bluuebunny/crossref_metadata_2025 huggingface dataset:
Processing of a Class I-Restricted Epitope from Tyrosinase Requires Peptide N-Glycanase and the Cooperative Action of Endoplasmic Reticulum Aminopeptidase 1 and Cytosolic Proteases
Authors: Altrich-VanLith Michelle L. , Ostankovitch Marina , Polefrone Joy M. , Mosse Claudio A. , Shabanowitz Jeffrey , Hunt Donald F. , Engelhard Victor H.
Published: April 2014

Although multiple components of the class I MHC processing pathway have been elucidated, the participation of nonproteasomal cytosolic enzymes has been largely unexplored. In this study, we provide evidence for multiple cytosolic mechanisms in the generation of an HLA-A*0201-associated epitope from tyrosinase. This epitope is presented in two isoforms containing either Asn or Asp, depending on the structure of the tyrosinase precursor. We show that deamidation of Asn to Asp is dependent on glycosylation in the endoplasmic reticulum (ER), and subsequent deglycosylation by peptide-N-glycanase in the cytosol. Epitope precursors with N-terminal extensions undergo a similar process. This is linked to an inability of ER aminopeptidase 1 to efficiently remove N-terminal residues, necessitating processing by nonproteasomal peptidases in the cytosol. Our work demonstrates that processing of this tyrosinase epitope involves recycling between the ER and cytosol, and an obligatory interplay between enzymes involved in proteolysis and glycosylation/deglycosylation located in both compartments.
Antibody Repertoire Development in Fetal and Neonatal Piglets. XVII. IgG Subclass Transcription Revisited with Emphasis on New IgG3
Authors: Butler John E. , Wertz Nancy
Published: April 2014

Fetal piglets offer an in vivo model for determining whether Ag-independent IgG subclass transcription proceeds in a manner that differs from subclass transcription in pigs exposed to environmental Ags and TLR ligands. Our data from ≃12,000 Cγ clones from >60 piglets provide the first report on the relative usage of all known porcine Cγ genes in fetal and young pigs. Studies revealed that among the six Cγ genes, allelic variants of IgG1 comprised 50–80% of the repertoire, and IgG2 alleles comprised <10% in nearly all tissues. However, relative transcription of allelic variants of IgG1 randomly deviate from the 1:1 ratio expected in heterozygotes. Most surprising was the finding that IgG3 accounted for half of all Cγ transcripts in the ileal Peyer’s patches (IPPs) and mesenteric lymph nodes but on average only ≃5% of the clones from the thymus, tonsil, spleen, peripheral blood, and bone marrow of newborns. Lymphoid tissues from late term fetuses revealed a similar expression pattern. Except for IgG3 in the IPPs and mesenteric lymph nodes, no stochastic pattern of Cγ expression during development was seen in animals from mid-gestation through 5 mo. The age and tissue dependence of IgG3 transcription paralleled the developmental persistence of the IPP, and its near disappearance corresponds to the diversification of the preimmune VDJ repertoire in young piglets. We hypothesize that long-hinged porcine IgG3 may be important in preadaptive responses to T cell-independent Ags similar to those described for its murine namesake.
Critical Role of Prostaglandin E2 Overproduction in Impaired Pulmonary Host Response following Bone Marrow Transplantation
Authors: Ballinger Megan N. , Aronoff David M. , McMillan Tracy R. , Cooke Kenneth R. , Olkiewicz Krystyna , Toews Galen B. , Peters-Golden Marc , Moore Bethany B.
Published: April 2014

The success of bone marrow transplantation (BMT) as a therapy for malignant and inherited disorders is limited by infectious complications. We previously demonstrated syngeneic BMT mice are more susceptible to Pseudomonas aeruginosa pneumonia due to defects in the ability of donor-derived alveolar macrophages (AMs), but not polymorphonuclear leukocytes (PMNs), to phagocytose bacteria. We now demonstrate that both donor-derived AMs and PMNs display bacterial killing defects post-BMT. PGE2 is a lipid mediator with potent immunosuppressive effects against antimicrobial functions. We hypothesize that enhanced PGE2 production post-BMT impairs host defense. We demonstrate that lung homogenates from BMT mice contain 2.8-fold more PGE2 than control mice, and alveolar epithelial cells (2.7-fold), AMs (125-fold), and PMNs (10-fold) from BMT animals all overproduce PGE2. AMs also produce increased prostacyclin (PGI2) post-BMT. Interestingly, the E prostanoid (EP) receptors EP2 and EP4 are elevated on donor-derived phagocytes post-BMT. Blocking PGE2 synthesis with indomethacin overcame the phagocytic and killing defects of BMT AMs and the killing defects of BMT PMNs in vitro. The effect of indomethacin on AM phagocytosis could be mimicked by an EP2 antagonist, AH-6809, and exogenous addition of PGE2 reversed the beneficial effects of indomethacin in vitro. Importantly, in vivo treatment with indomethacin reduced PGE2 levels in lung homogenates and restored in vivo bacterial clearance from the lung and blood in BMT mice. Genetic reduction of cyclooxygenase-2 in BMT mice also had similar effects. These data clearly demonstrate that overproduction of PGE2 post-BMT is a critical factor determining impaired host defense against pathogens.
Triosephosphate Isomerase- and Glyceraldehyde-3-Phosphate Dehydrogenase-Reactive Autoantibodies in the Cerebrospinal Fluid of Patients with Multiple Sclerosis
Authors: Kolln Johanna , Ren Hui-Min , Da Reng-Rong , Zhang Yiping , Spillner Edzard , Olek Michael , Hermanowicz Neal , Hilgenberg Lutz G. , Smith Martin A. , van den Noort Stanley , Qin Yufen
Published: April 2014

