bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the Long Form of Human FLIP by Retroviral Gene Transfer of Hemopoietic Stem Cells Exacerbates Experimental Autoimmune Encephalomyelitis
Authors: Djerbi Mounira , Abdul-Majid Khairul-Bariah , Abedi-Valugerdi Manuchehr , Olsson Tomas , Harris Robert A. , Grandien Alf
Published: April 2014

Subsidence of inflammation and clinical recovery in experimental autoimmune encephalomyelitis (EAE) is postulated to involve apoptosis of inflammatory cells. To test this concept, we examined the effects of overexpressing the long form of human FLICE-inhibitory protein, a potent inhibitor of death receptor-mediated apoptosis, in myelin oligodendrocyte glycoprotein-induced EAE in DBA/1 mice. We found that overexpression of the long form of human FLICE-inhibitory protein by retroviral gene transfer of hemopoietic stem cells led to a clinically more severe EAE in these mice compared with control mice receiving the retroviral vector alone. The exacerbated disease was evident by an enhanced and prolonged inflammatory reaction in the CNS of these animals compared with control mice. The acute phase of EAE was characterized by a massive infiltration of macrophages and granulocytes and a simultaneous increase in TNF-α production in the CNS. In the chronic phase of the disease, there was a prolonged inflammatory response in the form of persistent CD4+ T and B cells in the CNS and a peripheral Th1 cytokine bias caused by elevated levels of IFN-γ and reduced levels of IL-4 in the spleen. Our findings demonstrate that death receptor-mediated apoptosis can be important in the pathogenesis of EAE and further emphasize the need for effective apoptotic elimination of inflammatory cells to achieve disease remission.
Mechanisms of Inhibition of Collagen-Induced Arthritis by Murine IL-18 Binding Protein
Authors: Banda Nirmal K. , Vondracek Andrea , Kraus Damian , Dinarello Charles A. , Kim Soo-Hyun , Bendele Alison , Senaldi Giorgio , Arend William P.
Published: April 2014

IL-18 is an important cytokine in autoimmune and inflammatory diseases through the induction of IFN-γ, TNF-α, and IL-1. We report herein that collagen-induced arthritis (CIA) in mice is inhibited by treatment with murine IL-18 binding protein (mIL-18BP). CIA was induced in DBA/1J mice by the injection of bovine type II collagen (CII) in IFA with added Mycobacterium tuberculosis on days 0 and 21. The mice were then treated for 3 wk with PBS or with two doses of mIL-18BP (0.5 and 3 mg/kg) as a fusion protein with the Fc portion of murine IgG1. Both the clinical disease activity scores and the histological scores of joint damage were reduced 50% in mice treated with either dose of mIL-18BP. Proliferation of CII-stimulated spleen and lymph node cells as well as the change in serum levels of IgG1 and IgG2a Ab to collagen between days 21 and 42 were decreased in mice treated with mIL-18BP. The production of IFN-γ, TNF-α, and IL-1β in cultured spleen cells was reduced by in vivo treatment with low dose, but not high dose, mIL-18BP. FACS analysis showed a slight decrease in NK cells and an increase in CD4+ T cells in spleens of mice treated with mIL-18BP. The steady state mRNA levels of IFN-γ, TNF-α, and IL-1β in isolated joints were all decreased in mice treated with both doses of mIL-18BP. The mechanisms of mIL-18BP inhibition of CIA include reductions in cell-mediated and humoral immunity to collagen as well as decreases in production of proinflammatory cytokines in the spleen and joints.
Parenchymal, But Not Leukocyte, TNF Receptor 2 Mediates T Cell-Dependent Hepatitis in Mice
Authors: Schümann Jens , Mühlen Katrin , Kiemer Alexandra K. , Vollmar Angelika M. , Tiegs Gisa
Published: April 2014

TNF-α is a central mediator of T cell activation-induced hepatitis in mice, e.g., induced by Pseudomonas exotoxin A (PEA). In this in vivo mouse model of T cell-dependent hepatitis, liver injury depends on both TNFRs. Whereas TNFR1 can directly mediate hepatocyte death, the in vivo functions of TNFR2 in pathophysiology remained unclear. TNFR2 has been implicated in deleterious leukocyte activation in a transgenic mouse model and in enhancement of TNFR1-mediated cell death in cell lines. In this study, we clarify the role of hepatocyte- vs leukocyte-expressed TNFR2 in T cell-dependent liver injury in vivo, using the PEA-induced hepatitis model. Several types of TNFR2-expressing leukocytes, especially neutrophils and NK cells, accumulated within the liver throughout the pathogenic process. Surprisingly, only parenchymal TNFR2 expression, but not the TNFR2 expression on leukocytes, contributed to PEA-induced hepatitis, as shown by analysis of wild-type → tnfr2° and the reciprocal mouse bone marrow chimeras. Furthermore, PEA induced NF-κB activation and cytokine production in the livers of both wild-type and tnfr2° mice, whereas only primary mouse hepatocytes from wild-type, but not from tnfr2°, mice were susceptible to cell death induced by a combination of agonistic anti-TNFR1 and anti-TNFR2 Abs. Our results suggest that parenchymal, but not leukocyte, TNFR2 mediates T cell-dependent hepatitis in vivo. The activation of leukocytes does not appear to be disturbed by the absence of TNFR2.
Absence of CCR8 Does Not Impair the Response to Ovalbumin-Induced Allergic Airway Disease
Authors: Goya Íñigo , Villares Ricardo , Zaballos Ángel , Gutiérrez Julio , Kremer Leonor , Gonzalo José-Ángel , Varona Rosa , Carramolino Laura , Serrano Alfredo , Pallarés Pilar , Criado Luis Miguel , Kolbeck Roland , Torres Miguel , Coyle Anthony J. , Gutiérrez-Ramos José-Carlos , Martínez-A Carlos , Márquez Gabriel
Published: April 2014

