bluuebunny/crossref_metadata_2025 huggingface dataset:
Major histocompatibility complex class I related molecules control the development of CD4+8- and CD4-8- subsets of natural killer 1.1+ T cell receptor-alpha/beta+ cells in the liver of mice.
Authors: Ohteki T , MacDonald H R
Published: June 2004

Normal mouse liver contains prominent subsets of CD4+8- and CD4-8- T cell receptor (TCR)-alpha/beta+ cells with intermediate TCR levels. We show here that these cells express the natural killer (NK)1.1 surface antigen and have a restricted TCRV beta repertoire that is highly skewed to V beta 7 and V beta 8. Surprisingly, both CD4+8- and CD4-8- subsets of NK1.1+TCR-alpha/beta+ cells are absent in the liver of beta 2-microglobulin deficient mice, which do not express major histocompatibility complex (MHC) class I or "class I-like" molecules. Analysis of reciprocal radiation bone marrow chimeras established with beta 2-microglobulin deficient and wild-type mice demonstrates that MHC class I expression on radiosensitive (presumably hematopoietic) cells is required for the development of NK1.1+TCR-alpha/beta+ cells in the liver. In the liver of MHC class II deficient mice, the CD4+8- and CD4-8- subsets of NK1.1+TCR-alpha/beta+ cells develop normally. Collectively our data suggest that NK1.1+TCR-alpha/beta+ cells in liver require interaction with a MHC class I-related ligand on hematopoietic cells for their development. This unusual property of liver T cells is shared by a subset of CD4-8-NK1.1+TCR-alpha/beta+ thymocytes, suggesting a common lineage independent of the mainstream of T cell development.
Macrophage-colony-stimulating factor selectively enhances macrophage scavenger receptor expression and function.
Authors: de Villiers W J , Fraser I P , Hughes D A , Doyle A G , Gordon S
Published: June 2004

Regulation of macrophage scavenger receptor (MSR) activity may be an important determinant of the extent of atherogenesis. The effect of macrophage-colony-stimulating factor (M-CSF) on this pathway was studied using a recently developed monoclonal antibody to murine MSR. M-CSF markedly and selectively increased MSR synthesis in murine macrophages: posttranslationally, the receptor appeared more stable and shifted to a predominantly surface distribution. Functionally, M-CSF enhanced modified lipoprotein uptake and increased divalent cation-independent adhesion in vitro. These results suggest a plausible mechanism whereby M-CSF production in the atheromatous plaque microenvironment could promote the recruitment and retention of mononuclear phagocytes and subsequent foam cell formation.
Interleukin 10 inhibits lipopolysaccharide-induced survival and cytokine production by human peripheral blood eosinophils.
Authors: Takanaski S , Nonaka R , Xing Z , O'Byrne P , Dolovich J , Jordana M
Published: June 2004

In this study we have investigated the effects of interleukin 10 (IL-10) on human peripheral blood eosinophils stimulated with granulocyte/macrophage colony stimulating factor (GM-CSF) and lipopolysaccharide (LPS). We show that LPS was able to enhance eosinophil survival in a dose-dependent manner, as well as release of the cytokines GM-CSF, tumor necrosis factor alpha, and IL-8. LPS-induced eosinophil survival was largely inhibited by an anti-GM-CSF neutralizing antibody and completely blocked by polymyxin B, suggesting GM-CSF involvement in the survival enhancing mechanism and LPS specificity, respectively. IL-10 significantly inhibited survival of, and cytokine production from, eosinophils induced by LPS, but did not inhibit the survival induced by GM-CSF. These observations suggest a novel activation mechanism of eosinophils and, also, that IL-10 may participate in the regulation of diseases characterized by eosinophil infiltration.
Sequence homology of the diabetes-associated autoantigen glutamate decarboxylase with coxsackie B4-2C protein and heat shock protein 60 mediates no molecular mimicry of autoantibodies.
Authors: Richter W , Mertens T , Schoel B , Muir P , Ritzkowsky A , Scherbaum W A , Boehm B O
Published: June 2004

Molecular mimicry between viral antigens and host proteins was often suggested to be involved in induction of autoimmune diseases. In type 1 diabetes where pancreatic beta cells are destroyed by autoimmune phenomena, a linear sequence homology between a major autoantigen, glutamate decarboxylase (GAD), and the 2C protein of coxsackie B4 was identified. In addition, a sequence homology between GAD and the mycobacterial heat shock protein 60 was described and the suggestions were made that molecular mimicry between GAD, coxsackievirus B4-2C protein, and/or heat shock protein 60 (hsp60) may be actively involved in an autoimmune reaction towards the pancreatic beta-cells. Our group was the first to isolate human monoclonal autoantibodies to GAD (MICA 1-6) from a patient with newly diagnosed type 1 diabetes. The MICA allowed a detailed characterization of the diabetes associated self-epitopes in GAD and represent a set of GAD autoantibodies present in sera from patients with type 1 diabetes. Using deletion mutants of GAD we demonstrated that the regions of GAD covering the homology sequences to coxsackievirus B4 and to the hsp60 were absolutely required for binding of the MICA to GAD. We now designed an antibody-based analysis to ask whether molecular mimicry between GAD and coxsackie B4-2C or hsp60 is relevant in type 1 diabetes. Since part of the MICA recognize conformational epitopes, they allow to test for conformational molecular mimicry in viruses that have been incriminated in the development of type 1 diabetes. Our data reveal no crossreactivity between the diabetes associated GAD epitopes defined by the MICA and hsp60, rubellavirus, cytomegalovirus, and coxsackie B1-B6 virus antigens. Neither coxsackie B4-specific antibodies in sera from normal individuals nor GAD-positive sera from patients with type 1 diabetes indicated a crossreactivity between coxsackie B4-2C and GAD. Although the regions in GAD homologous to coxsackie B4-2C and hsp60 represented parts of GAD indispensible for binding of diabetes associated autoantibodies they did not mediate a crossreactivity of autoantibodies between GAD and these two proteins. No evidence for molecular mimicry between GAD and a whole panel of foreign antigens was detected by autoantibodies in type 1 diabetes.
Recognition of carbohydrate by major histocompatibility complex class I-restricted, glycopeptide-specific cytotoxic T lymphocytes.
Authors: Haurum J S , Arsequell G , Lellouch A C , Wong S Y , Dwek R A , McMichael A J , Elliott T
Published: June 2004

