bluuebunny/crossref_metadata_2025 huggingface dataset:
Outer Membrane Protein A from Klebsiella pneumoniae Activates Bronchial Epithelial Cells: Implication in Neutrophil Recruitment
Authors: Pichavant Muriel , Delneste Yves , Jeannin Pascale , Fourneau Catherine , Brichet Anne , Tonnel André-Bernard , Gosset Philippe
Published: April 2014

Aside from its mechanical barrier function, bronchial epithelium plays an important role both in the host defense and in the pathogenesis of inflammatory airway disorders. To investigate its role in lung defense, the effect of a bacterial cell wall protein, the outer membrane protein A from Klebsiella pneumoniae (kpOmpA) on bronchial epithelial cells (BEC) was evaluated on adhesion molecule expression and cytokine production. Moreover, the potential implication of this mechanism in kpOmpA-induced lung inflammation was also determined. Our in vitro studies demonstrated that kpOmpA strongly bound to BEAS-2B cells, a human BEC line, and to BEC primary cultures, resulting in NF-κB signaling pathway activation. Exposure to kpOmpA increased ICAM-1 mRNA and cell surface expression, as well as the secretion of IL-6, CXC chemokine ligand (CXCL)1, CXCL8, C-C chemokine ligand 2, CXCL10 by BEAS-2B cells, and BEC primary cultures (p < 0.005). We analyzed in vivo the consequences of intratracheal injection of kpOmpA to BALB/c mice. In kpOmpA-treated mice, a transient neutrophilia (with a maximum at 24 h) was observed in bronchoalveolar lavage and lung sections. In vivo kpOmpA priming induced bronchial epithelium activation as evaluated by ICAM-1 and CXCL1 expression, associated with the secretion of CXCL1 and CXCL5 in bronchoalveolar lavage fluids. In the lung, an increased level of the IL-6, CXCL1, CXCL5, CXCL10 mRNA was observed with a maximum at 6 h. These data showed that kpOmpA is involved in host defense mechanism by its ability to activate not only APC but also BEC, resulting in a lung neutrophilia.
Leishmania-Induced Inhibition of Macrophage Antigen Presentation Analyzed at the Single-Cell Level
Authors: Meier Courtney L. , Svensson Mattias , Kaye Paul M.
Published: April 2014

A number of studies have previously examined the capacity of intracellular Leishmania parasites to modulate the capacity of macrophages to process and present Ags to MHC class II-restricted CD4+ T cells. However, the bulk culture approaches used for assessing T cell activation make interpretation of some of these studies difficult. To gain a more precise understanding of the interaction between Leishmania-infected macrophages and effector T cells, we have analyzed various parameters of T cell activation in individual macrophage-T cell conjugates. Leishmania-infected macrophages efficiently stimulate Ag-independent as well as Ag-dependent, TCR-mediated capping of cortical F-actin in DO.11 T cells. However, infected macrophages are less efficient at promoting the sustained TCR signaling necessary for reorientation of the T cell microtubule organizing center and for IFN-γ production. A reduced ability to activate these T cell responses was not due to altered levels of surface-expressed MHC class II-peptide complexes. This study represents the first direct single-cell analysis of the impact of intracellular infection on the interaction of macrophages with T cells and serves to emphasize the subtle influence Leishmania has on APC function.
Salmonella Rapidly Kill Dendritic Cells via a Caspase-1- Dependent Mechanism
Authors: van der Velden Adrianus W. M. , Velasquez Marisela , Starnbach Michael N.
Published: April 2014

Dendritic cells provide a critical link between innate and acquired immunity. In this study, we demonstrate that the bacterial pathogen Salmonella enterica serovar Typhimurium can efficiently kill these professional phagocytes via a mechanism that is dependent on sipB and the Salmonella pathogenicity island 1-encoded type III protein secretion system. Rapid phosphatidylserine redistribution, caspase activation, and loss of plasma membrane integrity were characteristic of dendritic cells infected with wild-type Salmonella, but not sipB mutant bacteria. Caspase-1 was particularly important in this process because Salmonella-induced dendritic cell death was dramatically reduced in the presence of a caspase-1-specific inhibitor. Furthermore, dendritic cells obtained from caspase-1-deficient mice, but not heterozygous littermate control mice, were resistant to Salmonella-induced cytotoxicity. We hypothesize that Salmonella have evolved the ability to selectively kill professional APCs to combat, exploit, or evade immune defense mechanisms.
Role of Monocyte Chemotactic Protein-1/CC Chemokine Ligand 2 on γδ T Lymphocyte Trafficking during Inflammation Induced by Lipopolysaccharide orMycobacterium bovisBacille Calmette-Guérin
Authors: Penido Carmen , Vieira-de-Abreu Adriana , Bozza Marcelo T. , Castro-Faria-Neto Hugo C. , Bozza Patrícia T.
Published: April 2014

