bluuebunny/crossref_metadata_2025 huggingface dataset:
CD8+ CTL Priming by Exact Peptide Epitopes in Incomplete Freund’s Adjuvant Induces a Vanishing CTL Response, whereas Long Peptides Induce Sustained CTL Reactivity
Authors: Bijker Martijn S. , van den Eeden Susan J. F. , Franken Kees L. , Melief Cornelis J. M. , Offringa Rienk , van der Burg Sjoerd H.
Published: April 2014

Therapeutic vaccination trials, in which patients with cancer were vaccinated with minimal CTL peptide in oil-in-water formulations, have met with limited success. Many of these studies were based on the promising data of mice studies, showing that vaccination with a short synthetic peptide in IFA results in protective CD8+ T cell immunity. By use of the highly immunogenic OVA CTL peptide in IFA as a model peptide-based vaccine, we investigated why minimal CTL peptide vaccines in IFA performed so inadequately to allow full optimization of peptide vaccination. Injection of the minimal MHC class I-binding OVA257–264 peptide in IFA transiently activated CD8+ effector T cells, which eventually failed to undergo secondary expansion or to kill target cells, as a result of a sustained and systemic presentation of the CTL peptides gradually leaking out of the IFA depot without systemic danger signals. Complementation of this vaccine with the MHC class II-binding Th peptide (OVA323–339) restored both secondary expansion and in vivo effector functions of CD8+ T cells. Simply extending the CTL peptide to a length of 30 aa also preserved these CD8+ T cell functions, independent of T cell help, because the longer CTL peptide was predominantly presented in the locally inflamed draining lymph node. Importantly, these functional differences were reproduced in two additional model Ag systems. Our data clearly show why priming of CTL with minimal peptide epitopes in IFA is suboptimal, and demonstrate that the use of longer versions of these CTL peptide epitopes ensures the induction of sustained effector CD8+ T cell reactivity in vivo.
PD-1 Regulates Self-Reactive CD8+ T Cell Responses to Antigen in Lymph Nodes and Tissues
Authors: Keir Mary E. , Freeman Gordon J. , Sharpe Arlene H.
Published: April 2014

PD-1, an inhibitory receptor expressed on activated lymphocytes, regulates tolerance and autoimmunity. We tested the role of PD-1:PD-1 ligand (PD-L) interactions in cross-presentation and the generation and control of CD8+ responses against self-Ag. Ag-naive PD-1−/− OVA-specific OT-I CD8+ T cells exhibited exacerbated responses to cross-presented Ag in mice expressing soluble OVA under the control of the rat insulin promoter (RIP-ovahigh). Following adoptive transfer into RIP-ovahigh recipients, PD-1−/− OT-I T cells expanded in the pancreatic lymph node. In contrast to wild-type OT-I cells, PD-1−/− OT-I T cells secreted IFN-γ and migrated into the pancreas, ultimately causing diabetes. Loss of PD-1 affected CD8+ cells intrinsically, and did not significantly alter the responses of wild-type OT-I T cells adoptively transferred into the same RIP-ovahigh recipient mouse. PD-1:PD-L interactions also limited CD8+ effector cells, and PD-L1 expression on parenchymal tissues protected against effector OT-I T cell attack. Finally, we found that the loss of PD-1 on effector OT-I cells lowers the threshold for Ag recognition in peripheral tissues. These findings indicate two checkpoints where PD-1 attenuates self-reactive T cell responses: presentation of self-Ag to naive self-reactive T cells by dendritic cells in the draining lymph node and reactivation of pathogenic self-reactive T cells in the target organ.
Modulation of miR-155 and miR-125b Levels following Lipopolysaccharide/TNF-α Stimulation and Their Possible Roles in Regulating the Response to Endotoxin Shock
Authors: Tili Esmerina , Michaille Jean-Jacques , Cimino Amelia , Costinean Stefan , Dumitru Calin Dan , Adair Brett , Fabbri Muller , Alder Hannes , Liu Chang Gong , Calin George Adrian , Croce Carlo Maria
Published: April 2014

We report here that miR-155 and miR-125b play a role in innate immune response. LPS stimulation of mouse Raw 264.7 macrophages resulted in the up-regulation of miR-155 and down-regulation of miR-125b levels. The same changes also occurred when C57BL/6 mice were i.p. injected with LPS. Furthermore, the levels of miR-155 and miR-125b in Raw 264.7 cells displayed oscillatory changes in response to TNF-α. These changes were impaired by pretreating the cells with the proteasome inhibitor MG-132, suggesting that these two microRNAs (miRNAs) may be at least transiently under the direct control of NF-κB transcriptional activity. We show that miR-155 most probably directly targets transcript coding for several proteins involved in LPS signaling such as the Fas-associated death domain protein (FADD), IκB kinase ε (IKKε), and the receptor (TNFR superfamily)-interacting serine-threonine kinase 1 (Ripk1) while enhancing TNF-α translation. In contrast, miR-125b targets the 3′-untranslated region of TNF-α transcripts; therefore, its down-regulation in response to LPS may be required for proper TNF-α production. Finally, Eμ-miR-155 transgenic mice produced higher levels of TNF-α when exposed to LPS and were hypersensitive to LPS/d-galactosamine-induced septic shock. Altogether, our data suggest that the LPS/TNF-α-dependent regulation of miR-155 and miR-125b may be implicated in the response to endotoxin shock, thus offering new targets for drug design.
Role of CXCR5 and CCR7 in Follicular Th Cell Positioning and Appearance of a Programmed Cell Death Gene-1High Germinal Center-Associated Subpopulation
Authors: Haynes Nicole M. , Allen Christopher D. C. , Lesley Robin , Ansel K. Mark , Killeen Nigel , Cyster Jason G.
Published: April 2014