Cytotoxic T cells (CTL) recognize short peptide epitopes presented by class I glycoproteins encoded by the major histocompatibility complex (MHC). It is not yet known whether peptides containing posttranslationally modified amino acids can also be recognized by CTL. To address this issue, we have studied the immunogenicity and recognition of a glycopeptide carrying an O-linked N-acetylglucosamine (GlcNAc) monosaccharide-substituted serine residue. This posttranslational modification is catalyzed by a recently described cytosolic glycosyltransferase. We show that glycosylation does not affect peptide binding to MHC class I and that glycopeptides can elicit a strong CTL response that is glycopeptide specific. Furthermore, glycopeptide recognition by cytotoxic T cells is dependent on the chemical structure of the glycan as well as its position within the peptide.
Selective release of some invariant chain-derived peptides from HLA-DR1 molecules at endosomal pH.
Authors: Urban R G , Chicz R M , Strominger J L
Published: June 2004

The predominant peptides bound to major histocompatibility complex class II molecules expressed on human B cells are derived from a relatively limited number of self proteins. To determine whether any of the prebound self peptides might be released in endosomes during recycling, water-soluble HLA-DR1 molecules were incubated with a high affinity synthetic peptide at pH 4.0 and 7.0 at 37 degrees C. The resulting bound peptide repertoire was then acid extracted, and separated by reversed-phase high performance liquid chromatography. Using a combination of mass spectrometry and ultraviolet spectroscopy, prebound self peptides and newly bound synthetic peptide were characterized. Most self peptides bound to HLA-DR1 were not appreciably released during extended exposure to pH 4.0. However, some invariant chain-derived peptides were uniquely released at this pH.
Regulation of cytokine production by soluble CD23: costimulation of interferon gamma secretion and triggering of tumor necrosis factor alpha release.
Authors: Armant M , Ishihara H , Rubio M , Delespesse G , Sarfati M
Published: June 2004

Soluble CD23 (sCD23) has multiple IgE-independent biological activities. In the present study, we examined the regulatory effect of sCD23 on cytokine production by human peripheral blood mononuclear cells (PBMC). We show that sCD23 enhances by about 80-fold the interleukin 2 (IL-2)-induced interferon gamma (IFN-gamma) production and by about 10-fold the response to IL-12. This potentiating activity is time and dose dependent and is not associated with a significant effect on DNA synthesis. The sCD23 costimulatory activity for IFN-gamma synthesis is drastically reduced in monocyte-depleted PBMC, suggesting that monocytes may be the target for sCD23. This hypothesis was supported by the following observations. First, sCD23 alone is a potent inducer of tumor necrosis factor alpha (TNF-alpha) production by PBMC and this effect disappears after monocyte depletion. The triggering of TNF-alpha release is specifically inhibited by neutralizing anti-CD23 monoclonal antibody (mAb). In addition, IL-2 and IL-12 synergize with sCD23 to induce TNF-alpha production. Second, sCD23 triggers the release of other inflammatory mediators such as IL-1 alpha, IL-1 beta, and IL-6. Finally, TNF-alpha production in response to IL-2 and sCD23 precedes IFN-gamma and IFN-gamma secretion is significantly inhibited by anti-TNF-alpha mAb, indicating that the sCD23 costimulatory signal for IFN-gamma production may be partially mediated by TNF-alpha release. It is proposed that sCD23 is a proinflammatory cytokine that, in addition, may play an important role in the control of the immune response via the enhancement of IFN-gamma production.
The role of p21ras in CD28 signal transduction: triggering of CD28 with antibodies, but not the ligand B7-1, activates p21ras.
Authors: Nunès J A , Collette Y , Truneh A , Olive D , Cantrell D A
Published: June 2004

CD28 is a 44-kD homodimer expressed on the surface of the majority of human T cells that provides an important costimulus for T cell activation. The biochemical basis of the CD28 accessory signals is poorly understood. Triggering of the T cell antigen receptor (TCR) activates the p21ras proteins. Here we show that ligation of CD28 by a monoclonal antibody (mAb) also stimulates p21ras and induces Ras-dependent events such as stimulation of the microtubule-associated protein (MAP) kinase ERK2 and hyperphosphorylation of Raf-1. One physiological ligand for CD28 is the molecule B7-1. In contrast to the effect of CD28 mAb, the present studies show that interactions between CD28 and B7-1 do not stimulate p21ras signaling pathways. Two substrates for TCR-regulated protein tyrosine kinases (PTKs) have been implicated in p21ras activation in T cells: p95vav and a 36-kD protein that associates with a complex of Grb2 and the Ras exchange protein Sos. Triggering CD28 with both antibodies and B7-1 activates cellular PTKs, and we have exploited the differences between antibodies and B7-1 for p21ras activation in an attempt to identify critical PTK-controlled events for Ras activation in T cells. The data show that antibodies against TCR or CD28 induce tyrosine phosphorylation of both Vav and p36. B7-1 also induces Vav tyrosine phosphorylation but has no apparent effect on tyrosine phosphorylation of the Grb2-associated p36 protein. The intensity of the Vav tyrosine phosphorylation is greater in B7-1 than in TCR-stimulated cells. Moreover the kinetics of Vav tyrosine phosphorylation is prolonged in the B7-1-stimulated cells. These studies show that for CD28 signaling, the activation of p21ras correlates more closely with p36 tyrosine phosphorylation than with Vav tyrosine phosphorylation. However, the experiments demonstrate that Vav is a major substrate for B7-activated PTKs and hence could be important in CD28 signal transduction pathway.
Impaired cytotoxic T lymphocyte recognition due to genetic variations in the main immunogenic region of the human immunodeficiency virus 1 NEF protein.
Authors: Couillin I , Culmann-Penciolelli B , Gomard E , Choppin J , Levy J P , Guillet J G , Saragosti S
Published: June 2004

