bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokine Dysregulation Induced by Apoptotic Cells Is a Shared Characteristic of Macrophages from Nonobese Diabetic and Systemic Lupus Erythematosus-Prone Mice
Authors: Fan Hanli , Longacre Angelika , Meng Fanyong , Patel Vimal , Hsiao Kevin , Koh Jason S. , Levine Jerrold S.
Published: April 2014

Macrophages from nonobese diabetic (NOD) mice, which spontaneously develop type I diabetes, share a defect in elicited cytokine production with macrophages from multiple diverse strains of systemic lupus erythematosus (SLE)-prone mice. We have previously shown that, in SLE-prone mice, this defect is triggered by exposure to apoptotic cells. We report in this work that macrophages from prediseased NOD mice also respond abnormally to apoptotic cells, mimicking closely the apoptotic cell-dependent abnormality that we have observed in multiple SLE-prone strains. This defect is characterized by the underexpression of IL-1β and multiple other cytokines. In the presence of apoptotic cells or FBS, elicited expression of IL-1β by NOD macrophages is markedly reduced compared with that by macrophages from control mice, including three strains of mice that develop type II (nonautoimmune) diabetes. Given the increasing role of apoptotic cells in tolerance and autoimmunity, a macrophage defect triggered by apoptotic cells has broad potential to upset the balance between tolerance and immunity. The concordance of this defect among so many diverse autoimmune-prone strains suggests that the genetic basis for this abnormality may constitute a permissive background for autoimmunity.
Triggering Receptor Expressed on Myeloid Cells-1 in Neutrophil Inflammatory Responses: Differential Regulation of Activation and Survival
Authors: Radsak Markus P. , Salih Helmut R. , Rammensee Hans-Georg , Schild Hansjörg
Published: April 2014

Polymorphonuclear neutrophils (PMN) are crucial in the innate host defense by their ability to rapidly accumulate in inflamed tissues and clear a site of infection from microbial pathogens by their potent effector mechanisms. The triggering receptor expressed on myeloid cells (TREM)-1 is a recently described activating receptor on PMN with an important role in inflammation. However, the effects of TREM-1 stimulation on a cellular level remain to be further defined. To characterize TREM-1-mediated activation of human PMN, we evaluated the effect of receptor ligation on PMN effector functions. Activation via TREM-1 induces immediate degranulation of neutrophilic granules resulting in the release of IL-8, respiratory burst, and phagocytosis. TREM-1 ligation synergizes with the activation by the Toll-like receptors (TLR) ligands LPS, Pam3Cys, and R-848. In contrast, no synergy between TREM-1- and TLR-mediated stimulation was observed concerning PMN survival, whereas TLR-mediated stimuli protect PMN from apoptosis, concurrent TREM-1 activation neutralizes these anti-apoptotic effects. These results give a new perspective for the regulation of neutrophil inflammatory responses emphasizing the importance of TREM-1 in innate immunity.
Differential Recognition and Scavenging of Native and Truncated Macrophage-Derived Chemokine (Macrophage-Derived Chemokine/CC Chemokine Ligand 22) by the D6 Decoy Receptor
Authors: Bonecchi Raffaella , Locati Massimo , Galliera Emanuela , Vulcano Marisa , Sironi Marina , Fra Anna M. , Gobbi Marco , Vecchi Annunciata , Sozzani Silvano , Haribabu Bodduluri , Van Damme Jo , Mantovani Alberto
Published: April 2014

The promiscuous D6 receptor binds several inflammatory CC chemokines and has been recently proposed to act as a chemokine-scavenging decoy receptor. The present study was designed to better characterize the spectrum of CC chemokines scavenged by D6, focusing in particular on CCR4 ligands and analyzing the influence of NH2-terminal processing on recognition by this promiscuous receptor. Using D6 transfectants, it was found that D6 efficiently bound and scavenged most inflammatory CC chemokines (CCR1 through CCR5 agonists). Homeostatic CC chemokines (CCR6 and CCR7 agonists) were not recognized by D6. The CCR4 agonists CC chemokine ligand 17 (CCL17) and CCL22 bound to D6 with high affinity. CCL17 and CCL22 have no agonistic activity for D6 (chemotaxis and calcium fluxes), but were rapidly scavenged, resulting in reduced chemotactic activity on CCR4 transfectants. CD26 mediates NH2 terminus processing of CCL22, leading to the production of CCL22 (3–69) and CCL22 (5–69) that do not interact with CCR4. These NH2-terminal truncated forms of CCL22 were not recognized by D6. The results presented in this study show that D6 recognizes and scavenges a wide spectrum of inflammatory CC chemokines, including the CCR4 agonists CCL22 and CCL17. However, this promiscuous receptor is not engaged by CD26-processed, inactive, CCL22 variants. By recognizing intact CCL22, but not its truncated variants, D6 expressed on lymphatic endothelial cells may regulate the traffic of CCR4-expressing cells, such as dendritic cells.
A Novel P2X7 Receptor Activator, the Human Cathelicidin-Derived Peptide LL37, Induces IL-1β Processing and Release
Authors: Elssner Andreas , Duncan Michelle , Gavrilin Mikhail , Wewers Mark D.
Published: April 2014