Th cell access to primary B cell follicles is dependent on CXCR5. However, whether CXCR5 induction on T cells is sufficient in determining their follicular positioning has been unclear. In this study, we find that transgenic CXCR5 overexpression is not sufficient to promote follicular entry of naive T cells unless the counterbalancing influence of CCR7 ligands is removed. In contrast, the positioning of Ag-engaged T cells at the B/T boundary could occur in the absence of CXCR5. The germinal center (GC) response was 2-fold reduced when T cells lacked CXCR5, although these T cells were able to access the GC. Finally, CXCR5highCCR7low T cells were found to have elevated IL-4 transcript and programmed cell death gene-1 (PD-1) expression, and PD-1high cells were reduced in the absence of T cell CXCR5 or in mice compromised in GC formation. Overall, these findings provide further understanding of how the changes in CXCR5 and CCR7 expression regulate Th cell positioning during Ab responses, and they suggest that development and/or maintenance of a PD-1high follicular Th cell subset is dependent on appropriate interaction with GC B cells.
Mycobacterium bovis Bacillus Calmette-Guérin Secreting Active Cathepsin S Stimulates Expression of Mature MHC Class II Molecules and Antigen Presentation in Human Macrophages
Authors: Soualhine Hafid , Deghmane Ala-Eddine , Sun Jim , Mak Karen , Talal Amina , Av-Gay Yossef , Hmama Zakaria
Published: April 2014

A successful Th cell response to bacterial infections is induced by mature MHC class II molecules presenting specific Ag peptides on the surface of macrophages. In recent studies, we demonstrated that infection with the conventional vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) specifically blocks the surface export of mature class II molecules in human macrophages by a mechanism dependent on inhibition of cathepsin S (Cat S) expression. The present study examined class II expression in macrophages infected with a rBCG strain engineered to express and secrete biologically active human Cat S (rBCG-hcs). Cat S activity was completely restored in cells ingesting rBCG-hcs, which secreted substantial levels of Cat S intracellularly. Thus, infection with rBCG-hcs, but not parental BCG, restored surface expression of mature MHC class II molecules in response to IFN-γ, presumably as result of MHC class II invariant chain degradation dependent on active Cat S secreted by the bacterium. These events correlated with increased class II-directed presentation of mycobacterial Ag85B to a specific CD4+ T cell hybridoma by rBCG-hcs-infected macrophages. Consistent with these findings, rBCG-hcs was found to accelerate the fusion of its phagosome with lysosomes, a process that optimizes Ag processing in infected macrophages. These data demonstrated that intracellular restoration of Cat S activity improves the capacity of BCG-infected macrophages to stimulate CD4+ Th cells. Given that Th cells play a major role in protection against tuberculosis, rBCG-hcs would be a valuable tuberculosis vaccine candidate.
Inhibition of T Cell Activation by Cyclic Adenosine 5′-Monophosphate Requires Lipid Raft Targeting of Protein Kinase A Type I by the A-Kinase Anchoring Protein Ezrin
Authors: Ruppelt Anja , Mosenden Randi , Grönholm Mikaela , Aandahl Einar M. , Tobin Derek , Carlson Cathrine R. , Abrahamsen Hilde , Herberg Friedrich W. , Carpén Olli , Taskén Kjetil
Published: April 2014

cAMP negatively regulates T cell immune responses by activation of type I protein kinase A (PKA), which in turn phosphorylates and activates C-terminal Src kinase (Csk) in T cell lipid rafts. Using yeast two-hybrid screening, far-Western blot, immunoprecipitation and immunofluorescense analyses, and small interfering RNA-mediated knockdown, we identified Ezrin as the A-kinase anchoring protein that targets PKA type I to lipid rafts. Furthermore, Ezrin brings PKA in proximity to its downstream substrate Csk in lipid rafts by forming a multiprotein complex consisting of PKA/Ezrin/Ezrin-binding protein 50, Csk, and Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains. The complex is initially present in immunological synapses when T cells contact APCs and subsequently exits to the distal pole. Introduction of an anchoring disruptor peptide (Ht31) into T cells competes with Ezrin binding to PKA and thereby releases the cAMP/PKA type I-mediated inhibition of T cell proliferation. Finally, small interfering RNA-mediated knockdown of Ezrin abrogates cAMP regulation of IL-2. We propose that Ezrin is essential in the assembly of the cAMP-mediated regulatory pathway that modulates T cell immune responses.
Silent Development of Memory Progenitor B Cells
Authors: Aviszus Katja , Zhang Xianghua , Wysocki Lawrence J.
Published: April 2014

T cell-dependent immune responses generate long-lived plasma cells and memory B cells, both of which express hypermutated Ab genes. The relationship between these cell types is not entirely understood. Both appear to emanate from the germinal center reaction, but it is unclear whether memory cells evolve while obligatorily generating plasma cells by siblings under all circumstances. In the experiments we report, plasma cell development was functionally segregated from memory cell development by a series of closely spaced injections of Ag delivered during the period of germinal center development. The injection series elevated serum Ab of low affinity, supporting the idea that a strong Ag signal drives plasma cell development. At the same time, the injection series produced a distinct population of affinity/specificity matured memory B cells that were functionally silent, as manifested by an absence of corresponding serum Ab. These cells could be driven by a final booster injection to develop into Ab-forming cells. This recall response required that a rest period precede the final booster injection, but a pause of only 4 days was sufficient. Our results support a model of memory B cell development in which extensive affinity/specificity maturation can take place within a B cell clone under some circumstances in which a concomitant generation of Ab-forming cells by siblings does not take place.
High Susceptibility of Human Dendritic Cells to Avian Influenza H5N1 Virus Infection and Protection by IFN-α and TLR Ligands
Authors: Thitithanyanont Arunee , Engering Anneke , Ekchariyawat Peeraya , Wiboon-ut Suwimon , Limsalakpetch Amporn , Yongvanitchit Kosol , Kum-Arb Utaiwan , Kanchongkittiphon Watcharoot , Utaisincharoen Pongsak , Sirisinha Stitaya , Puthavathana Pilaipan , Fukuda Mark M. , Pichyangkul Sathit
Published: April 2014

