination and Outgrowth
Authors: Swiecki Melissa K. , Lisanby Mark W. , Shu Fengyu , Turnbough Charles L. , Kearney John F.
Published: April 2014

All members of the Bacillus genus produce endospores as part of their life cycle; however, it is not possible to determine the identity of spores by casual or morphological examination. The 2001 anthrax attacks demonstrated a need for fast, dependable methods for detecting Bacillus anthracis spores in vitro and in vivo. We have developed a variety of isotypes and specificities of mAbs that were able to distinguish B. anthracis spores from other Bacillus spores. The majority of Abs were directed toward BclA, a major component of the exosporium, although other components were also distinguished. These Abs did not react with vegetative forms. Some Abs distinguished B. anthracis spores from spores of distantly related species in a highly specific manner, whereas others discriminated among strains that are the closest relatives of B. anthracis. These Abs provide a rapid and reliable means of identifying B. anthracis spores, for probing the structure and function of the exosporium, and in the analysis of the life cycle of B. anthracis.
Cross-Neutralization of Human and Palm Civet Severe Acute Respiratory Syndrome Coronaviruses by Antibodies Targeting the Receptor-Binding Domain of Spike Protein
Authors: He Yuxian , Li Jingjing , Li Wenhui , Lustigman Sara , Farzan Michael , Jiang Shibo
Published: April 2014

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is considered as a protective Ag for vaccine design. We previously demonstrated that the receptor-binding domain (RBD) of S protein contains multiple conformational epitopes (Conf I-VI) that confer the major target of neutralizing Abs. Here we show that the recombinant RBDs derived from the S protein sequences of Tor2, GD03, and SZ3, the representative strains of human 2002–2003 and 2003–2004 SARS-CoV and palm civet SARS-CoV, respectively, induce in the immunized mice and rabbits high titers of cross-neutralizing Abs against pseudoviruses expressing S proteins of Tor2, GD03, and SZ3. We also demonstrate that the Tor2-RBD induced-Conf I-VI mAbs can potently neutralize both human SARS-CoV strains, Tor2 and GD03. However, only the Conf IV-VI, but not Conf I-III mAbs, neutralize civet SARS-CoV strain SZ3. All these mAbs reacted significantly with each of the three RBD variants (Tor2-RBD, GD03-RBD, and SZ3-RBD) that differ at several amino acids. Regardless, the Conf I-IV and VI epitopes were completely disrupted by single-point mutation of the conserved residues in the RBD (e.g., D429A, R441A, or D454A) and the Conf III epitope was significantly affected by E452A or D463A substitution. Interestingly, the Conf V epitope, which may overlap the receptor-binding motif and induce most potent neutralizing Abs, was conserved in these mutants. These data suggest that the major neutralizing epitopes of SARS-CoV have been apparently maintained during cross-species transmission, and that RBD-based vaccines may induce broad protection against both human and animal SARS-CoV variants.
B Cells Are Required for Generation of Protective Effector and Memory CD4 Cells in Response to Pneumocystis Lung Infection
Authors: Lund Frances E. , Hollifield Melissa , Schuer Kevin , Lines J. Louise , Randall Troy D. , Garvy Beth A.
Published: April 2014

B cell-deficient mice are susceptible to infection by Pneumocystis carinii f. sp. muris (PC). To determine whether this susceptibility is due to a requirement for B cells to prime T cells, we compared CD4 T cell responses to PC in bone marrow chimeric mice that express MHC class II (MHCII) on all APCs (wild-type (WT) chimeras) and in bone marrow chimeric mice that express MHCII on all APCs except B cells (MHCII−/− chimeras). Although PC was rapidly cleared by WT chimeric mice, PC levels remained high in chimeric mice that lacked MHCII on B cells. In addition, although T cells were primed in the draining lymph nodes of MHCII−/− chimeric mice, the number of activated CD4 T cells infiltrating the lungs of these mice was reduced relative to the number in the lungs of WT chimeras. We also adoptively transferred purified CD4 T cells from the draining lymph nodes of PC-infected normal or B cell-deficient mice into SCID mice. Mice that received CD4 cells from normal mice were able to mount a response to infection in the lungs and clear PC. However, mice that received CD4 cells from B cell-deficient mice had a delayed T cell response in the lungs and failed to control the infection. These data indicate that B cells play a vital role in generation of CD4+ memory T cells in response to PC infection in the lungs.
Anthrax Lethal Toxin Has Direct and Potent Inhibitory Effects on B Cell Proliferation and Immunoglobulin Production
Authors: Fang Hui , Xu Lixin , Chen Trina Y. , Cyr Julianne M. , Frucht David M.
Published: April 2014

