bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Five MAGE-A1 Epitopes Recognized by Cytolytic T Lymphocytes Obtained by In Vitro Stimulation with Dendritic Cells Transduced with MAGE-A1
Authors: Chaux Pascal , Luiten Rosalie , Demotte Nathalie , Vantomme Valérie , Stroobant Vincent , Traversari Catia , Russo Vincenzo , Schultz Erwin , Cornelis Guy R. , Boon Thierry , van der Bruggen Pierre
Published: December 2022

MAGE genes are expressed by many human tumors of different histological types but not by normal cells, except for male germline cells. The Ags encoded by MAGE genes and recognized by T cells are therefore strictly tumor-specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes recognized by CTL. Candidate peptides known to bind to a given HLA have been used to stimulate T lymphocytes in vitro. In some instances, CTL clones directed against these synthetic peptides have been obtained, but these clones often failed to recognize tumor cells expressing the relevant gene. Therefore, we designed a method to identify CTL epitopes that selects naturally processed peptides. Monocyte-derived dendritic cells infected with a recombinant canarypoxvirus (ALVAC) containing the entire MAGE-A1 gene were used to stimulate CD8+ T lymphocytes from the blood of individuals without cancer. Responder cell microcultures that specifically lysed autologous cells expressing MAGE-A1 were cloned using autologous stimulator cells either transduced with a retrovirus coding for MAGE-A1 or infected with recombinant Yersinia-MAGE-A1 bacteria. The CTL clones were tested for their ability to lyse autologous cells loaded with each of a set of overlapping MAGE-A1 peptides. This strategy led to the identification of five new MAGE-A1 epitopes recognized by CTL clones on HLA-A3, -A28, -B53, -Cw2, and -Cw3 molecules. All of these CTL clones recognized target cells expressing gene MAGE-A1.
Experimental Autoimmune Encephalomyelitis in NF-κB- Deficient Mice: Roles of NF-κB in the Activation and Differentiation of Autoreactive T Cells
Authors: Hilliard Brendan , Samoilova Elena B. , Liu Tzu-Shang T. , Rostami Abdolmohamad , Chen Youhai
Published: December 2022

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the CNS, which has long been used as an animal model for human multiple sclerosis. Development of autoimmune disease requires coordinated expression of a number of genes that are involved in the activation and effector functions of inflammatory cells. These include genes that encode costimulatory molecules, cytokines, chemokines, and adhesion molecules. Activation of these genes is regulated at the transcriptional level by several families of transcription factors. One of these is the NF-κB family, which is present in a variety of cell types and becomes highly activated at sites of inflammation. To test the roles of NF-κB in the development of autoimmune diseases, we studied EAE in mice deficient in one of the NF-κB isoforms, i.e., NF-κB1 (p50). We found that NF-κB1-deficient mice were significantly resistant to EAE induced by myelin oligodendrocyte glycoprotein. The resistance was primarily evidenced by a decrease in disease incidence, clinical score, and the degree of CNS inflammation. Furthermore, we established that the resistance to EAE in NF-κB1-deficient mice was associated with a deficiency of myelin oligodendrocyte glycoprotein-specific T cells to differentiate into either Th1- or Th2-type effector cells in vivo. These results strongly suggest that NF-κB1 plays crucial roles in the activation and differentiation of autoreactive T cells in vivo and that blocking NF-κB function can be an effective means to prevent autoimmune encephalomyelitis.
HIV-1 Tat Induces Monocyte Chemoattractant Protein-1-Mediated Monocyte Transmigration Across a Model of the Human Blood-Brain Barrier and Up-Regulates CCR5 Expression on Human Monocytes
Authors: Weiss Jonathan M. , Nath Avindra , Major Eugene O. , Berman Joan W.
Published: December 2022

AIDS dementia is characterized by neuronal loss in association with synaptic damage. A central predictor for clinical onset of these symptoms is the infiltration of monocytes and macrophages into CNS parenchyma. Chronic HIV-1 infection of monocytes also allows these cells to serve as reservoirs for persistent viral infection. Using a coculture of endothelial cells and astrocytes that models several aspects of the human blood-brain barrier, we examined the mechanism whereby the HIV-derived factor Tat may facilitate monocyte transmigration. We demonstrate that treatment of cocultures on the astrocyte side with HIV-1 Tat induced significant monocyte chemoattractant protein (MCP)-1 protein. Astrocytes, but not endothelial cells, were the source of this MCP-1 expression. Supernatants from Tat-treated cocultures induced significant monocyte transmigration, which was detected by 2.5 h after the addition of PBMC. Pretreatment of the supernatants from Tat-stimulated cocultures with an Ab to MCP-1 completely blocked monocyte transmigration. Flow cytometric analysis of Tat-stimulated PBMC demonstrated that Tat up-regulated expression of the chemokine receptor, CCR5, on monocytes in a time-dependent manner. Taken together, our data indicate that HIV-1 Tat may facilitate the recruitment of monocytes into the CNS by inducing MCP-1 expression in astrocytes. These recruited monocytes may contribute to the pathogenesis of HIV-1-associated AIDS encephalitis and dementia.
A Dominant Role for the Thymus and MHC Genes in Determining the Peripheral CD4/CD8 T Cell Ratio in the Rat
Authors: Damoiseaux Jan G. M. C. , Cautain Bastien , Bernard Isabelle , Mas Magali , van Breda Vriesman Peter J. C. , Druet Philippe , Fournié Gilbert , Saoudi Abdelhadi
Published: December 2022