γδ T lymphocytes are involved in a great variety of inflammatory and infectious responses. However, the mechanisms by which γδ T lymphocytes migrate to inflamed sites are poorly understood. In this study we investigate the role of monocyte chemotactic protein (MCP)-1 in regulating γδ T cell migration after LPS or Mycobacterium bovis bacille Calmette-Guérin (BCG) challenge. LPS-induced γδ T cell influx was significantly inhibited by either pretreatment with dexamethasone or vaccinia virus Lister 35-kDa chemokine binding protein, vCKBP, a CC chemokine neutralizing protein, suggesting a role for CC chemokines in this phenomenon. LPS stimulation increased the expression of MCP-1 mRNA and protein at the inflammation site within 6 h. It is noteworthy that LPS was unable to increase MCP-1 production or γδ T cell recruitment in C3H/HeJ, indicative of the involvement of Toll-like receptor 4. γδ T cells express MCP-1 receptor CCR2. Pretreatment with anti-MCP-1 mAb drastically inhibited LPS-induced in vivo γδ T cell mobilization. Indeed, MCP-1 knockout mice were unable to recruit γδ T cells to the pleural cavity after LPS stimulation, effect that could be restored by coadministration of MCP-1. In addition, BCG-induced γδ lymphocyte accumulation was significantly reduced in MCP-1 knockout mice when compared with wild-type mice. In conclusion, our results indicate that LPS-induced γδ T lymphocyte migration is dependent on Toll-like receptor 4 and sensitive to both dexamethasone and CC chemokine-binding protein inhibition. Moreover, by using MCP-1 neutralizing Abs and genetically deficient mice we show that LPS- and BCG-induced γδ T lymphocyte influx to the pleural cavity of mice is mainly orchestrated by the CC chemokine MCP-1.
Shedding of the Type II IL-1 Decoy Receptor Requires a Multifunctional Aminopeptidase, Aminopeptidase Regulator of TNF Receptor Type 1 Shedding
Authors: Cui Xinle , Rouhani Farshid N. , Hawari Feras , Levine Stewart J.
Published: April 2014

Proteolytic cleavage of the extracellular domain of the type II IL-1 decoy receptor (IL-1RII) generates soluble IL-1-binding proteins that prevent excessive bioactivity by binding free IL-1. In this study we report that an aminopeptidase, aminopeptidase regulator of TNFR1 shedding (ARTS-1), is required for IL-1RII shedding. Coimmunoprecipitation experiments demonstrate an association between endogenous membrane-associated ARTS-1 and a 47-kDa IL-1RII, consistent with ectodomain cleavage of the membrane-bound receptor. A direct correlation exists between ARTS-1 protein expression and IL-1RII shedding, as cell lines overexpressing ARTS-1 have increased IL-1RII shedding and decreased membrane-associated IL-1RII. Basal IL-1RII shedding is absent from ARTS-1 knockout cell lines, demonstrating that ARTS-1 is required for constitutive IL-1RII shedding. Similarly, PMA-mediated IL-1RII shedding is almost entirely ARTS-1-dependent. ARTS-1 expression also enhances ionomycin-induced IL-1RII shedding. ARTS-1 did not alter levels of membrane-associated IL-1RI or IL-1R antagonist release from ARTS-1 cell lines, which suggests that the ability of ARTS-1 to promote shedding of IL-1R family members may be specific for IL-1RII. Further, increased IL-1RII shedding by ARTS-1-overexpressing cells attenuates the biological activity of IL-1β. We conclude that the ability of ARTS-1 to enhance IL-1RII shedding represents a new mechanism by which IL-1-induced cellular events can be modulated. As ARTS-1 also promotes the shedding of the structurally unrelated 55-kDa, type I TNF receptor and the IL-6R, we propose that ARTS-1 may play an important role in regulating innate immune and inflammatory responses by increasing cytokine receptor shedding.
Among CXCR3 Chemokines, IFN-γ-Inducible Protein of 10 kDa (CXC Chemokine Ligand (CXCL) 10) but Not Monokine Induced by IFN-γ (CXCL9) Imprints a Pattern for the Subsequent Development of Autoimmune Disease
Authors: Christen Urs , McGavern Dorian B. , Luster Andrew D. , von Herrath Matthias G. , Oldstone Michael B. A.
Published: April 2014

Infection of the pancreas with lymphocytic choriomeningitis virus results in rapid and differential expression among CXCR3 chemokines. IFN-γ-inducible protein of 10 kDa (IP-10), in contrast with monokine induced by IFN-γ and IFN-inducible T cell-α chemoattractant, is strongly expressed within 24 h postinfection. Blocking of IP-10, but not monokine induced by IFN-γ, aborts severity of Ag-specific injury of pancreatic β cells and abrogates type 1 diabetes. Mechanistically, IP-10 blockade impedes the expansion of peripheral Ag-specific T cells and hinders their migration into the pancreas. IP-10 expression was restricted to viruses infecting the pancreas and that are capable of causing diabetes. Hence, virus-induced organ-specific autoimmune diseases may be dependent on virus tropism and its ability to alter the local milieu by selectively inducing chemokines that prepare the infected tissue for the subsequent destruction by the adaptive immune response.
A Novel Protein Highly Expressed in Testis Is Overexpressed in Systemic Sclerosis Fibroblasts and Targeted by Autoantibodies
Authors: Yasuoka Hidekata , Ihn Hironobu , Medsger Thomas A. , Hirakata Michito , Kawakami Yutaka , Ikeda Yasuo , Kuwana Masataka
Published: April 2014

Nearly all autoantibody specificities in sera from patients with systemic sclerosis (SSc) target proteins distributed ubiquitously, and Abs against proteins whose expression is restricted to the affected sites have not been identified. In this study we describe SSc-specific autoantibody to a novel testicular Ag, termed protein highly expressed in testis (PHET), which is ectopically overexpressed in SSc dermal fibroblasts. A partial cDNA encoding PHET was isolated by immunoscreening of a HepG2 cDNA library with an SSc serum. PHET appeared to be a member of the UniGene cluster Hs.129872, but had a unique exon composition and a characteristic mRNA expression profile restricted to the testis. Serum Abs to a recombinant PHET fragment were detected in nine (8.4%) of 107 SSc patients, but in none of 50 systemic lupus erythematosus patients or 77 healthy controls. In SSc patients, the presence of anti-PHET Abs was associated with diffuse cutaneous SSc and lung involvement (p = 0.02 and 0.01, respectively). PCR-based quantitative analysis of PHET mRNA expression in cultured dermal fibroblasts showed increased expression of PHET mRNA in SSc fibroblasts compared with control fibroblasts. PHET-reactive Abs purified from SSc sera stained the cytoplasm of SSc dermal fibroblasts, and the staining intensity tended to be more prominent on SSc compared with control fibroblasts. These findings suggest that the autoantibody response to PHET can be induced by ectopic overexpression of PHET in dermal fibroblasts in SSc patients.
Evasion from NK Cell Immunity by MHC Class I Chain-Related Molecules Expressing Colon Adenocarcinoma
Authors: Doubrovina Ekaterina S. , Doubrovin Mikhail M. , Vider Elena , Sisson Richard B. , O’Reilly Richard J. , Dupont Bo , Vyas Yatin M.
Published: April 2014

