bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthetic Prostacyclin Analogs Differentially Regulate Macrophage Function via Distinct Analog-Receptor Binding Specificities
Authors: Aronoff David M. , Peres Camila M. , Serezani Carlos H. , Ballinger Megan N. , Carstens Jennifer K. , Coleman Nicole , Moore Bethany B. , Peebles R. Stokes , Faccioli Lucia H. , Peters-Golden Marc
Published: April 2014

PGI2 (prostacyclin) is a lipid mediator with vasodilatory and antithrombotic effects used in the treatment of vasoconstrictive/ischemic diseases including pulmonary artery hypertension. However, emerging research supports a role for PGs, including PGI2, in the regulation of both innate and acquired immunity. As PGI2 is unstable, we sought to define the effects of various PGI2 analogs on resident alveolar macrophage (AM) and peritoneal macrophage (PM) innate immune functions. The effects of iloprost, carbaprostacyclin, and treprostinil on the regulation of phagocytosis, bacterial killing, and inflammatory mediator production were determined in both macrophage populations from rats. Iloprost failed to suppress AM functions to the same degree that it did in PMs, a characteristic shared by carbaprostacyclin. This difference reflected greater expression of the Gαs protein-coupled I prostanoid receptor and greater cAMP generation in PMs than AMs. Treprostinil inhibited phagocytosis, bacterial killing, and cytokine generation in AMs to a much greater degree than the other PGI2 analogs and more closely resembled the effects of PGE2. Studies with the E prostanoid (EP) 2 receptor antagonist AH-6809 and EP2-null macrophages indicated that this was due in part to the previously unknown ability of treprostinil to stimulate the EP2 receptor. The present investigation for the first time identifies differences in immunoregulatory properties of clinically administered PGI2 analogs. These studies are the first to explore the capacity of PGI2 to regulate bacterial killing and phagocytosis in macrophages, and our findings may hold important consequences regarding the risk of infection for patients receiving such agents.
Stimulation of Cell Surface CCR5 and CD40 Molecules by Their Ligands or by HSP70 Up-Regulates APOBEC3G Expression in CD4+ T Cells and Dendritic Cells
Authors: Pido-Lopez Jeffrey , Whittall Trevor , Wang Yufei , Bergmeier Lesley A. , Babaahmady Kaboutar , Singh Mahavir , Lehner Thomas
Published: April 2014

Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like-3G (A3G) is an intracellular innate antiviral factor that deaminates retroviral cytidine to uridine. In an attempt to harness the anti-HIV effect of A3G, we searched for an agent that would up-regulate A3G and identify the receptors involved. Stimulation of cell surface CCR5 with CCL3 and CD40 with CD40L or both molecules with microbial 70-kDa heat shock protein (HSP)70 up-regulated A3G mRNA and protein expression in human CD4+ T cells and monocyte-derived dendritic cells (DC), demonstrated by real-time PCR and Western blots, respectively. The specificity of CCR5 and CD40 stimulation was established by inhibition with TAK 779 and mAb to CD40, as well as using human embryonic kidney 293 cells transfected with CCR5 and CD40, respectively. A dose-dependent increase of A3G in CCL3- or HSP70-stimulated CD4+ T cells was associated with inhibition in HIV-1 infectivity. To differentiate between the inhibitory effect of HSP70-induced CCR5 binding and that of A3G, GFP-labeled pseudovirions were used to infect human embryonic kidney 293 cells, which showed inhibition of pseudovirion uptake, consistent with A3G being responsible for the inhibitory effect. Ligation of cell surface CCR5 receptors by CCL3 or CD40 by CD40L activated the ERK1/2 and p38 MAPK signaling pathways that induced A3G mRNA expression and production of the A3G protein. These in vitro results were corroborated by in vivo studies in rhesus macaques in which A3G was significantly up-regulated following immunization with SIVgp120 and p27 linked to HSP70. This novel preventive approach may in addition to adaptive immunity use the intracellular innate antiviral effect of A3G.
Correlates of Preserved CD4+ T Cell Homeostasis during Natural, Nonpathogenic Simian Immunodeficiency Virus Infection of Sooty Mangabeys: Implications for AIDS Pathogenesis
Authors: Sumpter Beth , Dunham Richard , Gordon Shari , Engram Jessica , Hennessy Margaret , Kinter Audrey , Paiardini Mirko , Cervasi Barbara , Klatt Nichole , McClure Harold , Milush Jeffrey M. , Staprans Silvija , Sodora Donald L. , Silvestri Guido
Published: April 2014

In contrast to HIV-infected humans, naturally SIV-infected sooty mangabeys (SMs) very rarely progress to AIDS. Although the mechanisms underlying this disease resistance are unknown, a consistent feature of natural SIV infection is the absence of the generalized immune activation associated with HIV infection. To define the correlates of preserved CD4+ T cell counts in SMs, we conducted a cross-sectional immunological study of 110 naturally SIV-infected SMs. The nonpathogenic nature of the infection was confirmed by an average CD4+ T cell count of 1,076 ± 589/mm3 despite chronic infection with a highly replicating virus. No correlation was found between CD4+ T cell counts and either age (used as a surrogate marker for length of infection) or viremia. The strongest correlates of preserved CD4+ T cell counts were a low percentage of circulating effector T cells (CD28−CD95+ and/or IL-7R/CD127−) and a high percentage of CD4+CD25+ T cells. These findings support the hypothesis that the level of immune activation is a key determinant of CD4+ T cell counts in SIV-infected SMs. Interestingly, we identified 14 animals with CD4+ T cell counts of <500/mm3, of which two show severe and persistent CD4+ T cell depletion (<50/mm3). Thus, significant CD4+ T cell depletion does occasionally follow SIV infection of SMs even in the context of generally low levels of immune activation, lending support to the hypothesis of multifactorial control of CD4+ T cell homeostasis in this model of infection. The absence of AIDS in these “CD4low” naturally SIV-infected SMs defines a protective role of the reduced immune activation even in the context of a significant CD4+ T cell depletion.
Perturbation of B Cell Activation in SLAM-Associated Protein-Deficient Mice Is Associated with Changes in Gammaherpesvirus Latency Reservoirs
Authors: Kim In-Jeong , Burkum Claire E. , Cookenham Tres , Schwartzberg Pamela L. , Woodland David L. , Blackman Marcia A.
Published: April 2014

Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP)) interactions with SLAM family proteins play important roles in immune function. SAP-deficient mice have defective B cell function, including impairment of germinal center formation, production of class-switched Ig, and development of memory B cells. B cells are the major reservoir of latency for both EBV and the homologous murine gammaherpesvirus, gammaherpesvirus 68. There is a strong association between the B cell life cycle and viral latency in that the virus preferentially establishes latency in activated germinal center B cells, which provides access to memory B cells, a major reservoir of long-term latency. In the current studies, we have analyzed the establishment and maintenance of γHV68 latency in wild-type and SAP-deficient mice. The results show that, despite SAP-associated defects in germinal center and memory B cell formation, latency was established and maintained in memory B cells at comparable frequencies to wild-type mice, although the paucity of memory B cells translated into a 10-fold reduction in latent load. Furthermore, there were defects in normal latency reservoirs within the germinal center cells and IgD+“naive” B cells in SAP-deficient mice, showing a profound effect of the SAP mutation on latency reservoirs.
Contribution of T Cells and Neutrophils in Protection of Young Susceptible Rats from Fatal Experimental Malaria
Authors: Pierrot Christine , Adam Estelle , Hot David , Lafitte Sophia , Capron Monique , George James D. , Khalife Jamal
Published: April 2014

In human malaria, children suffer very high rates of morbidity and mortality. To analyze the mechanisms involved in age-dependent protection against malaria, we developed an experimental model of infection in rats, where young rats are susceptible to Plasmodium berghei and adult rats control blood parasites and survive thereafter. In this study, we showed that protection of young rats could be achievable by adoptive transfer of spleen cells from adult protected rats, among which T cells could transfer partial protection. Transcriptome analysis of spleen cells transferring immunity revealed the overexpression of genes mainly expressed by eosinophils and neutrophils. Evaluation of the role of neutrophils showed that these cells were able to transfer partial protection to young rats. This antiparasitic effect was shown to be mediated, at least in part, through the neutrophil protein-1 defensin. Further adoptive transfer experiments indicated an efficient cooperation between neutrophils and T cells in protecting all young recipients. These observations, together with those from in vitro studies in human malaria, suggest that the failure of children to control infection could be related not only to an immaturity of their adaptive immunity but also to a lack in an adequate innate immune response.
The Chemokine Receptor CXCR3 Attenuates the Control of Chronic Mycobacterium tuberculosis Infection in BALB/c Mice
Authors: Chakravarty Soumya D. , Xu Jiayong , Lu Bao , Gerard Craig , Flynn JoAnne , Chan John
Published: April 2014

The chemokine receptor CXCR3 plays a significant role in regulating the migration of Th1 cells. Given the importance of Th1 immunity in the control of tuberculous infection, the results of the present study demonstrating that CXCR3-deficient BALB/c mice are more resistant to Mycobacterium tuberculosis, compared with wild-type mice, is surprising. This enhanced resistance manifests in the chronic but not the acute phase of infection. Remarkable differences in the cellular composition of the pulmonic granuloma of the CXCR3−/− and wild-type mice were found, the most striking being the increase in the number of CD4+ T cells in the knockout strain. In the chronic phase of infection, the number of CD69-expressing CD4+ T lymphocytes in the lungs of CXCR3−/− mice was higher than in wild-type mice. Additionally, at 1 mo postinfection, the number of IFN-γ-producing CD4+ T cells in the lungs and mediastinal lymph nodes of the CXCR3-deficient strain was elevated compared with wild-type mice. Pulmonic expression of IFN-γ, IL-12, TNF-α, or NO synthase 2, the principal antimycobacterial factors, were equivalent in the two mouse strains. These results indicate that: 1) CXCR3 plays a role in modulating the cellular composition of tuberculous granuloma; 2) CXCR3 impairs antimycobacterial activity in chronic tuberculosis; and 3) in the absence of CXCR3, mice exhibit a heightened state of CD4+ T lymphocyte activation in the chronic phase of infection that is associated with enhanced CD4+ T cell priming. Therefore, CXCR3 can attenuate the host immune response to M. tuberculosis by adversely affecting T cell priming.
A Novel Role for IκB Kinase (IKK) α and IKKβ in ERK-Dependent Up-Regulation of MUC5AC Mucin Transcription by Streptococcus pneumoniae
Authors: Ha Unhwan , Lim Jae Hyang , Jono Hirofumi , Koga Tomoaki , Srivastava Amit , Malley Richard , Pagès Gilles , Pouysségur Jacques , Li Jian-Dong
Published: April 2014

