bluuebunny/crossref_metadata_2025 huggingface dataset:
Live-Cell Dynamics and the Role of Costimulation in Immunological Synapse Formation
Authors: Wetzel Scott A. , McKeithan Timothy W. , Parker David C.
Published: April 2014

Using transfected fibroblasts expressing both wild-type I-Ek and green fluorescent protein-tagged I-Ek with covalently attached antigenic peptide, we have monitored movement of specific MHC:peptide complexes during CD4+ T cell-APC interactions by live-cell video microscopy. Ag recognition occurs within 30 s of T cell-APC contact, as shown by a sharp increase in cytoplasmic calcium ion concentration. Within 1 min, small MHC:peptide clusters form in the contact zone that coalesce into an immunological synapse over 3–20 min. When T cells conjugated to APC move across the APC surface, they appear to drag the synapse with them. This system was used to examine the role of costimulation in the formation of the immunological synapse. Blocking CD80/CD28 or ICAM-1/LFA-1 interactions alters synapse morphology and reduces the area and density of accumulated complexes. These reductions correlate with reduced T cell proliferation, while CD69 and CD25 expression and TCR down-modulation remain unaffected. Thus, costimulation is essential for normal mature immunological synapse formation.
NK Cell CD94/NKG2A Inhibitory Receptors Are Internalized and Recycle Independently of Inhibitory Signaling Processes
Authors: Borrego Francisco , Kabat Juraj , Sanni Tolib B. , Coligan John E.
Published: April 2014

Human CD94/NKG2A is an inhibitory receptor that recognizes HLA-E and is expressed by NK cells and a subset of T cells. We have analyzed the cellular trafficking of the CD94/NKG2A receptor using the NKL cell line and peripheral blood NK cells. Flow cytometric, confocal microscopic, and biochemical analyses show that CD94/NKG2A continuously recycles in an active process that requires the cytoskeleton between the cell surface and intracellular compartments that are distinguishable from recycling compartments used by well-characterized receptors, such as transferrin receptor (CD71). CD94/NKG2A, an inhibitory receptor, traffics differently from the closely related CD94/NKG2C molecule, an activating receptor. Using transfection/expression analyses of wild-type and mutant CD94/NKG2A molecules in the HLA-E negative rat basophilic cell line RBL-2H3, we demonstrate that CD94/NKG2A internalization is independent of ligand cross-linking or the presence of functional immunoreceptor tyrosine-based inhibition motifs. Thus, the mechanisms that control cell surface homeostasis of CD94/NKG2A are independent of functional signaling.
Bacterial-Reactive T Regulatory Cells Inhibit Pathogenic Immune Responses to the Enteric Flora
Authors: Cong Yingzi , Weaver Casey T. , Lazenby Audrey , Elson Charles O.
Published: April 2014

We showed previously that cecal bacterial Ag (CBA)-specific CD4+ T cells induce colitis when transferred into SCID mice. The purpose of this study was to generate and characterize CBA-specific regulatory T cells in C3H/HeJBir (Bir) mice. CD4+ T cells were stimulated with CBA-pulsed APC in the presence of IL-10 every 10–14 days. After four or more cycles, these T cells produced high levels of IL-10, low levels of IL-4 and IFN-γ, and no IL-2, consistent with the phenotype of T regulatory-1 (Tr1) cells. Bir Tr1 cells proliferated poorly, but their proliferation was dependent on CD28-B7 interactions and was MHC class II-restricted. Transfer of Bir Tr1 cells into SCID mice did not result in colitis, and cotransfer of Bir Tr1 T cells with pathogenic Bir CD4+ Th1 cells prevented colitis. Bir Tr1 cells inhibited proliferation and IFN-γ production of a CBA-specific Th1 cell line in vitro. Such inhibition was partly due to IL-10 and TGFβ1, but cognate interactions with either APCs or Th1 cells were also involved. Normal intestinal lamina propria CD4+ T cells had Tr1-like activity when stimulated with CBA-pulsed APCs. We conclude that CD4+ T cells with the properties of Tr1 cells are present in the intestinal lamina propria and hypothesize that these cells maintain intestinal immune homeostasis to the enteric flora.
Determinants of Autoantibody Induction by Conjugated Papillomavirus Virus-Like Particles
Authors: Chackerian Bryce , Lenz Petra , Lowy Douglas R. , Schiller John T.
Published: April 2014

Immunization of mice with self-Ag arrayed on the surface of papillomavirus-like particles induces long-lasting high-titer IgG production by autoreactive B cells. In contrast, immunization with disorganized self-Ag linked to foreign Th epitopes induces weak autoantibody responses that are predominantly of the IgM isotype. In this study, we evaluated the structural correlates of autoantibody induction to determine the basis of these disparate observations, using a system in which mice were vaccinated with a fusion protein containing self (TNF-α) and foreign (streptavidin) components, conjugated to biotinylated virus-like particles (VLPs). Similar titers of autoantibodies to TNF-α were elicited using conjugated polyomavirus VLPs and papillomavirus VLPs, indicating that acute activation of dendritic cells by the Ag is not required. Strong autoantibody responses were also induced by conjugated papillomavirus capsid pentamers, indicating that a higher order particulate structure is also not required. However, a reduction of self-Ag density on VLP surfaces dramatically reduced the efficiency of IgG autoantibody induction. In contrast, the negative effects of reductions in foreign Ag density were limited and could be overcome by dosage and adjuvant. These data suggest that the immune system has evolved to differentially recognize closely spaced repetitive Ags and that the signals generated upon interactions with high-density self-Ags can overwhelm the normal mechanisms for B cell tolerance.
Age-Associated Augmentation of the Synthetic Ligand- Mediated Function of Mouse NK1.1 Ag+ T Cells: Their Cytokine Production and Hepatotoxicity In Vivo and In Vitro
Authors: Inui Takuo , Nakagawa Ryusuke , Ohkura Shuri , Habu Yoshiko , Koike Yuji , Motoki Kazuhiro , Kuranaga Noritsugu , Fukasawa Masashi , Shinomiya Nariyoshi , Seki Shuhji
Published: April 2014

