bluuebunny/crossref_metadata_2025 huggingface dataset:
OX40-OX40 Ligand Interaction through T Cell-T Cell Contact Contributes to CD4 T Cell Longevity
Authors: Soroosh Pejman , Ine Shouji , Sugamura Kazuo , Ishii Naoto
Published: April 2014

Signals through the OX40 costimulatory receptor on naive CD4 T cells are essential for full-fledged CD4 T cell activation and the generation of CD4 memory T cells. Because the ligand for OX40 is mainly expressed by APCs, including activated B cells, dendritic cells, and Langerhans cells, the OX40-OX40 ligand (OX40L) interaction has been thought to participate in T cell-APC interactions. Although several reports have revealed the expression of OX40L on T cells, the functional significance of its expression on them is still unclear. In this study, we demonstrate that Ag stimulation induced an increase in the surface expression and transcript levels of OX40L in CD4 T cells. Upon contact with OX40-expressing T cells, the cell surface expression of OX40L on CD4 T cells was markedly down-regulated, suggesting that OX40-OX40L binding occurs through a novel T cell-T cell interaction. To investigate the function of this phenomenon, we examined the proliferative response and survival of OX40L-deficient CD4 T cells when challenged with Ag. In vitro studies demonstrated markedly less CD3-induced proliferation of OX40L-deficient CD4 T cells compared with wild-type CD4 T cells. When using TCR transgenic CD4 T cells upon Ag stimulation, survival of OX40L-deficient T cells was impaired. Furthermore, we show that upon antigenic stimulation, fewer OX40L-deficient CD4 T cells than wild-type cells survived following transfer into wild-type and sublethally irradiated recipient mice. Taken together, our findings indicate that OX40L-expressing T cells have an autonomous machinery that provides OX40 signals through a T cell-T cell circuit, creating an additional mechanism for sustaining CD4 T cell longevity.
Defective IgG2a/2b Class Switching in PKCα−/− Mice
Authors: Pfeifhofer Christa , Gruber Thomas , Letschka Thomas , Thuille Nikolaus , Lutz-Nicoladoni Christina , Hermann-Kleiter Natascha , Braun Uschi , Leitges Michael , Baier Gottfried
Published: April 2014

Using model tumor T cell lines, protein kinase C (PKC) α has been implicated in IL-2 cytokine promoter activation in response to Ag receptor stimulation. In this study, for the first time, PKCα null mutant mice are analyzed and display normal T and B lymphocyte development. Peripheral CD3+ PKCα-deficient T cells show unimpaired activation-induced IL-2 cytokine secretion, surface expression of CD25, CD44, and CD69, as well as transactivation of the critical transcription factors NF-AT, NF-κB, AP-1, and STAT5 in vitro. Nevertheless, CD3/CD28 Ab- and MHC alloantigen-induced T cell proliferation and IFN-γ production are severely impaired in PKCα−/− CD3+ T cells. Consistently, PKCα-deficient CD3+ T cells from OVA-immunized PKCα-deficient mice exhibit markedly reduced recall proliferation to OVA in in vitro cultures. In vivo, PKCα-deficient mice give diminished OVA-specific IgG2a and IgG2b responses following OVA immunization experiments. In contrast, OVA-specific IgM and IgG1 responses and splenic PKCα−/− B cell proliferation are unimpaired. Our genetic data, thus, define PKCα as the physiological and nonredundant PKC isotype in signaling pathways that are necessary for T cell-dependent IFN-γ production and IgG2a/2b Ab responses.
Trem-Like Transcript 2 Is Expressed on Cells of the Myeloid/Granuloid and B Lymphoid Lineage and Is Up-Regulated in Response to Inflammation
Authors: King R. Glenn , Herrin Brantley R. , Justement Louis B.
Published: April 2014

The triggering receptor expressed on myeloid cells (TREM) gene cluster encodes a group of transmembrane proteins that are emerging as important components in innate and adaptive immunity. In both mice and humans, the TREM gene cluster encodes eight receptors; only four of these, however, are direct homologs: TREM-1, TREM-2, TREM-like transcript 1 (TLT1), and TLT2. Of the transmembrane receptors encoded by the four conserved genes within this cluster, TLT2 has not been studied previously. Data presented in this study demonstrate that TLT2 is expressed early in B cell development in conjunction with B220 and is detected on all developing mouse B cell populations as well as B cells in the periphery. TLT2 expression on B cells in the periphery exhibits a distinct hierarchy with the highest detectable levels observed on B1 B cells in the peritoneum. The overall gradation of TLT2 expression on B cells is: B1 > marginal zone/transitional 2 > transitional 1 > follicular. Additionally, TLT2 expression was observed on mouse neutrophils throughout the body. Although monocytes were not observed to express TLT2, resident peritoneal and lung macrophages do express TLT2, suggesting that it is up-regulated in association with terminal differentiation of monocytes. Finally, both neutrophils and macrophages were observed to up-regulate TLT2 expression in vivo in response to inflammatory stimuli, whereas TLT2 expression on B cells remained unchanged. In conclusion, the data suggest that TLT2 may be involved in the innate immune response based on its expression profile and the fact that it is up-regulated in response to inflammation.
Costimulation of Chemokine Receptor Signaling by Matrix Metalloproteinase-9 Mediates Enhanced Migration of IFN-α Dendritic Cells
Authors: Hu Yang , Ivashkiv Lionel B.
Published: April 2014