The release of IL-1β is a tightly controlled process that requires induced synthesis of the precursor pro-IL-1β and a second stimulus that initiates cleavage and secretion of mature IL-1β. Although ATP as a second stimulus potently promotes IL-1β maturation and release via P2X7 receptor activation, millimolar ATP concentrations are needed. The human cathelicidin-derived peptide LL37 is a potent antimicrobial peptide produced predominantly by neutrophils and epithelial cells. In this study, we report that LL37 stimulation of LPS-primed monocytes leads to maturation and release of IL-1β via the P2X7 receptor. LL37 induces a transient release of ATP, membrane permeability, caspase-1 activation, and IL-1β release without cell cytotoxicity. IL-1β release and cell permeability are suppressed by pretreatment with the P2X7 inhibitors oxidized ATP, KN04, and KN62. In the presence of apyrase, which hydrolyzes ATP to AMP, the effect of LL37 was not altered, indicating that LL37 rather than autocrine ATP is responsible for the activation of the P2X7 receptor. We conclude that endogenous LL37 may promote IL-1β processing and release via direct activation of P2X7 receptors.
Flt-3 Ligand Reverses Late Allergic Response and Airway Hyper-Responsiveness in a Mouse Model of Allergic Inflammation
Authors: Edwan Jehad H. , Perry Greg , Talmadge James E. , Agrawal Devendra K.
Published: April 2014

Flt3 ligand (Flt3-L) is a growth factor for dendritic cells and induces type 1 T cell responses. We recently reported that Flt3-L prevented OVA-induced allergic airway inflammation and suppressed late allergic response and airway hyper-responsiveness (AHR). In the present study we examined whether Flt3-L reversed allergic airway inflammation in an established model of asthma. BALB/c mice were sensitized and challenged with OVA, and AHR to methacholine was established. Then mice with AHR were randomized and treated with PBS or 6 μg of Flt3-L i.p. for 10 days. Pulmonary functions and AHR to methacholine were examined after rechallenge with OVA. Treatment with Flt3-L of presensitized mice significantly suppressed (p < 0.001) the late allergic response, AHR, bronchoalveolar lavage fluid total cellularity, absolute eosinophil counts, and inflammation in the lung tissue. There was a significant decrease in proinflammatory cytokines (TNF-α, IL-4, and IL-5) in bronchoalveolar lavage fluid, with a significant increase in serum IL-12 and a decrease in serum IL-5 levels. There was no significant effect of Flt3-L treatment on serum IL-4 and serum total IgE levels. Sensitization with OVA significantly increased CD11b+CD11c+ cells in the lung, and this phenomenon was not significantly affected by Flt3-L treatment. These data suggest that Flt3-L can reverse allergic airway inflammation and associated changes in pulmonary functions in murine asthma model.
Helicobacter pylori-Specific Antibodies Impair the Development of Gastritis, Facilitate Bacterial Colonization, and Counteract Resistance against Infection
Authors: Akhiani Ali A. , Schön Karin , Franzén Lennart E. , Pappo Jacques , Lycke Nils
Published: April 2014

In recent years, Abs have been considered a correlate rather than an effector of resistance against Helicobacter pylori infection. However, it is still poorly understood to what extent Ab production correlates with gastric immunopathology. Here we report that Abs not only are dispensable for protection, but they are detrimental to elimination of the bacteria and appear to impair gastric inflammatory responses. We found that the initial colonization with H. pylori bacteria was normal in the B cell-deficient (μMT) mice, whereas at later times (>8 wk) most of the bacteria were cleared, concomitant with the development of severe gastritis. In contrast, wild-type (WT) mice exhibited extensive bacterial colonization and only mild gastric inflammation, even at 16 wk after inoculation. Oral immunizations with H. pylori lysate and cholera toxin adjuvant stimulated comparable levels of protection in μMT and WT mice. The level of protection in both strains correlated well with the severity of the postimmunization gastritis. Thus, T cells were responsible for the gastritis, whereas Abs, including potentially host cell cross-reactive Abs, were not involved in causing the gastritis. The T cells in μMT and WT mice produced high and comparable levels of IFN-γ to recall Ag at 2 and after 8 wk, whereas IL-4 was detected after 8 wk only, indicating that Th1 activity dominated the early phase of protection, whereas later a mixed Th1 and Th2 activity was seen.
CXCL1/Macrophage Inflammatory Protein-2-Induced Angiogenesis In Vivo Is Mediated by Neutrophil-Derived Vascular Endothelial Growth Factor-A
Authors: Scapini Patrizia , Morini Monica , Tecchio Cristina , Minghelli Simona , Di Carlo Emma , Tanghetti Elena , Albini Adriana , Lowell Clifford , Berton Giorgio , Noonan Douglas M. , Cassatella Marco A.
Published: April 2014

The angiogenic activity of CXC-ELR+ chemokines, including CXCL8/IL-8, CXCL1/macrophage inflammatory protein-2 (MIP-2), and CXCL1/growth-related oncogene-α in the Matrigel sponge angiogenesis assay in vivo, is strictly neutrophil dependent, as neutrophil depletion of the animals completely abrogates the angiogenic response. In this study, we demonstrate that mice deficient in the src family kinases, Hck and Fgr (hck−/−fgr−/−), are unable to develop an angiogenic response to CXCL1/MIP-2, although they respond normally to vascular endothelial growth factor-A (VEGF-A). Histological examination of the CXCL1/MIP-2-containing Matrigel implants isolated from wild-type or hck−/−fgr−/− mice showed the presence of an extensive neutrophil infiltrate, excluding a defective neutrophil recruitment into the Matrigel sponges. Accordingly, neutrophils from hck−/−fgr−/− mice normally migrated and released gelatinase B in response to CXCL1/MIP-2 in vitro, similarly to wild-type neutrophils. However, unlike wild-type neutrophils, those from hck−/−fgr−/− mice were completely unable to release VEGF-A upon stimulation with CXCL1/MIP-2. Furthermore, neutralizing anti-VEGF-A Abs abrogated the angiogenic response to CXCL1/MIP-2 in wild-type mice and CXCL1/MIP-2 induced angiogenesis in the chick embryo chorioallantoic membrane assay, indicating that neutrophil-derived VEGF-A is a major mediator of CXCL1/MIP-2-induced angiogenesis. Finally, in vitro kinase assays confirmed that CXCL1/MIP-2 activates Hck and Fgr in murine neutrophils. Taken together, these data demonstrate that CXCL1/MIP-2 leads to recruitment of neutrophils that, in turn, release biologically active VEGF-A, resulting in angiogenesis in vivo. Our observations delineate a novel mechanism by which CXCL1/MIP-2 induces neutrophil-dependent angiogenesis in vivo.
Human Intestinal Microvascular Endothelial Cells Express Toll-Like Receptor 5: A Binding Partner for Bacterial Flagellin
Authors: Maaser Christian , Heidemann Jan , von Eiff Christof , Lugering Andreas , Spahn Thomas W. , Binion David G. , Domschke Wolfram , Lugering Norbert , Kucharzik Torsten
Published: April 2014