Interaction of chemokines with their specific receptors results in tight control of leukocyte migration and positioning. CCR8 is a chemokine receptor expressed mainly in CD4+ single-positive thymocytes and Th2 cells. We generated CCR8-deficient mice (CCR8−/−) to study the in vivo role of this receptor, and describe in this study the CCR8−/− mouse response in OVA-induced allergic airway disease using several models, including an adoptive transfer model and receptor-blocking experiments. All CCR8−/− mice developed a pathological response similar to that of wild-type animals with respect to bronchoalveolar lavage cell composition, peripheral blood and bone marrow eosinophilia, lung infiltrates, and Th2 cytokine levels in lung and serum. The results contrast with a recent report using one of the OVA-induced asthma models studied here. Similar immune responses were also observed in CCR8−/− and wild-type animals in a different model of ragweed allergen-induced peritoneal eosinophilic inflammation, with an equivalent number of eosinophils and analogous increased levels of Th2 cytokines in peritoneum and peripheral blood. Our results show that allergic diseases course without critical CCR8 participation, and suggest that further work is needed to unravel the in vivo role of CCR8 in Th2-mediated pathologies.
Induction of Pemphigus Phenotype by a Mouse Monoclonal Antibody Against the Amino-Terminal Adhesive Interface of Desmoglein 3
Authors: Tsunoda Kazuyuki , Ota Takayuki , Aoki Miyo , Yamada Taketo , Nagai Tetsuo , Nakagawa Taneaki , Koyasu Shigeo , Nishikawa Takeji , Amagai Masayuki
Published: April 2014

Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering disease that is caused by IgG autoantibodies against the cadherin-type adhesion molecule desmoglein (Dsg)3. Previously, we have generated an active mouse model for PV by adoptive transfer of Dsg3−/− splenocytes. In this study, we isolated eight AK series, anti-Dsg3 IgG mAbs from the PV mouse model, and examined their pathogenic activities in induction of blister formation. Intraperitoneal inoculation of the AK23 hybridoma, but not the other AK hybridomas, induced the virtually identical phenotype to that of PV model mice or Dsg3−/− mice with typical histology of PV. Epitope mapping with domain-swapped and point-mutated Dsg1/Dsg3 molecules revealed that AK23 recognized a calcium-dependent conformational epitope on Dsg3, which consisted of the V3, K7, P8, and D59 Dsg3-specific residues that formed the adhesive interface between juxtaposed Dsg, as predicted by the crystal structure. The epitopes of the mAbs that failed to show apparent pathogenic activity were mapped in the middle to carboxyl-terminal extracellular region of Dsg3, where no direct intermolecular interaction was predicted. These findings demonstrate the pathogenic heterogeneity among anti-Dsg3 IgG Abs due to their epitopes, and suggest the direct inhibition of adhesive interaction of Dsg as an initial molecular event of blister formation in pemphigus.
LF 15-0195 Inhibits the Development of Rat Central Nervous System Autoimmunity by Inducing Long-Lasting Tolerance in Autoreactive CD4 T Cells
Authors: Duplan Valérie , Dutartre Patrick , Mars Lennart T. , Liblau Roland S. , Druet Philippe , Saoudi Abdelhadi
Published: April 2014

Experimental autoimmune encephalomyelitis (EAE) is a T cell-dependent autoimmune disease induced in susceptible animals by a single immunization with myelin basic protein (MBP). LF 15-0195 is a novel immunosuppressor that has been shown to have a potent immunosuppressive effect in several pathological manifestations. The purpose of this study was to investigate the effect of this drug on the induction and progression of established rat EAE and to dissect the mechanisms involved. We show that LF 15-0195 administration at the time of MBP immunization reduces the incidence and severity of EAE in Lewis rats. This drug also inhibits ongoing and passively induced EAE, indicating that LF 15-0195 affects already differentiated pathogenic lymphocytes. Compared with lymph node cells from untreated rats, lymphocytes from MBP-immunized rats treated with LF 15-0195 proliferated equally well in response to MBP in vitro, while their ability to produce effector cytokines and to transfer EAE into syngeneic recipients was significantly reduced. This phenomenon is stable and long-lasting. Indeed, neither IL-12 nor repeated stimulation with naive APC and MBP in vitro rendered MBP-specific CD4 T cells from protected rats encephalitogenic. In conclusion, LF 15-0195 treatment suppresses EAE by interfering with both the differentiation and effector functions of autoantigen-specific CD4 T cells.
Essential Roles of Perforin in Antigen-Specific Cytotoxicity Mediated by Human CD4+ T Lymphocytes: Analysis Using the Combination of Hereditary Perforin-Deficient Effector Cells and Fas-Deficient Target Cells
Authors: Yanai Fumio , Ishii Eiichi , Kojima Kensuke , Hasegawa Atsuhiko , Azuma Taichi , Hirose Shinichi , Suga Naohiro , Mitsudome Akihisa , Zaitsu Masafumi , Ishida Yasushi , Shirakata Yuji , Sayama Koji , Hashimoto Koji , Yasukawa Masaki
Published: April 2014