During their development, immature CD4CD8 double positive thymocytes become committed to either the CD4 or CD8 lineage. The final size of the peripheral CD4 and CD8 T cell compartments depends on thymic output and on the differential survival and proliferation of the respective T cell subsets in the periphery. Our results reveal that the development of the distinct peripheral CD4/CD8 T cell ratio between Lewis and Brown Norway rats originates in the thymus and, as shown by the use of radiation bone marrow chimeras, is determined by selection on radio-resistant stromal cells. Furthermore, this difference is strictly correlated with the MHC haplotype and is the result of a reduction in the absolute number of CD8 T cells in Brown Norway rats. These data suggest that the distinct CD4/CD8 T cell ratio between these two rat strains is the consequence of differential interactions of the TCR/CD8 coreceptor complex with the respective MHC class I haplotypes during selection in the thymus.
Differential Coupling of Second Signals for Cytotoxicity and Proliferation in CD8+ T Cell Effectors: Amplification of the Lytic Potential by B7
Authors: Krummel Matthew F. , Heath William R. , Allison Janette
Published: December 2022

The role of second signals delivered through B7/CD28 interactions in T cell activation is well documented. However, once CTLs are elicited, TCR-mediated cytotoxicity appears to be uncoupled from the requirement for costimulatory signals. In this study, we show an uncoupling across a broad range of concentrations of peptide, thus demonstrating that cytolysis is a TCR-mediated response that is fully independent of costimulatory signals. However, the same T cell effectors remain fully responsive to B7 engagement, which is able to amplify Ag-mediated proliferation and cytolytic capacity. B7 expression by targets results in an IL-2-mediated proliferative expansion of the effectors concurrent with the elimination of the targets. Thus, costimulation of effectors results in a vast expansion in lytic units over time, which does not occur in the absence of IL-2 or B7. Both TCR-derived and second signals appear to be necessary to achieve this result. These results suggest that B7-expressing APC or a cohort of IL-2-producing helper cells would functionally extend the duration and effectiveness of the cytotoxic response occurring in localized immune responses.
Intracellular Signaling of gp34, the OX40 Ligand: Induction of c-junand c-fosmRNA Expression Through gp34 upon Binding of Its Receptor, OX40
Authors: Matsumura Yumi , Hori Toshiyuki , Kawamata Shin , Imura Akihiro , Uchiyama Takashi
Published: December 2022

We investigated the intracellular signaling events of OX40 ligand (gp34), a member of the TNF family. To elucidate the intracellular signaling via gp34, we prepared a model system in which a human gp34-transfected mouse epithelial cell line was stimulated with a recombinant soluble form of OX40. We demonstrated that OX40 binding resulted in increase in c-jun and c-fos mRNA levels in this transfectant by Northern blot analysis, which was blocked by the pretreatment with anti-gp34 Ab. The studies with various gp34 deletion mutants showed that the cytoplasmic portion including the amino acid sequence 16–21 (RPRFER) was required for the induction of c-jun and c-fos mRNA expression. Furthermore, OX40 binding induced c-jun mRNA expression also in HUVECs, which in our previous study have been shown to express gp34 and interact with activated T cells through the OX40/gp34 pathway. On the other hand, c-fos mRNA was detectable neither in unstimulated HUVECs nor in gp34-stimulated HUVECs. These results indicate that the OX40/gp34 system generates two-way signals and may elicit biological effects on vascular endothelial cells.
Characterization of RANTES- and Aminooxypentane-RANTES- Triggered Desensitization Signals Reveals Differences in Recruitment of the G Protein-Coupled Receptor Complex
Authors: Vila-Coro Antonio J. , Mellado Mario , Martín de Ana Ana , Martínez-A. Carlos , Rodríguez-Frade José Miguel
Published: December 2022

The trafficking of lymphocyte populations is a complex process controlled by a vast array of molecules. In this process, cells must be able to sense small changes in chemoattractant gradients. Migration through a chemotactic gradient probably employs an on-off mechanism in which chemokine receptor desensitization, internalization, and recycling may be important steps. This multistep process requires the coordinated action of many factors, including G protein-coupled receptor kinases, arrestins, clathrin, and GTP-hydrolyzing proteins such as dynamin. In this report, we show that RANTES and its derivative, aminooxypentane (AOP)-RANTES, a potent RANTES antagonist as well as an inhibitor of HIV-1 infection, both promote CCR5 desensitization involving G protein-coupled receptor kinases-2 and β-arrestin equally well. An important difference between the two molecules is that (AOP)-RANTES is more efficient than RANTES in promoting Ser/Thr phosphorylation of the receptor and association of G protein-coupled receptor kinases-2, β-arrestin, and clathrin to the CCR5. After stimulation with either ligand, we observe rapid, transient association of dynamin to CCR5, implicating this protein in receptor sensitization, but this association is faster and longer-lasting following (AOP)-RANTES stimulation. In summary, we show that chemokine receptor internalization takes place through the formation of clathrin vesicles and involves dynamin activity. We provide compelling evidence that the differences between RANTES and (AOP)-RANTES in Gαi activation condition subsequent signaling events, including internalization and receptor recycling.
β-Glucan, a “Specific” Biologic Response Modifier That Uses Antibodies to Target Tumors for Cytotoxic Recognition by Leukocyte Complement Receptor Type 3 (CD11b/CD18)
Authors: Yan Jun , Větvička Václav , Xia Yu , Coxon Angela , Carroll Michael C. , Mayadas Tanya N. , Ross Gordon D.
Published: December 2022