Human immunodeficiency virus (HIV) induces strong responses from human histocompatibility leukocyte antigen (HLA) class I-restricted cytotoxic T lymphocytes (CTL). In a previous report we identified an immunodominant region (amino acids 73-144) in the NEF protein that was recognized by CD8+ class I-restricted CTL of most asymptomatic individuals. Analysis of the 73-144 region by peptide sensitization, experiments using overlapping peptides corresponding to the LAI isolate identified the peptide sequences located between residues 73 and 82 or 84 and 92 and the peptide sequence between residues 134 and 144 as cognate peptides for HLA-A11- and HLA-B18-restricted epitopes, respectively. This report describes the variable demonstrable reactivities of CTL obtained from HLA-A11 or HLA-B18 seropositive, asymptomatic patients who all had a response to the virus NEF protein, but who did not always recognize appropriate cognate peptides. The high mutation rate of HIV probably facilitates the selection of mutants that can avoid the cellular immune response. We therefore analyzed the variability of these epitopes restricted by HLA-A11 and HLA-B18. We sequenced several viral isolates from HLA-A11 and HLA-B18 donors who recognized certain HLA-peptide complexes and from those who did not. A CTL sensitization assay was used to show that some mutations led to a great reduction in CTL activity in vitro. This might be due to failure of the mutated epitope to bind major histocompatibility complex class I molecule. A simple assay was used to detect peptides that promoted the assembly of class I molecules. Some of these mutations at major anchor positions prevented HLA-A11/peptide binding, and consequently impaired recognition of the HLA-peptide complex by the T cell receptor.
Pertussis toxin inhibits activation-induced cell death of human thymocytes, pre-B leukemia cells and monocytes.
Authors: Ramírez R , Carracedo J , Zamzami N , Castedo M , Kroemer G
Published: June 2004

Activation of human thymocytes and pre-B cells via the CD3/T cell receptor (TCR) complex or the IgM/B cell receptor complex, respectively, results in apoptotic cell death. Similarly, cross-linking of the activation marker CD69, which belongs to the natural killer complex, causes apoptosis of lipopolysaccharide-preactivated monocytes. Here we show that pertussis toxin (PTX) inhibits the activation-induced apoptosis of these three cell types, though it fails to prevent the programmed cell death that follows exposure of cells to the synthetic glucocorticoid dexamethasone (thymocytes, pre-B cells) or to interleukin 4 (monocytes). The capacity of pertussis toxin to suppress activation-induced death is not due to quenching of the activation signal, because thymocytes exposed to PTX are still capable of mobilizing Ca2+ after TCR-alpha/beta cross-linking and proliferate in response to costimulation with PTX and CD3/TCR ligation. The apoptosis-inhibitory effect of PTX depends on the presence of an intact adenosine diphosphate (ADP)-ribosylating moiety, since a mutant pertussis toxin molecule that lacks enzymatic activity, but still possesses the membrane translocating activity, fails to interfere with activation-induced cell death. A toxin that induces a different spectrum of ADP ribosylation than PTX, cholera toxin, fails to inhibit apoptosis. To suppress apoptosis, the intact PTX holotoxin must be added to cells before the lethal activation step; its addition 30 min after initial activation remains without effect on apoptosis. These data unravel a PTX sensitive signal transduction event that intervenes during an early step of activation-induced cell death of immune cells.
Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major.
Authors: Stenger S , Thüring H , Röllinghoff M , Bogdan C
Published: June 2004

Previous studies with inhibitors of inducible nitric oxide synthase (iNOS) suggested that high-output production of nitric oxide (NO) is an important antimicrobial effector pathway in vitro and in vivo. Here, we investigated the tissue expression of iNOS in mice after infection with Leishmania major. Immunohistochemical staining with an iNOS-specific antiserum revealed that in the cutaneous lesion and draining lymph nodes (LN) of clinically resistant mice (C57BL/6), iNOS protein is found earlier during infection and in significantly higher amounts than in the nonhealing BALB/c strain. Similar differences were seen on the mRNA level as quantitated by competitive polymerase chain reaction. Anti-CD4 treatment of BALB/c mice not only induced resistance to disease, but also restored the expression of iNOS in the tissue. In situ, few or no parasites were found in those regions of the skin lesion and the draining LN which were highly positive for iNOS. By double labeling experiments, macrophages were identified as iNOS expressing cells in vivo. In the lesions of BALB/c mice, cells staining positively for transforming growth factor beta (TGF-beta), a potent inhibitor of iNOS in vitro, were strikingly more prominent than in C57BL/6, whereas no such difference was found for interleukin 4 or interferon gamma (IFN-gamma). In vitro, production of NO was approximately threefold higher in C57BL/6 than in BALB/c macrophages after stimulation with IFN-gamma. We conclude that the pronounced expression of iNOS in resistant mice is an important mechanism for the elimination of Leishmania in vivo. The relative lack of iNOS in susceptible mice might be a consequence of macrophage deactivation by TGF-beta and reduced responsiveness to IFN-gamma.
T lymphocytes adhere to airway smooth muscle cells via integrins and CD44 and induce smooth muscle cell DNA synthesis.
Authors: Lazaar A L , Albelda S M , Pilewski J M , Brennan B , Puré E , Panettieri R A
Published: June 2004