Although the cytotoxic mechanisms of murine CTLs have been investigated extensively using various mutant and knockout mice, those of human CTLs, especially CD4+ CTLs, are still obscure. To clarify the roles of perforin in Ag-specific cytotoxicity mediated by human CD4+ CTLs, alloantigen-specific and HSV-specific human CD4+ T lymphocyte bulk lines and clones were established from a patient with hereditary perforin deficiency and her healthy father, and their cytotoxic activities were investigated. Alloantigen-specific CD4+ T lymphocytes expressing perforin exerted cytotoxicity against Fas-negative as well as Fas-positive allogeneic B lymphoblastoid cell lines established from members of a family with hereditary Fas deficiency. Perforin-deficient, but not perforin-expressing, CD4+ T lymphocytes failed to show strong cytotoxicity against HSV-infected autologous B lymphoblastoid cells. Perforin-deficient CD4+ T lymphocytes could exert relatively low level cytotoxicity against allogeneic IFN-γ-treated keratinocytes. Although cytotoxicity mediated by perforin-expressing CD4+ CTLs was almost completely inhibited by concanamycin A, a potent inhibitor of the perforin-mediated cytotoxic pathway, cytotoxicity against IFN-γ-treated keratinocytes mediated by perforin-deficient CD4+ T lymphocytes was inhibited only partially by concanamycin A, but was inhibited significantly by antagonistic anti-Fas Ab and anti-Fas ligand Ab. The combination of perforin-deficient effector T lymphocytes and Fas-negative target cells used in the present study provides a novel experimental system for studying the detailed mechanisms of human CTL-mediated cytotoxicity. The present data demonstrate that perforin-negative CD4+ CTLs can exert cytotoxicity against Fas-sensitive target cells; however, perforin plays essential roles in Ag-specific cytotoxicity mediated by human CD4+ as well as CD8+ CTLs.
Perforin-Dependent Brain-Infiltrating Cytotoxic CD8+ T Lymphocytes Mediate Experimental Cerebral Malaria Pathogenesis
Authors: Nitcheu Josianne , Bonduelle Olivia , Combadiere Christophe , Tefit Maurel , Seilhean Danielle , Mazier Dominique , Combadiere Behazine
Published: April 2014

Experimental cerebral malaria (ECM) resulting from Plasmodium berghei ANKA infection involves T lymphocytes. However, the mechanisms of T cell-mediated pathogenesis remain unknown. We found that, in contrast to ECM-susceptible C57BL6 mice, perforin-deficient (PFP-KO) mice were resistant to ECM in the absence of brain lesions, whereas cytoadherence of parasitized erythrocytes and massive accumulation of activated/effector CD8 lymphocytes were observed in both groups of mice. ECM is induced in PFP-KO mice after adoptive transfer of cytotoxic CD8+ cells from infected C57BL6 mice, which were directed to the brain of PFP-KO mice. This specific recruitment might involve chemokine/chemokine receptors, since their expression was up-regulated on activated CD8 cells, and susceptibility to ECM was delayed in CCR5-KO mice. Thus, lymphocyte cytotoxicity and cell trafficking are key players in ECM pathogenesis.
T Cell Reactivity to MHC Class II-Bound Self Peptides in Systemic Lupus Erythematosus-Prone MRL/lpr Mice
Authors: Suh Chang-Hee , Freed John H. , Cohen Philip L.
Published: April 2014

The epitopes recognized by pathogenic T cells in systemic autoimmune disease remain poorly defined. Certain MHC class II-bound self peptides from autoimmune MRL/lpr mice are not found in eluates from class II molecules of MHC-identical C3H mice. Eleven of 16 such peptides elicited lymph node cell and spleen cell T cell proliferation in both MRL/lpr (stimulation index = 2.03–5.01) and C3H mice (stimulation index = 2.03–3.75). IL-2 and IFN-γ production were detected, but not IL-4. In contrast to what was seen after immunization, four self peptides induced spleen cell proliferation of T cells from naive MRL/lpr, but not from C3H and C57BL/6.H2k, mice. These peptides were derived from RNA splicing factor SRp20, histone H2A, β2-microglobulin, and MHC class II I-Akβ. The first three peptides were isolated from I-Ek molecules and the last peptide was bound to I-Ak. T cell responses, evident as early as 1 mo of age, depended on MHC class II binding motifs and were inhibited by anti-MHC class II Abs. Thus, although immunization can evoke peripheral self-reactive T cells in normal mice, the presence in MRL/lpr mice of spontaneous T cells reactive to certain MHC-bound self peptides suggests that these T cells actively participate in systemic autoimmunity. Peptides eluted from self MHC class II molecules may yield important clues to T cell epitopes in systemic autoimmunity.
Cutting Edge: Mast Cell Antimicrobial Activity Is Mediated by Expression of Cathelicidin Antimicrobial Peptide
Authors: Di Nardo Anna , Vitiello Antonella , Gallo Richard L.
Published: April 2014

Cathelicidins (caths) are peptides that are expressed at high levels in neutrophils and some epithelia and can act as natural antibiotics by directly killing a wide range of microorganisms. We hypothesized that caths are expressed in mast cells (MCs), because these cells have been previously associated with inherent antimicrobial activity. Cultured murine MCs contained abundant amounts of cathelin-related antimicrobial peptide (AMP), the murine cath, and this expression was inducible by LPS or lipoteichoic acid. Human skin MCs also expressed cath as detected by immunohistochemical analysis for the human cath LL-37. The functional significance of this expression was shown by comparing MCs cultured from normal mice to MCs from littermates deficient in the cathelin-related AMP gene (Cnlp−). MCs derived from Cnlp−/− animals had a 50% reduction in their ability to kill group A Streptococcus. These MCs expressed equivalent amounts of mRNA for murine β-defensin-4, a β-defensin AMP. Thus, different antimicrobials can be identified in MCs, and the presence of cath is necessary for efficient bacterial killing. These observations suggest that the presence of cath is vital to the ability of mammalian MCs to participate in antimicrobial defense.
The Yaa Mutation Promoting Murine Lupus Causes Defective Development of Marginal Zone B Cells
Authors: Amano Hirofumi , Amano Eri , Moll Thomas , Marinkovic Dragan , Ibnou-Zekri Nabila , Martinez-Soría Eduardo , Semac Isabelle , Wirth Thomas , Nitschke Lars , Izui Shozo
Published: April 2014