Our previous results revealed that Igs in lesions and single chain variable fragment Abs (scFv-Abs) generated from clonal B cells in the cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) bind to axons in MS brains. To study the axonal Ags involved in MS, we identified the glycolytic enzymes, triosephosphate isomerase (TPI) and GAPDH, using Igs from the CSF and scFv-Abs generated from clonal B cells in the CSF and in lesions from MS patients. Elevated levels of CSF-Abs to TPI were observed in patients with MS (46%), clinically isolated syndrome (CIS) suggestive of MS (40%), other inflammatory neurological diseases (OIND; 29%), and other noninflammatory neurological diseases (ONIND; 31%). Levels of GAPDH-reactive Abs were elevated in MS patients (60%), in patients with CIS (10%), OIND (14%), and ONIND (8%). The coexistence of both autoantibodies was detected in 10 MS patients (29%), and 1 CIS patient (3%), but not in patients with OIND/ONIND. Two scFv-Abs generated from the CSF and from lesions of a MS brain showed immunoreactivity to TPI and GAPDH, respectively. The findings suggest that TPI and GAPDH may be candidate Ags for an autoimmune response to neurons and axons in MS.
Differential Expression of CD11c by Peripheral Blood NK Cells Reflects Temporal Activity of Multiple Sclerosis
Authors: Aranami Toshimasa , Miyake Sachiko , Yamamura Takashi
Published: April 2014

Multiple sclerosis (MS) is an autoimmune disease, showing a great degree of variance in temporal disease activity. We have recently demonstrated that peripheral blood NK cells biased for secreting IL-5 (NK2 bias) are associated with the remission state of MS. In this study, we report that MS patients in remission differentially express CD11c on NK cell surface (operationally defined as CD11chigh or CD11clow). When we compared CD11chigh or CD11clow patients, the expression of IL-5 and GATA-3 in NK cells supposed to endow a disease-protective NK2 phenotype was observed in CD11clow but not in CD11chigh patients. In contrast, the CD11chigh group showed a higher expression of HLA-DR on NK cells. In vitro studies demonstrated that NK cell stimulatory cytokines such as IL-15 would up-regulate CD11c expression on NK cells. Given previous evidence showing an association between an increased level of proinflammatory cytokines and temporal disease activity in MS, we postulate that inflammatory signals may play a role in inducing the CD11chigh NK cell phenotype. Follow-up of a new cohort of patients showed that 6 of 10 CD11chigh MS patients developed a clinical relapse within 120 days after evaluation, whereas only 2 of 13 CD11clow developed exacerbated disease (p = 0.003). As such, a higher expression of CD11c on NK cells may reflect the temporal activity of MS as well as a loss of regulatory NK2 phenotype, which may allow us to use it as a potential biomarker to monitor the immunological status of MS patients.
Cutting Edge: Rapid Recovery of NKT Cells upon Institution of Highly Active Antiretroviral Therapy for HIV-1 Infection
Authors: van der Vliet Hans J. J. , van Vonderen Marit G. A. , Molling Johan W. , Bontkes Hetty J. , Reijm Martine , Reiss Peter , van Agtmael Michiel A. , Danner Sven A. , van den Eertwegh Alfons J. M. , von Blomberg B. Mary E. , Scheper Rik J.
Published: April 2014

CD1d-restricted NKT cells play important regulatory roles in various immune responses and are rapidly and selectively depleted upon infection with HIV-1. The cause of this selective depletion is incompletely understood, although it is in part due to the high susceptibility of CD4+ NKT cells to direct infection and subsequent cell death by HIV-1. Here, we demonstrate that highly active antiretroviral therapy (HAART) results in the rapid recovery of predominantly CD4− NKT cells with kinetics that are strikingly similar to those of mainstream T cells. As it is well known that the early recovery of mainstream T cells in response to HAART is due to their redistribution from tissues to the circulation, our data suggest that the selective depletion of circulating NKT cells is likely due to a combination of cell death and tissue sequestration and indicates that HAART can improve immune functions by reconstituting both conventional T cells and immunoregulatory NKT cells.
Accelerated Antigen Sampling and Transport by Airway Mucosal Dendritic Cells following Inhalation of a Bacterial Stimulus
Authors: Jahnsen Frode L. , Strickland Deborah H. , Thomas Jennifer A. , Tobagus Iriani T. , Napoli Sylvia , Zosky Graeme R. , Turner Debra J. , Sly Peter D. , Stumbles Philip A. , Holt Patrick G.
Published: April 2014

An increase in the tempo of local dendritic cell (DC)-mediated immune surveillance is a recognized feature of the response to acute inflammation at airway mucosal surfaces, and transient up-regulation of the APC functions of these DC preceding their emigration to regional lymph nodes has recently been identified as an important trigger for T cell-mediated airway tissue damage in diseases such as asthma. In this study, using a rat model, we demonstrate that the kinetics of the airway mucosal DC (AMDC) response to challenge with heat-killed bacteria is considerably more rapid and as a consequence more effectively compartmentalized than that in recall responses to soluble Ag. Notably, Ag-bearing AMDC expressing full APC activity reach regional lymph nodes within 30 min of cessation of microbial exposure, and in contrast to recall responses to nonpathogenic Ags, there is no evidence of local expression of APC activity within the airway mucosa preceding DC emigration. We additionally demonstrate that, analogous to that reported in the gut, a subset of airway intraepithelial DC extend their processes into the airway lumen. This function is constitutively expressed within the AMDC population, providing a mechanism for continuous immune surveillance of the airway luminal surface in the absence of “danger” signals.
T-bet Controls Pathogenicity of CTLs in the Heart by Separable Effects on Migration and Effector Activity
Authors: Taqueti Viviany R. , Grabie Nir , Colvin Richard , Pang Hong , Jarolim Petr , Luster Andrew D. , Glimcher Laurie H. , Lichtman Andrew H.
Published: April 2014