β-Glucans were identified 36 years ago as a biologic response modifier that stimulated tumor rejection. In vitro studies have shown that β-glucans bind to a lectin domain within complement receptor type 3 (CR3; known also as Mac-1, CD11b/CD18, or αMβ2-integrin, that functions as an adhesion molecule and a receptor for factor I-cleaved C3b, i.e., iC3b) resulting in the priming of this iC3b receptor for cytotoxicity of iC3b-opsonized target cells. This investigation explored mechanisms of tumor therapy with soluble β-glucan in mice. Normal mouse sera were shown to contain low levels of Abs reactive with syngeneic or allogeneic tumor lines that activated complement, depositing C3 onto tumors. Implanted tumors became coated with IgM, IgG, and C3, and the absent C3 deposition on tumors in SCID mice was reconstituted with IgM or IgG isolated from normal sera. Therapy of mice with glucan- or mannan-rich soluble polysaccharides exhibiting high affinity for CR3 caused a 57–90% reduction in tumor weight. In young mice with lower levels of tumor-reactive Abs, the effectiveness of β-glucan was enhanced by administration of a tumor-specific mAb, and in SCID mice, an absent response to β-glucan was reconstituted with normal IgM or IgG. The requirement for C3 on tumors and CR3 on leukocytes was highlighted by therapy failures in C3- or CR3-deficient mice. Thus, the tumoricidal function of CR3-binding polysaccharides such as β-glucan in vivo is defined by natural and elicited Abs that direct iC3b deposition onto neoplastic cells, making them targets for circulating leukocytes bearing polysaccharide-primed CR3. Therapy fails when tumors lack iC3b, but can be restored by tumor-specific Abs that deposit iC3b onto the tumors.
Identification of a New Type of Invariant Vα14+ T Cells and Responsiveness to a Superantigen,Yersinia pseudotuberculosis- Derived Mitogen
Authors: Yagi Junji , Dianzani Umberto , Kato Hidehito , Okamoto Toshihiro , Katsurada Tomoko , Buonfiglio Donatella , Miyoshi-Akiyama Tohru , Uchiyama Takehiko
Published: December 2022

We examined the expression of the H4 T cell activation marker in thymic T cell subpopulations and found that TCR-αβ+ CD4+ thymic T cells are segregated into three subpopulations based upon H4 levels. Thymic T cells with either no or low H4 expression differentiate via the mainstream differentiation pathway in the thymus. H4int thymic T cells, which express a skewed Vβ repertoire of Vβ2, -7, and -8 in their TCRs, show the phenotype of NKT cells: CD44high, Ly6Chigh, NK1.1+, and TCR-αβlow. H4high thymic T cells also show a skewed Vβ repertoire, Vβ2, -7, and -8, and predominantly express an invariant Vα14-Jα281+ α-chain in their TCRs but constitute a distinct population in that they are CD44int, Ly6C−, NK1.1−, and TCR-αβhigh. Thus, invariant Vα14+ thymic T cells consist of ordinary NKT cells and a new type of T cell population. Vβ7+ and Vβ8.1+ invariant Vα14+ thymic T cells are present in DBA/2 mice, which carry mammary tumor virus-7-encoded superantigens, in comparable levels to those in BALB/c mice. Furthermore, Vβ7+ invariant Vα14+ thymic T cells in DBA/2 mice are in the immunologically responsive state, and Yersinia pseudotuberculosis-derived mitogen-induced Vβ7+ invariant Vα14+ thymic T cell blasts from DBA/2 and BALB/c mice exhibited equally enhanced responses upon restimulation with Y. pseudotuberculosis-derived mitogen. Thus, invariant Vα14+ thymic T cells that escape negative selection in DBA/2 mice contain T cells as functionally mature as those in BALB/c mice.
TNF-α-Induced Growth Suppression of CD34+ Myeloid Leukemic Cell Lines Signals Through TNF Receptor Type I and Is Associated with NF-κB Activation
Authors: Hu Xiaotang , Tang Menque , Fisher Ariana Brown , Olashaw Nancy , Zuckerman Kenneth S.
Published: December 2022

Conflicting results have been reported regarding the effect of TNF-α on the growth of human primitive hemopoietic cells. In this study, we have examined the effect of TNF-α on the proliferation of several CD34+/CD38+ (KG-1, TF-1) and CD34+/CD38− (KG-1a, TF-1a) myeloid leukemic progenitor cell lines. Our data show that TNF-α markedly inhibits the growth of these cells in both liquid and soft agar cultures. Addition of GM-CSF or IL-3 does not prevent TNF-α-induced growth inhibition. Flow cytometry analyses of propidium iodide-stained cells demonstrated cell death of all four cell lines, as judged by the presence of cells with hypodiploid DNA content after exposure of cells to TNF-α for 4 days. Annexin V assays detected apoptosis in TF-1, but not in TF-1a, KG-1, and KG-1a cells in terms of translocation of phosphatidylserine shortly after TNF-α treatment. Neutralizing anti-TNF receptor type I (TNFR-I; p55) Ab almost completely reversed TNF-α-induced growth inhibition in both liquid and soft agar cultures, whereas anti-TNFR-II (p75) Ab had only a marginal effect. TNF-α rapidly induced marked activation of nuclear transcription factor NF-κB in all 4 cell lines. The majority of this effect was abolished by the type I receptor Ab, whereas the type II receptor neutralizing Ab had no effect. Our data also show that TNF-α is incapable of inducing activation of the mitogen-activated protein kinase pathway in these leukemic cell lines.
Differential Control of Autoantibodies and Lymphoproliferation by Fas Ligand Expression on CD4+ and CD8+ T Cells In Vivo
Authors: Maldonado Michael A. , MacDonald Glen C. , Kakkanaiah Vellalore N. , Fecho Karamarie , Dransfield Mark , Sekiguchi Debora , Cohen Philip L. , Eisenberg Robert A.
Published: December 2022

We have previously shown that the gld autoimmune syndrome is suppressed in lethally irradiated gld mice reconstituted with a mixture of normal and gld bone marrow (BM). Furthermore, in vivo depletion of normal Thy-1+ cells restores lymphoproliferation and autoantibody production in such chimeras, suggesting that T cells bearing Fas ligand are responsible for correcting the gld defect. In this study, mixed-BM chimeras lacking either normal CD4+ (B6CD4KO-B6gld) or normal CD8+ T cells (B6CD8KO-B6gld) were generated to determine the contribution of the normal T cell subsets to disease suppression. Lymphoproliferation was completely suppressed in B6CD4KO-B6gld chimeras but only modestly in B6CD8KO-B6gld chimeras. On the other hand, both types of mixed-BM chimeras had incomplete effects on the suppression of serum autoantibodies when compared with B6gld reconstituted with isologous BM. These results suggest that both T cell subsets provide Fas ligand to suppress immune cells responsible for autoantibody production; however, CD8+ T cells are mainly responsible for preventing lymphoproliferation.
Role of the Costimulatory Molecule CD28 in the Development of Lupus in MRL/lprMice
Authors: Tada Yoshifumi , Nagasawa Kohei , Ho Alexandra , Morito Fumitaka , Koarada Syuichi , Ushiyama Osamu , Suzuki Noriaki , Ohta Akihide , Mak Tak W.
Published: December 2022