Asthma is a disease of airway inflammation and hyperreactivity that is associated with a lymphocytic infiltrate in the bronchial submucosa. The interactions between infiltrating T lymphocytes with cellular and extracellular matrix components of the airway and the consequences of these interactions have not been defined. We demonstrate the constitutive expression of CD44 on human airway smooth muscle (ASM) cells in culture as well as in human bronchial tissue transplanted into severe combined immunodeficient mice. In contrast, basal levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression are minimal but are induced on ASM by inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha). Activated, but not resting T cells, adhere to cultured ASM; stimulation of the ASM with TNF-alpha enhanced this adhesion. Adhesion was partially blocked by monoclonal antibodies (mAb) specific for lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4) on T cells and ICAM-1 and VCAM-1 on ASM cells. The observed integrin-independent adhesion was mediated by CD44/hyaluronate interactions as it was inhibited by anti-CD44 mAb 5F12 and by hyaluronidase. Furthermore, the adhesion of activated T lymphocytes induced DNA synthesis in growth-arrested ASM cells. Thus, the interaction between T cells and ASM may provide insight into the mechanisms that induce bronchial inflammation and possibly ASM cell hyperplasia seen in asthma.
Experimental autoimmune panencephalitis and uveoretinitis transferred to the Lewis rat by T lymphocytes specific for the S100 beta molecule, a calcium binding protein of astroglia.
Authors: Kojima K , Berger T , Lassmann H , Hinze-Selch D , Zhang Y , Gehrmann J , Reske K , Wekerle H , Linington C
Published: June 2004

The pathogenic potential of autoimmune T cell responses to nonmyelin autoantigens was investigated in the Lewis rat using the astrocyte-derived calcium binding protein S100 beta, as a model nonmyelin autoantigen. The Lewis rat mounts a vigorous RT1B1 (major histocompatibility complex class II) restricted autoimmune response to an immunodominant S100 beta epitope (amino acid residues 76-91). The adoptive transfer of S100 beta-specific T cell lines induced a severe inflammatory response in the nervous system, but only minimal neurological dysfunction in naive syngeneic recipients. The inability of S100 beta-specific T cell transfer to induce severe disease was associated with a decreased recruitment of ED1+ macrophages into the central nervous system (CNS) in comparison with that seen in severe experimental autoimmune encephalomyelitis (EAE) induced by the adoptive transfer of myelin basic protein (MBP)-specific T line cells. Moreover, unlike encephalitogenic MBP-specific T cell lines, S100 beta-specific T cell lines exhibited no cytotoxic activity in vitro. Histopathological analysis also revealed striking differences in the distribution of inflammatory lesions in MBP- and S100 beta-specific T cell-mediated disease. In contrast to the MBP paradigm, S100 beta-specific T cell transfer induces intense inflammation not only in the spinal cord, but throughout the entire CNS and also in the uvea and retina of the eye. In view of the distribution of lesions throughout the grey and white matter of the CNS we propose to term this new model experimental autoimmune panencephalomyelitis (EAP) to differentiate it from EAE. These experiments demonstrate for the first time that nonmyelin CNS autoantigens can initiate a pathogenic autoimmune T cell response, although the nature of the target autoantigen profoundly influences the clinical and histopathological characteristics of the resulting autoimmune disease. This is not simply a consequence of the distribution of the autoantigen, as both MBP and S100 beta are coexpressed in many areas of the CNS, but reflects differences in the capacity of different regions of the CNS to process and present specific autoantigens. This new model of T cell-mediated autoimmune CNS disease exhibits a number of similarities to multiple sclerosis (MS), such as its mild clinical course and the involvement of areas of the brain and eye, which are absent in myelin-mediated models of EAE. Nonmyelin autoantigens may therefore play an unexpectedly important role in the immunopathogenesis of inflammatory diseases of the CNS.
Detection of human T lymphotrophic virus type I (HTLV-I) proviral DNA and analysis of T cell receptor V beta CDR3 sequences in spinal cord lesions of HTLV-I-associated myelopathy/tropical spastic paraparesis.
Authors: Hara H , Morita M , Iwaki T , Hatae T , Itoyama Y , Kitamoto T , Akizuki S , Goto I , Watanabe T
Published: June 2004

Identification of the localization of human T lymphotrophic virus type I (HTLV-I) proviral DNA in the central nervous system (CNS) is crucial to the understanding of the pathogenesis of HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) pathogenesis. We have developed a sensitive detection method, called two-step polymerase chain reaction (PCR) in situ hybridization, which enabled us to detect the HTLV-I proviral DNA in paraffin-embedded spinal cord tissue sections from HAM/TSP patients. HTLV-I proviral DNA was detected only in the nucleus of lymphocytes that had infiltrated into the spinal cord. However, no proviral DNA was amplified in any neuronal cells, including neurons and glial cells. This indicates that the demyelination of the spinal cord by HTLV-I as a result of viral infection of oligodendrocytes or neuronal cells is unlikely. The T cell receptor V beta gene sequence from lymphocytes in the spinal cord lesions taken from the same HAM/TSP autopsy cases revealed unique and restricted CDR3 motifs, CASSLXG(G) (one-letter amino acid. X is any amino acid), CASSPT(G), and CASSGRL which are similar to those described in T cells from brain lesions of multiple sclerosis (MS) and in a rat T cell clone derived from experimental allergic encephalomyelitis (EAE) lesions. The present results suggest that T cells containing restricted V beta CDR3 motifs, which are also found in MS and EAE, become activated upon HTLV-I infection and infiltrate into the spinal cord lesions of HAM/TSP patients.
Apoptosis is regulated by the rate of glucose transport in an interleukin 3 dependent cell line.
Authors: Kan O , Baldwin S A , Whetton A D
Published: June 2004

