bluuebunny/crossref_metadata_2025 huggingface dataset:
Unresponsiveness to Lymphoid-Mediated Signals at the Neonatal Follicular Dendritic Cell Precursor Level Contributes to Delayed Germinal Center Induction and Limitations of Neonatal Antibody Responses to T-Dependent Antigens
Authors: Pihlgren Maria , Tougne Chantal , Bozzotti Paola , Fulurija Alma , Duchosal Michel A. , Lambert Paul-Henri , Siegrist Claire-Anne
Published: April 2014

The factors limiting neonatal and infant IgG Ab responses to T-dependent Ags are only partly known. In this study, we assess how these B cell responses are influenced by the postnatal development of the spleen and lymph node microarchitecture. When BALB/c mice were immunized with alum-adsorbed tetanus toxoid at various stages of their immune development, a major functional maturation step for induction of serum IgG, Ab-secreting cells, and germinal center (GC) responses was identified between the second and the third week of life. This correlated with the development of the follicular dendritic cell (FDC) network, as mature FDC clusters only appeared at 2 wk of age. Adoptive transfer of neonatal splenocytes into adult SCID mice rapidly induced B cell follicles and FDC precursor differentiation into mature FDC, indicating effective recruitment and signaling capacity of neonatal B cells. In contrast, adoptive transfer of adult splenocytes into neonatal SCID mice induced primary B cell follicles without any differentiation of mature FDC and failed to correct limitations of tetanus toxoid-induced GC. Thus, unresponsiveness to lymphoid-mediated signals at the level of neonatal FDC precursors delays FDC maturation and GC induction, thus limiting primary Ab-secreting cell responses to T-dependent Ags in early postnatal life.
The Size of the Synaptic Cleft and Distinct Distributions of Filamentous Actin, Ezrin, CD43, and CD45 at Activating and Inhibitory Human NK Cell Immune Synapses
Authors: McCann Fiona E. , Vanherberghen Bruno , Eleme Konstantina , Carlin Leo M. , Newsam Ray J. , Goulding David , Davis Daniel M.
Published: April 2014

In this study, we report the organization of cytoskeletal and large transmembrane proteins at the inhibitory and activating NK cell immunological or immune synapse (IS). Filamentous actin accumulates at the activating, but not the inhibitory, NK cell IS. However, surprisingly, ezrin and the associated protein CD43 are excluded from the inhibitory, but not the activating, NK cell IS. This distribution of ezrin and CD43 at the inhibitory NK cell IS is similar to that previously seen at the activating T cell IS. CD45 is also excluded from the inhibitory, but not activating, NK cell IS. In addition, electron microscopy reveals wide and narrow domains across the synaptic cleft. Target cell HLA-C, located by immunogold labeling, clusters where the synaptic cleft spans the size of HLA-C bound to the inhibitory killer Ig-like receptor. These data are consistent with assembly of the NK cell IS involving a combination of cytoskeletal-driven mechanisms and thermodynamics favoring the organization of receptor/ligand pairs according to the size of their extracellular domains.
Vav-1 and the IKKα Subunit of IκB Kinase Functionally Associate to Induce NF-κB Activation in Response to CD28 Engagement
Authors: Piccolella Enza , Spadaro Francesca , Ramoni Carlo , Marinari Barbara , Costanzo Antonio , Levrero Massimo , Thomson Lesley , Abraham Robert T. , Tuosto Loretta
Published: April 2014

We have recently observed that CD28 engagement initiates a signaling pathway leading to the activation of IκB kinase (IKK) complex and, consequently, to NF-κB activation, and we identified Vav-1 as an important mediator of this function. Here we report for the first time that Vav-1 constitutively associates with IKKα in both Jurkat and primary CD4+ T cells. Vav-1/IKKα association is mediated by their helix-loop-helix domains, does not involve IKKβ, and is functionally relevant in that Vav-1-associated IKKα kinase activity is increased following CD28 engagement by B7. Moreover, we demonstrate that CD28-induced NF-κB activation is augmented by both IKKα and Vav-1, but not IKKβ. Confocal microscopy showed that endogenous Vav-1 and IKKα, but not IKKβ, were recruited to the membrane and colocalized in response to CD28 stimulation. Taken together, these data evidence that Vav-1 plays a key role in the control of NF-κB pathway by targeting IKKα in the T cell membrane and favoring its activation in response to CD28 stimulation.
Glucocorticoids Alter the Lipid and Protein Composition of Membrane Rafts of a Murine T Cell Hybridoma
Authors: Laethem François Van , Liang Xiquan , Andris Fabienne , Urbain Jacques , Vandenbranden Michel , Ruysschaert Jean-Marie , Resh Marilyn D. , Stulnig Thomas M. , Leo Oberdan
Published: April 2014

Glucocorticoids (GC) are widely used anti-inflammatory agents known to suppress T cell activation by interfering with the TCR activation cascade. The attenuation of early TCR signaling events by these compounds has been recently attributed to a selective displacement of key signaling proteins from membrane lipid rafts. In this study, we demonstrate that GC displace the acyl-bound adaptor proteins linker for activation of T cells and phosphoprotein associated with glycosphingolipid-enriched microdomains from lipid rafts of murine T cell hybridomas, possibly by inhibiting their palmitoylation status. Analysis of the lipid content of the membrane rafts revealed that GC treatment led to a significant decrease in palmitic acid content. Moreover, we found an overall decrease in the proportion of raft-associated saturated fatty acids. These changes were consistent with a decrease in fluorescence anisotropy of isolated lipid rafts, indicating an increase in their fluidity. These findings identify the mechanisms underlying the complex inhibitory effects of glucocorticoids on early TCR signaling and suggest that some of the inhibitory properties of GC on T cell responses may be related to their ability to affect the membrane lipid composition and the palmitoylation status of important signaling molecules.
The Cyclic Adenosine 5′-Monophosphate Response Element Modulator Suppresses IL-2 Production in Stimulated T Cells by a Chromatin-Dependent Mechanism
Authors: Tenbrock Klaus , Juang Yuang-Taung , Tolnay Mate , Tsokos George C.
Published: April 2014