MRL/Mpj-lpr/lpr (MRL/lpr) mice develop autoimmune disorders, including lymphoproliferation, glomerulonephritis, autoantibody production, and hypergammaglobulinemia. To investigate the role of the costimulatory molecule CD28 in the development of these disorders, MRL/lpr mice lacking CD28 were generated by gene targeting. Compared with CD28+/+ MRL/lpr mice, CD28−/− MRL/lpr mice showed decreased lymphadenopathy but increased splenomegaly associated with the expansion of abnormal B220+ TCRαβ+ T cells. Although levels of IgM Abs were unchanged in CD28−/− MRL/lpr mice, the production of anti-DNA IgG Abs and IgG rheumatoid factors were suppressed. IgG deposition in the glomeruli was markedly decreased, and the development of glomerulonephritis was significantly retarded. Furthermore, renal vasculitis and arthritis were absent in CD28−/− MRL/lpr mice. These results indicate that, although CD28 is not required for the generation of the abnormal T cell population in MRL/lpr mice, it does play an important role in the development of autoimmune disease in these animals.
Stochastic Acquisition of Qa1 Receptors During the Development of Fetal NK Cells In Vitro Accounts in Part But Not in Whole for the Ability of These Cells to Distinguish Between Class I-Sufficient and Class I-Deficient Targets
Authors: Toomey Jennifer A. , Salcedo Margarita , Cotterill Lisa A. , Millrain Margaret M. , Chrzanowska-Lightowlers Zosia , Lawry John , Fraser Karen , Gays Frances , Robinson John H. , Shrestha Sunil , Dyson P. Julian , Brooks Colin G.
Published: December 2022

Fetal mouse NK cells are grossly deficient in the expression of Ly49 molecules yet show a limited ability to distinguish between wild-type and MHC class I-deficient target cells. In this paper we report that during their development in vitro from immature thymic progenitors, a proportion of C57BL/6 fetal NK cells acquires receptors for a soluble form of the nonclassical class I molecule Qa1b associated with the Qdm peptide, but not for soluble forms of the classical class I molecules Kb and Db. The acquisition of these Qa1 receptors occurs in a stochastic manner that is strictly controlled by cytokines, and in particular is strongly inhibited by IL-4. All fetal NK clones tested, including those that lack detectable Qa1 receptors, express mRNA for CD94 and for both inhibitory and noninhibitory members of the NKG2 family. Fetal NK cells lacking receptors for Qa1 (and also for classical class I molecules) cannot distinguish between wild-type and class I-deficient blasts but, surprisingly, distinguish efficiently between certain wild-type and class I-deficient tumor cells. A variant line that lacks several members of the NKG2 family kills both types of tumor cell equally well, suggesting the existence of NKG2-containing inhibitory receptors that recognize as yet undefined nonclassical class I molecules of restricted distribution.
Survival, Maturation, and Function of CD11c− and CD11c+ Peripheral Blood Dendritic Cells Are Differentially Regulated by Cytokines
Authors: Kohrgruber Norbert , Halanek Nicole , Gröger Marion , Winter Dorian , Rappersberger Klemens , Schmitt-Egenolf Marcus , Stingl Georg , Maurer Dieter
Published: December 2022

Two types of dendritic cells (DC) are circulating in human blood and can be identified by their differential expression of the myeloid Ag CD11c. In this study, we show that CD11c− peripheral blood (PB)-DC correspond to plasmacytoid DC of lymphoid tissue not only by their surface Ag expression profile but, more impressively, by their peculiar ultramorphology. We also demonstrate that CD11c− and CD11c+ DC differ in the quality of their response to and in their requirement for certain cytokines. Freshly isolated CD11c− cells depend on IL-3 for survival and use autocrine or exogenous TNF-α as maturation signal, leading to the appearance of a highly dendritic phenotype, the up-regulation and redistribution of MHC class II from lysosomal compartments to the plasma membrane, the increased expression of costimulatory molecules, and the switch from a high Ag-processing to a low Ag-processing/potent accessory cell mode. Surprisingly, IL-4 efficiently killed freshly isolated CD11c− PB-DC, but did not impair the viability of CD11c+ PB-DC and, together with GM-CSF, induced maturation of these cells. A direct functional comparison revealed that neo-Ag-modified and subsequently matured CD11c− but to a lesser extent CD11c+ DC were able to prime naive Ag-specific CD4+ T cells. Our findings show that two diverse DC types respond to certain T cell-derived cytokines in a differential manner and, thus, suggest that suppression or activation of functionally diverse DC types may be a novel mechanism for the regulation of the quantity and quality of immune responses.
TNF-Mediated Activation of the Stress-Activated Protein Kinase Pathway: TNF Receptor-Associated Factor 2 Recruits and Activates Germinal Center Kinase Related
Authors: Shi Chong-Shan , Leonardi Antonio , Kyriakis John , Siebenlist Ulrich , Kehrl John H.
Published: December 2022