Type I IFNs induce differentiation of dendritic cells (DCs) with potent Ag-presenting capacity, termed IFN-α DCs, that have been implicated in the pathogenesis of systemic lupus erythematosus. In this study, we found that IFN-α DCs exhibit enhanced migration across the extracellular matrix (ECM) in response to chemokines CCL3 and CCL5 that recruit DCs to inflammatory sites, but not the lymphoid-homing chemokine CCL21. IFN-α DCs expressed elevated matrix metalloproteinase-9 (MMP-9), which mediated increased migration across ECM. Unexpectedly, MMP-9 and its cell surface receptors CD11b and CD44 were required for enhanced CCL5-induced chemotaxis even in the absence of a matrix barrier. MMP-9, CD11b, and CD44 selectively modulated CCL5-dependent activation of JNK that was required for enhanced chemotactic responses. These results establish the migratory phenotype of IFN-α DCs and identify an important role for costimulation of chemotactic responses by synergistic activation of JNK. Thus, cell motility is regulated by integrating signaling inputs from chemokine receptors and molecules such as MMP-9, CD11b, and CD44 that also mediate cell interactions with inflammatory factors and ECM.
Advanced Intercross Line Mapping of Eae5 Reveals Ncf-1 and CLDN4 as Candidate Genes for Experimental Autoimmune Encephalomyelitis
Authors: Becanovic Kristina , Jagodic Maja , Sheng Jian Rong , Dahlman Ingrid , Aboul-Enein Fahmy , Wallstrom Erik , Olofsson Peter , Holmdahl Rikard , Lassmann Hans , Olsson Tomas
Published: April 2014

Eae5 in rats was originally identified in two F2 intercrosses, (DA × BN) and (E3 × DA), displaying linkage to CNS inflammation and disease severity in experimental autoimmune encephalomyelitis (EAE), respectively. This region overlaps with an arthritis locus, Pia4, which was also identified in the (E3 × DA) cross. Two congenic strains, BN.DA-Eae5 and BN.DA-Eae5.R1, encompassing the previously described Eae5 and Pia4, were established. DA alleles within the chromosome 12 fragment conferred an increase in disease susceptibility as well as increased inflammation and demyelination in the CNS as compared with BN alleles. To enable a more precise fine mapping of EAE regulatory genes, we used a rat advanced intercross line between the EAE-susceptible DA strain and the EAE-resistant PVG.1AV1 strain. Linkage analysis performed in the advanced intercross line considerably narrowed down the myelin oligodendrocyte glycoprotein-EAE regulatory locus (Eae5) to a ∼1.3-megabase region with a defined number of candidate genes. In this study we demonstrate a regulatory effect of Eae5 on MOG-EAE by using both congenic strains as well as fine mapping these effects to a region containing Ncf-1, a gene associated with arthritis. In addition to structural polymorphisms in Ncf-1, both sequence polymorphisms and expression differences were identified in CLDN4. CLDN4 is a tight junction protein involved in blood-brain barrier integrity. In conclusion, our data strongly suggests Ncf-1 to be a gene shared between two organ-specific inflammatory diseases with a possible contribution by CLDN4 in encephalomyelitis.
Induction of Primary Human T Cell Responses against Hepatitis C Virus-Derived Antigens NS3 or Core by Autologous Dendritic Cells Expressing Hepatitis C Virus Antigens: Potential for Vaccine and Immunotherapy
Authors: Li Wen , Krishnadas Deepa K. , Li Jie , Tyrrell D. Lorne J. , Agrawal Babita
Published: April 2014

Hepatitis C virus (HCV)-specific T cell responses have been suggested to play significant role in viral clearance. Dendritic cells (DCs) are professional APCs that play a major role in priming, initiating, and sustaining strong T cell responses against pathogen-derived Ags. DCs also have inherent capabilities of priming naive T cells against given Ags. Recombinant adenoviral vectors containing HCV-derived Core and NS3 genes were used to endogenously express HCV Core and NS3 proteins in human DCs. These HCV Ags expressing DCs were used to prime and stimulate autologous T cells obtained from uninfected healthy donors. The DCs expressing HCV Core or NS3 Ags were able to stimulate T cells to produce various cytokines and proliferate in HCV Ag-dependent manner. Evidence of both CD4+ and CD8+ T cell responses against HCV Core and NS3 generated in vitro were obtained by flow cytometry and Ab blocking experiments. Further, in secondary assays, the T cells primed in vitro exhibited HCV Ag-specific proliferative responses against recombinant protein Ags and also against immunodominant permissive peptide epitopes from HCV Ags. In summary, we demonstrate that the dendritic cells expressing HCV Ags are able to prime the Ag-specific T cells from uninfected healthy individuals in vitro. These studies have implications in designing cellular vaccines, T cell adoptive transfer therapy or vaccine candidates for HCV infection in both prophylactic and therapeutic settings.
HIV-1 Epitope-Specific CD8+ T Cell Responses Strongly Associated with Delayed Disease Progression Cross-Recognize Epitope Variants Efficiently
Authors: Turnbull Emma L. , Lopes A. Ross , Jones Nicola A. , Cornforth David , Newton Phillipa , Aldam Diana , Pellegrino Pierre , Turner Jo , Williams Ian , Wilson Craig M. , Goepfert Paul A. , Maini Mala K. , Borrow Persephone
Published: April 2014

The ability of HIV-1-specific CD8+ T cell responses to recognize epitope variants resulting from viral sequence variation in vivo may affect the ease with which HIV-1 can escape T cell control and impact on the rate of disease progression in HIV-1-infected humans. Here, we studied the functional cross-reactivity of CD8 responses to HIV-1 epitopes restricted by HLA class I alleles associated with differential prognosis of infection. We show that the epitope-specific responses exhibiting the most efficient cross-recognition of amino acid-substituted variants were those strongly associated with delayed progression to disease. Not all epitopes restricted by the same HLA class I allele showed similar variant cross-recognition efficiency, consistent with the hypothesis that the reported associations between particular HLA class I alleles and rate of disease progression may be due to the quality of responses to certain “critical” epitopes. Irrespective of their efficiency of functional cross-recognition, CD8+ T cells of all HIV-1 epitope specificities examined showed focused TCR usage. Furthermore, interpatient variability in variant cross-reactivity correlated well with use of different dominant TCR Vβ families, suggesting that flexibility is not conferred by the overall clonal breadth of the response but instead by properties of the dominant TCR(s) used for epitope recognition. A better understanding of the features of T cell responses associated with long-term control of viral replication should facilitate rational vaccine design.
IL-17 Receptor Knockout Mice Have Enhanced Myelotoxicity and Impaired Hemopoietic Recovery Following Gamma Irradiation
Authors: Tan Weihong , Huang Weitao , Zhong Qiu , Schwarzenberger Paul
Published: April 2014

