bluuebunny/crossref_metadata_2025 huggingface dataset:
A Nonclassical MHC Class I Molecule Restricts CTL-Mediated Rejection of a Syngeneic Melanoma Tumor
Authors: Chiang Eugene Y. , Stroynowski Iwona
Published: April 2014

Although CTL and polymorphic, classical MHC class I molecules have well defined roles in the immune response against tumors, little is currently known regarding the participation of nonpolymorphic, nonclassical MHC class I in antitumor immunity. Using an MHC class I-deficient melanoma as a model tumor, we demonstrate that Q9, a murine MHC class Ib molecule from the Qa-2 family, expressed on the surface of tumor cells, protects syngeneic hosts from melanoma outgrowth. Q9-mediated protective immunity is lost or greatly diminished in mice deficient in CTL, including β2-microglobulin knockout (KO), CD8 KO, and SCID mice. In contrast, the Q9 antitumor effects are not detectably suppressed in CD4 KO mice with decreased Th cell activity. Killing by antitumor CTL in vitro is Q9 specific and can be blocked by anti-Q9 and anti-CD8 Abs. The adaptive Q9-restricted CTL response leads to immunological memory, because mice that resist the initial tumor challenge reject subsequent challenges with less immunogenic tumor variants and show expansion of CD8+ T cell populations with an activated/memory CD44high phenotype. Collectively, these studies demonstrate that a MHC class Ib molecule can serve as a restriction element for antitumor CTL and mediate protective immune responses in a syngeneic setting.
Perturbed Homeostasis of Peripheral T Cells Elicits Decreased Susceptibility to Anti-CD3-Induced Apoptosis in Prediabetic Nonobese Diabetic Mice
Authors: Yang Wen , Hussain Shabbir , Mi Qing-Sheng , Santamaria Pere , Delovitch Terry L.
Published: April 2014

Activation-induced cell death (AICD) plays a key role in the homeostasis of the immune system. Autoreactive T cells are eliminated through AICD both from the thymus and periphery. In this study, we show that NOD peripheral T cells, especially CD8+ T cells, display a decreased susceptibility to anti-CD3-induced AICD in vivo compared with T cells from diabetes-resistant B6, nonobese diabetes-resistant, and NOD.B6Idd4 mice. The susceptibility of NOD CD8+ T cells to AICD varies in an age- and dose-dependent manner upon stimulation in vivo with either a mitogenic or nonmitogenic anti-CD3. NOD T cells preactivated by anti-CD3 in vivo are less susceptible than B6 T cells to TCR-induced AICD. Treatment of NOD mice with a mitogenic anti-CD3 depletes CD4+CD25−CD62L+ but not CD4+CD25+CD62L+ T cells, thereby resulting in an increase of the latter subset in the spleen. Treatment with a nonmitogenic anti-CD3 mAb delays the onset of T1D in 8.3 TCR transgenic NOD mice. These results demonstrate that the capacity of anti-CD3 to protect NOD mice from T1D correlates with its ability to perturb T cell homeostasis by inducing CD8+ T cell AICD and increasing the number of CD4+CD25+CD62L+ T cells in the periphery.
DNA Fusion Vaccines Induce Targeted Epitope-Specific CTLs against Minor Histocompatibility Antigens from a Normal or Tolerized Repertoire
Authors: Rice Jason , Buchan Sarah , Dewchand Hamlata , Simpson Elizabeth , Stevenson Freda K.
Published: April 2014

We have designed DNA fusion vaccines able to induce high levels of epitope-specific CD8+ T cells, using linked CD4+ T cell help. Such vaccines can activate effective immunity against tumor Ags. To model performance against minor histocompatibility (H) Ags important in allogeneic hemopoietic stem cell transplantation, responses against the H2Db-restricted Uty and Smcy male HY epitopes have been investigated. Vaccination of females induced high levels of tetramer-specific, IFN-γ-producing CD8+ T cells against each epitope. Vaccines incorporating a single epitope primed effector CTL able to kill male splenocytes in vitro and in vivo, and HYDbUty-specific vaccination accelerated rejection of syngeneic male skin grafts. Priming against either epitope established long-term memory, expandable by injection of male cells. Expanded CD8+ T cells remained specific for the priming HY epitope, with responses to the second suppressed. To investigate vaccine performance in a tolerized repertoire, male mice were vaccinated with the fusion constructs. Strikingly, this also generated epitope-specific IFN-γ-producing CD8+ T cells with cytotoxic function. However, numbers and avidity were lower than in vaccinated females, and vaccinated males failed to reject CFSE-labeled male splenocytes in vivo. Nevertheless, these findings indicate that DNA fusion vaccines can mobilize CD8+ T cells against endogenous minor H Ags, even from a profoundly tolerized repertoire. In the transplantation setting, vaccination of donors could prime and expand specific T cells for in vivo transfer. For patients, vaccination could activate a potentially less tolerized repertoire against similar Ags that may be overexpressed by tumor cells, for focused immune attack.
Tracking of Vβ8.2-Positive Encephalitogenic T Cells by Complementarity-Determining Region 3 Spectratyping and Subsequent Southern Blot Hybridization in Lewis Rats after Neuroantigen Sensitization
Authors: Sakuma Hiroshi , Kohyama Kuniko , Jee Youngheun , Matsumoto Yoh
Published: April 2014

