bluuebunny/crossref_metadata_2025 huggingface dataset:
CCR4 Participation in Th Type 1 (Mycobacterial) and Th Type 2 (Schistosomal) Anamnestic Pulmonary Granulomatous Responses
Authors: Freeman Christine M. , Stolberg Valerie R. , Chiu Bo-Chin , Lukacs Nicholas W. , Kunkel Steven L. , Chensue Stephen W.
Published: April 2014

CCR4 is purported to be a Th type 2 (Th2) cell-biased receptor but its functional role is unclear. Recent studies suggest that chemokine receptor expression and function are more complex in vivo and raise doubts regarding restricted CCR4 expression by Th2 cells. To address these issues, we analyzed the role of CCR4 in highly polarized models of Th type 1 (Th1) and Th2 cell-mediated pulmonary granulomas, respectively, elicited by i.v. challenge of primed mice with either mycobacterial purified protein derivative or schistosomal egg Ag-coated beads. CCR4 agonists were expressed during both responses, correlating with a shift of CCR4+CD4+ T cells from blood to lungs. CCL22 dominated in draining nodes during the Th1 response. Analysis of CD4+ effector T cells revealed CCR4 expression and CCR4-mediated chemotaxis by both IFN-γ and IL-4 producers. Studies of CCR4 knockout (CCR4−/−) mice showed partial impairment of the local type-2 cytokine response and surprisingly strong impairment of the Th1 response with abrogated IFN-γ production during secondary but not primary challenge. Adoptive transfer indicated CCR4−/−CD4+ Th1 cell function was defective but this could not be reconstituted with wild-type (CCR4+/+) CD4+ T cells indicating involvement of another CCR4+ population. Coculture of CCR4+/+CD4+ T cells and CCR4−/− dendritic cells revealed intact IL-2 but impaired IFN-γ production, pointing to a role for CCR4+ dendritic cells in effector cell expression. Therefore, CCR4 is not Th2-restricted and was required for sustenance and expression of the Th1 effector/memory response to mycobacterial Ags.
Killing of Normal Melanocytes, Combined with Heat Shock Protein 70 and CD40L Expression, Cures Large Established Melanomas
Authors: Sanchez-Perez Luis , Kottke Timothy , Daniels Gregory A. , Diaz Rosa Maria , Thompson Jill , Pulido Jose , Melcher Alan , Vile Richard G.
Published: April 2014

Previously, we showed that nine intradermal injections of a plasmid in which the HSVtk suicide gene is expressed from a melanocyte-specific promoter (Tyr-HSVtk), combined with a plasmid expressing heat shock protein 70 (CMV-hsp70), along with systemic ganciclovir, kills normal melanocytes and raises a CD8+ T cell response that is potent enough to eradicate small, 3-day established B16 tumors. We show in this study that, in that regimen, hsp70 acts as a potent immune adjuvant through TLR-4 signaling and local induction of TNF-α. hsp70 is required for migration of APC resident in the skin to the draining lymph nodes to present Ags, derived from the killing of normal melanocytes, to naive T cells. The addition of a plasmid expressing CD40L increased therapeutic efficacy, such that only six plasmid injections were now required to cure large, 9-day established tumors. Generation of potent immunological memory against rechallenge in cured mice accompanied these therapeutic gains, as did induction of aggressive autoimmune symptoms. Expression of CD40L, along with hsp70, increased both the frequency and activity of T cells activated against melanocyte-derived Ags. In this way, addition of CD40L to the hsp70-induced inflammatory killing of melanocytes can be used to cure large established tumors and to confer immunological memory against tumor cells, although a concomitant increase in autoimmune sequelae also is produced.
Gliadin-Specific Type 1 Regulatory T Cells from the Intestinal Mucosa of Treated Celiac Patients Inhibit Pathogenic T Cells
Authors: Gianfrani Carmen , Levings Megan K. , Sartirana Claudia , Mazzarella Giuseppe , Barba Gianvincenzo , Zanzi Delia , Camarca Alessandra , Iaquinto Gaetano , Giardullo Nicola , Auricchio Salvatore , Troncone Riccardo , Roncarolo Maria-Grazia
Published: April 2014

Celiac disease (CD) results from a permanent intolerance to dietary gluten and is due to a massive T cell-mediated immune response to gliadin, the main component of gluten. In this disease, the regulation of immune responses to dietary gliadin is altered. Herein, we investigated whether IL-10 could modulate anti-gliadin immune responses and whether gliadin-specific type 1 regulatory T (Tr1) cells could be isolated from the intestinal mucosa of CD patients in remission. Short-term T cell lines were generated from jejunal biopsies, either freshly processed or cultured ex vivo with gliadin in the presence or absence of IL-10. Ex vivo stimulation of CD biopsies with gliadin in the presence of IL-10 resulted in suppression of Ag-specific proliferation and cytokine production, indicating that pathogenic T cells are susceptible to IL-10-mediated immune regulation. T cell clones generated from intestinal T cell lines were tested for gliadin specificity by cytokine production and proliferative responses. The majority of gliadin-specific T cell clones had a Th0 cytokine production profile with secretion of IL-2, IL-4, IFN-γ, and IL-10 and proliferated in response to gliadin. Tr1 cell clones were also isolated. These Tr1 cells were anergic, restricted by DQ2 (a CD-associated HLA), and produced IL-10 and IFN-γ, but little or no IL-2 or IL-4 upon activation with gliadin or polyclonal stimuli. Importantly, gliadin-specific Tr1 cell clones suppressed proliferation of pathogenic Th0 cells. In conclusion, dietary Ag-specific Tr1 cells are present in the human intestinal mucosa, and strategies to boost their numbers and/or function may offer new therapeutic opportunities to restore gut homeostasis.
Innate Immunity in Multiple Sclerosis: Myeloid Dendritic Cells in Secondary Progressive Multiple Sclerosis Are Activated and Drive a Proinflammatory Immune Response
Authors: Karni Arnon , Abraham Michal , Monsonego Alon , Cai Guifang , Freeman Gordon J. , Hafler David , Khoury Samia J. , Weiner Howard L.
Published: April 2014

