bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Vγ9‐Vδ2 cells are stimulated in a crossreactive fashion by a variety of phosphorylated metabolites
Authors: Bürk Martin R. , Mori Lucia , de Libero Gennaro
Published: March 2007

Many different pathogens stimulate cells bearing the Vγ9‐Vδ2 T cell receptor (TCR), which represent the most abundant population of human γδ cells. The antigens responsible for the stimulation of these γδ cells are not well characterized. Here, we describe six non‐peptidic molecules which share this property: isopentenylpyrophosphate, dimethylallylpyrophosphate, 2,3‐diphosphoglyceric acid, glycerol‐3‐phosphoric acid, xylose‐1‐phosphate, and ribose‐1‐phosphate. All these molecules are naturally occurring metabolites in prokaryotic and eukaryotic cells, and stimulate freshly isolated γδ cells from peripheral blood of different donors as well as established γδ clones. Comparison of their structure with that of similar but inactive molecules showed that both the number and position of the phosphate groups, as well as the residues connected with the carbon backbone are required for stimulation. The CD3‐TCR complex is involved in cell triggering as shown by inhibition with anti‐CD3 Fab fragments. However, all γδ clones were broadly cross‐reactive and we could not isolate cells specific for only one ligand. The capacity of this frequent subset of γδ cells to recognize common bacterial metabolites confers the advantage to react rapidly to different invading pathogens.
Functional analysis of avian class I (BFIV) glycoproteins by epitope tagging and mutagenesis in vitro
Authors: Fulton Janet Elizabeth , Thacker Eileen Lenore , Bacon Larry Dean , Hunt Henry Donald
Published: March 2007

Similarities between the physical structures of avian and mammalian major histocompatibility complex (MHC) class I glycoproteins have been proposed based on comparative alignment of their amino acid sequences. To investigate the physical structure of the chicken class I glycoprotein, we cloned the cDNA representing the BFIV locus of the B21 haplotype. A unique, chimeric class I glycoprotein was constructed by incorporating an epitope tag (FLAG) at the N terminus. Monoclonal antibodies to the FLAG epitope served to monitor cellsurface expression for functional analysis of the BFIV21 class I glycoprotein. The chimeric class I glycoprotein was expressed in target cells using an avian leukosis virus (ALV)‐derived retrovirus vector (RCASBP). The presence of the FLAG epitope did not interfere with either alloantibody recognition or cytotoxic T lymphocyte interaction. Functional analysis employing site‐directed mutagenesis identified BF amino acid residues forming serologic epitopes as well as residues important in antigen presentation to ALV‐induced cytotoxic T lymphocytes. BF residues 78 and 81, corresponding to HLA 79 and 82, form an antibody epitope with a slight effect on ALV antigen presentation, consistent with their predicted orientation based on the HLA‐A2 crystal structure. Alignment of the BFIV21 sequence with previously published BFIV sequences revealed polymorphisms at position 34 (HLA 34), a monomorphic residue in HLA and H‐2. Residue 34 is located in pocket B and is predicted to contact the main‐chain carbon of peptides bound in HLA‐A2. A site‐directed substitution in BFIV residue 34 dramatically alters ALV antigen presentation by the BFIV21 class I glycoprotein. These data indicate that the physical molecular structure of the chicken MHC class I glycoprotein is similar to HLA.
The peripheral T cell repertoire: independent homeostatic regulation of virgin and activated CD8+ T cell pools
Authors: Tanchot Corinne , Rocha Benedita
Published: March 2007

Mature T cells may be produced in the thymus, or by expansion in the periphery. While thymus output of virgin cells ensures repertoire diversity, peripheral expansion increases the size of rare clones, and thus the efficiency of immune responses. We studied the role of both phenomena in the generation of the CD8+ T cell pool using RAG−/− female mice expressing a transgenic T cell receptor specific for the male antigen; nude mice injected with peripheral T cells; and euthymic irradiated chimeras injected with bone marrow and mature T cells. Our results show that the total number of virgin and activated T cells, each constituting about half of the peripheral T cell pool, was regulated by homeostatic mechanisms controlling pool size. The size of each pool was regulated independently, revealing an efficient mechanism to maintain repertoire diversity while optimizing the immune response.
Expression of the early lymphocyte activation antigen CD69, a C‐type lectin, is regulated by mRNA degradation associated with AU‐rich sequence motifs
Authors: Santis Ana G. , López‐Cabrera Manuel , Sánchez‐Madrid Francisco , Proudfoot Nick
Published: March 2007

CD69 is the earliest inducible cell surface glycoprotein acquired during lymphoid activation both in vitro and in vivo under physiological conditions and inflammation. This molecule is involved in lymphocyte proliferation, and functions as a signal‐transmitting receptor in T and B lymphocytes, NK cells and platelets. Molecular cloning of CD69 cDNA revealed that this antigen is a type‐II integral membrane protein with a C‐type lectin domain in the extracellular region. The expression time course of CD69 mRNA has previously been reported to be transient, peaking around 3 h after induction in T lymphocytes, and declining to nearly resting levels by 8 h. We describe herein studies on the stability of the CD69 mRNA in phorbol ester‐activated T lymphocytes. The level of CD69 mRNA in these cells declined rapidly with a half‐life of less than 60 min. This finding is consistent with the presence of several AU‐rich sequence motifs in the 3′ untranslated region (3′UTR), which have been implicated in the selective destabilization of short‐lived mRNA of mammalian cytokines, and proto‐oncogenes. We have therefore introduced a fragment of the 3′UTR of the human CD69 cDNA, which contains the AU‐rich sequence motifs, into the 3′UTR of the rabbit β‐globin gene. This inserted sequence causes the otherwise stable β‐globin mRNA to become unstable in vivo. A similar destabilizing effect is observed when the 51‐nucleotide AU sequence from the mRNA of the human cytokine granulocyte/macrophage colony‐stimulating factor is used as a positive control. Furthermore, the introduction of 194‐bp fragment from the CD69 3′UTR containing most of the AU‐rich motifs was sufficient to induce the destabilizing effect. We propose that the selective degradation pathway involved in the regulation of the expression of cytokines and proto‐oncogenes is implicated in the rapid degradation of CD69 mRNA in activated T lymphocytes. This pathway may constitute a general mechanism to regulate the expression of inducible molecules involved in inflammatory processes.
Subtle differences in antibody responses and hypermutation of λ light chains in mice with a disrupted χ constant region
Authors: Zou Xiangang , Xian Jian , Popov Andrei V. , Rosewell Ian R. , Müller Marion , Brüggemann Marianne
Published: March 2007