IL-17A is a T cell-derived proinflammatory cytokine required for microbial host defense. In vivo expression profoundly stimulates granulopoiesis. At baseline, the hemopoietic system of IL-17R knockout mice (IL-17Ra−/−) is, with the exception of increased splenic progenitor numbers, indistinguishable from normal control mice. However, when challenged with gamma irradiation, hemopoietic toxicity is significantly more pronounced in IL-17Ra−/− animals, with the gamma irradiation-associated LD50 being reduced by 150 rad. In spleen-derived T cells, gamma irradiation induces significant murine IL-17A expression in vivo but not in vitro. After sublethal radiation injury (500 rad), the infusion of purified CD4+ T cells enhances hemopoietic recovery. This recovery is significantly impaired in IL-17Ra−/− animals or after in vivo blockade of IL-17Ra in normal mice, resulting in a reduction of hemopoietic precursors by 50% and of neutrophils by 43%. Following sublethal radiation-induced myelosuppression, in vivo overexpression of murine IL-17A in normal mice substantially enhanced granulopoietic restoration in mice with a 4-fold increase in neutrophils and splenic precursors on day 8 (CFU-granulocyte-macrophage/granulocyte-erythrocyte-megakaryocyte-monocyte, CFU-high proliferative potential), as well as 2- and 3-fold increases of bone marrow precursors, respectively. This establishes IL-17A as a hemopoietic response cytokine to radiation injury in mice and an inducible mechanism that is required for recovery of granulopoiesis after radiation injury.
Targeted Disruption of Leukotriene B4 Receptors BLT1 and BLT2: A Critical Role for BLT1 in Collagen-Induced Arthritis in Mice
Authors: Shao Wen-Hai , Del Prete Annalisa , Bock Cheryl B. , Haribabu Bodduluri
Published: April 2014

Leukotriene B4 mediates diverse inflammatory diseases through the G protein-coupled receptors BLT1 and BLT2. In this study, we developed mice deficient in BLT1 and BLT2 by simultaneous targeted disruption of these genes. The BLT1/BLT2 double-deficient mice developed normally and peritoneal exudate cells showed no detectable responses to leukotriene B4 confirming the deletion of the BLT1/BLT2 locus. In a model of collagen-induced arthritis on the C57BL/6 background, the BLT1/BLT2−/− as well as the previously described BLT1−/− animals showed complete protection from disease development. The disease severity correlated well with histopathology, including loss of joint architecture, inflammatory cell infiltration, fibrosis, pannus formation, and bone erosion in joints of BLT1/BLT2+/+ animals and a total absence of disease pathology in leukotriene receptor-deficient mice. Despite these differences, all immunized BLT1−/− and BLT1/BLT2−/− animals had similar serum levels of anti-collagen Abs relative to BLT1/BLT2+/+ animals. Thus, BLT1 may be a useful target for therapies directed at treating inflammation associated with arthritis.
Interleukin-17 Acts Independently of TNF-α under Arthritic Conditions
Authors: Koenders Marije I. , Lubberts Erik , van de Loo Fons A. J. , Oppers-Walgreen Birgitte , van den Bersselaar Liduine , Helsen Monique M. , Kolls Jay K. , Di Padova Franco E. , Joosten Leo A. B. , van den Berg Wim B.
Published: April 2014

The proinflammatory T cell cytokine IL-17 is a potent inducer of other cytokines such as IL-1 and TNF-α. The contribution of TNF in IL-17-induced joint inflammation is unclear. In this work we demonstrate using TNF-α-deficient mice that TNF-α is required in IL-17-induced joint pathology under naive conditions in vivo. However, overexpression of IL-17 aggravated K/B×N serum transfer arthritis to a similar degree in TNF-α-deficient mice and their wild-type counterparts, indicating that the TNF dependency of IL-17-induced pathology is lost under arthritic conditions. Also, during the course of the streptococcal cell wall-induced arthritis model, IL-17 was able to enhance inflammation and cartilage damage in the absence of TNF. Additional blocking of IL-1 during IL-17-enhanced streptococcal cell wall-induced arthritis did not reduce joint pathology in TNF-deficient mice, indicating that IL-1 is not responsible for this loss of TNF dependency. These data provide further understanding of the cytokine interplay during inflammation and demonstrate that, despite a strong TNF dependency under naive conditions, IL-17 acts independently of TNF under arthritic conditions.
The Sjögren’s Syndrome-Associated Autoantigen Ro52 Is an E3 Ligase That Regulates Proliferation and Cell Death
Authors: Espinosa Alexander , Zhou Wei , Ek Monica , Hedlund Malin , Brauner Susanna , Popovic Karin , Horvath Linn , Wallerskog Therese , Oukka Mohamed , Nyberg Filippa , Kuchroo Vijay K. , Wahren-Herlenius Marie
Published: April 2014