Epithelial cells represent the first line of host innate defense against invading microbes by elaborating a range of molecules involved in pathogen clearance. In particular, epithelial mucins facilitate the mucociliary clearance by physically trapping inhaled microbes. Up-regulation of mucin production thus represents an important host innate defense response against invading microbes. How mucin is induced in upper respiratory Streptococcus pneumoniae infections is unknown. In this study, we show that pneumolysin is required for up-regulation of MUC5AC mucin via TLR4-dependent activation of ERK in human epithelial cells in vitro and in mice in vivo. Interestingly, a “second wave” of ERK activation appears to be important in mediating MUC5AC induction. Moreover, IκB kinase (IKK) α and IKKβ are distinctly involved in MUC5AC induction via an ERK1-dependent, but IκBα-p65- and p100-p52-independent, mechanism, thereby revealing novel roles for IKKs in mediating up-regulation of MUC5AC mucin by S. pneumoniae.
Induction of the Formyl Peptide Receptor 2 in Microglia by IFN-γ and Synergy with CD40 Ligand
Authors: Chen Keqiang , Iribarren Pablo , Huang Jian , Zhang Lingzhi , Gong Wanghua , Cho Edward H. , Lockett Stephen , Dunlop Nancy M. , Wang Ji Ming
Published: April 2014

Human formyl peptide receptor (FPR)-like 1 (FPRL1) and its mouse homologue mFPR2 are functional receptors for a variety of exogenous and host-derived chemotactic peptides, including amyloid β 1–42 (Aβ42), a pathogenic factor in Alzheimer’s disease. Because mFPR2 in microglial cells is regulated by proinflammatory stimulants including TLR agonists, in this study we investigated the capacity of IFN-γ and the CD40 ligand (CD40L) to affect the expression and function of mFPR2. We found that IFN-γ, when used alone, induced mFPR2 mRNA expression in a mouse microglial cell line and primary microglial cells in association with increased cell migration in response to mFPR2 agonists, including Aβ42. IFN-γ also increased the endocytosis of Aβ42 by microglial cells via mFPR2. The effect of IFN-γ on mFPR2 expression in microglial cells was dependent on activation of MAPK and IκB-α. IFN-γ additionally increased the expression of CD40 by microglial cells and soluble CD40L significantly promoted cell responses to IFN-γ during a 6-h incubation period by enhancing the activation of MAPK and IκB-α signaling pathways. We additionally found that the effect of IFN-γ and its synergy with CD40L on mFPR2 expression in microglia was mediated in part by TNF-α. Our results suggest that IFN-γ and CD40L, two host-derived factors with increased concentrations in inflammatory central nervous system diseases, may profoundly affect microglial cell responses in the pathogenic process in which mFPR2 agonist peptides are elevated.
CD59, a Complement Regulatory Protein, Controls Choroidal Neovascularization in a Mouse Model of Wet-Type Age-Related Macular Degeneration
Authors: Bora Nalini S. , Kaliappan Sankaranarayanan , Jha Purushottam , Xu Qin , Sivasankar Baalasubramanian , Harris Claire L. , Morgan B. Paul , Bora Puran S.
Published: April 2014

We have shown that membrane attack complex (MAC) formation via the activation of the alternative pathway plays a central role in the laser-induced choroidal neovascularization (CNV). This study was undertaken to understand the role of a complement regulatory protein, CD59, which controls MAC assembly and function, in this model. CNV was induced by laser photocoagulation in C57BL/6 and Cd59a−/− mice using an argon laser. Animals from each group were sacrificed on day 1, 3, 5, and 7 postlaser. Retinal pigment epithelium-choroid-scleral tissue was examined to determine the incidence and size of CNV complex, and semiquantitative RT-PCR and Western blot analysis for CD59a was studied. Recombinant soluble mouse CD59a-IgG2a fusion (rsCD59a-Fc) protein was injected via i.p. or intravitreal routes 24 h before laser. Our results demonstrated that CD59a (both mRNA and protein) was down-regulated during laser-induced CNV. Cd59a−/− mice developed CNV complex early in the disease process. Increased MAC deposition was also observed in these Cd59a−/− mice. Administration of rsCD59a-Fc inhibited the development of CNV complex in the mouse model by blocking MAC formation and also inhibited expression of angiogenic growth factors. These data provide strong evidence that CD59a plays a crucial role in regulating complement activation and MAC formation essential for the release of growth factors that drive the development of laser-induced CNV in mice. Thus, our results suggest that the inhibition of complement by soluble CD59 may provide a novel therapeutic alternative to current treatment.
Commensal Bacteria Exacerbate Intestinal Inflammation but Are Not Essential for the Development of Murine Ileitis
Authors: Bamias Giorgos , Okazawa Akira , Rivera-Nieves Jesus , Arseneau Kristen O. , De La Rue Sarah A. , Pizarro Theresa T. , Cominelli Fabio
Published: April 2014

The pathogenesis of Crohn’s disease has been associated with a dysregulated response of the mucosal immune system against intraluminal Ags of bacterial origin. In this study, we have investigated the effects of germfree (GF) conditions in the SAMP1/YitFc murine model of Crohn’s disease-like ileitis. We show that the bacterial flora is not essential for ileitis induction, because GF SAMP1/YitFc mice develop chronic ileitis. However, compared with disease in specific pathogen-free (SPF) mice, ileitis in GF mice is significantly attenuated, and is associated with delayed lymphocytic infiltration and defective mucosal expression of Th2 cytokines. In addition, we demonstrate that stimulation with purified fecal Ags from SPF, but not GF mice leads to the generation of IL-4-secreting effector lymphocytes. This result suggests that commensal bacteria drive Th2 responses characteristic of the chronic phase of SAMP1/YitFc ileitis. Finally, adoptive transfer of CD4-positive cells from GF, but not SPF mice induces severe colitis in SCID recipients. These effects were associated with a decreased frequency of CD4+CD25+Foxp3+ T cells in the mesenteric lymph nodes of GF mice compared with SPF mice, as well as lower relative gene expression of Foxp3 in CD4+CD25+ T cells in GF mice. It is therefore apparent that, in the absence of live intraluminal bacteria, the regulatory component of the mucosal immune system is compromised. All together, our results indicate that in SAMP1/YitFc mice, bacterial flora exacerbates intestinal inflammation, but is not essential for the generation of the chronic ileitis that is characteristic of these mice.
Unique Human Tumor Antigens: Immunobiology and Use in Clinical Trials
Authors: Parmiani Giorgio , De Filippo Annamaria , Novellino Luisa , Castelli Chiara
Published: April 2014