There is worldwide concern that the avian influenza H5N1 virus, with a mortality rate of >50%, might cause the next influenza pandemic. Unlike most other influenza infections, H5N1 infection causes a systemic disease. The underlying mechanisms for this effect are still unclear. In this study, we investigate the interplay between avian influenza H5N1 and human dendritic cells (DC). We showed that H5N1 virus can infect and replicate in monocyte-derived and blood myeloid DC, leading to cell death. These results suggest that H5N1 escapes viral-specific immunity, and could disseminate via DC. In contrast, blood pDC were resistant to infection and produced high amounts of IFN-α. Addition of this cytokine to monocyte-derived DC or pretreatment with TLR ligands protected against infection and the cytopathic effects of H5N1 virus.
Induction of Indoleamine 2,3-Dioxygenase in Vascular Smooth Muscle Cells by Interferon-γ Contributes to Medial Immunoprivilege
Authors: Cuffy Madison C. , Silverio Amanda M. , Qin Lingfeng , Wang Yinong , Eid Raymond , Brandacher Gerald , Lakkis Fadi G. , Fuchs Dietmar , Pober Jordan S. , Tellides George
Published: April 2014

Atherosclerosis and graft arteriosclerosis are characterized by leukocytic infiltration of the vessel wall that spares the media. The mechanism(s) for medial immunoprivilege is unknown. In a chimeric humanized mouse model of allograft rejection, medial immunoprivilege was associated with expression of IDO by vascular smooth muscle cells (VSMCs) of rejecting human coronary artery grafts. Inhibition of IDO by 1-methyl-tryptophan (1-MT) increased medial infiltration by allogeneic T cells and increased VSMC loss. IFN-γ-induced IDO expression and activity in cultured human VSMCs was considerably greater than in endothelial cells (ECs) or T cells. IFN-γ-treated VSMCs, but not untreated VSMCs nor ECs with or without IFN-γ pretreatment, inhibited memory Th cell alloresponses across a semipermeable membrane in vitro. This effect was reversed by 1-MT treatment or tryptophan supplementation and replicated by the absence of tryptophan, but not by addition of tryptophan metabolites. However, IFN-γ-treated VSMCs did not activate allogeneic memory Th cells, even after addition of 1-MT or tryptophan. Our work extends the concept of medial immunoprivilege to include immune regulation, establishes the compartmentalization of immune responses within the vessel wall due to distinct microenvironments, and demonstrates a duality of stimulatory EC signals versus inhibitory VSMC signals to artery-infiltrating T cells that may contribute to the chronicity of arteriosclerotic diseases.
Role of Activation-Induced Deaminase Protein Kinase A Phosphorylation Sites in Ig Gene Conversion and Somatic Hypermutation
Authors: Chatterji Monalisa , Unniraman Shyam , McBride Kevin M. , Schatz David G.
Published: April 2014

Activation-induced deaminase (AID) is thought to initiate somatic hypermutation (SHM), gene conversion (GCV), and class switch recombination (CSR) by the transcription-coupled deamination of cytosine residues in Ig genes. Phosphorylation of AID by protein kinase A (PKA) and subsequent interaction of AID with replication protein A (RPA) have been proposed to play important roles in allowing AID to deaminate DNA during transcription. Serine 38 (S38) of mouse AID is phosphorylated in vivo and lies in a consensus target site for PKA, and mutation of this residue interferes with CSR and SHM. In this study, we demonstrate that S38 in mouse and chicken AID is phosphorylated in chicken DT40 cells and is required for efficient GCV and SHM in these cells. Paradoxically, zebra fish AID, which lacks a serine at the position corresponding to S38, has previously been shown to be active for CSR and we demonstrate that it is active for GCV/SHM. Aspartate 44 (D44) of zebra fish AID has been proposed to compensate for the absence of the S38 phosphorylation site but we demonstrate that mutation of D44 has no effect on GCV/SHM. Some features of zebra fish AID other than D44 might compensate for the absence of S38. Alternatively, the zebra fish protein might function in a manner that is independent of PKA and RPA in DT40 cells, raising the possibility that, under some circumstances, AID mediates efficient Ig gene diversification without the assistance of RPA.
A Role for EZH2 in Silencing of IFN-γ Inducible MHC2TA Transcription in Uveal Melanoma
Authors: Holling Tjadine M. , Bergevoet Marloes W. T. , Wilson Louis , Van Eggermond Marja C. J. A. , Schooten Erik , Steenbergen Renske D. M. , Snijders Peter J. F. , Jager Martine J. , Van den Elsen Peter J.
Published: April 2014