Multiple sclerosis (MS) is postulated to be a T cell-mediated autoimmune disease characterized clinically by a relapsing-remitting (RR) stage followed by a secondary progressive (SP) phase. The progressive phase is felt to be secondary to neuronal degenerative changes triggered by inflammation. The status of the innate immune system and its relationship to the stages of MS is not well understood. Dendritic cells (DCs) are professional APCs that are central cells of the innate immune system and have the unique capacity to induce primary immune responses. We investigated circulating myeloid DCs isolated directly from the blood to determine whether there were abnormalities in myeloid DCs in MS and whether they were related to disease stage. We found that SP-MS subjects had an increased percentage of DCs expressing CD80, a decreased percentage expressing PD-L1, and an increased percentage producing IL-12 and TNF-α compared with RR-MS or controls. A higher percentage of DCs from both RR and SP-MS patients expressed CD40 compared with controls. We then investigated the polarization effect of DCs from MS patients on naive T cells taken from cord blood using a MLR assay. Whereas DCs from RR-MS induced higher levels of Th1 (IFN-γ, TNF-α) and Th2 (IL-4, IL-13) cytokines compared with controls, DCs from SP-MS only induced a polarized Th1 response. These results demonstrate abnormalities of DCs in MS and may explain the immunologic basis for the different stages and clinical patterns of MS.
Increased Frequencies of Cochlin-Specific T Cells in Patients with Autoimmune Sensorineural Hearing Loss
Authors: Baek Moo-Jin , Park Hyun-Min , Johnson Justin M. , Altuntas Cengiz Z. , Jane-wit Daniel , Jaini Ritika , Solares C. Arturo , Thomas Dawn M. , Ball Edward J. , Robertson Nahid G. , Morton Cynthia C. , Hughes Gordon B. , Tuohy Vincent K.
Published: April 2014

Autoimmune sensorineural hearing loss (ASNHL) is the most common cause of sudden hearing loss in adults. Although autoimmune etiopathogenic events have long been suspected in ASNHL, inner ear-specific Ags capable of targeting T cell autoreactivity have not been identified in ASNHL. In this study, we show by ELISPOT analysis that compared with normal hearing age- and sex-matched control subjects, ASNHL patients have significantly higher frequencies of circulating T cells producing either IFN-γ (p = 0.0001) or IL-5 (p = 0.03) in response to recombinant human cochlin, the most abundant inner ear protein. In some patients, cochlin responsiveness involved both CD4+ and CD8+ T cells whereas other patients showed cochlin responsiveness confined to CD8+ T cells. ASNHL patients also showed significantly elevated cochlin-specific serum Ab titers compared with both normal hearing age- and sex-matched control subjects and patients with noise- and/or age-related hearing loss (p < 0.05 at all dilutions tested through 1/2048). Our study is the first to show T cell responsiveness to an inner ear-specific protein in ASNHL patients, and implicates cochlin as a prominent target Ag for mediating autoimmune inner ear inflammation and hearing loss.
CD45neg but Not CD45pos Human Myeloma Cells Are Sensitive to the Inhibition of IGF-1 Signaling by a Murine Anti-IGF-1R Monoclonal Antibody, mAVE1642
Authors: Descamps Géraldine , Wuillème-Toumi Soraya , Trichet Valérie , Venot Corinne , Debussche Laurent , Hercend Thierry , Collette Madeleine , Robillard Nelly , Bataille Régis , Amiot Martine
Published: April 2014

Insulin-like growth factor 1 (IGF-1) is a well-known growth factor for myeloma cells. Thus, therapeutic strategies targeting IGF-1R have been proposed for multiple myeloma treatment. In this study, we investigated the effect of the antagonistic anti-IGF-1R murineAVE1642 Ab (mAVE1642). We show that mAVE1642 selectively inhibits IGF-1R but not insulin signaling in human myeloma cell lines. Since we have previously shown the functional relevance of CD45 expression in the growth of myeloma cells and the association of CD45-negative (CD45neg) status with a less favorable clinical outcome, both CD45-positive (CD45pos) and CD45neg myeloma cell lines were selected for our study. We found that mAVE1642 strongly inhibits the growth of CD45neg myeloma cell lines, leading to a G1 growth arrest, whereas it has almost no effect on the growth of CD45pos myeloma cell lines. Furthermore, mAVE1642 binding induced a significant reduction of IGF-1R expression. We next demonstrated that the overexpression of IGF-1R in the CD45pos myeloma cell line increased Akt phosphorylation but was not sufficient to sensitize these cells to mAVE1642. In contrast, we generated a stable CD45-silencing XG-1 cell line and showed that it became sensitive to mAVE1642. Thus, for the first time, we provided direct evidence that the expression of CD45 renders cells resistant to mAVE1642. Taken together, these results support that therapy directed against IGF-1R can be beneficial in treating CD45neg patients.
Cutting Edge: Paracrine, but Not Autocrine, IL-2 Signaling Is Sustained during Early Antiviral CD4 T Cell Response
Authors: Long Meixiao , Adler Adam J.
Published: April 2014

IL-2 is expressed predominantly by activated T cells, and regulates T cell function by activating, via its receptor, the latent transcription factor STAT5. This signaling can occur in either a paracrine (between cells) or an autocrine (same cell) manner, although the kinetics by which these two signaling modes operate during in vivo T cell responses are unknown. In the current study, IL-2 expression and signaling in a clonotypic population of antiviral CD4+ T cells was analyzed by flow cytometry during the initial 24 h of priming. IL-2 expression and STAT5 activation peaked in parallel, but surprisingly, were almost completely mutually exclusive. Thus, only paracrine IL-2 signaling could be observed. As an additional indication of the efficiency of paracrine IL-2 signaling, polyclonal CD4+CD25+Foxp3+ regulatory T cells displayed detectable STAT5 activation under steady-state conditions, which was strongly enhanced by neighboring IL-2-expressing antiviral CD4 cells.
Cutting Edge: Rho Activation and Actin Polarization Are Dependent on Plexin-A1 in Dendritic Cells
Authors: Eun So-Young , O’Connor Brian P. , Wong Athena W. , van Deventer Hendrick W. , Taxman Debra J. , Reed William , Li Ping , Blum Janice S. , McKinnon Karen P. , Ting Jenny P.-Y.
Published: April 2014

