bluuebunny/crossref_metadata_2025 huggingface dataset:
The Duration of Nuclear Residence of NFAT Determines the Pattern of Cytokine Expression in Human SCID T Cells
Authors: Feske Stefan , Draeger Ruth , Peter Hans-Hartmut , Eichmann Klaus , Rao Anjana
Published: April 2014

The expression of cytokine genes and other inducible genes is crucially dependent on the pattern and duration of signal transduction events that activate transcription factor binding to DNA. Two infant patients with SCID and a severe defect in T cell activation displayed an aberrant regulation of the transcription factor NFAT. Whereas the expression levels of the NFAT family members NFAT1, -2, and -4 were normal in the patients’ T cells, dephosphorylation and nuclear translocation of these NFAT proteins occurred very transiently and incompletely upon stimulation. Only after inhibition of nuclear export with leptomycin B were we able to demonstrate a modest degree of nuclear translocation in the patients’ T cells. This transient activation of NFAT was not sufficient to induce the expression of several cytokines, including IL-2, IL-3, IL-4, and IFN-γ, whereas mRNA levels for macrophage inflammatory protein-1α, GM-CSF, and IL-13 were only moderately reduced. By limiting the time of NFAT activation in normal control cells using the calcineurin inhibitor cyclosporin A, we were able to mimic the cytokine expression pattern in SCID T cells, suggesting that the expression of different cytokine genes is differentially regulated by the duration of NFAT residence in the nucleus.
Tapasin-Mediated Retention and Optimization of Peptide Ligands During the Assembly of Class I Molecules
Authors: Barnden Megan J. , Purcell Anthony W. , Gorman Jeffrey J. , McCluskey James
Published: April 2014

The murine class I H-2Kb molecule achieves high level surface expression in tapasin-deficient 721.220 human cells. Compared with their behavior in wild-type cells, Kb molecules expressed on 721.220 cells are more receptive to exogenous peptide, undergo more rapid surface decay, and fail to form macromolecular peptide loading complexes. As a result, they are rapidly transported to the cell surface, reflecting a failure of endoplasmic reticulum retention mechanisms in the absence of loading complex formation. Despite the failure of Kb molecules to colocalize to the TAP and their rapid egress to the cell surface, Kb is still capable of presenting TAP-dependent peptides in the absence of tapasin. Furthermore, pool sequencing of peptides eluted from these molecules revealed strict conservation of their canonical H-2Kb-binding motif. There was a reduction in the total recovery of peptides associated with Kb molecules purified from the surface of tapasin-deficient cells. Comparison of the peptides bound to Kb in the presence and absence of tapasin revealed considerable overlap in peptide repertoire. These results indicate that in the absence of an interaction with tapasin, Kb molecules fail to assemble with calreticulin and TAP, yet they are still capable of acquiring a diverse array of peptides. However, a significant proportion of these peptides appear to be suboptimal, resulting in reduced cell surface stability of Kb complexes. Taken together, the findings indicate that tapasin plays an essential role in the formation of the class I loading complex, which retains class I heterodimers in the endoplasmic reticulum until optimal ligand selection is completed.
CD4+ T Cells Acting Independently of Antibody Contribute to Protective Immunity to Plasmodium chabaudi Infection After Apical Membrane Antigen 1 Immunization
Authors: Xu Huji , Hodder Anthony N. , Yan Huara , Crewther Pauline E. , Anders Robin F. , Good Michael F.
Published: April 2014

Apical membrane Ag 1 (AMA1) is a leading malaria vaccine candidate. Homologues of AMA1 can induce protection in mice and monkeys, but the mechanism of immunity is not understood. Mice immunized with a refolded, recombinant, Plasmodium chabaudi AMA1 fragment (AMA1B) can withstand subsequent challenge with P. chabaudi adami. Here we show that CD4+ T cell depletion, but not γδ T cell depletion, can cause a significant drop in antiparasite immunity in either immunized normal or immunized B cell KO mice. In normal mice, this loss of immunity is not accompanied by a decline in Ab levels. These observations indicate a role for AMA1-specific Ab-independent T cell-mediated immunity. However, the loss of immunity in normal CD4+ T cell-depleted mice is temporary. Furthermore, immunized B cell KO mice cannot survive infection, demonstrating the absolute importance of B cells, and presumably Ab, in AMA1-induced immunity. CD4+ T cells specific for a cryptic conserved epitope on AMA1 can adoptively transfer protection to athymic (nu/nu) mice, the level of which is enhanced by cotransfer of rabbit anti-AMA1-specific antisera. Recipients of rabbit antisera alone do not survive. Some protected recipients of T cells plus antisera do not develop their own AMA 1-specific Ab response, suggesting that AMA 1-specific CMI alone can protect mice. These data are the first to demonstrate the specificity of any protective CMI response in malaria and have important implications for developing a malaria vaccine.
Proteolysis of Human Monocyte CD14 by Cysteine Proteinases (Gingipains) fromPorphyromonas gingivalisLeading to Lipopolysaccharide Hyporesponsiveness
Authors: Sugawara Shunji , Nemoto Eiji , Tada Hiroyuki , Miyake Kensuke , Imamura Takahisa , Takada Haruhiko
Published: April 2014

