bluuebunny/crossref_metadata_2025 huggingface dataset:
Clonotypic Structure of the Human CD4+ Memory T Cell Response to Cytomegalovirus
Authors: Bitmansour Arlene D. , Waldrop Shar L. , Pitcher Christine J. , Khatamzas Elham , Kern Florian , Maino Vernon C. , Picker Louis J.
Published: April 2014

High steady-state frequencies of CMV-specific CD4+ memory T cells are maintained in CMV-exposed subjects, and these cells are thought to play a key role in the immunologic control of this permanent infection. However, the essential components of this response are poorly defined. Here, we report the use of a step-wise application of flow cytometric and molecular techniques to determine the number and size of the TCR Vβ-defined clonotypes within freshly obtained CMV-specific CD4+ memory T cell populations of four healthy, CMV-exposed human subjects. This analysis revealed a stable clonotypic hierarchy in which 1–3 dominant clonotypes are maintained in concert with more numerous subdominant and minor clonotypes. These dominant clonotypes accounted for 10–50% of the overall CMV response, and comprised from 0.3 to 4.0% of peripheral blood CD4+ T cells. Two subjects displayed immunodominant responses to single epitopes within the CMV matrix phosphoprotein pp65; these single epitope responses were mediated by a single dominant clonotype in one subject, and by multiple subdominant and minor clonotypes in the other. Thus, the CMV-specific CD4+ T cell memory repertoire in normal subjects is characterized by striking clonotypic dominance and the potential for epitope focusing, suggesting that primary responsibility for immunosurveillance against CMV reactivation rests with a handful of clones recognizing a limited array of CMV determinants. These data have important implications for the understanding of mechanisms by which a genetically stable chronic viral pathogen such as CMV is controlled, and offer possible insight into the failure of such control for a genetically flexible pathogen like HIV-1.
CD1d-Restricted NKT Cells: An Interstrain Comparison
Authors: Hammond Kirsten J. L. , Pellicci Daniel G. , Poulton Lynn D. , Naidenko Olga V. , Scalzo Anthony A. , Baxter Alan G. , Godfrey Dale I.
Published: April 2014

CD1d-restricted Vα14-Jα281 invariant αβTCR+ (NKT) cells are well defined in the C57BL/6 mouse strain, but they remain poorly characterized in non-NK1.1-expressing strains. Surrogate markers for NKT cells such as αβTCR+CD4−CD8− and DX5+CD3+ have been used in many studies, although their effectiveness in defining this lineage remains to be verified. Here, we compare NKT cells among C57BL/6, NK1.1-congenic BALB/c, and NK1.1-congenic nonobese diabetic mice. NKT cells were identified and compared using a range of approaches: NK1.1 expression, surrogate phenotypes used in previous studies, labeling with CD1d/α-galactosylceramide tetramers, and cytokine production. Our results demonstrate that NKT cells and their CD4/CD8-defined subsets are present in all three strains, and confirm that nonobese diabetic mice have a numerical and functional deficiency in these cells. We also highlight the hazards of using surrogate phenotypes, none of which accurately identify NKT cells, and one in particular (DX5+CD3+) actually excludes these cells. Finally, our results support the concept that NK1.1 expression may not be an ideal marker for CD1d-restricted NKT cells, many of which are NK1.1-negative, especially within the CD4+ subset and particularly in NK1.1-congenic BALB/c mice.
Rejection of Mouse Cardiac Allografts by Costimulation in trans
Authors: Mandelbrot Didier A. , Kishimoto Koji , Auchincloss Hugh , Sharpe Arlene H. , Sayegh Mohamed H.
Published: April 2014

The activation of T cells by B7 costimulation in trans has been demonstrated in vitro, but the in vivo relevance is unknown. To study costimulation in trans of CD4+ T cells in vivo, we performed cardiac transplants from B7-1/B7-2-deficient mice to recipients that do not express MHC class II molecules on peripheral APCs, but do have functional CD4+ T cells (II−/4+ mice). This model restricts the B7-dependent activation of CD4+ T cells to costimulation in trans and excludes any contribution from indirect Ag presentation. We find that II−/4+ recipients reject B7-deficient grafts as rapidly as wild-type grafts, suggesting that costimulation in trans can mediate rejection as potently as costimulation in cis. Treatment of II−/4+ recipients of B7-deficient grafts with depleting Abs to CD4 or CD8 demonstrates that indirect Ag presentation to CD8+ cells does not significantly contribute to rejection. This is the first demonstration that costimulation in trans can mediate an immune response in vivo and has important therapeutic implications.
IL-15 Is Expressed by Dendritic Cells in Response to Type I IFN, Double-Stranded RNA, or Lipopolysaccharide and Promotes Dendritic Cell Activation
Authors: Mattei Fabrizio , Schiavoni Giovanna , Belardelli Filippo , Tough David F.
Published: April 2014

Cytokines that are induced by infection may contribute to the initiation of immune responses through their ability to stimulate dendritic cells (DCs). In this paper, we have addressed the role of IL-15 in DC activation, investigating its expression by DCs in response to three different signals of infection and examining its ability to stimulate DCs. We report that the expression of both IL-15 and the IL-15 receptor α-chain are increased in splenic DCs from mice inoculated with dsRNA (poly(I:C)), LPS, or IFN-αβ, and in purified murine splenic DCs treated with IFN-αβ in vitro. Furthermore, IL-15 itself was able to activate DCs, as in vivo or in vitro exposure of splenic DCs to IL-15 resulted in an up-regulation of costimulatory molecules, markedly increased production of IFN-γ by DC and an enhanced ability of DCs to stimulate Ag-specific CD8+ T cell proliferation. The magnitude of all of the IL-15-induced changes in DCs was reduced in mice deficient for the IFN-αβ receptor, suggesting a role for IFN-αβ in the stimulation of DCs by IL-15. These results identify IL-15 as a stimulatory cytokine for DCs with the potential for autocrine activity and link its effects to expression of IFN-αβ.
Mature Dendritic Cells Prime Functionally Superior Melan-A-Specific CD8+ Lymphocytes as Compared with Nonprofessional APC
Authors: Salio Mariolina , Shepherd Dawn , Dunbar P. Rod , Palmowski Michael , Murphy Kristine , Wu Lijun , Cerundolo Vincenzo
Published: April 2014