Bacterial flagellin has recently been identified as a ligand for Toll-like receptor 5 (TLR5). Human sites known to specifically express TLR5 include macrophages and gastric and intestinal epithelium. Because infection of intestinal epithelial cells with Salmonella leads to an active transport of flagellin to the subepithelial compartment in proximity to microvessels, we hypothesized that human intestinal endothelial cells functionally express TLR5, thus enabling an active inflammatory response upon binding of translocated flagellin. Endothelial expression of TLR5 in human macro- and microvascular endothelial cells was examined by RT-PCR, immunoblot analysis, and immunofluorescence. Endothelial expression of TLR5 in vivo was verified by immunohistochemistry. Endothelial modulation of ICAM-1 expression was quantitated using flow cytometry, and leukocyte transmigration in vitro was assessed by an endothelial transmigration assay. Epithelial-endothelial cellular interactions upon infection with viable Salmonella were investigated using a coculture system in vitro. We found that Salmonella-infected intestinal epithelial cells induce endothelial ICAM-1 expression in cocultured human endothelial cells. Both macro- (HUVEC) and microvascular endothelial cells derived from human skin (human dermal microvascular endothelial cell 1) and human colon (human intestinal microvascular endothelial cells) were found to express high constitutive amounts of TLR5 mRNA and protein. These findings were paralleled by strong immunoreactivity for TLR5 of normal human colonic microvessels in vivo. Furthermore, incubation of human dermal microvascular endothelial cells with flagellin from clinical isolates of Escherichia and Salmonella strains led to a marked up-regulation of ICAM-1, as well as to an enhanced leukocyte transendothelial cell migration. These results suggest that endothelially expressed TLR5 might play a previously unrecognized role in the innate immune response toward bacterial Ags.
A Novel Locus Regulates Both Retroviral Glycoprotein 70 and Anti-Glycoprotein 70 Antibody Production in New Zealand Mice When Crossed with BALB/c
Authors: Rigby Robert J. , Rozzo Stephen J. , Gill Herpreet , Fernandez-Hart Timothy , Morley Bernard J. , Izui Shozo , Kotzin Brian L. , Vyse Timothy J.
Published: April 2014

Lupus-prone New Zealand Black and New Zealand White mice produce high serum levels of the endogenous retroviral envelope protein gp70 and develop an Ab response to this autoantigen as part of their autoimmune disease. Linkage analysis of two crosses involving New Zealand and BALB/c mice mapped these traits to a group of overlapping loci, including a novel locus on proximal chromosome 12. This locus was linked with serum gp70 and the autoimmune response against it. The linkage of serum gp70 levels to a previously described locus on distal chromosome 4 was also confirmed. Sequence analysis of a candidate gene on distal chromosome 4, Fv1, provided support that this gene may be associated with the control of serum gp70 levels in both New Zealand Black and New Zealand White mice. Linkage data and statistical analysis confirmed a close correlation between gp70 Ag and anti-gp70 Ab levels, and together gave support to the concept that a threshold level of gp70 is required for the production of anti-gp70 Abs. Serum levels of anti-gp70 Abs were closely correlated with the presence of renal disease, more so than anti-dsDNA Abs. Understanding the genetic basis of this complex autoantigen-autoantibody system will provide insight into the pathogenesis of lupus in mice, which may have implications for human disease.
Thalidomide Induces Apoptosis in Human Monocytes by Using a Cytochrome c-Dependent Pathway
Authors: Gockel Henning R. , Lügering Andreas , Heidemann Jan , Schmidt Michael , Domschke Wolfram , Kucharzik Torsten , Lügering Norbert
Published: April 2014

Thalidomide has been shown to be an effective treatment in various immunologic diseases such as Crohn’s disease and rheumatoid arthritis. Its major effect is thought to be mediated by the inhibition of TNF-α, but the exact mechanism of action is still uncertain. Recent observations could demonstrate that the induction of monocyte apoptosis is a common feature of a variety of anti-inflammatory agents. Therefore, we investigated the role of thalidomide on monocyte apoptosis. Treatment with thalidomide resulted in apoptosis of human peripheral blood monocytes in a time- and dose-dependent manner as demonstrated by annexin V staining. Monocyte apoptosis required the activation of caspases, as combined stimulation by thalidomide together with the broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone markedly prevented monocyte cell death. Apoptosis was triggered by a CD95/CD95 ligand, TNF-RI, and TRAIL-R1 independent pathway with an inhibition of AKT-1 kinase and consecutive mitochondrial release of cytochrome c, followed by the proteolytic activation of initiator caspase-9 and effector caspase-3. Our data suggest that thalidomide-induced monocyte apoptosis is at least partially mediated by a mitochondrial signaling pathway and might contribute to the complex immunomodulatory properties of the drug.
Increased CD8+ Cytotoxic T Cell Responses to Myelin Basic Protein in Multiple Sclerosis
Authors: Zang Ying C. Q. , Li Sufang , Rivera Victor M. , Hong Jian , Robinson Rachel R. , Breitbach Wini T. , Killian James , Zhang Jingwu Z.
Published: April 2014