The accelerated development of systemic lupus erythematosus (SLE) in BXSB male mice is associated with the presence of an as yet unidentified mutant gene, Yaa (Y-linked autoimmune acceleration). In view of a possible role of marginal zone (MZ) B cells in murine SLE, we have explored whether the expression of the Yaa mutation affects the differentiation of MZ and follicular B cells, thereby implicating the acceleration of the disease. In this study, we show that both BXSB and C57BL/6 Yaa mice, including two different substrains of BXSB Yaa males that are protected from SLE, displayed an impaired development of MZ B cells early in life. Studies in bone marrow chimeras revealed that the loss of MZ B cells resulted from a defect intrinsic to B cells expressing the Yaa mutation. The lack of selective expansion of MZ B cells in diseased BXSB Yaa males strongly argues against a major role of MZ B cells in the generation of pathogenic autoantibodies in the BXSB model of SLE. Furthermore, a comparative analysis with mice deficient in CD22 or expressing an IgM anti-trinitrophenyl/DNA transgene suggests that the hyperreactive phenotype of Yaa B cells, as judged by a markedly increased spontaneous IgM secretion, is likely to contribute to the enhanced maturation toward follicular B cells and the block in the MZ B cell generation.
A Role for Class A Scavenger Receptor in Dendritic Cell Nibbling from Live Cells
Authors: Harshyne Larry A. , Zimmer Michael I. , Watkins Simon C. , Barratt-Boyes Simon M.
Published: April 2014

Monocyte-derived dendritic cells (DC) possess the unique capacity to capture Ag from live cells through intimate cell contact, a process referred to as nibbling. We sought to define the receptor(s) mediating DC nibbling. Uptake of fluorescently labeled plasma membrane from live cells by DC was inhibited by protease treatment and by a panel of polyanionic ligands, implicating scavenger receptors (SR) in this process. Differential expression of SR on DC and macrophages correlated with the capacity to acquire membrane from live cells. Internalized membrane colocalized with SR ligand and entered the endosomal pathway. DC very efficiently acquired and internalized gp100 tumor Ag expressed at the surface of viable adenocarcinoma cells via recombinant adenoviral infection. Cross-presentation of gp100 by DC to MHC class I-restricted T cells was inhibited by polyanionic SR ligand and an Ab to type A SR (SR-A), whereas Ab to the class B SR CD36, which mediates uptake of apoptotic cells, induced no inhibition. DC capture of fluorescently labeled membrane from live cells was partially blocked by SR-A-specific Ab, suggesting that other SR may also be contributing to nibbling. DC maturation resulted in a switch in expression from type II SR-A (SR-AII) to the SR-AI splice variant. Finally, SR-A was identified on interdigitating DC isolated from monkey lymph nodes. These findings define a novel role for SR-A, and suggest that Ag uptake from live cells by DC may be important in the generation of immunity and in the maintenance of peripheral tolerance in vivo.
Inducible Costimulatory Molecule-B7-Related Protein 1 Interactions Are Important for the Clonal Expansion and B Cell Helper Functions of Naive, Th1, and Th2 T Cells
Authors: Smith Karen M. , Brewer James M. , Webb Phillip , Coyle Anthony J. , Gutierrez-Ramos Carlos , Garside Paul
Published: April 2014

Inducing T cell responses requires at least two distinct signals: 1) TCR engagement of MHC-peptide and 2) binding of CD28 to B7.1/2. However, the recent avalanche of newly described costimulatory molecules may represent additional signals which can modify events after the initial two-signal activation. Inducible costimulatory molecule (ICOS) is a CD28 family member expressed on T cells rapidly following activation that augments both Th1 and Th2 T cell responses and has been implicated in sustaining rather than initiating T cell responses. Although it is known that blockade of ICOS-B7-related protein 1 (B7RP-1) in vivo dramatically reduces germinal center formation and Ab production, the mechanism(s) remains unclear. An optimal T cell-dependent Ab response requires T and B cell activation, expansion, differentiation, survival, and migration, and the ICOS-B7RP-1 interaction could be involved in any or all of these processes. Understanding this will have important implications for targeting ICOS-B7RP-1 therapeutically. We have therefore used a double-adoptive transfer system, in which all of the above events can be analyzed, to assess the role of ICOS-B7RP-1 in T cell help for B cell responses. We have shown that ICOS signaling is involved in the initial clonal expansion of primary and primed Th1 and Th2 cells in response to immunization. Furthermore, while ICOS-B7RP-1 interactions have no effect on the migration of T cells into B cell follicles, it is essential for their ability to support B cell responses.
Human Heat Shock Protein 60 Induces Maturation of Dendritic Cells Versus a Th1-Promoting Phenotype
Authors: Flohé Stefanie B. , Brüggemann Jutta , Lendemans Sven , Nikulina Marina , Meierhoff Guido , Flohé Sascha , Kolb Hubert
Published: April 2014