Evasion of host immune responses is well documented for viruses and may also occur during tumor immunosurveillance. The mechanisms involve alterations in MHC class I expression, Ag processing and presentation, chemokine and cytokine production, and lymphocyte receptor expression. Epithelial tumors overexpress MHC class I chain-related (MIC) molecules, which are ligands for the activating receptor NKG2D on NK and T cells. We report that NK cells from patients with colorectal cancer lack expression of activating NKG2D and chemokine CXCR1 receptors, both of which are internalized. Serum levels of soluble MIC (sMIC) are elevated and are responsible for down-modulation of NKG2D and CXCR1. In contrast, high serum levels of CXC ligands, IL-8, and epithelial-neutrophil-activating peptide (ENA-78) do not down-modulate CXCR1. In vitro, internalization of NKG2D and CXCR1 occurs within 4 and 24 h, respectively, of incubating normal NK cells with sMIC-containing serum. Furthermore, natural cytotoxicity receptor NKp44 and chemokine receptor CCR7 are also down-modulated in IL-2-activated NK cells cocultured in MIC-containing serum—an effect secondary to the down-modulation of NKG2D and not directly caused by physical association with sMIC. The patients’ NK cells up-regulate expression of NKG2D, NKp44, CXCR1, and CCR7 when cultured in normal serum or anti-MIC Ab-treated autologous serum. NKG2D+ but not NKG2D− NK cells are tumoricidal in vitro, and in vivo they selectively traffic to the xenografted carcinoma, form immunological synapse with tumor cells, and significantly retard tumor growth in the SCID mice. These results suggest that circulating sMIC in the cancer patients deactivates NK immunity by down-modulating important activating and chemokine receptors.
Cross-Priming of Diabetogenic T Cells Dissociated from CTL-Induced Shedding of β Cell Autoantigens
Authors: Yamanouchi Jun , Verdaguer Joan , Han Bingye , Amrani Abdelaziz , Serra Pau , Santamaria Pere
Published: April 2014

Cross-presentation of self Ags by APCs is key to the initiation of organ-specific autoimmunity. As MHC class I molecules are essential for the initiation of diabetes in nonobese diabetic (NOD) mice, we sought to determine whether the initial insult that allows cross-presentation of β cell autoantigens in diabetes is caused by cognate interactions between naive CD8+ T cells and β cells. Naive splenic CD8+ T cells from transgenic NOD mice expressing a diabetogenic TCR killed peptide-pulsed targets in the absence of APCs. To ascertain the role of CD8+ T cell-induced β cell lysis in the initiation of diabetes, we expressed a rat insulin promoter (RIP)-driven adenovirus E19 transgene in NOD mice. RIP-E19 expression inhibited MHC class I transport exclusively in β cells and rendered these cells resistant to lysis by CD8+ (but not CD4+) T cells, both in vitro and in vivo. Surprisingly, RIP-E19 expression impaired the accumulation of CD8+ T cells in islets and delayed the onset of islet inflammation, without affecting the timing or magnitude of T cell cross-priming in the pancreatic lymph nodes, which is the earliest known event in diabetogenesis. These results suggest that access of β cell autoantigens to the cross-presentation pathway in diabetes is T cell independent, and reveal a previously unrecognized function of MHC class I molecules on target cells in autoimmunity: local retention of disease-initiating clonotypes.
Influence of MHC CLASS II in Susceptibility to Beryllium Sensitization and Chronic Beryllium Disease
Authors: Maier Lisa A. , McGrath Dierdre S. , Sato Hiroe , Lympany Penny , Welsh Ken , du Bois Roland , Silveira Lori , Fontenot Andrew P. , Sawyer Richard T. , Wilcox Eric , Newman Lee S.
Published: April 2014

A glutamic acid at residue 69(Glu69) in the HLA-DPB1 gene (Glu69) is associated with chronic beryllium disease (CBD) and possibly beryllium sensitization (BeS). This study tested the hypothesis that MHC class II polymorphisms are important in susceptibility to BeS and CBD and that the Glu69 variant is related to markers of disease severity. Genomic DNA was obtained from BeS (n = 50), CBD (n = 104), and beryllium-exposed nondiseased (Be-nondiseased) (n = 125) subjects. HLA-DPB1, -DRB1, and -DQB1 genotypes were determined by (sequence-specific primers) PCR. Disease severity was assessed by pulmonary function and exercise testing. A higher frequency of the DPB1 Glu69 gene was found in CBD and BeS compared with the Be-nondiseased subjects, with odds ratios of 10.1 for CBD vs Be-nondiseased and 9.5 for BeS vs Be-nondiseased. The majority of BeS and CBD subjects displayed non-0201 Glu69 alleles. Glu69 homozygosity was higher in the CBD subjects, while BeS subjects were intermediate and Be-nondiseased lowest. DRB1*01 and DQB1*05 phenotypes were reduced in CBD vs Be-nondiseased subjects, while DRB1*13 and DQB1*06 were associated with CBD in the absence of Glu69. Markers of disease severity, including a lower forced vital capacity, diffusion capacity for carbon monoxide, PaO2 at rest, maximum workload on exercise testing, and a higher arterial-alveolar gradient at rest, were associated with Glu69 homozygosity. We conclude that DPB1 Glu69 is a marker of sensitization and not specific for disease. Glu69 homozygosity acts as a functional marker associated with markers of CBD severity.
Paradoxical Dampening of Anti-Islet Self-Reactivity but Promotion of Diabetes by OX40 Ligand
Authors: Martin-Orozco Natalia , Chen Zhibin , Poirot Laurent , Hyatt Elzbieta , Chen Andy , Kanagawa Osami , Sharpe Arlene , Mathis Diane , Benoist Christophe
Published: April 2014