Pathogenic T cells in organ-specific autoimmune diseases use a limited number of TCR α- and β-chains. In experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats by immunization with myelin basic protein, encephalitogenic T cells mainly use Vβ8.2 TCR and clonal expansion of the Vβ8.2 spectratype containing the EAE-specific complementarity-determining region 3 (CDR3) sequence, DSSYEQYFGPG, is found in the spinal cord throughout the course of clinical EAE. In the present study we performed temporal and spatial analyses of Vβ8.2 spectratype expansion by CDR3 spectratyping and subsequent DNA hybridization with a probe specific for the encephalitogenic CDR3 sequence to elucidate the kinetics of encephalitogenic T cells during the induction phase after neuroantigen sensitization. It was demonstrated that Vβ8.2 spectratype expansion and/or the positive signal in Southern blot were first detected in the regional lymph nodes as early as day 3 postimmunization and was disseminated over the lymphoid organs by day 6. Because perfusion of immunized rats with PBS erased the positive signals on day 3 postimmunization, the majority of Vβ8.2-positive encephalitogenic T cells at the very early stage would reside within the lymphatic or blood vessels. Furthermore, removal of the draining lymph node 1, 3, and 6 days after immunization in the foot pad did not ameliorate clinical EAE. These findings strongly suggest that encephalitogenic T cells disseminate throughout the whole body very rapidly after sensitization. Analysis of pathogenic T cells at the clonal level provides useful information for designing effective immunotherapy.
CCAAT/Enhancer-Binding Protein β Isoforms and the Regulation of α-Smooth Muscle Actin Gene Expression by IL-1β
Authors: Hu Biao , Wu Zhe , Jin Hong , Hashimoto Naozumi , Liu Tianju , Phan Sem H.
Published: April 2014

The role of IL-1β in inflammation is amply documented, but its ability to inhibit myofibroblast differentiation and, in particular, the suppression of α-smooth muscle actin (α-SMA) gene expression is less well understood. Because IL-1β can induce C/EBPβ expression, the role of C/EBPβ isoforms in IL-1β regulation of α-SMA gene expression was investigated in rat lung myofibroblasts. The results showed that IL-1β inhibited α-SMA expression in a dose-dependent manner, which was associated with stimulation of the expression of both C/EBPβ isoforms, liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP). However, a greater increase in LIP relative to LAP expression resulted in a reduced LAP/LIP ratio after IL-1β treatment. Transfection with an LAP-expressing plasmid stimulated, whereas an LIP-expressing plasmid inhibited, α-SMA expression. Cells from C/EBPβ-deficient mice had reduced levels of α-SMA expression and promoter activity, which failed to respond to IL-1β treatment. Sequence analysis identified the presence of a C/EBPβ consensus binding sequence in the α-SMA promoter, which, when mutated, resulted in diminished promoter activity and abolished its responsiveness to IL-1β treatment. EMSA revealed binding of C/EBPβ to this C/EBPβ consensus binding sequence from the α-SMA promoter. Finally, IL-1β enhanced the expression of eukaryotic initiation factor 4E, a stimulator of LIP expression, which may account for a mechanism by which IL-1β could alter the LAP/LIP ratio. These data taken together suggest that C/EBPβ isoforms regulate α-SMA gene expression, and that its inhibition by IL-1β was due to preferential stimulation of LIP expression.
Deletion of the Complement Anaphylatoxin C3a Receptor Attenuates, Whereas Ectopic Expression of C3a in the Brain Exacerbates, Experimental Autoimmune Encephalomyelitis
Authors: Boos Laura , Campbell Iain L. , Ames Robert , Wetsel Rick A. , Barnum Scott R.
Published: April 2014

The C3aR is expressed throughout the CNS and is increased in expression on glial cells during CNS inflammation. However, the role that C3a and the C3aR play in chronic inflammation, such as in the demyelinating disease experimental autoimmune encephalomyelitis (EAE), remains unclear. We show in this study that deletion of the C3aR is protective in myelin oligodendrocyte glycoprotein-induced EAE in C57BL/6 mice. C3aR-deficient (C3aR−/−) mice had a significantly attenuated course of EAE compared with control mice during the chronic phase of the disease. Immunohistochemical analysis demonstrated modestly reduced macrophage and T cell infiltration in the spinal cords of C3aR−/− mice. To examine the role of C3a in EAE, we developed a transgenic mouse that expresses C3a exclusively in the CNS using the glial fibrillary acidic protein (GFAP) promoter. We observed that C3a/GFAP mice had exacerbated EAE during the chronic phase of the disease, with significant mortality compared with nontransgenic littermates. C3a/GFAP mice had massive meningeal and perivascular infiltration of macrophages and CD4+ T cells. These studies indicate that C3a may contribute to the pathogenesis of demyelinating disease by directly or indirectly chemoattracting encephalitogenic cells to the CNS.
Anti-Idiotype × Anti-CD44 Bispecific Antibodies Inhibit Invasion of Lymphoid Organs by B Cell Lymphoma
Authors: Avin Esther , Haimovich Joseph , Hollander Nurit
Published: April 2014

The demonstration that Abs to adhesion molecules can block tumor metastasis suggested their use for therapy. However, such Abs affect nonmalignant cells as well. To circumvent this adverse effect, we proposed the use of bispecific Abs that bind simultaneously to an adhesion receptor and to a tumor-specific Ag. Such bifunctional Abs bind more avidly to tumor cells that coexpress both target Ags than to normal cells. The Id of the surface Ig of malignant B lymphocytes is a tumor-specific Ag. Therefore, we produced bispecific Abs with specificity to the adhesion molecule, CD44, and to an idiotypic determinant of the murine B cell lymphoma, 38C-13. These anti-Id × anti-CD44 bispecific Abs blocked 38C-13 cell adhesion to hyaluronic acid, while not affecting adhesion of Id-negative cells. In vivo studies demonstrated that the bispecific Abs inhibited lymphoma cell dissemination to the lymph nodes, bone marrow, and spleen, and prolonged survival of tumor-bearing mice. Migration of 38C-13 cells to the lymphoid organs was inhibited by the bispecific Abs. Thus, the bispecific Ab-mediated reduction in metastasis resulted, at least in part, from reduced homing to these organs. In contrast to anti-CD44 monospecific Abs, the anti-Id × anti-CD44 bispecific Abs did not affect immune responses such as delayed-type hypersensitivity. Hence, bispecific Abs against adhesion molecules and tumor-specific Ags may selectively block tumor metastasis in a way which may leave at least part of the immune system intact.
Negative Role of Colony-Stimulating Factor-1 in Macrophage, T Cell, and B Cell Mediated Autoimmune Disease in MRL-Fas
lpr Mice
Authors: Lenda Deborah M. , Stanley E. Richard , Kelley Vicki R.
Published: April 2014