The production of IL-2 is tightly controlled by several transcription factors that bind to the IL-2 promoter. The cAMP response element modulator (CREM) is known to form complexes with CREB and bind to the −180 site of the IL-2 promoter in anergic and in systemic lupus erythematosus T cells. In this study we show that CREM is transcriptionally induced in T cells following stimulation through CD3 and CD28, binds to the IL-2 promoter in vivo, and suppresses IL-2 production. Transfection of an antisense CREM plasmid into T cells blocked the expression and binding of CREM to the IL-2 promoter and the decrease of IL-2 production, which follows the early increase after T cell stimulation with CD3 and CD28. In addition, as assessed by chromatin immunoprecipitation experiments, antisense CREM prevented the binding of protein 300 and cAMP response element binding protein and promoted the acetylation of histones. Antisense CREM also enhanced the accessibility of the IL-2 promoter to endonucleases and prevented the condensation of chromatin in vivo. Our data suggest that upon T cell activation, CREM gradually replaces phosphorylated CREB at the −180 site of the IL-2 promoter. CREM, in turn, binds protein 300 and cAMP response element binding protein, but CREM is unable to activate its histone acetyltransferase activity, which results in condensation of chromatin and down-regulation of IL-2 production.
Quantifying Recruitment of Cytosolic Peptides for HLA Class I Presentation: Impact of TAP Transport
Authors: Fruci Doriana , Lauvau Grégoire , Saveanu Loredana , Amicosante Massimo , Butler Richard H. , Polack Axel , Ginhoux Florent , Lemonnier François , Firat Hüseyin , van Endert Peter M.
Published: April 2014

MHC class I ligands are recruited from the cytosolic peptide pool, whose size is likely to depend on the balance between peptide generation by the proteasome and peptide degradation by downstream peptidases. We asked what fraction of this pool is available for presentation, and how the size of this fraction is modulated by peptide affinity for the TAP transporters. A model epitope restricted by HLA-A2 and a series of epitope precursors with N-terminal extensions by single residues modifying TAP affinity were expressed in a system that allowed us to monitor and modulate cytosolic peptide copy numbers. We show that presentation varies strongly according to TAP affinities of the epitope precursors. The fraction of cytosolic peptides recruited for MHC presentation does not exceed 1% and is more than two logs lower for peptides with very low TAP affinities. Therefore, TAP affinity has a substantial impact on MHC class I Ag presentation.
Regulatory T Cells Control Autoimmunity In Vivo by Inducing Apoptotic Depletion of Activated Pathogenic Lymphocytes
Authors: Madakamutil Loui Thomas , Maricic Igor , Sercarz Eli , Kumar Vipin
Published: April 2014

Clinical autoimmunity requires both activation of self-reactive T cells as well as a failure of peripheral tolerance mechanisms. We previously identified one such mechanism that involves regulatory T cells recognizing TCR Vβ8.2 chain-derived peptides in the context of MHC. How this regulation affects the fate of target Vβ8.2+ T lymphocytes in vivo that mediate experimental autoimmune encephalomyelitis has remained unknown. The present study using immunoscope and CFSE-labeling analysis demonstrates that the expansion of regulatory CD4 and CD8 T cells in vivo results in apoptotic depletion of the dominant, myelin basic protein-reactive Vβ8.2+ T cells, but not subdominant Vβ13+ T cells. The elimination of only activated T cells by this negative feedback mechanism preserves the remainder of the naive Vβ8.2+ T cell repertoire and at the same time results in protection from disease. These studies are the first in clearly elucidating the fate of myelin basic protein-specific encephalitogenic T cells in vivo following regulation.
Cu/Zn Superoxide Dismutase Plays Important Role in Immune Response
Authors: Marikovsky Moshe , Ziv Vered , Nevo Nava , Harris-Cerruti Catherine , Mahler Ori
Published: April 2014

Activation of macrophages leads to the secretion of cytokines and enzymes that shape the inflammatory response and increase metabolic processes. This, in turn, results in increased production of reactive oxygen species. The role of Cu/Zn superoxide dismutase (SOD-1), an important enzyme in cellular oxygen metabolism, was examined in activated peritoneal elicited macrophages (PEM) and in several inflammatory processes in vivo. LPS and TNF-α induced SOD-1 in PEM. SOD-1 induction by LPS was mainly via extracellular signal-regulated kinase-1 activation. Transgenic mice overexpressing SOD-1 demonstrated a significant increase in the release of TNF-α and of the metalloproteinases MMP-2 and MMP-9 from PEM. Disulfiram (DSF), an inhibitor of SOD-1, strongly inhibited the release of TNF-α, vascular endothelial growth factor, and MMP-2 and MMP-9 from cultured activated PEM. These effects were prevented by addition of antioxidants, further indicating involvement of reactive oxygen species. In vivo, transgenic mice overexpressing SOD-1 demonstrated a 4-fold increase in serum TNF-α levels and 2-fold stronger delayed-type hypersensitivity reaction as compared with control nontransgenic mice. Conversely, oral administration of DSF lowered TNF-α serum level by 4-fold, lowered the delayed-type hypersensitivity response in a dose-dependent manner, and significantly inhibited adjuvant arthritis in Lewis rats. The data suggest an important role for SOD-1 in inflammation, establish DSF as a potential inhibitor of inflammation, and raise the possibility that regulation of SOD-1 activity may be important in the treatment of immune-dependent pathologies.
CD28 Signal Enhances Apoptosis of CD8 T Cells After Strong TCR Ligation
Authors: Yu Xue-Zhong , Martin Paul J. , Anasetti Claudio
Published: April 2014

