bluuebunny/crossref_metadata_2025 huggingface dataset:
Interferon‐γ activates multiple pathways to regulate the expression of the genes for major histocompatibility class II I‐Aβ, tumor necrosis factor and complement component C3 in mouse macrophages
Authors: Celada Antonio , Klemsz Michael J. , Maki Richard A.
Published: March 2007

The purpose of this study was to obtain additional information on the mechanism by which interferon‐γ (IFN‐γ) is able to regulate gene expression in macrophages. The expression of the genes for class II histocompatibility I‐Aβ, tumor necrosis factor (TNF) and complement component C3 was assayed after treating bone marrow macrophages with IFN‐γ. Each gene displayed a characteristic pattern of regulation. First, the increase in the level of RNA for each gene followed different kinetics. The level of TNF RNA increased within 15 min after IFN‐γ treatment and reached a plateau after 4 h. In contrast, there was a lag of about 4 h before the level of I‐Aβ RNA began to rise and a plateau was not reached until 48 h after the IFN‐γ treatment began. C3 gene expression followed an intermediate time course between that for TNF and I‐Aβ. Second, the expression of I‐Aβ was inhibited when cells were treated with both IFN‐γ and cycloheximide, while the expression of TNF and C3 was not. Interestingly, the sensitivity to cycloheximide only lasted 30 min following the addition of IFN‐γ, after which cycloheximide had no effect on the expression of I‐Aβ. Third, lipopolysaccharide abolished the IFN‐γ‐induced expression of I‐Aβ, but enhanced the expression of TNF. Based on these observations, we conclude that IFN‐γ must activate multiple pathways to regulate gene expression in macrophages.
Increased cyclic AMP levels block interleukin 2‐induced protein kinase C substrate phosphorylation but not the mitogenic response
Authors: Friedrich Bengt , Cantrel Doreen A. , Gullberg Martin
Published: March 2007

Protein kinase C (PKC) has been implicated in the signaling of a number of cellular responses including activation of T cells. In the present report we have evaluated the effect of increased cAMP levels on PKC activation after stimulation of two distinct receptor systems on normal human T cells. PKC substrate phosphorylation can be induced via either the CD3 complex or, to a limited extent, the high affinity interleukin 2 (IL2) receptor. Substrate phosphorylation via both pathways is shown to be blocked by increased intracellular levels of cAMP. In accordance with previous reports, the CD3‐dependent autocrine proliferative response could also be blocked by a cAMP‐dependent mechanism. Since direct activation of PKC with a phorbol ester reversed this inhibition, a causal relationship between cAMP‐dependent PKC blockage and inhibition of the CD3 response is suggested. In contrast, however, initiation of IL2‐induced proliferation was essentially unaltered by cAMP and could progress in the apparent absence of PKC activity. Thus, this study indicates that IL2‐induced proliferation can under such conditions be completely uncoupled from IL2‐induced PKC activation in normal T cells.
Membrane IgM cross‐linking is not coupled to protein kinase C translocation in WEHI‐231 B lymphoma cells
Authors: Sarthou Pierre , Henry‐Toulmé Nelly , Cazenave Pierre‐André
Published: March 2007