We recently reported that the direct antitumor effectors in the liver induced by α-galactosylceramide (α-GalCer) are NK cells that are activated by the IFN-γ produced from NK1.1 Ag+ T cells (NKT cells) specifically stimulated with α-GalCer, whereas NKT cells cause hepatocyte injury through the Fas-Fas ligand pathway. In the present study, we investigated how mouse age affects the α-GalCer-induced effect using young (6-wk-old), middle-aged (30-wk-old), and old (75-wk-old) mice. The serum IFN-γ and IL-4 concentrations as well as alanine aminotransferase levels after the α-GalCer injection increased in an age-dependent manner. An α-GalCer injection also induced an age-dependent increase in the Fas ligand expression on liver NKT cells. Under the stimulus of α-GalCer in vitro, the liver mononuclear cells from old and middle-aged mice showed vigorous proliferation, remarkable antitumor cytotoxicity, and enhanced production of both IFN-γ and IL-4 in comparison to those of young mice, all of which were mediated mainly by NK1.1+ cells. Furthermore, liver mononuclear cells from old mice stimulated with α-GalCer showed a more potent Fas-Fas ligand-mediated cytotoxicity against primary cultured hepatocytes than did those from young mice. Most α-GalCer-injected old mice, but no young mice, died, while anti-IFN-γ Ab pretreatment completely inhibited mouse mortality. However, α-GalCer-induced hepatic injury did not improve at all by anti-IFN-γ Ab treatment, and the Fas-ligand expression of liver NKT cells did not change. Taken together, the synthetic ligand-mediated function of NKT cells is age-dependently up-regulated, and the produced IFN-γ is responsible for α-GalCer-induced antitumor immunity and the mouse mortality, while hepatic injury was unexpectedly found to be independent of IFN-γ.
Multilineage Engraftment with Minimal Graft-Versus-Host Disease Following In Utero Transplantation of S-59 Psoralen/Ultraviolet A Light-Treated, Sensitized T Cells and Adult T Cell-Depleted Bone Marrow in Fetal Mice
Authors: Bhattacharyya Swati , Chawla Anjulika , Smith Kristofer , Zhou Yungui , Talib Sohel , Wardwell Brian , Cowan Morton J.
Published: April 2014

Although engraftment following in utero stem cell transplantation can readily be achieved, a major limitation is the low level of donor chimerism. We hypothesized that a lack of space for donor cells in the recipient marrow was one of the primary reasons for failure to achieve significant engraftment, and that donor T cells could make space in an allogeneic mismatched setting. We found that 3 × 105 C57BL/6 (B6) naive CD3+ cells coinjected with B6 T cell-depleted bone marrow (TCDBM) into 14- to 15-day-old BALB/c fetuses resulted in multilineage engraftment (median, 68.3%) associated with severe graft-vs-host disease (GvHD; 62 vs 0% with TCDBM alone). When 1.5 × 105 CD4+ or CD8+ cells were used, low levels of engraftment were seen vs recipients of 1.5 × 105 CD3+ cells (2.4 ± 1.1 and 6.6 ± 3.9 vs 20.4 ± 10.4%, respectively). To test the hypothesis that proliferation of T cells in response to alloantigen resulted in GvHD and increased engraftment, we pretreated naive T cells with photochemical therapy (PCT) using S-59 psoralen and UVA light to prevent proliferation. GvHD was reduced (60–0%), but was also associated with a significant reduction in engrafted donor cells (53.4 ± 4.2 to 1.7 ± 0.5%). However, when B6 T cells were sensitized to BALB/c splenocytes, treated with PCT, and coinjected with TCDBM, there was a partial restoration of engraftment (13.3 ± 2.4% H2Kb+ cells) with only one of nine animals developing mild to moderate GvHD. In this study we have shown that PCT-treated T cells that are cytotoxic but nonproliferative can provide an engraftment advantage to donor cells, presumably by destroying host hemopoietic cells without causing GvHD.
Different Efficiency of Heat Shock Proteins (HSP) to Activate Human Monocytes and Dendritic Cells: Superiority of HSP60
Authors: Bethke Karen , Staib Frank , Distler Martin , Schmitt Ute , Jonuleit Helmut , Enk Alexander H. , Galle Peter R. , Heike Michael
Published: April 2014

One essential immunoregulatory function of heat shock protein (HSP) is activation of the innate immune system. We investigated the activation of human monocytes and monocyte-derived dendritic cells (DC) by recombinant human HSP60, human inducible HSP72, and preparations of human gp96 and HSP70 under stringent conditions, in the absence of serum and with highly purified monocytes. HSP60 induced human DC maturation and activated human DC to secrete proinflammatory cytokines. HSP72 induced DC maturation to a lesser extent, but activated human monocytes and immature DC as efficiently as HSP60 to release proinflammatory cytokines. The independence of the effects of HSP60 and HSP72 from endotoxin or another copurifying bacterial component was shown by the resistance of these effects to polymyxin B, their sensitivity to heat treatment, the inactivity of endotoxin controls at concentrations up to 100-fold above the endotoxin contents of the HSP, and the inactivity of a recombinant control protein. Preparations of HSP70, which consisted mainly of the constitutively expressed HSP73, induced only marginal cytokine release from monocytes. The gp96 preparations did not have significant effects on human monocytes and monocyte-derived DC, indicating that these human APC populations were not susceptible to gp96 signaling under the stringent conditions applied in this study. The biological activities of gp96 and HSP70 preparations were confirmed by their peptide binding activity. These findings show that HSP can differ considerably in the capacity to activate monocyte-derived APC under certain conditions and underline the potential of HSP60 and HSP72 as activation signals for the innate immune system.
Zwitterionic Polysaccharides Stimulate T Cells by MHC Class II-Dependent Interactions
Authors: Kalka-Moll Wiltrud M. , Tzianabos Arthur O. , Bryant Paula W. , Niemeyer Marcus , Ploegh Hidde L. , Kasper Dennis L.
Published: April 2014