Analysis of λ light chain use in normal mice is made difficult by the dominant x light chain repertoire. We produced mice rendered deficient in x light chain expression by gene targeting and focused on questions concerned with the generation of λ light chain diversity. Whilst these mice compensate the x deficiency with increased λ titers, and their Ig level is therefore not significantly reduced, they show major differences in immunization titers, germinal center (GC) development and somatic hypermutation. After immunization, using antigens that elicit a restricted IgL response in normal mice, we obtained in the x−/− mice elevated primary antibody titers but a subsequent lack in titer increase after repeated antigen challenge. Analysis of the Peyer's patches (PP) revealed a dramatically reduced cell content with rather small but highly active GC. Flow cytometric analysis showed different cell populations in the PP with enriched peanut agglutinin (PNA)hi/CD45R(B220)+ B cells, implying that the apparent compensation for the lack of x light chain expression involves the GC microenvironment in cell selection, the initiation of hypermutation and high affinity expansion. The three Vλ genes, V1, V2 and Vx, are mutated in the GC B cells, but show no junctional diversity. In contrast, a reduced rate of Vλ hypermutation is found in the hybridoma antibodies, which appears to reflect a selection bias rather than structural constraints. However, mechanisms of somatic mutation and specificity selection can operate with equal efficiency on the few Vλ genes.
Characterization of mouse CD53: Epitope mapping, cellular distribution and induction by T cell receptor engagement during repertoire selection
Authors: Tomlinson Michael G. , Hanke Thomas , Hughes Derralyn A. , Barclay A. Neil , Scholl Eva , Hünig Thomas , Wright Mark D.
Published: March 2007

The pan‐leukocyte antigen CD53 is a member of the poorly understood transmembrane 4 superfamily (TM4SF) of cell membrane glycoproteins. CD53 is proposed to play a role in thymopoiesis, since rat CD53 is expressed on immature CD4−8−thymocytes and the functionally mature single‐positive subset, but is largely absent from the intermediate CD4+8+ cells. We have characterized CD53 in the mouse through the production of two new monoclonal antibodies, MRC OX‐79 and OX‐80, which were raised against the RAW 264 cell line and screened on recombinant CD53 fusion proteins. The epitopes recognized by both antibodies are dependent on disulfide bonding and map to the major extracellular region of CD53, requiring the presence of a single threonine residue at position 154. Mouse CD53 has a molecular mass of 35‐45 kDa and is expressed on virtually all peripheral leukocytes, but not on cells outside the lymphoid or myeloid lineages. CD53 expression distinguishes subpopulations of thymocytes in the mouse and resembles the expression pattern of rat CD53. Amongst the immature CD4−8− thymocytes, mouse CD53 is clearly detectable on the earliest CD44high25− subset, but down‐regulated on the later CD44high25+, CD44low25+ and CD44low25− stages. Also, the subsequent transient TcR−/low CD4−8+ cells and most CD4+8+ thymocytes express little or no CD53. This is consistent with the idea that cells which are committed to enter the selectable CD4+8+ compartment switch off CD53. The effect of T cell receptor (TcR) engagement on the reexpression of CD53 on CD4+8+ thymocytes was studied both ex vivo and in vitro using F5 mice, transgenic for the H‐2b/influenza nucleoprotein‐peptide‐specific TcR, back‐crossed onto an H‐2q or H‐2b background of RAG‐2‐deficient mice. CD4+8+ thymocytes from non‐selecting H‐2q F5 mice are CD53 negative, but in vitro stimulation through the TcR dramatically induces CD53 expression. In contrast, a fraction of CD4+8+ thymocytes from positively selecting H‐2b F5 transgenic mice express CD53. Therefore TcR engagement by selecting major histo‐compatibility complex peptide complexes, or surrogate ligands, induces CD53 expression on otherwise CD53‐negative, non‐selected CD4+8+ thymocytes. Whether CD53 itself participates as a signaling molecule in further stages of thymic selection is still a matter of speculation.
Regulation of the function of the first component of complement by human C1q receptor
Authors: van den Berg Rocco H. , Faber‐Krol Maria , van Es Leendert A. , Daha Mohamed R.
Published: March 2007

A membrane‐associated receptor for the C1q subcomponent of complement is widely distributed among different cell types. While a number of possible physiological functions of the C1q receptor (C1qR) on different cell types have been described, the way in which C1qR regulates complement activity remains unclear. This report describes the mechanism by which C1qR regulates activation of the first component of complement, C1. Using purified components of complement, we were able to show that membrane‐associated C1qR as well as detergent‐solubilized C1qR, purified from polymorphonuclear leukocytes, human umbilical vein endothelial cells or an endothelial cell line, EA.hy 926, are able to inhibit complement‐mediated lysis of C1q‐sensitized erythrocytes. Using hemolytic assays, we were able to demonstrate that C1qR prevents the association of C1q with C1r and C1s to form macromolecular C1. In addition, incubation of C1qR with the collagen‐like stalks, but not with the globular heads of C1q, inhibits the effect of C1qR. This demonstrates that C1qR exerts its complement inhibitory effect by binding to the collagen‐like stalk of C1q. No complement regulatory effect of C1qR was observed on preformed macromolecular C1. These data suggest that besides such well‐known complement regulatory molecules as CD55 (DAF), CD46 (MCP), CD35 (CR1) and CD59 (HRF), C1qR too is able to regulate complement activity.
Compounds that induce autoimmunity in the Brown Norway rat sensitize mast cells for mediator release and interleukin‐4 expression
Authors: Oliveira David B. G. , Gillespie Kathleen , Wolfreys Karen , Mathieson Peter W. , Qasim Faieza , Coleman John W.
Published: March 2007