The early molecular events involved in the process of signal transduction via membrane immunoglobulins (mIg) include phosphatidyl inositol metabolism, intracellular Ca2+ mobilization, and protein kinase C (PKC) activation. Anti‐mIg antibodies exert either stimulating or inhibitory effects depending on the activation state and/or the differentiation stage of B cells. WEHI‐231 is a murine B lymphoma that becomes inactivated upon anti‐mIg treatment. This lymphoma has an immature B cell phenotype and is considered as a model for tolerance induction in B lymphocytes. In this study, we have investigated the relationship between mIg triggering, Ca2+ elevation, PKC translocation, and growth inhibition in WEHI‐231 cells. Monoclonal antibodies to μ and χ chains of the mIgM receptor promoted a rapid increase in intracytoplasmic Ca2+ and were potent inhibitors of cell growth. Ca2+ elevation and PKC translocation have been previously shown to be associated in B lymphocytes. To study the subcellular distribution of PKC in WEHI‐231 cells, we used enzymatic assays and immunodetection methods. Although phorbol 12‐myristate 13‐acetate induced a rapid and almost complete redistribution of cytosolic PKC to the membrane fraction, anti‐mIg treatment failed to modify the compartmentalization of PKC. These findings extend recent observations suggesting that B cell triggering through mIg receptors may involve additional pathways independent from PKC activation. PKC activation in normal B cells is also believed to provide a regulatory signal which limits the magnitude of the early signals produced by anti‐mIg. Such a regulatory control is unlikely in WEHI‐231 cells, due to the dissociation between Ca2+ mobilization and PKC translocation. Our findings therefore suggest that the sensitivity of immature B cells such as WEHI‐231 to the inhibitory effects of anti‐mIg antibodies may result in part from alterations of the phosphoinositide signal transduction pathway.
Coordinated Vγ and Vδ gene segment rearrangements in human T cell receptor γ/δ+ lymphocytes
Authors: Sturm Els , Braakman Eric , Bontrop Ronald E. , Chuchana Paul , Van Griend René J. De , Koning Frits , Lefranc Marie‐Paule , Bolhuis Reinder L. H.
Published: March 2007

Monoclonal antibodies (mAb) were used to characterize a panel (n = 46) of T cell receptor (TcR) γ/δ+ T cell clones. Three of these antibodies have been described to react with specific variable region‐encoded protein products and can therefore be used to detect functional gene rearrangements. The majority of peripheral blood‐derived clones (43 out of 45) expressed the epitopes recognized by mAb BB3, encoded by the Vδ2 gene segment and mAb TiγA, encoded by the Vγ9 gene segment. These clones lacked the antigenic determinant recognized by mAb δ‐TCS‐1, encoded by the Vδ1 gene segment. The other two peripheral blood‐derived clones and an ascites‐derived clone were TiγA‐, BB3‐ and δ‐TCS‐1+. Biochemical analysis revealed that all TiγA+, BB3+ T cell clones expressed the disulfide‐linked form of the receptor. The two peripheral blood‐derived δ‐TCS‐1+ T cell clones expressed the nondisulfide‐linked form whereas the ascites‐derived δ‐TCS‐1+ clone, AK119 expressed the disulfide‐linked form of the TcRγ/δ heterodimer. This indicates that Vδ1‐encoded δ chains can be associated either with a Cγ1‐ or a Cγ2‐encoded γ chain. The preferential use of certain Vγ and Vδ gene segments suggests the existence of a limited combinatorial diversity in TcRγ/δ heterodimers, i.e. TiγA+ (Vγ9), BB3+ (Vδ2) and δ‐TCS‐1‐ disulfide‐linked heterodimers and TiγA‐, BB3‐ and δ‐TCS‐1+ (Vδ1) disulfide‐ or non disulfide‐linked forms.
Specificity of human T lymphocytes expressing a γ/δ T cell antigen receptor. Recognition of a polymorphic determinant of HLA class I molecules by a γ/δ clone
Authors: Ciccone Ermanno , Viale Oriane , Pende Daniela , Malnati Mauro , Ferrara Giovan Battista , Barocci Sergio , Moretta Alessandro , Moretta Lorenzo
Published: March 2007