Costimulatory signals received by diabetogenic T cells during priming by or upon secondary encounter with autoantigen are decisive in determining the outcome of autoimmune attack. The OX40-OX40 ligand (OX40L) costimulatory pathway is known to influence T cell responses, prompting us to examine its role in autoimmune diabetes. A null allele at OX40L completely prevented diabetes development in nonobese diabetic mice and strongly reduced its incidence in a TCR transgenic model (BDC2.5). However, somewhat paradoxically, the initial activation of T cells responsive to islet β cell Ag was slightly faster and more efficient in the absence of OX40L, with an increased degree of cell proliferation and survival in the deficient hosts. Activated T cell migration into and retention within the islets was also slightly accelerated. When challenged in vitro, splenocytes from BDC2.5.OX40Lo/o mice showed no altered reactivity to exogenously added peptide, no bias to the Th1 or Th2 phenotype, and no alteration in T cell survival. Thus, the OX40/OX40L axis has the paradoxical effect of dampening the early activation and migration of autoimmune T cells, but sustains the long-term progression to autoimmune destruction.
Protective Immunity Induced with Malaria Vaccine, RTS,S, Is Linked to Plasmodium falciparum Circumsporozoite Protein-Specific CD4+ and CD8+ T Cells Producing IFN-γ
Authors: Sun Peifang , Schwenk Robert , White Katherine , Stoute Jose A. , Cohen Joe , Ballou W. Ripley , Voss Gerald , Kester Kent E. , Heppner D. Gray , Krzych Urszula
Published: April 2014

The Plasmodium falciparum circumsporozoite (CS) protein-based pre-erythrocytic stage vaccine, RTS,S, induces a high level of protection against experimental sporozoite challenge. The immune mechanisms that constitute protection are only partially understood, but are presumed to rely on Abs and T cell responses. In the present study we compared CS protein peptide-recalled IFN-γ reactivity of pre- and RTS,S-immune lymphocytes from 20 subjects vaccinated with RTS,S. We observed elevated IFN-γ in subjects protected by RTS,S; moreover, both CD4+ and CD8+ T cells produced IFN-γ in response to CS protein peptides. Significantly, protracted protection, albeit observed only in two of seven subjects, was associated with sustained IFN-γ response. This is the first study demonstrating correlation in a controlled Plasmodia sporozoite challenge study between protection induced by a recombinant malaria vaccine and Ag-specific T cell responses. Field-based malaria vaccine studies are in progress to validate the establishment of this cellular response as a possible in vitro correlate of protective immunity to exo-erythrocytic stage malaria vaccines.
Reconstitution of Virus-Specific CD4 Proliferative Responses in Pediatric HIV-1 Infection
Authors: Feeney Margaret E. , Draenert Rika , Roosevelt Kathleen A. , Pelton Stephen I. , McIntosh Kenneth , Burchett Sandra K. , Mao Charlotte , Walker Bruce D. , Goulder Philip J. R.
Published: April 2014

Gag-specific CD4 proliferative responses correlate inversely with HIV-1 RNA levels in infected adults, and robust responses are characteristic of long-term nonprogressive infection. However, strong responses are seldom detected in adult subjects with progressive infection and are not generally reconstituted on highly active antiretroviral therapy (HAART). To date, the role of HIV-1-specific Th responses in children has not been thoroughly examined. We characterized Gag-specific CD4 responses among 35 perinatally infected subjects, including 2 children who spontaneously control viremia without antiretroviral therapy, 21 children with viral loads (VL) of <400 on HAART, and 12 viremic children. Gag-specific Th activity was assessed by lymphoproliferative assay, and responses were mapped using overlapping Gag peptides in an IFN-γ ELISPOT. Robust proliferative responses were detected in the children exhibiting spontaneous control of viremia, and mapping of targeted Gag regions in one such subject identified multiple epitopes. Among children ≥5 years old, 14 of 17 subjects with VL of <400 on HAART demonstrated a significant p24 proliferative response (median p24 stimulation index, 20), in contrast with only 1 of 9 viremic children (median p24 stimulation index, 2.0; p = 0.0008). However, no subject younger than 5 years of age possessed a significant response, even when viremia was fully suppressed. When compared with adults with VL of <400 on HAART, Th responses among children with VL of <400 were both more frequent (p = 0.009) and of greater magnitude (p = 0.002). These data suggest that children may have a greater intrinsic capacity to reconstitute HIV-1-specific immunity than adults, and may be excellent candidates for immune-based therapies.
Cutting Edge: T Lymphocyte Activation by Repeated Immunological Synapse Formation and Intermittent Signaling
Authors: Faroudi Mustapha , Zaru Rossana , Paulet Pierre , Müller Sabina , Valitutti Salvatore
Published: April 2014

The activation of biological T cell responses requires prolonged contact with APCs and sustained signaling. We investigated whether signaling must be uninterrupted to commit T cells to cytokine production or whether T cell activation may also result from summation of interrupted signals. Upon periodic addition and removal of a src kinase inhibitor, human CD4+ T cells destroyed and re-formed immunological synapses while aborting and restarting signal transduction. Remarkably, under these conditions, T cells were eventually activated to IFN-γ production and the amount of IFN-γ produced was directly related to the total signaling time despite the repeated interruptions. Our results illustrate that T cell activation does not require a stable immunological synapse and can be achieved by interrupted signaling. It is implied that T cells can add activation signals, possibly collected on multiple APCs.
Differential Expression of Smad7 Transcripts Identifies the CD4+CD45RChigh Regulatory T Cells That Mediate Type V Collagen-Induced Tolerance to Lung Allografts
Authors: Mizobuchi Teruaki , Yasufuku Kazuhiro , Zheng Yan , Haque M. Azizul , Heidler Kathleen M. , Woods Kena , Smith Gerald N. , Cummings Oscar W. , Fujisawa Takehiko , Blum Janice S. , Wilkes David S.
Published: April 2014