Cysteine proteinases (gingipains) elaborated from Porphyromonas gingivalis exhibit enzymatic activities against a broad range of host proteins and are considered key virulence factors in the onset and development of adult periodontitis and host defense evasion. In this study, we examined the ability of arginine-specific gingipains (high molecular mass Arg-specific gingipain (HRGP) and Arg-specific gingipain 2) and lysine-specific gingipain (KGP) to cleave monocyte CD14, the main receptor for bacterial cell surface components such as LPS. Binding of anti-CD14 mAb MY4 to human monocytes was almost completely abolished by 0.3 μM HRGP and KGP treatments for 15 min, and 1 μM RGP2 for 30 min. In contrast, the expressions of Toll-like receptor 4, and CD18, CD54, CD59, and HLA-A, -B, -C on monocytes were slightly increased and decreased, respectively, by 0.3 μM HRGP and KGP. This down-regulation resulted from direct proteolysis, because 1) gingipains eliminated MY4 binding even to fixed monocytes, and 2) CD14 fragments were detected in the extracellular medium by immunoblot analysis. Human rCD14 was degraded by all three gingipains, which confirmed that CD14 was a substrate for gingipains. TNF-α production by monocytes after HRGP and KGP treatments was decreased at 1 ng/ml, but not at 20 μg/ml LPS, indicating that gingipains inhibited a CD14-dependent cell activation. These results suggest that gingipains preferentially cleave monocyte CD14, resulting in attenuation of the cellular recognition of bacteria, and as a consequence sustain chronic inflammation.
Group BStreptococcusInduces TNF-α Gene Expression and Activation of the Transcription Factors NF-κB and Activator Protein-1 in Human Cord Blood Monocytes
Authors: Vallejo Jesus G. , Knuefermann Pascal , Mann Douglas L. , Sivasubramanian Natarajan
Published: April 2014

It has been postulated that production of TNF-α is central to the pathogenesis of septic shock induced by group B Streptococcus (GBS). In vitro studies using human cord blood monocytes have demonstrated that GBS induces TNF-α secretion, but little is known about the intracellular signaling pathways of TNF-α induction. In this report we show that heat-killed serotype III GBS induces host cell signal transduction pathways that lead to activation of the transcription factors NF-κB and AP-1. Using adenoviral transfer of IκBα (IκBα overexpression), the production of TNF-α induced by whole GBS was inhibited by only 20%. We also show that the p38 mitogen-activated protein kinase (MAPK) pathway is involved in GBS-induced TNF-α secretion, because TNF-α protein and mRNA levels in the presence of a specific inhibitor of p38 MAPK, SB 202190, were dramatically diminished. EMSAs showed that SB 202190 inhibited GBS-induced AP-1 activation, but had no effect on NF-κB-DNA binding activity. These results indicate that both NF-κB and AP-1 (via p38 MAPK) are involved in the regulation of TNF-α production in GBS-stimulated neonatal monocytes. Therefore, disrupting the signal transduction pathways induced by GBS has the potential to attenuate the production of immune response mediators, thereby halting or possibly reversing the course of this potentially fatal disease.
The LFA-1 Integrin Supports Rolling Adhesions on ICAM-1 Under Physiological Shear Flow in a Permissive Cellular Environment
Authors: Sigal Alex , Bleijs Diederik A. , Grabovsky Valentin , van Vliet Sandra J. , Dwir Oren , Figdor Carl G. , van Kooyk Yvette , Alon Ronen
Published: April 2014

The LFA-1 integrin is crucial for the firm adhesion of circulating leukocytes to ICAM-1-expressing endothelial cells. In the present study, we demonstrate that LFA-1 can arrest unstimulated PBL subsets and lymphoblastoid Jurkat cells on immobilized ICAM-1 under subphysiological shear flow and mediate firm adhesion to ICAM-1 after short static contact. However, LFA-1 expressed in K562 cells failed to support firm adhesion to ICAM-1 but instead mediated K562 cell rolling on the endothelial ligand under physiological shear stress. LFA-1-mediated rolling required an intact LFA-1 I-domain, was enhanced by Mg2+, and was sharply dependent on ICAM-1 density. This is the first indication that LFA-1 can engage in rolling adhesions with ICAM-1 under physiological shear flow. The ability of LFA-1 to support rolling correlates with decreased avidity and impaired time-dependent adhesion strengthening. A β2 cytoplasmic domain-deletion mutant of LFA-1, with high avidity to immobilized ICAM-1, mediated firm arrests of K562 cells interacting with ICAM-1 under shear flow. Our results suggest that restrictions in LFA-1 clustering mediated by cytoskeletal attachments may lock the integrin into low-avidity states in particular cellular environments. Although low-avidity LFA-1 states fail to undergo adhesion strengthening upon contact with ICAM-1 at stasis, these states are permissive for leukocyte rolling on ICAM-1 under physiological shear flow. Rolling mediated by low-avidity LFA-1 interactions with ICAM-1 may stabilize rolling initiated by specialized vascular rolling receptors and allow the leukocyte to arrest on vascular endothelium upon exposure to stimulatory endothelial signals.
Polyethylene Glycol-Modified GM-CSF Expands CD11bhighCD11chigh But Not CD11blowCD11chigh Murine Dendritic Cells In Vivo: A Comparative Analysis with Flt3 Ligand
Authors: Daro Elizabeth , Pulendran Bali , Brasel Kenneth , Teepe Mark , Pettit Dean , Lynch David H. , Vremec David , Robb Lorraine , Shortman Ken , McKenna Hilary J. , Maliszewski Charles R. , Maraskovsky Eugene
Published: April 2014

Dendritic cells (DC) are potent APCs that can be characterized in the murine spleen as CD11bhighCD11chigh or CD11blowCD11chigh. Daily injection of mice of Flt3 ligand (FL) into mice transiently expands both subsets of DC in vivo, but the effect of administration of GM-CSF on the expansion of DC in vivo is not well defined. To gain further insight into the role of GM-CSF in DC development and function in vivo, we treated mice with polyethylene glycol-modified GM-CSF (pGM-CSF) which has an increased half-life in vivo. Administration of pGM-CSF to mice for 5 days led to a 5- to 10-fold expansion of CD11bhighCD11chigh but not CD11blowCD11chigh DC. DC from pGM-CSF-treated mice captured and processed Ag more efficiently than DC from FL-treated mice. Although both FL- and pGM-CSF-generated CD11bhighCD11chigh DC were CD8α−, a greater proportion of these DC from pGM-CSF-treated mice were 33D1+ than from FL-treated mice. CD11blowCD11chigh DC from FL-treated mice expressed high levels of intracellular MHC class II. DC from both pGM-CSF- and FL-treated mice expressed high levels of surface class II, low levels of the costimulatory molecules CD40, CD80, and CD86 and were equally efficient at stimulating allogeneic and Ag-specific T cell proliferation in vitro. The data demonstrate that treatment with pGM-CSF in vivo preferentially expands CD11bhighCD11chigh DC that share phenotypic and functional characteristics with FL-generated CD11bhighCD11chigh DC but can be distinguished from FL-generated DC on the basis of Ag capture and surface expression of 33D1.
Tetramer-Guided Analysis of TCR β-Chain Usage Reveals a Large Repertoire of Melan-A-Specific CD8+ T Cells in Melanoma Patients
Authors: Valmori Danila , Dutoit Valérie , Liénard Danielle , Lejeune Ferdy , Speiser Daniel , Rimoldi Donata , Cerundolo Vincenzo , Dietrich Pierre-Yves , Cerottini Jean-Charles , Romero Pedro
Published: April 2014