Priming of melan-A26/27–35-specific CTL occurs only in a fraction of late stage melanoma patients, whereas during the early stages of the disease and in healthy volunteers, melan-A CTL have functional and phenotypic markers consistent with a naive phenotype. To study the requirements for expansion of naive melan-A CTL from healthy donors, we set up an in vitro priming protocol and, using tetramer assays, we demonstrate that the activity and phenotype of the expanded melan-A CTL are profoundly influenced by the type of APC used. Priming by nonprofessional APC leads to expansion of melan-A CTL with reduced cytolytic activity and low level of IFN-γ secretion. In contrast, mature dendritic cells (DC) expand cytolytic and IFN-γ-producing melan-A CTL. Priming by mature DC is also efficient at low peptide concentration and requires only one round of stimulation. Finally, we observed that a significant fraction of CD45RO+ melan-A CTL primed by mature DC expresses high levels of the homing receptor CD62L, whereas CTL primed by nonprofessional APC express CD62L in lower percentages and at lower levels. These results suggest that suboptimal priming by nonprofessional APC could account for the presence in vivo of dysfunctional cells and strongly support the immunotherapeutic use of mature DC for expansion of effector and memory Ag-specific CTL.
Differences in the Expression of Human Class I MHC Alleles and Their Associated Peptides in the Presence of Proteasome Inhibitors
Authors: Luckey Chance John , Marto Jarrod A. , Partridge Megan , Hall Ed , White Forest M. , Lippolis John D. , Shabanowitz Jeffrey , Hunt Donald F. , Engelhard Victor H.
Published: April 2014

We have studied the contributions of proteasome inhibitor-sensitive and -insensitive proteases to the generation of class I MHC-associated peptides. The cell surface expression of 13 different human class I MHC alleles was inhibited by as much as 90% or as little as 40% when cells were incubated with saturating concentrations of three different proteasome inhibitors. Inhibitor-resistant class I MHC expression was not due to TAP-independent expression or preexisting internal stores of peptides. Furthermore, it did not correlate with the amount or specificity of residual proteasome activity as determined in in vitro proteolysis assays and was not augmented by simultaneous incubation with multiple inhibitors. Mass spectrometry was used to directly characterize the peptides expressed in the presence and absence of proteasome inhibitors. The number of peptide species detected correlated with the levels of class I detected by flow cytometry. Thus, for many alleles, a significant proportion of associated peptide species continue to be generated in the presence of saturating levels of proteasome inhibitors. Comparison of the peptide-binding motifs of inhibitor-sensitive and -resistant class I alleles further suggested that inhibitor-resistant proteolytic activities display a wide diversity of cleavage specificities, including a trypsin-like activity. Sequence analysis demonstrated that inhibitor-resistant peptides contain diverse carboxyl termini and are derived from protein substrates dispersed throughout the cell. The possible contributions of inhibitor-resistant proteasome activities and nonproteasomal proteases residing in the cytosol to the peptide profiles associated with many class I MHC alleles are discussed.
IL-4 Influences Apoptosis of Mycobacterium-Reactive Lymphocytes in the Presence of TNF-α
Authors: Seah Geok Teng , Rook Graham A. W.
Published: April 2014

T cell apoptosis is associated with defective cell-mediated effector functions in several infectious diseases. In tuberculosis, there is evidence that T cell apoptosis may be cytokine mediated, but the mechanisms are not clearly understood. Type 2 cytokines have recently been associated with disease extent in human tuberculosis, but they have not previously been linked to apoptosis in mycobacterium-reactive T cells. This study presents evidence that PBLs from healthy donors respond to sonicated Mycobacterium tuberculosis Ags with increased IL-4 gene activation, CD30 expression, and apoptosis. The changes were significantly greater than those observed when cells were stimulated with Ags from nonpathogenic Mycobacterium vaccae. A hypothesis linking these observations was tested. CD30 expression and TNF-α-mediated lymphocyte apoptosis were both down-regulated by inhibiting IL-4 in this model. TNFR-associated factor 2 (TRAF2) expression was down-regulated in CD30+ cells, and addition of anti-TNF-α Ab significantly reduced apoptosis in the CD30+ but not the CD30− population. These observations support the hypothesis that increased IL-4 expression in M. tuberculosis-activated lymphocytes promotes CD30 expression, which sensitizes the lymphocytes to TNF-α-mediated apoptosis via TRAF2 depletion. This may be one mechanism by which IL-4 is associated with immunopathological consequences in human tuberculosis.
Human IL-18 Receptor and ST2L Are Stable and Selective Markers for the Respective Type 1 and Type 2 Circulating Lymphocytes
Authors: Chan Woon Ling , Pejnovic Nada , Lee Christine A. , Al-Ali Nadia A.
Published: April 2014