Polysaccharides of pathogenic extracellular bacteria commonly have negatively charged groups or no charged groups at all. These molecules have been considered classic T cell-independent Ags that do not elicit cell-mediated immune responses in mice. However, bacterial polysaccharides with a zwitterionic charge motif (ZPSs), such as the capsular polysaccharides of many strains of Bacteroides fragilis, Staphylococcus aureus, and Streptococcus pneumoniae type 1 elicit potent CD4+ T cell responses in vivo and in vitro. The cell-mediated response to ZPS depends on the presence of both positively charged and negatively charged groups on each repeating unit of the polysaccharide. In this study, we define some of the requirements for the presentation of ZPS to CD4+ T cells. We provide evidence that direct interactions of T cells with APCs are essential for T cell activation by ZPS. Monocytes, dendritic cells, and B cells are all able to serve as APCs for ZPS-mediated T cell activation. APCs lacking MHC class II molecules do not support this activity. Furthermore, mAb to HLA-DR specifically blocks ZPS-mediated T cell activation, while mAbs to other MHC class II and class I molecules do not. Immunoprecipitation of lysates of MHC class II-expressing cells following incubation with ZPS shows binding of ZPS and HLA-DR. Electron microscopy reveals colocalization of ZPS with HLA-DR on the cell surface and in compartments of the endocytic pathway. These results indicate that MHC class II molecules expressing HLA-DR on professional APCs are required for ZPS-induced T cell activation. The implication is that binding of ZPS to HLA-DR may be required for T cell activation.
Mouse Vascular Endothelium Activates CD8+ T Lymphocytes in a B7-Dependent Fashion
Authors: Kreisel Daniel , Krupnick Alexander S. , Balsara Keki R. , Riha Markus , Gelman Andrew E. , Popma Sicco H. , Szeto Wilson Y. , Turka Laurence A. , Rosengard Bruce R.
Published: April 2014

Despite several studies examining the contribution of allorecognition pathways to acute and chronic rejection of vascularized murine allografts, little data describing activation of alloreactive T cells by mouse vascular endothelium exist. We have used primary cultures of resting or IFN-γ-activated C57BL/6 (H-2b) vascular endothelial cells as stimulators and CD8+ T lymphocytes isolated from CBA/J (H-2k) mice as responders. Resting endothelium expressed low levels of MHC class I, which was markedly up-regulated after activation with IFN-γ. It also expressed moderate levels of CD80 at a resting state and after activation. Both resting and activated endothelium were able to induce proliferation of unprimed CD8+ T lymphocytes, with proliferation noted at earlier time points after coculture with activated endothelium. Activated endothelium was also able to induce proliferation of CD44low naive CD8+ T lymphocytes. Activated CD8+ T lymphocytes had the ability to produce IFN-γ and IL-2, acquired an effector phenotype, and showed up-regulation of the antiapoptotic protein Bcl-xL. Treatment with CTLA4-Ig led to marked reduction of T cell proliferation and a decrease in expression of Bcl-xL. Moreover, we demonstrate that nonhemopoietic cells such as vascular endothelium induce proliferation of CD8+ T lymphocytes in a B7-dependent fashion in vivo. These results suggest that vascular endothelium can act as an APC for CD8+ direct allorecognition and may, therefore, play an important role in regulating immune processes of allograft rejection.
Acquisition of CD80 by Human T Cells at Early Stages of Activation: Functional Involvement of CD80 Acquisition in T Cell to T Cell Interaction
Authors: Tatari-Calderone Zohreh , Semnani Roshanak Tolouei , Nutman Thomas B. , Schlom Jeffrey , Sabzevari Helen
Published: April 2014

The interaction between CD28 on T cells and CD80 on APCs intensifies the linkage between TCR and MHC at the site of contact between T cells and APCs. In this study, we demonstrate that during human T cell/human APC interaction, the autologous or allogeneic human CD4+ T cells become positive for the detection of CD80 at an early stage of activation (24 h). This detection of CD80 is attributable to the acquisition of CD80 from APCs, as opposed to the up-regulation of endogenous CD80, as demonstrated by CD4+ T cells treated with cyclohexamide. Furthermore, no CD80 mRNA could be detected at 24 h in T cells that had acquired CD80 from APCs. CD80 acquisition by T cells from APCs was enhanced upon TCR engagement. The amount of CD80 acquisition by CD4+ T cells was shown to be related to the expression of CD80 on APCs. Using soluble fusion proteins (soluble CTLA-4, CD28, and CD80) to block either CD28 on the surface of T cells or CD80 on the surface of APCs, it was demonstrated that CD80 acquisition by T cells is mediated through its receptors, possibly CD28 interaction. Moreover, we demonstrate that T cells that have acquired CD80 have the ability to stimulate other T cells. These data thus suggest that CD80 acquisition by human T cells might play a role in the immunoregulation of T cell responses.
Kinetics of Gene Induction After FcεRI Ligation of Atopic Monocytes Identified by Suppression Subtractive Hybridization
Authors: von Bubnoff Dagmar , Matz Heike , Cazenave Jean-Pierre , Hanau Daniel , Bieber Thomas , de la Salle Henri
Published: April 2014