Regulatory T cells (Tregs) induced by oral tolerance may suppress immunity by production of TGF-β that could also enhance Treg activity. However, all cells that are phenotypically Tregs in rats (CD4+CD45RChigh-RChigh) may not have regulatory function. Because Smad7 expression in T cells is associated with inflammation and autoimmunity, then lack of Smad7 may identify those cells that function as Tregs. We reported that feeding type V collagen (col(V)) to WKY rats (RT1l) induces oral tolerance to lung allografts (F344-RT1lvl) by T cells that produce TGF-β. The purpose of the current study was to identify the Tregs that mediate col(V)-induced tolerance, and determine Smad7 expression in these cells. RChigh cells from tolerant rats were unresponsive to allogeneic stimulation and abrogated rejection after adoptive transfer. In contrast, CD4+CD45RClow (RClow) cells from tolerant rats and RChigh or RClow cells from normal rats or untreated allograft recipients proliferated vigorously in response to donor Ags, and did not suppress rejection after adoptive transfer. TGF-β enhanced proliferation in response to col(V) presented to tolerant RChigh, but not other cells. In contrast to other cells, only RChigh cells from tolerant rats did not express Smad7. Collectively, these data show that the Tregs that mediate col(V)-induced tolerance to lung allografts do not express SMAD7 and, therefore, are permissive to TGF-β-mediated signaling.
Antibody Repertoire and Gene Expression Profile: Implications for Different Developmental and Functional Traits of Splenic and Peritoneal B-1 Lymphocytes
Authors: Kretschmer Karsten , Jungebloud Anke , Stopkowicz Jana , Stoermann Britta , Hoffmann Reinhard , Weiss Siegfried
Published: April 2014

In L2 mice, a high expression level of the transgenic λ2315 L chain results in nearly complete exclusion of endogenous L chains and a predominance of B-1a cells. In this study, we show that splenic and peritoneal B-1a cells differ considerably in their Ab repertoire and gene expression profile. Splenic B-1a cells exhibit a more diversified repertoire under L chain limitation. Despite oligoclonal overlaps between both B-1a compartments, some B cell receptor specificities are clearly restricted to the peritoneum. The capacity of peritoneal B-1a cells to enter the splenic B-1a compartment was found to be very limited. Gene expression profiling revealed genes up-regulated in splenic B-1a cells that are involved in mediating specialized first-line-of-defense effector functions and interaction with T cells. Thus, splenic and peritoneal B-1a cells differ not only in their developmental program but also in functional properties.
Hemopoietic Function After Use of IL-1 with Chemotherapy or Irradiation
Authors: Gardner Renee V. , McKinnon Evangeline , Poretta Connie , Leiva Lily
Published: April 2014

IL-1 has putative chemo- and radioprotective properties, but its effects on primitive hemopoietic stem cell (PHSC) and early multilineage precursor function when given with these modalities is unknown. C57BL6/J (B6) mice, given IL-1 20 h before cyclophosphamide (200 mg/kg for four biweekly doses) or before irradiation (500 cGy), were sacrificed after 4 wk. Their marrow was used as donor cells, and that from B6-HbbdGpi1a (B6-GPI) mice was used as competitor cells in competitive repopulation. Percentages of B6 cells were measured at 30 and 150 days. Stem cell numbers were estimated using binomial statistics. IL-1 alone did not affect stem cell function. As expected, significant declines in early multilineage precursor and PHSC function occurred with chemotherapy and radiation alone. IL-1 with chemotherapy led to exacerbation of these losses in function and numbers (p < 0.05). A similar reduction in function occurred using IL-1 before irradiation. In summary, IL-1 with chemotherapy or radiation worsened chemotherapy- and radiation-induced functional damage to PHSC and other hemopoietic precursors, suggesting that improvements in survival do not necessarily translate into preservation of hemopoietic function.
Theileria parva-Transformed T Cells Show Enhanced Resistance to Fas/Fas Ligand-Induced Apoptosis
Authors: Küenzi Peter , Schneider Pascal , Dobbelaere Dirk A. E.
Published: April 2014

Lymphocyte homeostasis is regulated by mechanisms that control lymphocyte proliferation and apoptosis. Activation-induced cell death is mediated by the expression of death ligands and receptors, which, when triggered, activate an apoptotic cascade. Bovine T cells transformed by the intracellular parasite Theileria parva proliferate in an uncontrolled manner and undergo clonal expansion. They constitutively express the death receptor Fas and its ligand, FasL but do not undergo apoptosis. Upon elimination of the parasite from the host cell by treatment with a theilericidal drug, cells become increasingly sensitive to Fas/FasL-induced apoptosis. In normal T cells, the sensitivity to death receptor killing is regulated by specific inhibitor proteins. We found that anti-apoptotic proteins such as cellular (c)-FLIP, which functions as a catalytically inactive form of caspase-8, and X-chromosome-linked inhibitor of apoptosis protein (IAP) as well as c-IAP, which can block downstream executioner caspases, are constitutively expressed in T. parva-transformed T cells. Expression of these proteins is rapidly down-regulated upon parasite elimination. Antiapoptotic proteins of the Bcl-2 family such as Bcl-2 and Bcl-xL are also expressed but, in contrast to c-FLIP, c-IAP, and X-chromosome-linked IAP, do not appear to be tightly regulated by the presence of the parasite. Finally, we show that, in contrast to the situation in tumor cells, the phosphoinositide 3-kinase/Akt pathway is not essential for c-FLIP expression. Our findings indicate that by inducing the expression of antiapoptotic proteins, T. parva allows the host cell to escape destruction by homeostatic mechanisms that would normally be activated to limit the continuous expansion of a T cell population.
Enhanced Priming of Multispecific, Murine CD8+ T Cell Responses by DNA Vaccines Expressing Stress Protein-Binding Polytope Peptides
Authors: Schirmbeck Reinhold , Fissolo Nicolas , Chaplin Paul , Reimann Jörg
Published: April 2014