CD4+ (Th) and CD8+ (Tc) T and NK lymphocytes can be divided into type 1 and 2 subsets according to their cytokine secretion profile. Studies on the role of lymphocyte subsets in human diseases have been hampered by the lack of stable surface markers to define them. Recently, we reported that ST2L and IL-18R are stably expressed on murine Th2 and Th1 cells, respectively. In this study, we generated Abs to human homologues of ST2L and IL-18R and tested them against Th1/Th2, Tc1/Tc2, and NK1/NK2 lines and PBMCs from healthy individuals. We show for the first time that ST2L and IL-18R are stable selective cell surface markers for human Th2/Tc2/NK2 and Th1/Tc1/NK1 lymphocytes, respectively. We then investigated PBMCs from HIV-infected patients and HIV-negative individuals, to test whether Abs to these two surface markers could be used directly to monitor lymphocyte subset distribution in human diseases. We found a clear Th1 to Th2 shift in the HIV-infected individuals, thus settling a long-standing controversy and include, for the first time, Tc and NK cells as well. Therefore, these cell surface molecules could serve as important determinants of the immune status of human diseases in general, and thereby could be useful for therapeutic monitoring and intervention.
CD4+CD25high Regulatory Cells in Human Peripheral Blood
Authors: Baecher-Allan Clare , Brown Julia A. , Freeman Gordon J. , Hafler David A.
Published: April 2014

Thymectomy in mice on neonatal day 3 leads to the development of multiorgan autoimmune disease due to loss of a CD+CD25+ T cell regulatory population in their peripheral lymphoid tissues. Here, we report the identification of a CD4+ population of regulatory T cells in the circulation of humans expressing high levels of CD25 that exhibit in vitro characteristics identical with those of the CD4+CD25+ regulatory cells isolated in mice. With TCR cross-linking, CD4+CD25high cells did not proliferate but instead totally inhibited proliferation and cytokine secretion by activated CD4+CD25− responder T cells in a contact-dependent manner. The CD4+CD25high regulatory T cells expressed high levels of CD45RO but not CD45RA, akin to the expression of CD45RBlow on murine CD4+CD25+ regulatory cells. Increasing the strength of signal by providing either costimulation with CD28 cross-linking or the addition of IL-2 to a maximal anti-CD3 stimulus resulted in a modest induction of proliferation and the loss of observable suppression in cocultures of CD4+CD25high regulatory cells and CD4+CD25− responder cells. Whereas higher ratios of CD4+CD25high T cells are required to suppress proliferation if the PD-L1 receptor is blocked, regulatory cell function is shown to persist in the absence of the PD-1/PD-L1 or CTLA-4/B7 pathway. Thus, regulatory CD4 T cells expressing high levels of the IL-2 receptor are present in humans, providing the opportunity to determine whether alterations of these populations of T cells are involved in the induction of human autoimmune disorders.
Processing of Exogenous Antigens for Presentation by Class I MHC Molecules Involves Post-Golgi Peptide Exchange Influenced by Peptide-MHC Complex Stability and Acidic pH
Authors: Chefalo Peter J. , Harding Clifford V.
Published: April 2014

Vacuolar alternate class I MHC (MHC-I) Ag processing allows presentation of exogenous Ag by MHC-I molecules with binding of antigenic peptides to post-Golgi MHC-I molecules. We investigated the role of previously bound peptides and their dissociation in generating peptide-receptive MHC-I molecules. TAP1-knockout macrophages were incubated overnight with an initial exogenous peptide, producing a large cohort of peptide-Kb complexes that could influence subsequent peptide dissociation/exchange. Initial incubation with FAPGNYPAL, KVVRFDKL, or RGYVYQGL enhanced rather than reduced subsequent binding and presentation of a readout peptide (SIINFEKL or FAPGNYPAL) to T cells. Thus, Kb molecules may be stabilized by an initial (stabilizing) peptide, enhancing their ability to bind readout peptide and implicating peptide dissociation/exchange. In contrast, incubation with SIINFEKL as stabilizing peptide reduced presentation of readout peptide. SIINFEKL-Kb complexes were more stable than other peptide-Kb complexes, which may limit their contribution to peptide exchange. Stabilizing peptides (FAPGNYPAL, KVVRFDKL, or RGYVYQGL) enhanced alternate MHC-I processing of HB101.Crl-OVA (Escherichia coli expressing an OVA fusion protein), indicating that alternate MHC-I Ag processing involves peptide dissociation/exchange. Stabilizing peptide enhanced processing of HB101.Crl-OVA more than presentation of exogenous OVA peptide (SIINFEKL), suggesting that peptide dissociation/exchange may be enhanced in the acidic phagosomal processing environment. Furthermore, exposure of cells to acidic pH increased subsequent binding and presentation of readout peptide. Thus, peptide dissociation/exchange contributes to alternate MHC-I Ag processing and may be influenced by both stability of peptide-MHC-I complexes and pH.
Heterogeneity of Channel Catfish CTL with Respect to Target Recognition and Cytotoxic Mechanisms Employed
Authors: Zhou He , Stuge Tor B. , Miller Norman W. , Bengten Eva , Naftel John P. , Bernanke Jayne M. , Chinchar V. Gregory , Clem L. William , Wilson Melanie
Published: April 2014