Autoreactive T cells of CD4 and CD8 subsets recognizing myelin basic protein (MBP), a candidate myelin autoantigen, are thought to contribute to and play distinct roles in the pathogenesis of multiple sclerosis (MS). In this study we identified four MBP-derived peptides that had high binding affinity to HLA-A2 and HLA-A24 and characterized the CD8+ T cell responses and their functional properties in patients with MS. There were significantly increased CD8+ T cell responses to 9-mer MBP peptides, in particular MBP111–119 and MBP87–95 peptides that had high binding affinity to HLA-A2, in patients with MS compared with healthy individuals. The resulting CD8+ T cell lines were of the Th1 phenotype, producing TNF-α and IFN-γ and belonged to a CD45RA−/CD45RO+ memory T cell subset. Further characterization indicated that the CD8+ T cell lines obtained were stained with MHC class I tetramer (HLA-A2/MBP111–119) and exhibited specific cytotoxicity toward autologous target cells pulsed with MBP-derived peptides in the context of MHC class I molecules. These cytotoxic CD8+ T cell lines derived from MS patients recognized endogenously processed MBP and lysed COS cells transfected with genes encoding MBP and HLA-A2. These findings support the potential role of CD8+ CTLs recognizing MBP in the injury of oligodendrocytes expressing both MHC class I molecules and MBP.
Accommodation: Preventing Injury in Transplantation and Disease
Authors: Koch Cody A. , Khalpey Zain I. , Platt Jeffrey L.
Published: April 2014

Humoral immunity, as a cause of damage to blood vessels, poses a major barrier to successful transplantation of organs. Under some conditions, humoral immunity causes little or no damage to an organ graft. We have referred to this condition, in which a vascularized graft functions in the face of humoral immunity directed against it, as “accommodation.” In this paper, we review changes in the graft and in the host that may account for accommodation, and we consider that what we call accommodation of organ grafts may occur widely in the context of immune responses, enabling immune responses to target infectious organisms without harming self-tissues.
Cutting Edge: IL-21 Is a Switch Factor for the Production of IgG1 and IgG3 by Human B Cells
Authors: Pène Jérôme , Gauchat Jean-François , Lécart Sandrine , Drouet Elodie , Guglielmi Paul , Boulay Vera , Delwail Adriana , Foster Don , Lecron Jean-Claude , Yssel Hans
Published: April 2014

IL-21 is a cytokine that regulates the activation of T and NK cells and promotes the proliferation of B cells activated via CD40. In this study, we show that rIL-21 strongly induces the production of all IgG isotypes by purified CD19+ human spleen or peripheral blood B cells stimulated with anti-CD40 mAb. Moreover, it was found to specifically induce the production of IgG1 and IgG3 by CD40-activated CD19+CD27− naive human B cells. Although stimulation of CD19+ B cells via CD40 alone induced γ1 and γ3 germline transcripts, as well as the expression of activation-induced cytidine deaminase, only stimulation with both anti-CD40 mAb and rIL-21 resulted in the production of Sγ/Sμ switch circular DNA. These results show that IL-21, in addition to promoting growth and differentiation of committed B cells, is a specific switch factor for the production of IgG1 and IgG3.
Inflammatory Cytokines Overcome Age-Related Defects in CD4 T Cell Responses In Vivo
Authors: Haynes Laura , Eaton Sheri M. , Burns Eve M. , Rincon Mercedes , Swain Susan L.
Published: April 2014

Age-related decreases in immune function are thought to contribute to the reduced efficacy of vaccinations seen in elderly populations. Our previous in vitro studies demonstrated that naive CD4 T cells from aged TCR-transgenic mice proliferate less than young cells and generate poorly functioning effectors due to decreased IL-2 production. In this current study, we show that this age-related defect in CD4 T cell response also occurs in vivo and that it is correlated with reduced NF-κB activation. After transfer to young hosts, CD4 T cells from aged transgenic mice proliferate less and produce reduced levels of IL-2 upon immunization with Ag and alum. Introducing a combination of the inflammatory cytokines TNF-α, IL-1, and IL-6, or the use of an adjuvant such as CFA that induces these cytokines, markedly enhanced responses of these aged CD4 T cells, so that they proliferated and produced IL-2 similar to young cells. This enhancement is correlated with the enhanced activation of the transcription factor NF-κB in aged cells. We suggest that induction of inflammatory cytokines via adjuvants may enhance the efficacy of vaccinations in elderly populations.
CTL-Dependent and -Independent Antitumor Immunity Is Determined by the Tumor Not the Vaccine
Authors: Leitch Jaina , Fraser Katie , Lane Cecilia , Putzu Kelley , Adema Gosse J. , Zhang Qian-Jin , Jefferies Wilfred A. , Bramson Jonathan L. , Wan Yonghong
Published: April 2014

Previously, we compared the efficiency of direct injection with an adenovirus (Ad) expressing human gp100 (hgp100) to immunization with dendritic cells (DC) loaded with the same vector ex vivo. The DC vaccine provided the greatest protection against challenge with B16F10 melanoma, and antitumor immunity was found to be CD8+ T cell-independent. In the current study, we sought to determine whether lack of CD8+ T cell-mediated antitumor immunity was a function of the vaccine platform or the tumor line. Both Ad and DC/Ad vaccines elicited CD8+ CTL reactive against hgp100 and provided protection against B16F10 engineered to express hgp100 demonstrating that both vaccination platforms can effectively generate protective CD8+ T cell-mediated immunity. The hgp100-induced CTL cross-reacted with murine gp100 (mgp100) and lysed B16F10 cells pulsed with mgp100 peptide indicating that the resistance of B16F10 cells to CTL elicited by hgp100 vaccination may be due to a defect in processing of the endogenous mgp100. Indeed, introduction of the TAP-1 cDNA into B16F10 rendered the cells sensitive to lysis by gp100-specific CTL. Furthermore, gp100-immunized mice were protected from challenge with B16F10-TAP1 cells through a mechanism dependent upon CD8+ T cells. These results demonstrate that tumor phenotype, not the vaccination platform, ultimately determines CD8+ or CD4+ T cell-mediated tumor clearance.
Dihydropyridine Receptors Are Selective Markers of Th2 Cells and Can Be Targeted to Prevent Th2-Dependent Immunopathological Disorders
Authors: Savignac Magali , Gomes Bruno , Gallard Alexandra , Narbonnet Stéphane , Moreau Marc , Leclerc Catherine , Paulet Pierre , Mariamé Bernard , Druet Philippe , Saoudi Abdelhadi , Fournié Gilbert J. , Guéry Jean-Charles , Pelletier Lucette
Published: April 2014