The assessment of the TCR repertoire expressed by tumor-specific CD8+ T lymphocytes has been hampered to date by the difficulty of targeting the analysis to lymphocytes directed against a single epitope. In the present study we have used fluorescent A2/Melan-A tetramers in conjunction with anti-CD8 and anti-TCR β-chain variable (BV) mAbs to analyze by flow cytometry the BV segment usage by Melan-A-specific CD8+ T cells in tumor-infiltrated lymph nodes (TILN) and tumor-infiltrating lymphocytes (TIL) from A2 melanoma patients. Analysis of TILN populations revealed small proportions of A2/Melan-A tetramer+ cells expressing many different BV together with over-representation of A2/Melan-A tetramer+ cells expressing certain BVs. The BV usage by A2/Melan-A tetramer+ lymphocytes in TIL was more restricted than that in TILN. Moreover, the predominant BV segments were quite distinct in populations derived from different patients. A2/Melan-A tetramer+ cells expressing the dominant BVs found in TILN could also be found in the corresponding peptide-stimulated autologous PBMC, although A2/Melan-A tetramer+ lymphocytes expressing additional BVs were also identified. Together, these results suggest that a large and diverse repertoire of Melan-A-specific T cells using different BV TCR segments is available in A2 melanoma patients.
IFN-β-1b Inhibits IL-12 Production in Peripheral Blood Mononuclear Cells in an IL-10-Dependent Mechanism: Relevance to IFN-β-1b Therapeutic Effects in Multiple Sclerosis
Authors: Wang Xin , Chen Man , Wandinger Klaus Peter , Williams Gary , Dhib-Jalbut Suhayl
Published: April 2014

IL-12 is a proinflammatory cytokine secreted by dendritic cells in response to microbial Ags and mitogens. IL-12 is thought to contribute to the pathogenesis of autoimmune diseases such as multiple sclerosis (MS). This is based on studies in experimental allergic encephalomyelitis and the demonstration that PBMC IL-12 production correlates with disease progression in MS. IFN-β-1b is an effective treatment for MS, which is thought to involve in part inhibition of proinflammatory cytokines. In this study we examined the effect of in vitro treatment with IFN-β-1b, on mitogen-induced IL-12 production in human PBMC and myelin basic protein-specific T cell lines obtained from healthy donors and MS patients. We demonstrate that IFN-β-1b significantly inhibits inducible IL-12 p40 up to 80% and biologically active IL-12 p70 up to 70% beginning at a dose of 10 IU/ml. This inhibition is IL-10 dependent, as it could be blocked by anti-IL-10 but not anti-IL-4 or control Abs. Thus, endogenously produced IL-10 is a required cofactor for the IFN-β-1b inhibitory effect on IL-12 to occur. We conclude that IFN-β-1b has a profound inhibitory effect on PBMC IL-12 production in vitro, and that this effect is IL-10 dependent. These findings are potentially relevant to the therapeutic mechanism of IFN-β-1b in MS.
Positive Impact of Inhibitory Ly49 Receptor-MHC Class I Interaction on NK Cell Development
Authors: Lowin-Kropf Bente , Held Werner
Published: April 2014

NK cells can kill MHC-different or MHC-deficient but not syngeneic MHC-expressing target cells. This MHC class I-specific tolerance is acquired during NK cell development. MHC recognition by murine NK cells largely depends on clonally distributed Ly49 family receptors, which inhibit NK cell function upon ligand engagement. We investigated whether these receptors play a role for the development of NK cells and provide evidence that the expression of a Ly49 receptor transgene on developing NK cells endowed these cells with a significant developmental advantage over NK cells lacking such a receptor, but only if the relevant MHC ligand was present in the environment. The data suggest that the transgenic Ly49 receptor accelerates and/or rescues the development of NK cells which would otherwise fail to acquire sufficient numbers of self-MHC-specific receptors. Interestingly, the positive effect on NK cell development is most prominent when the MHC ligand is simultaneously present on both hemopoietic and nonhemopoietic cells. These findings correlate with functional data showing that MHC class I ligand on all cells is required to generate functionally mature NK cells capable of reacting to cells lacking the respective MHC ligand. We conclude that the engagement of inhibitory MHC receptors during NK cell development provides signals that are important for further NK cell differentiation and/or maturation.
Evidence for Functional Relevance of CTLA-4 in Ultraviolet-Radiation-Induced Tolerance
Authors: Schwarz Agatha , Beissert Stefan , Grosse-Heitmeyer Karin , Gunzer Matthias , Bluestone Jeffrey A. , Grabbe Stephan , Schwarz Thomas
Published: April 2014