The high-affinity receptor for IgE, FcεRI, on APCs plays an important role in the initiation and chronicity of inflammatory atopic diseases. To understand the molecular regulation of FcεRI-mediated processes, differentially expressed genes are of great interest to be identified. Suppression subtractive cDNA hybridization has been used to identify genes induced after FcεRI stimulation on atopic monocytes. Overexpression of the identified genes was determined by semiquantitative RT-PCR analysis of transcripts from the tester (stimulated) and driver (unstimulated) monocytes. Results were confirmed and kinetics of the transcripts established using blood cells from additional atopics at 4 and 24 h of FcεRI induction. The following sequences were identified: monocyte chemoattractant protein 1, macrophage-inflammatory protein 1β, IL-6, βA subunit of inhibin/activin, IFN-stimulated gene of 54 kDa, IL-1R antagonist, and kynurenine 3-monooxygenase. Chemokines are highly expressed during the early and late phase after FcεRI cross-linking, whereas proinflammatory and differentiation stimuli rapidly decline after an initial overexpression. Kynurenine 3-monooxygenase, an enzyme involved in the degradation of the amino acid tryptophan, is significantly up-regulated during the late phase after 24 h of FcεRI induction. These results demonstrate that the analysis of the profile of gene induction following activation of FcεRI on atopic monocytes may reveal how these cells might participate in the regulation of atopic disorders.
Clonal Anergy Is Maintained Independently of T Cell Proliferation
Authors: Colombetti Sara , Benigni Fabio , Basso Veronica , Mondino Anna
Published: April 2014

Ag encounter in the absence of proliferation results in the establishment of T cell unresponsiveness, also known as T cell clonal anergy. Anergic T cells fail to proliferate upon restimulation because of the inability to produce IL-2 and to properly regulate the G1 cell cycle checkpoint. Because optimal TCR and CD28 engagement can elicit IL-2-independent cell cycle progression, we investigated whether CD3/CD28-mediated activation of anergic T cells could overcome G1 cell cycle block, drive T cell proliferation, and thus reverse clonal anergy. We show here that although antigenic stimulation fails to elicit G1-to-S transition, anti-CD3/CD28 mAbs allow proper cell cycle progression and proliferation of anergic T cells. However, CD3/CD28-mediated cell division does not restore Ag responsiveness. Our data instead indicate that reversal of clonal anergy specifically requires an IL-2-dependent, rapamycin-sensitive signal, which is delivered independently of cell proliferation. Thus, by tracing proliferation and Ag responsiveness of individual cells, we show that whereas both TCR/CD28 and IL-2-generated signals can drive T cell proliferation, only IL-2/IL-2R interaction regulates Ag responsiveness, indicating that proliferation and clonal anergy can be independently regulated.
Autocrine IL-15 Mediates Intestinal Epithelial Cell Death Via the Activation of Neighboring Intraepithelial NK Cells
Authors: Kinoshita Naotoshi , Hiroi Takachika , Ohta Noriyuki , Fukuyama Satoshi , Park Eun Jeong , Kiyono Hiroshi
Published: April 2014

Intestinal intraepithelial lymphocytes (IELs), which reside between the basolateral faces of intestinal epithelial cells (IECs), provide a first-line defense against pathogens via their cytotoxic activity. Although IEC-derived IL-7 and IL-15 are key regulatory cytokines for the development and activation of IELs, we report here that IL-15 but not IL-7 mediates the reciprocal interaction between IELs and IECs, an important interaction for the regulation of appropriate mucosal immunohomeostasis. IL-15-treated IELs induced cell death in IECs via the cytotoxic activity in vitro. Among the different subsets of IL-15-treated IELs, CD4−CD8−TCR− IELs, which express NK marker (DX5 or NK1.1), showed the most potent syngenic IEC killing activity. These intraepithelial NK cells expressed Ly-49 molecules, NKG2 receptors, and perforin. These results suggest the possibility that the cell death program of IECs could be regulated by self-produced IL-15 through the activation of intraepithelial NK cells.
Bone Marrow B Cell Apoptosis During In Vivo Influenza Virus Infection Requires TNF-α and Lymphotoxin-α
Authors: Sedger Lisa M. , Hou Sam , Osvath Sarah R. , Glaccum Moira B. , Peschon Jacques J. , van Rooijen Nico , Hyland Lisa
Published: April 2014

Suppression of bone marrow myeloid and erythroid progenitor cells occurs after infection with a variety of different viruses. In this study, we characterize the alterations in bone marrow (BM) lymphocytes after influenza virus infection in mice. We found a severe loss of BM B cells, particularly CD43low/−B220+ pre-B and immature B cells, in influenza virus-infected mice. Depletion of BM B lineage cells resulted primarily from cell cycle arrest and most likely apoptosis within the BM environment, rather than from increased trafficking of BM emigrants to peripheral lymphoid tissues. Use of gene-knockout mice indicates that depletion of BM B cells is dependent on TNF-α, lymphotoxin-α, and both TNF receptors, TNFR1-p55 and TNFR2-p75. Thus, TNF-α and lymphotoxin-α are required for loss of BM B lineage cells during respiratory infection with influenza virus.
CTLA-4 Differentially Regulates T Cell Responses to Endogenous Tissue Protein Versus Exogenous Immunogen
Authors: Walker Lucy S. K. , Ausubel Lara J. , Chodos Anna , Bekarian Nyree , Abbas Abul K.
Published: April 2014