Brown Norway (BN) rats given mercuric chloride (HgCl2), gold (Au) salts or D‐penicillamine develop a T helper 2 (Th2) cell‐mediated autoimmune syndrome. The recent observation of tissue injury within 24 h of HgCl2 treatment suggested the involvement of a non‐T cell. We therefore examined the effect of these compounds on rat mast cells in vitro. Incubation of BN rat peritoneal mast cells with HgCl2 enhanced the release of serotonin in response to IgE cross‐linking agents. Mast cells from Lewis rats, a strain not susceptible to the autoimmune syndrome in vivo, were affected to a lesser extent. The effect was observed with purified BN mast cells, suggesting a direct action. Similar effects were seen with D‐penicillamine in the presence of copper ions, a combination that produces hydrogen peroxide, and Au. HgCl2 caused significant induction of interleukin (IL)‐4 mRNA in mast cells from BN, but not Lewis rats. The data demonstrate a novel enhancing effect of a number of compounds on mast cell mediator release, and an inducing effect of HgCl2 on mast cell IL‐4 expression. These findings are consistent with our hypotheses that mast cells may contribute to early tissue injury, and also, via production of IL‐4, may initiate and/or augment, the Th2 response in the BN rat model of chemical‐induced autoimmunity.
The effect of carrier and carrier priming on the kinetics and pattern of somatic mutation in the VϰOx1 gene
Authors: Källberg Eva , Gray David , Leanderson Tomas
Published: March 2007

Priming mice with a chicken gamma globulin (CGG) carrier protein significantly accelerated the onset of somatic mutation in the VϰOx1 gene when the mice were subsequently immunized with 2‐phenyl‐5‐oxazolone (phOx) coupled to CGG. The first mutations were already detected 7 days after immunization, while in the true primary response, they are not apparent until day 10. It was also found that comparing the mutation pattern of VϰOx1 genes from hybridomas derived after immunization with phOx coupled to different carriers revealed quite distinct patterns of somatic mutation. Analysis of hybridoma sequences from the primary immune response to phOx‐ovalbumin showed that the codons for Ser29, Ser31 and Lys45 were hot‐spots for somatic mutation. Thus, the frequency and pattern of somatic mutations in the VϰOx1 gene depends on the available T cell help as well as on the complex structure of the immunizing antigen.
Modulation of peptide binding by HLA‐B27 polymorphism in pockets A and B, and peptide specificity of B*2703
Authors: Villadangos José A. , Galocha Begoña , García Fernando , Albar Juan P. , de Castro José A. López
Published: March 2007

The results in this study address three aspects of peptide binding to the disease‐associated antigen HLA‐B27 and its modulation by polymorphism: the contribution of major anchor residues 2 and 9, the role of pocket B polymorphism in modulating peptide specificity, and the binding properties of B*2703, a subtype not found to be associated with spondyloarthropathy. Synthetic analogs of peptides naturally presented by B*2705 were used to demonstrate that residue 2 is essential, since Ala2 analogs bound marginally to B*2705, but the specificity of B*2705 for Arg2 is not absolute, and show that the contribution of basic residue 9 to binding was significant, but less than Arg2. The effect of single mutations in the B pocket was to decrease or ‐ with the Glu > Met‐45 mutation ‐ totally shift pocket B specificity for Arg2 towards other residues at this position. This was shown by quantitating the relative binding of Gln2 and Ala2 analogs, and by pool‐sequencing of the peptides bound in vivo to these mutants. Peptides naturally presented by B*2705 apparently bound with a lower affinity to pocket A variants with altered hydrogen bonding to the peptide N terminus, including B*2703. Binding of peptide analogs with changes at positions 2 or 9 suggested that in B*2703 pocket A, interactions are weaker and pocket B interactions are stronger than in B*2705. This can be explained by the effect of the unique His59 change in B*2703 in both pockets. Thus, B*2703 is probably the HLA‐B27 subtype with the most stringent specificity for the Arg2 peptide motif.
Soluble interleukin‐6 receptor strongly increases the production of acute‐phase protein by hepatoma cells but exerts minimal changes on human primary hepatocytes
Authors: Gabay Cem , Silacci Paolo , Genin Bernard , Mentha Gilles , le Coultre Claude , Guerne Pierre‐André
Published: March 2007