High avidity ligation of the TCR induces negative selection in the thymus and can also induce apoptosis of peripheral T cells. Costimulation through CD28 enhances T cell activation and facilitates negative selection in the thymus, but the role of CD28 in peripheral T cell deletional tolerance has not been investigated. We used 2C CD28 wild-type and 2C CD28-deficient strains to assess the effects of CD28 and TCR avidity on peripheral T cell expansion and apoptosis. We compared the activation, division, expansion, and apoptosis of CD28+/+ and CD28−/− 2C cells in response to self-Ag (Kb), alloantigens with intermediate (Kbm3), high (Ld), or very high (Ld + QL9 peptide) avidity. With intermediate avidity alloantigen, the CD28 signal enhanced T cell activation and expansion. However, when T cells encountered high avidity alloantigen, the CD28 signal reduced T cell expansion and increased apoptosis. These results indicate that the CD28 signal can down-regulate peripheral T cell responses by increasing apoptosis when TCR ligation exceeds a critical threshold.
Activation of Alloreactive CD8+ T Cells Operates Via CD4-Dependent and CD4-Independent Mechanisms and Is CD154 Blockade Sensitive
Authors: Zhai Yuan , Meng Lingzhong , Busuttil Ronald W. , Sayegh Mohamed H. , Kupiec-Weglinski Jerzy W.
Published: April 2014

CD154, one of the most extensively studied T cell costimulation molecules, represents a promising therapeutic target in organ transplantation. However, the immunological mechanisms of CD154 blockade that result in allograft protection, particularly in the context of alloreactive CD4/CD8 T cell activation, remain to be elucidated. We now report on the profound inhibition of alloreactive CD8+ T cells by CD154 blockade via both CD4-dependent and CD4-independent activation pathways. Using CD154 KO recipients that are defective in alloreactive CD8+ T cell activation and unable to reject cardiac allografts, we were able to restore CD8 activation and graft rejection by adoptively transferring CD4+ or CD8+ T cells from wild-type syngeneic donor mice. CD4-independent activation of alloreactive CD8+ T cells was confirmed following treatment of wild-type recipients with CD4-depleting mAb, and by using CD4 KO mice. Comparable levels of alloreactive CD8+ T cell activation was induced by allogenic skin engraftment in both animal groups. CD154 blockade inhibited CD4-independent alloreactive CD8+ T cell activation. Furthermore, we analyzed whether disruption of CD154 signaling affects cardiac allograft survival in skin-sensitized CD4 KO and CD8 KO recipients. A better survival rate was observed consistently in CD4 KO, as compared with CD8 KO recipients. Our results document CD4-dependent and CD4-independent activation pathways for alloreactive CD8+ T cells that are both sensitive to CD154 blockade. Indeed, CD154 blockade was effective in preventing CD8+ T cell-mediated cardiac allograft rejection.
Ca2+ Stores and Ca2+ Entry Differentially Contribute to the Release of IL-1β and IL-1α from Murine Macrophages
Authors: Brough David , Le Feuvre Rosalind A. , Wheeler Rachel D. , Solovyova Natasha , Hilfiker Sabine , Rothwell Nancy J. , Verkhratsky Alex
Published: April 2014

Interleukin-1 is a primary mediator of immune responses to injury and infection, but the mechanism of its cellular release is unknown. IL-1 exists as two agonist forms (IL-1α and IL-1β) present in the cytosol of activated monocytes/macrophages. IL-1β is synthesized as an inactive precursor that lacks a signal sequence, and its trafficking does not use the classical endoplasmic reticulum-Golgi route of secretion. Using primary cultured murine peritoneal macrophages, we demonstrate that P2X7 receptor activation causes release of IL-1β and IL-1α via a common pathway, dependent upon the release of Ca2+ from endoplasmic reticulum stores and caspase-1 activity. Increases in intracellular Ca2+ alone do not promote IL-1 secretion because a concomitant efflux of K+ through the plasmalemma is required. In addition, we demonstrate the existence of an alternative pathway for the secretion of IL-1α, independent of P2X7 receptor activation, but dependent upon Ca2+ influx. The identification of these mechanisms provides insight into the mechanism of IL-1 secretion, and may lead to the identification of targets for the therapeutic modulation of IL-1 action in inflammation.
Mast Cell-Derived Exosomes Induce Phenotypic and Functional Maturation of Dendritic Cells and Elicit Specific Immune Responses In Vivo
Authors: Skokos Dimitris , Botros Hany Goubran , Demeure Christian , Morin Joelle , Peronet Roger , Birkenmeier Gerd , Boudaly Sarah , Mécheri Salaheddine
Published: April 2014

Mast cells (MCs) are considered major players in IgE-mediated allergic responses, but have also recently been recognized as active participants in innate as well as specific immune responses. Recent work provided evidence that MCs are able to activate B and T lymphocytes through the release of vesicles called exosomes. Here we demonstrate that exosomes, which are located in the endocytic pathway, harbor exogenous Ags that associate with other molecules endowed with immunomodulatory functions, including 60- and 70-kDa heat shock proteins. Administration to naive mice of Ag-containing exosomes in the absence of conventional adjuvants elicits specific Ab responses across the MHC II haplotype barrier. We demonstrate that MC-exosomes induce immature dendritic cells (DCs) to up-regulate MHC class II, CD80, CD86, and CD40 molecules and to acquire potent Ag-presenting capacity to T cells. Uptake and processing of Ag-associated exosomes by endogenous DCs were also demonstrated. Finally, exosome-associated heat shock proteins are critical for the acquisition by DCs of the Ag-presenting function. This work demonstrates a heretofore unrecognized collaborative interaction between MCs and DCs leading to the elicitation of specific immune responses.
Elimination of Leukemia in the Absence of Lethal Graft-Versus-Host Disease After Allogenic Bone Marrow Transplantation
Authors: Drobyski William R. , Gendelman Maria , Vodanovic-Jankovic Sanja , Gorski Jack
Published: April 2014