Two types of catfish alloantigen-dependent cytotoxic T cells were cloned from PBL from a fish immunized in vivo and stimulated in vitro with the allogeneic B cell line 3B11. Because these are the first clonal cytotoxic T cell lines derived from an ectothermic vertebrate, studies were undertaken to characterize their recognition and cytotoxic mechanisms. The first type of CTL (group I) shows strict alloantigen specificity, i.e., they specifically kill and proliferate only in response to 3B11 cells. The second type (group II) shows broad allogeneic specificity, i.e., they kill and proliferate in response to several different allogeneic cells in addition to 3B11. “Cold” target-inhibition studies suggest that group II CTL recognize their targets via a single receptor, because the killing of one allotarget can be inhibited by a different allotarget. Both types of catfish CTL form conjugates with and kill targets by apoptosis. Killing by Ag-specific cytotoxic T cells (group I) was completely inhibited by treatment with EGTA or concanamycin A, and this killing is sensitive to PMSF inhibition, suggesting that killing was mediated exclusively by the secretory perforin/granzyme mechanism. In contrast, killing by the broadly specific T cytotoxic cells (group II) was only partially inhibited by either EGTA or concanamycin A, suggesting that these cells use a cytotoxic mechanism in addition to that involving perforin/granzyme. Consistent with the presumed use of a secretory pathway, both groups of CTL possess putative lytic granules. These results suggest that catfish CTL show heterogeneity with respect to target recognition and cytotoxic mechanisms.
The Role of p53 in Regulating Antiviral T Cell Responses
Authors: Grayson Jason M. , Lanier J. Gibson , Altman John D. , Ahmed Rafi
Published: April 2014

It is now well established that viral infections can induce large expansions of Ag-specific CD8+ T cells. These cells divide very rapidly with an estimated doubling time of ∼6 h. When virus is cleared, the vast majority of these effector CD8 T cells undergo apoptosis. The remaining memory cells persist at constant levels and provide the basis for the accelerated recall response upon rechallenge. The molecular mechanisms that control the rapid proliferation and death of Ag-specific T cells are poorly understood. Because of its important role in controlling cell proliferation and death, we examined antiviral immune responses in p53−/− mice using lymphocytic choriomeningitis virus. We found that effector CD8 and CD4 responses were comparable but that memory levels were slightly higher in −/− mice compared with +/+ mice. The lack of a major difference in virus-specific T cell responses between +/+ and −/− mice suggests that p53 only plays a minor role in regulating the proliferation, apoptosis, and maintenance of Ag-specific T cells. Thus, it appears that the primary function of p53 is in controlling “illegitimate” proliferation and tumor development and not in regulating Ag-specific T cell responses.
Transgenic Expression of CD95 Ligand on Thyroid Follicular Cells Confers Immune Privilege upon Thyroid Allografts
Authors: Tourneur Léa , Malassagne Benoit , Batteux Frédéric , Fabre Monique , Mistou Sylvie , Lallemand Eliette , Lores Patrick , Chiocchia Gilles
Published: April 2014

Constitutive Fas ligand (FasL) expression by specialized cells in the body participates in the immune privilege status of tissues containing these cells. This property has been used to prevent rejection of allogeneic grafts. Nevertheless, the mechanism responsible for such protection has not been fully elucidated. Unfortunately, grafting of FasL transgenic (TG) tissues has been unsuccessful. We have generated TG mice expressing FasL (soluble + membrane bound) on thyroid follicular cells (TFC), and used them to show that ectopic FasL expression prevents thyroid allograft rejection. FasL expression on TFC led to markedly decreased anti-allogeneic, cytotoxic, and helper T lymphocyte activities. The alloantibody response in TG thyroid recipients was either completely inhibited or switched toward a T2-Ab response. Surprisingly, the beneficial effect of FasL on TG thyroid grafts was abolished by host CD4+ T cell depletion. Host CD8+ T cell depletion improved nontransgenic (NTG), but not TG graft survival. Altogether, our results suggest that FasL-induced tolerance is concomitant with a move away from a T1 type response, and a CD4 T cell-mediated regulation of the allocytotoxic T cell response. These results were dependent upon the level of FasL expression on TFC, in that low expression of FasL led to a less marked effect compared with the effect observed with high expression of FasL. These results provide some insight into the role of FasL in regulating destructive alloimmune responses in the case of whole organ grafting, and they have important implications for the development of FasL-based immunotherapy in organ transplantation.
A Common Pathway for Dendritic Cell and Early B Cell Development
Authors: Izon David , Rudd Kristina , DeMuth William , Pear Warren S. , Clendenin Cynthia , Lindsley R. Coleman , Allman David
Published: April 2014

B cells and dendritic cells (DCs) each develop from poorly described progenitor cells in the bone marrow (BM). Although a subset of DCs has been proposed to arise from lymphoid progenitors, a common developmental pathway for B cells and BM-derived DCs has not been clearly identified. To address this possibility, we performed a comprehensive analysis of DC differentiative potential among lymphoid and B lymphoid progenitor populations in adult mouse BM. We found that both the common lymphoid progenitors (CLPs), shown here and elsewhere to give rise exclusively to lymphocytes, and a down-stream early B-lineage precursor population devoid of T and NK cell precursor potential each give rise to DCs when exposed to the appropriate cytokines. This result contrasts with more mature B-lineage precursors, all of which failed to give rise to detectable numbers of DCs. Significantly, both CLP and early B-lineage-derived DCs acquired several surface markers associated with functional DCs, and CLP-derived DCs readily induced proliferation of allogeneic CD4+ T cells. Surprisingly, however, DC differentiation from both lymphoid-restricted progenitors was accompanied by up-regulation of CD11b expression, a cell surface molecule normally restricted to myeloid lineage cells including putative myeloid DCs. Together, these data demonstrate that loss of DC developmental potential is the final step in B-lineage commitment and thus reveals a previously unrecognized link between early B cell and DC ontogeny.
Activating Immunity in the Liver. I. Liver Dendritic Cells (but Not Hepatocytes) Are Potent Activators of IFN-γ Release by Liver NKT Cells
Authors: Trobonjaca Zlatko , Leithäuser Frank , Möller Peter , Schirmbeck Reinhold , Reimann Jörg
Published: April 2014