We investigated the contribution of epigenetic mechanisms in MHC2TA transcriptional silencing in uveal melanoma. Although no correlation was observed between impaired CIITA transcript levels after IFN-γ induction and DNA methylation of MHC2TA promoter IV (CIITA-PIV), an association was found with high levels of trimethylated histone H3-lysine 27 (3Me-K27-H3) in CIITA-PIV chromatin. The 3Me-K27-H3 modification correlated with a strong reduction in RNA polymerase II-recruitment to CIITA-PIV. Interestingly, we observed that none of these epigenetic modifications affected recruitment of activating transcription factors to this promoter. Subsequently, we demonstrated the presence of the histone methyltransferase EZH2 in CIITA-PIV chromatin, which is known to be a component of the Polycomb repressive complex 2 and able to triple methylate histone H3-lysine 27. RNA interference-mediated down-regulation of EZH2 expression resulted in an increase in CIITA transcript levels after IFN-γ induction. Our data therefore reveal that EZH2 contributes to silencing of IFN-γ-inducible transcription of MHC2TA in uveal melanoma cells.
Expression Profiling of Immature Thymocytes Revealed a Novel Homeobox Gene That Regulates Double-Negative Thymocyte Development
Authors: Kawazu Masahito , Yamamoto Go , Yoshimi Mayumi , Yamamoto Kazuki , Asai Takashi , Ichikawa Motoshi , Seo Sachiko , Nakagawa Masahiro , Chiba Shigeru , Kurokawa Mineo , Ogawa Seishi
Published: April 2014

Intrathymic development of CD4/CD8 double-negative (DN) thymocytes can be tracked by well-defined chronological subsets of thymocytes, and is an ideal target of gene expression profiling analysis to clarify the genetic basis of mature T cell production, by which differentiation of immature thymocytes is investigated in terms of gene expression profiles. In this study, we show that development of murine DN thymocytes is predominantly regulated by largely repressive rather than inductive activities of transcriptions, where lineage-promiscuous gene expression in immature thymocytes is down-regulated during their differentiation. Functional mapping of genes showing common temporal expression profiles implicates previously uncharacterized gene regulations that may be relevant to early thymocytes development. A small minority of genes is transiently expressed in the CD44lowCD25+ subset of DN thymocytes, from which we identified a novel homeobox gene, Duxl, whose expression is up-regulated by Runx1. Duxl promotes the transition from CD44highCD25+ to CD44lowCD25+ in DN thymocytes, while constitutive expression of Duxl inhibits expression of TCR β-chains and leads to impaired β selection and greatly reduced production of CD4/CD8 double-positive thymocytes, indicating its critical roles in DN thymocyte development.
Regulation of Tumor Cell Sensitivity to TRAIL-Induced Apoptosis by the Metastatic Suppressor Raf Kinase Inhibitor Protein via Yin Yang 1 Inhibition and Death Receptor 5 Up-Regulation
Authors: Baritaki Stavroula , Katsman Alina , Chatterjee Devasis , Yeung Kam C. , Spandidos Demetrios A. , Bonavida Benjamin
Published: April 2014

Raf-1 kinase inhibitor protein (RKIP) has been implicated in the regulation of cell survival pathways and metastases, and is poorly expressed in tumors. We have reported that the NF-κB pathway regulates tumor resistance to apoptosis by the TNF-α family via inactivation of the transcription repressor Yin Yang 1 (YY1). We hypothesized that RKIP overexpression may regulate tumor sensitivity to death ligands via inhibition of YY1 and up-regulation of death receptors (DRs). The TRAIL-resistant prostate carcinoma PC-3 and melanoma M202 cell lines were examined. Transfection with CMV-RKIP, but not with control CMV-EV, sensitized the cells to TRAIL-mediated apoptosis. Treatment with RKIP small interfering RNA (siRNA) inhibited TRAIL-induced apoptosis. RKIP overexpression was paralleled with up-regulation of DR5 transcription and expression; no change in DR4, decoy receptor 1, and decoy receptor 2 expression; and inhibition of YY1 transcription and expression. Inhibition of YY1 by YY1 siRNA sensitized the cells to TRAIL apoptosis concomitantly with DR5 up-regulation. RKIP overexpression inhibited several antiapoptotic gene products such as X-linked inhibitor of apoptosis (XIAP), c-FLIP long, and Bcl-xL that were accompanied with mitochondrial membrane depolarization. RKIP overexpression in combination with TRAIL resulted in the potentiation of these above effects and activation of caspases 8, 9, and 3, resulting in apoptosis. These findings demonstrate that RKIP overexpression regulates tumor cell sensitivity to TRAIL via inhibition of YY1, up-regulation of DR5, and modulation of apoptotic pathways. We suggest that RKIP may serve as an immune surveillance cancer gene, and its low expression or absence in tumors allows the tumor to escape host immune cytotoxic effector cells.
OX40 Controls Functionally Different T Cell Subsets and Their Resistance to Depletion Therapy
Authors: Kroemer Alexander , Xiao Xiang , Vu Minh Diem , Gao Wenda , Minamimura Keisuke , Chen Ming , Maki Takashi , Li Xian Chang
Published: April 2014

T cell depletion is a widely used approach in clinical transplantation. However, not all T cells are equally sensitive to depletion therapies and a significant fraction of T cells persists even after aggressive treatment. The functional attributes of such T cells and the mechanisms responsible for their resistance to depletion are poorly studied. In the present study, we showed that CD4+ T cells that are resistant to polyclonal anti-lymphocyte serum (ALS) mediated depletion exhibit phenotypic features of memory cells and uniformly express OX40 on the cell surface. Studies using the foxp3gfp knockin mice revealed that the remaining CD4+OX40+ cells consist of Foxp3+ Tregs and Foxp3− T effector/memory cells. The ALS-resistant CD4+OX40+ cells failed to mediate skin allograft rejection upon adoptive transferring into congenic Rag−/− mice, but removal of Foxp3+ Tregs from the OX40+ cells resulted in prompt skin allograft rejection. Importantly, OX40 is critical to survival of both Foxp3+ Tregs and T effector/memory cells. However, OX40 exhibits opposing effects on the functional status of Foxp3+ Tregs and T effector/memory cells, as stimulation of OX40 on T effector cells induced amplified cell proliferation but stimulation of OX40 on the Foxp3+ Tregs impaired their suppressor functions. Our study demonstrates that OX40 is a critical molecule in regulating survival and functions of depletion-resistant T cells; and these findings may have important clinical implications.
Cutting Edge: Two Distinct Motifs within the Fas Ligand Tail Regulate Fas Ligand-Mediated Costimulation
Authors: Sun Mingyi , Lee Shinhee , Karray Saoussen , Levi-Strauss Matthieu , Ames Kristina T. , Fink Pamela J.
Published: April 2014