A polytope DNA vaccine (pCI/pt10) was used that encodes within a 106-residue sequence 10-well characterized epitopes binding MHC class I molecules encoded by the K, D, or L locus (of H-2d, H-2b, and H-2k haplotype mice). The pCI/pt10 DNA vaccine efficiently primed all four Kb/Db-restricted CD8+ T cell responses in H-2b mice, but was deficient in stimulating most CD8+ T cell responses in H-2d mice. Comparing CD8+ T cell responses elicited with the pCI/pt10 DNA vaccine in Ld+ BALB/c and Ld− BALB/cdm2 (dm2) mice revealed that Ld-restricted CD8+ T cell responses down-regulated copriming of CD8+ T cell responses to other epitopes regardless of their restriction or epitope specificity. Although the pt10 vaccine could thus efficiently co prime multispecific CD8+ T cell responses, this priming was impaired by copriming Ld-restricted CD8+ T cell responses. When the pt10 sequence was fused to a 77-residue DnaJ-homologous, heat shock protein 73-binding domain (to generate a 183-residue cT77-pt10 fusion protein), expression and immunogenicity (for CD8+ T cells) of the chimeric Ag were greatly enhanced. Furthermore, priming of multispecific CD8+ T cell responses was readily elicited even under conditions in which the suppressive, Ld-dependent immunodominance operated. The expression of polytope vaccines as chimeric peptides that endogenously capture stress proteins during in situ production thus facilitates copriming of CD8+ T cell populations with a diverse repertoire.
CD4+ NKT Cells, But Not Conventional CD4+ T Cells, Are Required to Generate Efferent CD8+ T Regulatory Cells Following Antigen Inoculation in an Immune-Privileged Site
Authors: Nakamura Takahiko , Sonoda Koh-Hei , Faunce Douglas E. , Gumperz Jenny , Yamamura Takashi , Miyake Sachiko , Stein-Streilein Joan
Published: April 2014

Following inoculation of Ag into the anterior chamber (a.c.), systemic tolerance develops that is mediated in part by Ag-specific efferent CD8+ T regulatory (Tr) cells. This model of tolerance is called a.c.-associated immune deviation. The generation of the efferent CD8+ Tr cell in a.c.-associated immune deviation is dependent on IL-10-producing, CD1d-restricted, invariant Vα14+ NKT (iNKT) cells. The iNKT cell subpopulations are either CD4+ or CD4−CD8− double negative. This report identifies the subpopulation of iNKT cells that is important for induction of the efferent Tr cell. Because MHC class II−/− (class II−/−) mice generate efferent Tr cells following a.c. inoculation, we conclude that conventional CD4+ T cells are not needed for the development of efferent CD8+ T cells. Furthermore, Ab depletion of CD4+ cells in both wild-type mice (remove both conventional and CD4+ NKT cells) and class II−/− mice (remove CD4+ NKT cells) abrogated the generation of Tr cells. We conclude that CD4+ NKT cells, but not the class II molecule or conventional CD4+ T cells, are required for generation of efferent CD8+ Tr cells following Ag introduction into the eye. Understanding the mechanisms that lead to the generation of efferent CD8+ Tr cells may lead to novel immunotherapy for immune inflammatory diseases.
The Lyn Tyrosine Kinase Negatively Regulates Neutrophil Integrin Signaling
Authors: Pereira Shalini , Lowell Clifford
Published: April 2014

The Src family kinase Lyn has been shown to play both stimulatory and inhibitory roles within several hemopoietic cell types. In this study, we investigated the role played by Lyn in neutrophil integrin signaling. Loss of Lyn resulted in a hyperresponsive phenotype on engagement of surface integrins at low valency. Lyn−/− neutrophils displayed enhanced respiratory burst, secondary granule release, and a hyperadhesive phenotype when adherent to surfaces coated with either cellular counterreceptors or extracellular matrix proteins. In contrast, Lyn-deficient and wild-type cells expressed similar levels of surface integrins and responded equivalently to activating agents in suspension, indicating that the enhanced responses of lyn−/− cells was specific to the integrin signaling pathways. Lyn-deficient macrophages also displayed a hyperadhesive phenotype. Biochemical analysis of macrophages from lyn−/− mice revealed that Lyn plays an essential role in the adhesion-dependent phosphorylation of the immunoreceptor tyrosine-based inhibitory motif of the inhibitory receptors SIRP1α and PIR-B, which in turn recruit the phosphatase SHP-1. These observations suggest that reduced mobilization of SHP-1 to the membrane in lyn−/− neutrophils results in a hyperadhesive and hyperactive phenotype. This hypothesis is further supported by the fact that neutrophils from mev/mev mice, which have significantly reduced SHP-1 activity, are also hyperresponsive following integrin engagement. This is the first direct evidence using primary leukocytes from lyn−/− mice that this kinase functions as a negative regulator in integrin signaling.
B Cell Receptor-Mediated Syk-Independent Activation of Phosphatidylinositol 3-Kinase, Ras, and Mitogen-Activated Protein Kinase Pathways
Authors: Yokozeki Takeaki , Adler Kristin , Lankar Danielle , Bonnerot Christian
Published: April 2014