Interleukin‐6 (IL‐6) interacts with a system of receptors, which include a 80‐kDa IL‐6‐binding subunit (IL‐6R) and a transducing element (gp130). The soluble form of IL‐6R (sIL‐6R) can bind its ligand and induce cellular responses by association with gp130, thus acting as an IL‐6 agonist. We and others have previously shown that the responsiveness to IL‐6 is different in hepatoma and human primary hepatocytes. We therefore compared the effects of sIL‐6R on the two types of cells, and on the B9 hybridoma, another IL‐6‐sensitive cell line. Human primary hepatocytes, hepatoma cells PLC/PRF/5, and B9 cells were incubated with different concentrations of IL‐6, sIL‐6‐R, or both. The hepatocyte culture supernatants were tested for their content of acute‐phase proteins (APP). The proliferation of B9 cells was assessed by a colorimetric method. Results showed that sIL‐6R alone markedly increases the production of APP by hepatoma cells in a dose‐dependent manner, but affects only minimally primary hepatocytes and the proliferation of B9 cells. The combinations of IL‐6R and its ligand enhanced the effects of IL‐6 alone in both PLC/PRF/5 and B9 cells, but had no effect on primary hepatocytes. An immunohistochemical study indicated that the cell‐surface expression of IL‐6R was dramatically lower in hepatoma cells than in primary hepatocytes. In conclusion, our results show that the expression of IL‐6R is low in the hepatoma cell PLC/PRF/5 when compared with primary hepatocytes and that this difference can, at least partly, explain their deficient responsiveness to IL‐6. On the other hand, it appears that IL‐6R expression by primary hepatocytes is sufficient and that circulating sIL‐6R is unlikely to play a significant role in the modulation of IL‐6 effects.
Systemic immunization induces protective CD4+ and CD8+ T cell‐mediated immune responses in murine Listeria monocytogenes meningoencephalitis
Authors: Schlüter Dirk , Oprisiu Simona B. , Chahoud Soraya , Weiner Daniela , Wiestler Otmar D. , Hof Herbert , Deckert‐Schlüter Martina
Published: March 2007

The immune mechanisms underlying immunization‐induced protection of mice from lethal central nervous system (CNS) listeriosis were evaluated by immuno‐histochemistry, flow cytometry of leukocytes isolated from the brain, reverse transcription‐polymerase chain reaction analysis of intracerebral (i.c.) tumor‐necrosis factor‐α, interferon‐γ, interleukin (IL)‐2, IL‐1β, IL‐10, granulocyte/macrophage colony‐stimulating factor, and inducible nitric oxide synthase mRNA expression, and T cell depletion experiments. The data demonstrate that active immunization of mice prior to an i.c. infection with Listeria monocytogenes prevents the development of a fatal necrotizing encephalitis and accelerates the recruitment of an increased number of αβ T cell receptor (TcR)+ CD4+ and CD8+ T cells, γδ TcR+ T cells, B cells, granulocytes and macrophages to the brain compared to non‐immunized animals. In addition, immunization induced a pronounced activation of i.c. macrophages and microglial cells as shown by an increased expression of MHC class II antigens. In parallel, transcript levels for all cytokine mRNA analyzed were higher in the brains of immunized mice. The protective effects of immunization were completely abolished by depletion of CD4+, CD8+, or both T cell subsets. All groups of T cell‐depleted immunized mice developed a fatal necrotizing encephalitis with an increased i.c. bacterial load. In addition, cytokine mRNA synthesis was significantly impaired. The severity of disease was only slightly different between CD4+, CD8+ and CD4+/CD8+ T cell depleted mice, indicating that both subsets of T cells are required for an effective i.c. immune response to L. monocytogenes. This is in marked contrast to systemic listeriosis, and points to CNS‐specific features of the immune response.
Interactions between the amino‐terminal domain of p56lck and cytoplasmic domains of CD4 and CD8α in yeast
Authors: Campbell Kerry S. , Buder Annie , Deuschle Ulrich
Published: March 2007

The interactions between CD4 or CD8 and p56lck were tested using the twohybrid protein interaction system in yeast. Plasmid constructs were created which fuse the cytoplasmic domains of either CD4 or CD8α to the DNA‐binding protein LexA, and the unique amino‐terminal domain of p56lck fused to a transcriptional activation domain. These constructs were transfected into yeast bearing lacZ and LEU2 reporter genes controlled by upstream LexA operator sequences. Yeast transfectants bearing either CD4 or CD8α hybrid proteins in combination with the amino terminal p56lck hybrid protein exhibited increased β‐galactosidase activity and growth on leucine‐deficient medium, indicating interactions between these protein domains.

Quantitation of reporter activation indicated that the interaction of p56lck with CD8α is at least 18‐fold weaker than the interaction with CD4 in this assay. This reduced interactive capacity is apparently not due to competition by CD8α interacting with itself, since homotypic or heterotypic interactions between CD8α and/or CD4 could not be detected. Truncation and point mutants demonstrated that the interactions of p56lck with CD4 or CD8α were dependent on the integrity of a pair of cysteines on each protein. The results indicate that these interactions do not require any additional proteins. Additionally, expression of the entire p56lck molecule as a hybrid with LexA resulted in dramatic reduction in the growth of yeast. Though the two‐hybrid system is a powerful tool for examining protein interactions, this result indicates potential limitations in studying full‐length src family tyrosine kinases in yeast.
Interleukin‐4 and interleukin‐5 are rarely co‐expressed by human T cells
Authors: Jung Thomas , Schauer Uwe , Rieger Christian , Wagner Kathrin , Einsle Karin , Neumann Christine , Heusser Christoph
Published: March 2007

Interleukin (IL)‐4 and IL‐5 are two cytokines which synergize in the induction of several biological effector functions. They are produced by mouse and human T helper 2 (Th2) and T helper 0 (Th0) cells. Little is known about the regulation of the two cytokines at the single‐cell level. Here we show, using a flow cytometric intracellular staining technique, that IL‐4 and IL‐5 are predominantly produced by different human peripheral CD4+ and CD8+ T cells, whereas interferon (IFN)‐γ and IL‐2 are produced by the same cells. In contrast, cloned human Th0 and Th2 cells were able to produce IL‐4 and IL‐5 simultaneously. The segregation of IL‐4 and IL‐5 in activated peripheral T cells was found within 72 h of activation upon anti‐CD3 or phorbol ester + ionomycin stimulation. The kinetics of IL‐4 and IL‐5 production were different at the mRNA and the intra‐and extracellular protein level, indicating that the cytokines are regulated differently. T cells from three patients with hyper‐IgE syndrome did not display a substantial proportion of IL‐4/IL‐5 double‐positive cells. However, simultaneous production could be induced in normal human T cells after prolonged stimulation with a minimum of two restimulation cycles. We conclude that the simultaneous production of IL‐4 and IL‐5 is a feature of repetitively activated human T cells.
Role of N‐linked glycosylation in expression of E‐selectin on human endothelial cells
Authors: Påhlsson Peter , Strindhall Jan , Srinivas Uppugunduri , Lundblad Arne
Published: March 2007