Donor T cells are able to effect a graft-vs-leukemia (GVL) response but also induce graft-vs-host disease (GVHD) after allogeneic bone marrow transplantation. We used an AKR leukemia murine transplant model, analogous to human acute lymphoblastic leukemia, in which donor T cells expressed a thymidine kinase suicide gene, to test whether separation of GVL and graft-vs-host (GVH) responses was feasible by selectively eliminating alloactivated donor T cells at defined time points posttransplant. Under experimental conditions where untreated mice could not be cured of disease without dying from GVHD, mice transplanted with thymidine kinase-positive T cells and subsequently administered ganciclovir (GCV) could eliminate leukemia without lethal GVHD. Timing of GCV administration, donor T cell dose, and preexisting leukemia burden were observed to be critical variables. Eradication of leukemia without lethal GVHD in GCV-treated mice implied that the kinetics of GVL and GVH responses were asynchronous and could therefore be temporally dissociated by timely GCV administration. That this strategy was feasible in a murine leukemia model in which GVHD and GVL reactivity are tightly linked suggests that this approach may be relevant to the treatment of selected human leukemias where similar constraints exist. This strategy represents an alternative approach to separating GVL and GVH reactivity and challenges the current paradigm that separation of these responses is dependent upon the administration of donor T cells with restricted specificity for leukemia as opposed to host Ags.
IgE-Mediated Activation of NK Cells Through FcγRIII
Authors: Arase Noriko , Arase Hisashi , Hirano Satoshi , Yokosuka Tadashi , Sakurai Daiju , Saito Takashi
Published: April 2014

NK cells express FcγRIII (CD16), which is responsible for IgG-dependent cell cytotoxicity and for production of several cytokines and chemokines. Whereas FcγRIII on NK cells is composed of both FcγRIIIα and FcRγ chains, that on mast cells is distinct from NK cells and made of FcγRIIIα, FcRβ, and FcRγ. Mast cells show degranulation and release several mediators, which cause anaphylactic responses upon cross-linking of FcγRIII as well as FcεRI with aggregated IgE. In this paper, we examined whether IgE activates NK cells through FcγRIII on their cell surface. We found that NK cells produce several cytokines and chemokines related to an allergic reaction upon IgE stimulation. Furthermore, NK cells exhibited cytotoxicity against IgE-coated target cells in an FcγRIII-dependent manner. These effects of IgE through FcγRIII were not observed in NK cells from FcRγ-deficient mice lacking FcγRIII expression. Collectively, these results demonstrate that NK cells can be activated with IgE through FcγRIII and exhibit both cytokine/chemokine production and Ab-dependent cell cytotoxicity. These data imply that not only mast cells but also NK cells may contribute to IgE-mediated allergic responses.
Proline-Rich Tyrosine Kinase 2 and Rac Activation by Chemokine and Integrin Receptors Controls NK Cell Transendothelial Migration
Authors: Gismondi Angela , Jacobelli Jordan , Strippoli Raffaele , Mainiero Fabrizio , Soriani Alessandra , Cifaldi Loredana , Piccoli Mario , Frati Luigi , Santoni Angela
Published: April 2014

Protein tyrosine kinase activation is an important requisite for leukocyte migration. Herein we demonstrate that NK cell binding to endothelium activates proline-rich tyrosine kinase 2 (Pyk-2) and the small GTP binding protein Rac that are coupled to integrin and chemokine receptors. Chemokine-mediated, but not integrin-mediated, Pyk-2 and Rac activation was sensitive to pretreatment of NK cells with pertussis toxin, a pharmacological inhibitor of Gi protein-coupled receptors. Both Pyk-2 and Rac are functionally involved in chemokine-induced NK cell migration through endothelium or ICAM-1 or VCAM-1 adhesive proteins, as shown by the use of recombinant vaccinia viruses encoding dominant negative mutants of Pyk-2 and Rac. Moreover, we found that Pyk-2 is associated with the Rac guanine nucleotide exchange factor Vav, which undergoes tyrosine phosphorylation upon integrin triggering. Finally, we provide direct evidence for the involvement of Pyk-2 in the control of both chemokine- and integrin-mediated Rac activation. Collectively, our results indicate that Pyk-2 acts as a receptor-proximal link between integrin and chemokine receptor signaling, and the Pyk-2/Rac pathway plays a pivotal role in the control of NK cell transendothelial migration.
Differential Distribution of HLA-DQβ/DRβ Epitopes in the Two Forms of Guillain-Barré Syndrome, Acute Motor Axonal Neuropathy and Acute Inflammatory Demyelinating Polyneuropathy (AIDP): Identification of DQβ Epitopes Associated with Susceptibility to and Protection from AIDP
Authors: Magira Eleni E. , Papaioakim Miltiadis , Nachamkin Irving , Asbury Arthur K. , Li Chun Y. , Ho Tony W. , Griffin John W. , McKhann Guy M. , Monos Dimitri S.
Published: April 2014