The individual, unique tumor Ags, which characterize each single tumor, were described 50 years ago in rodents but their molecular characterization was limited to few of them and obtained during the last 20 years. Here we summarize the evidence for the existence and the biological role of such Ags in human tumors, although such evidence was provided only during the last 10 years and by a limited number of studies, a fact leading to a misrepresentation of unique Ags in human tumor immunology. This was also due to the increasing knowledge on the shared, self-human tumor Ags, which have been extensively used as cancer vaccines. In this review, we highlight the biological and clinical importance of unique Ags and suggest how they could be used in clinical studies aimed at assessing their immunogenic and clinical potential both in active and adoptive immunotherapy of human tumors.
Cutting Edge: The PI3K p110δ Is Required for Down-Regulation of RAG Expression in Immature B Cells
Authors: Llorian Miriam , Stamataki Zania , Hill Susan , Turner Martin , Mårtensson Inga-Lill
Published: April 2014

At the immature B cell stage the BCR signals the down-regulation of the RAG genes and Ig L chain (LC) allelic and isotype exclusion. The signaling pathway that regulates these events is poorly characterized. We demonstrate that immature B cells from mice deficient in the PI3K catalytic subunit p110δ fail to suppress RAG expression and inappropriately recombine κ and λ LC loci. In addition, in the presence of the autoantigen, clonal deletion and receptor editing still takes place, demonstrating that these processes are independent of p110δ. These results demonstrate a role for p110δ in the regulation of RAG gene expression and thereby LC allelic/isotype exclusion.
Distinct Differentiation Potential of Blood Monocyte Subsets in the Lung
Authors: Landsman Limor , Varol Chen , Jung Steffen
Published: April 2014

Peripheral blood monocytes are a population of circulating mononuclear phagocytes that harbor potential to differentiate into macrophages and dendritic cells. As in humans, monocytes in the mouse comprise two phenotypically distinct subsets that are Gr1highCX3CR1int and Gr1lowCX3CR1high, respectively. The question remains whether these populations contribute differentially to the generation of peripheral mononuclear phagocytes. In this study, we track the fate of adoptively transferred, fractionated monocyte subsets in the lung of recipient mice. We show that under inflammatory and noninflammatory conditions, both monocyte subsets give rise to pulmonary dendritic cells. In contrast, under the conditions studied, only Gr1lowCX3CR1high monocytes, but not Gr1highCX3CR1int cells, had the potential to differentiate into lung macrophages. However, Gr1highCX3CR1int monocytes could acquire this potential upon conversion into Gr1lowCX3CR1high cells. Our results therefore indicate an intrinsic dichotomy in the differentiation potential of the two main blood monocyte subsets.
Stromal Derived Growth Factor-1α: Another Mediator in Neural-Emerging Immune System through Tac1 Expression in Bone Marrow Stromal Cells
Authors: Corcoran Kelly E. , Patel Nitixa , Rameshwar Pranela
Published: April 2014

Stromal cell-derived growth factor-1α (SDF-1α) is a member of the CXC chemokines and interacts with the G protein, seven-transmembrane CXCR4 receptor. SDF-1α acts as a chemoattractant for immune and hemopoietic cells. The Tac1 gene encodes peptides belonging to the tachykinin family with substance P being the predominant member. Both SDF-1α and Tac1 peptides are relevant hemopoietic regulators. This study investigated the effects of SDF-1α on Tac1 expression in the major hemopoietic supporting cells, the bone marrow stroma, and addresses the consequence to hemopoiesis. Reporter gene assays with the 5′ flanking region of Tac1 showed a bell-shaped effect of SDF-1α on luciferase activity with 20 ng/ml SDF-1α acting as stimulator, whereas 50 and 100 ng/ml SDF-1α acted as inhibitors. Gel shift assays and transfection with wild-type and mutant IκB indicate NF-κB as a mediator in the repressive effects at 50 and 100 ng/ml SDF-1α. Northern analyses and ELISA showed correlations among reporter gene activities, mRNA (β-preprotachykinin I), and protein levels for substance P. Of relevance is the novel finding by long-term culture-initiating cell assays that showed an indirect effect of SDF-1α on hemopoiesis through substance P production. The results also showed neurokinin 1 and not neurokinin 2 as the relevant receptor. Another crucial finding is that substance P does not regulate the production of SDF-1α in stroma. The studies indicate that SDF-1α levels above baseline production in bone marrow stroma induce the production of substance P to stimulate hemopoiesis. Substance P, however, does not act as autocrine stimulator to induce the production of SDF-1α. This study adds SDF-1α as a mediator within the neural-immune-hemopoietic axis.
Regulation of CD8+ T Lymphocyte Effector Function and Macrophage Inflammatory Cytokine Production by Retinoic Acid Receptor γ
Authors: Dzhagalov Ivan , Chambon Pierre , He You-Wen
Published: April 2014

Vitamin A and its derivatives regulate a broad array of immune functions. The effects of these retinoids are mediated through members of retinoic acid receptors (RARs) and retinoid X receptors. However, the role of individual retinoid receptors in the pleiotropic effects of retinoids remains unclear. To dissect the role of these receptors in the immune system, we analyzed immune cell development and function in mice conditionally lacking RARγ, the third member of the RAR family. We show that RARγ is dispensable for T and B lymphocyte development, the humoral immune response to a T-dependent Ag and in vitro Th cell differentiation. However, RARγ-deficient mice had a defective primary and memory CD8+ T cell response to Listeria monocytogenes infection. Unexpectedly, RARγ-deficient macrophages exhibited impaired inflammatory cytokine production upon TLR stimulation. These results suggest that under physiological condition, RARγ is a positive regulator of inflammatory cytokine production.
Bacterial c-di-GMP Is an Immunostimulatory Molecule
Authors: Karaolis David K. R. , Means Terry K. , Yang De , Takahashi Munehisa , Yoshimura Teizo , Muraille Eric , Philpott Dana , Schroeder John T. , Hyodo Mamoru , Hayakawa Yoshihiro , Talbot Brian G. , Brouillette Eric , Malouin François
Published: April 2014