Alloreactive clones expressing T cell receptor (TcR) γ/δ were derived by limiting dilution from CD3+CD4‐CD8‐WT31‐ populations stimulated in allogeneic mixed lymphocyte culture. These clones specifically lysed phytohemagglutinin‐induced blast cells bearing the stimulating alloantigens, whereas they had no effect on autologous or allogeneic unrelated target cells. Analysis of the reactivity with monoclonal antibodies (mAb) specific for two different subsets of TcR γ/δ (BB3 and δ‐TCS‐1) showed that five out of nine clones were BB3+, whereas the remaining reacted with δ‐TCS‐1. Therefore, we can conclude that both subsets of TcR γ/δ+ cells are able to specifically recognize and lyse allogeneic cells. mAb directed against the CD3‐TcR γ/δ molecular complex strongly inhibited the specific cytolytic activity of TcR γ/δ+ clones, whereas they had no effect on the lysis of the natural killer‐sensitive K‐562 target cells mediated by the same clones. An alloreactive δ‐TCS‐1+ clone (LM12) was further characterized for its specificity. LM12 clone had been derived after stimulation in mixed lymphocyte culture against donor M. M. (HLA typing: Aw68, 24; B35, w55; DR1, 7). The analysis of a large panel of phytohemagglutinin‐induced target cells revealed that only the HLA‐A24+ target cells were lysed. The direct evidence that the A24 molecule represented the restriction element was provided by experiments using A24‐transfected murine P815 target cells. Thus, clone LM12 efficiently lysed A24‐transfected P815 cells, but not the same cells untransfected or transfected with the Cw3 gene. Therefore, it appears that polymorphic determinants of class I major histocompatibility complex molecules can be the target of TcR γ/δ+ alloreactive cell recognition.
Mechanism of recovery from acute virus infection. VIII. Treatment of lymphocytic choriomeningitis virus‐infected mice with anti‐interferon‐γ monoclonal antibody blocks generation of virus‐specific cytotoxic T lymphocytes and virus elimination
Authors: Wille Andreas , Gessner André , Lother Heinz , Lehmann‐Grube Fritz
Published: March 2007

In acutely infected mice the lymphocytic choriomeningitis (LCM) virus multiplies to high titers in essentially all tissues. Around day 6, virus clearance sets in, which has previously been shown to be mediated by CD8+ cytotoxic T lymphocytes (CTL), probably by releasing (or inducing other cells to release) anti‐viral cytokines. To ascertain whether interferon‐γ plays a role, infected mice were injected once i.v. with monoclonal antibody known to neutralize this lymphokine, and the effect this had on both termination of the infection and development of LCM virus‐specific CTL was determined. Administration 1 day after infection blocked virus elimination from spleen and liver and decreased the generation of CTL; also, limiting dilution analysis revealed absence of activation of CTL precursors. In contrast, when the antibody was given 3 days after or 1 day before the virus, neither clearance nor generation of CTL was measurably affected. Furthermore, the antiviral effect of immune spleen cells after their transfer into infected recipients was not altered by treatment of the latter with monoclonal antibody. We conclude that in the generation of LCM virus‐specific CTL an early event is dependent on constitutively produced interferon‐γ; when its activity is blocked, CTL do not mature, resulting in the mouse's inability to terminate the infection.
Biased immunoglobulin variable region gene expression by Ly‐1 B cells due to clonal selection
Authors: Pennell Christopher A. , Mercolino Thomas J. , Grdina Therese A. , Arnold Larry W. , Haughton Geoffrey , Clarke Stephen H.
Published: March 2007

Most, if not all, autoantibodies specific for bromelain‐treated mouse erythrocytes recognize the common membrane phospholipid, phosphatidyl choline (PtC). Anti‐PtC antibodies are produced by 5%‐15% of CD5+ Ly‐1 B cells of normal unimmunized mice, but not by detectable numbers of conventional CD5– B cells. At 1 week of age PtC‐specific B cells are undetectable but then increase dramatically over the next 3 to 4 weeks to reach adult numbers. We report here that PtC‐specific Ly‐1 B cells in B10.H‐2aH‐4bp/Wts mice predominantly express either of two heavy and χ chain variable (V) region gene combinations. In addition, the sequence and length of DH genes are conserved among cells expressing the same V gene combination, and the Vχ‐Jχ junctions of one group involve unusual splice sites. Preferential V gene rearrangement models are insufficient to explain the DH and Vχ‐Jχ junctional sequences or the delayed appearance of this specificity, and so they cannot solely account for the high frequency of PtC‐specific cells. These characteristics are more consistent with antigen selection. We therefore attribute the frequent use of the two V region gene combinations to selection for cells that express them and conclude that the expressed V gene repertoire of Ly‐1 B cells in adult mice is influenced by antigen selection. Apparently, there is no selection for mutant anti‐PtC antibodies of higher affinity during the formation of the Ly‐1 B repertoire because the V region genes expressed by PtC‐specific cells are unmutated. Our findings are consistent with an important, germ line‐encoded function for the immunoglobulin products of these gene combinations.
Interleukin 4 induces synthesis of IgE and IgG4 in human B cells
Authors: Lundgren Mats , Persson Ulla , Larsson Per , Magnusson Carl , Smith C. I. Edvard , Hammarström Lennart , Severinson Eva
Published: March 2007