Patients affected by Sjögren’s syndrome and systemic lupus erythematosus (SLE) carry autoantibodies to an intracellular protein denoted Ro52. Although the serologic presence of Ro52 autoantibodies is used clinically for diagnostic purposes, the function of the protein or why it is targeted as an autoantigen in several rheumatic conditions has not been elucidated. In this study, we show that the expression of Ro52 is significantly increased in PBMC of patients with Sjögren’s syndrome and SLE, and demonstrate that Ro52 is a RING-dependent E3 ligase involved in ubiquitination. Overexpression of Ro52, but not of Ro52 lacking the RING domain, in a mouse B cell line lead to decreased growth in steady state and increased cell death after activation via the CD40 pathway. The role of Ro52 in activation-mediated cell death was further confirmed as a reduction in Ro52 expression restored cell viability. These findings suggest that the increased expression of the Ro52 autoantigen in patients may be directly involved in the reduced cellular proliferation and increased apoptotic cell death observed in Sjögren’s syndrome and SLE, and may thus contribute to the autoantigenic load and induction of autoimmune B and T cell responses observed in rheumatic patients.
CXCR3+CD4+ T Cells Mediate Innate Immune Function in the Pathophysiology of Liver Ischemia/Reperfusion Injury
Authors: Zhai Yuan , Shen Xiu-da , Hancock Wayne W. , Gao Feng , Qiao Bo , Lassman Charles , Belperio John A. , Strieter Robert M. , Busuttil Ronald W. , Kupiec-Weglinski Jerzy W.
Published: April 2014

Ischemia-reperfusion injury (IRI), an innate immune-dominated inflammatory response, develops in the absence of exogenous Ags. The recently highlighted role of T cells in IRI raises a question as to how T lymphocytes interact with the innate immune system and function with no Ag stimulation. This study dissected the mechanism of innate immune-induced T cell recruitment and activation in rat syngeneic orthotopic liver transplantation (OLT) model. Liver IRI was induced after cold storage (24–36 h) at 4°C in University of Wisconsin solution. Gene products contributing to IRI were identified by cDNA microarray at 4-h posttransplant. IRI triggered increased intrahepatic expression of CXCL10, along with CXCL9 and 11. The significance of CXCR3 ligand induction was documented by the ability of neutralizing anti-CXCR3 Ab treatment to ameliorate hepatocellular damage and improve 14-day survival of 30-h cold-stored OLTs (95 vs 40% in controls; p < 0.01). Immunohistology analysis confirmed reduced CXCR3+ and CD4+ T cell infiltration in OLTs after treatment. Interestingly, anti-CXCR3 Ab did not suppress innate immune activation in the liver, as evidenced by increased levels of IL-1β, IL-6, inducible NO synthase, and multiple neutrophil/monokine-targeted chemokine programs. In conclusion, this study demonstrates a novel mechanism of T cell recruitment and function in the absence of exogenous Ag stimulation. By documenting that the execution of innate immune function requires CXCR3+CD4+ T cells, it highlights the critical role of CXCR3 chemokine biology for the continuum of innate to adaptive immunity in the pathophysiology of liver IRI.
Influence of CD4+CD25+ Regulatory T Cells on Low/High-Avidity CD4+ T Cells following Peptide Vaccination
Authors: Nishikawa Hiroyoshi , Qian Feng , Tsuji Takemasa , Ritter Gerd , Old Lloyd J. , Gnjatic Sacha , Odunsi Kunle
Published: April 2014

We have recently reported that NY-ESO-1-specific naive CD4+ T cell precursors exist in most individuals but are suppressed by CD4+CD25+ regulatory T cells (Tregs), while memory CD4+ T cell effectors against NY-ESO-1 are found only in cancer patients with spontaneous Ab responses to NY-ESO-1. In this study, we have analyzed mechanisms of CD4+ T cell induction following peptide vaccination in relation to susceptibility to Tregs. Specific HLA-DP4-restricted CD4+ T cell responses were elicited after vaccination with NY-ESO-1157–170 peptide (emulsified in IFA) in patients with NY-ESO-1-expressing epithelial ovarian cancer. These vaccine-induced CD4+ T cells were detectable from effector/memory populations without requirement for in vitro CD4+CD25+ T cell depletion. However, they were only able to recognize NY-ESO-1157–170 peptide but not naturally processed NY-ESO-1 protein and had much lower avidity compared with NY-ESO-1-specific pre-existing naive CD4+CD25− T cell precursors or spontaneously induced CD4+ T cell effectors of cancer patients with NY-ESO-1 Ab. We propose that vaccination with NY-ESO-1157–170 peptide recruits low-avidity T cells with low sensitivity to Tregs and fails to modulate the suppressive effect of Tregs on high-avidity NY-ESO-1-specific T cell precursors.
Experimental Models to Study Development and Function of the Human Immune System In Vivo
Authors: Legrand Nicolas , Weijer Kees , Spits Hergen
Published: April 2014

The study of development and function of the immune system in vivo has made intensive use of animal models, but performing such work in humans is difficult for experimental, practical, and ethical reasons. Confronted with this scientific challenge, several pioneering groups have developed in the late 1980s mouse models of human immune system development. Although these experimental approaches were proven successful and useful, they were suffering from limitations due to xenograft transplantation barriers. By reviewing the characteristics of the successive models over the last 20 years, it becomes apparent that screening of potentially interesting mouse strains and usage of combinations of genetic deficiencies has led to major advances. This is particularly true for human T cell development in the murine thymus. This review will focus on these advances and the potential future improvements that remain to be accomplished.
Cutting Edge: An Alternative Pathway of CD4+ T Cell Differentiation Is Induced Following Activation in the Absence of γ-Chain-Dependent Cytokine Signals
Authors: Mayack Shane R. , Berg Leslie J.
Published: April 2014