The cytoplasmic domain of Fas ligand is sufficient to costimulate CD8+ T cells by driving Fas ligand recruitment into lipid rafts and association with select Src homology 3-containing proteins, activating PI3K and MAPK pathways, mediating nuclear translocation of the transcription factors NFAT and AP-1, and enhancing IFN-γ production and Ag-specific CD8+ T cell proliferation. We now show that Fas ligand molecules lacking amino acids 45–54 in the proline-rich region of the cytoplasmic domain fail to costimulate but serve as effective death inducers. Death induction and costimulation by Fas ligand are therefore clearly separable functions. Further, upon Fas ligand-mediated costimulation, casein kinase I phosphorylates Fas ligand, in which two conserved casein kinase I binding sites regulate NFAT activation and costimulation. These results help resolve how one molecule can serve as a double-edged immunomodulator by directing discrete biological consequences.
Cutting Edge: Modulation of Intestinal Autoimmunity and IL-2 Signaling by Sphingosine Kinase 2 Independent of Sphingosine 1-Phosphate
Authors: Samy Eileen T. , Meyer Claas A. , Caplazi Patrick , Langrish Claire L. , Lora Jose M. , Bluethmann Horst , Peng Stanford L.
Published: April 2014

Sphingosine kinase (Sphk) phosphorylates sphingosine into sphingosine-1-phosphate (S1P), but its recently identified isoform Sphk2 has been suggested to have distinct subcellular localization and substrate specificity. We demonstrate here that, surprisingly, Sphk2−/− CD4+ T cells exhibit a hyperactivated phenotype with significantly enhanced proliferation and cytokine secretion in response to IL-2 as well as reduced sensitivity to regulatory T cell-mediated suppression in vitro, apparently independent of effects upon S1P. Such findings appear to reflect a requirement for Sphk2 to suppress IL-2 signaling because, in Sphk2−/− CD4+ T cells, IL-2 induced abnormally accentuated STAT5 phosphorylation and small interfering RNA knockdown of STAT5 abrogated their hyperactive phenotype. This pathway physiologically modulates autoinflammatory responses, because Sphk2−/− T cells induced more rapid and robust inflammatory bowel disease in scid recipients. Thus, Sphk2 regulates IL-2 pathways in T cells, and the modulation of Sphk2 activity may be of therapeutic utility in inflammatory and/or infectious diseases.
Cutting Edge: Atypical PKCs Regulate T Lymphocyte Polarity and Scanning Behavior
Authors: Real Eliana , Faure Sophie , Donnadieu Emmanuel , Delon Jérôme
Published: April 2014

Leukocyte locomotion is a polarized process with diverse regulatory assemblies segregating along an anterior-posterior axis that defines two regions within the cell, the leading edge and the uropod. However, the mechanisms that generate T cell asymmetry downstream of chemokine receptors are ill defined. In this study we show that the atypical protein kinases C (aPKCs), PKCι and PKCζ, are required for an early symmetry breaking step. Once the polarity is established, aPKCs also drive uropod formation. These effects depend on the interaction between Par6 and aPKCs. Finally, failure to transduce aPKC-dependent signals reduces T cell motility and their ability to scan dendritic cells. Altogether, our findings suggest that lymphocyte motor activity is regulated by a signaling cascade that relays chemokinetic input to aPKCs.
Histone Acetylation at theIfngPromoter in Tolerized CD4 Cells Is Associated with Increased IFN-γ Expression during Subsequent Immunization to the Same Antigen
Authors: Long Meixiao , Slaiby Aaron M. , Wu Shuang , Hagymasi Adam T. , Mihalyo Marianne A. , Bandyopadhyay Suman , Vella Anthony T. , Adler Adam J.
Published: April 2014

When naive CD4+ Th cells encounter cognate pathogen-derived Ags they expand and develop the capacity to express the appropriate effector cytokines for neutralizing the pathogen. Central to this differentiation process are epigenetic modifications within the effector cytokine genes that allow accessibility to the transcriptional machinery. In contrast, when mature self-reactive CD4 cells encounter their cognate epitopes in the periphery they generally undergo a process of tolerization in which they become hyporesponsive/anergic to antigenic stimulation. In the current study, we used a TCR transgenic adoptive transfer system to demonstrate that in a dose-dependent manner parenchymal self-Ag programs cognate naive CD4 cells to acetylate histones bound to the promoter region of the Ifng gene (which encodes the signature Th1 effector cytokine) during peripheral tolerization. Although the Ifng gene gains transcriptional competence, these tolerized CD4 cells fail to express substantial amounts of IFN-γ in response to antigenic stimulation apparently because a blockage in TCR-mediated signaling also develops. Nevertheless, responsiveness to antigenic stimulation is partially restored when self-Ag-tolerized CD4 cells are retransferred into mice infected with a virus expressing the same Ag. Additionally, there is preferential boosting in the ability of these CD4 cells to express IFN-γ relative to other cytokines with expression that also becomes impaired. Taken together, these results suggest that epigenetic modification of the Ifng locus during peripheral CD4 cell tolerization might allow for preferential expression of IFN-γ during recovery from tolerance.
Evidence for In Vivo Primed and Expanded Autoreactive T Cells as a Specific Feature of Patients with Type 1 Diabetes
Authors: Monti Paolo , Scirpoli Miriam , Rigamonti Andrea , Mayr Anya , Jaeger Annika , Bonfanti Riccardo , Chiumello Giuseppe , Ziegler Anette G. , Bonifacio Ezio
Published: April 2014