A prominent subset of the hepatic innate immune system is α-galactosylceramide (αGalCer)-reactive, (CD4+ and CD4−CD8−) CD1d-restricted NKT cells. We investigated in C57BL/6 (B6) mice which hepatic cell type stimulates hepatic NKT cell activation. Surface expression of CD1d but not CD40, CD80, or CD86 costimulator molecules was detected in hepatocytes. Pulsed in vitro or in vivo with αGalCer, hepatocytes triggered IL-4 release by liver NKT cells but required exogenous IL-12 to trigger IFN-γ release by NKT cells. Liver dendritic cells (DC) isolated from nontreated mice showed low surface expression of MHC, CD1d, and CD40, CD80, or CD86 costimulator molecules that were strikingly up-regulated after αGalCer injection. Although liver CD11c+ DC displayed lower CD1d surface expression than hepatocytes, they were potent stimulators of IFN-γ and IL-4 release by liver NKT when pulsed with αGalCer in vitro or in vivo. Liver DC are thus potent stimulators of proinflammatory cytokine release by NKT cells, are activated themselves in the process of NKT cell activation, and express an activated phenotype after the NKT cell population is eliminated following αGalCer stimulation.
Cellular Activation of Leukocyte Function-Associated Antigen-1 and Its Affinity Are Regulated at the I Domain Allosteric Site
Authors: Lupher Mark L. , Harris Edith A. S. , Beals Chan R. , Sui LiMing , Liddington Robert C. , Staunton Donald E.
Published: April 2014

The I domain of the integrin LFA-1 possesses a ligand binding interface that includes the metal ion-dependent adhesion site. Binding of the LFA-1 ligand, ICAM-1 to the metal ion-dependent adhesion site is regulated by the I domain allosteric site (IDAS). We demonstrate here that intracellular signaling leading to activation of LFA-1 binding to ICAM-1 is regulated at the IDAS. Inhibitory mutations in or proximal to the IDAS are dominant to cytoplasmic signals that activate binding to ICAM-1. In addition, mutational activation at the IDAS greatly increases the binding of lymphocyte-expressed LFA-1 to ICAM-1 in response to PMA, but does not result in constitutive binding. Binding of a novel CD18 activation epitope mAb to LFA-1 in response to soluble ICAM-1 binding was also blocked by inhibitory and was enhanced by activating IDAS mutations. Surface plasmon resonance using soluble wild-type LFA-1 and an IDAS mutant of LFA-1 indicate that the IDAS can regulate a 6-fold change in the Kd of ICAM-1 binding. The Kd of wild-type LFA-1 (1.2 × 10−1 s−1) differed with that of the activating IDAS mutant (1.9 × 10−2 s−1), but their Ka values were identical (2.2 × 105 M−1s−1). We propose that IDAS regulates the binding of LFA-1 to ICAM-1 activated by intracellular signals. IDAS can control the affinity state of LFA-1 with concomitant I domain and CD18 conformational changes.
Two Novel IL-1 Family Members, IL-1δ and IL-1ε, Function as an Antagonist and Agonist of NF-κB Activation Through the Orphan IL-1 Receptor-Related Protein 2
Authors: Debets Reno , Timans Jackie C. , Homey Bernhard , Zurawski Sandra , Sana Theodore R. , Lo Sylvia , Wagner Janet , Edwards Gina , Clifford Teresa , Menon Satish , Bazan J. Fernando , Kastelein Robert A.
Published: April 2014

IL-1 is of utmost importance in the host response to immunological challenges. We identified and functionally characterized two novel IL-1 ligands termed IL-1δ and IL-1ε. Northern blot analyses show that these IL-1s are highly abundant in embryonic tissue and tissues containing epithelial cells (i.e., skin, lung, and stomach). In extension, quantitative real-time PCR revealed that of human skin-derived cells, only keratinocytes but not fibroblasts, endothelial cells, or melanocytes express IL-1δ and ε. Levels of keratinocyte IL-1δ are ∼10-fold higher than those of IL-1ε. In vitro stimulation of keratinocytes with IL-1β/TNF-α significantly up-regulates the expression of IL-1ε mRNA, and to a lesser extent of IL-1δ mRNA. In NF-κB-luciferase reporter assays, we demonstrated that IL-1δ and ε proteins do not initiate a functional response via classical IL-1R pairs, which confer responsiveness to IL-1α and β or IL-18. However, IL-1ε activates NF-κB through the orphan IL-1R-related protein 2 (IL-1Rrp2), whereas IL-1δ, which shows striking homology to IL-1 receptor antagonist, specifically and potently inhibits this IL-1ε response. In lesional psoriasis skin, characterized by chronic cutaneous inflammation, the mRNA expression of both IL-1 ligands as well as IL-1Rrp2 are increased relative to normal healthy skin. In total, IL-1δ and ε and IL-1Rrp2 may constitute an independent signaling system, analogous to IL-1αβ/receptor agonist and IL-1R1, that is present in epithelial barriers of our body and takes part in local inflammatory responses.
Genomic Structure and Transcriptional Regulation of the Early B Cell GenechB1
Authors: Goitsuka Ryo , Mamada Hiroshi , Kitamura Daisuke , Cooper Max D. , Chen Chen-lo H.
Published: April 2014