The Syk tyrosine kinase is a key molecule in the development of the B cell lineage and the activation of B lymphocytes after Ag recognition by the B cell Ag receptor (BCR). Several genetic studies with chicken B cells have reported that the recruitment of Syk by BCR is essential for activation of a cascade of signaling molecules including phosphatidylinositol 3-kinase, mitogen-activated protein kinases, Ras signaling pathways, phospholipase C-γ2 activation, and calcium mobilization. The identification of a Syk-deficient mouse IIA1.6/A20 B cell line provided us the opportunity to investigate Syk-mediated signaling in mouse. Surprisingly, phosphatidylinositol 3-kinase, Ras, and mitogen-activated protein kinases were activated upon BCR cross-linking in these Syk-deficient mouse B cells, whereas, as expected from results obtained in chicken B cells, phospholipase C-γ2 activation and calcium mobilization were impaired as well as the NF-κB pathway. These results indicate that BCR signaling is not strictly dependent on Syk expression in mouse IIA1.6/A20 B cells. Thus, B lymphocyte activation may be initiated by Syk-dependent and Syk-independent signaling cascades.
Identification and Characterization of a Novel Regulatory Factor: IgA-Inducing Protein
Authors: Austin Amy S. , Haas Karen M. , Naugler Sasha M. , Bajer Anna A. , Garcia-Tapia David , Estes D. Mark
Published: April 2014

IgA is the predominant Ig isotype in mucosal secretions and thus plays a pivotal role in host defense. The mechanisms by which IgA expression is regulated may differ among species and involve multiple pathways. Various cytokines and costimulators have been identified which regulate expression of this isotype, including IL-10, IL-2, vasoactive intestinal peptide, and TGF-β. We have tested a wide array of known factors, but only under very limited conditions do these factors mediate substantial IgA production in vitro from bovine B cells. In response to these findings, we generated a cDNA library in a mammalian expression vector from activated cells derived from bovine gut-associated lymphoid tissues (Peyer’s patch and mesenteric lymph node cells) as a source of soluble factor(s) that may regulate IgA production. We have identified a novel factor, IgA-inducing protein, which stimulates relatively high levels of IgA production in vitro following CD40 stimulation in coculture with IL-2. Our data suggest that IgA-inducing protein regulates IgA by acting as a switch or differentiation factor and is expressed in a variety of lymphoid and nonlymphoid tissues.
Positive Modulation of IL-12 Signaling by Sphingosine Kinase 2 Associating with the IL-12 Receptor β1 Cytoplasmic Region
Authors: Yoshimoto Takayuki , Furuhata Masae , Kamiya Sadahiro , Hisada Masayuki , Miyaji Hiroko , Magami Yasushi , Yamamoto Koh , Fujiwara Hiromi , Mizuguchi Junichiro
Published: April 2014

IL-12 is a key immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses. IL-12 stimulation results in the activation of Janus kinase 2 and tyrosine kinase 2 and, subsequently, STAT4 and STAT3. In addition, mitogen-activated protein kinase kinase 6/p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways have been recently demonstrated to be activated by IL-12 and play an important role in IL-12 signaling. To further elucidate the molecular mechanism underlying IL-12 signaling, we have performed a yeast two-hybrid screening and identified mouse sphingosine kinase 2 (SPHK2) as a molecule associating with the mouse IL-12Rβ1 cytoplasmic region. Analyses of various mutants of each molecule revealed that the region including the proline-rich domain in SPHK2 is probably responsible for the binding to IL-12Rβ1, while the regions including the carboxyl terminus and Box II in the IL-12Rβ1 cytoplasmic region appear to be involved in the binding to SPHK2. Transient expression of wild-type SPHK2 in T cell hybridoma augmented IL-12-induced STAT4-mediated transcriptional activation. Ectopic expression of dominant-negative SPHK2 in Th1 cell clone significantly reduced IL-12-induced IFN-γ production, while that of wild-type SPHK2 enhanced it. In contrast, the expression minimally affected IL-12-induced proliferation. A similar decrease in IL-12-induced IFN-γ production was observed when dominant-negative SPHK2 was expressed in activated primary T cells using a retroviral expression system. These results suggest that SPHK2 associates with the IL-12Rβ1 cytoplasmic region and probably plays a role in modulating IL-12 signaling.
SLP-76 Coordinates Nck-Dependent Wiskott-Aldrich Syndrome Protein Recruitment with Vav-1/Cdc42-Dependent Wiskott-Aldrich Syndrome Protein Activation at the T Cell-APC Contact Site
Authors: Zeng Rong , Cannon Judy L. , Abraham Robert T. , Way Michael , Billadeau Daniel D. , Bubeck-Wardenberg Julie , Burkhardt Janis K.
Published: April 2014

We have shown previously that Wiskott-Aldrich syndrome protein (WASP) activation at the site of T cell-APC interaction is a two-step process, with recruitment dependent on the proline-rich domain and activation dependent on binding of Cdc42-GTP to the GTPase binding domain. Here, we show that WASP recruitment occurs through binding to the C-terminal Src homology 3 domain of Nck. In contrast, WASP activation requires Vav-1. In Vav-1-deficient T cells, WASP recruitment proceeds normally, but localized activation of Cdc42 and WASP is disrupted. The recruitment and activation of WASP are coordinated by tyrosine-phosphorylated Src homology 2 domain-containing leukocyte protein of 76 kDa, which functions as a scaffold, bringing Nck and WASP into proximity with Vav-1 and Cdc42-GTP. Taken together, these findings reconstruct the signaling pathway leading from TCR ligation to localized WASP activation.
Analysis of HLA-E Peptide-Binding Specificity and Contact Residues in Bound Peptide Required for Recognition by CD94/NKG2
Authors: Miller Joseph D. , Weber Dominique A. , Ibegbu Chris , Pohl Jan , Altman John D. , Jensen Peter E.
Published: April 2014