Hapten sensitization through UV-exposed skin induces hapten-specific tolerance that can be adoptively transferred by injecting T lymphocytes into naive recipients. The exact phenotype of T cells responsible for inhibiting the immune response and their mode of action remain unclear. Evidence exists that CTLA-4 negatively regulates T cell activation. We addressed whether CTLA-4 is involved in the transfer of UV-induced tolerance. Injection of lymph node cells from mice that were sensitized with dinitrofluo-robenzene (DNFB) through UV-irradiated skin inhibited induction of contact hypersensitivity against DNFB in the recipient animals. When CTLA-4+ cells were depleted, transfer of suppression was lost. Likewise, significantly fewer lymphocytes enriched for CTLA-4+ cells were necessary to transfer suppression than unfractionated cells. Expression of CTLA-4 appears to be functionally relevant, since in vivo injection of a blocking anti-CTLA-4 Ab was able to break UV-induced tolerance and inhibited transfer of suppression. Upon stimulation with dendritic cells in the presence of the water-soluble DNFB analogue, DNBS, CTLA-4+ T cells from DNFB-tolerized mice secreted high levels of IL-10, TGF-β, and IFN-γ; low levels of IL-2; and no IL-4, resembling the cytokine pattern of T regulatory 1 cells. Ab blocking of CTLA-4 resulted in inhibition of IL-10 release. Accordingly, transfer of tolerance was not observed when recipients were treated with an anti-IL-10 Ab. Hence we propose that T cells, possibly of the T regulatory 1 type, transfer UV-mediated suppression through the release of IL-10. Activation of CTLA-4 appears to be important in this process.
Normal Thymic Architecture and Negative Selection Are Associated with Aire Expression, the Gene Defective in the Autoimmune-Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED)
Authors: Zuklys Saulius , Balciunaite Gina , Agarwal Anni , Fasler-Kan Elizaveta , Palmer Ed , Holländer Georg A.
Published: April 2014

T cell development is tightly controlled by thymic stromal cells. Alterations in stromal architecture affect T cell maturation and the development of self-tolerance. The monogenic autoimmune syndrome APECED (autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy) is characterized by the loss of self-tolerance to multiple organs. Although mutations in the autoimmune regulator (AIRE) gene are responsible for this disease, the function of AIRE is not known. Here we report on the spatial and temporal pattern of murine Aire expression during thymic ontogeny and T cell selection. Early during development, thymic Aire transcription is critically dependent on RelB and occurs in epithelial cells in response to lymphocyte-mediated signals. In adult tissue, Aire expression is confined to the medulla and the corticomedullary junction, where it is modulated by thymocytes undergoing negative selection. Aire may determine thymic stromal organization and with it the induction of self-tolerance.
Intestinal Microflora and Diversification of the Rabbit Antibody Repertoire
Authors: Lanning Dennis , Sethupathi Periannan , Rhee Ki-Jong , Zhai Shi-Kang , Knight Katherine L.
Published: April 2014

The rabbit establishes its primary Ab repertoire by somatically diversifying an initial repertoire that is limited by restricted VH gene segment usage during VDJ gene rearrangement. Somatic diversification occurs in gut-associated lymphoid tissue (GALT), and by about 1–2 mo of age nearly all Ig VDJ genes are somatically diversified. In other species that are known to establish their primary Ab repertoire by somatic diversification, such as chicken, sheep, and cattle, diversification appears to be developmentally regulated: it begins before birth and occurs independent of exogenous factors. Because somatic diversification in rabbit occurs well after birth in GALT, the diversification process may not be developmentally regulated, but may require interaction with exogenous factors derived from the gut. To test this hypothesis, we examined Ab repertoire diversification in rabbits in which the appendix was ligated shortly after birth to prevent microbial colonization and all other organized GALT was surgically removed. We found that by 12 wk of age nearly 90% of the Ig VDJ genes in PBL were undiversified, indicating that intestinal microflora are required for somatically diversifying the Ab repertoire. We also examined repertoire diversification in sterilely derived remote colony rabbits that were hand raised away from contact with conventional rabbits and thereby acquired a different gut microflora. In these remote colony rabbits, GALT was underdeveloped, and 70% of the Ig VDJ genes in PBL were undiversified. We conclude that specific, currently unidentified intestinal microflora are required for Ab repertoire diversification.
Differentiation of Monocytes to Macrophages Switches theMycobacterium tuberculosisEffect on HIV-1 Replication from Stimulation to Inhibition: Modulation of Interferon Response and CCAAT/Enhancer Binding Protein β Expression
Authors: Weiden Michael , Tanaka Naohiko , Qiao Yaming , Zhao Ben Yang , Honda Yoshihiro , Nakata Koh , Canova Antony , Levy David E. , Rom William N. , Pine Richard
Published: April 2014

HIV-1 replication is inhibited in uninflamed lung macrophages and is stimulated during tuberculosis. Attempts to recapitulate activation of HIV-1 replication in primary monocytes and macrophages ex vivo and in the untreated and PMA-treated THP-1 cell line model in vitro have produced opposite results depending on the state of differentiation of the cells. After infection with Mycobacterium tuberculosis, monocytes enhanced HIV-1 replication and produced a stimulatory 37-kDa CCAAT/enhancer binding protein β (C/EBPβ) transcription factor, whereas macrophages suppressed HIV-1 replication and produced an inhibitory 16-kDa C/EBPβ transcription factor. IFN-β induced inhibitory 16-kDa C/EBPβ in macrophages, but had no effect on C/EBPβ expression in monocytes. Macrophages, but not monocytes, were able to activate IFN-stimulated gene factor-3 (ISGF-3), a transcription factor composed of STAT-1, STAT-2, and IFN regulatory factor (IRF)-9, after infection with M. tuberculosis or stimulation with type I IFN. Macrophages expressed IRF-9 DNA-binding activity, but monocytes did not, and addition of the IRF-9 component reconstituted ISGF-3 in extracts of IFN-treated monocytes. Modulation of IFN responsiveness upon differentiation occurred at least in part through a post-transcriptionally regulated increase in IRF-9 expression. Both monocytes and macrophages maintained IFN responsiveness, activating STAT-1 homodimer formation and transcription of the STAT-1 gene after IFN stimulation. In addition, both monocytes and macrophages were able to activate NF-κB upon infection with M. tuberculosis. These results show that induction of ISGF-3, expression of the inhibitory 16-kDa C/EBPβ, and suppression of HIV-1 replication via a transcriptional mechanism are macrophage-specific responses to infection with M. tuberculosis.
Impaired CTL Recognition of Cells Latently Infected with Kaposi’s Sarcoma-Associated Herpes Virus
Authors: Brander Christian , Suscovich Todd , Lee Yun , Nguyen Phuong Thi , O’Connor Paula , Seebach Joerg , Jones Norman G. , van Gorder Mark , Walker Bruce D. , Scadden David T.
Published: April 2014