Inflammation in the kidney and other tissues (lung, and salivary and lacrimal glands) is characteristic of MRL-Faslpr mice with features of lupus. Macrophages (Mφ) are prominent in these tissues. Given that 1) Mφ survival, recruitment, proliferation, and activation during inflammation is dependent on CSF-1, 2) Mφ mediate renal resident cell apoptosis, and 3) CSF-1 is up-regulated in MRL-Faslpr mice before, and during nephritis, we hypothesized that CSF-1-deficient MRL-Faslpr mice would be protected from Mφ-mediated nephritis, and the systemic illness. To test this hypothesis, we compared CSF-1-deficient MRL-Faslpr with wild-type strains. Renal pathology is suppressed and function improved in CSF-1-deficient MRL-Faslpr mice. There are far fewer intrarenal Mφ and T cells in CSF-1-deficient MRL-Faslpr vs wild-type kidneys. This leukocytic reduction results from suppressed infiltration, and intrarenal proliferation, but not enhanced apoptosis. The CSF-1-deficient MRL-Faslpr kidneys remain preserved as indicated by greatly reduced indices of injury (nephritogenic cytokines, tubular apoptosis, and proliferation). The renal protective mechanism in CSF-1-deficient mice is not limited to reduced intrarenal leukocytes; circulating Igs and autoantibodies, and renal Ig deposits are decreased. This may result from enhanced B cell apoptosis and fewer B cells in CSF-1-deficient MRL-Faslpr mice. Furthermore, the systemic illness including, skin, lung, and lacrimal and salivary glands pathology, lymphadenopathy, and splenomegaly are dramatically suppressed in CSF-1-deficient MRL-Faslpr as compared with wild-type mice. These results indicate that CSF-1 is an attractive therapeutic target to combat Mφ-, T cell-, and B cell-mediated autoimmune lupus.
Essential Role of LFA-1 in Activating Th2-Like Responses by α-Galactosylceramide-Activated NKT Cells
Authors: Matsumoto Goichi , Kubota Eiro , Omi Yasushi , Lee Ushaku , Penninger Josef M.
Published: April 2014

NKT cells produce large amounts of cytokines associated with both the Th1 (IFN-γ) and Th2 (IL-4) responses following stimulation of their invariant Vα14 Ag receptor. The role of adhesion molecules in the activation of NKT cells by the Vα14 ligand α-galactosylceramide (α-GalCer) remains unclear. To address this issue, LFA-1−/− (CD11a−/−) mice were used to investigate IL-4 and IFN-γ production by NKT cells following α-GalCer stimulation. Intriguingly, LFA-1−/− mice showed increased IL-4, IL-5, and IL-13 production and polarized Th2-type responses in response to α-GalCer in vitro and in vivo. Furthermore, the Th2-specific transcription factor GATA-3 was up-regulated in α-GalCer-activated NKT cells from LFA-1−/− mice. These results provide the first genetic evidence that the adhesion receptor LFA-1 has a crucial role in Th2-polarizing functions of NKT cells.
Suppressive Oligodeoxynucleotides Inhibit Th1 Differentiation by Blocking IFN-γ- and IL-12-Mediated Signaling
Authors: Shirota Hidekazu , Gursel Mayda , Klinman Dennis M.
Published: April 2014

Repetitive TTAGGG motifs present at high frequency in mammalian telomeres can suppress Th1-mediated immune responses. Synthetic oligonucleotides (ODN) containing TTAGGG motifs mimic this activity and have proven effective in the prevention/treatment of certain Th1-dependent autoimmune diseases. This work explores the mechanism by which suppressive ODN block the induction of Th1 immunity. Findings indicate that these ODN inhibit IFN-γ-induced STAT1 phosphorylation and IL-12-induced STAT3 and STAT4 phosphorylation. As a result, T-bet expression is reduced as is the maturation of naive CD4+ cells into Th1 effectors. These changes indirectly support the generation of Th2-dominated immune responses. Suppressive ODN may thus represent a novel approach to influence the Th1:Th2 balance in vivo.
Engagement of Glucocorticoid-Induced TNFR Family-Related Receptor on Effector T Cells by its Ligand Mediates Resistance to Suppression by CD4+CD25+ T Cells
Authors: Stephens Geoffrey L. , McHugh Rebecca S. , Whitters Matthew J. , Young Deborah A. , Luxenberg Deborah , Carreno Beatriz M. , Collins Mary , Shevach Ethan M.
Published: April 2014

Nonactivated CD4+CD25+ regulatory T cells constitutively express glucocorticoid-induced TNFR family-related receptor (GITR), a TNFR family member whose engagement was presumed to abrogate regulatory T cell-mediated suppression. Using GITR−/− mice, we report that GITR engagement on CD25−, not CD25+ T cells abrogates T cell-mediated suppression. Mouse APCs constitutively express GITR ligand (GITR-L), which is down-regulated following TLR signaling in vivo. Although GITR−/−CD25− T cells were capable of mounting proliferative responses, they were incapable of proliferation in the presence of physiological numbers of CD25+ T cells. Thus, GITR-L provides an important signal for CD25− T cells, rendering them resistant to CD25+-mediated regulation at the initiation of the immune response. The down-regulation of GITR-L by inflammatory stimuli may enhance the susceptibility of effector T cells to suppressor activity during the course of an infectious insult.
Novel Negative Regulator of Expression in Fas Ligand (CD178) Cytoplasmic Tail: Evidence for Translational Regulation and against Fas Ligand Retention in Secretory Lysosomes
Authors: Xiao Sheng , Deshmukh Umesh S. , Jodo Satoshi , Koike Takao , Sharma Rahul , Furusaki Akiro , Sung Sun-sang J. , Ju Shyr-Te
Published: April 2014