Interleukin (IL) 4 has been shown to regulate the IgG subclasses and induce IgE production in splenic mouse B cells. Here we show that IL 4 and phorbol 12‐myristate 13‐acetate (PMA) induce, on a per cell basis, very high IgE secretion in purified human B cells by using a mouse thymoma (EL4) co‐culture method. In addition, a marked increase in the number of IgG4‐producing cells was also observed. Furthermore, IL 2 could synergize with IL4 and PMA in the production of IgE. By using limiting dilution analysis, a considerable increase in the precursor frequency for IgE was found when IL 4 and PMA were added to cultures as compared to cultures with PMA only. This indicates that IL4 induces an isotype switch in human B cells.
The intermediate‐affinity interleukin (IL) 2 receptor expressed on Theileria annulata‐infected cells comprises a single IL 2‐binding protein. Partial characterization of bovine IL 2 receptors
Authors: Herrmann Thomas , Ahmed Jabbar S. , Diamantstein Tibor
Published: March 2007

Bovine high‐, intermediate‐ and low‐affinity interleukin 2 receptors (IL2R) were studied by ligand binding and affinity labeling using 125I‐labeled IL 2 and homobifunctional chemical cross‐linking reagents. High‐ (Kd = 17 pM) and low‐affinity (Kd > 6 nM) IL2R were detected on concanavalin A‐activated peripheral blood lymphocytes (PBL). Theileria annulata (TA)‐infected autonomously growing PBL (TA‐PBL) express predominantly intermediate‐affinity IL2R (Kd = 1 nM). Affinity‐labeling studies revealed that the high‐affinity IL2R comprises a 55‐kDa (L chain) and an additional 90‐kDa IL 2‐binding protein (H chain). TA‐PBL express predominantly the H chain. In contrast to the human chain, the bovine form was not separable by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis/urea into the two distinct bands H1 and H2 and ran in parallel to the human H2 band. These results suggest (a) that the bovine intermediate‐affinity IL 2R comprises a single H chain and (b) that the single H chain and the L chain are sufficient to form the functional high‐affinity bovine IL2R.
Alloantigen pretreatment induces a down‐regulation of the in vivo subsequent development of cytotoxic T lymphocytes directed against linked alloantigens
Authors: Schutze Marie‐Paule , Langlade‐Demoyen Pierre , Przewlocki Grzegorz , Leclerc Claude
Published: March 2007

In the present study, we describe a new regulatory system that influences the in vivo development of cytotoxic T lymphocytes (CTL) and that could be related to epitopic suppression. Epitopic suppression has been previously shown to occur when carrier‐primed mice are subsequently immunized with a “new” epitope coupled to the priming carrier. The suppression specifically inhibited the antibody response to the “new” epitope without affecting the secondary antibody response to the carrier.