Guillain-Barré syndrome (GBS), an acute, immune-mediated paralytic disorder affecting the peripheral nervous system, is the most common cause of acute flaccid paralysis in the postpolio era. GBS is classified into several subtypes based on clinical and pathologic criteria, with acute inflammatory demyelinating polyneuropathy (AIDP) and acute motor axonal neuropathy (AMAN) being the most common forms observed. To better understand the pathogenesis of GBS and host susceptibility to developing the disease, the distribution of HLA class II Ags along with the seroreactivity to Campylobacter jejuni were investigated in a population of GBS patients from northern China. Using DNA-based typing methods, 47 patients with AMAN, 25 patients with AIDP, and 97 healthy controls were studied for the distribution of class II alleles. We found that the DQβ RLD55–57/ED70–71 and DRβ E9V11H13 epitopes were associated with susceptibility to AIDP (p = 0.009 and p = 0.004, respectively), and the DQβ RPD55–57 epitope was associated with protection (p = 0.05) from AIDP. These DQβ/DRβ positional residues are a part of pockets 4 (DQβ 70, 71, DRβ13), 6 (DRβ11), and 9 (DQβ 56, 57, DRβ9); have been demonstrated to be important in peptide binding and T cell recognition; and are associated with other diseases that have a pathoimmunological basis. Class II HLA associations were not identified with AMAN, suggesting a different immunological mechanism of disease induction in the two forms of GBS. These findings provide immunogenetic evidence for differentiating the two disease entities (AMAN and AIDP) and focuses our attention on particular DRβ/DQβ residues that may be instrumental in understanding the pathophysiology of AIDP.
The Four TCR Genes of Teleost Fish: The cDNA and Genomic DNA Analysis of Japanese Flounder (Paralichthys olivaceus) TCR α-, β-, γ-, and δ-Chains
Authors: Nam Bo-Hye , Hirono Ikuo , Aoki Takashi
Published: April 2014

We have isolated and identified all four TCR α, β, γ, and δ cDNAs and genomic clones from a Japanese flounder leukocyte cDNA library and bacterial artificial chromosomal genomic library. Numerous TCR transcripts were sequenced to examine the variability against antigenic peptide, and were shown hypervariability on their complementarity-determining region 3 (CDR3) loops. Among CDR3s, CDR3δ showed a long and broad length distribution, indicating greater similarity to that of Ig. From cDNA sequences and genomic gene analysis of each chain, we found that flounder TCR β, γ, and δ have two different C gene segments, while the TCR α C region exists as a single segment. The flounder Cγs and Cδs showed different lengths in the connecting peptide (CP) region between the different types of polypeptides. The Cδ1 gene consists of two exons, one that encodes an extracellular Ig-like domain (exon 1) and the other that encodes either a very short or possibly a lacking CP region, a transmembrane region, and a cytoplasmic tail (exon 2); these are located within TCR α gene locus. Southern blot analysis, using the bacterial artificial chromosomal genomic DNA clones, revealed that the Cδ2 gene segment, which has a long CP region and different genomic organization to the Cδ1 gene, exists on same gene locus as the TCR γ-chain. This suggests that the flounder possesses very unique genomic DNA organization and gene loci for TCR, Cα/Cδ1, and Cγ/Cδ2.
Ikaros Isoform X Is Selectively Expressed in Myeloid Differentiation
Authors: Payne Kimberly J. , Huang Grace , Sahakian Eva , Zhu Judy Y. , Barteneva Natasha S. , Barsky Lora W. , Payne Marvin A. , Crooks Gay M.
Published: April 2014

The Ikaros gene is alternately spliced to generate multiple DNA-binding and nonbinding isoforms that have been implicated as regulators of hematopoiesis, particularly in the lymphoid lineages. Although early reports of Ikaros mutant mice focused on lymphoid defects, these mice also show significant myeloid, erythroid, and stem cell defects. However, the specific Ikaros proteins expressed in these cells have not been determined. We recently described Ikaros-x (Ikx), a new Ikaros isoform that is the predominant Ikaros protein in normal human hematopoietic cells. In this study, we report that the Ikx protein is selectively expressed in human myeloid lineage cells, while Ik1 predominates in the lymphoid and erythroid lineages. Both Ik1 and Ikx proteins are expressed in early human hematopoietic cells (Lin−CD34+). Under culture conditions that promote specific lineage differentiation, Ikx is up-regulated during myeloid differentiation but down-regulated during lymphoid differentiation from human Lin−CD34+ cells. We show that Ikx and other novel Ikaros splice variants identified in human studies are also expressed in murine bone marrow. In mice, as in humans, the Ikx protein is selectively expressed in the myeloid lineage. Our studies suggest that Ikaros proteins function in myeloid, as well as lymphoid, differentiation and that specific Ikaros isoforms may play a role in regulating lineage commitment decisions in mice and humans.
NF-κB Is Required for Surface Ig-Induced Fas Resistance in B Cells
Authors: Schram Brian R. , Rothstein Thomas L.
Published: April 2014

The susceptibility of primary murine B cells to Fas-mediated apoptosis is regulated in a receptor-specific fashion. Whereas CD40 engagement produces marked sensitivity to Fas killing, engagement of the B cell Ag receptor blocks Fas signaling for cell death in otherwise Fas-sensitive, CD40-stimulated targets and thus induces Fas resistance. The signaling pathway that leads from B cell Ag receptor to Fas resistance has not been fully characterized, but has been shown to depend on new gene expression. NF-κB is activated following B cell Ag receptor engagement and is associated with antiapoptosis; thus, it would seem a likely candidate to mediate transcriptional activation for inducible Fas resistance. Inhibition of B cell Ag receptor signaling for NF-κB activation completely blocked induction of Fas resistance by anti-Ig, and this same phenotype was observed both with chemical inhibitors such as lactacystin and pyrrolidinedithiocarbamate as well as with an IκBα dominant negative TAT fusion protein. Antiapoptotic, NF-κB-responsive transcripts include two gene products previously implicated in mediating anti-Ig-induced Fas resistance, Bcl-xL and FLIP. B cell Ag receptor-induced up-regulation of both these gene products was blocked by NF-κB inhibition, suggesting a mechanism by which the loss of nuclear NF-κB alters the sensitivity of B cell Ag receptor-stimulated B cells to Fas-mediated apoptosis. These results indicate that activation of NF-κB plays a key role in mediating Fas resistance produced by B cell Ag receptor engagement.
Identification of a Functionally Impaired Positive Regulatory Domain I Binding Factor 1 Transcription Repressor in Myeloma Cell Lines
Authors: Györy Ildikó , Fejér György , Ghosh Nilanjan , Seto Ed , Wright Kenneth L.
Published: April 2014