CD8+ CTL contribute to the pathogenesis of myocarditis and cardiac allograft rejection. Using a transgenic model of myocarditis, we examined the role of the transcription factor T-bet in the differentiation of pathogenic cardiac Ag-specific CTL. We demonstrate that T-bet-deficient CTL are significantly impaired in their ability to cause disease, despite intact proliferation and activation phenotypes. In the absence of T-bet, there is markedly reduced expression of the chemokine receptor CXCR3, and CXCR3-gene knockout CTL are significantly less pathogenic than control CTL. Retroviral-mediated CXCR3 expression in T-bet-deficient CD8+ T cells reconstitutes their ability to infiltrate but not to damage the heart, establishing that CD8+ T cell pathogenicity is related to T-bet-dependent CXCR3 expression, reduced cytotoxicity, and enhanced regulation. These findings highlight the potential therapeutic benefit of targeting T-bet-regulated gene expression and CXCR3-dependent migration in immune-mediated heart disease.
Impact of Bronchial Epithelium on Dendritic Cell Migration and Function: Modulation by the Bacterial Motif KpOmpA
Authors: Pichavant Muriel , Taront Solenne , Jeannin Pascale , Breuilh Laëtitia , Charbonnier Anne-Sophie , Spriet Corentin , Fourneau Catherine , Corvaia Nathalie , Héliot Laurent , Brichet Anne , Tonnel André-Bernard , Delneste Yves , Gosset Philippe
Published: April 2014

Mucosal immune response depends on the surveillance network established by dendritic cells (DC), APC localized within the epithelium. Bronchial epithelial cells (BEC) play a pivotal role both in the host defense and in the pathogenesis of inflammatory airway disorders. We previously showed that the outer membrane protein A from Klebsiella pneumoniae (KpOmpA), a pathogen-associated molecular pattern (PAMP) derived from Klebsiella pneumoniae, activates BEC. In this study, we evaluated the consequences of this activation on DC traffic and functions. KpOmpA significantly increased the production of CCL2, CCL5, CXCL10, and CCL20 by BEC. Stimulation of BEC increased their chemotactic activity for monocyte-derived DC (MDDC) precursors, through CCL5 and CXCL10 secretion. BEC/MDDC precursor coculture leads to an ICAM-1-dependent accelerated differentiation and enhanced maturation of MDDC. BEC/DC interactions did not affect the capacity of DC to induce T cell proliferation. However, DC preincubated with BEC increased significantly the IL-10 production by autologous T cells. Basolateral and intraepithelial DC differently enhance IL-4 and/or IL-10 synthesis according to the condition of stimulation. In vivo, intranasal injections of KpOmpA into BALB/c mice induced the recruitment of CD11c+ and I-Ad+ myeloid DC associated with bronchial epithelium activation as evidenced by CCL20 expression. These data show that KpOmpA-exposed BEC participate in the homeostasis of myeloid DC network, and regulate the induction of local immune response.
Heme Oxygenase-1-Mediated CD4+CD25high Regulatory T Cells Suppress Allergic Airway Inflammation
Authors: Xia Zhen-Wei , Zhong Wen-Wei , Xu Li-Qing , Sun Jian-Le , Shen Qing-Xiang , Wang Ji-Guang , Shao Jie , Li Yun-Zhu , Yu Shan-Chang
Published: April 2014

Heme oxygenase-1 (HO-1) has anti-inflammatory effects in asthma. CD4+CD25high regulatory T cells (Treg) are a potent immunoregulator that suppresses the immune response. We studied the effects of HO-1-mediated CD4+CD25high Treg on suppression of allergic airway inflammation by comparing mice treated with hemin, OVA, Sn-protoporphyrin (SnPP), and hemin plus SnPP. Airway responsiveness, airway eosinophil infiltration, the level of OVA-specific IgE, and the numbers of cells in general and eosinophils in particular in bronchial alveolar lavage fluid were lower in the hemin group than in the OVA, SnPP, and hemin plus SnPP groups. The expressions of HO-1 mRNA and protein in the lung were increased by repeated administrations of hemin and SnPP. However, the activity of HO-1 was highest in hemin mice. The percentage and suppressive function of CD4+CD25high Treg and the expression of Foxp3 mRNA were obviously enhanced after treatment with hemin. This increase was diminished by the administration of SnPP. The concentration of serum IL-10 was higher in the hemin group than in the other groups, whereas the level of serum TGF-β did not significantly differ across groups. Furthermore, the ratio of IFN-γ/IL-4 mRNA in the lung was higher in hemin-treated mice than in OVA and SnPP mice. The suppressive capacity of CD4+CD25high Treg was not enhanced in the IL-10-deficient mice treated with hemin. In conclusion, our experiments in the animal model demonstrated that HO-1 has anti-inflammatory effects, probably via enhancement of the secretion of IL-10 and promotion of the percentage of CD4+CD25high Treg.
Conditional Deletion of Shp2 Tyrosine Phosphatase in Thymocytes Suppresses Both Pre-TCR and TCR Signals
Authors: Nguyen Thanh V. , Ke Yuehai , Zhang Eric E. , Feng Gen-Sheng
Published: April 2014

It is well known that T cell differentiation and maturation in the thymus is tightly controlled at multiple checkpoints. However, the molecular mechanism for the control of this developmental program is not fully understood. A number of protein tyrosine kinases, such as Zap-70, Lck, and Fyn, have been shown to promote signals required for thymocyte development, whereas a tyrosine phosphatase Src homology domain-containing tyrosine phosphatase (Shp)1 has a negative effect in pre-TCR and TCR signaling. We show in this study that Shp2, a close relative of Shp1, plays a positive role in T cell development and functions. Lck-Cre-mediated deletion of Shp2 in the thymus resulted in a significant block in thymocyte differentiation/proliferation instructed by the pre-TCR at the β selection step, and reduced expansion of CD4+ T cells. Furthermore, mature Shp2−/− T cells showed decreased TCR signaling in vitro. Mechanistically, Shp2 acts to promote TCR signaling through the ERK pathway, with impaired activation of ERK kinase observed in Shp2−/− T cells. Thus, our results provide physiological evidence that Shp2 is a common signal transducer for pre-TCR and TCR in promoting T cell maturation and proliferation.
A CD8/Lck Transgene Is Able to Drive Thymocyte Differentiation
Authors: Fragoso Ruben C. , Pyarajan Saiju , Irie Hanna Yoko , Burakoff Steven J.
Published: April 2014