E‐selectin is a cytokine‐inducible membrane glycoprotein capable of mediating adhesion of leukocytes to endothelial cells. It is highly glycosylated, containing 11 sites for N‐linked glycosylation. N‐Glycosylation of E‐selectin was analyzed by endoglycosidase treatment. Analysis of immunoprecipitated E‐selectin from human umbilical vein endothelial cells (HUVEC) by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that E‐selectin was completely resistant to endoglycosidase H, but sensitive to peptide N‐glycanase F digestion. This suggested that all N‐linked oligosaccharide chains were of the complex type. The role of N‐linked glycosylation in surface expression and secretion of E‐selectin was studied using interleukin‐1‐stimulated HUVEC, cultured in the presence of the soluble glycosylation inhibitors tunicamycin or castanospermine. Cell surface expression was analyzed by indirect flow cytometry. N‐Glycosylation was blocked by tunicamycin, and resulted in a significantly reduced surface expression of E‐selectin, whereas castanospermine only marginally reduced E‐selectin expression. The deglycosylated forms of E‐selectin were also found to be fully capable of mediating adhesion of HT‐29 cells in vitro. In conclusion, these studies show that E‐selectin is heavily glycosylated with complex type N‐linked oligosaccharides and that N‐glycosylation is important for expression of E‐selectin on human endothelial cells.
Analysis of the natural human IgG antibody repertoire: life‐long stability of reactivities towards self antigens contrasts with age‐dependent diversification of reactivities against bacterial antigens
Authors: Lacroix‐Desmazes Sébastien , Mouthon Luc , Coutinho Antonio , Kazatchkine Michel D.
Published: March 2007

We used a quantitative immunoblotting technique to analyze the repertoires of IgG antibody reactivities in the serum of healthy young children, young adult males and aged males with self and non‐self antigens. Densitometric patterns of reactivity of purified IgG with self antigens were highly conserved between individuals within a given age group and across age groups. Inter‐individual differences were observed, however, upon analysis of self reactivities of IgG in whole serum. A striking heterogeneity between individuals within a given age group and across age groups characterized the reactivity of purified IgG and of IgG in whole serum with bacterial antigens. Inter‐individual differences were more marked among aged individuals than among individuals of other age groups. Analysis of variances of reactivities of IgG with bacterial antigens further demonstrated an increased diversity of repertoires of aged donors compared with those of young adults and children. Our results document the stability of the self‐reactive repertoires of IgG throughout life, which contrasts with the diversification of the repertoire of IgG antibody reactivities directed toward foreign antigens with aging. These findings support the concept that self‐reactive antibody repertoires are positively selected throughout life by a restricted set of self antigens shared by all individuals.
Incorporation of major histocompatibility complex – encoded subunits LMP2 and LMP7 changes the quality of the 20S proteasome polypeptide processing products independent of interferon‐γ
Authors: Kuckelkorn Ulrike , Frentzel Stefan , Kraft Regine , Kostka Susanne , Groettrup Marcus , Kloetzel Peter‐M.
Published: March 2007

The 20S proteasome is the enzyme complex responsible for the processing of antigens bound by major histocompatibility complex class I molecules. The role of the interferon‐γ (IFN‐γ)‐inducible proteasome subunits LMP2 and LMP7 in this process is, however, still controversial. We have studied the effects of IFN‐γ‐independent LMP incorporation on the quality of peptides processed from the murine cytomegalovirus IE pp89 25‐mer polypeptide substrate through dual cleavages by 20S proteasomes. The incorporation of a single LMP subunit or both LMP2 and LMP7 induces changes in 20S proteasome subunit stoichiometry, alters its cleavage site preference and in consequence, the quality of the generated peptides. When the several hydrolytic activities are tested with short fluorogenic peptide substrates, the Vmax, S0.5 (Km), or both values of 20S proteasomes are altered, depending on the combination of LMP. There exists, however, no obvious correlation between the observed changes in hydrolytic activities against short fluorogenic peptides and the changes in dual cleavage site usage within the 25‐mer polypeptide substrate. As judged from the calculated Hill coefficients, the presence of both LMP subunits induces a drastic increase in positive cooperativity between the proteasome subunits.
Interleukin‐10 differentially regulates B7‐1 (CD80) and B7‐2 (CD86) expression on human peripheral blood dendritic cells
Authors: Buelens Christel , Willems Fabienne , Delvaux Anne , Piérard Géraldine , Delville Jean‐Pierre , Velu Thierry , Goldman Michel
Published: March 2007