Cyclic diguanylate (c-di-GMP) is a bacterial intracellular signaling molecule. We have shown that treatment with exogenous c-di-GMP inhibits Staphylococcus aureus infection in a mouse model. We now report that c-di-GMP is an immodulator and immunostimulatory molecule. Intramammary treatment of mice with c-di-GMP 12 and 6 h before S. aureus challenge gave a protective effect and a 10,000-fold reduction in CFUs in tissues (p < 0.001). Intramuscular vaccination of mice with c-di-GMP coinjected with S. aureus clumping factor A (ClfA) Ag produced serum with significantly higher anti-ClfA IgG Ab titers (p < 0.001) compared with ClfA alone. Intraperitoneal injection of mice with c-di-GMP activated monocyte and granulocyte recruitment. Human immature dendritic cells (DCs) cultured in the presence of c-di-GMP showed increased expression of costimulatory molecules CD80/CD86 and maturation marker CD83, increased MHC class II and cytokines and chemokines such as IL-12, IFN-γ, IL-8, MCP-1, IFN-γ-inducible protein 10, and RANTES, and altered expression of chemokine receptors including CCR1, CCR7, and CXCR4. c-di-GMP-matured DCs demonstrated enhanced T cell stimulatory activity. c-di-GMP activated p38 MAPK in human DCs and ERK phosphorylation in human macrophages. c-di-GMP is stable in human serum. We propose that cyclic dinucleotides like c-di-GMP can be used clinically in humans and animals as an immunomodulator, immune enhancer, immunotherapeutic, immunoprophylactic, or vaccine adjuvant.
Peripheral Tolerance Induction Using Ethylenecarbodiimide-Fixed APCs Uses both Direct and Indirect Mechanisms of Antigen Presentation for Prevention of Experimental Autoimmune Encephalomyelitis
Authors: Turley Danielle M. , Miller Stephen D.
Published: April 2014

MHC class II (MHC II)-restricted T cell responses are a common driving force of autoimmune disease. Accordingly, numerous therapeutic strategies target CD4+ T cells with the hope of attenuating autoimmune responses and restoring self-tolerance. We have previously reported that i.v. treatment with Ag-pulsed, ethylenecarbodiimide (ECDI)-fixed splenocytes (Ag-SPs) is an efficient protocol to induce Ag-specific tolerance for prevention and treatment of experimental autoimmune encephalomyelitis (EAE). Ag-SPs coupled with peptide can directly present peptide:MHC II complexes to target CD4+ T cells in the absence of costimulation to induce anergy. However, Ag-SPs coupled with whole protein also efficiently attenuates Ag-specific T cell responses suggesting the potential contribution of alternative indirect mechanisms/interactions between the Ag-SPs and target CD4+ T cells. Thus, we investigated whether MHC II compatibility was essential to the underlying mechanisms by which Ag-SP induces tolerance during autoimmune disease. Using MHC-deficient, allogeneic, and/or syngeneic donor Ag-SPs, we show that MHC compatibility between the Ag-SP donor and the host is not required for tolerance induction. Interestingly, we found that ECDI treatment induces apoptosis of the donor cell population which promotes uptake and reprocessing of donor cell peptides by host APCs resulting in the apparent MHC II-independent induction of tolerance. However, syngeneic donor cells are more efficient at inducing tolerance, suggesting that Ag-SPs induce functional Ag-SP tolerance via both direct and indirect (cross-tolerance) mechanisms leading to prevention and effective treatment of autoimmune disease.
The N Terminus of the Non-T Cell Activation Linker (NTAL) Confers Inhibitory Effects on Pre-B Cell Differentiation
Authors: Herzog Sebastian , Jumaa Hassan
Published: April 2014

SLP-65 and the linker for activation of T cells (LAT) are central adaptor proteins that link the activated pre-BCR to downstream events in pre-B cells. Recently, a new transmembrane adaptor called NTAL/LAB/LAT2 (hereafter called NTAL for non-T cell activation linker) with striking functional and structural similarity to LAT has been identified in B cells. In this study, we compare the function of NTAL and LAT in pre-BCR signaling and show that, in contrast to LAT, NTAL does not induce pre-BCR down-regulation, calcium flux, or pre-B cell differentiation. To test whether differences between NTAL-mediated and LAT-mediated signaling are caused by the missing phospholipase C (PLC)-γ binding motif in NTAL, we inserted the PLC-γ1/2 binding motif of LAT into NTAL. This insertion rendered NTAL capable of activating pre-BCR down-regulation and calcium flux. Unexpectedly however, the ability of NTAL to induce calcium flux was not sufficient to promote pre-B cell differentiation, suggesting that the PLC-γ binding motif has only partial effects on NTAL-mediated pre-BCR signaling. By generating chimeric swap mutants, we identified the N terminus of NTAL as an inhibitory domain that prevents pre-B cell differentiation while allowing pre-BCR down-regulation and receptor-mediated calcium flux. Our data suggest that, in addition to the missing PLC-γ1/2 binding motif, the N terminus is responsible for the functional differences between NTAL and LAT in pre-B cells.
Mucosal HIV-1 Pox Virus Prime-Boost Immunization Induces High-Avidity CD8+ T Cells with Regime-Dependent Cytokine/Granzyme B Profiles
Authors: Ranasinghe Charani , Turner Stephen J. , McArthur Craig , Sutherland Duncan B. , Kim Jee-Hye , Doherty Peter C. , Ramshaw Ian A.
Published: April 2014