Efficient development of thymocytes requires participation of a CD8 or CD4 coreceptor in the TCR:MHC interaction. Both CD8 and CD4 coreceptor cytoplasmic domains associate with Lck. In this study, we attempted to delineate the role of CD8α-associated Lck in driving CD8 single positive (SP) thymocyte development. We used a chimeric molecule encoding the extracellular and transmembrane domains of CD8α fused to full-length Lck. In mice deficient for CD8α and transgenic for 2C, a MHC class I-restricted TCR, robust reconstitution of CD8 SP thymocytes occurred both centrally and peripherally. The reconstituted CD8 SP population was phenotypically and functionally comparable to 2C wild-type counterparts expressing endogenous CD8α. A CD8α/Lck kinase-dead chimera also resulted in reconstitution of CD8 SP thymocytes. Our results suggest that CD8α-associated Lck is sufficient to drive CD8 SP thymocyte development. Furthermore, this CD8 SP development may not necessarily depend on Lck kinase activity.
Rapid Selective Priming of FcαR on Eosinophils by Corticosteroids
Authors: ten Hove Willem , Houben Leo A. , Raaijmakers Jan A. M. , Koenderman Leo , Bracke Madelon
Published: April 2014

Preactivation or priming of eosinophils by (proinflammatory) cytokines is important in the pathogenesis of allergic diseases. Several priming-dependent eosinophil responses, such as migration and adhesion, are reduced by treatment with corticosteroids. Many inhibitory effects of corticosteroids are mediated by the glucocorticoid receptor via genomic mechanisms, which are evident only after prolonged interaction (>30 min). However, also faster actions of corticosteroids have been identified, which occur in a rapid, nongenomic manner. In this study, fast effects of corticosteroids were investigated on the function of eosinophil opsonin receptors. Short term corticosteroid treatment of eosinophils for maximal 30 min with dexamethasone (Dex) did not influence eosinophil cell surface CD11b/CD18 expression, adhesion, and/or chemokinesis. In marked contrast, incubation with Dex resulted in a rapid increase in binding of IgA-coated beads to human eosinophils, showing that Dex can up-regulate the activation of FcαR (CD89). This priming response by Dex was dose dependent and optimal between 10−8 and 10−6 M and was mediated via the glucocorticoid receptor as its selective antagonist RU38486 (10−6 M) blocked the priming effect. In contrast to FcαR, eosinophil FcγRII (CD32) was not affected by Dex. Further characterization of the Dex-induced inside-out regulation of FcαR revealed p38 MAPK as the central mediator. Dex dose dependently enhanced p38 MAPK phosphorylation and activation in situ as measured by phosphorylation of its downstream target mitogen-activated protein kinase-activated protein kinase 2. The dose responses of the Dex-induced activation of these kinases were similar as seen for the priming of FcαR. This work demonstrates that corticosteroids selectively activate the FcαR on eosinophils by activation of p38 MAPK.
Prion Protein Expression by Mouse Dendritic Cells Is Restricted to the Nonplasmacytoid Subsets and Correlates with the Maturation State
Authors: Martínez del Hoyo Gloria , López-Bravo María , Metharom Patraporn , Ardavín Carlos , Aucouturier Pierre
Published: April 2014

Expression of the physiological cellular prion protein (PrPC) is remarkably regulated during differentiation and activation of cells of the immune system. Among these, dendritic cells (DCs) display particularly high levels of membrane PrPC, which increase upon maturation, in parallel with that of molecules involved in Ag presentation to T cells. Freshly isolated mouse Langerhans cells, dermal DCs, and DCs from thymus, spleen, and mesenteric lymph nodes expressed low to intermediate levels of PrPC. Highest levels of both PrPC and MHC class II molecules were displayed by lymph node CD8αint DCs, which represent fully mature cells having migrated from peripheral tissues. Maturation induced by overnight culture resulted in increased levels of surface PrPC, as did in vivo DC activation by bacterial LPS. Studies on Fms-like tyrosine kinase 3 ligand bone marrow-differentiated B220− DCs confirmed that PrPC expression followed that of MHC class II and costimulatory molecules, and correlated with IL-12 production in response to TLR-9 engagement by CpG. However, at variance with conventional DCs, B220+ plasmacytoid DCs isolated from the spleen, or in vitro differentiated, did not significantly express PrPC, both before and after activation by TLR-9 engagement. PrP knockout mice displayed higher numbers of spleen CD8α+ DCs, but no significant differences in their maturation response to stimulation through TLR-4 and TLR-9 were noticed. Results are discussed in relation to the functional relevance of PrPC expression by DCs in the induction of T cell responses, and to the pathophysiology of prion diseases.
The Transcriptional Repressor cAMP Response Element Modulator α Interacts with Histone Deacetylase 1 to Repress Promoter Activity
Authors: Tenbrock Klaus , Juang Yuang-Taung , Leukert Nadja , Roth Johannes , Tsokos George C.
Published: April 2014