Most of the immunosuppressive effects of interleukin‐10 (IL‐10) are related to functional inhibition of antigen‐presenting cells (APC). Herein, we investigate the influence of recombinant (r)IL‐10 on human dendritic cells (DC) purified from peripheral blood of healthy volunteers. First, we found that rIL‐10 inhibited in a dose‐dependent manner the proliferative responses as well as the production of IL‐2 and interferon‐γ (IFN‐γ) in mixed lymphocyte reaction (MLR) between purified T cells and DC. This rIL‐10 effect could be attributed to a direct effect on DC, as DC preincubated with rIL‐10 were found to be deficient in the induction of alloreactive T cells even when anti‐IL‐10 neutralizing mAb was added at the time of MLR. Flow cytometric analysis indicated that rIL‐10 did not modify the expression of ICAM‐1 (CD54) and B7‐1 (CD80), but decreased HLA‐DR and B7‐2 (CD86) expression at the DC surface. We conclude that the inhibitory effect of rIL‐10 on primary alloreactive T cell responses involves down‐regulation of class II MHC and B7‐2 expression at the DC surface.
Human naive CD4 T cells produce interleukin‐4 at priming and acquire a Th2 phenotype upon repetitive stimulations in neutral conditions
Authors: Demeure Christian E. , Yang Liang‐Peng , Byun Dae Gyoo , Ishihara Hideki , Vezzio Nadia , Delespesse Guy
Published: March 2007

The maturation of naive CD4 T cells into interleukin (IL)‐4‐producing effectors was shown to require the presence of IL‐4 at priming, the cellular origin of which remains unclear. We demonstrate here that naive T cells themselves release IL‐4 at very low levels that are nevertheless sufficient to promote their development into Th2‐like cells. This conclusion is based on three observations: (1) highly purified human naive CD4T cells, of neonatal or adult origin, develop into Th2 effectors upon repetitive cycles of stimulation with anti‐CD3 monoclonal antibody (mAb) cross‐linked to CD32‐B7 transfected L fibroblasts followed by IL‐2 expansion; (2) IL‐4 protein is readily detectable in the concentrated supernatant fluids of priming cultures performed in the presence of anti‐IL‐4 receptor mAb; and (3) addition of anti‐IL‐4 or anti‐IL‐4 receptor mAb at priming markedly inhibits the acquisition of IL‐4‐ and IL‐5‐producing capacity while enhancing that of interferon‐γ.
Antibody‐targeted superantigen therapy induces tumor‐infiltrating lymphocytes, excessive cytokine production, and apoptosis in human colon carcinoma
Authors: Litton Mark J. , Dohlsten Mikael , Lando Peter A. , Kalland Terje , Ohlsson Lennart , Andersson Jan , Andersson Ulf
Published: March 2007

Bacterial superantigens are the most potent known activators of human T lymphocytes. To engineer superantigens for immunotherapy of human colon carcinoma, the superantigen, staphylococcal enterotoxin A (SEA) was genetically fused to the Fab region of the colon carcinoma‐reactive monoclonal antibody C242. In the present study the effector mechanisms involved in the anti‐tumor response to C242 Fab‐SEA were characterized. Immunohistochemistry and computer‐aided image analysis were used in studies of cryopreserved tumor tissue to evaluate the phenotype of infiltrating cells and their cytokine profiles in response to therapy. Human T cells and monocytes were recruited to the tumor area and penetrated the entire tumor mass within hours after injection of C242 Fab‐SEA. The production of cytokines at the single‐cell level was found to be dominated by tumor necrosis factor (TNF)‐α, interleukin (IL)‐2, IL‐4, IL‐5, IL‐10, IL‐12, interferon (IFN)‐γ, granulocyte‐macrophage colony‐stimulating factor, and transforming growth factor‐β, whereas IL‐1‐α, IL‐1ra, IL‐1β, TNF‐β, IL‐3, IL‐6, and IL‐8 were undetectable. Most of the TNF‐α, IL‐2, IL‐12, and IFN‐γ were made by the infiltrating human leukocytes, while the colon carcinoma cells were induced to produce IL‐4, IL‐10, and TNF‐α. Up‐regulation of IFN‐γ receptors and TNF R p60 receptors was found, while the TNF R p80 receptor was absent. The cytokine production, T cell infiltration, and CD95 Fas receptor expression concomitantly occurred to induce programmed cell death in the tumor cells. This was followed by a strong reduction of the tumor mass that was seen within 24 h after C242 Fab‐SEA infusion. These findings demonstrate that antibody‐superantigen proteins efficiently recruit tumor‐infiltrating lymphocytes actively producing a variety of cytokines likely to be essential for the therapeutic effects observed in the model. Although the humanized SCID model has obvious limitations in its predictive value for treatment of human cancer, we believe that these results encourage clinical evaluation of antibody‐targeted superantigens.
T cell recognition of penicillin G: Structural features determining antigenic specificity
Authors: Padovan Elisabetta , Mauri‐Hellweg Daniela , Pichler Werner J. , Weltzien Hans Ulrich
Published: March 2007

Penicillin G (Pen G) and other beta‐lactam antibiotics frequently induce allergic reactions constituting typical examples of human immune responses to haptens. In fact, penicillins represent a unique set of haptens with outstanding structural variability on the basis of an identical protein‐reactive beta‐lactam containing backbone. Although both cellular and humoral responses are involved in drug‐induced allergies, little is known about the T cell reactivity to penicillins. To understand which structural features determine antigenic specificity, we isolated a panel of MHC‐restricted, Pen G‐reactive T cell clones from different penicillin‐allergic patients and tested them for their capacity to proliferate in the presence of other penicillin derivatives. We found that the antigenic epitope consists of both the amide‐linked side chain, which is different in every member of the penicillin family, as well as the thiazolidine ring common to all penicillin derivatives. We also demonstrated the presence of two different types of penicillin‐specific T cells, one dependent, and the other independent of antigen processing by autologous antigen‐presenting cells. Our data strongly suggest that penicillins form part of the epitopes contacting the antigen receptors of T cells.
Characterization of cytokine production in murine Trypanosoma cruzi infection by in situ immunocytochemistry: Lack of association between susceptibility and type 2 cytokine production
Authors: Zhang Lei , Tarleton Rick L.
Published: March 2007