TNF-induced activation of stress activated protein kinases (SAPKs, Jun NH2-terminal kinases) requires TNF receptor associated factor 2 (TRAF2). TRAF2 is a potent activator of a 95-kDa serine/threonine kinase termed germinal center kinase related (GCKR, also referred to as KHS1), which signals activation of the SAPK pathway. Consistent with a role for GCKR in TNF- induced SAPK activation, a kinase-inactive mutant of GCKR is a dominant negative inhibitor of TRAF2-induced SAPK activation. Here we show that TRAF2 interacts with GCKR. This interaction depended upon the TRAF domain of TRAF2 and the C-terminal 150 aa of GCKR. The full activation of GCKR by TRAF2 required the TRAF2 RING finger domain. TNF treatment of a T cell line, Jurkat, increased both GCRK and SAPK activity and enhanced the coimmunoprecipitation of GCKR with TRAF2. Similar results were found with the B cell line HS-Sultan. These findings are consistent with a model whereby TNF signaling results in the recruitment and activation of GCKR by TRAF2, which leads to SAPK activation.
An Activation-Responsive Element in Single C Motif-1/Lymphotactin Promoter Is a Site of Constitutive and Inducible DNA-Protein Interactions Involving Nuclear Factor of Activated T Cell
Authors: Yoshida Tetsuya , Ishikawa Izumi , Ono Yuichi , Imai Toshio , Suzuki Ryuji , Yoshie Osamu
Published: December 2022

Single C motif-1 (SCM-1)/lymphotactin is a C-type chemokine whose expression is activation dependent, cyclosporin A sensitive and restricted to CD8+ T cells, double-negative thymocytes, γδ-type T cells, and NK cells. In humans, there are two highly homologous genes encoding SCM-1α and SCM-1β. Here we examined the regulatory mechanism of the SCM-1 genes. The luciferase reporter gene under the control of the 5′ flanking region of 0.7 kb was strongly induced upon activation with anti-CD3 or PHA plus PMA only in SCM-1-producer T cell lines through a cyclosporin A-sensitive mechanism. An element termed E1 located at −108 to −95 nt relative to the major transcription start site was found to be critical for the promoter activity. In electrophoretic mobility shift assays using the E1 oligonucleotide as probe, nuclear extracts from unstimulated T and B cell lines formed a constitutive complex termed complex I, while nuclear extracts from stimulated SCM-1-producer T cell lines formed a higher mobility complex termed complex II with a concomitant decrease in complex I. The shift from complex I to complex II seen only in SCM-1-producer T cell lines upon activation was completely suppressed by cyclosporin A. Both complexes were critically dependent on the NF-AT core sequence TTTCC in the E1 element and were partially supershifted by anti-NF-ATp. One-hybrid assays in yeast isolated NF-ATp as an E1 binding protein, and transfection of NF-ATp into T and B cell lines strongly enhanced the activation-dependent SCM-1 promoter activity. Collectively, a unique mechanism involving NF-ATp appears to regulate the cell type-specific and activation-dependent expression of the SCM-1 genes.
The Destabilization of IL-2 mRNA by a Premature Stop Codon and Its Differential Stabilization byTrans-Acting Inhibitors of Protein Synthesis Do Not Support a Role for Active Translation in mRNA Stability
Authors: Ragheb Jack A. , Deen Mary , Schwartz Ronald H.
Published: December 2022

To investigate the role that translation plays in the stabilization of the IL-2 mRNA, we inhibited protein synthesis in both cis and trans. To block translation in trans, we utilized the inhibitors puromycin (PUR) and cycloheximide (CHX), which differentially effect polysome structure. We found that CHX enhances the stability of IL-2 mRNA in cells stimulated with anti-TCR Ab alone, but it inhibits CD28-induced message stabilization in costimulated cells. In contrast, PUR had a minimal effect on IL-2 mRNA stability in either the presence or absence of costimulation. The differential effects of these two inhibitors suggest that: 1) CHX is unlikely to stabilize the IL-2 mRNA by inhibiting the expression of a labile RNase; 2) CD28-mediated IL-2 mRNA stabilization does not require translation; and 3) IL-2 mRNA decay is not coupled to translation. To block translation in cis, we generated sequence-tagged IL-2 genomic reporters that contain a premature termination codon (PTC). In both the presence and absence of costimulation, these PTC-containing mRNAs exhibit drastically diminished stability. Interestingly, the addition of CHX but not PUR completely restored CD28-mediated stabilization, suggesting that CHX can block the enhanced decay induced by a PTC. Finally, CHX was able to superinduce IL-2 mRNA levels in anti-TCR Ab-stimulated cells but not in CD28-costimulated cells, suggesting that CHX may also act by other mechanisms.
Visualization of Polyoma Virus-Specific CD8+ T Cells In Vivo During Infection and Tumor Rejection
Authors: Lukacher Aron E. , Moser Janice M. , Hadley Annette , Altman John D.
Published: December 2022

T cells are critical for clearing infection and preventing tumors induced by polyoma virus, a natural murine papovavirus. We previously identified the immunodominant epitope for polyoma virus-specific CTL in tumor-resistant H-2k mice as the Dk-restricted peptide, MT389–397, derived from the polyoma middle T oncoprotein. In this study, we developed tetrameric Dk complexes containing the MT389–397 peptide to directly visualize and enumerate MT389–397-specific CTL during polyoma virus infection. We found that Dk/MT389 tetramer+CD8+ T cells undergo a massive expansion during primary infection such that by day 7 postinfection these Ag-specific CD8+ T cells constitute ∼20% of the total and ∼40% of the activated CD8+ T cells in the spleen. This expansion of Dk/MT389 tetramer+CD8+ T cells parallels the emergence of MT389–397-specific ex vivo cytolytic activity and clearance of polyoma virus. Notably, Dk/MT389 tetramer+CD8+ T cells are maintained in memory at very high levels. The frequencies of Dk/MT389 tetramer+CD8+ effector and memory T cells in vivo match those of CD8+ T cells producing intracellular IFN-γ after 6-h in vitro stimulation by MT389–397 peptide. Consistent with preferential Vβ6 expression by MT389–397-specific CD8+CTL lines and clones, Dk/MT389 tetramer+CD8+ T cells exhibit biased expression of this Vβ gene segment. Finally, we show that Dk/MT389 tetramer+CD8+ T cells efficiently infiltrate a polyoma tumor challenge to virus-immune mice. Taken together, these findings strongly implicate virus-induced MT389–397-specific CD8+ T cells as essential effectors in eliminating polyoma-infected and polyoma-transformed cells in vivo.
Inverted Immunodominance and Impaired Cytolytic Function of CD8+ T Cells During Viral Persistence in the Central Nervous System
Authors: Bergmann Cornelia C. , Altman John D. , Hinton David , Stohlman Stephen A.
Published: December 2022