Transcriptional repression is a fundamental mechanism of gene regulation. cAMP response element (CRE) modulator (CREM)α is an ubiquitously expressed transcription factor and a counterpart of the activator CREB. In T cells, CREM is responsible for the termination of the IL-2 expression by a chromatin-dependent mechanism. We demonstrate in this study that CREMα associates with histone deacetylase (HDAC)1 through its H domain, which is located between the kinase inducible and DNA binding domains. The CREMα-mediated recruitment of HDAC1 to the CRE sites of the IL-2 and c-Fos promoter causes histone deacetylation and inaccessibility to restriction enzymes and limited transcriptional activity. Importantly, the CRE sites of these promoters are crucial for the activity and binding of HDAC1. Therefore, CREMα exerts its repressor activity by a mechanism that involves recruitment of HDAC1, increased deacetylation of histones, and repression of promoter activity.
Critical Role of the C-Terminal Domains of Factor H in Regulating Complement Activation at Cell Surfaces
Authors: Ferreira Viviana P. , Herbert Andrew P. , Hocking Henry G. , Barlow Paul N. , Pangburn Michael K.
Published: April 2014

The plasma protein factor H primarily controls the activation of the alternative pathway of complement. The C-terminal of factor H is known to be involved in protection of host cells from complement attack. In the present study, we show that domains 19–20 alone are capable of discriminating between host-like and complement-activating cells. Furthermore, although factor H possesses three binding sites for C3b, binding to cell-bound C3b can be almost completely inhibited by the single site located in domains 19–20. All of the regulatory activities of factor H are expressed by the N-terminal four domains, but these activities toward cell-bound C3b are inhibited by isolated recombinant domains 19–20 (rH 19–20). Direct competition with the N-terminal site is unlikely to explain this because regulation of fluid phase C3b is unaffected by domains 19–20. Finally, we show that addition of isolated rH 19–20 to normal human serum leads to aggressive complement-mediated lysis of normally nonactivating sheep erythrocytes and moderate lysis of human erythrocytes, which possess membrane-bound regulators of complement. Taken together, the results highlight the importance of the cell surface protective functions exhibited by factor H compared with other complement regulatory proteins. The results may also explain why atypical hemolytic uremic syndrome patients with mutations affecting domains 19–20 can maintain complement homeostasis in plasma while their complement system attacks erythrocytes, platelets, endothelial cells, and kidney tissue.
Endothelial Cell Cortactin Coordinates Intercellular Adhesion Molecule-1 Clustering and Actin Cytoskeleton Remodeling during Polymorphonuclear Leukocyte Adhesion and Transmigration
Authors: Yang Lin , Kowalski Jennifer R. , Yacono Patrick , Bajmoczi Milan , Shaw Sunil K. , Froio Richard M. , Golan David E. , Thomas Sheila M. , Luscinskas Francis W.
Published: April 2014

Endothelial cell ICAM-1 interacts with leukocyte β2 integrins to mediate adhesion and transmit outside-in signals that facilitate leukocyte transmigration. ICAM-1 redistribution and clustering appear necessary for leukocyte transmigration, but the mechanisms controlling ICAM-1 redistribution and clustering have not been identified. We recently reported that Src kinase phosphorylation of endothelial cortactin regulates polymorphonuclear cell (PMN) transmigration. In this study, we tested the hypotheses that the Src family kinase-cortactin pathway mediates association of ICAM-1 with the actin cytoskeleton and that this association is required for ICAM-1 clustering and leukocyte transmigration. Cross-linking ICAM-1 induced cytoskeletal remodeling and a decrease in ICAM-1 lateral mobility, as assessed by fluorescence recovery after photobleaching. Cytoskeletal remodeling after ICAM-1 cross-linking was reduced by knockdown of cortactin by small interfering RNA, by expression of a cortactin mutant deficient in Src phosphorylation sites (cortactin3F), and by the Src kinase inhibitor PP2. Pretreatment of cytokine-activated human endothelial monolayers with cortactin small interfering RNA significantly decreased both actin and ICAM-1 clustering around adherent PMN and the formation of actin-ICAM-1 clusters required for PMN transmigration. Our data suggest a model in which tyrosine phosphorylation of cortactin dynamically links ICAM-1 to the actin cytoskeleton, enabling ICAM-1 to form clusters and facilitate leukocyte transmigration.
Immunization with an Apoptotic Cell-Binding Protein Recapitulates the Nephritis and Sequential Autoantibody Emergence of Systemic Lupus Erythematosus
Authors: Levine Jerrold S. , Subang Rebecca , Nasr Samih H. , Fournier Sylvie , Lajoie Ginette , Wither Joan , Rauch Joyce
Published: April 2014

The initial events predisposing to loss of tolerance in patients with systemic lupus erythematosus (SLE) are largely unknown, as are the events that precipitate the transition from preclinical to overt disease. We hypothesized that induction of murine SLE would require tipping the balance between tolerance and immunity in two ways: 1) an immunogen that could take advantage of apoptotic cells as a scaffold for epitope spread, and 2) an immune activator that would generate a strong and persistent T cell response to the inciting immunogen. We show that immunization of C57BL/6 and BALB/c mice with human β2-glycoprotein I, an apoptotic cell-binding protein, in the presence of LPS induces a long-lived, potent response to β2-glycoprotein I that results in epitope spread to multiple SLE autoantigens. SLE-specific autoantibodies emerged in a sequential manner that recapitulated the order seen in human SLE. Moreover, immunized mice developed overt glomerulonephritis closely resembling human lupus nephritis.
Genetic Manipulation of CD74 in Mouse Strains of Different Backgrounds Can Result in Opposite Responses to Central Nervous System Injury
Authors: Schori Hadas , Shechter Ravid , Shachar Idit , Schwartz Michal
Published: April 2014