Protective host immune responses to anthrax infection in humans and animal models are characterized by the development of neutralizing Abs against the receptor-binding anthrax protective Ag (PA), which, together with the lethal factor (LF) protease, composes anthrax lethal toxin (LT). We now report that B cells, in turn, are targets for LT. Anthrax PA directly binds primary B cells, resulting in the LF-dependent cleavage of the MAPK kinases (MAPKKs) and disrupted signaling to downstream MAPK targets. Although not directly lethal to B cells, anthrax LT treatment causes severe B cell dysfunction, greatly reducing proliferative responses to IL-4-, anti-IgM-, and/or anti-CD40 stimulation. Moreover, B cells treated with anthrax LT in vitro or isolated from mice treated with anthrax LT in vivo have a markedly diminished capacity to proliferate and produce IgM in response to TLR-2 and TLR-4 ligands. The suppressive effects of anthrax LT on B cell function occur at picomolar concentrations in vitro and at sublethal doses in vivo. These results indicate that anthrax LT directly inhibits the function of B cells in vitro and in vivo, revealing a potential mechanism through which the pathogen could bypass protective immune responses.
Modulation of Dendritic Cell Function by Naive and Regulatory CD4+ T Cells
Authors: Veldhoen Marc , Moncrieffe Halima , Hocking Richard J. , Atkins Christopher J. , Stockinger Brigitta
Published: April 2014

The consequences of interactions between dendric cells (DCs) and either naive CD4+ T cells or regulatory CD4+CD25+ T cells on the expression of proinflammatory IL-6 and anti-inflammatory IL-10 in DC were examined over a period of 12 h, spanning the time frame during which stable T cell–DC interactions shape the development of tolerance and immunity in vivo. We demonstrate that the basal production of IL-6 and IL-10, which is initiated following DC stimulation with LPS, is modified in distinctly different ways by interaction with the two T cell populations. Naive CD4 T cells skew DC cytokine production toward IL-6 and suppress IL-10, whereas CD4+CD25+ T cells have the opposite effect. CD8 T cells or memory CD4 T cells do not influence basal cytokine production by stimulated DC. The effect of CD4+CD25+ T cells is dominant in coculture with naive CD4 T cells as long as inflammatory LPS is absent; the addition of LPS abrogates the suppression of IL-6. However, the modulating influence of CD4+CD25+ T cells remains evident in the enhancement of IL-10 production. Thus, mutual interactions between DC and CD4+ T cell subpopulations following contact with pathogens are likely to influence the strength and quality of incipient immune responses in the local microenvironment.
Regulatory Roles for MD-2 and TLR4 in Ligand-Induced Receptor Clustering
Authors: Kobayashi Makiko , Saitoh Shin-ichiroh , Tanimura Natsuko , Takahashi Koichiro , Kawasaki Kiyoshi , Nishijima Masahiro , Fujimoto Yukari , Fukase Koichi , Akashi-Takamura Sachiko , Miyake Kensuke
Published: April 2014