We recently identified expression of the semaphorin receptor, plexin-A1, in dendritic cells (DCs); however, its function in these cells remains to be elucidated. To investigate function and maximize physiological relevance, we devised a retroviral approach to ablate plexin-A1 gene expression using small hairpin RNA (shRNA) in primary bone marrow-derived DCs. We show that plexin-A1 localizes within the cytoplasm of immature DCs, becomes membrane-associated, and is enriched at the immune synapse in mature DCs. Reducing plexin-A1 expression with shRNA greatly reduced actin polarization as well as Rho activation without affecting Rac or Cdc42 activation. A Rho inhibitor, C3, also reduced actin polarization. These changes were accompanied by the near-ablation of T cell activation. We propose a mechanism of adaptive immune regulation in which plexin-A1 controls Rho activation and actin cytoskeletal rearrangements in DCs that is associated with enhanced DC-T cell interactions.
Cutting Edge: Uniqueness of Lymphoid Chemokine Requirement for the Initiation and Maturation of Nasopharynx-Associated Lymphoid Tissue Organogenesis
Authors: Fukuyama Satoshi , Nagatake Takahiro , Kim Dong-Young , Takamura Kaoru , Park Eun Jeong , Kaisho Tsuneyasu , Tanaka Norimitsu , Kurono Yuichi , Kiyono Hiroshi
Published: April 2014

CD3−CD4+CD45+ inducer cells are required for the initiation of mucosa-associated organogenesis of both nasopharynx-associated lymphoid tissues (NALT) and Peyer’s patches (PP) in the aerodigestive tract. CXCL13−/− mice and mice carrying the paucity of lymph node T cell (plt) mutation and lacking expression of CCL19 and CCL21 accumulate CD3−CD4+CD45+ cells at the site of NALT but not of PP genesis. Although NALT was observed to develop in adult CXCL13−/− and plt/plt mice, the formation of germinal centers in CXCL13−/− mice was affected, and their population of B cells was much lower than in the NALT of CXCL13+/− mice. Similarly, fewer T cells were observed in the NALT of plt/plt mice than in control mice. These findings indicate that the initiation of NALT organogenesis is independent of CXCL13, CCL19, and CCL21. However, the expression of these lymphoid chemokines is essential for the maturation of NALT microarchitecture.
Preferential Involvement of Tim-3 in the Regulation of Hepatic CD8+ T Cells in Murine Acute Graft-versus-Host Disease
Authors: Oikawa Tsunekazu , Kamimura Yosuke , Akiba Hisaya , Yagita Hideo , Okumura Ko , Takahashi Hiroki , Zeniya Mikio , Tajiri Hisao , Azuma Miyuki
Published: April 2014

Tim-3, a member of the T cell Ig mucin (TIM) family regulates effector Th1 responses. We examined Tim-3 and its ligand expression as well as the effects of anti-Tim-3 mAb treatment in a murine model of acute graft-vs-host disease (aGVHD). In mice with aGVHD, Tim-3 expression was markedly up-regulated on splenic and hepatic CD4+ and CD8+ T cells, dendritic cells (DCs), and macrophages, and this was especially dramatic in hepatic CD8+ T cells. Both donor- and host-derived CD8+ T cells induced similar levels of Tim-3. Tim-3 ligand expression was also up-regulated in splenic T cells, DCs, and macrophages, but not in the hepatic lymphocytes. The administration of anti-Tim-3 mAbs accelerated aGVHD, as demonstrated by body weight loss, reduction in total splenocyte number, and infiltration of lymphocytes in the liver. IFN-γ expression by splenic and hepatic CD4+ and CD8+ T cells was significantly augmented by anti-Tim-3 mAb treatment. In addition, the cytotoxicity against host alloantigen by donor CD8+ T cells was enhanced. These results demonstrate that the anti-Tim-3 treatment in aGVHD augmented the activation of effector T cells expressing IFN-γ or exerting cytotoxicity. Our results suggest that Tim-3 may play a crucial role in the regulation of CD8+ T cells responsible for the maintenance of hepatic homeostasis and tolerance.
Dual Ligand Stimulation of RAW 264.7 Cells Uncovers Feedback Mechanisms That Regulate TLR-Mediated Gene Expression
Authors: Zhu Xiaocui , Chang Mi Sook , Hsueh Robert C. , Taussig Ron , Smith Kelly D. , Simon Melvin I. , Choi Sangdun
Published: April 2014

To characterize how signaling by TLR ligands can be modulated by non-TLR ligands, murine RAW 264.7 cells were treated with LPS, IFN-γ, 2-methyl-thio-ATP (2MA), PGE2, and isoproterenol (ISO). Ligands were applied individually and in combination with LPS, for 1, 2, and 4 h, and transcriptional changes were measured using customized oligo arrays. We used nonadditive transcriptional responses to dual ligands (responses that were reproducibly greater or less than the expected additive responses) as a measure of pathway interaction. Our analysis suggests that cross-talk is limited; <24% of the features with significant responses to the single ligands responded nonadditively to a dual ligand pair. PGE2 and ISO mainly attenuated, while 2MA enhanced, LPS-induced transcriptional changes. IFN-γ and LPS cross-regulated the transcriptional response induced by each other: while LPS preferentially enhanced IFN-γ-induced changes in gene expression at 1 h, IFN-γ signaling primarily attenuated LPS-induced changes at 4 h. Our data suggest specific cross-talk mechanisms: 1) LPS enhances the expression of IFN-γ- response genes by augmenting STAT1 activity and by activating NF-κB, which synergizes with IFN-γ-induced transcriptional factors; 2) IFN-γ attenuates the late LPS transcriptional response by increasing the expression of suppressor of cytokine signaling 1 and cytokine-inducible SH2-containing protein expression; 3) 2MA modulates LPS secondary transcriptional response by increasing IFN-β and inhibiting IL-10 gene expression; 4) PGE2 and ISO similarly regulate the LPS transcriptional response. They increase IL-10 transcription, resulting in attenuated expression of known IL-10-suppressed genes.
Transient Receptor Potential Vanilloid Subtype 1 Mediates Microglial Cell Death In Vivo and In Vitro via Ca2+-Mediated Mitochondrial Damage and CytochromecRelease
Authors: Kim Sang R. , Kim Seung U. , Oh Uhtaek , Jin Byung K.
Published: April 2014