CTLA-4 is critical to the regulation of CD4 T cell homeostasis in vivo. However, whether CTLA-4 regulates responses to both self and foreign proteins is not clear. We have directly compared the role of CTLA-4 in controlling T cell responses to the same protein presented as an endogenous tissue Ag vs a foreign immunizing Ag. We show that CTLA-4 only modestly reduces responses to Ag administered with adjuvant, but dramatically inhibits responses to the same Ag expressed transgenically as a tissue self protein. The critical consequence of CTLA-4 engagement is to inhibit T cell accumulation in the local lymph node draining the Ag-bearing tissue, and failure of this control leads to the onset of autoimmune tissue destruction. Thus, CTLA-4 may preferentially dampen pathologic immune responses to self proteins while permitting protective immunity to foreign agents.
Inhibition of Human CD4+CD25+high Regulatory T Cell Function
Authors: Baecher-Allan Clare , Viglietta Vissia , Hafler David A.
Published: April 2014

CD4+CD25+high T cells are potent regulators of autoreactive T cells. However, it is unclear how regulatory CD4+CD25+high cells discriminate between desirable inflammatory immune responses to microbial Ags and potentially pathologic responses by autoreactive T cells. In this study, an in vitro model was created that allowed differential activation of regulatory CD4+CD25+high and responder CD4+ T cells. If CD4+CD25+high regulatory cells were strongly activated, they maintained suppressive effector function for only 15 h, while stimulation with weaker TCR stimuli produced regulatory cells that were suppressive until 60 h after activation. In contrast, strongly activated CD4+ responder T cells were resistant to regulation at all time points, while weakly stimulated CD4+ cells were sensitive to suppression until 38 or 60 h after activation depending upon the strength of the stimulus. The extent of suppression mediated by CD4+CD25+high cells also depended on the strength of stimulation in an Ag-specific system. Thus, the stronger the TCR signal, the more rapidly and more completely the responder cells become refractory to suppression.
T Cell Receptor Ligation Triggers Novel Nonapoptotic Cell Death Pathways That Are Fas-Independent or Fas-Dependent
Authors: Davidson Wendy F. , Haudenschild Christian , Kwon Jaeyul , Williams Mark S.
Published: April 2014

Short-term culture of activated T cells with IL-2 renders them highly susceptible to apoptotic death triggered by TCR cross-linking. Activation-induced apoptosis is contingent upon caspase activation and this is mediated primarily by Fas/Fas ligand (FasL) interactions that, in turn, are optimized by p38 mitogen-activated protein kinase (MAPK)-regulated signals. Although T cells from mice bearing mutations in Fas (lpr) or FasL (gld) are more resistant to activation-induced cell death (AICD) than normal T cells, a significant proportion of CD8+ T cells and to a lesser extent CD4+ T cells from mutant mice die after TCR religation. Little is known about this Fas-independent death process. In this study, we demonstrate that AICD in lpr and gld CD4+ and CD8+ T cells occurs predominantly by a novel mechanism that is TNF-α-, caspase-, and p38 MAPK-independent and has morphologic features more consistent with oncosis/primary necrosis than apoptosis. A related Fas- and caspase-independent, nonapoptotic death process is revealed in wild-type (WT) CD8+ T cell blasts following TCR ligation and treatment with caspase inhibitors, the p38 MAPK inhibitor, SB203580, or neutralizing anti-FasL mAb. In parallel studies with WT CD4+ T cells, two minor pathways leading to nonapoptotic, caspase-independent AICD were identified, one contingent upon Fas ligation and p38 MAPK activation and the other Fas- and p38 MAPK-independent. These data indicate that TCR ligation can activate nonapoptotic death programs in WT CD8+ and CD8+ T blasts that normally are masked by Fas-mediated caspase activation. Selective use of potentially proinflammatory oncotic death programs by activated lpr and gld T cells may be an etiologic factor in autosensitization.
Cyclic Adenosine 5′-Monophosphate and Calcium Induce CD152 (CTLA-4) Up-Regulation in Resting CD4+ T Lymphocytes
Authors: Vendetti Silvia , Riccomi Antonella , Sacchi Alessandra , Gatta Lucia , Pioli Claudio , De Magistris Maria Teresa
Published: April 2014

The CTLA-4 (CD152) molecule is up-regulated upon T cell activation and proliferation, and plays a critical role in the inhibition of immune responses. We show in this study that cAMP induces up-regulation of CD152 in human CD4+ T lymphocytes. This effect occurs in the absence of the up-regulation of CD69 and CD25 activation markers and T cell proliferation. In addition, we found that the Ca2+ ionophore ionomycin also up-regulates CD152, and that the combination of a cAMP analog or cAMP inducers with ionomycin further enhances the expression of CD152 in resting CD4+ T lymphocytes. However, cyclosporin A, which inhibits Ca2+/calcineurin signaling pathway, fully prevented the ionomycin- but not the cAMP-induced up-regulation of CD152. The effects of cAMP and ionomycin involve increase of both CD152 mRNA transcripts, coding for the membrane and the soluble forms of CD152. Furthermore, we show that CD152 molecules are translocated to the membrane and are functional, as their engagement by specific mAbs prevented NF-κB activation by anti-CD3/CD28 stimulation. These findings demonstrate that at least two novel signal pathways regulate CTLA-4 gene expression and CD152 molecule up-regulation in human CD4+ T lymphocytes, in the absence of full T cell activation.
NF-κB Regulates the Expression of the Human Complement Receptor 2 Gene
Authors: Tolnay Mate , Vereshchagina Lyudmila A. , Tsokos George C.
Published: April 2014