The ability to recover from CNS injuries is strain dependent. Transgenic mice that weakly express the p41 CD74 isoform (an integral membrane protein functioning as a MHC class II chaperone) on an I-Ab genetic background have normal CD4+ T cell populations and normal surface expression of MHC class II, but their B cell development is arrested while the cells are still immature. After a CNS injury, these mice recover better than their matched wild-type controls. We generated p41-transgenic mice on an I-Ad background (p41-I-Ad mice), and found that their recovery from CNS injuries was worse than that of controls. A correlative inverse effect was seen with respect to the kinetics of T cell and B cell recruitment to the injured CNS and the expression of insulin-like growth factor at the lesion site. These results, besides verifying previous findings that B cells function in the damaged CNS, demonstrate that the outcome of a particular genetic manipulation may be strain dependent.
α1β1 Integrin+ and Regulatory Foxp3+ T Cells Constitute Two Functionally Distinct Human CD4+ T Cell Subsets Oppositely Modulated by TNFα Blockade
Authors: Goldstein Itamar , Ben-Horin Shomron , Koltakov Alexander , Chermoshnuk Helly , Polevoy Victor , Berkun Yakov , Amariglio Ninette , Bank Ilan
Published: April 2014

The expression of the collagen receptor α1β1 integrin (VLA-1) on CD4+ T cells is largely restricted to CCR7−CD45RO+ cells that localize to inflamed tissues. Moreover, neutralizing α1 integrin, in vivo, has been shown to compromise cell-mediated immunity. Our current study shows that the expression of VLA-1 on human CD4+ T cells is restricted to conventional effectors. In contrast, Foxp3+ T regulatory cells (Tregs) do not express this receptor. Moreover, Foxp3 or VLA-1 expression remained a mutually exclusive event in CD4+ T cells even upon polyclonal anti-CD3-induced activation. Because TNFα blockade ameliorates certain T cell-dependent autoimmune disorders in humans, we investigated, in vitro, whether neutralizing TNFα affected the balance between the proinflammatory VLA-1+ effectors and the counteracting Tregs. We found that anti-CD3 stimulation of freshly isolated PBL from healthy individuals, coupled with continuous TNFα blockade, inhibited the typical activation-dependent generation of CD4+VLA-1+ Th1 cells. In contrast, it augmented the outgrowth of VLA-1neg/dimCD25high and Foxp3+CD4+ T cells. Indeed, repeated anti-CD3 stimulation coupled with TNFα blockade generated CD4+ T cell lines enriched for VLA-1−Foxp3+ Tregs. Importantly, these CD4+ T cells displayed potent suppressive functions toward autologous CD4+ PBL, including the suppression of the activation-dependent induction of VLA-1+ effectors. Thus, we propose a novel mechanism by which anti-TNFα therapy may restore self-tolerance, by shifting the balance between VLA-1+ effectors and Foxp3+ Tregs, during immune activation, in favor of the latter suppressor cell population.
Multiple NF-κB and IFN Regulatory Factor Family Transcription Factors Regulate CCL19 Gene Expression in Human Monocyte-Derived Dendritic Cells
Authors: Pietilä Taija E. , Veckman Ville , Lehtonen Anne , Lin Rongtuan , Hiscott John , Julkunen Ilkka
Published: April 2014

CCL19 chemokine has a central role in dendritic cell (DC) biology regulating DC traffic and recruitment of naive T cells to the vicinity of activated DCs. In this study, we have analyzed the regulation of CCL19 gene expression in human monocyte-derived DCs. DCs infected with Salmonella enterica or Sendai virus produced CCL19 at late times of infection. The CCL19 promoter was identified as having two putative NF-κB binding sites and one IFN-stimulated response element (ISRE). Transcription factor binding experiments demonstrated that Salmonella or Sendai virus infection increased the binding of classical p50+p65 and alternative p52+RelB NF-κB proteins to both of the CCL19 promoter NF-κB elements. Interestingly, Salmonella or Sendai virus infection also increased the binding of multiple IFN regulatory factors (IRFs), STAT1, and STAT2, to the ISRE element. Enhanced binding of IRF1, IRF3, IRF7, and IRF9 to the CCL19 promoter ISRE site was detected in Salmonella or Sendai virus-infected cell extracts. The CCL19 promoter in a luciferase reporter construct was activated by the expression of NF-κB p50+p65 or p52+RelB dimers. IRF1, IRF3, and IRF7 proteins also activated CCL19 promoter in the presence of Sendai virus infection. CCL19 promoter constructs mutated at NF-κB and/or ISRE sites were only weakly activated. Ectopic expression of RIG-I (ΔRIG-I, CARDIF) or TLR3/4 (TRIF, MyD88, IKKε, or TBK1) signaling pathway components induced CCL19 promoter activity, suggesting that these pathways are important in CCL19 gene expression. Our experiments reveal that expression of the CCL19 gene is regulated by a combined action of several members of the NF-κB, IRF, and STAT family transcription factors.
IL-7/STAT5 Cytokine Signaling Pathway Is Essential but Insufficient for Maintenance of Naive CD4 T Cell Survival in Peripheral Lymphoid Organs
Authors: Seki Yoh-ichi , Yang Jianying , Okamoto Mariko , Tanaka Shinya , Goitsuka Ryo , Farrar Michael A. , Kubo Masato
Published: April 2014

Constitutive expression of suppressors of cytokine signaling (SOCS)1 in T lineage in vivo attenuated cytokine signaling and resulted in a dramatic reduction in the number of naive CD44lowCD62Lhigh CD4 T cells in the spleen. After adoptive transfer of thymocytes from SOCS1 transgenic mice into normal recipients, naive CD4 T cells rapidly disappeared from the spleen within 1 wk. Likewise, T cell-specific deletion of STAT5a/b in vivo resulted in a similar phenotype characterized by loss of naive CD4 T cells. Thus, STAT5-mediated signaling is crucial for promoting naive T cell survival. However, forced expression of constitutively active STAT5 failed to rescue CD4 T cells in SOCS1 transgenic mice, implying that STAT5 activation is necessary but not sufficient for naive CD4 T cell survival. Although blockade of the IL-7R, a SOCS1 target, resulted in clear inhibition of naive T cell survival, the effect occurred 3 wk after anti-IL-7R Ab treatment, but not at earlier time points. These results suggest that IL-7-mediated STAT5 activation is essential for long-term survival of naive CD4 cells after export from thymus, and that another SOCS1-sensitive cytokine is critical for short-term naive T cell survival.
The Net Effect of Costimulatory Blockers Is Dependent on the Subset and Activation Status of the Autoreactive T Cells
Authors: Zhang Ping , Sun Deming , Ke Yan , Kaplan Henry J. , Shao Hui
Published: April 2014