In the absence of a survival stimulus, the interleukin 3 (IL-3)-dependent IC.DP cell line undergoes a process termed programmed cell death or apoptosis. Survival can be induced by IL-3, which can also stimulate proliferation of IC.DP cells. IC.DP cells have been stably transfected with the p160v-abl protein tyrosine kinase, activation of the kinase at the permissive temperature permits cell survival in the absence of IL-3 by suppression of apoptosis, although the growth factor is still required for proliferation. Both IL-3 and activation of the v-ABL tyrosine kinase stimulated glucose transport, which may in part be due to a translocation of transporters to the cell surface. Inhibition of glucose uptake markedly increased the rate of apoptosis in these cells, an effect that could be reversed by the provision of alternative energy sources such as glutamine. Growth factor- or oncogene-mediated increases in glucose uptake may therefore represent an important regulatory point in the suppression of apoptosis.
Cytotoxic T lymphocyte response to a wild type hepatitis B virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope.
Authors: Bertoletti A , Costanzo A , Chisari F V , Levrero M , Artini M , Sette A , Penna A , Giuberti T , Fiaccadori F , Ferrari C
Published: June 2004

Mutations that abrogate recognition of a viral epitope by class I-restricted cytotoxic T lymphocyte (CTL) can lead to viral escape if the CTL response against that epitope is crucial for viral clearance. The likelihood of this type of event is low when the CTL response is simultaneously directed against multiple viral epitopes, as has been recently reported for patients with acute self-limited hepatitis B virus (HBV) infection. The CTL response to HBV is usually quite weak, however, during chronic HBV infection, and it is generally acknowledged that this is a major determinant of viral persistence in this disease. If such individuals were to produce a mono- or oligospecific CTL response, however, negative selection of the corresponding mutant viruses might occur. We have recently studied two HLA-A2-positive patients with chronic hepatitis B who, atypically, developed a strong HLA-A2-restricted CTL response against an epitope (FLPSDFFPSV) that contains an HLA-A2-binding motif located between residues 18-27 of the viral nucleocapsid protein, hepatitis B core antigen (HBcAg). These patients failed, however, to respond to any of other HLA-A2-restricted HBV-derived peptides that are generally immunogenic in acutely infected patients who successfully clear the virus. Interestingly, DNA sequence analysis of HBV isolates from these two patients demonstrated alternative residues at position 27 (V --> A and V --> I) and position 21 (S --> N, S --> A, and S --> V) that reduced the HLA and T cell receptor-binding capacities of the variant sequences, respectively. Synthetic peptides containing these alternative sequences were poorly immunogenic compared to the prototype HBc18-27 sequence, and they could not be recognized by CTL clones specific for the prototype peptide. While we do not know if the two patients were originally infected by these variant viruses or if the variants emerged subsequent to infection because of immune selection, the results are most consistent with the latter hypothesis. If this is correct, the data suggest that negative selection of mutant viral genomes might contribute to viral persistence in a subset of patients with chronic HBV infection who express a narrow repertoire of anti-HBV CTL responses.
Isolation of a nitric oxide inhibitor from mammary tumor cells and its characterization as phosphatidyl serine.
Authors: Calderón C , Huang Z H , Gage D A , Sotomayor E M , Lopez D M
Published: June 2004

Macrophages from mice bearing large D1-DMBA-3 mammary tumors have a decreased capacity to kill tumor targets. This effect is due to an impaired ability to produce nitric oxide (NO) in response to lipopolysaccharide (LPS) stimulation. Here we report that the DA-3 tumor cell line, derived from the in vivo adenocarcinoma D1-DMBA-3, produces a factor that inhibits both NO production/release and cytotoxicity of LPS-activated peritoneal exudate macrophages (PEM). However, other complex macrophage functions such as phagocytosis, superoxide production, mitochondrial dehydrogenase activity, and synthesis of proteins were not reduced by this factor. The NO inhibitor has been found to be lipid in nature. Lipid extracts from DA-3 cell culture supernatants were purified by repeated silica gel column chromatography. The active molecule was unambiguously characterized as phosphatidyl serine (PS) by fast atom bombardment tandem mass spectrometry. Preliminary results indicate a lack of induced NO synthase (iNOS) activity in the lysates of LPS-activated PEM pretreated with PS. The ubiquity of PS in the inner leaflet of biological membranes and its NO inhibitory property, suggest that this phospholipid may be one of the long elusive molecules responsible for regulating physiological levels of NO in the host and hence preventing cellular dysfunction and/or tissue damage. Furthermore, the possible overexpression and shedding of PS by DA-3 tumor cells may represent a novel mechanism to impair macrophage cytotoxicity, a host function that contributes to the protection against developing neoplasms.
Reduced thymic maturation but normal effector function of CD8+ T cells in CD8 beta gene-targeted mice.
Authors: Fung-Leung W P , Kündig T M , Ngo K , Panakos J , De Sousa-Hitzler J , Wang E , Ohashi P S , Mak T W , Lau C Y
Published: June 2004