Identifying β cell autoantigen-reactive T cells that are involved in the pathogenesis of type 1 diabetes has been troublesome for many laboratories. Disease-relevant autoreactive T cells should be in vivo Ag experienced. The aim of this study was to test this hypothesis and then use this principle as a strategy for identifying diabetes-relevant autoreactive T cells. In this study, a CSFE dilution assay was used to detect glutamic acid decarboxylase 65 (GAD65)- and insulin-responsive T cells and HLA-0201*-GAD65114–122 pentamers were used to detect CD8+ GAD-responsive T cells in memory CD45RO+ and naive CD45RO− cell populations from patients with type 1 diabetes and healthy control subjects. T cell proliferative history was evaluated by flow cytometry telomere length measurement. CD4+ and CD8+ T cells specific for GAD65 and insulin were present in patients with type 1 diabetes and control subjects. Within the naive CD45RO− cells, CD4+ and CD8+ T cell responses were similar between patients and controls. Within the memory CD45RO+ cells, CD4+ T cell responses against whole GAD65 and insulin and HLA-0201*-GAD65114–122 pentamer-positive CD8+ T cells were found in patients with type 1 diabetes, but not in control subjects (p < 0.05 for all). Responding cells from the CD45RO+ T cell population had substantially shorter telomere lengths than responding cells from the CD45RO− cell population. Diabetes-specific autoreactive T cells in the circulation have uniquely undergone sustained in vivo proliferation and differentiation into memory T cells. Prior selection of these cells is possible and is a way to identify diabetes-relevant target Ags and epitopes.
IL-6 and Maturation Govern TLR2 and TLR4 Induced TLR Agonist Tolerance and Cross-Tolerance in Dendritic Cells
Authors: Geisel Julia , Kahl Frauke , Müller Martina , Wagner Hermann , Kirschning Carsten J. , Autenrieth Ingo B. , Frick Julia-Stefanie
Published: April 2014

Stimulation of naive mouse dendritic cells (DC) with LPS or Pam3CSK4 (P3C) induces production of TNF-α via TLR4- or TLR2-signaling. Although tolerance in macrophages has been studied in detail, we investigated the role of TLR agonist concentration and IL-6 for tolerance in DC. P3C- or LPS-primed DC were nonresponsive to P3C or LPS restimulation in terms of TNF-α but not IL-6 production. The mechanisms involved in tolerance were dependent on the concentration of the TLR ligand used for DC priming. DC primed with LPS or P3C at high concentrations developed a maturation dependent, IL-6 independent tolerance associated with inhibition of TLR signaling upstream of IκB as indicated by decreased IκB degradation. In contrast, priming of DC with LPS or P3C at low concentrations resulted in IL-6-dependent tolerance, which was abolished in IL-6 deficient DC, and was not accompanied by maturation of DC or by down-regulation of TLR2 or TLR4. In homotolerogenic DC primed with LPS or P3C at high concentrations, degradation of IκB upon restimulation with LPS or P3C was inhibited suggesting tolerance mechanism(s) upstream of IκB; in contrast, cross-tolerance in DC primed with LPS or P3C at low concentrations was not associated with reduced IκB degradation suggesting tolerance mechanisms downstream of IκB. Our data indicate that in naive DC TLR4- and TLR2-stimulation results in homo- and cross-tolerance; the mechanisms involved in tolerance depend on the concentration of the TLR agonist used for DC priming and are governed by IL-6 and maturation.
Quantitative Time-Resolved Phosphoproteomic Analysis of Mast Cell Signaling
Authors: Cao Lulu , Yu Kebing , Banh Cindy , Nguyen Vinh , Ritz Anna , Raphael Benjamin J. , Kawakami Yuko , Kawakami Toshiaki , Salomon Arthur R.
Published: April 2014

Mast cells play a central role in type I hypersensitivity reactions and allergic disorders such as anaphylaxis and asthma. Activation of mast cells, through a cascade of phosphorylation events, leads to the release of mediators of the early phase allergic response. Understanding the molecular architecture underlying mast cell signaling may provide possibilities for therapeutic intervention in asthma and other allergic diseases. Although many details of mast cell signaling have been described previously, a systematic, quantitative analysis of the global tyrosine phosphorylation events that are triggered by activation of the mast cell receptor is lacking. In many cases, the involvement of particular proteins in mast cell signaling has been established generally, but the precise molecular mechanism of the interaction between known signaling proteins often mediated through phosphorylation is still obscure. Using recently advanced methodologies in mass spectrometry, including automation of phosphopeptide enrichments and detection, we have now substantially characterized, with temporal resolution as short as 10 s, the sites and levels of tyrosine phosphorylation across 10 min of FcεRI-induced mast cell activation. These results reveal a far more extensive array of tyrosine phosphorylation events than previously known, including novel phosphorylation sites on canonical mast cell signaling molecules, as well as unexpected pathway components downstream of FcεRI activation. Furthermore, our results, for the first time in mast cells, reveal the sequence of phosphorylation events for 171 modification sites across 121 proteins in the MCP5 mouse mast cell line and 179 modification sites on 117 proteins in mouse bone marrow-derived mast cells.
Glucocorticoid-Induced TNFR-Related Protein Lowers the Threshold of CD28 Costimulation in CD8+ T Cells
Authors: Ronchetti Simona , Nocentini Giuseppe , Bianchini Rodolfo , Krausz L. Tibor , Migliorati Graziella , Riccardi Carlo
Published: April 2014