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) clear infectious virus; nevertheless, virus persists in the CNS as noninfectious RNA, resulting in ongoing primary demyelination. Phenotypic and functional analysis of CNS infiltrating cells during acute infection revealed a potent regional CD8+ T cell response comprising up to 50% virus-specific T cells. The high prevalence of virus-specific T cells correlated with ex vivo cytolytic activity and efficient reduction in viral titers. Progressive viral clearance coincided with the loss of cytolytic activity, but retention of IFN-γ secretion and increased expression of the early activation marker CD69, indicating differential regulation of effector function. Although the total number of infiltrating T cells declined following clearance of infectious virus, CD8+ T cells, both specific for the dominant viral epitopes and of unknown specificity, were retained within the CNS, suggesting an ongoing T cell response during persistent CNS infection involving a virus-independent component. Reversed immunodominance within the virus-specific CD8+ T cell population further indicated epitope-specific regulation, supporting ongoing T cell activation. Even in the absence of infectious virus, the CNS thus provides an environment that maintains both unspecific and Ag-specific CD8+ T cells with restricted effector function. Chronic T cell stimulation may thus play a role in preventing viral recrudescence, while increasing the risk of pathological conditions, such as demyelination.
Requirement for CD4+ T Cells in Vβ4+CD8+ T Cell Activation Associated with Latent Murine Gammaherpesvirus Infection
Authors: Flaño Emilio , Woodland David L. , Blackman Marcia A.
Published: December 2022

A CD8+ T cell lymphocytosis in the peripheral blood is associated with the establishment of latency following intranasal infection with murine gammaherpesvirus-68. Remarkably, a large percentage of the activated CD8+ T cells of mice expressing different MHC haplotypes express Vβ4+ TCR. Identification of the ligand driving the Vβ4+CD8+ T cell activation remains elusive, but there is a general correlation between Vβ4+CD8+ T cell stimulatory activity and establishment of latency in the spleen. In the current study, the role of CD4+ T cells in the Vβ4+CD8+ T cell expansion has been addressed. The results show that CD4+ T cells are essential for expansion of the Vβ4+CD8+ subset, but not other Vβ subsets, in the peripheral blood. CD4+ T cells are required relatively late in the antiviral response, between 7 and 11 days after infection, and mediate their effect independently of IFN-γ. Assessment of Vβ4+CD8+ T cell stimulatory activity using murine gammaherpesvirus-68-specific T cell hybridomas generated from latently infected mice supports the idea that CD4+ T cells control levels of the stimulatory ligand that drives the Vβ4+CD8+ T cells. As Vβ4+CD8+ T cell expansion also correlates with levels of activated B cells, these data raise the possibility that CD4+ T cell-mediated B cell activation is required for optimal expression of the stimulatory ligand. In addition, in cases of low ligand expression, there may also be a direct role for CD4+ T cell-mediated help for Vβ4+CD8+ T cells.
A Novel Mechanism of Action of Chemically Modified Tetracyclines: Inhibition of COX-2-Mediated Prostaglandin E2 Production
Authors: Patel Rajesh N. , Attur Mukundan G. , Dave Mandar N. , Patel Indravadan V. , Stuchin Steven A. , Abramson Steven B. , Amin Ashok R.
Published: December 2022

Tetracyclines (doxycycline and minocycline) inhibit inducible NO synthase expression and augment cyclooxygenase (COX)-2 expression and PGE2 production. In contrast, chemically modified tetracyclines (CMTs), such as CMT-3 and -8 (but not CMT-1, -2, and -5), that lack antimicrobial activity, inhibit both NO and PGE2 production in LPS-stimulated murine macrophages, bovine chondrocytes, and human osteoarthritis-affected cartilage, which spontaneously produces NO and PGE2 in ex vivo conditions. Furthermore, CMT-3 augments COX-2 protein expression but inhibits net PGE2 accumulation. This coincides with the ability of CMT-3 and -8 to inhibit COX-2 enzyme activity in vitro. The action of CMTs is distinct from that observed with tetracyclines because 1) CMT-3-mediated inhibition of PGE2 production coincides with modification of COX-2 protein, which is distinct from the nonglycosylated COX-2 protein generated in the presence of tunicamycin, as observed by Western blot analysis and 2) CMT-3 and -8 have no significant effect on COX-2 mRNA accumulation. In contrast, CMT-3 and -8 do not inhibit COX-1 expression in A549 human epithelial cells at the level of protein and mRNA accumulation or modification of COX-1 protein. CMT-3 and -8 inhibit the sp. act. of COX-2 (but not COX-1) in cell-free extracts. These results demonstrate differential action of CMT-3 (Metastat) on COX-1 and -2 expression, which is distinct from other tetracyclines.
A Defective Protein Kinase C Anchoring System Underlying Age-Associated Impairment in TNF-α Production in Rat Macrophages
Authors: Corsini Emanuela , Battaini Fiorenzo , Lucchi Laura , Marinovich Marina , Racchi Marco , Govoni Stefano , Galli Corrado L.
Published: December 2022