Fas ligand ((FasL) CD178), a type II transmembrane protein, induces apoptosis of cells expressing the Fas receptor. It possesses a unique cytoplasmic tail (FasLCyt) of 80 aa. As a type II transmembrane protein, the early synthesis of FasLCyt could affect FasL translation by impacting FasL endoplasmic reticulum translocation and/or endoplasmic reticulum retention. Previous studies suggest that the proline-rich domain (aa 43–70) in FasLCyt (FasLPRD) inhibits FasL membrane expression by retaining FasL in the secretory lysosomes. This report shows that deletion of aa 2–33 of FasLCyt dramatically increased total FasL levels and FasL cell surface expression. This negative regulator of FasL expression is dominant despite the presence of FasLPRD. In addition, retention of proline-rich domain-containing FasL in the cytoplasm was not observed. Moreover, we demonstrated that FasLCyt regulates FasL expression by controlling the rate of de novo synthesis of FasL. Our study demonstrated a novel negative regulator of FasL expression in the FasLCyt region and its mechanism of action.
The Host Resistance Locussst1Controls Innate Immunity toListeria monocytogenesInfection in Immunodeficient Mice
Authors: Boyartchuk Victor , Rojas Mauricio , Yan Bo-Shiun , Jobe Ousman , Hurt Nicholas , Dorfman David M. , Higgins Darren E. , Dietrich William F. , Kramnik Igor
Published: April 2014

Epidemiological, clinical, and experimental approaches have convincingly demonstrated that host resistance to infection with intracellular pathogens is significantly influenced by genetic polymorphisms. Using a mouse model of infection with virulent Mycobacterium tuberculosis (MTB), we have previously identified the sst1 locus as a genetic determinant of host resistance to tuberculosis. In this study we demonstrate that susceptibility to another intracellular pathogen, Listeria monocytogenes, is also influenced by the sst1 locus. The contribution of sst1 to anti-listerial immunity is much greater in immunodeficient scid mice, indicating that this locus controls innate immunity and becomes particularly important when adaptive immunity is significantly depressed. Similar to our previous observations using infection with MTB, the resistant allele of sst1 prevents formation of necrotic infectious lesions in vivo. We have shown that macrophages obtained from sst1-resistant congenic mice possess superior ability to kill L. monocytogenes in vitro. The bactericidal effect of sst1 is dependent on IFN-γ activation and reactive oxygen radical production by activated macrophages after infection, but is independent of NO production. It is possible that there is a single gene that controls common IFN-dependent macrophage function, which is important in the pathogenesis of infections caused by both MTB and L. monocytogenes. However, host resistance to the two pathogens may be controlled by two different polymorphic genes encoded within the sst1 locus. The polymorphic gene(s) encoded within the sst1 locus that controls macrophage interactions with the two intracellular pathogens remains to be elucidated.
Bacterial Heat Shock Proteins Enhance Class II MHC Antigen Processing and Presentation of Chaperoned Peptides to CD4+ T Cells
Authors: Tobian Aaron A. R. , Canaday David H. , Harding Clifford V.
Published: April 2014

APCs process heat shock protein (HSP):peptide complexes to present HSP-chaperoned peptides on class I MHC molecules, but the ability of HSPs to contribute chaperoned peptides for class II MHC (MHC-II) Ag processing and presentation is unclear. Our studies revealed that exogenous bacterial HSPs (Escherichia coli DnaK and Mycobacterium tuberculosis HSP70) delivered an extended OVA peptide for processing and MHC-II presentation, as detected by T hybridoma cells. Bacterial HSPs enhanced MHC-II presentation only if peptide was complexed to the HSP, suggesting that the key HSP function was enhanced delivery or processing of chaperoned peptide Ag rather than generalized enhancement of APC function. HSP-enhanced processing was intact in MyD88 knockout cells, which lack most TLR signaling, further suggesting the effect was not due to TLR-induced induction of accessory molecules. Bacterial HSPs enhanced uptake of peptide, which may contribute to increased MHC-II presentation. In addition, HSPs enhanced binding of peptide to MHC-II molecules at pH 5.0 (the pH of vacuolar compartments), but not at pH 7.4, indicating another mechanism for enhancement of MHC-II Ag processing. Bacterial HSPs are a potential source of microbial peptide Ags during phagocytic processing of bacteria during infection and could potentially be incorporated in vaccines to enhance presentation of peptides to CD4+ T cells.
Transendothelial Migration of Human Basophils
Authors: Iikura Motoyasu , Ebisawa Motohiro , Yamaguchi Masao , Tachimoto Hiroshi , Ohta Ken , Yamamoto Kazuhiko , Hirai Koichi
Published: April 2014