CR2 is a key regulator of the B cell response to Ag. Here we show that NF-κB enhances the expression of the human CR2 gene. Promoter truncation, deletion, and mutagenesis studies indicated a functional role for a consensus NF-κB promoter element, as well as a heterogeneous nuclear ribonucleoprotein D element and an overlapping X box/E box. By supershift analysis, the first two elements bound NF-κB p50 and p65 and heterogeneous nuclear ribonucleoprotein RNP D, respectively. The X box/E box bound regulatory factor X5 and, surprisingly, NF-κB p50 and p65. Overexpression of NF-κB p50 enhanced the activity of the CR2 promoter in B cell lines and primary B cells, suggesting a direct role for NF-κB in regulating promoter activity. Importantly, mutation of the NF-κB element or the X box/E box rendered the promoter unresponsive to NF-κB p50. Using chromatin immunoprecipitation in live B cell lines and primary B cells, we found that NF-κB proteins p50, p65, and c-Rel bound to the genomic promoter at two locations that overlap with the consensus NF-κB element or the X box/E box. Finally, stimuli that activate NF-κB enhanced the activity of the CR2 promoter, and LPS rapidly increased the number of CR2 proteins on the surface of primary B cells. We propose that the NF-κB signaling pathway enhances the expression of the CR2 gene, as a result of NF-κB proteins binding to two CR2 promoter elements. Thus, at the onset of an infection, LPS could sensitize the B cell to Ag by enhancing the level of CR2-costimulatory molecules on the cell surface.
Primary Structure and Complementarity-Determining Region (CDR) 3 Spectratyping of Rainbow Trout TCRβ Transcripts Identify Ten Vβ Families with Vβ6 Displaying Unusual CDR2 and Differently Spliced Forms
Authors: Boudinot Pierre , Boubekeur Samira , Benmansour Abdenour
Published: April 2014

VDJ rearrangement at the teleost TCRβ locus leads to a highly diverse repertoire of junctions for each VβJβ combination. From a rainbow trout 5′ RACE library of TCRβ transcripts, 47 clones encompassing a full Vβ-Dβ-Jβ-Cβ sequence were selected and analyzed. A similarity analysis of the sequences evidenced 10 Vβ families, of which 6 were not previously described. Immunoscope and sequence analysis of the Vβ-Dβ-Jβ junctions of the new families confirmed that they create a polyclonal and diverse repertoire. Multiple alignments showed that rainbow trout Vβs possess most of the conserved residues typical of Vβ segments. However, this study revealed a high complementarity-determining region 2 (CDR2) and CDR1 length diversity among rainbow trout Vβ families, suggesting that the spatial orientation of the TCR could fluctuate in the TCR/peptide/MHC complex, depending on the Vβ expressed. Among the new Vβ families, Vβ6 displayed the strongest deviance from typical hypervariable CDR1 and CDR2 loops, with an unusually short CDR2. Moreover, the Vβ6 sequence is overall divergent from typical Vβ sequence, raising the question of its functional relevance. Immunoscope experiments identified a Vβ6-Jβ3 junction, which was amplified during the response against viral hemorrhagic septicemia virus, a fish rhabdovirus. Vβ6 seems therefore to be expressed functionally in a selected TCR. However, the shorter Vβ6 transcripts produced through an alternative splicing lack the C′, C″, D, and E strands of the Vβ domain and are probably nonfunctional.
Dominant TCR-α Requirements for a Self Antigen Recognition in Humans
Authors: Mantovani Stefania , Palermo Belinda , Garbelli Silvia , Campanelli Rita , Robustelli della Cuna Gioacchino , Gennari Roberto , Benvenuto Federica , Lantelme Erica , Giachino Claudia
Published: April 2014

TCR-α and -β chains are composed of somatically rearranged V, D, and J germline-encoded gene segments that confer Ag specificity. Recent crystallographic analyses revealed that TCR-α has more contacts with peptide than TCR-β, suggesting the possibility that peptide recognition predominantly relies on TCR-α. T cells specific for the self Ag Melan-A/MART-1 possess an exceptionally high precursor frequency in human histocompatibility leukocyte Ag-A2 individuals. This provided a unique situation for assessment of the structural relationship between TCR and peptide/MHC ligand at both the pre- and postimmune levels. Molecular and phenotypic analysis of many different Melan-A-specific T cell populations revealed that a structural constraint is imposed on the TCR for engagement with Melan-A peptides presented by HLA-A2, namely the highly preferential use of a particular TCRAV segment, AV2. Examination of CD8 single-positive thymocytes indicated that this preferential use in forming the Melan-A-specific TCR is mainly imposed by intrathymic positive selection. Our data demonstrate a dominant function of TCRAV2 segment in forming the TCR repertoire specific for the human self Ag Melan-A/MART-1 and support the view that Ag recognition is mediated predominantly by TCR-α.
CD4 Dimers Constitute the Functional Component Required for T Cell Activation
Authors: Moldovan Maria-Cristina , Yachou Abdelkader , Lévesque Karine , Wu Hao , Hendrickson Wayne A. , Cohen Eric A. , Sékaly Rafick-Pierre
Published: April 2014

The CD4 molecule plays a key role in the development and activation of helper T cells. Dimerization and oligomerization is often a necessary step in the function of several cell surface receptors. Herein, we provide direct biochemical evidence confirming the presence of CD4 as dimers in transfected cells from hemopoetic and fibroblastic origin as well as in primary T cells. Such dimers are also observed with murine CD4 confirming selective pressure during evolution to maintain such a structure. Using a series of point mutations, we have precisely mapped the dimerization site at residues K318 and Q344 within the fourth extracellular domain of CD4. These residues are highly conserved and their mutation results in interference with dimer formation. More importantly, we demonstrate that dimer formation is essential for the coligand and coreceptor functions of CD4 in T cell activation. These data strongly suggest that CD4 dimerization is necessary for helper T cell function.
Regulation of Constitutive TCR Internalization by the ζ-Chain
Authors: D’Oro Ugo , Munitic Ivana , Chacko George , Karpova Tatiana , McNally James , Ashwell Jonathan D.
Published: April 2014