Kaposi’s sarcoma-associated herpes virus (KSHV) is a recently identified human γ2-herpesvirus associated with Kaposi’s sarcoma, primary effusion lymphoma, and Castleman’s disease. We reasoned that CTL responses may provide host defense against this virus, and consequently, KSHV may have evolved strategies to evade the CTL-mediated immune surveillance. In this study six B cell lines latently infected with KSHV were found to express reduced levels of HLA class I surface molecules compared with B cell lines transformed by the related γ-herpesvirus EBV. KSHV-infected cells also required higher concentrations of soluble peptides to induce efficient CTL-mediated lysis than control cell lines and were unable to process and/or present intracellularly expressed Ag. Incubation of the KSHV-infected cell lines with high concentrations of soluble HLA class I binding peptides did not restore the deficient HLA class I surface expression. To assess the underlying mechanisms of these phenomena, TAP-1 and TAP-2 gene expression was analyzed. While no attenuation in TAP-2 expression was observed, TAP-1 expression was significantly reduced in all KSHV cell lines compared with that in controls. These results indicate that KSHV can modulate HLA class I-restricted Ag presentation to CTL, which may allow latently infected cells to escape CTL recognition and persist in the infected host.
Monocyte Tissue Factor Induction by Activation of β2-Glycoprotein-I-Specific T Lymphocytes Is Associated with Thrombosis and Fetal Loss in Patients with Antiphospholipid Antibodies
Authors: Visvanathan Sudha , Geczy Carolyn L. , Harmer Jason A. , McNeil H. Patrick
Published: April 2014

Antiphospholipid (aPL) syndrome (APS) is characterized by thromboembolic events, thrombocytopenia, or recurrent miscarriage associated with aPL Abs with specificity for β2-glycoprotein-I (β2GPI). We recently reported that at least 44% of patients with the APS possess circulating type 1 (Th1) CD4+ T cells that proliferate and secrete IFN-γ when stimulated with β2GPI in vitro. In this study, we show that stimulation of PBMCs from 20 APS patients with β2GPI induced substantial monocyte tissue factor (TF) (80 ± 11 TF stimulation index (TF-SI)), whereas no induction was observed using PBMCs from 13 patients with aPL Abs without APS (6 ± 1 TF-SI) or 7 normal and 7 autoimmune controls (5 ± 1 and 3 ± 1 TF-SI, respectively) (p < 0.0001). TF induction on monocytes by β2GPI was dose dependent and required CD4+ T lymphocytes and class II MHC molecules. Because monocyte TF induction by β2GPI was observed in all patients with APS, but not in any patient with aPL Abs without APS, this response is a potentially useful predictor for APS in patients with aPL Abs, as well as providing mechanistic insight into thrombosis and fetal loss in these patients.
Proteolytic Cleavage of a Self-Antigen Following Xenobiotic-Induced Cell Death Produces a Fragment with Novel Immunogenic Properties
Authors: Pollard K. Michael , Pearson Deborah L. , Blüthner Martin , Tan Eng M.
Published: April 2014

The heavy metal mercury elicits a genetically restricted autoantibody response in mice that targets the nucleolar autoantigen fibrillarin. HgCl2-induced cell death of macrophages resulted in the proteolytic cleavage of fibrillarin. A prominent feature of mercury-induced cell death was the generation of a 19-kDa fragment of fibrillarin that was not found following apoptotic or nonapoptotic cell death induced by stimuli other than mercury. Proteolysis of fibrillarin lacking cysteines, and therefore unable to bind mercury, also produced the 19-kDa fragment, suggesting that a mercury-fibrillarin interaction was not necessary for the unique cleavage pattern of this self-Ag. In contrast to immunization with full-length fibrillarin, the 19-kDa fragment produced anti-fibrillarin Abs with some of the properties of the HgCl2-induced anti-fibrillarin response. We propose that cell death following exposure to an autoimmunity-inducing xenobiotic can lead to the generation of novel protein fragments that may serve as sources of antigenic determinants for self-reactive T lymphocytes.
CD4 and CD8 T Cells Have Redundant But Not Identical Roles in Virus-Induced Demyelination
Authors: Wu Gregory F. , Dandekar Ajai A. , Pewe Lecia , Perlman Stanley
Published: April 2014

A chronic demyelinating disease results from murine infection with the neurotropic strain JHM of mouse hepatitis virus (MHV-JHM). Demyelination is largely immune mediated. In this study, the individual roles of CD4 and CD8 T cells in MHV-induced demyelination were investigated using recombination-activating gene 1−/− (RAG1−/−) mice infected with an attenuated strain of MHV-JHM. These animals develop demyelination only after adoptive transfer of splenocytes from mice previously immunized to MHV. In this study, we show that, following adoptive transfer, virus-specific CD4 and CD8 T cells rapidly infiltrate the CNS of MHV-JHM-infected RAG1−/− mice. Adoptive transfer of CD4 T cell-enriched donors resulted in more severe clinical disease accompanied by less demyelination than was detected in the recipients of undepleted cells. Macrophage infiltration into the gray matter of CD4 T cell-enriched recipients was greater than that observed in mice receiving undepleted splenocytes. In contrast, CD8 T cell-enriched recipients developed delayed disease with extensive demyelination of the spinal cord. MHV-JHM-infected RAG1−/− mice receiving donors depleted of both CD4 and CD8 T cells did not develop demyelination. These results demonstrate that the development of demyelination following MHV infection may be initiated by either CD4 or CD8 T cells. Furthermore, they show that CD4 T cells contribute more prominently than CD8 T cells to the severity of clinical disease, and that this correlates with increased macrophage infiltration into the gray matter.
Cutting Edge: A Novel Chemokine Ligand for CCR10 And CCR3 Expressed by Epithelial Cells in Mucosal Tissues
Authors: Pan Junliang , Kunkel Eric J. , Gosslar Uwe , Lazarus Nicole , Langdon Patricia , Broadwell Kim , Vierra Mark A. , Genovese Mark C. , Butcher Eugene C. , Soler Dulce
Published: April 2014