This report addresses the role of γ-chain cytokine signals in regulating CD4+ T cell differentiation following activation. Using murine CD4+ T cells lacking the Jak3 tyrosine kinase, we show that activation of these cells in the absence of γ-chain-dependent cytokine signals induces an alternative pathway of T cell differentiation. Specifically, activated Jak3−/− CD4+ T cells produce IL-10, TGF-β, and IFN-γ, but not IL-2 or IL-4, and are unable to proliferate in vitro. In addition, Jak3−/− CD4+ T cells express high levels of programmed death-1 and lymphocyte activation gene-3 and modestly suppress the proliferation of wild-type CD4+ T cells in coculture assays. Together, these features demonstrate a striking similarity between Jak3−/− CD4+ T cells and the regulatory T cells that have been shown to suppress immune responses in vitro and in vivo. We conclude that Jak3 is a critical component of signaling pathways that regulate T cell differentiation into effector vs regulatory lineages.
Cutting Edge: Influence of the TCR Vβ Domain on the Selection of Semi-Invariant NKT Cells by Endogenous Ligands
Authors: Schümann Jens , Mycko Marcin P. , Dellabona Paolo , Casorati Giulia , MacDonald H. Robson
Published: April 2014

Invariant Vα14 (Vα14i) NKT cells are a murine CD1d-dependent regulatory T cell subset characterized by a Vα14-Jα18 rearrangement and expression of mostly Vβ8.2 and Vβ7. Whereas the TCR Vβ domain influences the binding avidity of the Vα14i TCR for CD1d-α-galactosylceramide complexes, with Vβ8.2 conferring higher avidity binding than Vβ7, a possible impact of the TCR Vβ domain on Vα14i NKT cell selection by endogenous ligands has not been studied. In this study, we show that thymic selection of Vβ7+, but not Vβ8.2+, Vα14i NKT cells is favored in situations where endogenous ligand concentration or TCRα-chain avidity are suboptimal. Furthermore, thymic Vβ7+ Vα14i NKT cells were preferentially selected in vitro in response to CD1d-dependent presentation of endogenous ligands or exogenously added self ligand isoglobotrihexosylceramide. Collectively, our data demonstrate that the TCR Vβ domain influences the selection of Vα14i NKT cells by endogenous ligands, presumably because Vβ7 confers higher avidity binding.
IL-3-Mediated TNF Production Is Necessary for Mast Cell Development
Authors: Wright Harry V. , Bailey Daniel , Kashyap Mohit , Kepley Christopher L. , Drutskaya Marina S. , Nedospasov Sergei A. , Ryan John J.
Published: April 2014

Mouse mast cell development and survival are largely controlled by the cytokines IL-3 and stem cell factor (SCF). We have found that IL-3 stimulation of bone marrow cells induces the production of TNF via a PI3K- and MAPK kinase/ERK-dependent pathway. Specifically, Mac-1-positive cells were responsible for TNF production, which peaked on days 7–10 of culture and decreased rapidly thereafter. The importance of IL-3-induced TNF secretion was demonstrated by the failure of TNF-deficient bone marrow cells to survive for >3 wk when cultured in IL-3 and SCF, a defect that was reversed by the addition of soluble TNF. The development of human mast cells from bone marrow progenitors was similarly hampered by the addition of TNF-blocking Abs. Cell death was due to apoptosis, which occurred with changes in mitochondrial membrane potential and caspase activation. Apoptosis appeared to be due to loss of IL-3 signaling, because TNF-deficient cells were less responsive than their wild-type counterparts to IL-3-mediated survival. In vitro cultured mast cells from TNF-deficient mice also demonstrated reduced expression of the high affinity IgE receptor, which was restored to normal levels by the addition of soluble TNF. Finally, TNF-deficient mice demonstrated a 50% reduction in peritoneal mast cell numbers, indicating that TNF is an important mast cell survival factor both in vitro and in vivo.
Role of MHC Class II on Memory B Cells in Post-Germinal Center B Cell Homeostasis and Memory Response
Authors: Shimoda Michiko , Li Tao , Pihkala Jeanene P. S. , Koni Pandelakis A.
Published: April 2014

We investigated the role of B cell Ag presentation in homeostasis of the memory B cell compartment in a mouse model where a conditional allele for the β-chain of MHC class II (MHC-II) is deleted in the vast majority of all B cells by cd19 promoter-mediated expression of Cre recombinase (IA-B mice). Upon T cell-dependent immunization, a small number of MHC-II+ B cells in IA-B mice dramatically expanded and restored normal albeit delayed levels of germinal center (GC) B cells with an affinity-enhancing somatic mutation to Ag. IA-B mice also established normal levels of MHC-II+ memory B cells, which, however, subsequently lost MHC-II expression by ongoing deletion of the conditional iab allele without significant loss in their number. Furthermore, in vivo Ag restimulation of MHC-II− memory B cells of IA-B mice failed to cause differentiation into plasma cells (PCs), even in the presence of Ag-specific CD4+ T cells. In addition, both numbers and Ag-specific affinity of long-lived PCs during the late post-GC phase, as well as post-GC serum affinity maturation, were significantly reduced in IA-B mice. These results support a notion that MHC-II-dependent T cell help during post-GC phase is not absolutely required for the maintenance of memory B cell frequency but is important for their differentiation into PCs and for the establishment of the long-lived PC compartment.
Human Mast Cells Undergo TRAIL-Induced Apoptosis
Authors: Berent-Maoz Beata , Piliponsky Adrian M. , Daigle Isabelle , Simon Hans-Uwe , Levi-Schaffer Francesca
Published: April 2014