During allergic reactions, basophils migrate from the blood compartment to inflammatory sites, where they act as effector cells in concert with eosinophils. Because transendothelial migration (TEM) represents an essential step for extravasation of cells, for the first time we have studied basophil TEM using HUVEC. Treatment of HUVEC with IL-1β significantly enhanced basophil TEM, which was further potentiated by the presence of a CCR3-specific ligand, eotaxin/CCL11. In addition to CCR3 ligands, MCP-1/CCL2 was also active on basophil TEM. Although stromal cell-derived factor-1/CXCL12, a CXCR4 ligand, failed to induce TEM in freshly isolated basophils, it caused strong TEM in 24-h cultured cells. IL-3 enhanced basophil TEM by increasing the chemokinetic response. Spontaneous TEM across activated HUVEC was inhibited by treatment of cells with anti-CD18 mAb, but not with anti-CD29 mAb, and also by treatment of HUVEC with anti-ICAM-1 mAb. Anti-VCAM-1 mAb alone failed to inhibit TEM, but showed an additive inhibitory effect in combination with anti-ICAM-1 mAb. In contrast, eotaxin- and IL-3-mediated TEM was significantly inhibited by anti-CD29 mAb as well as anti-CD18 mAb. These results indicate that β2 integrins play the primary role in basophil TEM, but β1 integrins are also involved, especially in TEM of cytokine/chemokine-stimulated basophils. In conclusion, the regulatory profile of basophil TEM is very similar to that reported for eosinophils. Our results thus support the previous argument for a close relationship between basophils and eosinophils and suggest that the in vivo kinetics of these two cell types are similar.
Role of L-Selectin in the Vascular Homing of Peripheral Blood-Derived Endothelial Progenitor Cells
Authors: Biancone Luigi , Cantaluppi Vincenzo , Duò Debora , Deregibus Maria Chiara , Torre Carlo , Camussi Giovanni
Published: April 2014

Ex vivo expanded endothelial progenitor cells (EPCs) represent a new potential approach for the revascularization of ischemic sites. However, local accumulation of infused EPCs in these sites is poor, and the mechanisms responsible for their homing are largely unknown. We observed the expression of L-selectin, an adhesion receptor that regulates lymphocyte homing and leukocyte rolling and migration, on ex vivo expanded blood-derived human EPCs. When EPCs were subcloned in SV40-T large Ag-transfected isolates, the copresence of L-selectin and endothelial lineage markers was confirmed. We therefore demonstrated that the expression of L-selectin by EPCs was functional because it mediates interaction with a murine endothelial cell line (H.end) expressing L-selectin ligands by way of transfection with α(1,3/4)-fucosyltransferase. Indeed, adhesion of EPCs after incubation at 4°C on a rotating platform was enhanced on α(1,3/4)-fucosyltransferase-transfected H.end cells compared with control vector-transfected cells, and treatment with anti-L-selectin Abs prevented this event. We then studied the role of L-selectin in EPC homing in vivo. H.end cells were implanted s.c. in SCID mice to form endothelioma tumors, and EPCs were subsequently i.v. injected. L-selectin+ EPCs localized into α(1,3/4)-fucosyltransferase-transfected endothelial tumors to a greater extent than in control tumors, and they were able to directly contribute to tumor vascularization by forming L-selectin+ EPC-containing vessels. In conclusion, our results showed that a mechanism typical of leukocyte adhesion is involved in the vascular homing of EPCs within sites of selectin ligand expression. This observation may provide knowledge about the substrate to design strategies to improve EPC localization in damaged tissues.
Cutting Edge: Identification of Hepatitis C Virus-Specific CD8+ T Cells Restricted by Donor HLA Alleles following Liver Transplantation
Authors: Rosen Hugo R. , Hinrichs David J. , Leistikow Rachel L. , Callender Glenda , Wertheimer Anne M. , Nishimura Michael I. , Lewinsohn David M.
Published: April 2014

By necessity, human liver transplantation is performed across HLA barriers. As a result, intracellular infection of the allograft presents a unique immunologic challenge for the recipient’s immune system. In this study, we describe the presence of HLA-A2-restricted, hepatitis C virus (HCV)-specific CD8+ T cells in liver transplant recipients in whom the allograft is HLA-A2 positive and the recipient is HLA-A2 negative. These memory-effector T cells are recipient derived and recognize HCV peptide uniquely in the context of HLA-A2. Furthermore, these cells were absent before the transplant, suggesting that the allograft is capable of selectively expanding naive CD8+ T cells. The in vitro specificity to donor HLA allele-restricted CD8+ T cells suggests that these cells may function to control HCV spread in the allograft.
Vaccination in Humans Generates Broad T Cell Cytokine Responses
Authors: De Rosa Stephen C. , Lu Fabien X. , Yu Joanne , Perfetto Stephen P. , Falloon Judith , Moser Susan , Evans Thomas G. , Koup Richard , Miller Christopher J. , Roederer Mario
Published: April 2014

In recent years, the quantification of T cell responses to pathogens or immunogens has become a common tool in the evaluation of disease pathogenesis or vaccine immunogenicity. Such measurements are usually limited to enumerating IFN-γ-producing cells after ex vivo stimulation with Ag, but little is known about the phenotype or complete functional repertoire of the Ag-specific cells. We used 12-color flow cytometry to characterize Ag-specific T cells elicited by vaccines or natural infection to determine lineage and differentiation status as well as the capacity to produce four cytokines (IFN-γ, TNF-α, IL-2, and IL-4) and a chemokine (MIP1β). As expected, responding cells had a typical memory phenotype; however, the cytokine profiles associated with the responses were highly complex. The pattern of cytokine coexpression in response to specific Ags was a skewed subset of the complete repertoire (revealed by polyclonal stimulation). We found significant differences in the patterns of cytokines elicited by vaccination (where IFN-γ was by far a subdominant response) vs natural infection; in addition, there was fairly significant intersubject variation. Our findings illustrate the limitation of the evaluation of immune responses using single functional measurements (such as IFN-γ); in fact, it is likely that sensitive evaluation of Ag-specific T cells will require the coordinate measurement of several cytokines. The presence and variability of these complex response profiles introduce the possibility that selective functional expression patterns may provide correlates for vaccine efficacy or disease progression.
Delineation of Signals Required for Thymocyte Positive Selection
Authors: Santori Fabio R. , Vukmanović Stanislav
Published: April 2014