Th1 cells that produce IFN-γ are essential in the elimination of intracellular pathogens, and Th2 cells that synthetize IL-4 control the eradication of helminths. However, highly polarized Th1 or Th2 responses may be harmful and even lethal. Thus, the development of strategies to selectively down-modulate Th1 or Th2 responses is of therapeutic importance. Herein, we demonstrate that dihydropyridine receptors (DHPR) are expressed on Th2 and not on Th1 murine cells. By using selective agonists and antagonists of DHPR, we show that DHPR are involved in TCR-dependent calcium response in Th2 cells as well as in IL-4, IL-5, and IL-10 synthesis. Nicardipine, an inhibitor of DHPR, is beneficial in experimental models of Th2-dependent pathologies in rats. It strongly inhibits the Th2-mediated autoimmune glomerulonephritis induced by injecting Brown Norway (BN) rats with heavy metals. This drug also prevents the chronic graft vs host reaction induced by injecting CD4+ T cells from BN rats into (LEW × BN)F1 hybrids. By contrast, treatment with nicardipine has no effect on the Th1-dependent experimental autoimmune encephalomyelitis triggered in LEW rats immunized with myelin. These data indicate that 1) DHPR are a selective marker of Th2 cells, 2) these calcium channels contribute to calcium signaling in Th2 cells, and 3) blockers of these channels are beneficial in the treatment of Th2-mediated pathologies.
Natural and Induced CD4+CD25+ Cells Educate CD4+CD25− Cells to Develop Suppressive Activity: The Role of IL-2, TGF-β, and IL-10
Authors: Zheng Song Guo , Wang Ju Hua , Gray J. Dixon , Soucier Harold , Horwitz David A.
Published: April 2014

Thymus-derived, natural CD4+CD25+ regulatory T cells can educate peripheral CD4+CD25− cells to develop suppressive activity by poorly understood mechanisms. TGF-β has IL-2-dependent costimulatory effects on alloactivated naive, human CD4+ T cells and induces them ex vivo to become potent contact-dependent, cytokine-independent suppressor cells. In this study, we report that CD4+CD25+ cells are the targets of the costimulatory effects of IL-2 and TGF-β. These cells do not divide, but, instead, greatly increase the numbers of CD4+CD25− cells that become CD25+ cytokine-independent suppressor cells. These CD4+CD25+ regulatory cells, in turn, induce other alloactivated CD4+CD25− cells to become potent suppressor cells by mechanisms that, surprisingly, require both cell contact and TGF-β and IL-10. The suppressive effects of these secondary CD4+CD25+ cells depend upon TGF-β and IL-10. Moreover, both the naive CD4+ cells induced by IL-2 and TGF-β to become suppressor cells, and the subsequent CD4+CD25− cells educated by them to become suppressors express FoxP3. We suggest that the long-term effects of adoptively transferred natural-like CD4+CD25+ regulatory cells induced ex vivo are due to their ability to generate new cytokine-producing CD4+ regulatory T cells in vivo.
Passive and Active Mechanisms Trap Activated CD8+ T Cells in the Liver
Authors: John Beena , Crispe Ian Nicholas
Published: April 2014

The liver is a site where activated CD8+ T cells are trapped and destroyed at the end of an immune response. The intrahepatic accumulation of activated murine TCR transgenic CD8+ T cells was significantly reduced when either ICAM-1 or VCAM-1 was blocked by specific Ab. These two adhesion mechanisms account for essentially all the trapping of activated CD8+ T cells in the mouse liver. Although the ICAM-1-mediated trapping depends on the capacity of the vasculature and/or the parenchymal cells to present Ag, the accumulation of cells through VCAM-1 does not require Ag recognition. Thus, Ags expressed by the non-bone marrow-derived cells in the liver actively cause CD8+ T cell accumulation through TCR-activated ICAM-1 adhesion, but the liver can also passively sequester activated CD8+ T cells that do not recognize intrahepatic Ag, through VCAM-1 adhesion.
Antigen-Specific T Cell Repertoire Modification of CD4+CD25+ Regulatory T Cells
Authors: Hayashi Yuki , Tsukumo Shin-ichi , Shiota Hiroshi , Kishihara Kenji , Yasutomo Koji
Published: April 2014

T cell immune responses are regulated by the interplay between effector and suppressor T cells. Immunization with Ag leads to the selective expansion and survival of effector CD4+ T cells with high affinity TCR against the Ag and MHC. However, it is not known if CD4+CD25+ regulatory T cells (Treg) recognize the same Ag as effector T cells or whether Ag-specific TCR repertoire modification occurs in Treg. In this study, we demonstrate that after a primary Ag challenge, Treg proliferate and TCR repertoire modification is observed although both of these responses were lower than those in conventional T cells. The repertoire modification of Ag-specific Treg after primary Ag challenge augmented the total suppressive function of Treg against TCR repertoire modification but not against the proliferation of memory CD4+ T cells. These results reveal that T cell repertoire modification against a non-self Ag occurs in Treg, which would be crucial for limiting excess primary and memory CD4+ T cell responses. In addition, these studies provide evidence that manipulation of Ag-specific Treg is an ideal strategy for the clinical use of Treg.
The HIV-1 HLA-A2-SLYNTVATL Is a Help-Independent CTL Epitope
Authors: Kan-Mitchell June , Bisikirska Brygida , Wong-Staal Flossie , Schaubert Keri L. , Bajcz Melissa , Bereta Michal
Published: April 2014