Mast cells (MC), supposedly long-lived cells, play a key role in allergy and are important contributors to other inflammatory conditions in which they undergo hyperplasia. In humans, stem cell factor (SCF) is the main regulator of MC growth, differentiation, and survival. Although human MC numbers may also be regulated by apoptotic cell death, there have been no reports concerning the role of the extrinsic apoptotic pathway mediated by death receptors in these cells. We examined expression and function of death receptors for Fas ligand and TRAIL in human MC. Although the MC leukemia cell line HMC-1 and human lung-derived MC expressed both Fas and TRAIL-R, MC lines derived from cord blood (CBMC) expressed only TRAIL-R. Activation of TRAIL-R resulted in caspase 3-dependent apoptosis of CBMC and HMC-1. IgE-dependent activation of CBMC increased their susceptibility to TRAIL-mediated apoptosis. Results suggest that TRAIL-mediated apoptosis may be a mechanism of regulating MC survival in vivo and, potentially, for down-regulating MC hyperplasia in pathologic conditions.
Adaptive Tolerance and Clonal Anergy Are Distinct Biochemical States
Authors: Chiodetti Lynda , Choi Seeyoung , Barber Daniel L. , Schwartz Ronald H.
Published: April 2014

Adaptive tolerance is a process by which T cells become desensitized when Ag stimulation persists following an initial immune response in vivo. To examine the biochemical changes in TCR signaling present in this state, we used a mouse model in which Rag2−/− TCR-transgenic CD4+ T cells were transferred into CD3ε−/− recipients expressing their cognate Ag. Compared with naive T cells, adaptively tolerant T cells had normal levels of TCR and slightly increased levels of CD4. Following activation with anti-TCR and anti-CD4 mAbs, the predominant signaling block in the tolerant cells was at the level of Zap70 kinase activity, which was decreased 75% in vitro. Phosphorylations of the Zap70 substrates (linker of activated T cells and phospholipase Cγ1 were also profoundly diminished. This proximal defect impacted mostly on the calcium/NFAT and NF-κB pathways, with only a modest decrease in ERK1/2 phosphorylation. This state was contrasted with T cell clonal anergy in which the RAS/MAPK pathway was preferentially impaired and there was much less inhibition of Zap70 kinase activity. Both hyporesponsive states manifested a block in IκB degradation. These results demonstrate that T cell adaptive tolerance and clonal anergy are distinct biochemical states, possibly providing T cells with two molecular mechanisms to curtail responsiveness in different biological circumstances.
Thymocyte Negative Selection Is Mediated by Protein Kinase C- and Ca2+-Dependent Transcriptional Induction of Bim
Authors: Canté-Barrett Kirsten , Gallo Elena M. , Winslow Monte M. , Crabtree Gerald R.
Published: April 2014

The processes of positive and negative selection in the thymus both determine the population of T cells that will enter the peripheral immune system and eliminate self-reactive T cells by apoptosis. Substantial evidence indicates that TCR signal intensity mediates this cell fate choice: low-intensity signals lead to survival and differentiation, whereas high-intensity signals generated by self-Ag lead to cell death. The molecular mechanism by which these graded signals are converted to discrete outcomes is not understood. Positive selection requires the Ca2+-dependent phosphatase calcineurin, whereas negative selection requires the proapoptotic Bcl-2 family member Bcl-2-interacting mediator of cell death (Bim). In this study, we investigated the regulation of Bim expression and the role of Ca2+ in mediating negative selection. Our results show that transcription is necessary for both negative selection and Bim induction. Surprisingly, we also found that Ca2+ is necessary for Bim induction. Induction of bim transcription appears to involve protein kinase C, but not calcineurin, JNK, p38 MAPK, or MEK. These results localize the decision point in positive vs negative selection to a step downstream of Ca2+ signaling and suggest that negative selection signals induce Ca2+-dependent bim transcription through PKC.
Frequency of Natural Regulatory CD4+CD25+ T Lymphocytes Determines the Outcome of Tolerance across Fully Mismatched MHC Barrier through Linked Recognition of Self and Allogeneic Stimuli
Authors: Fucs Rita , Jesus Joszilene T. , Souza Junior Paulo H. N. , Franco Larissa , Verícimo Mauricio , Bellio Maria , Nobrega Alberto
Published: April 2014

We show in this study that long-term tolerance to allogeneic skin grafts can be established in the absence of immunosuppression by the combination of the following elements: 1) augmenting the frequency of regulatory CD4+CD25+ T cells (Treg) and 2) presentation of the allogeneic stimuli through linked recognition of allo- and self-epitopes on semiallogeneic F1 APCs. BALB/c spleen cells enriched for CD4+CD25+ T lymphocytes were transferred either to BALB/c nu/nu mice or to BALB/c nu/nu previously injected with F1(BALB/c × B6.Ba) spleen cells, or else grafted with F1(BALB/c × B6.Ba) skin (chimeric BALB/c nu/nu-F1). Chimeric BALB/c nu/nu-F1 reconstituted with syngeneic CD25+-enriched spleen cells were unable to reject the previously transferred F1(BALB/c × B6.Ba) spleen cells or F1(BALB/c × B6.Ba) skin grafts, and a specific tolerance to a secondary B6 graft was obtained, with rejection of third-party CBA grafts. BALB/c nu/nu mice reconstituted only with syngeneic CD25+-enriched spleen cells rejected both B6 and CBA skin grafts. In contrast, when chimeric BALB/c nu/nu-F1 were reconstituted with spleen populations comprising normal frequencies of Treg cells, the linked recognition of allo and self resulted in breaking of self tolerance and rejection of syngeneic grafts, strongly suggesting that linked recognition works in both directions, either to establish tolerance to allo, or to break tolerance to self, the critical parameter being the relative number of Treg cells.
Ex Vivo Characterization of Allo-MHC-Restricted T Cells Specific for a Single MHC-Peptide Complex
Authors: Pittet Mikaël J. , Gati Asma , Le Gal Frederic-Anne , Bioley Gilles , Guillaume Philippe , de Smedt Magda , Plum Jean , Speiser Daniel E. , Cerottini Jean-Charles , Dietrich Pierre-Yves , Romero Pedro , Zippelius Alfred
Published: April 2014