The quality of virus-specific CD8+ CTL immune responses generated by mucosal and systemic poxvirus prime-boost vaccines were evaluated in terms of T cell avidity and single-cell analysis of effector gene expression. Intranasal (I.N.) immunization regimes generated higher avidity CTL responses specific for HIV KdGag197–205 (amino acid sequence AMQMLKETI; H-2Kd binding) compared with i.m. immunization regime. Single-cell RT-PCR of KdGag197–205-specific mucosal and systemic CTL revealed that the cytokine and granzyme B expression profiles were dependent on both the route and time after immunization. The I.N./i.m.-immunized group elicited elevated number of CTL-expressing granzyme B mRNA from the genitomucosal sites compared with the i.m./i.m. regime. Interestingly, CTL generated after both I.N. or i.m. immunization demonstrated expression of Th2 cytokine IL-4 mRNA that was constitutively expressed over time, although lower numbers were observed after I.N./I.N. immunization. Results suggest that after immunization, Ag-specific CTL expression of IL-4 may be an inherent property of the highly evolved poxvirus vectors. Current observations indicate that the quality of CTL immunity generated after immunization can be influenced by the inherent property of vaccine vectors and route of vaccine delivery. A greater understanding of these factors will be crucial for the development of effective vaccines in the future.
Rapid Clonal Expansion and Prolonged Maintenance of Memory CD8+ T Cells of the Effector (CD44highCD62Llow) and Central (CD44highCD62Lhigh) Phenotype by an Archaeosome Adjuvant Independent of TLR2
Authors: Krishnan Lakshmi , Gurnani Komal , Dicaire Chantal J. , van Faassen Henk , Zafer Ahmed , Kirschning Carsten J. , Sad Subash , Sprott G. Dennis
Published: April 2014

Vaccines capable of eliciting long-term T cell immunity are required for combating many diseases. Live vectors can be unsafe whereas subunit vaccines often lack potency. We previously reported induction of CD8+ T cells to Ag entrapped in archaeal glycerolipid vesicles (archaeosomes). In this study, we evaluated the priming, phenotype, and functionality of the CD8+ T cells induced after immunization of mice with OVA-Methanobrevibacter smithii archaeosomes (MS-OVA). A single injection of MS-OVA evoked a profound primary response but the numbers of H-2KbOVA257–264-specific CD8+ T cells declined by 14–21 days, and <1% of primarily central phenotype (CD44highCD62Lhigh) cells persisted. A booster injection of MS-OVA at 3–11 wk promoted massive clonal expansion and a peak effector response of ∼20% splenic/blood OVA257–264-specific CD8+ T cells. Furthermore, contraction was protracted and the memory pool (IL-7Rαhigh) of ∼5% included effector (CD44highCD62Llow) and central (CD44highCD62Lhigh) phenotype cells. Recall response was observed even at >300 days. CFSE-labeled naive OT-1 (OVA257–264 TCR transgenic) cells transferred into MS-OVA-immunized recipients cycled profoundly (>90%) within the first week of immunization indicating potent Ag presentation. Moreover, ∼25% cycling of Ag-specific cells was seen for >50 days, suggesting an Ag depot. In vivo, CD8+ T cells evoked by MS-OVA killed >80% of specific targets, even at day 180. MS-OVA induced responses similar in magnitude to Listeria monocytogenes-OVA, a potent live vector. Furthermore, protective CD8+ T cells were induced in TLR2-deficient mice, suggesting nonengagement of TLR2 by archaeal lipids. Thus, an archaeosome adjuvant vaccine represents an alternative to live vectors for inducing CD8+ T cell memory.
Signaling in Lipopolysaccharide-Induced Stabilization of Formyl Peptide Receptor 1 mRNA in Mouse Peritoneal Macrophages
Authors: Mandal Palash , Hamilton Thomas
Published: April 2014

To identify the TLR4-initiated signaling events that couple to formyl peptide receptor (FPR)1 mRNA stabilization, macrophages were treated with LPS along with a selection of compounds targeting several known signaling pathways. Although inhibitors of protein tyrosine kinases, MAPKs, and stress-activated kinases had little or no effect on the response to LPS, LY294002 (LY2) and parthenolide (an IκB kinase inhibitor) were both potent inhibitors. LY2 but not parthenolide blocked the LPS-induced stabilization of FPR1 mRNA. Although both LY2 and wortmannin effectively blocked PI3K activity, wortmannin had little effect on FPR1 expression and did not modulate the decay of FPR1 mRNA. Moreover, although LY2 was demonstrated to be a potent inhibitor of PI3K activity, a structural analog of LY2, LY303511 (LY3), which did not inhibit PI3K, was equally effective at preventing LPS-stimulated FPR1 expression. The mammalian target of rapamycin activity (measured as phospho-p70S6 kinase) was activated by LPS but not significantly blocked by LY2. In addition, although rapamycin blocked mTOR activity, it did not inhibit FPR1 mRNA expression. Finally, the mechanisms involved in stabilization of FPR1 by LPS could be distinguished from those involved in stabilization of AU-rich mRNAs because the prolonged half-life of FPR1 mRNA was insensitive to the inhibition of p38 MAPK. These findings demonstrate that LY2/LY3 targets a novel TLR4-linked signaling pathway that selectively couples to the stabilization of FPR1 mRNA.
A SmD Peptide Induces Better Antibody Responses to Other Proteins within the Small Nuclear Ribonucleoprotein Complex than to SmD Protein via Intermolecular Epitope Spreading
Authors: Deshmukh Umesh S. , Bagavant Harini , Sim Davis , Pidiyar Vyankatesh , Fu Shu Man
Published: April 2014