The present study examined the expression of transient receptor potential vanilloid subtype 1 (TRPV1) in microglia, and its association with microglial cell death. In vitro cell cultures, RT-PCR, Western blot analysis, and immunocytochemical staining experiments revealed that rat microglia and a human microglia cell line (HMO6) showed TRPV1 expression. Furthermore, exposure of these cells to TRPV1 agonists, capsaicin (CAP) and resiniferatoxin (RTX), triggered cell death. This effect was ameliorated by the TRPV1 antagonists, capsazepine and iodo-resiniferatoxin (I-RTX), suggesting that TRPV1 is directly involved. Further examinations revealed that TRPV1-induced toxicity was accompanied by increases in intracellular Ca2+, and mitochondrial damage; these effects were inhibited by capsazepine, I-RTX, and the intracellular Ca2+ chelator BAPTA-AM. Treatment of cells with CAP or RTX led to increased mitochondrial cytochrome c release and enhanced immunoreactivity to cleaved caspase-3. In contrast, the caspase-3 inhibitor z-DEVD-fmk protected microglia from CAP- or RTX-induced toxicity. In vivo, we also found that intranigral injection of CAP or 12-hydroperoxyeicosatetraenoic acid, an endogenous agonist of TRPV1, into the rat brain produced microglial damage via TRPV1 in the substantia nigra, as visualized by immunocytochemistry. To our knowledge, this study is the first to demonstrate that microglia express TRPV1, and that activation of this receptor may contribute to microglial damage via Ca2+ signaling and mitochondrial disruption.
Three Memory Subsets of Human CD8+ T Cells Differently Expressing Three Cytolytic Effector Molecules
Authors: Takata Hiroshi , Takiguchi Masafumi
Published: April 2014

Multicolor flow cytometric analysis for the expression of three effector molecules, i.e., perforin (Per), granzyme A (GraA), and granzyme B (GraB), in human CD8+ T cells demonstrated that they included five subpopulations, implying the following pathway for the differentiation of CD8+ T cells: Per−GraA−GraB−→Per−GraA+GraB−→PerlowGraA+GraB−→ PerlowGraA+GraB+→PerhighGraA+GraB+. The analysis of the expression of these molecules in the subsets classified by the combination of the expression of CCR7 and CD45RA or by that of CD27, CD28, and CD45RA showed that functional CD8+ T cell subsets could be partially identified by these phenotypic classifications. However, the functional subsets could be precisely identified by the classification using five cell surface markers or three cell surface markers and three cytolytic molecules. Per−GraA−GraB− and Per−/lowGraA+GraB− cells were predominantly found in CCR5−CCR7+ and CCR5high/lowCCR7− subsets, respectively, of CD8+ T cells expressing the CD27+CD28+CD45RA− phenotype, whereas PerlowGraA+GraB+ cells were found in the CCR5lowCCR7− subset of those expressing this phenotype and in a part of the CCR5−/lowCCR7− subset of those expressing the CD27−/lowCD28−CD45RA−/+ phenotype. Ex vivo EBV-specific CD8+ T cells, which were Perlow/−GraA+GraB−/+ cells, hardly or very weakly killed the target cells, indicating that these were not effector T cells. These findings suggest that the Per−GraA−GraB−, Per−/lowGraA+GraB−, and PerlowGraA+GraB+ cells were central memory, early effector memory, and late effector memory T cells, respectively. Per−/lowGraA+GraB− cells gained GraB expression after TCR stimulation, indicating that early effector memory T cells could differentiate into late effector and effector T cells. The present study showed the existence of three memory subsets and the pathway for their differentiation.
Cigarette Smoke-Induced Pulmonary Inflammation and Emphysema Are Attenuated in CCR6-Deficient Mice
Authors: Bracke Ken R. , D’hulst An I. , Maes Tania , Moerloose Katrien B. , Demedts Ingel K. , Lebecque Serge , Joos Guy F. , Brusselle Guy G.
Published: April 2014

Chronic obstructive pulmonary disease (COPD) is mainly caused by cigarette smoking, and is characterized by an increase in inflammatory cells in the airways and pulmonary tissue. The chemokine receptor CCR6 and its ligand MIP-3α/CCL20 may be involved in the recruitment of these inflammatory cells. To investigate the role of CCR6 in the pathogenesis of COPD, we analyzed the inflammatory responses of CCR6 knockout (KO) and wild-type mice upon cigarette smoke (CS) exposure. Both subacute and chronic exposure to CS induced an increase in cells of the innate and adaptive immune system in the bronchoalveolar lavage, both in CCR6 KO and wild-type mice. However, the accumulation of dendritic cells, neutrophils, and T lymphocytes, which express CCR6, was significantly attenuated in the CCR6 KO mice, compared with their wild-type littermates. In the lung tissue of CCR6 KO mice, there was an impaired increase in dendritic cells, activated CD8+ T lymphocytes, and granulocytes. Moreover, this attenuated inflammatory response in CCR6 KO mice offered a partial protection against pulmonary emphysema, which correlated with an impaired production of MMP-12. Importantly, protein levels of MIP-3α/CCL20, the only chemokine ligand of the CCR6 receptor, and MCP-1/CCL2 were significantly increased upon CS exposure in wild-type, but not in CCR6 KO mice. In contrast, CCR6 deficiency had no effect on the development of airway wall remodeling upon chronic CS exposure. These results indicate that the interaction of CCR6 with its ligand MIP-3α contributes to the pathogenesis of CS-induced pulmonary inflammation and emphysema in this murine model of COPD.
Inhibitory Role of IFN-γ-Inducible Lysosomal Thiol Reductase in T Cell Activation
Authors: Barjaktarević Igor , Rahman Ayman , Radoja Sasa , Bogunović Branka , Vollmer Alison , Vukmanović Stanislav , Marić Maja
Published: April 2014