B cell differentiation into a plasma cell requires expression of the positive regulatory domain zinc finger protein 1 gene (PRDM1) that encodes the positive regulatory domain I binding factor 1 (PRDI-BF1 or Blimp-1) protein. It represses the transcription of specific target genes, including c-myc, the MHC class II trans-activator, Pax-5, and CD23b. In this study we demonstrate the presence of an alternative protein product of the PRDM1 gene. The new protein, PRDI-BF1β, has a disrupted PR domain and lacks the amino-terminal 101 aa of the originally described protein. PRDI-BF1β has a dramatic loss of repressive function on multiple target genes, but maintains normal DNA-binding activity, nuclear localization, and association with histone deacetylases and deacetylase activity. Myeloma cell lines express the highest levels of PRDM1β mRNA relative to the full-length form, while primary cells and several other cell lines have very low, but detectable, levels of PRDM1β. RNA analysis and analysis of the PRDM1 promoters demonstrate that PRDI-BF1β is generated from the same gene by alternative transcription initiation using an internal promoter. These newly described features of the PRDM1 gene are highly analogous to the PRDM2 (RIZ) and PRDM3 (MDS1-EVI1) genes, in which each express a truncated protein missing the PR domain. The expression of each of the truncated proteins is elevated in cancerous cells and may play an important role in the disease.
Inhibition of NF-κB Activity in T and NK Cells Results in Defective Effector Cell Expansion and Production of IFN-γ Required for Resistance to Toxoplasma gondii
Authors: Tato Cristina M. , Villarino Alejandro , Caamaño Jorge H. , Boothby Mark , Hunter Christopher A.
Published: April 2014

To define the role of NF-κB in the development of T cell responses required for resistance to Toxoplasma gondii, mice in which T cells are transgenic for a degradation-resistant (ΔN) form of IκBα, an inhibitor of NF-κB, were challenged with T. gondii and their response to infection compared with control mice. IκBα(ΔN)-transgenic (Tg) mice succumbed to T. gondii infection between days 12 and 35, and death was associated with an increased parasite burden compared with wild-type (Wt) controls. Analysis of the responses of infected mice revealed that IL-12 responses were comparable between strains, but Tg mice had a marked reduction in systemic levels of IFN-γ, the major mediator of resistance to T. gondii. In addition, the infection-induced increase in NK cell activity observed in Wt mice was absent from Tg mice and this correlated with NK cell expression of the transgene. Infection-induced activation of CD4+ T cells was similar in Wt and Tg mice, but expansion of activated CD4+T cells was markedly reduced in the Tg mice. This difference in T cell numbers correlated with a reduced capacity of these cells to proliferate after stimulation and was associated with a major defect in the ability of CD4+ T cells from infected mice to produce IFN-γ. Together, these studies reveal that inhibition of NF-κB activity in T and NK cells results in defective effector cell expansion and production of IFN-γ required for resistance to T. gondii.
Role of CD1d in Coxsackievirus B3-Induced Myocarditis
Authors: Huber Sally , Sartini Danielle , Exley Mark
Published: April 2014

The myocarditic (H3) variant of Coxsackievirus B3 (CVB3) causes severe myocarditis in BALB/c mice and BALB/c mice lacking the invariant Jα281 gene, but minimal disease in BALB/c CD1d−/− animals. This indicates that CD1d expression is important in this disease but does not involve the invariant NKT cell often associated with CD1d-restricted immunity. The H3 variant of the virus increases CD1d expression in vitro in neonatal cardiac myocytes whereas a nonmyocarditic (H310A1) variant does not. Vγ4+ T cells show increased activation in both H3-infected BALB/c and Jα281−/− mice compared with CD1d−/− animals. The activated BALB/c Vγ4+ T cells from H3-infected mice kill H3-infected BALB/c myocytes and cytotoxicity is blocked with anti-CD1d but not with anti-MHC class I (Kd/Dd) or class II (IA/IE) mAbs. In contrast, H3 virus-infected CD1d−/− myocytes are not killed. These studies demonstrate that CD1d expression is essential for pathogenicity of CVB3-induced myocarditis, that CD1d expression is increased early after infection in vivo in CD1d+ mice infected with the myocarditic but not with the nonmyocarditic CVB3 variant, and that Vγ4+ T cells, which are known to promote myocarditis susceptibility, appear to recognize CD1d expressed by CVB3-infected myocytes.
Dendritic Cell (DC)-Based Protection Against an Intracellular Pathogen Is Dependent Upon DC-Derived IL-12 and Can Be Induced by Molecularly Defined Antigens
Authors: Berberich Christof , Ramírez-Pineda José R. , Hambrecht Christine , Alber Gottfried , Skeiky Yasir A. W. , Moll Heidrun
Published: April 2014