In this report, using a carrier/hapten‐carrier type of immunization protocol, we have demonstrated that a similar regulatory system could also affect the induction of CTL directed against allogeneic cells. Priming mice with an alloantigen 1 (carrier) inhibits the induction of alloantigen 2 (hapten)‐specific cytotoxic responses when the alloantigen 2 is presented in association with the alloantigen 1 on an F1 stimulator cell (hapten‐carrier conjugate). This has been demonstrated by the specific decrease of anti‐H‐2b or anti‐H‐2d CTL responses generated in C3H/He mice (H‐2k) previously primed with, respectively, H‐2d or H‐2b spleen cells before immunization with F1 (H‐2d x b) spleen cells. This suppression of the CTL responses against the second immunizing alloantigen is associated with a strong CTL response against the first priming alloantigen. The induction of the suppression is dependent on the dose of H‐2d spleen cells administered before immunization with F1 spleen cells and is not related to antigen elimination since a strong suppression of the CTL response against H‐2b antigens is shown following immunization with a mixture of F1 cells and H‐2b‐bearing cells of H‐2d‐primed animals.
Class switch from μ to δ is mediated by homologous recombination between σμ and σμ sequences in human immunoglobulin gene loci
Authors: Yasui Hisashi , Akahori Yasushi , Hirano Masami , Yamada Kazumasa , Kurosawa Yoshikazu
Published: March 2007

Class switch of immunoglobulin from μ to γ occurs by recombination between two repetitive switch sequences: Sμ and Sγ. However, there are no such sequences in the μ‐δ introns of human and mouse genomes. Although the frequency of IgD‐secreting cells is extremely low in mouse about 1% of patients with myeloma produce IgD in human. In a previous report (Nucleic Acids Res. 1988. 16: 9497) we reported that a 442‐bp DNA sequence located in the JH‐μ intron (defined as σμ) was inserted into the μ‐δ intron (defined as σμ) in the human genome. There is no such insertion in mouse. We analyzed Ig H chain gene loci of two human IgD myelomas: one was analyzed by cloning and sequencing and the other by Southern hybridization. We found that recombination had occurred between these two homologous DNA sequences, resulting in loss of the DNA segment from σμ to σμ On the other hand, in a Burkitt lymphoma, Daudi, the DNA fragment from σμ to σμ was duplicated. These results suggest that homologous recombination between σμ and σμ sequences mediates class switch from μ to δ in human and that it occurs via unequal crossing‐over between sister chromatids or daughter chromosomes.
Heat shock protects WEHI‐164 target cells from the cytolysis by tumor necrosis factors α and β
Authors: Jäättelä Marja , Saksela Kalle , Saksela Eero
Published: March 2007

Elevated temperatures and a number of other types of stress induce synthesis of a small number of highly conserved proteins, the heat shock proteins, in a wide variety of cells. The structure and regulation of these proteins have been intensively studied but the question of the function of this universal response has remained unanswered. We studied the effect of heat shock on tumor necrosis factor‐α (TNF‐α)‐ and ‐β (TNF‐β)‐mediated cytolysis of WEHI‐164 clone 13 target cells. One hour pretreatment of target cells at 42°C decreased rTNF‐α‐mediated lysis by 65.3%, 50.5% and 44.8% and TNF‐β‐mediated lysis by 61.9%, 43.2% and 38.9% at cytokine concentrations of 0.5 ng/ml, 5 ng/ml and 50 ng/ml, respectively, in an 18‐h Cr‐release assay. The effect was maximal when TNF‐α was added 1 h after the heat shock and then gradually declined, being almost undetectable after 2 days. This pattern was found to roughly coincide with the kinetics of hsp68, the major heat‐induced protein in murine cells. Heat shock treatment had no protective effect when given 1 h after addition of recombinant TNF‐α. The heat‐induced target cell resistance was not associated with decreased binding of recombinant TNF‐α to its receptor. Inhibition of protein synthesis by cycloheximide diminished this effect by 76% and inhibition of transcription by actinomycin D abolished it completely, suggesting that de novo synthesized, heat‐induced proteins protect target cells from TNF‐mediated lysis in heat shock‐treated WEHI cells.
Suppression of antibody responses to the acetylcholine receptor by natural antibodies
Authors: Sundblad Anne , Hauser Stephen , Holmberg Dan , Cazenave Pierre‐André , Coutinho Antõnio
Published: March 2007