IFN-γ-inducible lysosomal thiol reductase (GILT) is a unique thiol reductase with optimal enzymatic activity at low pH. GILT plays a crucial role in unfolding the antigenic proteins in preparation for their proteolytic cleavage and presentation of resulting peptides by MHC class II. In this study, we demonstrate that GILT is expressed in T lymphocytes and that it has an APC-nonrelated role in the regulation of T cell activation. Surprisingly, comparison of wild-type and GILT-deficient T cell activation in vitro revealed stronger responsiveness in the absence of GILT. The effect of GILT in reducing the proliferative and cytotoxic responses was endogenous to T cells and resulted from decreased sensitivity at the individual cell level. Therefore, a molecule with primarily lysosomal localization suppresses T cell activation, a process characterized by signal transmission from plasma membrane to cytoplasm and nucleus.
Induction of Autoimmunity by Expansion of Autoreactive CD4+CD62Llow Cells In Vivo
Authors: Amend Bastian , Doster Hong , Lange Christian , Dubois Evelyn , Kalbacher Hubert , Melms Arthur , Bischof Felix
Published: April 2014

The prerequisites of peripheral activation of self-specific CD4+ T cells that determine the development of autoimmunity are incompletely understood. SJL mice immunized with myelin proteolipid protein (PLP) 139–151 developed experimental autoimmune encephalomyelitis (EAE) when pertussis toxin (PT) was injected at the time of immunization but not when injected 6 days later, indicating that PT-induced alterations of the peripheral immune response lead to the development of autoimmunity. Further analysis using IAs/PLP139–151 tetramers revealed that PT did not change effector T cell activation or regulatory T cell numbers but enhanced IFN-γ production by self-specific CD4+ T cells. In addition, PT promoted the generation of CD4+CD62Llow effector T cells in vivo. Upon adoptive transfer, these cells were more potent than CD4+CD62Lhigh cells in inducing autoimmunity in recipient mice. The generation of this population was paralleled by higher expression of the costimulatory molecules CD80, CD86, and B7-DC, but not B7-RP, PD-1, and B7-H1 on CD11c+CD4+ dendritic cells whereas CD11c+CD8α+ dendritic cells were not altered. Collectively, these data demonstrate the induction of autoimmunity by specific in vivo expansion of CD4+CD62Llow cells and indicate that CD4+CD62Llow effector T cells and CD11c+CD4+ dendritic cells may be attractive targets for immune interventions to treat autoimmune diseases.
The Effects of Age, Thymectomy, and HIV Infection on α and β TCR Excision Circles in Naive T Cells
Authors: van den Dool Carline , de Boer Rob J.
Published: April 2014

Due to homeostasis total naive T cell numbers remain fairly constant over life despite a gradual involution of the thymus. The contribution of the thymus to maintaining naive T cell pools is typically measured with TCR excision circles (TRECs) that are formed in thymocytes. The mechanisms underlying thymic involution are poorly understood. Some data suggest that thymocytes undergo fewer divisions in old (small) than young (large) thymi, and other data suggest that the number of TRECs per thymocyte is independent of age. If thymic involution were associated with a decreased number of divisions of the thymocytes, this would markedly complicate the interpretation of TREC data. To study this we develop a mathematical model in which the division rate of thymocytes decreases with increasing age. We describe the dilution of TRECs formed during the arrangement of both chains of the TCR by division of thymocytes, recent thymic emigrants, and mature naive T cells. The model behavior is complicated as TREC contents in naive T cells can increase with age due to decreased dilution in the thymus. Because our model is consistent with current data on the effects of age and thymectomy on TRECs in peripheral T cells, we conclude that aging may well affect thymocyte division, which markedly complicates the interpretation of TREC data. It is possible, but more difficult, to let the model be consistent with the rapid changes in α and β TRECs observed shortly after HIV infection.
Host-Derived Langerhans Cells Persist after MHC-Matched Allografting Independent of Donor T Cells and Critically Influence the Alloresponses Mediated by Donor Lymphocyte Infusions
Authors: Durakovic Nadira , Bezak Karl B. , Skarica Mario , Radojcic Vedran , Fuchs Ephraim J. , Murphy George F. , Luznik Leo
Published: April 2014

Mouse models of minor histocompatibility Ag-mismatched bone marrow transplantation were used to study donor dendritic cell (DC) reconstitution after conditioning, variables influencing the persistence of residual host DCs in different compartments, their phenotype, and their role in governing donor lymphocyte infusion (DLI)-mediated alloresponses. Reconstitution of all splenic DC subsets occurred rapidly after bone marrow transplantation and before T cell reconstitution. However, in contrast to MHC-mismatched chimeras, residual host-derived DCs persisted in the cutaneous lymph nodes (CLNs) of MHC-matched chimeras despite the presence or addition of donor T cells to the graft. The phenotype of these residual host-derived DCs in CLNs was consistent with Langerhans’ cells (LCs). We confirmed their skin origin and found near-complete preservation of host-derived LCs in the skin. Host-derived LCs retained their ability to continuously traffic to the CLNs, expressed homogeneously increased levels of costimulatory molecules, and could capture and carry epicutaneously applied Ags. To determine the role of residual host LCs in governing DLI-mediated alloresponses, we administered DLI alone or after topical application of the TLR7 ligand imiquimod, which is known to enhance the LC emigration from the skin. DLI administration resulted in a decrease in host-derived DCs in the CLNs and increased recruitment of donor-derived DCs to the skin, whereas imiquimod augmented their alloreactivity. These results suggest uniqueness of the MHC-matched setting in relation to the persistence of host-derived DCs in the skin and points to a previously unrecognized role of host-derived LCs in the induction of DLI-mediated graft-vs-host alloresponses.
Regulatory Role of γδ T Cells in the Recruitment of CD4+ and CD8+ T Cells to Lung and Subsequent Pulmonary Fibrosis
Authors: Simonian Philip L. , Roark Christina L. , Diaz del Valle Fernando , Palmer Brent E. , Douglas Ivor S. , Ikuta Koichi , Born Willi K. , O’Brien Rebecca L. , Fontenot Andrew P.
Published: April 2014