Cytokine production in the spleens of mice infected with the protozoan parasite Trypanosoma cruzi was analyzed in three models which differ in the outcome of the infection. Using immunocytochemical techniques to detect cytokine‐producing cells, the production of type 1 [interleukin‐2 (IL‐2) and interferon (IFN)‐γ], type 2 (IL‐4, IL‐5, IL‐10), inflammatory [tumor necrosis factor (TNF)‐α, IL‐1α, IL‐6] and regulatory (transforming growth factor‐β) cytokines were examined. With the exception of IL‐4 and IL‐5, cells producing all of the cytokines assayed were detected in both the resistant and susceptible models of T. cruzi infection. Cells producing IL‐4 and IL‐5 were not detected until later in infection in the resistant mice (>34 days), at about the time animals of the susceptible strain succumb to the infection. Mice of the susceptible model showed a slight delay in the appearance of cells producing the type 1 cytokines IL‐2 and IFN‐γ and an earlier appearance of TNF‐producing cells, in comparison to resistant mice. Cells producing IL‐2 or IL‐10 were transient in their appearance in the spleen while cells producing IL‐1, IL‐4, IL‐5, IL‐6, IFN‐γ, TNF, or TGF‐β were first detectable in either the acute or post‐acute stage of the infection and persisted up to 700 days post infection in two different resistant models of the infection. Cells producing IFN‐γ, TNF‐α and TGF‐β were particularly numerous even very late in the infection. Double‐staining techniques were used to show that the vast majority of the IFN‐γ‐producing cells in the spleen were CD4−, CD8− α/β TCR+ T cells. This study confirms the transience of IL‐2 production in the acute stage of T. cruzi infection and the persistent and simultaneous production of type 1 and type 2 cytokines during the late‐acute and chronic stages of the infection. Susceptibility or resistance to T. cruzi infection does not associate with a Th2 pattern of cytokine production in the three models examined in this study. The overlapping pattern of type 1 and type 2 cytokine‐producing cells in both the acute and chronic stages of T. cruzi infection demonstrates that longterm infections do not necessarily lead to a dominance of either type 1 or type 2 cytokine production.
Peripheral tolerance in T cell receptor‐transgenic mice: Evidence for T cell anergy
Authors: Falb Dean , Briner Thomas J. , Sunshine Geoffrey H. , Bourque Cheryl R. , Luqman Mohammad , Gefter Malcolm L. , Kamradt Thomas
Published: March 2007

T cell tolerance can be induced in adult mice by injection of soluble antigenic peptide. The underlying mechanism has been difficult to establish in normal mice due to the low precursor frequency of T cells specific for any given antigen. Therefore, we examined peripheral tolerance in mice transgenic for a T cell receptor specific for a cytochrome c peptide bound to I‐Ek. Antigen‐specific hyporesponsiveness could be induced in the transgenic mice. We followed the transgene‐bearing T cells with a clonotypic monoclonal antibody and found similar numbers of clonotypic T cells in tolerized and control mice. To prevent de novo differentiation of T cells we analyzed thymectomized mice in which antigen‐specific hyporesponsiveness was induced. Our analysis of thymectomized transgenic mice showed that antigen‐specific T cell hyporesponsiveness following injection of peptide intravenously is not caused by gross elimination of T cells. These data provide evidence for the role of anergy in peripheral tolerance.
Tissue‐specific deoxyribonuclease I‐hypersensitive sites in the vicinity of the immunoglobulin Cλ cluster of man
Authors: Asenbauer Hildegard , Klobeck H.‐Gustav
Published: March 2007

During B cell development, the onset of DNA rearrangements, expression, and somatic hypermutation of Ig genes are regulated through the complex interaction of cis‐acting elements with trans‐acting factors. Our aim is to identify DNA elements required during activation of the human Igδ light chain genes. Determination of deoxyribonuclease (DNase) I‐hypersensitive sites in complex regulated genes can lead to the identification of sequence elements which would have been overlooked by employing transient transfection protocols. We have therefore investigated the chromatin structure of human J‐Cλ genes and identified three DNase I‐hypersensitive sites (HSS‐1, −2, and −3) within an 8‐kb region downstream of the J‐Cλ7 gene. HSS‐2 and HSS‐3 are B cell specific. The DNase I‐hypersensitive sites are also present in X‐producing cell lines which have not rearranged the Igδ locus and produce germ‐line J‐Cλ transcripts. We conclude that in mature B cells, both x and λ loci are in an active structure regardless of the type of light chain they produce. This suggests that the chromatin structure of both loci is opened early in B cell development and that the active structure persists in mature B cells. The observed temporal order (first x, then δ) of activation can be explained by consecutive synthesis of the appropriate regulating factors and the tight regulation of the recombination machinery through the products of L chain gene rearrangements.
Major histocompatibility complex genes determine natural killer cell tolerance
Authors: Dorfman Jeffrey R. , Raulet David H.
Published: March 2007