Upon loading with microbial Ag and adoptive transfer, dendritic cells (DC) are able to induce immunity to infections. This offers encouragement for the development of DC-based vaccination strategies. However, the mechanisms underlying the adjuvant effect of DC are not fully understood, and there is a need to identify Ag with which to arm DC. In the present study, we analyzed the role of DC-derived IL-12 in the induction of resistance to Leishmania major, and we evaluated the protective efficacy of DC loaded with individual Leishmania Ag. Using Ag-pulsed Langerhans cells (LC) from IL-12-deficient or wild-type mice for immunization of susceptible animals, we showed that the inability to release IL-12 completely abrogated the capacity of LC to mediate protection against leishmaniasis. This suggests that the availability of donor LC-derived IL-12 is a requirement for the development of protective immunity. In addition, we tested the protective effect of LC loaded with Leishmania homolog of receptor for activated C kinase, gp63, promastigote surface Ag, kinetoplastid membrane protein-11, or Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a. The results show that mice vaccinated with LC that had been pulsed with selected molecularly defined parasite proteins are capable of controlling infection with L. major. Moreover, the protective potential of DC pulsed with a given Leishmania Ag correlated with the level of their IL-12 expression. Analysis of the cytokine profile of mice after DC-based vaccination revealed that protection was associated with a shift toward a Th1-type response. Together, these findings emphasize the critical role of IL-12 produced by the sensitizing DC and suggest that the development of a DC-based subunit vaccine is feasible.
CD4+ T Cell-Induced Differentiation of EBV-Transformed Lymphoblastoid Cells Is Associated with Diminished Recognition by EBV-Specific CD8+ Cytotoxic T Cells
Authors: Khanolkar Aaruni , Fu Zheng , Underwood L. Joey , Bondurant Kristy L. , Rochford Rosemary , Cannon Martin J.
Published: April 2014

EBV transformation of human B cells in vitro results in establishment of immortalized cell lines (lymphoblastoid cell lines (LCL)) that express viral transformation-associated latent genes and exhibit a fixed, lymphoblastoid phenotype. In this report, we show that CD4+ T cells can modify the differentiation state of EBV-transformed LCL. Coculture of LCL with EBV-specific CD4+ T cells resulted in an altered phenotype, characterized by elevated CD38 expression and decreased proliferation rate. Relative to control LCL, the cocultured LCL were markedly less susceptible to lysis by EBV-specific CD8+ CTL. In contrast, CD4+ T cell-induced differentiation of LCL did not diminish sensitivity of LCL to lysis by CD8+ CTL specific for an exogenously loaded peptide Ag or lysis by alloreactive CD8+ CTL, suggesting that differentiation is not associated with intrinsic resistance to CD8+ T cell cytotoxicity and that evasion of lysis is confined to EBV-specific CTL responses. CD4+ T cell-induced differentiation of LCL and concomitant resistance of LCL to lysis by EBV-specific CD8+ CTL were associated with reduced expression of viral latent genes. Finally, transwell cocultures, in which direct LCL-CD4+ T cell contact was prevented, indicated a major role for CD4+ T cell cytokines in the differentiation of LCL.
The Dendritic Cell-Specific Chemokine, Dendritic Cell-Derived CC Chemokine 1, Enhances Protective Cell-Mediated Immunity to Murine Malaria
Authors: Bruna-Romero Oscar , Schmieg John , Del Val Margarita , Buschle Michael , Tsuji Moriya
Published: April 2014

Cell-mediated immunity plays a crucial role in the control of many infectious diseases, necessitating the need for adjuvants that can augment cellular immune responses elicited by vaccines. It is well established that protection against one such disease, malaria, requires strong CD8+ T cell responses targeted against the liver stages of the causative agent, Plasmodium spp. In this report we show that the dendritic cell-specific chemokine, dendritic cell-derived CC chemokine 1 (DC-CK1), which is produced in humans and acts on naive lymphocytes, can enhance Ag-specific CD8+ T cell responses when coadministered with either irradiated Plasmodium yoelii sporozoites or a recombinant adenovirus expressing the P. yoelii circumsporozoite protein in mice. We further show that these enhanced T cell responses result in increased protection to malaria in immunized mice challenged with live P. yoelii sporozoites, revealing an adjuvant activity for DC-CK1. DC-CK1 appears to act preferentially on naive mouse lymphocytes, and its adjuvant effect requires IL-12, but not IFN-γ or CD40. Overall, our results show for the first time an in vivo role for DC-CK1 in the establishment of primary T cell responses and indicate the potential of this chemokine as an adjuvant for vaccines against malaria as well as other diseases in which cellular immune responses are important.
Serum-Resistant Strains of Borrelia burgdorferi Evade Complement-Mediated Killing by Expressing a CD59-Like Complement Inhibitory Molecule
Authors: Pausa Mario , Pellis Valentina , Cinco Marina , Giulianini Piero G. , Presani Gianni , Perticarari Sandra , Murgia Rossella , Tedesco Francesco
Published: April 2014

Borrelia burgdorferi, the etiological agent of Lyme disease, comprises three genospecies, Borrelia garinii, afzelii, and burgdorferi sensu strictu, that exhibit different pathogenicity and differ in the susceptibility to C-mediated killing. We examined C-sensitive and C-resistant strains of B. burgdorferi for deposition of C3 and late C components by fluorescence microscope and flow cytometry. Despite comparable deposition of C3 on the two strains, the resistant strain exhibited reduced staining for C6 and C7, barely detectable C9, and undetectable poly C9. Based on these findings, we searched for a protein that inhibits assembly of C membrane attack complex and documented an anti-human CD59-reactive molecule on the surface of C-resistant spirochetes by flow cytometry and electron microscopy. A molecule of 80 kDa recognized by polyclonal and monoclonal anti-CD59 Abs was identified in the membrane extract of C-resistant strains by SDS-PAGE and Western blot analysis. The molecule was released from the bacterial wall using deoxycholate and trypsin, suggesting its insertion into the bacterial membrane. The CD59-like molecule acts as C inhibitor on Borrelia because incubation with F(ab′)2 anti-CD59 renders the serum-resistant strain exquisitely susceptible to C-mediated killing and guinea pig erythrocytes bearing C5b-8, unlike the RBC coated with C5b-7, are protected from reactive lysis by the bacterial extract. Western blot analysis revealed preferential binding of the C inhibitory molecule to C9 and weak interaction with C8β.
Lupus-Prone Mice Have an Abnormal Response to Thioglycolate and an Impaired Clearance of Apoptotic Cells
Authors: Potter Paul K. , Cortes-Hernandez Josefina , Quartier Pierre , Botto Marina , Walport Mark J.
Published: April 2014