The MHC class Ib molecule HLA-E is the primary ligand for CD94/NKG2A-inhibitory receptors expressed on NK cells, and there is also evidence for TCR-mediated recognition of this molecule. HLA-E preferentially assembles with a homologous set of peptides derived from the leader sequence of class Ia molecules, but its capacity to bind and present other peptides remains to be fully explored. The peptide-binding motif of HLA-E was investigated by folding HLA-E in vitro in the presence of peptide libraries derived from a nonameric leader peptide sequence randomized at individual anchor positions. A high degree of selectivity was observed at four of five total anchor positions, with preference for amino acids present in HLA-E-binding peptides from class Ia leader sequences. Selectivity was also observed at the nonanchor P5 position, with preference for positively charged amino acids, suggesting that electrostatic interactions involving the P5 side chain may facilitate assembly of HLA-E peptide complexes. The observed HLA-E peptide-binding motif was strikingly similar to that previously identified for the murine class Ib molecule, Qa-1. Experiments with HLA-E tetramers bearing peptides substituted at nonanchor positions demonstrated that P5 and P8 are primary contact residues for interaction with CD94/NKG2 receptors. A conservative replacement of Arg for Lys at P5 completely abrogated binding to CD94/NKG2. Despite conservation of peptide-binding specificity in HLA-E and Qa-1, cross-species tetramer-staining experiments demonstrated that the interaction surfaces on CD94/NKG2 and the class Ib ligands have diverged between primates and rodents.
Protein Expression and Peptide Binding Suggest Unique and Interacting Functional Roles for HLA-E, F, and G in Maternal-Placental Immune Recognition
Authors: Ishitani Akiko , Sageshima Noriko , Lee Ni , Dorofeeva Natalia , Hatake Katsuhiko , Marquardt Hans , Geraghty Daniel E.
Published: April 2014

In this study we focused on the structure and expression of the HLA-E, F, and G class I complexes in placental tissue. Structural analysis included an examination of the peptides bound to soluble and membrane forms of the HLA-G complex isolated directly from placenta. An important distinction was observed from HLA-G bound peptides previously isolated from transfectant cells. Thus, the number of distinct moieties bound to placental-derived proteins was substantially lower than that bound to transfectant-derived HLA-G. Indeed, a single peptide species derived from a cytokine-related protein alone accounted for 15% of the molar ratio of HLA-G bound peptide. To further examine HLA-E and its potential to bind peptide, notably that derived from HLA-G, we combined new Abs to examine expression in placental tissues for all the known forms of the nonclassical class I molecules. Whereas membrane HLA-G was found in extravillous trophoblasts, soluble HLA-G was found in all placental trophoblasts, including villous cytotrophoblasts and syncitiotrophoblasts. Further, HLA-E was found in all cells that expressed either form of HLA-G, consistent with HLA-E being complexed with the HLA-G signal sequence-derived nonamer in these cells. Finally, using new reagents specific for HLA-F, a restricted pattern of expression was observed, primarily on extravillous trophoblasts that had invaded the maternal decidua. Comparative staining indicated that HLA-F was on the surface of these cells, defining them as the first to demonstrate surface expression of this Ag and the first cell type identified to express all three nonclassical HLA class I Ags simultaneously.
Inhibition of IFN-γ-Inducible Protein-10 Abrogates Colitis in IL-10−/− Mice
Authors: Singh Udai P. , Singh Shailesh , Taub Dennis D. , Lillard James W.
Published: April 2014

A deficiency in understanding the steps responsible for colitis is the lack of comprehension for the role chemokines play in mucosal inflammation. IFN-γ-inducible protein-10 (IP-10) and CXCR3 are highly expressed at sites of colitis. Our findings show that IP-10 significantly contributes to the development of Th1 and inflammatory responses. Specifically, IP-10 inhibition in IL-10−/− mice attenuates the associated increases in serum and/or local amyloid A, IL-2, IL-6, TNF-α, IFN-γ, IL-1α, and IL-1β with colitis as compared with IL-10−/− mice that develop colitis similar to human Crohn’s disease. Correspondingly, the rate or intensity of inflammation in IL-10−/− mice treated with anti-IP-10 Abs showed improved scoring of inflammation, compared with control IL-10−/− mice. This study provides important and novel information regarding IP-10 as a target for the treatment of colitis.
Glycosylinositolphosphate Soluble Variant Surface Glycoprotein Inhibits IFN-γ-Induced Nitric Oxide Production Via Reduction in STAT1 Phosphorylation in African Trypanosomiasis
Authors: Coller Susan P. , Mansfield John M. , Paulnock Donna M.
Published: April 2014

Macrophages are centrally involved in the host immune response to infection with Trypanosoma brucei rhodesiense, a protozoan parasite responsible for human sleeping sickness in Africa. During trypanosome infections, the host is exposed to parasite-derived molecules that mediate macrophage activation, specifically GPI anchor substituents associated with the shed variant surface glycoprotein (VSG), plus the host-activating agent IFN-γ, which is derived from activated T cells and is essential for resistance to trypanosomes. In this study, we demonstrate that the level and timing of exposure of macrophages to IFN-γ vs GPI ultimately determine the macrophage response at the level of induced gene expression. Treatment of macrophages with IFN-γ followed by GIP-sVSG (the soluble form of VSG containing the glycosylinositolphosphate substituent that is released by parasites) stimulated the induction of gene expression, including transcription of TNF-α, IL-6, GM-CSF, and IL-12p40. In contrast, treatment of macrophages with GIP-sVSG before IFN-γ stimulation resulted in a marked reduction of IFN-γ-induced responses, including transcription of inducible NO synthase and secretion of NO. Additional experiments revealed that the inhibitory activity of GIP-sVSG was associated with reduction in the level of STAT1 phosphorylation, an event required for IFN-γ-induced macrophage activation. These results suggest that modulation of specific aspects of the IFN-γ response may be one mechanism by which trypanosomes overcome host resistance during African trypanosomiasis.