Heat shock protein (HSP) 60 nonspecifically activates cells of the innate immune system. In the present study, we characterized the effects of human HSP60 maturation, cytokine release, and T cell-activating capacity of bone marrow-derived dendritic cells (DC). Furthermore, we analyzed HSP60-induced signal transduction in DC. HSP60 strongly stimulated DC for maturation and release of TNF-α, IL-12, and IL-1β. However, HSP60 elicited only a weak IL-10 response in DC suggesting a Th1 bias. HSP60-treated DC induced proliferation of allogeneic T cells. Again, a Th1 bias was noted in that cocultures of allogeneic T cells and HSP60-treated DC released IFN-γ but only small amounts of IL-10 and no detectable IL-4. Signaling via Toll-like receptor 4 was involved in HSP60-induced cytokine release and maturation because DC of C3H/HeJ mice with a mutant Toll-like receptor 4 showed deficient response to HSP60. HSP60 was found to rapidly activate the mitogen-activated protein kinases p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase as well as IκB in DC. Phosphorylation of these signaling molecules was also mediated by LPS, but with much slower kinetics. Thus, HSP60 stimulates DC more rapidly than LPS and elicits a Th1-promoting phenotype. These results suggest that DC play a pivotal role in priming for destructive Th1-type responses at sites of local HSP60 release.
CD7 Is a Differentiation Marker That Identifies Multiple CD8 T Cell Effector Subsets
Authors: Aandahl Einar M. , Sandberg Johan K. , Beckerman Karen P. , Taskén Kjetil , Moretto Walter J. , Nixon Douglas F.
Published: April 2014

The adaptive immune response of human CD8 T cells to invading pathogens involves the differentiation of naive cells into memory and effector cells. However, the lineage relationship between memory and effector cells and the differentiation of CD8 T cells into distinct subsets of effector cell subpopulations are subjects of considerable debate. CD7 identifies three populations of CD8 T cells: CD7 high (CD7high), low (CD7low), and negative (CD7neg) that translate into subsets with distinct functional properties. The CD7high subset contains naive and memory cells and the CD7low and CD7neg subsets contain effector cells. The effector cells can functionally be divided into cytokine-secreting effector CD8 T cells and lytic effector CD8 T cells. These data provide a model of human CD8 T cell differentiation in which specialized distinct subpopulations can be identified by expression of CD7.
Human Invariant Vα24-JαQ TCR Supports the Development of CD1d-Dependent NK1.1+ and NK1.1− T Cells in Transgenic Mice
Authors: Capone Myriam , Cantarella Daniela , Schümann Jens , Naidenko Olga V. , Garavaglia Claudio , Beermann Friederich , Kronenberg Mitchell , Dellabona Paolo , MacDonald H. Robson , Casorati Giulia
Published: April 2014

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR Vα24-JαQ and Vα14-Jα18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. Vα24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. Vα24-JαQ TCR chain in all T cells. The expression of the human inv. Vα24 TCR in TCR Cα−/− mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand α-galactosylceramide (α-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in Vα24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR Vβ7 and a corresponding decrease in TCR Vβ8.2 use. Despite the forced expression of the human CD1d-restricted TCR in Cα−/− mice, staining with mCD1d-α-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1− T cells that bind CD1d-α-GalCer tetramers. These findings indicate that human inv. Vα24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. Vα24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.
Distinct Thresholds for CD8 T Cell Activation Lead to Functional Heterogeneity: CD8 T Cell Priming Can Occur Independently of Cell Division
Authors: Auphan-Anezin Nathalie , Verdeil Grégory , Schmitt-Verhulst Anne-Marie
Published: April 2014

To examine the bases for CD8 T cell functional heterogeneity, we analyzed responses to partial vs full agonist Ag. An extended period of interaction with APCs was required to set the threshold required for cell division in response to partial as compared with full agonist Ag. Acquisition of cytolytic function was restricted to the divided T cell population. In contrast, the threshold for commitment to produce IFN-γ and express some activation markers appeared lower and independent of cell division. Indeed, we characterized a T cell population stimulated in response to the partial agonist that was committed to produce IFN-γ, but failed to divide or secrete IL-2. Importantly, this activated nondivided population behaved as “primed” rather than “anergized,” indicating 1) that priming of CD8 T cells may be induced by suboptimal stimulation independent of cell division and 2) that encounter with Ag does not always induce a complete differentiation program in naive CD8 T cells, as previously reported.
HIV Envelope Induces Virus Expression from Resting CD4+ T Cells Isolated from HIV-Infected Individuals in the Absence of Markers of Cellular Activation or Apoptosis
Authors: Kinter Audrey L. , Umscheid Craig A. , Arthos James , Cicala Claudia , Lin Yin , Jackson Robert , Donoghue Eileen , Ehler Linda , Adelsberger Joseph , Rabin Ronald L. , Fauci Anthony S.
Published: April 2014

Resting CD4+ T cells containing integrated HIV provirus constitute one of the long-lived cellular reservoirs of HIV in vivo. This cellular reservoir of HIV had been thought to be quiescent with regard to virus replication based on the premise that HIV production in T cells is inexorably linked to cellular activation as determined by classical activation markers. The transition of T cells within this HIV reservoir from a resting state to an activated HIV-producing state is believed to be associated with a shorten life span due to susceptibility to activation-associated apoptosis. Evidence is mounting, however, that HIV production may occur in T cells that have not undergone classic T cell activation. HIV encodes several proteins, including envelope and Nef, which trigger a variety of signaling pathways associated with cellular activation, thereby facilitating HIV replication in nondividing cells. The present study demonstrates that production of infectious virus from resting CD4+ T cells isolated from HIV-infected individuals can be induced following exposure of these cells to HIV-1 recombinant (oligomeric gp140) envelope protein. Envelope-mediated induction of HIV expression occurs in the presence of reverse transcriptase inhibitors and is not associated with markers of classic T cell activation, proliferation, or apoptosis. The ability of HIV envelope to induce virus replication in HIV-infected resting CD4+ T cells without triggering apoptosis provides a mechanism for the virus itself to directly participate in the maintenance of HIV production from this cellular reservoir.
Turnover Rates of B Cells, T Cells, and NK Cells in Simian Immunodeficiency Virus-Infected and Uninfected Rhesus Macaques
Authors: De Boer Rob J. , Mohri Hiroshi , Ho David D. , Perelson Alan S.
Published: April 2014