Some relationships between “natural” physiological autoreactivity and antibody responses against the acetylcholine receptor (AChR) were investigated. Collections of unselected monoclonal antibodies were derived from nonmanipulated newborn mice of susceptible (BALB.B) and resistant (BALB/c) mouse strains, and screened for reactivity with heterologous AChR. Five in 200 BALB/c but none in 200 BALB.B clones were found reactive. Four of these anti‐AChR antibodies were analyzed for connectivity to nine other antibodies in the same collection, and three of them found to react with a particular natural anti‐idiotype. The functional in vivo significance of such natural idiotypic complementarities in the anti‐AChR response could then be investigated. Treatment of BALB/c newborn animals with low (100 ng) or high (100 μg) doses of natural anti‐idiotype significantly suppressed their anti‐AChR antibody response when immunized as adults. The utilization of natural anti‐idiotype reagents with broad reactivity patterns might overcome the known idiotype heterogeneity of the autoimmune antibody response to AChR and provide a strategy in prophylactic and therapeutic manipulation of disease development.
Schistosoma mansoni‐specific rat T cell clones. II. Different effects of adult worm‐specific T cell clones in immunocompetent and nude infected rats
Authors: Pestel Joël , Dissous Colette , Louis Jacques , Kusnierz Jean‐Pierre , Damonneville Martine , Dessaint Jean‐Paul , Capron André
Published: March 2007

The in vivo functional activities of two highly proliferating helper rat T cell clones (E23 and G5) specific for the excretory‐secretory antigens of Schistosoma mansoni adult worms were investigated. When injected into infected immunocompetent rats, both clones increased the antibody response against the 30–40‐kDa schistosomulum surface antigens, but failed to induce an immune protection. In contrast, when the same clones were injected into infected nude rats, a high degree of protection was obtained. In this latter case the absence of detectable specific antibody response, whether of IgE or IgG isotype, suggested that parasites were destroyed by an antibody‐independent mechanism, i.e. macrophages activation by lymphokines. Indeed supernatants obtained from T cell clones specifically restimulated with schistosome antigens expressed a macrophage activated activity similar to interferon‐γ. Following incubation with these supernatants or with the active fractions, macrophages exhibited a significant schistosomulicidal activity and both clones were shown to transfer an antigen‐specific delayed‐type hypersensitivity reaction to normal rats. Taken together these results demonstrate that, depending on the immune status of the host, antigen‐specific T cell clones can function differently and consequently that one function associated with one type of lymphokine could be favored.
Regulation of immunoglobulin gene expression in trans by phorbol esters
Authors: Mårtensson Inga‐Lill , Iglesias Antonio , Leanderson Tomas
Published: March 2007

Lipopolysaccharide‐stimulated B cell cultures treated with phorbol‐12,13‐dibutyrate have been shown to down‐regulate their steady state levels of μs RNA while the μm RNA remained constant. In contrast, the steady state levels of δ RNA was upregulated by the same treatment. By using mice transgenic for a rearranged μ or δ gene, it could be shown that the down‐regulatory effect acts in trans on a defined DNA stretch and is independent of isotype. Thus, the positive effect on endogenous δ RNA steady‐state levels must be regulated at the level of RNA processing. In addition, a DNA construct containing 1.5 kb of a ϵ promoter 5′ of, and an IgH enhancer 3′ of, a chloramphenicol acetyl transferase reporter gene could be down‐regulated in trans by anti‐μ or phorbol‐12,13‐dibutyrate treatment, showing that transcriptional control of Ig promoter elements can readily be observed in non‐transformed B lymphocytes.
Physical linkage of variable, diversity and joining gene segments in the immunoglobulin heavy chain locus of the mouse
Authors: Christoph Thomas , Krawinkel Ulrich
Published: March 2007

The variable region of antibody heavy chains is encoded by multiple variable (V), diversity (D) and joining (J) gene segments which seem to be assembled in a programed developmental pathway during the maturation of B lymphocytes. A physical map linking V, D and J gene segments in the a haplotype of the murine Igh locus is presented in this report. The V gene located next to the known D and J gene segments maps 80 kb upstream of the JH locus and is a member of the VGAM3.8 V gene family. There is evidence for as yet unknown D gene segments located 5′ to this V gene.
Differences in the synthesis and kinetics of release of interleukin 1α, interleukin 1β and tumor necrosis factor from human mononuclear cells
Authors: Lonnemann Gerhard , Endres Stefan , Meer Jos W. M. Van Der , Cannon Joseph G. , Koch Karl M. , Dinarello Charles A.
Published: March 2007