Murine natural killer (NK) cell subsets, as defined by expression of members of the Ly49 gene family, discriminate target cells expressing different major histocompatibility complex (MHC) class I alleles. For example, Ly49A+ NK cells lyse H‐2b but not H‐2d tumor target cells. The specificity arises because Dd on target cells binds to Ly49A, transducing an inhibitory signal into the Ly49A+ NK cells. The capacity of NK cells to discriminate allelic class I determinants raises a key issue: are NK cells self‐tolerant, and if so what are the mechanisms that lead to self‐tolerance? As previously reported, potentially autoaggressive Ly49A+ NK cells are not clonally deleted in H‐2b mice. However, IL‐2‐ cultured Ly49A+ effector cells from H‐2b mice exhibit reduced lysis of H‐2b (self) concanavalin A blast target cells, compared to Ly49A+ effector cells from H‐2d mice. Possible mechanisms accounting for this self‐tolerance are addressed in this report. Self‐tolerance was not due to anergy of the cells, because the Ly49A+ effector cells from both types of mice lysed β2‐microglobulin‐deficient target cells efficiently and equivalently. These results also suggest that tolerance results from inhibition mediated by β2m‐dependent H‐2b class I molecules. Significantly, blockade of Ly49A on Ly49A+ effector cells from H‐2b mice did not restore lysis of H‐2b target cells, suggesting that inhibition is not mediated through the Ly49A receptor. Additional experiments suggest that inhibition is also not mediated primarily through the Ly49C receptor. These results suggest that Ly49A+ effector cells from H‐2b mice, unlike those from H‐2d mice, express inhibitory receptors specific for H‐2b molecules that are distinct from Ly49A and Ly49C.
Analysis of major histocompatibility complex class I‐restricted hapten recognition by mutation of the V‐J joining of T cell receptor α chains
Authors: Von Bonin Arne , Plaga Sandra , Ruh Helga , Hebbelmann Sabine , Pflugfelder Ulrike , Martin Stefan , Weltzien Hans Ulrich
Published: March 2007

Hapten‐specific T cell responses are responsible for chemically induced immune disorders. However, the molecular details of hapten interactions with T cell receptors (TCR) are poorly understood. Recent studies of trinitrophenyl (TNP)‐specific responses revealed major histocompatibility complex‐associated TNP‐peptides as dominant epitopes for CD8+ and CD4+ T cells. The present study is based on the observation that two H‐2Kb/TNP‐specific CTL clones (II/7 and III/1), differing exclusively in two amino acids of their TCR α chains, also differed in their carrier specificities for various TNP‐peptides. The genes of the two α chains and the common β chain were cloned into expression vectors. Transfection of the TCR α chain of clone III/1 into a hybridoma of clone II/7 also transferred the fine specificity of clone III/1, indicating that the small α chain variations were indeed responsible for the different carrier specificities. Point mutations bridging the difference between the α chains of clones II/7 and III/1 and functional studies of the respective TCRαβ transfectants into a TCR‐negative hybridoma revealed an unexpected result: the two receptors did not represent examples of structural complementarity for different sets of hapten‐peptide conjugates; rather, they resembled two structures of principally similar specificity but of significantly different overall affinity. This was demonstrated more directly by comparing the fine specificities of III/1 transfectants expressing or not expressing the co‐receptor CD8: the CD8‐negative III/1 transfectant assumed a specificity pattern indistinguishable from that of a CD8‐expressing, II/7‐derived transfectant. Hence, comparable alterations of antigen recognition may be induced either by subtle TCR alterations or by removal of CD8, i.e. by the presence or absence of a non‐polymorphic adhesion molecule.
B cell‐B cell interaction through intercellular adhesion molecule‐1 and lymphocyte functional antigen‐1 regulates immunoglobulin E synthesis by B cells stimulated with interleukin‐4 and anti‐CD40 antibody
Authors: Katada Yoshinori , Tanaka Toshio , Ochi Hiroshi , Aitani Masakazu , Yokota Akira , Kikutani Hitoshi , Suemura Masaki , Kishimoto Tadamitsu
Published: March 2007

IgE synthesis by purified human B cells is induced by two signals: a class switching factor, most commonly interleukin (IL)‐4, and the engagement of CD40, which is activated through its interaction with CD40 ligand (CD40L) expressed on activated T cells. Thus, the combination of IL‐4 and anti‐CD40 monoclonal antibody (mAb) has been shown to stimulate IgE production in vitro by highly purified B cells. In this T cell‐independent system, strong homotypic aggregation of B cells is observed prior to the production of IgE. Flow cytometric analysis and cell binding assays showed that the stimulation of purified B cells with anti‐CD40 mAb plus IL‐4 resulted in a striking increase of intercellular adhesion molecule (ICAM)‐1(CD54) expression, an induction of CD43 and an avidity change of lymphocyte functional antigen (LFA)‐1(CD11a/CD18), with little augmentation of CD 18 expression. Addition of anti‐ICAM‐1 mAb caused an inhibition of homotypic aggregation but augmented IgE synthesis by B cells stimulated with anti‐CD40 mAb and IL‐4, although it did not affect B cell proliferation or IL‐6 production by the B cells. Among the mAb against counter‐receptors for ICAM‐1 tested, anti‐CD11a mAb suppressed IgE synthesis, while anti‐CD18 mAb and anti‐CD43 mAb had little effect. The enhancing or inhibitory effect of anti‐ICAM‐1 mAb or anti‐CD11a mAb on IgE production was achieved by the increased or decreased expression of germline Cεe transcripts by B cells stimulated with anti‐CD40 mAb and IL‐4. These results indicate that B cell‐B cell interaction through ICAM‐1 and one of its counter receptors, LFA‐1, regulates IgE synthesis by modulating Cεe germ‐line transcription.
The β2 integrin Mac‐1 but not p150,95 associates with FcγRIIA
Authors: Annenkov Alexander , Ortlepp Susan , Hogg Nancy
Published: March 2007

In this study we have compared the ligand binding activity of the two closely related β2 integrins, Mac‐1 and p150,95, which are expressed separately as receptors permanently transfected into K562 cells. Mac‐1 has previously been shown to associate with FcγR, particularly FcγRIII, but K562 cells express only endogenous FcγRIIA. We have, therefore, taken advantage of this situation to examine a possible relationship between FcγRIIA with Mac‐1 and p150,95 in the absence of other FcγR. The main finding is that anti‐FcγRII mAb have a profound inhibitory effect on cell adhesion mediated by Mac‐1, but not on the adhesion mediated by p150,95. Thus, in spite of the fact that Mac‐1 and p150,95 bind to the same or at least a very similar selection of ligands, their association with other receptors on the cellular membrane, and therefore their mode of regulation may be different.