Alloreactive T cells are thought to be a potentially rich source of high-avidity T cells with therapeutic potential since tolerance to self-Ags is restricted to self-MHC recognition. Given the particularly high frequency of alloreactive T cells in the peripheral immune system, we used numerous MHC class I multimers to directly visualize and isolate viral and tumor Ag-specific alloreactive CD8 T cells. In fact, all but one specificities screened were undetectable in ex vivo labeling. In this study, we report the occurrence of CD8 T cells specifically labeled with allo-HLA-A*0201/Melan-A/MART-126–35 multimers at frequencies that are in the range of 10−4 CD8 T cells and are thus detectable ex vivo by flow cytometry. We report the thymic generation and shaping of tumor Ag-specific, alloreactive T cells as well as their fate once seeded in the periphery. We show that these cells resemble their counterparts in HLA-A*0201-positive individuals, based on their structural and functional attributes.
Position-Dependent Repression and Promotion of DQB1 Intron 3 Splicing by GGGG Motifs
Authors: Královic̆ová Jana , Vor̆echovský Igor
Published: April 2014

Alternative splicing of HLA-DQB1 exon 4 is allele-dependent and results in variable expression of soluble DQβ. We have recently shown that differential inclusion of this exon in mature transcripts is largely due to intron 3 variants in the branch point sequence (BPS) and polypyrimidine tract. To identify additional regulatory cis-elements that contribute to haplotype-specific splicing of DQB1, we systematically examined the effect of guanosine (G) repeats on intron 3 removal. We found that the GGG or GGGG repeats generally improved splicing of DQB1 intron 3, except for those that were adjacent to the 5′ splice site where they had the opposite effect. The most prominent splicing enhancement was conferred by GGGG motifs arranged in tandem upstream of the BPS. Replacement of a G-rich segment just 5′ of the BPS with a series of random sequences markedly repressed splicing, whereas substitutions of a segment further upstream that lacked the G-rich elements and had the same size did not result in comparable splicing inhibition. Systematic mutagenesis of both suprabranch guanosine quadruplets (G4) revealed a key role of central G residues in splicing enhancement, whereas cytosines in these positions had the most prominent repressive effects. Together, these results show a significant role of tandem G4NG4 structures in splicing of both complete and truncated DQB1 intron 3, support position dependency of G repeats in splicing promotion and inhibition, and identify positively and negatively acting sequences that contribute to the haplotype-specific DQB1 expression.
Regulation of IgH Gene Assembly: Role of the Intronic Enhancer and 5′DQ52 Region in Targeting DHJH Recombination
Authors: Afshar Roshi , Pierce Steven , Bolland Daniel J. , Corcoran Anne , Oltz Eugene M.
Published: April 2014

The assembly of Ag receptor genes by V(D)J recombination is regulated by transcriptional promoters and enhancers which control chromatin accessibility at Ig and TCR gene segments to the RAG-1/RAG-2 recombinase complex. Paradoxically, germline deletions of the IgH enhancer (Eμ) only modestly reduce DH→JH rearrangements when assessed in peripheral B cells. However, deletion of Eμ severely impairs recombination of VH gene segments, which are located over 100 kb away. We now test two alternative explanations for the minimal effect of Eμ deletions on primary DH→JH rearrangement: 1) Accessibility at the DHJH cluster is controlled by a redundant cis-element in the absence of Eμ. One candidate for this element lies 5′ to DQ52 (PDQ52) and exhibits promoter/enhancer activity in pre-B cells. 2) In contrast to endpoint B cells, DH→JH recombination may be significantly impaired in pro-B cells from enhancer-deficient mice. To elucidate the roles of PDQ52 and Eμ in the regulation of IgH locus accessibility, we generated mice with targeted deletions of these elements. We report that the defined PDQ52 promoter is dispensable for germline transcription and recombination of the DHJH cluster. In contrast, we demonstrate that Eμ directly regulates accessibility of the DHJH region. These findings reveal a significant role for Eμ in the control mechanisms that activate IgH gene assembly and suggest that impaired VH→DHJH rearrangement in enhancer-deficient cells may be a downstream consequence of the primary block in DH→JH recombination.
Activation of Invariant NKT Cells by the Helminth Parasite Schistosoma mansoni
Authors: Mallevaey Thierry , Zanetta Jean Pierre , Faveeuw Christelle , Fontaine Josette , Maes Emmanuel , Platt Frances , Capron Monique , de-Moraes Maria Leite- , Trottein François
Published: April 2014

Mouse CD1d-restricted NKT cells, including invariant (i)NKT cells, are innate cells activated by glycolipid Ags and play important roles in the initiation and regulation of immune responses. Through their ability to promptly produce large amounts of Th1 and/or Th2 cytokines upon TCR engagement, iNKT cells exert crucial functions in the immune/inflammatory system during bacterial, protozoan, fungal, and viral infections. However, their roles during metazoan parasite infection, which are generally associated with strong Th2 responses, still remain elusive. In this study, we show that during the course of murine schistosomiasis, iNKT cells exhibit an activated phenotype and that following schistosome egg encounter in the liver, hepatic iNKT cells produce both IFN-γ and IL-4 in vivo. We also report that schistosome egg-sensitized dendritic cells (DCs) activate, in a CD1d-dependent manner, iNKT cells to secrete IFN-γ and IL-4 in vitro. Interestingly, transfer of egg-sensitized DCs promotes a strong Th2 response in recipient wild-type mice, but not in mice that lack iNKT cells. Engagement of TLRs in DCs is not necessary for iNKT cell stimulation in response to egg-sensitized DCs, suggesting an alternative pathway of activation. Finally, we propose that self, rather than parasite-derived, CD1d-restricted ligands are implicated in iNKT cell stimulation. Taken together, our data show for the first time that helminths can activate iNKT cells to produce immunoregulatory cytokines in vivo, enabling them to influence the adaptive immune response.
Induction of Enhanced Immunity to Intestinal Nematodes Using IL-9-Producing Dendritic Cells
Authors: Leech Melanie D. , Grencis Richard K.
Published: April 2014