We determined average cellular turnover rates by fitting mathematical models to 5-bromo-2′-deoxyuridine measurements in SIV-infected and uninfected rhesus macaques. The daily turnover rates of CD4+ T cells, CD4− T cells, CD20+ B cells, and CD16+ NK cells in normal uninfected rhesus macaques were 1, 1, 2, and 2%, respectively. Daily turnover rates of CD45RA− memory T cells were 1%, and those of CD45RA+ naive T cells were 0.5% for CD4+ T cells and ∼1% for CD4−CD45RA+ T cells. In SIV-infected monkeys with high viral loads, the turnover rates of T cells were increased ∼2-fold, and that of memory T cells ∼3-fold. The turnover of CD4+CD45RA+ naive T cells was increased 2-fold, whereas that of CD4−CD45RA+ naive T cells was marginally increased. B cells and NK cells also had increased turnover in SIV-infected macaques, averaging 3 and 2.5% per day, respectively. For all cell types studied here the daily turnover rate increased with the decrease of the CD4 count that accompanied SIV infection. As a consequence, the turnover rates of CD4+ T cells, CD4− T cells, B cells, and NK cells within each monkey are strongly correlated. This suggests that the cellular turnover of different lymphocyte populations is governed by a similar process which one could summarize as “generalized immune activation.” Because the viral load and the CD4 T cell count are negatively correlated we cannot determine which of the two plays the most important role in this generalized immune activation.
Long-Term Maintenance of gp120-Specific Immune Responses by Genetic Vaccination with the HIV-1 Envelope Genes Linked to the Gene Encoding Flt-3 Ligand
Authors: Sailaja Gangadhara , Husain Sajid , Nayak Bishnu P. , Jabbar Abdul M.
Published: April 2014

DNA vaccines target dendritic cells (DC) to induce Ag-specific immune responses in animals. Potent HIV-specific immunity could be achieved by efficient priming of the immune system by DNA vaccines. We investigated a novel DNA vaccine approach based on the role of growth factors in DC expansion and differentiation. To this end, we constructed chimeric genes encoding the HIV envelope glycoproteins physically linked to the extracellular domain of Fms-like tyrosine kinase receptor-3 ligand (FLex; a DC growth factor; both mouse (m)FLex and human (h)FLex). These chimeric gene constructs synthesized biologically active, oligomeric FLex:gp120 fusion proteins and induced DC expansion (CD11c+CD11b+) when injected i.v. into mice. This DC expansion is comparable to that achieved by FLex DNA encoding native FLex protein. When delivered intramuscularly as DNA vaccines, hFLex:gp120 induced high frequencies of gp120-specific CD8+ T cells in the presence or absence of FLex DNA-induced DC expansion, but gp120 and mFLex:gp120 elicited only low to moderate levels of Ag-specific CD8+ T cells. In contrast, mFLex:gp120 induced high levels of anti-gp120 Abs under identical conditions of DNA vaccination. However, the Ab levels in mice immunized with DNA vaccines encoding hFLex:gp120 and gp120 proteins were low without DC expansion, but reached high levels comparable to that elicited by mFLex:gp120 only after the second boost in the presence of DC expansion. Importantly, the gp120-specific CD8+ T cells persisted at high frequency for 114 days (16 wk) after a booster injection. These experiments provide insight into the importance of modulating DC function in vivo for effective genetic vaccination in animals.
Regulatory CD8+ T Cells Control Neonatal Tolerance to a Th2-Mediated Autoimmunity
Authors: Field Anne-Christine , Caccavelli Laure , Bloch Marie-Françoise , Bellon Blanche
Published: April 2014

Exposure of newborn animals to a foreign Ag may result in immunological tolerance to that specific Ag, a phenomenon called neonatal tolerance. We have previously reported that neonatal administration to Brown-Norway rats of mercury, a heavy metal toxicant, induces a dominant tolerance, specific for the chemical otherwise responsible for Th2 cell-mediated autoimmune responses in this susceptible strain of rats. Neonatal exposure to Ags can prime immunity, rather than inactivate or delete responses, and sustain regulatory functions effective against autoreactive T cells. Here, we address whether such a tolerant response is due to the generation of regulatory cells. The results suggest that the CD8+ T cell subset is involved in neonatal tolerance to mercuric salt-induced Th2 autoimmune disease. Thus, we demonstrate that in vivo CD8 depletion breaks tolerance following mercury recall in animals under a neonatal tolerance protocol. Furthermore, adoptive cotransfer of splenocytes from naive and tolerant rats as well as transfer of CD8+ T cells from tolerant animals prevent naive syngeneic rats from developing pathologic Th2 immune responses. These observations indicate that CD8+ T cells are endowed with regulatory functions in neonatal tolerance and mediate active suppression. Moreover, neonatal tolerance induced the expansion of CD8+CD45RChigh T cells and the emergence of a high percentage of IFN-γ-synthesizing CD8+ T cells, which probably reflects the implication of regulatory Tc1 cells. Thus, in vivo induction of neonatal tolerance suppresses Th2 autoimmune responses via generation of a CD8+ cell-mediated regulatory response.
Selective Induction of High Avidity CTL by Altering the Balance of Signals from APC
Authors: Oh SangKon , Hodge James W. , Ahlers Jeffrey D. , Burke Donald S. , Schlom Jeffrey , Berzofsky Jay A.
Published: April 2014