CD28 is well characterized as a costimulatory molecule in T cell activation. Recent evidences indicate that TNFR superfamily members, including glucocorticoid-induced TNFR-related protein (GITR), act as costimulatory molecules. In this study, the relationship between GITR and CD28 has been investigated in murine CD8+ T cells. When suboptimal doses of anti-CD3 Ab were used, the absence of GITR lowered CD28-induced activation in these cells whereas the lack of CD28 did not affect the response of CD8+ T cells to GITR costimulus. In fact, costimulation of CD28 in anti-CD3-activated GITR−/− CD8+ T cells resulted in an impaired increase of proliferation, impaired protection from apoptosis, and an impaired rise of activation molecules such as IL-2R, IL-2, and IFN-γ. Most notably, CD28-costimulated GITR−/− CD8+ T cells revealed lower NF-κB activation. As a consequence, up-regulation of Bcl-xL, one of the major target proteins of CD28-dependent NF-κB activation, was defective in costimulated GITR−/− CD8+ T cells. What contributed to the response to CD28 ligation in CD8+ T cells was the early up-regulation of GITR ligand on the same cells, the effect of which was blocked by the addition of a recombinant GITR-Fc protein. Our results indicate that GITR influences CD8+ T cell response to CD28 costimulation, lowering the threshold of CD8+ T cell activation.
In Vitro and In Vivo Activation Induces BAFF and APRIL Expression in B Cells
Authors: Chu Van Trung , Enghard Philipp , Riemekasten Gabriela , Berek Claudia
Published: April 2014

B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) play key roles in peripheral B cell survival, maturation, and differentiation. BAFF and APRIL are produced by a variety of cell types such as macrophages/monocytes and dendritic cells. Our analysis shows that BAFF mRNA is also expressed in all B cell subsets isolated from bone marrow, spleen, and peritoneal cavity of BALB/c mice. APRIL expression is restricted to early stages of B cell development in the bone marrow and the peritoneal B1 subset. Stimulation of B2 and B1 cells with LPS or CpG-oligodeoxynucleotides induced MyD88-dependent plasma cell differentiation and intracellular expression of BAFF and APRIL. Furthermore, activation of B cells up-regulated membrane expression of BAFF. The finding that in vitro activation of B cells is inhibited by the antagonist transmembrane activator and calcium modulator ligand interactor Ig, indicates that BAFF and/or APRIL are released into the culture supernatants. It shows that B cell survival, proliferation, and differentiation are supported by an autocrine pathway. In vivo activation of B cells with a T-dependent Ag- induced BAFF expression in germinal center B cells. In (NZB × NZW)F1 mice with established autoimmune disease, marginal zone, germinal center B cells, as well as splenic plasma cells expressed high levels of BAFF. In (NZB × NZW)F1 mice, the continuous activation of B cells and thus overexpression of BAFF and APRIL may contribute to the development of autoimmune disease.
Molecular Mechanism of Lipopolysaccharide-Induced SOCS-3 Gene Expression in Macrophages and Microglia
Authors: Qin Hongwei , Roberts Kevin L. , Niyongere Sandrine A. , Cong Yingzi , Elson Charles O. , Benveniste Etty N.
Published: April 2014

Immunological activation of macrophages/microglia within the CNS leads to the production of cytokines and chemokines that ultimately impact on glial and neuronal function. Suppressor of cytokine signaling (SOCS) proteins are negative regulators of adaptive and innate immune responses. Our previous studies demonstrated that SOCS-3 attenuates macrophage/microglial activation in vitro, suggesting that SOCS-3 may exert beneficial effects for immune-mediated CNS diseases in vivo. In this study, we describe LPS as a potent inducer of SOCS-3 transcription and expression in macrophages/microglia. An analysis of the SOCS-3 promoter indicates that AP-1 and IFN-γ activation sequence (GAS) elements are involved in LPS-induced SOCS-3 transcription. LPS-induced SOCS-3 expression was diminished in IL-10-deficient macrophages at later time points, indicating the involvement of endogenous IL-10 in this response. Blocking STAT-3 expression and activation using STAT-3 small interfering RNA reduced LPS-induced SOCS-3 gene expression. LPS activated the MAPK-ERK1/2, JNK, and p38 pathways that, in addition to STAT-3, were also involved in LPS-induced SOCS-3 expression. LPS treatment of cells led to the acetylation of histones H3 and H4 on the SOCS-3 promoter and the recruitment of STAT-3, c-Jun, c-Fos, CREB-binding protein, p300, and RNA polymerase II to the endogenous SOCS-3 promoter in a time-dependent manner. These results indicate that LPS-induced MAPK activation, the production of endogenous IL-10, and STAT-3 activation play critical roles in SOCS-3 expression, which provides for feedback attenuation of cytokine-induced immune and inflammatory responses in macrophages and microglia.
Hierarchy of the Human Natural Killer Cell Response Is Determined by Class and Quantity of Inhibitory Receptors for Self-HLA-B and HLA-C Ligands
Authors: Yu Junli , Heller Glenn , Chewning Joseph , Kim Sungjin , Yokoyama Wayne M. , Hsu Katharine C.
Published: April 2014