The ability of macrophages to secrete cytokines is important in host responses to infections inflammatory stimuli, both of which are altered with aging. In this study, age-associated changes in the release of TNF-α from LPS-stimulated rat alveolar macrophages were determined and correlated with a decrease in the level of RACK1, the anchoring protein involved in protein kinase C translocation and activation. Macrophages from aged rats produced ∼50% less TNF-α than those from young rats. This effect was observed independently from the concentration of LPS used and the time considered. The decrease observed was associated with a defective PKC translocation, due to a reduction in the expression of RACK1, whereas no differences were detected in the expression of LPS receptor (CD14) or total PKC isoforms (α and βΙΙ) in old and young rats. Use of RACK1 antisense oligonucleotide reduced the ability of young macrophages to respond to LPS, further supporting the idea that a deficit in RACK1 contributes to the functional impairment in aged macrophages and that age-induced macrophage immunodeficiencies are associated with alteration in signal transduction pathways.
Curcumin Blocks Cytokine-Mediated NF-κB Activation and Proinflammatory Gene Expression by Inhibiting Inhibitory Factor I-κB Kinase Activity
Authors: Jobin Christian , Bradham Cynthia A. , Russo Maria Pia , Juma Booker , Narula Acharan S. , Brenner David A. , Sartor R. Balfour
Published: December 2022

NF-κB plays a critical role in the transcriptional regulation of proinflammatory gene expression in various cells. Cytokine-mediated activation of NF-κB requires activation of various kinases, which ultimately leads to the phosphorylation and degradation of IκB, the NF-κB cytoplasmic inhibitor. The food derivative curcumin has been shown to inhibit NF-κB activity in some cell types. In this report we investigate the mechanism of action of curcumin on cytokine-induced proinflammatory gene expression using intestinal epithelial cells (IEC). Curcumin inhibited IL-1β-mediated ICAM-1 and IL-8 gene expression in IEC-6, HT-29, and Caco-2 cells. Cytokine-induced NF-κB DNA binding activity, RelA nuclear translocation, IκBα degradation, IκB serine 32 phosphorylation, and IκB kinase (IKK) activity were blocked by curcumin treatment. Wound-induced p38 phosphorylation was not inhibited by curcumin treatment. In addition, mitogen-activated protein kinase/ERK kinase kinase-1-induced IL-8 gene expression and 12-O-tetraphorbol 12-myristate 13-acetate-responsive element-driven luciferase expression were inhibited by curcumin. However, IκBα degradation induced by ectopically expressed NF-κB-inducing kinase or IKK was not inhibited by curcumin treatment. Therefore, curcumin blocks a signal upstream of NF-κB-inducing kinase and IKK. We conclude that curcumin potently inhibits cytokine-mediated NF-κB activation by blocking a signal leading to IKK activity.
Glucocorticoids Induce Apoptosis in Human Monocytes: Potential Role of IL-1β
Authors: Schmidt Michael , Pauels Hans-Gerd , Lügering Norbert , Lügering Andreas , Domschke Wolfram , Kucharzik Torsten
Published: December 2022

Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive agents that act on a variety of immune cells, including monocytes and macrophages. However, the exact cellular mechanisms underlying this anti-inflammatory capacity are still unknown. In our study, we determined the induction of apoptosis by GC in human monocytes. Peripheral blood monocytes were isolated by density centrifugation methods with a purity of >90% and were cultured in RPMI 1640 medium. Monocyte apoptosis was determined by four independent methods, including annexin-V staining, TUNEL, DNA-laddering, and typical morphology by means of transmission electron microscopy. TNF-α and IL-1β were measured by ELISA. GC receptor was blocked with mifepristone. Caspase 3 was inhibited with caspase-3 inhibitor (DEVD-CHO). Stimulation with different GC at therapeutic concentrations resulted in monocyte apoptosis in a time- and dose-dependent manner. Necrosis was excluded by propidium iodide staining. Proinflammatory cytokines such as IL-1β and TNF-α were down-regulated by GC treatment. Continuous treatment of monocytes with IL-1β, but not with TNF-α, could almost completely prevent GC-induced cell death. The addition of mifepristone or caspase-3 inhibitor could partially abrogate GC-induced apoptosis as well as GC-induced inhibition of IL-1β. This is the first study to demonstrate induction of apoptosis by GC in human monocytes. GC-induced monocyte apoptosis may be partially mediated through effects on IL-1β production. It is conceivable that GC exert their anti-inflammatory capacity in various diseases, at least in part, by the induction of apoptosis in monocytes.
Enhanced Autoimmune Arthritis in IFN-γ Receptor-Deficient Mice Is Conditioned by Mycobacteria in Freund’s Adjuvant and by Increased Expansion of Mac-1+ Myeloid Cells
Authors: Matthys Patrick , Vermeire Kurt , Mitera Tania , Heremans Hubertine , Huang Sui , Schols Dominique , De Wolf-Peeters Chris , Billiau Alfons
Published: December 2022

Induction of experimental autoimmune diseases often relies on immunization with the organ-specific autoantigens in CFA, which contains heat-killed mycobacteria. In several of these models, including collagen-induced arthritis, endogenous IFN-γ acts as a disease-limiting factor in the pathogenesis of the disease. Here we show that in collagen-induced arthritis the protective effect of IFN-γ depends on the presence of mycobacteria in the adjuvant. Omission of mycobacteria inverts the role of endogenous IFN-γ to a disease-promoting factor. Thus, the mycobacterial component of CFA opens a pathway by which endogenous IFN-γ exerts a protective effect that supersedes its otherwise disease-promoting effect. Extramedullary hemopoiesis and expansion of the Mac-1+ cell population accompanied the accelerated and more severe disease course in the IFN-γ receptor knockout mice immunized with CFA. Treatment of such mice with Abs against the myelopoietic cytokines IL-6 or IL-12 inhibited both disease development and the expansion of the Mac-1+ population. We postulate that mycobacteria in CFA stimulate the expansion of the Mac-1+ cell population by a hemopoietic process that is restrained by endogenous IFN-γ. These results have important implications for the validity of animal models of autoimmunity to study the pathogenesis and to evaluate cytokine-based therapy of autoimmune diseases.
Polarized Type 1 Cytokine Profile in Bronchoalveolar Lavage T Cells of Patients with Hypersensitivity Pneumonitis
Authors: Yamasaki Hisato , Ando Masayuki , Brazer William , Center David M. , Cruikshank William W.
Published: December 2022