The CTL response to the HLA-A*0201-restricted, HIV-1 p17 Gag77–85 epitope (SLYNTVATL; SL9) has been extensively studied in patients. Although this reactivity is exceptionally prominent in chronically infected patients and inversely correlated to viral load, SL9-specific CTLs (SL9-CTLs) are rarely detected in acute infection. To explore the cellular basis for this unusual manifestation, SL9-CTLs primed ex vivo from naive circulating CD8+ T cells of healthy, seronegative donors were generated and characterized. SL9 appeared to differ from other well-studied A*0201-restricted epitopes in several significant respects. In contrast to published reports for influenza and melanoma peptides and the HIV gag IV9 epitope studied here in parallel, SL9-CTLs were primed by immature but not mature autologous dendritic cells. Highly activated SL9-CTLs produce sufficient autocrine mediators to sustain clonal expansion and CTL differentiation for months without CD4+ T cells or exogenous IL-2. Moreover, SL9-CTLs were sensitive to paracrine IL-2-induced apoptosis. IL-2 independence and sensitivity to paracrine IL-2 were also characteristic of SL9-CTLs immunized by dendritic cells transduced by a nonreplicating lentiviral vector encoding full-length Gag. In vitro-primed SL9-CTLs resembled those derived from patients in degeneracy of recognition and functional avidities for both SL9 and its natural mutations. Together, these data show that SL9 is a highly immunogenic, help-independent HIV epitope. The scarcity of SL9-CTLs in acute infection may result from cytokine-induced apoptosis with the intense activation of the innate immunity. In contrast, SL9-CTLs that constitutively produce autocrine help would predominate during CD4-diminished chronic infection.
IgG2a-Mediated Enhancement of Antibody and T Cell Responses and Its Relation to Inhibitory and Activating Fcγ Receptors
Authors: Getahun Andrew , Dahlström Jörgen , Wernersson Sara , Heyman Birgitta
Published: April 2014

A number of studies in experimental animal models point to an important role of FcγRs in autoimmunity and allergy. In this study, we investigate how the production of IgG, an early step in the chain of events leading to inflammation, is regulated by activating and inhibitory FcγRs. IgG Abs are known to feedback-enhance Ab responses to soluble Ags, and this effect requires activating FcγRs. To test proliferation of Th cells, mice were adoptively transferred with CD4+ T cells expressing a transgenic OVA-specific TCR before immunization with IgG2a anti-2,4,6-trinitrophenyl (TNP) plus OVA-TNP or with OVA-TNP alone. IgG2a induced a significant increase in OVA-specific T cell numbers, which preceded the OVA-specific Ab response and was dependent on the FcRγ chain. The role of the inhibitory FcγRIIB in Ab responses was studied in mice lacking this receptor. Although IgG2a enhanced primary Ab responses, development of germinal centers, and immunological memory in wild-type mice, enhancement was markedly stronger in FcγRIIB−/− mice. The presented data are compatible with the hypothesis that the mechanism behind IgG2a-mediated up-regulation of Ab responses involves increased Ag presentation to CD4+ T cells by FcγR+ APCs. Our observations also illustrate the intricate immunoregulatory role of IgG Abs. On the one hand, they enhance Ab responses via activating FcγRs, and on the other hand, they set an upper limit for the same Ab response via FcγRIIB.
Distinct IL-2 Receptor Signaling Pattern in CD4+CD25+ Regulatory T Cells
Authors: Bensinger Steven J. , Walsh Patrick T. , Zhang Jidong , Carroll Martin , Parsons Ramon , Rathmell Jeffrey C. , Thompson Craig B. , Burchill Matthew A. , Farrar Michael A. , Turka Laurence A.
Published: April 2014

Despite expression of the high-affinity IL-2R, CD4+CD25+ regulatory T cells (Tregs) are hypoproliferative upon IL-2R stimulation in vitro. However the mechanisms by which CD4+CD25+ T cells respond to IL-2 signals are undefined. In this report, we examine the cellular and molecular responses of CD4+CD25+ Tregs to IL-2. IL-2R stimulation results in a G1 cell cycle arrest, cellular enlargement and increased cellular survival of CD4+CD25+ T cells. We find a distinct pattern of IL-2R signaling in which the Janus kinase/STAT pathway remains intact, whereas IL-2 does not activate downstream targets of phosphatidylinositol 3-kinase. Negative regulation of phosphatidylinositol 3-kinase signaling and IL-2-mediated proliferation of CD4+CD25+ T cells is inversely associated with expression of the phosphatase and tensin homologue deleted on chromosome 10, PTEN.
CD1d1 Displayed on Cell Size Beads Identifies and Enriches an NK Cell Population Negatively Regulated by CD1d1
Authors: Huang Michael M. S. , Borszcz Peter , Sidobre Stephane , Kronenberg Mitchell , Kane Kevin P.
Published: April 2014