Mucosae-associated epithelial chemokine (MEC) is a novel chemokine whose mRNA is most abundant in salivary gland, with strong expression in other mucosal sites, including colon, trachea, and mammary gland. MEC is constitutively expressed by epithelial cells; MEC mRNA is detected in cultured bronchial and mammary gland epithelial cell lines and in epithelia isolated from salivary gland and colon using laser capture microdissection, but not in the endothelial, hemolymphoid, or fibroblastic cell lines tested. Although MEC is poorly expressed in skin, its closest homologue is the keratinocyte-expressed cutaneous T cell-attracting chemokine (CTACK; CCL27), and MEC supports chemotaxis of transfected lymphoid cells expressing CCR10, a known CTACK receptor. In contrast to CTACK, however, MEC also supports migration through CCR3. Consistent with this, MEC attracts eosinophils in addition to memory lymphocyte subsets. These results suggest an important role for MEC in the physiology of extracutaneous epithelial tissues, including diverse mucosal organs.
Th1 and Th2 CD4+ T Cells Provide Help for B Cell Clonal Expansion and Antibody Synthesis in a Similar Manner In Vivo
Authors: Smith Karen M. , Pottage Lindsay , Thomas Elaine R. , Leishman Andrew J. , Doig Tamsin N. , Xu Damo , Liew Foo Y. , Garside Paul
Published: April 2014

The relative ability of Th1 and Th2 T cells to help B cells remains controversial as do the mechanisms by which both T cell subsets provide help in vivo. Whether this help affects the clonal expansion and/or differentiation of B cells has been difficult to assess due to the low frequency of Ag-specific T and B lymphocytes. We have employed a novel technique to directly monitor the clonal expansion of Ag-specific T and B lymphocytes in vivo. OVA-specific TCR transgenic T lymphocytes were polarized toward a Th1 or Th2 phenotype in vitro. These cells were then transferred into syngeneic recipients, along with B cell receptor transgenic hen egg lysozyme-specific B lymphocytes. Our results indicate that Th1 and Th2 cells support B cell responses to a similar extent in vivo and that they achieve this in the same manner by migrating into B cell follicles to promote CD154-dependent B cell clonal expansion and Ab production.
Porcine CTLA4-Ig Lacks a MYPPPY Motif, Binds Inefficiently to Human B7 and Specifically Suppresses Human CD4+ T Cell Responses Costimulated by Pig But Not Human B7
Authors: Vaughan Andrew N. , Malde Prupti , Rogers Nicola J. , Jackson Ian M. , Lechler Robert I. , Dorling Anthony
Published: April 2014

The CTLA4 receptor (CD152) on activated T lymphocytes binds B7 molecules (CD80 and CD86) on APC and delivers a signal that inhibits T cell proliferation. Several regions involved in binding to B7 are known, but the relative importance of these is not clear. We have cloned porcine CTLA4 (pCTLA4). Although highly homologous to human CTLA4 (hCTLA4), the predicted protein sequence contains a leucine for methionine substitution at position 97 in the MYPPPY sequence. A fusion protein constructed from the extracellular regions of pCTLA4 and the constant regions of human IgG1 (pCTLA4-Ig) bound porcine CD86 with equivalent affinity to that of hCTLA4-Ig. However, pCTLA4-Ig bound poorly to human CD80 and CD86 expressed on transfectants and EBV-transformed human B cells. In functional assays with MHC class II-expressing porcine endothelial cells and human B cells, pCTLA4-Ig blocked human CD4+ T cell responses to pig but not human cells, whereas control hCTLA4-Ig inhibited responses to both. Comparison between mouse, human, and porcine CTLA4-Ig suggests that the selective binding of pCTLA4-Ig to porcine CD86 molecules is due to the L for M substitution at position 97. Our results indicate that pCTLA4-Ig may be a useful reagent to define the precise nature of the interaction between B7 and CTLA4. By failing to inhibit the delivery of costimulatory signals provided by human B7, it may also prove to be a relatively specific inhibitor of the direct human T cell response to immunogenic pig tissue.
Independent Selection by I-Ak Molecules of Two Epitopes Found in Tandem in an Extended Polypeptide Antigen
Authors: Gugasyan Raffi , Velazquez Carlos , Vidavsky Ilan , Deck Brian M. , van der Drift Koen , Gross Michael L. , Unanue Emil R.
Published: April 2014

The protein hen egg white lysozyme (HEL) contains two segments, in tandem, from which two families of peptides are selected by the class II molecule I-Ak, during processing. These encompass peptides primarily from residues 31–47 and 48–63. Mutant HEL proteins were created with changes in residues 52 and 55, resulting in a lack of binding and selection of the 48–63 peptides to I-Ak molecules. Such mutant HEL proteins donated the same amount of 31–47 peptide as did the unmodified protein. Other mutant HEL molecules containing proline residues at residue 46, 47, or 48 resulted in extensions of the selected 31–47 or 48–62 families to their overlapping regions (in the carboxyl or amino termini, respectively). However, the amount of each family of peptide selected was not changed. We conclude that the presence or absence of the major peptide from HEL does not influence the selection of other epitopes, and that these two families are selected independently of each other.
β57-Asp Plays an Essential Role in the Unique SDS Stability of HLA-DQA1*0102/DQB1*0602 αβ Protein Dimer, the Class II MHC Allele Associated with Protection from Insulin-Dependent Diabetes Mellitus
Authors: Ettinger Ruth A. , Liu Andrew W. , Nepom Gerald T. , Kwok William W.
Published: April 2014