The avian B cell differentiation Ag chB1 is a membrane glycoprotein relative of the mammalian B cell differentiation Ag CD72. Unlike CD72, this C-type lectin is expressed in relatively high levels on immature B cells in the bursa of Fabricius and is down-regulated on mature B cells in the periphery. An immunoreceptor tyrosine-based inhibitory motif in the chB1 cytoplasmic tail suggests a potential regulatory role in intrabursal B cell development. To gain further insight into the selective expression and function of chB1, we determined the genomic organization of chB1 and examined the mechanism of its transcriptional regulation. The 8-exon chB1 gene proved to have very similar organization to that of mouse CD72, further supporting the idea that chB1 is a CD72 relative. As for mouse CD72, the chB1 promoter region lacks a TATA box but contains a conserved initiator element. The 131-bp region (−161 to −30) proximal to the transcriptional start site, which contains a potential early B cell factor binding site, is essential for the B lineage stage-specific transcription of chB1, whereas PU.1 and B cell-specific activator protein/Pax5 have been shown to play important roles in CD72 promoter activity and cell-type specificity. This analysis suggests that differences in transcriptional regulation of these phylogenetically related genes may determine the differences in expression pattern and, therefore, the function of avian chB1 and mammalian CD72 during B cell development.
The Inhibitory Effect of IL-1β on IL-6-Induced α2-Macroglobulin Expression Is Due to Activation of NF-κB
Authors: Bode Johannes G. , Fischer Richard , Häussinger Dieter , Graeve Lutz , Heinrich Peter C. , Schaper Fred
Published: April 2014

The cross-talk between the signal transduction of simultaneous acting cytokines largely determines the final impact of cytokines on their target genes. Both NF-κB and STAT3 are transcription factors well known to be activated by many stimuli and to mediate transcriptional activation by binding to specific enhancer sequences. In this study, it is analyzed how IL-1β inhibits IL-6-induced transcriptional activation of the α2-macroglobulin promoter. It is shown that IL-1β prevents STAT3 binding to the two STAT3-responsive sites within the α2-macroglobulin promoter by association of IL-1β-activated NF-κB to this region. The observation that inhibition of IL-6-induced transcriptional activation of this promoter by IL-1β is reversed by cotransfection with I-κBα provides evidence that NF-κB activation by IL-1β is responsible for inhibition of IL-6-mediated trans activation of the α2-macroglobulin gene. Accordingly, cotransfection of the NF-κB subunits p50 or p65 themselves inhibited activation of the α2-macroglobulin promoter by IL-6. Introduction of point mutations in each of the two NF-κB sites overlapping the two STAT3 binding sites within the α2-macroglobulin promoter provides evidence that each of these two sites counteracts transcriptional activation via STAT3. Most interestingly, at least one functional NF-κB consensus site is essential for the IL-6-induced transcriptional activation of the α2-macroglobulin promoter. Additional data are provided indicating that the activation of NF-κB by IL-1β is also responsible for the inhibition of other IL-6-inducible genes, such as the α1-antichymotrypsin gene as well as the suppressor of cytokine signaling 3 gene, suggesting a more general relevance of this mechanism for transcriptional regulation.
Human IFN-α Protein Engineering: The Amino Acid Residues at Positions 86 and 90 Are Important for Antiproliferative Activity
Authors: Hu Renqiu , Bekisz Joseph , Schmeisser Hana , McPhie Peter , Zoon Kathryn
Published: April 2014

Human IFN-α is a family of structurally related proteins that exhibit a wide range of antiproliferative activities. To understand the structural basis for these different antiproliferative activities, eight recombinant human IFN-α hybrids (HY) of α21a/α2c (HY-4, HY-5) and mutants (site-directed mutagenesis (SDM)-1, 2 and cassette mutagenesis (CM)-1, 2, 3, and 4) have been expressed, purified, and characterized. The data showed that the amino acid region 81–95 is important for antiproliferative activity. Site-directed mutagenesis and cassette mutagenesis studies showed that if serine (S) 86 and asparagine (N) 90 were replaced by tyrosine (Y), the antiproliferative activity was increased. We have also observed that if Y86 was replaced by isoleucine (I), the antiproliferative activity was comparable. However, if Y86 was replaced by aspartic acid (D), lysine (K), or alanine (A), the antiproliferative activity was substantially decreased. Our results indicate that Y and/or I at position 86 and Y at position 90 are very important in antiproliferative activity of human IFN-α. Circular dichroism spectra showed that the amino acid replacements at position 86 did not change the secondary structure. Thus the biological activity changes among those mutants do not appear to be due to conformational changes. The results also suggest that hydrophobic residue(s) at position 86 may be important for the interaction of the molecule with its receptor. The competitive binding data correlated with the antiproliferative activity. The N-terminal region of the molecule and the hydrophobic residues (including Y and I) on the C-helix region at positions 86 and/or 90 are important for binding and antiproliferative activities of human IFN-αs.
Kinetic Analysis of the Interactions of Complement Receptor 2 (CR2, CD21) with Its Ligands C3d, iC3b, and the EBV Glycoprotein gp350/220
Authors: Sarrias Maria Rosa , Franchini Silvia , Canziani Gabriela , Argyropoulos Emelia , Moore William T. , Sahu Arvind , Lambris John D.
Published: April 2014

The molecular mechanisms involved in the interaction of complement receptor 2 (CR2) with its natural ligands iC3b and C3d are still not well understood. In addition, studies regarding the binding site(s) of the receptor on C3 as well as the affinities of the C3 fragments for CR2 have produced contradictory results. In the present study, we have used surface plasmon resonance technology to study the interaction of CR2 with its ligands C3d, iC3b, and the EBV surface glycoprotein gp350/220. We measured the kinetics of binding of the receptor to its ligands, examined the influence of ionic contacts on these interactions, and assessed whether immobilized and soluble iC3b bound with similar kinetics to CR2. Our results indicate that 1) gp350 binding to CR2 follows a simple 1:1 interaction, whereas that of the C3 fragments is more complex and involves more than one intramolecular component; 2) kinetic differences exist between the binding of C3d and iC3b to CR2, which may be due to an additional binding site found on the C3c region of iC3b; and 3) iC3b binds to CR2 with different kinetics, depending on whether the iC3b is in solution or immobilized on the surface. These findings suggest that binding of CR2 to iC3b and C3d is more complex than previously thought.
The Murine Cytomegalovirus pp89 Immunodominant H-2Ld Epitope Is Generated and Translocated into the Endoplasmic Reticulum as an 11-Mer Precursor Peptide
Authors: Knuehl Christine , Spee Pieter , Ruppert Thomas , Kuckelkorn Ulrike , Henklein Peter , Neefjes Jacques , Kloetzel Peter-M.
Published: April 2014