CD8 is a cell surface glycoprotein on major histocompatibility complex class I-restricted T cells. Thymocytes and most peripheral T cells express CD8 as heterodimers of CD8 alpha and CD8 beta. The intestinal intraepithelial lymphocytes (IEL), which have been suggested to be generated extrathymically, express CD8 predominantly as homodimers of CD8 alpha. We have generated CD8 beta gene-targeted mice. CD8 alpha+ T cell population in the thymus and in most peripheral lymphoid organs was reduced to 20-30% of that in wild-type littermates. CD8 alpha expression on thymocytes and peripheral T cells also decreased to 44 and 53% of the normal levels, respectively. In contrast, neither the population size nor the CD8 alpha expression level of CD8 alpha+ IEL was reduced. This finding indicates that CD8 beta is important only for thymic-derived CD8+ T cells. The lack of CD8 beta reduces but does not completely abolish thymic maturation of CD8+ T cells. Our result also reveals the role of CD8 beta in regulating CD8 alpha expression on thymic derived T cells. Peripheral T cells in these mice were efficient in cytotoxic activity against lymphocytic choriomeningitis virus and vesicular stomatitis virus, suggesting that CD8 beta is not essential for the effector function of CD8+ T cells.
Iron regulates nitric oxide synthase activity by controlling nuclear transcription.
Authors: Weiss G , Werner-Felmayer G , Werner E R , Grünewald K , Wachter H , Hentze M W
Published: June 2004

Recently, it was reported that nitric oxide (NO) directly controls intracellular iron metabolism by activating iron regulatory protein (IRP), a cytoplasmic protein that regulates ferritin translation. To determine whether intracellular iron levels themselves affect NO synthase (NOS), we studied the effect of iron on cytokine-inducible NOS activity and mRNA expression in the murine macrophage cell line J774A.1. We show here that NOS activity is decreased by about 50% in homogenates obtained from cells treated with interferon gamma plus lipopolysaccharide (IFN-gamma/LPS) in the presence of 50 microM ferric iron [Fe(3+)] as compared with extracts from cells treated with IFN-gamma/LPS alone. Conversely, addition of the iron chelator desferrioxamine (100 microM) at the time of stimulation with IFN-gamma/LPS increases NOS activity up to 2.5-fold in J774 cells. These effects of changing the cellular iron state cannot be attributed to a general alteration of the IFN-gamma/LPS signal, since IFN-gamma/LPS-mediated major histocompatibility complex class II antigen expression is unaffected. Furthermore, neither was the intracellular availability of the NOS cofactor tetrahydrobiopterin altered by treatment with Fe(3+) or desferrioxamine, nor do these compounds interfere with the activity of the hemoprotein NOS in vitro. We demonstrate that the mRNA levels for NOS are profoundly increased by treatment with desferrioxamine and reduced by Fe(3+). The half-life of NOS mRNA appeared not to be significantly altered by administration of ferric ion, and NOS mRNA stability was only slightly prolonged by desferrioxamine treatment. Nuclear run-off experiments demonstrate that nuclear transcription of cytokine-inducible NOS mRNA is strongly increased by desferrioxamine whereas it is decreased by Fe(3+). Thus, this transcriptional response appears to account quantitatively for the changes in enzyme activity. Our results suggest the existence of a regulatory loop between iron metabolism and the NO/NOS pathway.
Role of interferon regulatory factor 1 in induction of nitric oxide synthase.
Authors: Martin E , Nathan C , Xie Q W
Published: June 2004

Interferon gamma (IFN-gamma) interacts synergistically with bacterial lipopolysaccharide (LPS) to induce transcription of iNOS, the isoform of nitric oxide synthase whose activity is independent of elevated Ca2+ and exogenous calmodulin. To define a cis-acting element mediating IFN-gamma-dependent synergy, we made deletions in iNOS promoter constructs fused to reporter genes, transfected RAW 264.7 macrophages, and treated the cells with IFN-gamma and/or LPS. This analysis implicated the region from positions -951 to -911, a cluster of four enhancer elements known to bind IFN-gamma-responsive transcription factors, including an interferon regulatory factor binding site (IRF-E) at nucleotides -913 to -923. Site-specific substitution of two conserved nucleotides within IRF-E in the context of the full-length iNOS promoter ablated IFN-gamma's contribution to synergistic enhancement of transcription. Electromobility shift assays performed with a probe containing IRF-E revealed the existence of a complex in nuclei of RAW 264.7 macrophages that was present only after treatment with IFN-gamma, which reacted specifically with anti-IRF-1 immunoglobulin G and which included a species migrating at 40-45 kD, consistent with the apparent molecular weight of murine IRF-1. Thus, the synergistic contribution of IFN-gamma to transcription of iNOS in RAW 264.7 macrophages requires that IRF-1 bind to IRF-E in the iNOS promoter. In conjunction with the work of Kamijo et al. (Kamijo, R., H. Harada, T. Matsuyama, M. Bosland, J. Gerecitano, D. Shapiro, J. Le, K. S. Im, T. Kimura, S. Green et al. 1994. Science [Wash. DC]. 263:1612), these findings identify iNOS as the first gene that requires IRF-1 for IFN-gamma-dependent transcriptional regulation.
Th2 cell clonal anergy as a consequence of partial activation.
Authors: Sloan-Lancaster J , Evavold B D , Allen P M
Published: June 2004