Peptide/MHC complexes capable of inducing positive selection in mouse fetal thymic organ cultures fail to do so in suspension culture. Furthermore, this type of culture does not promote initial stages of differentiation, such as coreceptor down-modulation, unless peptides used for stimulation have (at least) weak agonist activity. We show in this study that signals provided in suspension culture by nonagonist peptide/MHC complexes on the surface of macrophages, even though apparently silent, are sufficient to promote complete phenotypic differentiation when CD4+CD8+ thymocytes are subsequently placed in a proper anatomical setting. Furthermore, the synergistic actions of suboptimal concentrations of phorbol esters and nonagonist peptide/MHC complexes can make the initial stages of positive selection visible, without converting maturation into negative selection. Thus, the correlation between efficiency of positive selection and the degree of coreceptor down-modulation on CD4+CD8+ thymocytes is not linear. Furthermore, these results suggest that the unique role of thymic stromal cells in positive selection is related not to presentation of self-peptide/MHC complexes, but most likely to another ligand.
Reduced Apoptosis and Ameliorated Listeriosis in TRAIL-Null Mice
Authors: Zheng Shi-Jun , Jiang Jiu , Shen Hao , Chen Youhai H.
Published: April 2014

Listeriosis is an infectious disease caused by the bacterium Listeria monocytogenes. Although it is well recognized that apoptosis plays a critical role in the pathogenesis of the disease, the molecular mechanisms of cell death in listeriosis remain to be established. We report in this study that mice deficient in TRAIL were partially resistant to primary listeriosis, and blocking TRAIL with a soluble death receptor 5 markedly ameliorated the disease. The numbers of Listeria in the liver and spleen of TRAIL+/+ mice were 10–100 times greater than those in TRAIL−/− mice following primary Listeria infection. This was accompanied by a significant increase in the survival rate of TRAIL−/− mice. Lymphoid and myeloid cell death was significantly inhibited in TRAIL−/− mice, which led to marked enlargement of the spleen. These results establish a critical role for TRAIL in apoptosis during listeriosis.
N-Terminal Residues of the Chemotaxis Inhibitory Protein of Staphylococcus aureus Are Essential for Blocking Formylated Peptide Receptor but Not C5a Receptor
Authors: Haas Pieter-Jan , de Haas Carla J. C. , Kleibeuker Wendy , Poppelier Miriam J. J. G. , van Kessel Kok P. M. , Kruijtzer John A. W. , Liskamp Rob M. J. , van Strijp Jos A. G.
Published: April 2014

Staphylococcus aureus excretes a factor that specifically and simultaneously acts on the C5aR and the formylated peptide receptor (FPR). This chemotaxis inhibitory protein of S. aureus (CHIPS) blocks C5a- and fMLP-induced phagocyte activation and chemotaxis. Monoclonal anti-CHIPS Abs inhibit CHIPS activity against one receptor completely without affecting the other receptor, indicating that two distinct sites are responsible for both actions. A CHIPS-derived N-terminal 6 aa peptide is capable of mimicking the anti-FPR properties of CHIPS but has no effect on the C5aR. Synthetic peptides in which the first 6 aa are substituted individually for all other naturally occurring amino acids show that the first and third residue play an important role in blocking the FPR. Using an Escherichia coli expression system, we created mutant CHIPS proteins in which these amino acids are substituted. These mutant proteins have impaired or absent FPR- but still an intact C5aR-blocking activity, indicating that the loss of the FPR-blocking activity is not caused by any structural impairment. This identifies the first and third amino acid, both a phenylalanine, to be essential for CHIPS blocking the fMLP-induced activation of phagocytes. The unique properties of CHIPS to specifically inhibit the FPR with high affinity (kd = 35.4 ± 7.7 nM) could be an important new tool to further stimulate the fundamental research on the mechanisms underlying the FPR and its role in disease processes.
Interference with Heparin Binding and Oligomerization Creates a Novel Anti-Inflammatory Strategy Targeting the Chemokine System
Authors: Johnson Zoë , Kosco-Vilbois Marie H. , Herren Suzanne , Cirillo Rocco , Muzio Valeria , Zaratin Paola , Carbonatto Michela , Mack Matthias , Smailbegovic Amir , Rose Mark , Lever Rebecca , Page Clive , Wells Timothy N. C. , Proudfoot Amanda E. I.
Published: April 2014

A hallmark of autoimmunity and other chronic diseases is the overexpression of chemokines resulting in a detrimental local accumulation of proinflammatory immune cells. Chemokines play a pivotal role in cellular recruitment through interactions with both cell surface receptors and glycosaminoglycans (GAGs). Anti-inflammatory strategies aimed at neutralizing the chemokine system have to-date targeted inhibition of the receptor-ligand interaction with receptor antagonists. In this study, we describe a novel strategy to modulate the inflammatory process in vivo through mutation of the essential heparin-binding site of a proinflammatory chemokine, which abrogates the ability of the protein to form higher-order oligomers, but retains receptor activation. Using well-established protocols to induce inflammatory cell recruitment into the peritoneal cavity, bronchoalveolar air spaces, and CNS in mice, this non-GAG binding variant of RANTES/CCL5 designated [44AANA47]-RANTES demonstrated potent inhibitory capacity. Through a combination of techniques in vitro and in vivo, [44AANA47]-RANTES appears to act as a dominant-negative inhibitor for endogenous RANTES, thereby impairing cellular recruitment, not through a mechanism of desensitization. [44AANA47]-RANTES is unable to form higher-order oligomers (necessary for the biological activity of RANTES in vivo) and importantly forms nonfunctional heterodimers with the parent chemokine, RANTES. Therefore, although retaining receptor-binding capacity, altering the GAG-associated interactive site of a proinflammatory chemokine renders it a dominant-negative inhibitor, suggesting a powerful novel approach to generate disease-modifying anti-inflammatory reagents.
Crucial Role of Inhibitor of DNA Binding/Differentiation in the Vascular Endothelial Growth Factor-Induced Activation and Angiogenic Processes of Human Endothelial Cells
Authors: Sakurai Daisuke , Tsuchiya Naoyuki , Yamaguchi Akihiro , Okaji Yurai , Tsuno Nelson H. , Kobata Tetsuji , Takahashi Koki , Tokunaga Katsushi
Published: April 2014