Autoantibody response against the small nuclear ribonucleoprotein (snRNP) complex is a characteristic feature of systemic lupus erythematosus. The current investigation was undertaken to determine whether activation of SmD-reactive T cells by synthetic peptides harboring T cell epitopes can initiate a B cell epitope spreading cascade within the snRNP complex. T cell epitopes on SmD were mapped in A/J mice and were localized to three regions on SmD, within aa 26–55, 52–69, and 86–115. Immunization with synthetic peptides SmD31–45, SmD52–66, and SmD91–110 induced T and B cell responses to the peptides, with SmD31–45 inducing the strongest response. However, only SmD52–66 immunization induced T cells capable of reacting with SmD. Analysis of sera by immunoprecipitation assays showed that intermolecular B cell epitope spreading to U1RNA-associated A ribonucleoprotein and SmB was consistently observed only in the SmD52–66-immunized mice. Surprisingly, in these mice, Ab responses to SmD were at low levels and transient. In addition, the sera did not react with other regions on SmD, indicating a lack of intramolecular B cell epitope spreading within SmD. Our study demonstrates that T cell responses to dominant epitope on a protein within a multiantigenic complex are capable of inducing B cell responses to other proteins within the complex. This effect can happen without generating a good Ab response to the protein from which the T epitope was derived. Thus caution must be taken in the identification of Ags responsible for initiating autoimmune responses based solely on serological analysis of patients and animals with systemic autoimmune disorders.
Deficient CD4+CD25high T Regulatory Cell Function in Patients with Active Systemic Lupus Erythematosus
Authors: Valencia Xavier , Yarboro Cheryl , Illei Gabor , Lipsky Peter E.
Published: April 2014

CD4+CD25+ T regulatory cells (Tregs) play an essential role in maintaining immunologic homeostasis and preventing autoimmunity. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a loss of tolerance to nuclear components. We hypothesized that altered function of CD4+CD25high Tregs might play a role in the breakdown of immunologic self-tolerance in patients with SLE. In this study, we report a significant decrease in the suppressive function of CD4+CD25high Tregs from peripheral blood of patients with active SLE as compared with normal donors and patients with inactive SLE. Notably, CD4+CD25high Tregs isolated from patients with active SLE expressed reduced levels of FoxP3 mRNA and protein and poorly suppressed the proliferation and cytokine secretion of CD4+ effector T cells in vitro. In contrast, the expression of FoxP3 mRNA and protein and in vitro suppression of the proliferation of CD4+ effector T cells by Tregs isolated from inactive SLE patients, was comparable to that of normal individuals. In vitro activation of CD4+CD25high Tregs from patients with active SLE increased FoxP3 mRNA and protein expression and restored their suppressive function. These data are the first to demonstrate a reversible defect in CD4+CD25high Treg function in patients with active SLE, and suggest that strategies to enhance the function of these cells might benefit patients with this autoimmune disease.
The JAK-STAT Signaling Pathway: Input and Output Integration
Authors: Murray Peter J.
Published: April 2014

Universal and essential to cytokine receptor signaling, the JAK-STAT pathway is one of the best understood signal transduction cascades. Almost 40 cytokine receptors signal through combinations of four JAK and seven STAT family members, suggesting commonality across the JAK-STAT signaling system. Despite intense study, there remain substantial gaps in understanding how the cascades are activated and regulated. Using the examples of the IL-6 and IL-10 receptors, I will discuss how diverse outcomes in gene expression result from regulatory events that effect the JAK1-STAT3 pathway, common to both receptors. I also consider receptor preferences by different STATs and interpretive problems in the use of STAT-deficient cells and mice. Finally, I consider how the suppressor of cytokine signaling (SOCS) proteins regulate the quality and quantity of STAT signals from cytokine receptors. New data suggests that SOCS proteins introduce additional diversity into the JAK-STAT pathway by adjusting the output of activated STATs that alters downstream gene activation.
Cutting Edge: Regulatory T Cells Prevent Efficient Clearance of Mycobacterium tuberculosis
Authors: Kursar Mischo , Koch Markus , Mittrücker Hans-Willi , Nouailles Geraldine , Bonhagen Kerstin , Kamradt Thomas , Kaufmann Stefan H. E.
Published: April 2014

Mycobacterium tuberculosis remains one of the top microbial killers of humans causing ∼2 million deaths annually. More than 90% of the 2 billion individuals infected never develop active disease, indicating that the immune system is able to generate mechanisms that control infection. However, the immune response generally fails to achieve sterile clearance of bacilli. Using adoptive cell transfer into C57BL/6J-Rag1tm1Mom mice (Rag1−/−), we show that regulatory T cells prevent eradication of tubercle bacilli by suppressing an otherwise efficient CD4+ T cell response. This protective CD4+ T cell response was not correlated with increased numbers of IFN-γ- or TNF-α-expressing cells or general expression levels of IFN-γ or inducible NO synthase in infected organs compared with wild-type C57BL/6 animals. Furthermore, suppression of protection by cotransferred regulatory T cells was neither accompanied by a general increase of IL-10 expression nor by higher numbers of IL-10-producing CD4+ T cells.
Murine Neonatal CD4+ Cells Are Poised for Rapid Th2 Effector-Like Function
Authors: Rose Shawn , Lichtenheld Mathias , Foote Monica R. , Adkins Becky
Published: April 2014