NK cells destroy microbe-infected cells while sparing healthy cells, and are controlled, in part, by inhibitory receptors specific for class I Ag-presenting molecules. CD1d1, a β2-microglobulin-associated class I-like molecule, binds glycolipids and stimulates NKT cells. We previously demonstrated that target cell lysis by IL-2-activated mouse NK cells is inhibited by target cell expression of CD1d1, suggesting that IL-2-activated NK cells may express a CD1d1-specific inhibitory receptor. We now report that a significant subset of mouse IL-2-activated NK cells specifically binds cell size beads displaying either naturally expressed or recombinant CD1d1. In contrast, although tetramers of soluble recombinant CD1d1 loaded with α-galactosylceramide identify NKT cells, binding of this reagent to resting or IL-2-activated NK cells was undetectable, even with activated NK cells sorted with CD1d1 beads. Cytotoxicity by the CD1d1 bead-separated NK subset was strongly inhibited by CD1d1, compared with the NK cell subset not bound to CD1d1 beads. An Ab that blocks NKT cell recognition of CD1d1 also reverses CD1d1 inhibition of NK lysis, suggesting that TCRs of NKT cells and NK inhibitory receptor(s) may interact with a similar site on CD1d1. These results provide direct evidence for a physical interaction of NK cells with CD1d1, mediated by a functional, CD1d1-specific low-affinity inhibitory NK receptor. Display of ligands on cell size beads to maximize multivalent interaction may offer an alternative approach to examine NK cell receptor-ligand interactions, particularly those of lower expression and/or lower affinity/avidity that may go undetected using tetrameric reagents.
Latent Membrane Protein 2A, a Viral B Cell Receptor Homologue, Induces CD5+ B-1 Cell Development
Authors: Ikeda Akiko , Merchant Mark , Lev Lori , Longnecker Richard , Ikeda Masato
Published: April 2014

The latent membrane protein 2A (LMP2A) of EBV plays a key role in regulating viral latency and EBV pathogenesis by functionally mimicking a constitutively active B cell Ag receptor. When expressed as a B cell-specific transgene in mice, LMP2A drives B cell development, resulting in the bypass of normal developmental checkpoints. In this study, we have demonstrated that expression of LMP2A in transgenic mice results in B cell development that exclusively favors B-1 cells. This switch to B-1 cell development occurs at the pre-B-cell stage of normal B cell development in the bone marrow, a B cell stage much earlier than appreciated for B-1 commitment. This finding indicates that all pre-B cells have the capacity to assume a B-1 cell phenotype if they encounter the appropriate signal during normal development. Furthermore, these studies offer insight into EBV latency and pathogenesis in the human host.
Deficiencies in Gut NK Cell Number and Function Precede Diabetes Onset in BB Rats
Authors: Todd Derrick J. , Forsberg Eric M. , Greiner Dale L. , Mordes John P. , Rossini Aldo A. , Bortell Rita
Published: April 2014

Defects in the intestinal immune system may contribute to the pathogenesis of autoimmune diseases. Intraepithelial lymphocytes represent a substantial fraction of gut-associated lymphocytes, but their function in mucosal immunity is unclear. A newly described population of NK cells that spontaneously secrete IL-4 and IFN-γ is present in the intraepithelial lymphocyte compartment of the rat. We hypothesized that defects in the number or function of these cells would be present in rats susceptible to autoimmunity. We report that the number of NKR-P1A+CD3− intraepithelial NK (IENK) cells is deficient before onset of spontaneous autoimmune diabetes in diabetes-prone BB (BBDP) rats. The absolute number of recoverable IENK cells was only ∼8% of that observed in WF rats. Bone marrow transplantation from histocompatible WF donors reversed the IENK cell deficiency (and prevented diabetes) in these animals, suggesting a hemopoietic origin for their IENK cell defect. Analysis of diabetes-resistant BB rats, which develop autoimmune diabetes only after perturbation of the immune system, revealed IENK cell numbers intermediate between that of BBDP and WF rats. IENK cells were selectively depleted during treatment to induce diabetes. Prediabetic BBDP and diabetes-resistant BB animals also exhibited defective IENK cell function, including decreased NK cell cytotoxicity and reduced secretion of IL-4 and IFN-γ. IENK functional defects were also observed in LEW and BN rats, which are susceptible to induced autoimmunity, but not in WF, DA, or F344 rats, which are resistant. Defects in IENK cell number and function may contribute to the pathogenesis of autoimmune diseases including type 1 diabetes.
Control of TCR-Mediated Activation of β1 Integrins by the ZAP-70 Tyrosine Kinase Interdomain B Region and the Linker for Activation of T Cells Adapter Protein
Authors: Goda Seiji , Quale Angie C. , Woods Melody L. , Felthauser Alicia , Shimizu Yoji
Published: April 2014

One of the earliest functional responses of T lymphocytes to extracellular signals that activate the Ag-specific CD3/TCR complex is a rapid, but reversible, increase in the functional activity of integrin adhesion receptors. Previous studies have implicated the tyrosine kinase ζ-associated protein of 70 kDa (ZAP-70) and the lipid kinase phosphatidylinositol 3-kinase, in the activation of β1 integrins by the CD3/TCR complex. In this report, we use human ZAP-70-deficient Jurkat T cells to demonstrate that the kinase activity of ZAP-70 is required for CD3/TCR-mediated increases in β1 integrin-mediated adhesion and activation of phosphatidylinositol 3-kinase. A tyrosine to phenylalanine substitution at position 315 in the interdomain B of ZAP-70 inhibits these responses, whereas a similar substitution at position 292 enhances these downstream signals. These mutations in the ZAP-70 interdomain B region also specifically affect CD3/TCR-mediated tyrosine phosphorylation of residues 171 and 191 in the cytoplasmic domain of the linker for activation of T cells (LAT) adapter protein. CD3/TCR signaling to β1 integrins is defective in LAT-deficient Jurkat T cells, and can be restored with expression of wild-type LAT. Mutant LAT constructs with tyrosine to phenylalanine substitutions at position 171 and/or position 191 do not restore CD3/TCR-mediated activation of β1 integrins in LAT-deficient T cells. Thus, these studies demonstrate that the interdomain B region of ZAP-70 regulates β1 integrin activation by the CD3/TCR via control of tyrosine phosphorylation of tyrosine residues 171 and 191 in the LAT cytoplasmic domain.
The Distribution of Antigen in Lymphoid Tissues following Its Injection into the Anterior Chamber of the Rat Eye
Authors: Camelo Serge , Shanley Adam , Voon Angel S. P. , McMenamin Paul G.
Published: April 2014