Angiogenesis plays a pivotal role in the aggressive proliferation of synovial cells in rheumatoid arthritis. We have previously reported the overexpression of inhibitor of DNA binding/differentiation (Id) in the endothelial cells within the synovial tissues of rheumatoid arthritis. In this study, we investigated the role of Id in inflammation and angiogenesis in an in vitro model using HUVECs. Vascular endothelial growth factor (VEGF) and TGFβ induced the expression of Id1 and Id3 in HUVECs. Forced expression of Id induced proliferative activity in HUVECs accompanied by down-regulation of p16INK4a. Overexpression of Id enhanced expression of ICAM-1 and E-selectin, and induced angiogenic processes such as transmigration, matrix metalloproteinase-2 and -9 expression, and tube formation. In contrast, knockdown of Id1 and Id3 with RNA interference abolished proliferation, activation, and angiogenic processes of HUVECs induced by VEGF. These results indicated that Id plays a crucial role in VEGF-induced signals of endothelial cells by causing activation and potentiation of angiogenic processes. Based on these findings, it was proposed that inhibition of expression and/or function of Id1 and Id3 may potentially be of therapeutic value for conditions associated with pathological angiogenesis.
CC Chemokine Ligand 18, An Atopic Dermatitis-Associated and Dendritic Cell-Derived Chemokine, Is Regulated by Staphylococcal Products and Allergen Exposure
Authors: Pivarcsi Andor , Gombert Michael , Dieu-Nosjean Marie-Caroline , Lauerma Antti , Kubitza Robert , Meller Stephan , Rieker Juliane , Muller Anja , Da Cunha Ludivine , Haahtela Anna , Sonkoly Eniko , Fridman Wolf-Herman , Alenius Harri , Kemeny Lajos , Ruzicka Thomas , Zlotnik Albert , Homey Bernhard
Published: April 2014

Atopic dermatitis is a chronic inflammatory skin disease with a steadily increasing prevalence. Exposure to allergens or bacterial superantigens triggers T and dendritic cell (DC) recruitment and induces atopic skin inflammation. In this study, we report that among all known chemokines CCL18/DC-CK1/PARC represents the most highly expressed ligand in atopic dermatitis. Moreover, CCL18 expression is associated with an atopic dermatitis phenotype when compared with other chronic inflammatory skin diseases. DCs either dispersed within the dermis or clustering at sites showing perivascular infiltrates are abundant sources of CCL18. In vitro, microbial products including LPS, peptidoglycan, and mannan, as well as the T cell-derived activation signal CD40L, induced CCL18 in monocytes. In contrast to monocytes, monocyte-derived, interstitial-type, and Langerhans-type DCs showed a constitutive and abundant expression of CCL18. In comparison to Langerhans cells, interstitial-type DCs produced higher constitutive levels of CCL18. In vivo, topical exposure to the relevant allergen or the superantigen staphylococcal enterotoxin B, resulted in a significant induction of CCL18 in atopic dermatitis patients. Furthermore, in nonatopic NiSO4-sensitized individuals, only relevant allergen but not irritant exposure resulted in the induction of CCL18. Taken together, findings of the present study demonstrate that CCL18 is associated with an atopy/allergy skin phenotype, and is expressed at the interface between the environment and the host by cells constantly screening foreign Ags. Its regulation by allergen exposure and microbial products suggests an important role for CCL18 in the initiation and amplification of atopic skin inflammation.
Targeting IL-15 Receptor-Bearing Cells with an Antagonist Mutant IL-15/Fc Protein Prevents Disease Development and Progression in Murine Collagen-Induced Arthritis
Authors: Ferrari-Lacraz Sylvie , Zanelli Eric , Neuberg Manfred , Donskoy Elina , Kim Yon Su , Zheng Xin Xiao , Hancock Wayne W. , Maslinski Wlodzimierz , Li Xian Chang , Strom Terry B. , Moll Thomas
Published: April 2014

It has been suggested that the inflammatory cytokine IL-15 plays an important role in the development of several autoimmune diseases, including rheumatoid arthritis. We have generated a unique lytic and antagonistic IL-15 mutant/Fcγ2a fusion protein (CRB-15) that targets the IL-15R. In the present study we examined the effects of targeting the IL-15R on the prevention and treatment of collagen-induced arthritis (CIA) in mice and probed the possible mechanisms of action of this IL-15 mutant/Fcγ2a protein. Upon immunization with type II collagen, DBA/1 mice develop severe articular inflammation and destruction. Treatment of DBA/1 mice with a brief course of CRB-15 at the time of type II collagen challenge markedly inhibited the incidence and severity of arthritis. Moreover, in animals with ongoing established arthritis, treatment with CRB-15 effectively blocked disease progression compared with that in control-treated animals. The therapeutic effect of CRB-15 on either disease development or disease progression is remarkably stable, because withdrawal of treatment did not lead to disease relapse. A detailed analysis revealed that treatment with CRB-15 decreased synovitis in the joints; reduced bone erosion and cartilage destruction; reduced in situ production of the proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-17; and decreased the responder frequency of autoreactive T cells. Our study suggests that the effective targeting of IL-15R-triggered events with CRB-15 can be of therapeutic importance in the treatment of rheumatoid arthritis.
Reciprocal Activating Interaction Between Dendritic Cells and Pamidronate-Stimulated γδ T Cells: Role of CD86 and Inflammatory Cytokines
Authors: Conti Lucia , Casetti Rita , Cardone Marco , Varano Barbara , Martino Angelo , Belardelli Filippo , Poccia Fabrizio , Gessani Sandra
Published: April 2014