The mechanisms by which T cells accumulate in the lungs of patients with pulmonary fibrosis are poorly understood. Because the lung is continually exposed to microbial agents from the environment, we repeatedly exposed C57BL/6 mice to the ubiquitous microorganism, Bacillus subtilis, to determine whether chronic exposure to an inhaled microorganism could lead to T cell accumulation in the lungs and subsequent pulmonary fibrosis. C57BL/6 mice repeatedly treated with B. subtilis for 4 consecutive weeks developed a 33-fold increase in the number of CD4+ T cells and a 354-fold increase in γδ T cells in the lung. The γδ T cells consisted almost entirely of Vγ6/Vδ1+ cells, a murine subset bearing an invariant TCR the function of which is still unknown. Treatment of C57BL/6 mice with heat-killed vs live B. subtilis resulted in a 2-fold increase in the number of CD4+ T cells in the lung but no expansion of γδ T cells indicating that γδ cells accumulate in response to live microorganisms. In addition, mice treated with heat-killed B. subtilis developed significantly increased pulmonary fibrosis compared with mice treated with the live microorganism. Mice deficient in Vγ6/Vδ1+ T cells when treated with B. subtilis had a 231-fold increase in lung CD4+ T cells and significantly increased collagen deposition compared with wild-type C57BL/6 mice, consistent with an immunoregulatory role for the Vγ6/Vδ1 T cell subset. These findings indicate that chronic inhalation of B. subtilis can result in T cell accumulation in the lung and fibrosis, constituting a new model of immune-mediated pulmonary fibrosis.
Functional Dichotomy between OX40 and 4-1BB in Modulating Effector CD8 T Cell Responses
Authors: Lee Seung-Woo , Park Yunji , Song Aihua , Cheroutre Hilde , Kwon Byoung S. , Croft Michael
Published: April 2014

Members of the TNFR family are thought to deliver costimulatory signals to T cells and modulate their function and survival. In this study, we compare the role of two closely related TNFR family molecules, OX40 and 4-1BB, in generating effector CD8 T cells to Ag delivered by adenovirus. OX40 and 4-1BB were both induced on responding naive CD8 T cells, but 4-1BB exhibited faster and more sustained kinetics than OX40. OX40-deficient CD8 T cells initially expanded normally; however, their accumulation and survival at late times in the primary response was significantly impaired. In contrast, 4-1BB-deficient CD8 T cells displayed hyperresponsiveness, expanding more than wild-type cells. The 4-1BB-deficient CD8 T cells also showed enhanced maturation attributes, whereas OX40-deficient CD8 T cells had multiple defects in the expression of effector cell surface markers, the synthesis of cytokines, and in cytotoxic activity. These results suggest that, in contrast to current ideas, OX40 and 4-1BB can have a clear functional dichotomy in modulating effector CD8 T cell responses. OX40 can positively regulate effector function and late accumulation/survival, whereas 4-1BB can initially operate in a negative manner to limit primary CD8 responses.
CpG DNA Activates Survival in Murine Macrophages through TLR9 and the Phosphatidylinositol 3-Kinase-Akt Pathway
Authors: Sester David P. , Brion Kristian , Trieu Angela , Goodridge Helen S. , Roberts Tara L. , Dunn Jasmyn , Hume David A. , Stacey Katryn J. , Sweet Matthew J.
Published: April 2014

Bacterial CpG-containing (CpG) DNA promotes survival of murine macrophages and triggers production of proinflammatory mediators. The CpG DNA-induced inflammatory response is mediated via TLR9, whereas a recent study reported that activation of the Akt prosurvival pathway occurs via DNA-dependent protein kinase (DNA-PK) and independently of TLR9. We show, in this study, that Akt activation and survival of murine bone marrow-derived macrophages (BMM) triggered by CpG-containing phosphodiester oligodeoxynucleotides or CpG-containing phosphorothioate oligodeoxynucleotides was completely dependent on TLR9. In addition, survival triggered by CpG-containing phosphodiester oligodeoxynucleotides was not compromised in BMM from SCID mice that express a catalytically inactive form of DNA-PK. CpG DNA-induced survival of BMM was inhibited by the PI3K inhibitor, LY294002, but not by the MEK1/2 inhibitor, PD98059. The effect of LY294002 was specific to survival, because treatment of BMM with LY294002 affected CpG DNA-induced TNF-α production only modestly. Therefore, CpG DNA activates macrophage survival via TLR9 and the PI3K-Akt pathway and independently of DNA-PK and MEK-ERK.
The Majority of Human Peripheral Blood CD4+CD25highFoxp3+ Regulatory T Cells Bear Functional Skin-Homing Receptors
Authors: Hirahara Kazuki , Liu Luzheng , Clark Rachael A. , Yamanaka Kei-ichi , Fuhlbrigge Robert C. , Kupper Thomas S.
Published: April 2014