High avidity CTL are most effective at clearing viruses and cancer cells. Therefore, understanding the mechanisms involved in induction of high avidity CTL is critical for effective vaccines. However, no vaccine approach to selectively induce high avidity CTL in vivo has been discovered. In a new approach, signals from MHC class I (signal 1) and costimulatory molecules (signal 2) were adjusted by varying Ag dose and by use of recombinant poxvirus expressing a triad of costimulatory molecules (B7-1, ICAM-1, and LFA-3), respectively. Independent of CTL avidity, a strong signal 1 resulted in an increased frequency of CD8+ CTL. However, a strong signal 2 was necessary for the induction of high avidity CD8+ CTL that killed target cells more efficiently, and signal 2 played a more crucial role in the absence of a strong signal 1. Only CTL induced with strong signal 2 killed tumor cells endogenously expressing low levels of Ag. Signal 2 contributed to the induction of high avidity CD8+ CTL in both primary and secondary responses. Thus, although signal 2 has been known to increase the quantity of CTL response, in this study we show that it also improves the quality of CTL response. Our data also suggested that dendritic cells play an important role in induction of high avidity CD8+ CTL in vivo. This strategy to selectively induce higher avidity CTL may lead to more effective vaccines for viruses and cancer.
NKT Cells Provide Help for Dendritic Cell-Dependent Priming of MHC Class I-Restricted CD8+ T Cells In Vivo
Authors: Stober Detlef , Jomantaitė Ieva , Schirmbeck Reinhold , Reimann Jörg
Published: April 2014

Dendritic cells (DC) are potent APCs for naive T cells in vivo. This is evident by inducing T cell responses through adoptive DC transfer. Priming specific CTL responses in vivo often requires “help”. We study alternative sources of help in DC-dependent priming of MHC class I-restricted CTL. Priming an anti-viral CTL response in naive B6 mice by adoptive transfer of antigenic peptide-pulsed DC required CD4+ T cell help. CTL priming was facilitated by providing MHC class II-dependent specific help. Furthermore, transfers of MHC class II-deficient pulsed DC into naive, normal hosts, or DC transfers into naive, CD4+ T cell-depleted hosts primed CTL inefficiently. Pretreatment of DC with immune-stimulating oligodeoxynucleotides rendered them more efficient for CD4+ T cell-independent priming of CTL. DC copresenting a Kb-binding antigenic peptide and the CD1d-binding glycolipid α-galactosyl-ceramide efficiently primed CTL in a class II-independent way. To obtain NKT cell-dependent help in CTL priming, the same DC had to present both the peptide and the glycolipid. CTL priming by adoptive DC transfer was largely NK cell-dependent. The requirement for NK cells was only partially overcome by recruiting NKT cell help into DC-dependent CTL priming. NKT cells thus are potent helper cells for DC-dependent CTL priming.
Prime Boost Vaccination Strategies: CD8 T Cell Numbers, Protection, and Th1 Bias
Authors: Woodberry Tonia , Gardner Joy , Elliott Suzanne L. , Leyrer Sonja , Purdie David M. , Chaplin Paul , Suhrbier Andreas
Published: April 2014

Vaccination strategies involving priming with DNA and boosting with a poxvirus vector have emerged as a preferred combination for the induction of protective CD8 T cell immunity. Using IFN-γ ELISPOT and a series of DNA plasmid, peptide, and modified vaccinia Ankara (MVA) vaccine combinations, we demonstrate that the DNA/MVA combination was uniquely able to enhance IFN-γ secretion by Ag-specific CD8 T cells. However, CD8 T cell populations induced by DNA/MVA vaccination failed to show an enhanced capability to mediate protection in an IFN-γ-independent influenza challenge model. The DNA/MVA vaccine strategy was also not unique in its ability to induce high numbers of CD8 T cells, with optimal strategies simply requiring the use of vaccine modalities that individually induce high numbers of CD8 T cells. These experiments argue that rivals to DNA/poxvirus vaccination strategies for the induction of optimal protective CD8 T cell responses are likely to emerge.
Severe Impairment in Early Host Defense Against Listeria monocytogenes in Mice Deficient in Acid Sphingomyelinase
Authors: Utermöhlen Olaf , Karow Ulrike , Löhler Jürgen , Krönke Martin
Published: April 2014

The phagolysosomal compartment is crucial for the defense against infection with intracellular pathogens. Within this compartment, the TNF- and IFN-γ-responsive acid sphingomyelinase (ASMase) generates the signaling molecule ceramide, resulting in the activation of proteases like cathepsin D. To investigate the possible role of ASMase as a mediator of the antibacterial effects of TNF and IFN-γ, ASMase−/− mice were infected with Listeria monocytogenes. ASMase−/− mice showed a dramatically increased susceptibility to L. monocytogenes (LD50 ∼100 CFU) when compared with syngeneic wild-type mice (LD50 ∼10,000 CFU). In L. monocytogenes-challenged ASMase−/− mice, IFN-γ serum levels as well as IL-1β and IL-6 secretion by macrophages were similar to those observed in wild-type C57BL/6 mice. Although macrophages and granulocytes from ASMase−/− mice showed intact production of reactive nitrogen intermediates and oxidative burst, ASMase−/− macrophages proved completely incapable of restricting the growth of L. monocytogenes in vitro. The results of this study suggest that ASMase is crucially required for the intracellular control of L. monocytogenes in macrophages and granulocytes by nonoxidative mechanisms.
Synthesis and Secretion of Monocyte Chemotactic Protein-1 Stimulated by the High Affinity Receptor for IgE
Authors: Eglite Santa , Morin Juan M. , Metzger Henry
Published: April 2014