The 20S proteasome is involved in the processing of MHC class I-presented Ags. A number of epitopes is known to be generated as precursor peptides requiring trimming either before or after translocation into the endoplasmic reticulum (ER). In this study, we have followed the proteasomal processing and TAP-dependent ER translocation of the immunodominant epitope of the murine CMV immediate early protein pp89. For the first time, we experimentally linked peptide generation by the proteasome system and TAP-dependent ER translocation. Our experiments show that the proteasome generates both an N-terminally extended 11-mer precursor peptide as well as the correct H2-Ld 9-mer epitope, a process that is accelerated in the presence of PA28. Our direct peptide translocation assays, however, demonstrate that only the 11-mer precursor peptide is transported into the ER by TAPs, whereas the epitope itself is not translocated. In consequence, our combined proteasome/TAP assays show that the 11-mer precursor is the immunorelevant peptide product that requires N-terminal trimming in the ER for MHC class I binding.
Impaired T Cell Immunity in B Cell-Deficient Mice Following Viral Central Nervous System Infection
Authors: Bergmann Cornelia C. , Ramakrishna Chandran , Kornacki Margaret , Stohlman Stephen A.
Published: April 2014

CD8+ T cells are required to control acute viral replication in the CNS following infection with neurotropic coronavirus. By contrast, studies in B cell-deficient (μMT) mice revealed Abs as key effectors in suppressing virus recrudescence. The apparent loss of initial T cell-mediated immune control in the absence of B cells was investigated by comparing T cell populations in CNS mononuclear cells from infected μMT and wild-type mice. Following viral recrudescence in μMT mice, total CD8+ T cell numbers were similar to those of wild-type mice that had cleared infectious virus; however, virus-specific T cells were reduced at least 3-fold by class I tetramer and IFN-γ ELISPOT analysis. Although overall T cell recruitment into the CNS of μMT mice was not impaired, discrepancies in frequencies of virus-specific CD8+ T cells were most severe during acute infection. Impaired ex vivo cytolytic activity of μMT CNS mononuclear cells, concomitant with reduced frequencies, implicated IFN-γ as the primary anti viral factor early in infection. Reduced virus-specific CD8+ T cell responses in the CNS coincided with poor peripheral expansion and diminished CD4+ T cell help. Thus, in addition to the lack of Ab, limited CD8+ and CD4+ T cell responses in μMT mice contribute to the ultimate loss of control of CNS infection. Using a model of virus infection restricted to the CNS, the results provide novel evidence for a role of B cells in regulating T cell expansion and differentiation into effector cells.
Essential Role of Neutrophils in the Initiation and Progression of a Murine Model of Rheumatoid Arthritis
Authors: Wipke Brian T. , Allen Paul M.
Published: April 2014

Neutrophils are prominent participants in the joint inflammation of human rheumatoid arthritis (RA) patients, but the extent of their role in the inductive phase of joint inflammation is unknown. In the K/B×N mouse RA model, transfer of autoreactive Ig from the K/B×N mouse into mice induces a rapid and profound joint-specific inflammatory response reminiscent of human RA. We observed that after K/B×N serum transfer, the earliest clinical signs of inflammation in the ankle joint correlated with the presence of neutrophils in the synovial regions of recipient mouse ankle joints. In this study, we investigated the role of neutrophils in the early inflammatory response to transferred arthritogenic serum from the K/B×N transgenic mouse. Mice were treated with a neutrophil-depleting mAb before and following transfer of arthritogenic serum and scored for clinical indications of inflammation and severity of swelling in ankle joints and front paws. In the absence of neutrophils, mice were completely resistant to the inflammatory effects of K/B×N serum. Importantly, depletion of neutrophils in diseased recipient mice up to 5 days after serum transfer reversed the inflammatory reaction in the joints. Transfer of serum into mice deficient in the generation of nitrogen or oxygen radicals (inducible NO synthase 2 or gp91phox genes, respectively) gave normal inflammatory responses, indicating that neither pathway is essential for disease induction. These studies have identified a critical role for neutrophils in initiating and maintaining inflammatory processes in the joint.
Decreased Expression of Toll-Like Receptor-4 and MD-2 Correlates with Intestinal Epithelial Cell Protection Against Dysregulated Proinflammatory Gene Expression in Response to Bacterial Lipopolysaccharide
Authors: Abreu Maria T. , Vora Puja , Faure Emmanuelle , Thomas Lisa S. , Arnold Elizabeth T. , Arditi Moshe
Published: April 2014