CD4+CD25+ T regulatory cells (Treg) are thought to be important in the peripheral tolerance. Recent evidence suggests that human peripheral blood CD4+CD25+ T cells are heterogeneous and contain both CD4+CD25high T cells with potent regulatory activity and many more CD4+CD25low/med nonregulatory T cells. In this study, we found that virtually all peripheral blood CD4+CD25highFoxp3+ Treg expressed high levels of the chemokine receptor CCR4. In addition, 80% of Treg expressed cutaneous lymphocyte Ag (CLA) and 73% expressed CCR6. These molecules were functional, as CLA+ Treg showed CD62E ligand activity and demonstrable chemotactic responses to the CCR4 ligands CCL22 and CCL17 and to the CCR6 ligand CCL20. The phenotype and chemotactic response of these Treg were significantly different from those of CD4+CD25med nonregulatory T cells. We further demonstrated that blood CLA+ Treg inhibited CD4+CD25− T cell proliferation induced by anti-CD3. Based on homing receptor profile, CLA+ Treg should enter normal skin. We next isolated CD4+CD25high T cells directly from normal human skin; these cells suppressed proliferation of skin CD4+CD25− T cells. Therefore, the majority of true circulating Treg express functional skin-homing receptors, and human Treg may regulate local immune responses in normal human skin.
Trauma-Hemorrhage Induces Depressed Splenic Dendritic Cell Functions in Mice
Authors: Kawasaki Takashi , Hubbard William J. , Choudhry Mashkoor A. , Schwacha Martin G. , Bland Kirby I. , Chaudry Irshad H.
Published: April 2014

Although Kupffer cell, splenic, and peritoneal macrophage functions are markedly altered following trauma-hemorrhage (T-H), it remains unclear whether T-H also affects splenic dendritic cell (sDC) functions. We hypothesized that sDC functions will also be compromised following T-H. Male C3H/HeN (6- to 8-wk) mice were randomly assigned to sham operation or T-H. T-H was induced by midline laparotomy and ∼90 min of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (four times the shed blood volume in the form of Ringer’s lactate). Two hours later, the mice were sacrificed; sDC were isolated; and the changes in their apoptosis, MHC class II expression, and ability to produce costimulatory cytokines and Ag presentation were measured. The results indicate that sDC Ag presentation capacity was significantly decreased and MHC class II expression was also significantly decreased following T-H. Moreover, LPS-induced IL-12 production and LPS- or IL-12-induced IFN-γ production following T-H were significantly decreased. Thus, the markedly decreased MHC class II expression and cytokine (IL-12, IFN-γ) production following T-H may be the cause for the depressed sDC Ag presentation under those conditions. This depression in Ag presentation could contribute to the host’s enhanced susceptibility to sepsis following T-H.
STAT1-Independent Cell Type-Specific Regulation of Antiviral APOBEC3G by IFN-α
Authors: Sarkis Phuong Thi Nguyen , Ying Songcheng , Xu Rongzhen , Yu Xiao-Fang
Published: April 2014

APOBEC3G (A3G) has broad antiviral activity against retroviruses and hepatitis B virus. However, the role of IFNs in regulating A3G during innate immunity has not been established. In this study, we show that the A3G gene is uniquely regulated by IFNs in a cell type-dependent manner. A3G was up-regulated by IFN-α in liver cells and macrophages, but not in T lymphoid cells or epithelial 293T cells. In contrast, other IFN-α-stimulated genes such as dsRNA-activated protein kinase were induced in all these cells, suggesting additional cellular factors may regulate IFN-α-induced A3G expression. Consistent with this idea, IFN-α-mediated induction of A3G, but not other IFN-α-stimulated genes, was potently inhibited by the drug Rottlerin, through a mechanism independent of STAT1 activation. The canonical IFN-α-mediated pathway of gene transcription requires both STAT1 and STAT2. Surprisingly, induction of A3G was STAT1 independent, but STAT2 dependent in liver cells. However, STAT1 signaling was functional and required for IFN-γ induction of A3G in these cells. Our results indicate that A3G may participate in antiviral cellular defenses through a novel IFN-mediated signaling pathway.
Intracellular Protein Modification Associated with Altered T Cell Functions in Autoimmunity
Authors: Yang Mei-Ling , Doyle Hester A. , Gee Renelle J. , Lowenson Jonathan D. , Clarke Steven , Lawson Brian R. , Aswad Dana W. , Mamula Mark J.
Published: April 2014

Posttranslational protein modifications influence a number of immunologic responses ranging from intracellular signaling to protein processing and presentation. One such modification, termed isoaspartyl (isoAsp), is the spontaneous nonenzymatic modification of aspartic acid residues occurring at physiologic pH and temperature. In this study, we have examined the intracellular levels of isoAsp residues in self-proteins from MRL+/+, MRL/lpr, and NZB/W F1 mouse strains compared with nonautoimmune B10.BR mice. In contrast to control B10.BR or NZB/W mice, the isoAsp content in MRL autoimmune mice increased and accumulated with age in erythrocytes, brain, kidney, and T lymphocytes. Moreover, T cells that hyperproliferate to antigenic stimulation in MRL mice also have elevated intracellular isoAsp protein content. Protein l-isoaspartate O-methyltransferase activity, a repair enzyme for isoAsp residues in vivo, remains stable with age in all strains of mice. These studies demonstrate a role for the accumulation of intracellular isoAsp proteins associated with T cell proliferative defects of MRL autoimmune mice.
PKB Rescues Calcineurin/NFAT-Induced Arrest of Rag Expression and Pre-T Cell Differentiation
Authors: Patra Amiya K. , Drewes Thomas , Engelmann Swen , Chuvpilo Sergei , Kishi Hiroyuki , Hünig Thomas , Serfling Edgar , Bommhardt Ursula H.
Published: April 2014