Deficiency of complement in humans and mice is associated with the development of lupus and with abnormal repair of inflammatory and immune complex-mediated tissue injury. Here we ask whether similar defects in the resolution of inflammation are found in mice prone to spontaneous lupus. We compared the response to an i.p. injection of thioglycolate between two lupus-prone strains (MRL/Mp and NZB/W) and two non lupus-prone strains of mice (C57BL/6 and BALB/c). In all four strains the influx of polymorphonuclear neutrophils (PMN) was similar. However, by 96 h clearance of PMN in the control strains was complete, whereas in the autoimmune-prone strains PMN were still detectable. The number of mononuclear cells recruited was markedly reduced in the lupus-prone strains compared with the controls, and their phenotype was different. The lupus-prone strains had significantly fewer elicited macrophages that were CD11b-high and Ly6C-negative. In lupus-prone mice at 24 h there was a significantly increased number of apoptotic PMN free in the peritoneum, accompanied by a reduced percentage of macrophages containing apoptotic bodies, suggesting a defect in their uptake. An impaired ability of resident peritoneal macrophages from lupus-prone mice to engulf apoptotic cells was demonstrated by in vivo and in vitro cell clearance assays. These observations indicate that lupus-prone strains have an abnormal inflammatory response to thioglycolate and an intrinsic impairment in apoptotic cell uptake. These findings have implications for the initiation of autoimmunity, as lupus autoantigens are expressed on dying cells, and impaired disposal of these could enhance the development of autoimmunity.
Proinflammatory Activities of S100: Proteins S100A8, S100A9, and S100A8/A9 Induce Neutrophil Chemotaxis and Adhesion
Authors: Ryckman Carle , Vandal Karen , Rouleau Pascal , Talbot Mariève , Tessier Philippe A.
Published: April 2014

S100A8 and S100A9 are small calcium-binding proteins that are highly expressed in neutrophil and monocyte cytosol and are found at high levels in the extracellular milieu during inflammatory conditions. Although reports have proposed a proinflammatory role for these proteins, their extracellular activity remains controversial. In this study, we report that S100A8, S100A9, and S100A8/A9 caused neutrophil chemotaxis at concentrations of 10−12–10−9 M. S100A8, S100A9, and S100A8/A9 stimulated shedding of L-selectin, up-regulated and activated Mac-1, and induced neutrophil adhesion to fibrinogen in vitro. Neutralization with Ab showed that this adhesion was mediated by Mac-1. Neutrophil adhesion was also associated with an increase in intracellular calcium levels. However, neutrophil activation by S100A8, S100A9, and S100A8/A9 did not induce actin polymerization. Finally, injection of S100A8, S100A9, or S100A8/A9 into a murine air pouch model led to rapid, transient accumulation of neutrophils confirming their activities in vivo. These studies 1) show that S100A8, S100A9, and S100A8/A9 are potent stimulators of neutrophils and 2) strongly suggest that these proteins are involved in neutrophil migration to inflammatory sites.
Pre-Existing Glomerular Immune Complexes Induce Polymorphonuclear Cell Recruitment Through an Fc Receptor-Dependent Respiratory Burst: Potential Role in the Perpetuation of Immune Nephritis
Authors: Suzuki Yusuke , Gómez-Guerrero Carmen , Shirato Isao , López-Franco Oscar , Gallego-Delgado Julio , Sanjuán Guillermo , Lázaro Alberto , Hernández-Vargas Purificación , Okumura Ko , Tomino Yasuhiko , Ra Chisei , Egido Jesús
Published: April 2014

In immune complex (IC) diseases, FcR are essential molecules facilitating polymorphonuclear cell (PMN) recruitment and effector functions at the IC site. Although FcR-dependent initial tethering and FcR/integrin-dependent PMN accumulation were postulated, their underlying mechanisms remain unclear. We here addressed potential mechanisms involved in PMN recruitment in acute IC glomerulonephritis (nephrotoxic nephritis). Since some renal cells may be recruited from bone marrow (BM) lineages, reconstitution studies with BM chimeras and PMN transfer between wild-type (WT) and FcR-deficient mice (γ−/−) were performed. Severe glomerular damage was induced in WT and Wγ chimeras (BM from WT to irradiated γ−/−), while it was absent in γ−/− and γW chimeras (γ−/− BM to WT). Moreover, WT PMN transfer, but not γ−/− PMN, reconstituted the disease in γ−/−, indicating that FcR on resident cells is not a prerequisite for PMN recruitment in this disease. Surprisingly, transferred WT PMN were recruited coincidentally with NF-κB activation and TNF-α overexpression even in glomeruli with preformed IC (nephrotoxic Ab administered 3 days previously), suggesting that PMN can initially be recruited via its own FcR without previous chemoattractant release. Furthermore, H2O2 inhibition by catalase attenuated the acute WT PMN recruitment and the induction of NF-κB and TNF-α much more than integrin (CD18) blockade, indicating a role for the respiratory burst before integrin-dependent accumulation. In coculture experiments with IC-stimulated PMN and glomeruli, PMN caused acute glomerular TNF-α expression predominantly via FcR-mediated H2O2 production. In conclusion, glomerular IC, even preformed, can cause PMN recruitment and injury through PMN FcR-mediated respiratory burst during initial PMN tethering to IC.