The interaction of NK inhibitory killer Ig-like receptors (KIRs) with self-MHC class I molecules mediates NK tolerance to self while conferring functional competence. Through single-cell analysis of intracellular IFN-γ production and NK clone cytotoxicity we evaluated the resting NK repertoire, analyzing the responsiveness of NK subgroups expressing discrete combinations of non-KIR and KIR class I-specific receptors. CD94:NKG2A and Ig-like transcript 2 (ILT2)-expressing cells have a modest response to class I-negative target cells, but NK cells expressing inhibitory KIRs to self-MHC class I ligands, both HLA-B and HLA-C ligands, achieve significantly higher effector capacity. There is a dose effect of KIR for self-MHC on effector capacity, but even in the most highly responsive NK cells expressing more than one inhibitory KIR for self-MHC the presentation of only one cognate MHC ligand is sufficient to abolish response. Among KIR+ cells there is preferential expression for inhibitory KIR for self-MHC. The likelihood of KIR expression is influenced by whether other KIRs are already expressed on the same cell, supporting a model of serial acquisition of KIR expression. These findings define how inhibitory receptor and autologous HLA interactions impact single-cell function and demonstrate that the resting human NK repertoire is highly attuned but variegated in response. These findings have important implications for the resting NK response to viral pathogens and malignancy, for donor selection in allogeneic hemopoietic cell transplantation, and for models of NK tolerance.
A Crucial Role for Dendritic Cell (DC) IL-10 in Inhibiting Successful DC-Based Immunotherapy: Superior Antitumor Immunity against Hepatocellular Carcinoma Evoked by DC Devoid of IL-10
Authors: Chen Yu-Xiao , Man Kwan , Ling Guang Sheng , Chen Yichen , Sun Bai-Shun , Cheng Qiao , Wong On Hong , Lo Chi-Kin , Ng Irene Oi-Lin , Chan Li Chong , Lau George K. , Lin Chen-Lung Steve , Huang Fanglu , Huang Fang-Ping
Published: April 2014

The dendritic cell (DC)-based tumor immunotherapy has been a new promise of cure for cancer patients, but animal studies and clinical trials have thus far only shown limited success, especially in treating established tumors. Certain immunosuppressive mechanisms triggered by tumor cells or the derivatives are believed to be a major obstacle. We studied the role of DC-derived IL-10 and its negative impact on vaccine efficacy in mouse models. Liver tumor cells were injected via the portal vein, giving rise to disseminated intrahepatic tumors, or s.c. to form solid but extrahepatic tumors. Bone marrow-derived DCs were generated from normal or IL-10-deficient mice and used as the vector to deliver tumor Ags. We demonstrate here that DCs devoid of IL-10, a potent immunosuppressive cytokine, are superior over conventional DCs in triggering antitumor immunity. The IL-10−/−DCs were highly immunogenic, expressed enhanced levels of surface MHC class II molecules, and secreted increased amounts of Th1-related cytokines. By inducing tumor-specific killing and through the establishment of immunological memory, the vaccines delivered by IL-10−/−DCs could evoke strong therapeutic and protective immunity against hepatocellular carcinoma in the mouse models. These findings will have great clinical impact once being translated into the treatment of malignant, and potentially infectious, diseases in humans.
Geranylgeranylation but Not GTP Loading Determines Rho Migratory Function in T Cells
Authors: Waiczies Sonia , Bendix Ivo , Prozorovski Timour , Ratner Maya , Nazarenko Irina , Pfueller Caspar F. , Brandt Alexander U. , Herz Josephine , Brocke Stefan , Ullrich Oliver , Zipp Frauke
Published: April 2014

Rho GTPases orchestrate signaling pathways leading to cell migration. Their function depends on GTP loading and isoprenylation by geranylgeranyl pyrophosphate (GGpp). In this study, we show that in human T cells, geranylgeranylation—and not GTP loading—is necessary for RhoA-mediated downstream events. As a result of GGpp depletion with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor atorvastatin, RhoA was sequestered from the membrane to the cytosol and, notwithstanding increased GTP loading, the constitutive activation of its substrate Rho-associated coiled-coil protein kinase-1 was blocked. In line with this, T cells expressing increased GTP-RhoA failed to form an intact cytoskeleton and to migrate toward a chemokine gradient. In vivo treatment with atorvastatin in the rodent model of multiple sclerosis markedly decreased the capacity of activated T cells to traffic within the brain, as demonstrated by multiphoton analysis. Thus, tethering of RhoA to the membrane by GGpp is determinant for T cell migration and provides a mechanism for preventing T cell infiltration into inflamed compartments by 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors,
The 3′ IgH Locus Control Region Is Sufficient to Deregulate a c-myc Transgene and Promote Mature B Cell Malignancies with a Predominant Burkitt-Like Phenotype
Authors: Truffinet Véronique , Pinaud Eric , Cogné Nadine , Petit Barbara , Guglielmi Laurence , Cogné Michel , Denizot Yves
Published: April 2014

Burkitt lymphoma (BL) features translocations linking c-myc to an Ig locus. Breakpoints in the H chain locus (IgH) stand either close to JH or within switch regions and always link c-myc to the 3′ IgH locus control region (3′ LCR). To test the hypothesis that the 3′ LCR alone was sufficient to deregulate c-myc, we generated mice carrying a 3′ LCR-driven c-myc transgene and specifically up-regulating c-myc in B cells. Splenic B cells from mice proliferated exaggeratedly in response to various signals had an elevated apoptosis rate but normal B220/IgM/IgD expression. Although all Ig levels were lowered in vivo, class switching and Ig secretion proved normal in vitro. Beginning at the age of 12 wk, transgenic mice developed clonal lymphoblastic lymphomas or diffuse anaplastic plasmacytomas with an overall incidence of 80% by 40 wk. Lymphoblastic lymphomas were B220+IgM+IgD+ with the BL “starry sky” appearance. Gene expression profiles revealed broad alterations in the proliferation program and the Ras-p21 pathway. Our study demonstrates that 3′ IgH enhancers alone can deregulate c-myc and initiate the development of BL-like lymphomas. The rapid and constant occurrence of lymphoma in this model makes it valuable for the understanding and the potential therapeutic manipulation of c-myc oncogenicity in vivo.