Injection of Ag into the anterior chamber (AC) of the eye induces deviant immune responses. It has been proposed that Ag internalized by ocular APCs is presented in a tolerogenic fashion in the spleen. However, the nature and distribution of the Ag-bearing cells in the lymphoid organs remain unclear. Fluorescent-labeled Ag (dextran, BSA) injected into the AC of Lewis rats was detected in the subcapsular sinus of the right submandibular lymph nodes (LNs) and cervical LNs, the marginal zone of the spleen, and the medulla of the mesenteric LNs. In the spleen, Ag-bearing cells were CD1+, CD11b+, ED1+, ED2low, ED3+, CD86low, OX6+, CD11c−, ED5− and in the LNs were CD4+, CD8+, CD80+, and OX41+ suggesting these were lymphoid organ resident macrophages. These Ag-bearing macrophages were located adjacent to CD4+ cells, CD8+ cells, and NK cells in the LNs and spleen and to marginal zone B cells in the spleen. No interaction with γδ T cells was observed. The data demonstrates that Ag derived from the AC of the eye is mainly internalized by resident macrophages in the LNs and spleen which are ideally placed to interact with cells involved in the induction of deviant ocular immune responses. The extensive distribution of Ag in LNs draining the upper airway and gastrointestinal tracts, together with the phenotype of Ag-bearing cells in the lymphoid organs, suggests that Ag leaves the eye predominantly in a soluble form and implies other mechanisms of tolerance may contribute to ocular-specific immune responses.
Cathepsin-Dependent Apoptosis Triggered by Supraoptimal Activation of T Lymphocytes: A Possible Mechanism of High Dose Tolerance
Authors: Michallet Marie-Cécile , Saltel Frédéric , Flacher Monique , Revillard Jean-Pierre , Genestier Laurent
Published: April 2014

High doses of Ag can paradoxically suppress immune responses in vivo. This Ag-specific unresponsiveness (termed high dose tolerance) involves extrathymic mechanisms in mature T lymphocytes. To investigate these mechanisms, we used the in vitro model of PBL activated with anti-CD3 or PHA. In these conditions, increasing mitogen concentrations resulted in a reduction of the proliferative response, associated with an increased percentage of apoptotic cells. Apoptosis did not require prior exposure to IL-2, it was not the consequence of CD178/CD95 or TNF/TNFR interactions, and was therefore clearly distinct from activation-induced cell death. Although the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) decreased DNA fragmentation, cytochrome c release and caspase-9 and caspase-3 activation were not implicated, suggesting that this apoptosis did not primarily involve the intrinsic mitochondrial pathway. E64d, a cysteine protease inhibitor, as well as specific inhibitors of cathepsin B and cathepsin L conferred protection. We further demonstrated that cathepsin B and cathepsin L were released from the lysosomes and catalytically active in the cytosol. Release of cathepsin B and cathepsin L was the consequence of lysosomal membrane permeabilization without complete disruption of the cytosol-lysosome pH gradient. These results demonstrate a role for cathepsins in supraoptimal activation-induced apoptosis in vitro and suggest their possible participation in high dose tolerance in vivo.
In Vivo Helper Functions of Alloreactive Memory CD4+ T Cells Remain Intact Despite Donor-Specific Transfusion and Anti-CD40 Ligand Therapy
Authors: Chen Yifa , Heeger Peter S. , Valujskikh Anna
Published: April 2014

Memory T cells have specific properties that are beneficial for rapid and efficient protection from pathogens previously encountered by a host. These same features of memory T cells may be deleterious in the context of a transplanted organ. Consistent with this contention is the accumulating evidence in experimental transplantation that previously sensitized animals are resistant to the effects of costimulatory blockade. Using a model of murine cardiac transplantation, we now demonstrate that alloreactive memory CD4+ T cells prevent long-term allograft survival induced through donor-specific cell transfusion in combination with anti-CD40 ligand Ab (DST/anti-CD40L). We show that memory donor-reactive CD4+ T cells responding through the direct or indirect pathways of allorecognition provide help for the induction of antidonor CD8+ T effector cells and for Ab isotype switching, despite DST/anti-CD40L. The induced pathogenic antidonor immunity functions in multiple ways to subsequently mediate graft destruction. Our findings show that the varied functions of alloreactive memory CD4+ T cells remain intact despite DST/anti-CD40L-based costimulatory blockade, a finding that will likely have important implications for designing approaches to induce tolerance in human transplant recipients.
Determination of Structural Principles Underlying Three Different Modes of Lymphocytic Choriomeningitis Virus Escape from CTL Recognition
Authors: Velloso Lucas Malard , Michaëlsson Jakob , Ljunggren Hans-Gustaf , Schneider Gunter , Achour Adnane
Published: April 2014

Lymphocytic choriomeningitis virus infection of H-2b mice generates a strong CD8+ CTL response mainly directed toward three immunodominant epitopes, one of which, gp33, is presented by both H-2Db and H-2Kb MHC class I molecules. This CTL response acts as a selective agent for the emergence of viral escape variants. These variants generate altered peptide ligands (APLs) that, when presented by class I MHC molecules, antagonize CTL recognition and ultimately allow the virus to evade the cellular immune response. The emergence of APLs of the gp33 epitope is particularly advantageous for LCMV, as it allows viral escape in the context of both H-2Db and H-2Kb MHC class I molecules. We have determined crystal structures of three different APLs of gp33 in complex with both H-2Db and H-2Kb. Comparison between these APL/MHC structures and those of the index gp33 peptide/MHC reveals the structural basis for three different strategies used by LCMV viral escape mutations: 1) conformational changes in peptide and MHC residues that are potential TCR contacts, 2) impairment of APL binding to the MHC peptide binding cleft, and 3) introduction of subtle changes at the TCR/pMHC interface, such as the removal of a single hydroxyl group.