Cell‐associated and secreted interleukin 1α (IL 1α), IL 1β and tumor necrosis factor α (TNF‐α), produced by human mononuclear cells (MNC) in vitro in response to lipopolysaccharide, were measured by radioimmunoassay. After 18 h of incubation, total production of IL 1α in medium containing 1% heat‐inactivated serum was two‐to‐three times higher than IL 1β. However, in the presence of 1% serum and 5% fresh plasma, IL 1α and IL 1β were produced in similar amounts. Independent of the culture conditions, 90% of the IL 1α remained cell associated whereas 80% of IL 1β was extracellular. The kinetics of production and release of IL 1α, β and TNF‐α were also studied. IL 1α and TNF‐α reached maximal levels within 6 h of stimulation, whereas IL 1β reached maximal levels between 12 and 16 h. IL 1α remained primarily cell associated (80%) for the first 24 h. After 48 h, extracellular IL 1α exceeded cell‐associated levels. IL 1β was primarily secreted (80%), appearing in the extracellular fluid within 6 h. TNF‐α appeared in the extracellular fluid within 1 h of incubation, with < 10% cell associated at any time during the 48 h of incubation. Although the three cytokines share many biological activities, this study provides evidence that MNC IL 1α is predominantly a cell‐associated cytokine acting on a cell‐cell basis, whereas IL 1β and TNF‐α are secreted as paracrine mediators.
Multiple subsets of HIV‐specific cytotoxic T lymphocytes in humans and in mice
Authors: Chenciner Nicole , Michel Frédérique , Dadaglio Gilles , Langlade‐Demoyen Pierre , Hoffenbach AgnèS , Leroux AléNa , Garcia‐Pons Francisco , Rautmann Guy , Guy Bruno , Guillon Jean‐Marcel , Mayaud Charles , Girard Marc , Autran Brigitte , Kieny Marie‐Paule , Plata Fernando
Published: March 2007

The human immunodeficiency virus type 1 (HIV‐1) induces a strong cytotoxic T lymphocyte (CTL) response in humans following infection. HIV‐specific CTL can be detected directly in the blood and lungs of infected patients, and can be expanded in vitro by stimulation with autologous HIV‐infected lymphoblasts. Furthermore, CTL specific for HIV envelope glycoprotein gp160 have been obtained in mice by immunization with recombinant vaccinia virus (VV) that carry the HIV env gene. In this study, we show that mice also produce strong CTL responses to gag and nef proteins following immunization with VV recombinants, thus providing a convenient model system to study T lymphocyte immunity to defined HIV antigens. To determine the specificity of circulating HIV‐immune CTL in humans, a panel of doubly transfected mouse P815 tumor cells was produced which express the human HLA‐A2 or HLA‐A3 transplantation antigen gene and one HIV‐1 gene (env, gag or nef). Using these cells as targets to CTL, we show that HIV‐infected humans carry co‐existing CTL sub‐populations of different specificities. Each subpopulation appears to vary in intensity among different individuals. Surprisingly, CTL specific for regulatory, non‐structural nef protein appear to be a major constituent of the human immune response to HIV.
cDNA cloning of functional T cell receptor γ/δ chains expressed in human peripheral blood lymphocytes
Authors: Mami‐Chouaib Fathia , Jitsukawa Setsuko , Faure Florence , Vasina Bruno , Genevee Catherine , Hercend Thierry , Triebel FréDéRic
Published: March 2007