We investigated the interactions between human monocyte-derived dendritic cells (DCs) and Ag-activated circulating TCR-γδ-expressing lymphocytes (Vδ2). Coculture of immature DCs (iDCs) with peripheral blood Vδ2 T cells activated with either pyrophosphomonoesters (isopentenyl pyrophosphate; IPP) or aminobiphosphonates (pamidronate; PAM) led to a significant up-modulation of CD86 and MHC class I molecules and to the acquisition of functional features typical of activated DCs. DC activation induced by both IPP- and PAM-stimulated γδ T cells was mostly mediated by TNF-α and IFN-γ secreted by activated lymphocytes. However, the effect of PAM-activated γδ T cells, but not that of IPP-activated cells, required cell-to-cell contact. Reciprocally, activation of Vδ2 T cells by PAM, but not by IPP, was dependent on cell contact with iDCs. In fact, when PAM-stimulated DC-γδ T cell cocultures were separated by a semipermeable membrane or treated with blocking anti-CD86 Abs, induction of CD25 and CD69 as well as IFN-γ and TNF-α secretion by Vδ2 cells were strongly reduced. These results demonstrate for the first time a bidirectional activating interaction between iDCs and PAM-stimulated γδ T lymphocytes, thus suggesting a potential adjuvant role of this early cross-talk in the therapeutic activity of aminobiphosphonate drugs.
IL-2 Overcomes the Unresponsiveness but Fails to Reverse the Regulatory Function of Antigen-Induced T Regulatory Cells
Authors: Anderson Per O. , Sundstedt Anette , Yazici Zihni , Minaee Sophie , Woolf Richard , Nicolson Kirsty , Whitley Nathaniel , Li Li , Li Suling , Wraith David C. , Wang Ping
Published: April 2014

Intranasal administration of peptide Ac1–9[4Y], based on the N-terminal epitope of myelin basic protein, can induce CD4+ T cell tolerance, and suppress experimental autoimmune encephalomyelitis induction. The peptide-induced regulatory T (PI-TReg) cells failed to produce IL-2, but expressed IL-10 in response to Ag and could suppress naive T cell responses in vitro. Analysis of Jak-STAT signaling pathways revealed that the activation of Jak1, STAT3, and STAT5 were induced in tolerant T cells after Ag stimulation in vivo. In addition, the expression of suppressor of cytokine signaling 3 was induced in tolerant T cells, suggesting that cytokines regulate the tolerant state of the PI-TReg cells. Stimulation of PI-TReg cells in vitro with IL-10 induced Jak1 and STAT3 activation, but not STAT5, suggesting that IL-10 is important, but not the only cytokine involved in the development of T cell tolerance. Although IL-2 expression was deficient, stimulation with IL-2 in vitro induced Jak1 and STAT5 activation in PI-TReg cells, restored their proliferative response to antigenic stimulation, and abrogated PI-TReg-mediated suppression in vitro. However, the addition of IL-2 could not suppress IL-10 expression, and the IL-2 gene remained inactive. After withdrawal of IL-2, the PI-TReg cells regained their nonproliferative state and suppressive ability. These results underline the ability of the immune system to maintain tolerance to autoantigens, but at the same time having the ability to overcome the suppressive phenotype of tolerant T cells by cytokines, such as IL-2, during the protective immune response to infection.
Tracing the Pre-B to Immature B Cell Transition in Human Leukemia Cells Reveals a Coordinated Sequence of Primary and Secondary IGK Gene Rearrangement, IGK Deletion, and IGL Gene Rearrangement
Authors: Klein Florian , Feldhahn Niklas , Mooster Jana L. , Sprangers Mieke , Hofmann Wolf-Karsten , Wernet Peter , Wartenberg Maria , Müschen Markus
Published: April 2014

The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant transformation of pre-B cells and prevents further development. We studied whether inhibition of BCR-ABL1 kinase activity using STI571 can relieve this differentiation block. STI571 treatment of leukemia patients induced expression of the Ig L chain-associated transcription factors IRF4 and SPIB, up-regulation of RAG1 and RAG2, Cκ and Cλ germline transcription, and rearrangement of Ig κ L chain (IGK) and Ig λ L chain (IGL) genes. However, STI571-treated pre-B ALL cells expressed λ L, but almost no κ L chains. This could be explained by STI571-induced rearrangement of the κ-deleting element (KDE), which can delete productively rearranged Vκ-Jκ joints. Amplifying double-strand breaks at recombination signal sequences within the IGK, KDE, and IGL loci revealed a coordinated sequence of rearrangement events induced by STI571: recombination of IGK gene segments was already initiated within 1 h after STI571 treatment, followed by KDE-mediated deletion of Vκ-Jκ joints 6 h later and, ultimately, IGL gene rearrangement after 12 h. Consistently, up-regulation of Cκ and Cλ germline transcripts, indicating opening of IGK and IGL loci, was detected after 1 and 6 h for IGK and IGL, respectively. Continued activity of the recombination machinery induced secondary IGK gene rearrangements, which shifted preferential usage of upstream located Jκ- to downstream Jκ-gene segments. Thus, inhibition of BCR-ABL1 in pre-B ALL cells 1) recapitulates early B cell development, 2) directly shows that IGK, KDE, and IGL genes are rearranged in sequential order, and 3) provides a model for Ig L chain gene regulation in the human.