In this study, we investigated whether CD4 and CD8 autoreactive T cells have different costimulatory requirements for their activation in vitro by testing the effect of a panel of Abs specific for various costimulatory molecules. Our results showed that CD8 interphotoreceptor retinoid-binding protein-specific T cells are more dependent on costimulatory molecules for activation than their CD4 counterparts. Interphotoreceptor retinoid-binding protein-specific T cells are less dependent on costimulatory molecules in the secondary response than the primary response. We also showed that blockade of costimulatory molecules can either promote or inhibit the proliferation of autoreactive T cells, depending on the degree of activation of the cells. Our results show that anti-costimulatory molecule treatment can have diverse actions on autoreactive T cell subsets, the net effect being determined by the subset of immune cells affected and the type and dose of treatment used.
Hepatitis B Virus Sensitizes Hepatocytes to TRAIL-Induced Apoptosis through Bax
Authors: Liang Xiaohong , Liu Yugang , Zhang Qiu , Gao Lifen , Han Lihui , Ma Chunhong , Zhang Lining , Chen Youhai H. , Sun Wensheng
Published: April 2014

Hepatitis B virus (HBV) infection afflicts >300 million people worldwide and is a leading cause of hepatocyte death, cirrhosis, and hepatocellular carcinoma. While the morphological characteristics of dying hepatocytes are well documented, the molecular mechanisms leading to the death of hepatocytes during HBV infection are not well understood. TRAIL, the TNF-related apoptosis-inducing ligand, has recently been implicated in the death of hepatocytes under certain inflammatory but not normal conditions. To determine the potential roles of TRAIL in HBV-induced hepatitis, we examined the effects of HBV and its X protein (HBx) on TRAIL-induced hepatocyte apoptosis both in vivo and in vitro. We found that hepatitis and hepatic cell death in HBV transgenic mice were significantly inhibited by a soluble TRAIL receptor that blocks TRAIL function. We also found that HBV or HBx transfection of a hepatoma cell line significantly increased its sensitivity to TRAIL-induced apoptosis. The increase in TRAIL sensitivity were associated with a dramatic up-regulation of Bax protein expression. Knocking down Bax expression using Bax-specific small interference RNA blocked HBV-induced hepatitis and hepatocyte apoptosis. The degradation of caspases 3 and 9, but not that of Bid or caspase-8, was preferentially affected by Bax knockdown. These results establish that HBV sensitizes hepatocytes to TRAIL-induced apoptosis through Bax and that Bax-specific small interference RNA can be used to inhibit HBV-induced hepatic cell death.
Th1 and Type 1 Cytotoxic T Cells Dominate Responses in T-bet Overexpression Transgenic Mice That Develop Contact Dermatitis
Authors: Ishizaki Kazusa , Yamada Akiko , Yoh Keigyou , Nakano Takako , Shimohata Homare , Maeda Atsuko , Fujioka Yuki , Morito Naoki , Kawachi Yasuhiro , Shibuya Kazuko , Otsuka Fujio , Shibuya Akira , Takahashi Satoru
Published: April 2014

Contact dermatitis in humans and contact hypersensitivity (CHS) in animal models are delayed-type hypersensitivity reactions mediated by hapten-specific T cells. Recently, it has become clear that both CD4+ Th1 and CD8+ type 1 cytotoxic T (Tc1) cells can act as effectors in CHS reactions. T-bet has been demonstrated to play an important role in Th1 and Tc1 cell differentiation, but little is known about its contribution to CHS. In the present study, we used C57BL/6 mice transgenic (Tg) for T-bet to address this issue. These Tg mice, which overexpressed T-bet in their T lymphocytes, developed dermatitis characterized by swollen, flaky, and scaly skin in regions without body hair. Skin histology showed epidermal hyperkeratosis, neutrophil, and lymphocyte infiltration similar to that seen in contact dermatitis. T-bet overexpression in Tg mice led to elevated Th1 Ig (IgG2a) and decreased Th2 Ig (IgG1) production. Intracellular cytokine analyses demonstrated that IFN-γ was increased in both Th1 and Tc1 cells. Furthermore, Tg mice had hypersensitive responses to 2,4-dinitrofluorobenzene, which is used for CHS induction. These results suggest that the level of expression of T-bet might play an important role in the development of contact dermatitis and that these Tg mice should be a useful model for contact dermatitis.
Streptococcal Preparation OK-432 Promotes Fusion Efficiency and Enhances Induction of Antigen-Specific CTL by Fusions of Dendritic Cells and Colorectal Cancer Cells
Authors: Koido Shigeo , Hara Eiichi , Homma Sadamu , Torii Akira , Mitsunaga Makoto , Yanagisawa Satoru , Toyama Yoichi , Kawahara Hidejiro , Watanabe Michiaki , Yoshida Seiya , Kobayashi Susumu , Yanaga Katsuhiko , Fujise Kiyotaka , Tajiri Hisao
Published: April 2014