Murine neonates typically mount Th2-biased immune responses. This entails a cell-intrinsic component whose molecular basis is unknown. We found that neonatal CD4+ T cells are uniquely poised for rapid Th2 function. Within 24 h of activation, neonatal CD4+ cells made high levels of IL-4 and IL-13 mRNA and protein. The rapid high-level IL-4 production arose from a small subpopulation of cells, did not require cell cycle entry, and was unaffected by pharmacologic DNA demethylation. CpG methylation analyses in resting neonatal cells revealed pre-existing hypomethylation at a key Th2 cytokine regulatory region, termed conserved noncoding sequence 1 (CNS-1). Robust Th2 function and increased CNS-1 demethylation was a stable property that persisted in neonatal Th2 effectors. The transcription factor STAT6 was not required for CNS-1 demethylation and this state was already established in neonatal CD4 single-positive thymocytes. CNS-1 demethylation levels were much greater in IL-4-expressing CD4 single-positive thymocytes compared with unactivated cells. Together, these results indicate that neonatal CD4+ T cells possess distinct qualities that could predispose them toward rapid, effector-like Th2 function.
Invariant NKT Cells Amplify the Innate Immune Response to Lipopolysaccharide
Authors: Nagarajan Niranjana A. , Kronenberg Mitchell
Published: April 2014

NKT cells are thought of as a bridge between innate and adaptive immunity. In this study, we demonstrate that mouse NKT cells are activated in response to Escherichia coli LPS, and produce IFN-γ, but not IL-4, although activation through their TCR typically induces both IL-4 and IFN-γ production. IFN-γ production by NKT cells is dependent on LPS-induced IL-12 and IL-18 from APC. LPS induced IFN-γ production by NKT cells does not require CD1d-mediated presentation of an endogenous Ag and exposure to a combination of IL-12 and IL-18 is sufficient to activate them. In mice that are deficient for NKT cells, innate immune cells are activated less efficiently in response to LPS, resulting in the reduced production of TNF and IFN-γ. We propose that in addition to acting as a bridge to adaptive immunity, NKT cells act as an early amplification step in the innate immune response and that the rapid and complete initiation of this innate response depends on the early production of IFN-γ by NKT cells.
IL-2/Neuroantigen Fusion Proteins as Antigen-Specific Tolerogens in Experimental Autoimmune Encephalomyelitis (EAE): Correlation of T Cell-Mediated Antigen Presentation and Tolerance Induction
Authors: Mannie Mark D. , Clayson Barbara A. , Buskirk Elizabeth J. , DeVine Jarret L. , Hernandez Jose J. , Abbott Derek J.
Published: April 2014

The purpose of this study was to assess whether the Ag-targeting activity of cytokine/neuroantigen (NAg) fusion proteins may be associated with mechanisms of tolerance induction. To assess this question, we expressed fusion proteins comprised of a N-terminal cytokine domain and a C-terminal NAg domain. The cytokine domain comprised either rat IL-2 or IL-4, and the NAg domain comprised the dominant encephalitogenic determinant of the guinea pig myelin basic protein. Subcutaneous administration of IL2NAg (IL-2/NAg fusion protein) into Lewis rats either before or after an encephalitogenic challenge resulted in an attenuated course of experimental autoimmune encephalomyelitis. In contrast, parallel treatment of rats with IL4NAg (IL-4/NAg fusion protein) or NAg lacked tolerogenic activity. In the presence of IL-2R+ MHC class II+ T cells, IL2NAg fusion proteins were at least 1,000 times more potent as an Ag than NAg alone. The tolerogenic activity of IL2NAg in vivo and the enhanced potency in vitro were both dependent upon covalent linkage of IL-2 and NAg. IL4NAg also exhibited enhanced antigenic potency. IL4NAg was ∼100-fold more active than NAg alone in the presence of splenic APC. The enhanced potency of IL4NAg also required covalent linkage of cytokine and NAg and was blocked by soluble IL-4 or by a mAb specific for IL-4. Other control cytokine/NAg fusion proteins did not exhibit a similar enhancement of Ag potency compared with NAg alone. Thus, the IL2NAg and IL4NAg fusion proteins targeted NAg for enhanced presentation by particular subsets of APC. The activities of IL2NAg revealed a potential relationship between NAg targeting to activated T cells, T cell-mediated Ag presentation, and tolerance induction.
Modulation of CD103 Expression on Human Colon Carcinoma-Specific CTL
Authors: Ling Khoon-Lin , Dulphy Nicolas , Bahl Pru , Salio Mariolina , Maskell Kevin , Piris Juan , Warren Bryan F. , George Bruce D. , Mortensen Neil J. , Cerundolo Vincenzo
Published: April 2014

Recent results have shown a correlation between survival and frequency of tumor-infiltrating T cells in colorectal cancer patients. However, the mechanisms controlling the ability of human T lymphocytes to infiltrate colon carcinoma remain unclear. Although, it is known that expression of the integrin CD103αE/β7 by intraepithelial lymphocytes controls the retention of lymphocytes in epithelial layers, very little is known about the expression of intestinal homing receptors in human T lymphocytes. In particular, it remains unknown whether expression of CD103/β7 by human colon cancer-specific T lymphocytes is controlled by recognition of tumor Ags and is imprinted during T cell priming, facilitating its expression during memory T cell activation. In this study, we demonstrate that expression of CD103/β7 in human colon carcinoma-specific CTL is synergistically enhanced by the simultaneous TGF-β1 stimulation and Ag recognition. These results were confirmed by using a panel of human CTL clones. Finally, we show that priming of naive CD8+ T cells in the presence of TGF-β1 ensures up-regulation of CD103/β7 in recall responses, at concentrations of TGF-β1 significantly lower than those required by memory T cells primed in the absence of TGF-β1. These results indicate a role of TGF-β1 during T cell priming in modulating expression of CD103/β7 and controlling retention of human memory CD8+ T cells into tumor epithelium.