The ability of a T cell to be activated is critically regulated by the number of TCRs expressed on the plasma membrane. Cell surface TCR expression is influenced by dynamic processes such as synthesis and transport of newly assembled receptors, endocytosis of surface TCR, and recycling to the plasma membrane of internalized receptors. In this study, the internalization of fluorescently labeled anti-TCR Abs was used to analyze constitutive endocytosis of TCRs on T cells, and to investigate the role of the ζ-chain in this process. We found that cell surface TCRs lacking ζ were endocytosed more rapidly than completely assembled receptors, and that reexpression of full-length ζ led to a dose-dependent decrease in the rate of TCR internalization. Rapid TCR internalization was also observed with CD4+CD8+ thymocytes from ζ-deficient mice, whereas TCR internalization on thymocytes from CD3-δ deficient animals was slow, similar to that of wild-type thymocytes. This identifies a specific role for ζ in the regulation of constitutive receptor internalization. Furthermore, chimeric ζ molecules containing non-native intracellular amino acid sequences also led to high levels of TCR expression and reduced TCR cycling. These effects were dependent solely on the length of the intracellular tail, ruling out a role for intracellular ζ-specific interactions with other molecules as a mechanism for regulating TCR internalization. Rather, these findings strongly support a model in which the ζ-chain stabilizes TCR residency on the cell surface, and functions to maintain cell surface receptor expression by sterically blocking internalization sequences in other TCR components.
Making Sense of the Diverse Ligand Recognition by NKG2D
Authors: Radaev Sergei , Kattah Michael , Zou Zhongcheng , Colonna Marco , Sun Peter D.
Published: April 2014

NKG2D recognizes multiple diverse ligands. Despite recent efforts in determining the crystal structures of NKG2D-ligand complexes, the principle governing this receptor-ligand recognition and hence the criteria for identifying unknown ligands of NKG2D remain central issues to be resolved. Here we compared the molecular recognition between NKG2D and three of the known ligands, UL16 binding protein (ULBP), MHC class I-like molecule, and retinoic acid early inducible gene as observed in the ligand-complexed crystal structures. The comparison shows that while the receptor uses a common interface region to bind the three diverse ligands, each ligand forms a distinct, but overlapping, set of hydrogen bonds, hydrophobic interactions, and salt bridges, illustrating the underlying principle of NKG2D-ligand recognition being the conservation in overall shape complementarity and binding energy while permitting variation in ligand sequence through induced fit recognition. To further test this hypothesis and to distinguish between diverse recognition and promiscuous ligand binding, four ULBP3 interface mutations, H21A, E76A, R82M, and D169A, were generated to each disrupt a single hydrogen bond or salt bridge. All mutant ULBP3 displayed reduced receptor binding, suggesting a specific, rather than promiscuous, receptor-ligand recognition. Mutants with severe loss of binding affect the receptor interactions that are mostly buried. Finally, a receptor-ligand recognition algorithm was developed to assist the identification of diverse NKG2D ligands based on evaluating the potential hydrogen bonds, hydrophobic interactions, and salt bridges at the receptor-ligand interface.
Mycobacterium aviumInfection and Modulation of Human Macrophage Gene Expression
Authors: Greenwell-Wild Teresa , Vázquez Nancy , Sim Davis , Schito Marco , Chatterjee Delphi , Orenstein Jan M. , Wahl Sharon M.
Published: April 2014

Mycobacterium avium is a facultative intracellular pathogen cleared rapidly via intact host defense mechanisms. In the absence of adequate T cell function, as occurs in HIV-1-induced immunodeficiency, M. avium becomes an opportunistic infection with uncontrolled replication and reinfection of macrophage hosts. How M. avium infects, survives, and replicates in macrophages without signaling an effective microbicidal counterattack is unresolved. To address whether M. avium signals the expression of molecules, which influence mycobacterial survival or clearance, human monocyte-derived macrophage cultures were exposed to M. avium. Within minutes, M. avium, or its cell wall lipoarabinomannan, binds to the adherent macrophages and induces a spectrum of gene expression. In this innate response, the most abundant genes detected within 2 h by cDNA expression array involved proinflammatory chemokines, cytokines including TNF-α and IL-1, and adhesion molecules. Associated with this rapid initial up-regulation of recruitment and amplification molecules was enhanced expression of transcription factors and signaling molecules. By 24 h, this proinflammatory response subsided, and after 4 days, when some bacteria were being degraded, others escaped destruction to replicate within intracellular vacuoles. Under these conditions, inducible NO synthase was not up-regulated and increased transferrin receptors may facilitate iron-dependent mycobacterial growth. Sustained adhesion molecule and chemokine expression along with the formation of multinucleated giant cells appeared consistent with in vivo events. Thus, in the absence of T lymphocyte mediators, macrophages are insufficiently microbicidal and provide a nonhostile environment in which mycobacteria not only survive and replicate, but continue to promote recruitment of new macrophages to perpetuate the infection.
B7/CD28-Dependent CD4+CD25+ Regulatory T Cells Are Essential Components of the Memory-Protective Immunity to Candida albicans
Authors: Montagnoli Claudia , Bacci Angela , Bozza Silvia , Gaziano Roberta , Mosci Paolo , Sharpe Arlene H. , Romani Luigina
Published: April 2014