We have demonstrated Th2 clonal anergy as a consequence of partial T cell activation by immunogenic peptide and chemically fixed APC, as well as by altered peptide ligand and live antigen-presenting cells (APC). Either stimulation resulted in a profound inability of the T cells to proliferate upon restimulation with antigen and functional APC, a similar phenomenon to that found with Th1 cells. The anergic state was long lasting and was restricted to proliferation, since the T cells retained the ability to produce cytokines upon restimulation, albeit at slightly reduced levels. Th2 anergy induction was inhibited by cyclosporine A, but not by provision of exogenous costimulation or growth factors. The data presented unify Th1 and Th2 cells with regard to anergy and suggest that the fundamental control during anergy for both subsets is prevention of clonal expansion, thus blocking amplification of the immune response.
Neonatal deletion and selective expansion of mouse T cells by exposure to rabies virus nucleocapsid superantigen.
Authors: Lafon M , Scott-Algara D , Marche P N , Cazenave P A , Jouvin-Marche E
Published: June 2004

The nucleocapsid (NC) of the rabies virus behaves as an exogenous superantigen (SAg) in humans. In the present report, we analyzed whether it is also a SAg in mice by studying the effect of NC on T cell receptor (TCR) V beta expression in BALB/c mice. Repeated injection of NC in newborn BALB/c mice led to a marked reduction by two- to sixfold of V beta 6 expressing CD4+ T cells in spleen and in peripheral blood. Decrease of V beta 6-expressing CD3+ mature T cells was also observed in thymus. Single NC injection in footpad resulted in a three- to sixfold expansion of V beta 6 CD4+ T cells, but not of CD8+ T cells, in the draining lymph nodes of BALB/c mice. The intensity of the stimulation was dose dependent and was maximal 3 d after the NC injection. The clonal deletion of T cells bearing a particular V beta demonstrates that NC is a SAg in mice. T cells, especially CD4+ T cells, are an essential factor in host resistance to rabies virus and also in the pathophysiology of paralysis; thus, we postulate that a rabies virus component, which stimulates T cells, such as a SAg, may increase virus immunopathogenicity. To evaluate this hypothesis, we compared the course of rabies in adult BALB/c lacking V beta 6, 7, 8.1, and 9 T cells and in normal BALB/c. Immune-related paralysis was decreased in BALB/c missing the NC target V beta T cells. Transfer of V beta 6 but not of V beta 8.1-3 T cells into recipient mice lacking V beta 6, 7, 8.1, and 9 allowed the immune-related paralysis to evolve. Taken together, these results strongly support the hypothesis that T cells expressing rabies SAg-specific V beta 6 T cells, are involved in the genesis of the immunopathology that is characteristic of paralytic rabies.
Activation of human dendritic cells through CD40 cross-linking.
Authors: Caux C , Massacrier C , Vanbervliet B , Dubois B , Van Kooten C , Durand I , Banchereau J
Published: June 2004

Dendritic cells, the professional antigen-presenting cells (APC) involved in T cell priming, express CD40, a molecule which triggering plays a key role in B cell growth and differentiation as well as monocyte activation. Herein we demonstrate that dendritic Langerhans cells (D-Lc) generated by culturing cord blood CD34+ progenitor cells with granulocyte/macrophage colony-stimulating and tumor necrosis factor alpha (TNF-alpha) express functional CD40 at a density higher than that found on B cells. Culturing D-Lc on CD40-ligand (CD40L) transfected L cells allowed D-Lc survival as 50 +/- 15% of seeded cells were recovered after 4 d while only 5% survived over control L cells. CD40 activation induced important morphological changes with a reduction of cytoplasmic content and a remarkable increase of dendrite development as well as an altered phenotype. In particular, CD40 triggering induced maintenance of high levels of major histocompatibility complex class II antigens and upregulation of accessory molecules such as CD58, CD80 (B7-1) and CD86 (B7-2). CD40 engagement also seems to turn on D-Lc maturation as illustrated by upregulation of CD25, a molecule usually expressed on interdigitating dendritic cells of secondary lymphoid organs. Finally, CD40 activated D-Lc secreted a limited set of cytokines (TNF-alpha, IL-8, and macrophage inflammatory protein 1 alpha [MIP-1 alpha]) whereas a similar activation induced elutriated monocytes to secrete IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, TNF-alpha, and MIP-1 alpha. As D-Lc activated T cells upregulated CD40L, it is likely that CD40 activation of D-Lc observed herein with a fibroblast cell line stably expressing CD40L, mimics physiological interactions between dendritic cells and T cells.
Naturally processed viral peptides recognized by cytotoxic T lymphocytes on cells chronically infected by human immunodeficiency virus type 1.
Authors: Tsomides T J , Aldovini A , Johnson R P , Walker B D , Young R A , Eisen H N
Published: June 2004

We have established long-term cultures of several cell lines stably and uniformly expressing human immunodeficiency virus type 1 (HIV-1) in order to (a) identify naturally processed HIV-1 peptides recognized by cytotoxic T lymphocytes (CTL) from HIV-1-seropositive individuals and (b) consider the hypothesis that naturally occurring epitope densities on HIV-infected cells may limit their lysis by CTL. Each of two A2-restricted CD8+ CTL specific for HIV-1 gag or reverse transcriptase (RT) recognized a single naturally processed HIV-1 peptide in trifluoroacetic acid (TFA) extracts of infected cells: gag 77-85 (SLYNTVATL) or RT 476-484 (ILKEPVHGV). Both processed peptides match the synthetic peptides that are optimally active in cytotoxicity assays and have the consensus motif described for A2-associated peptides. Their abundances were approximately 400 and approximately 12 molecules per infected Jurkat-A2 cell, respectively. Other synthetic HIV-1 peptides active at subnanomolar concentrations were not present in infected cells. Except for the antigen processing mutant line T2, HIV-infected HLA-A2+ cell lines were specifically lysed by both A2-restricted CTL, although infected Jurkat-A2 cells were lysed more poorly by RT-specific CTL than by gag-specific CTL, suggesting that low cell surface density of a natural peptide may limit the effectiveness of some HIV-specific CTL despite their vigorous activity against synthetic peptide-treated target cells.
Class II transactivator (CIITA) is sufficient for the inducible expression of major histocompatibility complex class II genes.
Authors: Chang C H , Fontes J D , Peterlin M , Flavell R A
Published: June 2004