Protein kinase B (PKB), an Ag receptor activated serine-threonine kinase, controls various cellular processes including proliferation and survival. However, PKB function in thymocyte development is still unclear. We report PKB as an important negative regulator of the calcineurin (CN)-regulated transcription factor NFAT in early T cell differentiation. Expression of a hyperactive version of CN induces a profound block at the CD25+CD44− double-negative (DN) 3 stage of T cell development. We correlate this arrest with up-regulation of Bcl-2, CD2, CD5, and CD27 proteins and constitutive activation of NFAT but a severe impairment of Rag1, Rag2, and intracellular TCR-β as well as intracellular TCR-γδ protein expression. Intriguingly, simultaneous expression of active myristoylated PKB inhibits nuclear NFAT activity, restores Rag activity, and enables DN3 cells to undergo normal differentiation and expansion. A correlation between the loss of NFAT activity and Rag1 and Rag2 expression is also found in myristoylated PKB-induced CD4+ lymphoma cells. Furthermore, ectopic expression of NFAT inhibits Rag2 promoter activity in EL4 cells, and in vivo binding of NFATc1 to the Rag1 and Rag2 promoter and cis-acting transcription regulatory elements is verified by chromatin immunoprecipitation analysis. The regulation of CN/NFAT signaling by PKB may thus control receptor regulated changes in Rag expression and constitute a signaling pathway important for differentiation processes in the thymus and periphery.
Molecular Mechanisms for Transcriptional Regulation of Human High-Affinity IgE Receptor β-Chain Gene Induced by GM-CSF
Authors: Takahashi Kyoko , Hayashi Natsuko , Kaminogawa Shuichi , Ra Chisei
Published: April 2014

The β-chain of the high-affinity receptor for IgE (FcεRI) plays an important role in regulating activation of FcεRI-expressing cells such as mast cells in allergic reactions. We already reported that the transcription factor myeloid zinc finger (MZF) 1 which formed a high m.w. complex including four and a half LIM-only protein (FHL)3 in the nucleus repressed human β-chain gene expression through an element in the fourth intron. We also found that GM-CSF induced expression of MZF-1 and nuclear translocation of FHL3. We screened a human cDNA library and identified NFY which was reported to bind histone deacetylases (HDACs) as a constituent of the complex. The C-subunit of NFY was demonstrated to form a ternary complex with MZF-1/FHL3 and interact with a β-chain gene region including the element in the fourth intron. HDAC1 and HDAC2 were also shown to interact with the fourth intron region of the β-chain gene. In a human mast cell line HMC-1 cultured with GM-CSF, both β-chain expression and acetylation of histones interacting with the fourth intron region of the β-chain gene were decreased. Collectively, these results indicated that HDACs, which were recruited to the β-chain gene through the element in the fourth intron by MZF-1/FHL3/NFY, repressed β-chain gene transcription by deacetylation of histones in the presence of GM-CSF. These mechanisms will be involved in not only the cell type-specific repression of β-chain gene expression in differentiating hemopoietic cells but also the repression of β-chain gene expression in the peripheral cells under specific circumstances.
MHC Class II Molecules Control Murine B Cell Responsiveness to Lipopolysaccharide Stimulation
Authors: Rodo Joana , Gonçalves Lígia A. , Demengeot Jocelyne , Coutinho António , Penha-Gonçalves Carlos
Published: April 2014

LPS is a strong stimulator of the innate immune system and inducer of B lymphocyte activation. Two TLRs, TLR4 and RP105 (CD180), have been identified as mediators of LPS signaling in murine B cells, but little is known about genetic factors that are able to control LPS-induced cell activation. We performed a mouse genome-wide screen that aside from identifying a controlling locus mapping in the TLR4 region (logarithm of odds score, 2.77), also revealed that a locus closely linked to the MHC region (logarithm of odds score, 3.4) governed B cell responsiveness to LPS stimulation. Using purified B cells obtained from MHC congenic strains, we demonstrated that the MHCb haplotype is accountable for higher cell activation, cell proliferation, and IgM secretion, after LPS stimulation, when compared with the MHCd haplotype. Furthermore, B cells from MHC class II−/− mice displayed enhanced activation and proliferation in response to LPS. In addition, we showed that the MHC haplotype partially controls expression of RP105 (a LPS receptor molecule), following a pattern that resembles the LPS responsiveness phenotype. Together, our results strongly suggest that murine MHC class II molecules play a role in constraining the B cell response to LPS and that genetic variation at the MHC locus is an important component in controlling B cell responsiveness to LPS stimulation. This work raises the possibility that constraining of B cell responsiveness by MHC class II molecules may represent a functional interaction between adaptive and innate immune systems.
Restoration of Natural IgM Production from Liver B Cells by Exogenous IL-18 Improves the Survival of Burn-Injured Mice Infected with Pseudomonas aeruginosa
Authors: Kinoshita Manabu , Shinomiya Nariyoshi , Ono Satoshi , Tsujimoto Hironori , Kawabata Toshinobu , Matsumoto Atsushi , Hiraide Hoshio , Seki Shuhji
Published: April 2014

Pseudomonas aeruginosa is the most common bacterium of postburn infection. In the present study we investigated the immune mechanism of susceptibility to this type of postburn infection and also examined the efficacy of IL-18 treatment. C57BL/6 mice were challenged with P. aeruginosa on day 7 after burn injury. Although the burn-injured mice showed a poor survival rate after bacterial challenge, they retained their IFN-γ production. The burned mice showed lower serum IgM levels and a poor IgM response following P. aeruginosa challenge in comparison with the sham mice, whereas IL-18 treatment after burn injury (alternate day injections for 1 wk) greatly improved the serum IgM levels, which are P. aeruginosa-independent natural IgM before bacterial challenge, thereby increasing the survival rate after the challenge. IL-18 treatment also induced specific IgM to P. aeruginosa in the sera 5 days after bacterial challenge in the burned mice. Interestingly, CD43+CD5−CD23−B220dim cells, namely B-1b cells, increased in the liver after the IL-18 treatment and were found to actively produce IgM in vitro without any additional stimulation. Furthermore, the IL-18 treatment up-regulated the neutrophil count and the C3a levels in the blood as a result of the increased IgM level, which may thus play a critical role in the opsonization and elimination of any invading bacteria. IL-18 treatment for the burned mice and their resultant natural IgM production were thus found to strengthen the host defense against P. aeruginosa infection.