Protective immunity to the fungus Candida albicans is mediated by Ag-specific Th1 cells. Paradoxically, some Th2 cytokines are required for the maintenance of Th1-mediated immune resistance to the fungus. Therefore, in addition to the Th1/Th2 balance, other mechanisms seem to be involved in the regulation of Th1 immunity to the fungus. Here we show that CD4+CD25+ T cells, negatively regulating antifungal Th1 reactivity, are generated in mice with candidiasis. CD4+CD25+ T cells were not generated in B7-2- or CD28-deficient mice or in condition of IL-10 signaling deficiency. Accordingly, although capable of efficiently restricting the fungal growth, these mice experienced inflammatory pathology and were incapable of resistance to reinfection. CD4+CD25+ T cells poorly proliferated in vitro; were highly enriched for cells producing IL-4, IL-10, and TGF-β; and required IL-10-producing, Candida hypha-activated dendritic cells for generation. Adoptive transfer of CD4+CD25+ T cells or IL-10-producing dendritic cells restored resistance to reinfection and decreased inflammation in B7-2-deficient mice. These results show that oral tolerance induced by Candida hyphae is required for the occurrence of long-lasting protective immunity after yeast priming. The implication is that preventing reactivation rather than favoring sterilizing immunity to ubiquitous fungal pathogens may represent the ultimate expectation of vaccine-based strategies.
IFN-γ-Induced Apoptosis and Microbicidal Activity in Monocytes Harboring the Intracellular Bacterium Coxiella burnetii Require Membrane TNF and Homotypic Cell Adherence
Authors: Dellacasagrande Jérôme , Ghigo Eric , Raoult Didier , Capo Christian , Mege Jean-Louis
Published: April 2014

IFN-γ is critical for the protection against intracellular bacteria through activation of the antimicrobial machinery of phagocytes. Coxiella burnetii, the etiological agent of Q fever, is a strictly intracellular bacterium that inhabits monocytes/macrophages. We previously showed that IFN-γ induced C. burnetii killing by promoting the apoptosis of infected monocytes. We show in this study that IFN-γ-induced apoptosis of infected monocytes was characterized by a time- and dose-dependent activation of caspase-3. IFN-γ-mediated caspase-3 activation and C. burnetii killing depend on the expression of membrane TNF. Indeed, TNF was transiently expressed on the cell surface of infected monocytes a few hours after IFN-γ treatment. In addition, anti-TNF Abs inhibited IFN-γ-mediated caspase-3 activation whereas soluble TNF had no effect on infected cells. Concomitantly, IFN-γ induced homotypic adherence of C. burnetii-infected monocytes. The latter required the interaction of β2 integrins with CD54. When adherence was disrupted by pipetting, by a combination of Abs specific for CD11b, CD18, and CD54, or by an antisense oligonucleotide targeting CD18 mRNA, both cell apoptosis and bacterial killing induced by IFN-γ were inhibited. Thus, adherence via CD54/β2 integrins together with membrane TNF are required to eliminate C. burnetii-infected cells through cell contact-dependent apoptosis. Our results reveal a new component of the antimicrobial arsenal mobilized by IFN-γ against infection by intracellular bacteria.
Evolution of the Cutaneous Immune Response to Experimental Haemophilus ducreyi Infection and Its Relevance to HIV-1 Acquisition
Authors: Humphreys Tricia L. , Schnizlein-Bick Carol T. , Katz Barry P. , Baldridge Lee Ann , Hood Antoinette F. , Hromas Robert A. , Spinola Stanley M.
Published: April 2014

Haemophilus ducreyi causes the sexually transmitted disease chancroid, which facilitates HIV-1 transmission. Skin biopsies were obtained from subjects experimentally infected with H. ducreyi to study the evolution of the immune response and immunophenotypes relevant to transmission of HIV-1. Compared with peripheral blood, there was an enrichment of T cells and macrophages after 48 h of infection in the skin. Neutrophils became the predominant cell type by 7–9 days. By immunohistochemistry, macrophage-inflammatory protein-1α was not present early in infection, but was abundant at later stages. RANTES was present throughout the papular and pustular stages of experimental infection, but not present in uninfected control skin. Stromal cell-derived factor-1 was present at low levels in all samples examined. Macrophages in lesions had significantly increased expression of CCR5 and CXCR4 compared with peripheral blood cells, and CD4 T cells had significant up-regulation of CCR5. The magnitude of increased expression of these receptors was not replicated when PBMCs were incubated with H. ducreyi or H. ducreyi lipooligosaccharide in vitro. Together with the disruption of mucosal and skin barriers, the presence of cells with up-regulated HIV-1 coreceptors in H. ducreyi-infected lesions may provide an environment that facilitates the acquisition of R5 (CCR5), X4 (CXCR4), and dual-tropic HIV-1 strains.
GM-CSF Transgene-Based Adjuvant Allows the Establishment of Protective Mucosal Immunity Following Vaccination with Inactivated Chlamydia trachomatis
Authors: Lu Hang , Xing Zhou , Brunham Robert C.
Published: April 2014

Cellular and humoral immune responses induced following murine Chlamydia trachomatis infection confer almost sterile protection against homologous reinfection. On the other hand, immunization with inactivated organism induces little protective immunity in this model system. The underlying mechanism(s) that determines such divergent outcome remains unclear, but elucidating the mechanism will probably be important for chlamydial vaccine development. One of the distinct differences between the two forms of immunization is that chlamydia replication in epithelial cells causes the secretion of a variety of proinflammatory cytokines and chemokines, such as GM-CSF, that may mobilize and mature dendritic cells and thereby enhance the induction of protective immunity. Using a murine model of C. trachomatis mouse pneumonitis lung infection and intrapulmonary adenoviral GM-CSF transfection, we demonstrate that the expression of GM-CSF in the airway compartment significantly enhanced systemic Th1 cellular and local IgA immune responses following immunization with inactivated organisms. Importantly, immunized mice had significantly reduced growth of chlamydia and exhibited less severe pulmonary inflammation following challenge infection. The site of GM-CSF transfection proved important, since mice immunized with inactivated organisms after GM-CSF gene transfer by the i.p. route exhibited little protection against pulmonary challenge, although i.p. immunization generated significant levels of systemic Th1 immune responses. The obvious difference between i.p. and intrapulmonary immunization was the absence of lung IgA responses following i.p. vaccination. In aggregate, the findings demonstrate that the local cytokine environment is critical to the induction of protective immunity following chlamydial vaccination and that GM-CSF may be a useful adjuvant for a chlamydial vaccine.