Dendritic cells can be considered natural adjuvants and are able to act as cellular vaccines to protect against disease. Adoptive transfer of Ag-pulsed bone marrow-derived dendritic cells (BMDCs) enhanced expulsion of the intestinal nematode, Trichinella spiralis, from the small intestine. IL 9 is a critical cytokine in protective immunity to intestinal nematode infection and is believed to enhance Th2 immune responses. Deriving dendritic cells from an IL-9 transgenic (IL-9t) mouse has enabled a detailed investigation of the importance of IL-9 during Ag presentation. Indeed, IL-9t dendritic cells significantly enhanced T cell proliferation and Th2 responses and, after adoptive transfer, enhanced parasite-specific IgG1 and intestinal mastocytosis in vivo, leading to accelerated expulsion of adult worms from the intestine. Overall, this paper demonstrates that dendritic cell vaccination can be used to successfully protect the host against intestinal nematode infection and suggests that IL-9 can act as a potent type 2 adjuvant during Ag presentation and the early stages of Th2 activation.
Ceramide-Dependent Regulation of Human Epidermal Keratinocyte CD1d Expression during Terminal Differentiation
Authors: Fishelevich Rita , Malanina Alla , Luzina Irina , Atamas Sergei , Smyth Miriam J. , Porcelli Steven A. , Gaspari Anthony A.
Published: April 2014

Human keratinocytes (KC), when cultured under conditions to remain undifferentiated or to terminally differentiate, changed their cellular distribution of CD1d. As studied by confocal microscopy, undifferentiated KC had a pool of cytoplasmic CD1d, whereas after terminal differentiation, this molecule localized in the cell membrane, which recapitulates CD1d expression in vivo. A comparison of undifferentiated and differentiated cultured KC did not reveal any differences in the association with β2-microglobulin, invariant chain of class II MHC, or patterns of glycosylation, suggesting that these biochemical properties are not regulating the cellular distribution of CD1d. Time-course studies of CD1d gene expression indicated that KC slowly increased gene expression with CaCl2-induced terminal differentiation. Increased CD1d gene expression was dependent on ceramide synthesis, because fumonisin B1, a ceramide synthetase inhibitor, blocked the increase in CD1d gene expression during terminal differentiation. Similarly, exogenous ceramide or the ceramidase inhibitor, B13, induced CD1d gene expression by undifferentiated, but not terminally differentiated, KC. A protein kinase C-ζ (PKC-ζ) inhibitor (a pseudosubstrate oligopeptide), but not a PKC-αβ inhibitor, significantly decreased CD1d gene expression by undifferentiated or ceramide-stimulated cultured, undifferentiated KC. As expected, downstream signaling events of PKC-ζ (JNK phosphorylation and NF-κΒ accumulation in the nucleus) were also attenuated. The calcineurin phosphatase inhibitor cyclosporine A, which blocks KC terminal differentiation, also blocked CD1d gene expression by cultured KC. In conclusion, this novel function of cellular ceramides extends the importance of this class of biologically active lipids beyond that of terminal differentiation and barrier function in normal human skin.
TNF-α Blockade Down-Regulates the CD40/CD40L Pathway in the Mucosal Microcirculation: A Novel Anti-Inflammatory Mechanism of Infliximab in Crohn’s Disease
Authors: Danese Silvio , Sans Miquel , Scaldaferri Franco , Sgambato Alessandro , Rutella Sergio , Cittadini Achille , Piqué Josep M. , Panes Julian , Katz Jeffry A. , Gasbarrini Antonio , Fiocchi Claudio
Published: April 2014

The CD40/CD40 ligand (CD40L) pathway is involved in Crohn’s disease (CD) pathogenesis. In the patients’ circulation, soluble CD40L (sCD40L) levels are elevated and surface CD40L is increased in platelets and T cells, whereas in the intestine CD40 is overexpressed in the microvasculature and CD40L in platelets and T cells. The therapeutic effects of infliximab in CD are attributed to its systemic anti-TNF-α action, but because TNF-α modulates both CD40 and CD40L, we investigated whether infliximab affects the CD40/CD40L pathway in the intestine. Eighteen CD patients were evaluated before and after infliximab therapy. Plasma sCD40L was measured by ELISA and platelet and peripheral blood T cell (PBT) CD40L expression by flow cytometry. Microvascular CD40 and VCAM-1 expression were assessed in mucosal biopsies by immunohistochemistry and by flow cytometry in human intestinal microvascular endothelial cells (HIMEC). Cell cultures were performed in the presence and absence of infliximab. Infliximab treatment significantly reduced plasma sCD40L levels and eliminated CD40 and VCAM-1 from mucosal microvessels. In vitro infliximab prevented TNF-α-induced CD40 and VCAM-1 expression by HIMEC, and reduced PBT, but not platelet, surface CD40L expression and sCD40L release. In addition, infliximab decreased T cell-induced VCAM-1 expression in HIMEC by down-regulating CD40L in T cells and promoting T cells apoptosis. These findings point to a novel mechanism of action of infliximab, i.e., the disruption of CD40/CD40L-dependent cognate interactions between intestinal microvessels and T cells. Thus, in addition to neutralizing TNF-α and inducing T cell death, the therapeutic effects of infliximab in CD appear to be also mediated by inhibition of vascular inflammation in the gut.