Studies of the stability of HLA-DQ have revealed a correlation between SDS stability of MHC class II αβ dimers and insulin-dependent diabetes mellitus (IDDM) susceptibility. The MHC class II αβ dimer encoded by HLA-DQA1*0102/DQB1*0602 (DQ0602), which is a dominant protective allele in IDDM, exhibits the greatest SDS stability among HLA-DQ molecules in EBV-transformed B-lymphoblastoid cells and PBLs. DQ0602 is also uniquely SDS stable in the HLA-DM-deficient cell line, BLS-1. We addressed the molecular mechanism of the stability of DQ0602 in BLS-1. A panel of mutants based on the polymorphic differences between HLA-DQA1*0102/DQB1*0602 and HLA-DQA1*0102/DQB1*0604 were generated and expressed in BLS-1. An Asp at β57 was found to be critical for SDS stability, whereas Tyr at β30, Gly at β70, and Ala at β86 played secondary roles. Furthermore, the level of class II-associated invariant chain peptide bound to HLA-DQ did not correlate with SDS stability, suggesting that class II-associated invariant chain peptide does not play a direct role in the unique SDS stability of DQ0602. These results support a role for DQB1 codon 57 in HLA-DQ αβ dimer stability and IDDM susceptibility.
Clustering of Urokinase Receptors (uPAR; CD87) Induces Proinflammatory Signaling in Human Polymorphonuclear Neutrophils
Authors: Sitrin Robert G. , Pan Pauline M. , Harper Hollie A. , Todd Robert F. , Harsh Donna M. , Blackwood R. Alexander
Published: April 2014

Leukocytes use urokinase receptors (uPAR; CD87) in adhesion, migration, and proteolysis of matrix proteins. Typically, uPAR clusters at cell-substratum interfaces, at focal adhesions, and at the leading edges of migrating cells. This study was undertaken to determine whether uPAR clustering mediates activation signaling in human polymorphonuclear neutrophils. Cells were labeled with fluo-3/AM to quantitate intracellular Ca2+ ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by Ab cross-linking. Aggregating uPAR induced a highly reproducible increase in [Ca2+]i (baseline to peak) of 295 ± 37 nM (p = 0.0002). Acutely treating cells with high m.w. urokinase (HMW-uPA; 4000 IU/ml) produced a response of similar magnitude but far shorter duration. Selectively aggregating uPA-occupied uPAR produced smaller increases in [Ca2+]i, but saturating uPAR with HMW-uPA increased the response to approximate that of uPAR cross-linking. Cross-linking uPAR induced rapid and significant increases in membrane expression of CD11b and increased degranulation (release of β-glucuronidase and lactoferrin) to a significantly greater degree than cross-linking control Abs. The magnitude of degranulation correlated closely with the difference between baseline and peak [Ca2+]i, but was not dependent on the state of uPA occupancy. By contrast, selectively cross-linking uPA-occupied uPAR was capable of directly inducing superoxide release as well as enhancing FMLP-stimulated superoxide release. These results could not be duplicated by preferentially cross-linking unoccupied uPAR. We conclude that uPAR aggregation initiates activation signaling in polymorphonuclear neutrophils through at least two distinct uPA-dependent and uPA-independent pathways, increasing their proinflammatory potency (degranulation and oxidant release) and altering expression of CD11b/CD18 to favor a firmly adherent phenotype.
Role of NF-κB in Cytokine Production Induced from Human Airway Epithelial Cells by Rhinovirus Infection
Authors: Kim Jean , Sanders Scherer P. , Siekierski Edward S. , Casolaro Vincenzo , Proud David
Published: April 2014

Infection of human epithelial cells with human rhinovirus (HRV)-16 induces rapid production of several proinflammatory cytokines, including IL-8, IL-6, and GM-CSF. We evaluated the role of NF-κB in HRV-16-induced IL-8 and IL-6 production by EMSA using oligonucleotides corresponding to the binding sites for NF-κB in the IL-6 and IL-8 gene promoters. Consistent with the rapid induction of mRNA for IL-8 and IL-6, maximal NF-κB binding to both oligonucleotides was detected at 30 min after infection. NF-κB complexes contained p65 and p50, but not c-Rel. The IL-8 oligonucleotide bound recombinant p50 with only about one-tenth the efficiency of the IL-6 oligonucleotide, even though epithelial cells produced more IL-8 protein than IL-6. Neither the potent glucocorticoid, budesonide (10−7 M), nor a NO donor inhibited NF-κB binding to either cytokine promoter or induction of mRNA for either IL-8 or IL-6. Sulfasalazine and calpain inhibitor I, inhibitors of NF-κB activation, blocked HRV-16-induced formation of NF-κB complexes with oligonucleotides from both cytokines, but did not inhibit mRNA induction for either cytokine. By contrast, sulfasalazine clearly inhibited HRV-16 induction of mRNA for GM-CSF in the same cells. Thus, HRV-16 induces epithelial expression of IL-8 and IL-6 by an NF-κB-independent pathway, whereas induction of GM-CSF is at least partially dependent upon NF-κB activation.
Mice with Spontaneous Pancreatic Cancer Naturally Develop MUC-1-Specific CTLs That Eradicate Tumors When Adoptively Transferred
Authors: Mukherjee Pinku , Ginardi Amelia R. , Madsen Cathy S. , Sterner Christopher J. , Adriance Melissa C. , Tevethia Mary J. , Gendler Sandra J.
Published: April 2014