Hypersensitivity pneumonitis (HP) is characterized by an inflammatory lymphocytic alveolitis comprised of both CD8+ and CD4+ T cells. Animal models suggest that HP is facilitated by overproduction of IFN-γ, and that IL-10 ameliorates severity of the disease, indicating a Th1-type response. To determine whether a Th1 phenotype in HP also exists clinically, bronchoalveolar lavage (BAL) and peripheral blood (PB) T cells were obtained from HP individuals and analyzed for Th1 vs Th2 cytokine profiles. It was determined that soluble OKT3-stimulated BAL T cells cocultured with alveolar macrophages produced more IFN-γ and less IL-10 than PB T cells cocultured with monocytes, but no difference was observed in IL-4 production. The monocytic cells did not account for this difference, as CD80 and CD86 expressions were similar, and coculturing PB T cells with alveolar macrophages resulted in no difference in IFN-γ production. Similarly, there was no difference in IL-12 production between stimulated BAL or PB T cells; however, addition of rIL-12 significantly increased production of IFN-γ by BAL T cells, but not by PB T cells. This effect was due to a difference in IL-12R expression. High affinity IL-12R were only present in association with BAL T cells. These studies indicate that clinical HP is characterized by a predominance of IFN-γ-producing T cells, perhaps resulting from a reduction in IL-10 production and an increase in high affinity IL-12R compared with blood T cells.
Up-Regulation of CCR2 Chemokine Receptor Expression and Increased Susceptibility to the Multitropic HIV Strain 89.6 in Monocytes Exposed to Glucocorticoid Hormones
Authors: Penton-Rol Giselle , Cota Manuela , Polentarutti Nadia , Luini Walter , Bernasconi Sergio , Borsatti Alessandro , Sica Antonio , LaRosa Gregory J. , Sozzani Silvano , Poli Guido , Mantovani Alberto
Published: December 2022

Glucocorticoid hormones (GC) are potent antiinflammatory agents widely used in the treatment of diverse human diseases. The present study was aimed at assessing the effect of GC on chemokine receptor expression in human monocytes. Dexamethasone (Dex) up-regulated mRNA expression of the monocyte chemotactic protein (MCP-1, CCL2) chemokine receptor CCR2. The effect was selective in that other chemokine receptors were not substantially affected. Stimulation by Dex was observed after 4 h of exposure at concentrations of 10−7 to 10−5 M. Steroids devoid of GC activity were inactive, and the GC receptor antagonist, RU486, inhibited stimulation. Dex did not affect the rate of nuclear transcription, but augmented the CCR2 mRNA half-life. Augmentation of CCR2 expression by Dex was associated with increased chemotaxis. Finally, Dex treatment induced productive replication of the HIV strain 89.6, which utilizes CCR2 as entry coreceptor, in freshly isolated monocytes. Together with previous findings, these results indicate that at least certain pro- and antiinflammatory molecules have reciprocal and divergent effects on expression of a major monocyte chemoattractant, MCP-1, and of its receptor (CCR2). Augmentation of monocyte CCR2 expression may underlie unexplained in vivo effects of GC as well as some of their actions on HIV infection.
Cutting Edge: NKT Cell Development Is Selectively Impaired in Fyn- Deficient Mice
Authors: Eberl Gérard , Lowin-Kropf Bente , MacDonald H. Robson
Published: December 2022

Most NK1.1+ T (NKT) cells express a biased TCRαβ repertoire that is positively selected by the monomorphic MHC class I-like molecule CD1d. The development of CD1d-dependent NKT cells is thymus dependent but, in contrast to conventional T cells, requires positive selection by cells of hemopoietic origin. Here, we show that the Src protein tyrosine kinase Fyn is required for development of CD1d-dependent NKT cells but not for the development of conventional T cells. In contrast, another Src kinase, Lck, is required for the development of both NKT and T cells. Impaired NKT cell development in Fyn-deficient mice cannot be rescued by transgenic expression of CD8, which is believed to increase the avidity of CD1d recognition by NKT cells. Taken together, our data reveal a selective and nonredundant role for Fyn in NKT cell development.
Cutting Edge: Bacterial DNA and LPS Act in Synergy in Inducing Nitric Oxide Production in RAW 264.7 Macrophages
Authors: Gao Jian Jun , Zuvanich Eleanor G. , Xue Qiao , Horn David L. , Silverstein Richard , Morrison David C.
Published: December 2022

LPS is well recognized for its potent capacity to activate mouse macrophages to produce NO, an important inflammatory mediator in innate host defense. We demonstrate here that, although inducing little NO alone, DNA from both Gram-negative and Gram-positive bacteria synergizes with subthreshold concentrations of LPS (0.3 ng/ml) to induce NO in cultures of RAW 264.7 macrophages. The effects of the DNA are mimicked by synthetic CpG-containing oligodeoxynucleotides but not by non-CpG-containing oligodeoxynucleotides. This synergistic activity is not inhibited by neutralizing Abs against IFN. Preincubation of macrophages with DNA for 8–24 h suppresses subsequent synergistic macrophage responses to DNA/LPS, whereas prolonged pretreatment with LPS enhances synergy. RT-PCR analysis indicates that the mRNA levels of the inducible NO synthase gene are also coordinately suppressed or induced. These findings indicate that temporally controlled, synergistic interactions exist between microbial DNA and LPS in the induction of macrophage NO via enhanced inducible NO synthase gene expression.