LPS, a principal membrane component in Gram-negative bacteria, is recognized by a receptor complex consisting of TLR4 and MD-2. MD-2 is an extracellular molecule that is associated with the extracellular domain of TLR4 and has a critical role in LPS recognition. MD-2 directly interacts with LPS, and the region from Phe119 to Lys132 (Arg132 in mice) has been shown to be important for interaction between LPS and TLR4/MD-2. With mouse MD-2 mutants, we show in this study that Gly59 was found to be a novel critical amino acid for LPS binding outside the region 119–132. LPS signaling is thought to be triggered by ligand-induced TLR4 clustering, which is also regulated by MD-2. Little is known, however, about a region or an amino acid in the MD-2 molecule that regulates ligand-induced receptor clustering. MD-2 mutants substituting alanine for Phe126 or Gly129 impaired LPS-induced TLR4 clustering, but not LPS binding to TLR4/MD-2, demonstrating that ligand-induced receptor clustering is differentially regulated by MD-2 from ligand binding. We further show that dissociation of ligand-induced receptor clustering and of ligand-receptor interaction occurs in a manner dependent on TLR4 signaling and requires endosomal acidification. These results support a principal role for MD-2 in LPS recognition.
Involvement of CCL18 in Allergic Asthma
Authors: Nadaï Patricia de , Charbonnier Anne-Sophie , Chenivesse Cécile , Sénéchal Stéphanie , Fournier Clément , Gilet Jules , Vorng Han , Chang Ying , Gosset Philippe , Wallaert Benoı̂t , Tonnel André-Bernard , Lassalle Philippe , Tsicopoulos Anne
Published: April 2014

Allergic asthma is associated with a pulmonary recruitment of Th type 2 cells, basophils, and eosinophils, mainly linked to chemokine production. CCL18 is a chemokine preferentially expressed in the lung, secreted by APCs, induced by Th2-type cytokines, and only present in humans. Therefore, CCL18 may be involved in allergic asthma. PBMC from asthmatics allergic to house dust mite cultured in the presence of Dermatophagoides pteronyssinus 1 (Der p 1) allergen secreted CCL18, 48 and 72 h after stimulation, whereas those from healthy donors did not. Part of CCL18 was directly derived from Der p 1-stimulated plasmacytoid dendritic cells, whereas the other part was linked to monocyte activation by IL-4 and IL-13 produced by Der p 1-stimulated T cells. In bronchoalveolar lavages from untreated asthmatic allergic patients, CCL18 was highly increased compared with controls. Functionally, CCL18 preferentially attracted in vitro-polarized Th2 cells and basophils, but not eosinophils and Th1 cells, and induced basophil histamine and intracellular calcium release. These data show a new function for CCL18, i.e., the recruitment of Th2 cells and basophils, and suggest that CCL18 may play a predominant role in allergic asthma.
T Cell Responses Are Better Correlates of Vaccine Protection in the Elderly
Authors: McElhaney Janet E. , Xie Dongxu , Hager W. David , Barry Mary Beth , Wang Yazhen , Kleppinger Alison , Ewen Catherine , Kane Kevin P. , Bleackley R. Chris
Published: April 2014

It is commonly held that increased risk of influenza in the elderly is due to a decline in the Ab response to influenza vaccination. This study prospectively evaluated the relationship between the development of influenza illness, and serum Ab titers and ex vivo cellular immune responses to influenza vaccination in community dwelling older adults including those with congestive heart failure (CHF). Adults age 60 years and older (90 subjects), and 10 healthy young adult controls received the 2003-04 trivalent inactivated influenza vaccine. Laboratory diagnosed influenza (LDI) was documented in 9 of 90 older adults. Pre- and postvaccination Ab titers did not distinguish between subjects who would subsequently develop influenza illness (LDI subjects) and those who would not (non-LDI subjects). In contrast, PBMC restimulated ex vivo with live influenza virus preparations showed statistically significant differences between LDI and non-LDI subjects. The mean IFN-γ:IL-10 ratio in influenza A/H3N2-stimulated PBMC was 10-fold lower in LDI vs non-LDI subjects. Pre-and postvaccination granzyme B levels were significantly lower in CHF subjects with LDI compared with subjects without LDI. In non-CHF subjects with LDI, granzyme B levels increased to high levels at the time of influenza infection. In conclusion, measures of the ex vivo cellular immune response to influenza are correlated with protection against influenza while serum Ab responses may be limited as a sole measure of vaccine efficacy in older people. Ex vivo measures of the cell-mediated immune response should be incorporated into evaluation of new vaccines for older adults.