Pancreatic cancer is a highly aggressive, treatment refractory cancer and is the fourth leading cause of death in the United States. In humans, 90% of pancreatic adenocarcinomas overexpress altered forms of a tumor-specific Ag, mucin 1 (MUC1; an epithelial mucin glycoprotein), which is a potential target for immunotherapy. We have established a clinically relevant animal model for pancreatic cancer by developing a double transgenic mouse model (called MET) that expresses human MUC1 as self molecule and develops spontaneous tumors of the pancreas. These mice exhibit acinar cell dysplasia at birth, which progresses to microadenomas and acinar cell carcinomas. The tumors express large amounts of underglycosylated MUC1 similar to humans. Tumor-bearing MET mice develop low affinity MUC1-specific CTLs that have no effect on the spontaneously occurring pancreatic tumors in vivo. However, adoptive transfer of these CTLs was able to completely eradicate MUC1-expressing injectable tumors in MUC1 transgenic mice, and these mice developed long-term immunity. These CTLs were MHC class I restricted and recognized peptide epitopes in the immunodominant tandem repeat region of MUC1. The MET mice appropriately mimic the human condition and are an excellent model with which to elucidate the native immune responses that develop during tumor progression and to develop effective antitumor vaccine strategies.
Coronary Arteries from Human Cardiac Allografts with Chronic Rejection Contain Oligoclonal T Cells: Persistence of Identical Clonally Expanded TCR Transcripts from the Early Post-Transplantation Period (Endomyocardial Biopsies) to Chronic Rejection (Coronary Arteries)
Authors: Slachta C. A. , Jeevanandam V. , Goldman B. , Lin W. L. , Platsoucas C. D.
Published: April 2014

Chronic cardiac allograft rejection presents pathologically as graft arteriosclerosis (GA) characterized by recipient T cell and monocyte infiltration. To determine whether oligoclonal T cells are present in coronary arteries of cardiac allografts from patients with GA, we conducted sequencing analysis of β-chain TCR transcripts from these explanted coronary arteries using the nonpalindromic adaptor-PCR. Substantial proportions of identical β-chain TCR transcripts in three of five patients were observed, clearly demonstrating the presence of oligoclonal T cells. TCR transcripts from the arteries of two other patients were relative heterogeneous. High proportions of identical CDR3 β-chain TCR motifs were found in each patient. GENEBANK/EMBL/SWISS PROT database comparison of all sequences revealed that these β-chain TCR transcripts were novel. Using Vβ-specific PCR (independent amplification), we found in patient GA03 that the TCR transcript that was clonally expanded in the left anterior descending artery after nonpalindromic adaptor-PCR was also clonally expanded in the right coronary artery of the same allograft. These results demonstrate that this TCR transcript was clonally expanded at different anatomic sides of the cardiac allograft in a systemic manner. In two patients identical β-chain TCR transcripts that were found to be clonally expanded in the coronary arteries of their explanted cardiac allografts were also found to be clonally explanted in endomyocardial biopsies collected 17 and 21 mo earlier from each patient. The presence of oligoclonal populations of T cells in the rejected graft suggest that these T cells have undergone specific Ag-driven proliferation and clonal expansion early on within the graft and persist throughout the post-transplantation period.
IL-10, But Not IL-4, Suppresses Infection-Stimulated Bone Resorption In Vivo
Authors: Sasaki Hajime , Hou Linda , Belani Anita , Wang Cun-Yu , Uchiyama Toru , Müller Ralph , Stashenko Philip
Published: April 2014

Periapical bone resorption occurs following infection of the dental pulp and is mediated mainly by IL-1α in the murine model. The production and activity of IL-1α is modulated by a network of regulatory cytokines, including those produced by Th1 (pro-inflammatory) and Th2 (anti-inflammatory) subset T cells. This study was designed to assess the functional role of the Th2-type cytokines IL-4 and IL-10 in infection-stimulated bone resorption in vivo. The dental pulps of the first molars were exposed and infected with a mixture of four common endodontic pathogens, and bone destruction was determined by micro-computed tomography at sacrifice on day 21. The results demonstrate that IL-10−/− mice had significantly greater infection-stimulated bone resorption in vivo compared with wild-type mice (p < 0.001), whereas IL-4−/− exhibited no increased resorption. IL-10−/− had markedly elevated IL-1α production within periapical inflammatory tissues (>10-fold) compared with wild type (p < 0.01), whereas IL-4−/− exhibited decreased IL-1α production (p < 0.05). IL-10 also suppressed IL-1α production by macrophages in a dose-dependent fashion in vitro, whereas IL-4 had weak and variable effects. We conclude that IL-10, but not IL-4, is an important endogenous suppressor of infection-stimulated bone resorption in vivo, likely acting via inhibition of IL-1α expression.
CD1d on Myeloid Dendritic Cells Stimulates Cytokine Secretion from and Cytolytic Activity of Vα24JαQ T Cells: A Feedback Mechanism for Immune Regulation
Authors: Yang Otto O. , Racke Frederick K. , Nguyen Phuong Thi , Gausling Rudolf , Severino Michael E. , Horton Heidi F. , Byrne Michael C. , Strominger Jack L. , Wilson S. Brian
Published: April 2014

The precise immunologic functions of CD1d-restricted, CD161+ AV24AJ18 (Vα24JαQ) T cells are not well defined, although production of IL-4 has been suggested as important for priming Th2 responses. However, activation of human Vα24JαQ T cell clones by anti-CD3 resulted in the secretion of multiple cytokines notably important for the recruitment and differentiation of myeloid dendritic cells. Specific activation of Vα24JαQ T cells was CD1d restricted. Expression of CD1d was found on monocyte-derived dendritic cells in vitro, and immunohistochemical staining directly revealed CD1d preferentially expressed on dendritic cells in the paracortical T cell zones of lymph nodes. Moreover, myeloid dendritic cells both activated Vα24JαQ T cells and were susceptible to lysis by these same regulatory T cells. Because myeloid dendritic cells are a major source of IL-12 and control Th1 cell differentiation, their elimination by lysis is a mechanism for limiting the generation of Th1 cells and thus regulating Th1/Th2 responses.