The lumenal surface of the colonic epithelium is continually exposed to Gram-negative commensal bacteria and LPS. Recognition of LPS by Toll-like receptor (TLR)-4 results in proinflammatory gene expression in diverse cell types. Normally, however, commensal bacteria and their components do not elicit an inflammatory response from intestinal epithelial cells (IEC). The aim of this study is to understand the molecular mechanisms by which IEC limit chronic activation in the presence of LPS. Three IEC lines (Caco-2, T84, HT-29) were tested for their ability to activate an NF-κB reporter gene in response to purified, protein-free LPS. No IEC line responded to LPS, whereas human dermal microvessel endothelial cells (HMEC) did respond to LPS. IEC responded vigorously to IL-1β in this assay, demonstrating that the IL-1 receptor signaling pathway shared by TLRs was intact. To determine the reason for LPS hyporesponsiveness in IEC, we examined the expression of TLR4 and MD-2, a critical coreceptor for TLR4 signaling. IEC expressed low levels of TLR4 compared with HMEC and none expressed MD-2. To determine whether the low level of TLR4 expression or absent MD-2 was responsible for the LPS signaling defect in IEC, the TLR4 or MD-2 gene was transiently expressed in IEC lines. Transient transfection of either gene individually was not sufficient to restore LPS signaling, but cotransfection of TLR4 and MD-2 in IEC led to synergistic activation of NF-κB and IL-8 reporter genes in response to LPS. We conclude that IEC limit dysregulated LPS signaling by down-regulating expression of MD-2 and TLR4. The remainder of the intracellular LPS signaling pathway is functionally intact.
The Cytoplasmic Domain of L-Selectin Participates in Regulating L-Selectin Endoproteolysis
Authors: Matala Erik , Alexander Shelia R. , Kishimoto Takashi K. , Walcheck Bruce
Published: April 2014

Neutrophil recruitment at sites of inflammation is regulated by a series of adhesion and activation events. L-selectin (CD62L) is a leukocyte expressed adhesion protein that is important for neutrophil accumulation and rolling along the vascular endothelium. L-selectin is unique from other adhesion molecules involved in leukocyte transmigration in that its adhesiveness appears to be regulated partly by rapid endoproteolysis. Cleavage of L-selectin occurs within a membrane-proximal region that results in ectodomain shedding and retention of a 6-kDa transmembrane fragment. The cleavage domain of L-selectin has been well characterized through mutational analysis. Whether the cytoplasmic domain of L-selectin also plays a role in regulating shedding is controversial. We have previously shown that the Ca2+-sensing protein calmodulin (CaM) constitutively associates with the cytoplasmic domain of L-selectin in transfected cell lines. However, in the absence of mapping and mutational analysis of the CaM-binding region of L-selectin, there remains no direct evidence that this interaction affects shedding. Using synthesized peptides and expressed L-selectin constructs, we demonstrate that CaM binding activity occurs in the membrane-proximal region of the cytoplasmic domain. Mutations engineered in this region that prevent CaM binding increase the proteolytic turnover of L-selectin. Moreover, we demonstrate that CaM binding to the 6-kDa transmembrane fragment is greatly reduced compared with intact L-selectin in neutrophils, suggesting that CaM binding is regulated. These data imply that the cytoplasmic domain of L-selectin can regulate shedding by a mechanism in which bound CaM may operate as a negative effector.
The Essential Role of Lipopolysaccharide-Binding Protein in Protection of Mice Against a Peritoneal Salmonella Infection Involves the Rapid Induction of an Inflammatory Response
Authors: Heinrich Jan-Michael , Bernheiden Martin , Minigo Gabriela , Yang Kang Kang , Schütt Christine , Männel Daniela N. , Jack Robert S.
Published: April 2014

Acute and chronic hyperinflammation are of major clinical concern, and many treatment strategies are therefore directed to inactivating parts of the inflammatory system. However, survival depends on responding quickly to pathogen attack, and since the adaptive immune system requires several days to adequately react, we rely initially on a range of innate defenses, many of which operate by activating parts of the inflammatory network. For example, LPS-binding protein (LBP) can transfer the LPS of Gram-negative bacteria to CD14 on the surface of macrophages, and this initiates an inflammatory reaction. However, the importance of this chain of events in infection is unclear. First, the innate system is redundant, and bacteria have many components that may serve as targets for it. Second, LBP can transfer LPS to other acceptors that do not induce inflammation. In this study, we show that innate defense against a lethal peritoneal infection with Salmonella requires a direct proinflammatory involvement of LBP, and that this is a major nonredundant function of LBP in this infection model. This emphasizes that blocking the LBP-initiated inflammatory cascade disables an essential defense pathway. Any anti-inflammatory protection that may be achieved must be balanced against the risks inherent in blinding the innate system to the presence of Gram-negative pathogens.
Evidence of IL-18 as a Novel Angiogenic Mediator
Authors: Park Christy C. , Morel Jacques C. M. , Amin M. Asif , Connors Matthew A. , Harlow Lisa A. , Koch Alisa E.
Published: April 2014

Angiogenesis, or new blood vessel growth, is a key process in the development of synovial inflammation in rheumatoid arthritis (RA). Integral to this pathologic proliferation are proinflammatory cytokines. We hypothesized a role for IL-18 as an angiogenic mediator in RA. We examined the effect of human IL-18 on human microvascular endothelial cell (HMVEC) migration. IL-18 induced HMVEC migration at 1 nM (p < 0.05). RA synovial fluids potently induced endothelial cell migration, but IL-18 immunodepletion resulted in a 68 ± 5% decrease in HMVEC migration (p < 0.05). IL-18 appears to act on HMVECs via αvβ3 integrin. To test whether IL-18 induced endothelial cell tube formation in vitro, we quantitated the degree of tube formation on Matrigel matrix. IL-18, 1 or 10 nM, resulted in a 77% or 87% increase in tube formation compared with control (p < 0.05). To determine whether IL-18 may be angiogenic in vivo, we implanted IL-18 in Matrigel plugs in mice, and IL-18 at 1 and 10 nM induced angiogenesis (p < 0.05). The angiogenesis observed appears to be independent of the contribution of local TNF-α, as evidenced by adding neutralizing anti-TNF-α Ab to the Matrigel plugs. In an alternative in vivo model, sponges embedded with IL-18 or control were implanted into mice. IL-18 (10 nM) induced a 4-fold increase in angiogenesis vs the control (p < 0.05). These findings support a novel function for IL-18 as an angiogenic factor in RA and may elucidate a potential therapeutic target for angiogenesis-directed diseases.