In prior studies aggregation of the high affinity receptors for IgE, FcεRI, on a rat mast cell line, RBL-2H3, stimulated transcription of the gene for monocyte chemotactic protein-1 (MCP-1) and secretion of the protein. Unexpectedly, those delayed events appeared much less constrained by kinetic proofreading than had been documented for other receptor-initiated responses. The results of the present experiments are consistent with the proposal that the biosynthesis and secretion of MCP-1 result from a soluble messenger formed in the reaction cascades initiated by the receptor, and that Ca2+ could serve as that messenger. Interestingly, whereas receptor-mediated signals were required for transcription of the gene for MCP-1 and secretion of the chemokine, such signals were not required for the intervening step of translation of its mRNA.
Lipoxin A4 and Aspirin-Triggered 15-epi-Lipoxin A4 Inhibit Human Neutrophil Migration: Comparisons Between Synthetic 15 Epimers in Chemotaxis and Transmigration with Microvessel Endothelial Cells and Epithelial Cells
Authors: Fierro Iolanda M. , Colgan Sean P. , Bernasconi Giovanni , Petasis Nicos A. , Clish Clary B. , Arita Makoto , Serhan Charles N.
Published: April 2014

Lipoxins (LX) are bioactive eicosanoids that can be formed during cell to cell interactions in human tissues to self limit key responses in host defense and promote resolution. Aspirin treatment initiates biosynthesis of carbon 15 epimeric LXs, and both series of epimers (LX and aspirin-triggered 15-epi-LX) display counter-regulatory actions with neutrophils. In this study, we report that synthetic lipoxin A4 (LXA4) and 15-epi-LXA4 (i.e., 15(R)-LXA4 or aspirin-triggered LXA4) are essentially equipotent in inhibiting human polymorphonuclear leukocytes (PMN) in vitro chemotaxis in response to leukotriene B4, with the maximum inhibition (∼50% reduction) obtained at 1 nM LXA4. At higher concentrations, 15-epi-LXA4 proved more potent than LXA4 as its corresponding carboxyl methyl ester. Also, exposure of PMN to LXA4 and 15-epi-LXA4 markedly decreased PMN transmigration across both human microvessel endothelial and epithelial cells, where 15-epi-LXA4 was more active than LXA4 at “stopping” migration across epithelial cells. Differences in potency existed between LXA4 and 15-epi-LXA4 as their carboxyl methyl esters appear to arise from cell type-specific conversion of their respective carboxyl methyl esters to their corresponding carboxylates as monitored by liquid chromatography tandem mass spectrometry. Both synthetic LXA4 and 15-epi-LXA4 as free acids activate recombinant human LXA4 receptor (ALXR) to regulate gene expression, whereas the corresponding methyl ester of LXA4 proved to be a partial ALXR antagonist and did not effectively regulate gene expression. These results demonstrate the potent stereospecific actions shared by LXA4 and 15-epi-LXA4 for activating human ALXR-regulated gene expression and their ability to inhibit human PMN migration during PMN vascular as well as mucosal cell to cell interactions.
Studies Investigating Pretransplant Donor-Specific Blood Transfusion, Rapamycin, and the CD154-Specific Antibody IDEC-131 in a Nonhuman Primate Model of Skin Allotransplantation
Authors: Xu He , Montgomery Sean P. , Preston Edwin H. , Tadaki Douglas K. , Hale Douglas A. , Harlan David M. , Kirk Allan D.
Published: April 2014

Anti-CD154 variably prolongs allograft survival in nonhuman primates. Rodent studies suggest that adding pretransplant donor-specific transfusion (DST) and/or rapamycin to anti-CD154 improves survival. The CD154-specific Ab IDEC-131 was tested alone and in combination with rapamycin for its ability to inhibit rhesus MLRs. The ability of the Ab to block endothelial activation was also assessed. IDEC-131 was then tested alone and in combination with DST and/or rapamycin for its ability to prevent rejection of full-thickness, MHC-mismatched rhesus skin allografts. Animals were monitored for donor-specific hyporesponsiveness by MLR and alloantibody determination. IDEC-131 modestly inhibited rhesus MLRs and inhibited CD154-dependent endothelial cell activation. Rapamycin combined with IDEC-131 additively inhibited MLRs. IDEC-131 modestly prolonged allograft survival when compared with no treatment, rapamycin alone, or DST plus rapamycin. Adding DST to IDEC-131 did not prolong survival beyond IDEC-131 alone. IDEC-131 plus rapamycin was effective in prolonging graft survival, although animals had episodes of acute rejection before graft demise. Therapy with IDEC-131, rapamycin, and DST induced long-term allograft survival without intermittent acute rejection. However, no evidence for MLR inhibition was seen, and most animals eventually developed alloantibody. All animals ultimately rejected their grafts after drug withdrawal. IDEC-131 modestly prolongs rhesus skin allograft survival. Rapamycin and rapamycin plus DST improves the efficacy of IDEC-131 in prolonging allograft survival. IDEC-131, rapamycin, and DST are a promising combination for clinical evaluation in allotransplantation.
Cutting Edge: Impairment of Dendritic Cells and Adaptive Immunity by Ebola and Lassa Viruses
Authors: Mahanty Siddhartha , Hutchinson Karen , Agarwal Sudhanshu , Mcrae Michael , Rollin Pierre E. , Pulendran Bali
Published: April 2014

Acute infection of humans with Ebola and Lassa viruses, two principal etiologic agents of hemorrhagic fevers, often results in a paradoxical pattern of immune responses: early infection, characterized by an outpouring of inflammatory mediators such as TNF-α, IL-1β, and IL-6, vs late stage infections, which are associated with poor immune responses. The mechanisms underlying these diverse outcomes are poorly understood. In particular, the role played by cells of the innate immune system, such as dendritic cells (DC), is not known. In this study, we show that Ebola and Lassa viruses infect human monocyte-derived DC and impair their function. Monocyte-derived DC exposed to either virus fail to secrete proinflammatory cytokines, do not up-regulate costimulatory molecules, and are poor stimulators of T cells. These data represent the first evidence for a mechanism by which Ebola and Lassa viruses target DC to impair adaptive immunity.