The class II transactivator (CIITA) has been shown to be required for major histocompatibility complex (MHC) class II gene expression in B cells and its deficiency is responsible for a hereditary MHC class II deficiency. Here we show that CIITA is also involved in the inducible expression of class II genes upon interferon gamma (IFN-gamma) treatment. The expression of CIITA is also inducible with IFN-gamma before the induction of MHC class II mRNA. In addition, CIITA mRNA expression does not require new protein synthesis, although new protein synthesis is necessary for the transcription of class II. This suggests that synthesis of new CIITA protein may be essential to induce class II gene expression. We also showed that the JAK1 protein tyrosine kinase activity is required to induce the expression of CIITA upon IFN-gamma stimulation. This finding indicates that CIITA is part of the signaling cascade from the IFN-gamma receptor to the activation of class II genes. In addition, the expression of CIITA is sufficient to activate class II genes in the absence of IFN-gamma stimulation suggesting that CIITA is the major regulatory factor for the inducible expression of class II genes. Together, these data suggest that CIITA is the IFN-inducible cycloheximide sensitive factor previously shown to be required for the induction of MHC class II gene expression.
Determinant selection of major histocompatibility complex class I-restricted antigenic peptides is explained by class I-peptide affinity and is strongly influenced by nondominant anchor residues.
Authors: Chen W , Khilko S , Fecondo J , Margulies D H , McCluskey J
Published: June 2004

The contribution of major histocompatibility complex (MHC) class I-peptide affinity to immunodominance of particular peptide antigens (Ags) in the class I-restricted cytotoxic T lymphocyte (CTL) response is not clearly established. Therefore, we have compared the H-2Kb-restricted binding and presentation of the immunodominant ovalbumin (OVA)257-264 (SIINFEKL) determinant to that of a subdominant OVA determinant OVA55-62 (KVVRFDKL). Immunodominance of OVA257-264 was not attributable to the specific T cell repertoire but correlated instead with more efficient Ag presentation. This enhanced Ag presentation could be accounted for by the higher affinity of Kb/OVA257-264 compared with Kb/OVA55-62 despite the presence of a conserved Kb-binding motif in both peptides. Kinetic binding studies using purified soluble H-2Kb molecules (Kbs) and biosensor techniques indicated that the Kon for association of OVA257-264-C6 and Kbs at 25 degrees C was integral of 10-fold faster (5.9 x 10(3) M-1 s-1 versus 6.5 x 10(2) M-1 s-1), and the Koff approximately twofold slower (9.1 x 10(-6) s-1 versus 1.6 x 10(-5) s-1), than the rate constants for interaction of OVA55-62-C6 and Kbs. The association of these peptides with Kb was significantly influenced by multiple residues at presumed nonanchor sites within the peptide sequence. The contribution of each peptide residue to Kb-binding was dependent upon the sequence context and the summed contributions were not additive. Thus the affinity of MHC class I-peptide binding is a critical factor controlling presentation of peptide Ag and immunodominance in the class I-restricted CTL response.
Positive and negative regulation of the composite octamer motif of the interleukin 2 enhancer by AP-1, Oct-2, and retinoic acid receptor.
Authors: de Grazia U , Felli M P , Vacca A , Farina A R , Maroder M , Cappabianca L , Meco D , Farina M , Screpanti I , Frati L , Gulino A
Published: June 2004

The differentiating agent retinoic acid (RA) has been previously reported to interfere with 12-O-tetradecanoyl-phorbol-13-acetate (TPA)/Ca2+-induced signals for the regulation of the -96 to -66-bp octamer motif found in the enhancer for the interleukin (IL)-2 gene, which encodes a major T lymphocyte growth factor. The IL-2 octamer motif is a composite cis-element which binds Oct-1 and Oct-2 as well as a TPA/Ca2+-inducible nuclear factor, previously termed octamer-associated protein (OAP40). We show here that Oct-2, despite the presence of an active transcriptional activation domain, requires TPA/Ca2+-induced signals to strongly transactivate the IL-2 octamer motif in Jurkat T cells. This Oct-2-dependent transactivation is inhibited by RA. The presence of an intact COOH-terminal domain of Oct-2 contributes to both TPA/Ca2+-induced transactivation and the RA-mediated repression. We also show that both Fos and Jun components of the AP-1 factors participate in the OAP40 complex. Furthermore, transfected c-jun, jun-B, jun-D, c-fos, or Fos-B expression vectors partially substitute for TPA and Ca2+ and cooperate with Oct-2 for the transactivation of the combined OAP/octamer cis-element. Mutations of the genuine octamer-binding site abrogate both the binding of Oct-1 and Oct-2 and the TPA/Ca2+-induced transactivation of the OAP/octamer motif. OAP confers to Oct-2 responsivity to both TPA/Ca2+ and RA, since specific mutations of the AP-1/OAP-binding site significantly reduce the transactivation by Oct-2 in response to TPA and Ca2+ and abolish the inhibition by RA. Furthermore, retinoic acid receptor (RAR) alpha is able to inhibit in vitro the formation of the complex between the nuclear AP-1/OAP and its specific binding site, resulting in the interference with Oct-2-dependent cis-regulatory function of this AP-1 element. Therefore, we propose that the TPA/calcium-activated AP-1/OAP element is the main target of positive or negative regulatory signals influencing the IL-2 octamer motif, through synergism with Oct-2 and antagonism by RAR.