Dendritic/tumor fusion cell (FC) vaccine is an effective approach for various types of cancer but has not yet been standardized. Antitumor activity can be modulated by different mechanisms such as dendritic cell (DC) maturation state. This study addressed optimal strategies for FC preparations to enhance Ag-specific CTL activity. We have created three types of FC preparations by alternating fusion cell partners: 1) immature DCs fused with autologous colorectal carcinoma cells (Imm-FCs); 2) Imm-FCs followed by stimulation with penicillin-inactivated Streptococcus pyogenes (OK-432) (Imm-FCs/OK); and 3) OK-432-stimulated DCs directly fused to autologous colorectal carcinoma cells (OK-FCs). Both OK-FCs and Imm-FCs/OK coexpressed the CEA, MUC1, and significantly higher levels of CD86, CD83, and IL-12 than those obtained with Imm-FCs. Short-term culture of fusion cell preparations promoted the fusion efficiency. Interestingly, OK-FCs were more efficient in stimulating CD4+ and CD8+ T cells capable of high levels of IFN-γ production and cytolysis of autologous tumor or semiallogeneic targets. Moreover, OK-FCs are more effective inducer of CTL activation compared with Imm-FCs/OK on a per fusion cell basis. The pentameric assay confirmed that CEA- and MUC1-specific CTL was induced simultaneously by OK-FCs at high frequency. Furthermore, the cryopreserved OK-FCs retained stimulatory capacity for inducing antitumor immunity. These results suggest that OK-432 promotes fusion efficiency and induction of Ag-specific CTL by fusion cells. We conclude that DCs fused after stimulation by OK-432 may have the potential applicability to the field of antitumor immunotherapy and may provide a platform for adoptive immunotherapy in the clinical setting.
Anti-TCR Antibody Treatment Activates a Novel Population of Nonintestinal CD8αα+TCRαβ+ Regulatory T Cells and Prevents Experimental Autoimmune Encephalomyelitis
Authors: Tang Xiaolei , Maricic Igor , Kumar Vipin
Published: April 2014

CD8αα+CD4−TCRαβ+ T cells are a special lineage of T cells found predominantly within the intestine as intraepithelial lymphocytes and have been shown to be involved in the maintenance of immune homeostasis. Although these cells are independent of classical MHC class I (class Ia) molecules, their origin and function in peripheral lymphoid tissues are unknown. We have recently identified a novel subset of nonintestinal CD8αα+CD4−TCRαβ+ regulatory T cells (CD8αα Tregs) that recognize a TCR peptide from the conserved CDR2 region of the TCR Vβ8.2-chain in the context of a class Ib molecule, Qa-1a, and control- activated Vβ8.2+ T cells mediating experimental autoimmune encephalomyelitis. Using flow cytometry, spectratyping, and real-time PCR analysis of T cell clones and short-term lines, we have determined the TCR repertoire of the CD8αα regulatory T cells (Tregs) and found that they predominantly use the TCR Vβ6 gene segment. In vivo injection of anti-TCR Vβ6 mAb results in activation of the CD8αα Tregs, inhibition of the Th1-like pathogenic response to the immunizing Ag, and protection from experimental autoimmune encephalomyelitis. These data suggest that activation of the CD8αα Tregs present in peripheral lymphoid organs other than the gut can be exploited for the control of T cell-mediated autoimmune diseases.
The Insulin-Specific T Cells of Nonobese Diabetic Mice Recognize a Weak MHC-Binding Segment in More Than One Form
Authors: Levisetti Matteo G. , Suri Anish , Petzold Shirley J. , Unanue Emil R.
Published: April 2014

Several naturally occurring anti-insulin CD4 T cells were isolated from islet infiltrates of NOD mice. In accordance with the results of others, these T cells recognized the segment of the β-chain from residues 9–23. Peptides encompassing the B:(9–23) sequence bound weakly to I-Ag7 in two main contiguous registers in which two residues at the carboxyl end, P20Gly and P21Glu, influenced binding and T cell reactivity. Naturally occurring insulin-reactive T cells exhibited differing reactivities with the carboxyl-terminal amino acids, although various single residue changes in either the flanks or the core segments affected T cell responses. The insulin peptides represent another example of a weak MHC-binding ligand that is highly immunogenic, giving rise to distinct populations of autoimmune T cells.
Unexpected Role of B and T Lymphocyte Attenuator in Sustaining Cell Survival during Chronic Allostimulation
Authors: Hurchla Michelle A. , Sedy John R. , Murphy Kenneth M.
Published: April 2014

B and T lymphocyte attenuator (BTLA; CD272) can deliver inhibitory signals to B and T cells upon binding its ligand herpesvirus entry mediator. Because CD28, CTLA-4, programmed death-1, and ICOS regulate the development of acute graft-vs-host disease (GVHD), we wished to assess if BTLA also played a role in this T cell-mediated response. In the nonirradiated parental-into-F1 model of acute GVHD, BTLA+/+ and BTLA−/− donor lymphocytes showed equivalent engraftment and expansion during the first week of the alloresponse. Unexpectedly, BTLA−/− donor T cells failed to sustain GVHD, showing a decline in surviving donor cell numbers beginning at day 9 and greatly reduced by day 11. Similarly, inhibition of BTLA-herpesvirus entry mediator engagement by in vivo administration of a blocking anti-BTLA Ab also caused reduced survival of donor cells. Microarray analysis revealed several genes that were differentially expressed by BTLA−/− and BTLA+/+ donor CD4+ T cells preceding the decline in BTLA−/− donor T cells. Several genes influencing Th cell polarization were differentially expressed by BTLA+/+ and BTLA−/− donor cells. Additionally, the re-expression of the IL-7Rα subunit that occurs in BTLA+/+ donor cells after 1 wk of in vivo allostimulation was not observed in BTLA−/− donor CD4+ cells. The striking loss of BTLA−/− T cells in this model indicates a role for BTLA activity in sustaining CD4+ T cell survival under the conditions of chronic stimulation in the nonirradiated parental-into-F1 GVHD.