We have identified in earlier studies two Vδ rearrangements corresponding to a 4.5‐kb Eco RI fragment detected with a Vδ1 probe and to a 7‐kb Eco RI band detected with a Vδ2 probe. These rearrangements have been found in two human T cell clones, F6C7 and G6, displaying surface phenotypes unfrequent in human peripheral blood, namely TiγA+ BB3‐ (F6C7) and TiγA‐ BB3+ (G6). Herein, we report the sequences of the functional transcripts encoded by these rearranged genes and show that the 4.5‐and the 7‐kb Eco RI fragments correspond to V1/D3/Jδ3 and to V2/D3/Jδ3 recombinations, respectively. In addition, we have sequenced the V2/D3/J1/Cδ transcripts expressed in two clones, AB12 and VTC, which have a TiγA+ BB3+ surface phenotype corresponding to that of most γ/δ peripheral lymphocytes. Analyses of the δ transcripts expressed by these four cells further strengthen the hypothesis that anti‐BB3 and anti‐δ‐TCS‐1 monoclonal antibodies recognize a Vδ2‐ and a V1/(D)/Jδ1‐encoded epitope, respectively. Sequence of the γ transcripts expressed by AB12 and F6C7 cells shows that they encode a V9/JP/Cγ1 chain. Finally, we confirm that non‐combinatorial diversity in the γ and δ proteins is generated by both junctional flexibility and N‐region addition without any somatic mutation.
Characterization of three functional sites in αβ1 DR of DRw13. all three sites are potentially involved in major histocompatibility complex‐peptide interaction
Authors: Sterkers Ghislaine , Tiercy Jean‐Marie , Zeliszewski Dominique , Levy Jean‐Paul , Mach Bernard
Published: March 2007

An HLA‐DR product encoded by the HLA‐DRw13/Dw19 haplotype has been identified as the HLA class II molecule involved in antigen presentation to several influenza‐specific helper T cell clones. Three different functional sites were identified on this molecule by comparing the structure of HLA‐DR products of known sequences and their ability to efficiently present foreign antigen to the T cell clones. These functional sites were mapped on the recently proposed three‐dimensional structure of HLA class II molecules. From their position, these sites are all potentially involved in HLA‐peptide interaction and capable of affecting the binding and/or the conformation of the foreign peptide. This suggests that polymorphic residues essential in major histocompatibility complex restriction are mostly involved in peptide binding.
Identification of interleukin 4 receptor‐associated proteins and expression of both high‐ and low‐affinity binding on human lymphoid cells
Authors: Foxwell Brian M. J. , Woerly Gaetane , Ryffel Bernhard
Published: March 2007

Interleukin 4 (IL 4) produced by activated T cells expresses its biological effects on T and B lymphocytes by binding to specific membrane receptors. Cross‐linking of human recombinant 125I‐IL 4 to peripheral blood mononuclear cells identifies a trimolecular complex consisting of a 65/70‐kDa doublet and a 110‐kDa protein. Scatchard analysis reveals about 300 IL 4 binding sites/cell on resting cells with an equilibrium binding constant (Kd) of ∼ 100 pM. Stimulation by anti‐CD3 antibodies causes an up‐regulation of IL 4 receptors by a factor of 2 to 3 without any change in binding affinity. In addition to this high‐affinity binding site a second class of a previously unidentified, low‐affinity receptor (Kd ∼ 30 nM, ∼ 9000 sites/cell) is expressed on resting lymphocytes. The number of low‐affinity binding sites for IL 4 also increases twofold upon cell activation. Exogenous IL 4 enhances the expression of its receptor on resting lymphocytes and this effect is further increased by anti‐CD3 activation. Binding of IL 4 to its receptor is specific, being only inhibited by IL 4 and not by IL 2.

By contrast, the gibbon leukemia cell line MLA 144 expresses only high‐affinity receptors for IL 4. Cross‐linking studies reveal a 45/50‐kDa IL4 receptor‐associated doublet in addition to the three proteins identified in human peripheral blood mononuclear cells. The functional significance of the different proteins composing the receptor for IL 4 is discussed.
Major histocompatibility complex – dependent T cell epitopes of lymphocytic choriomeningitis virus nucleoprotein and their protective capacity against viral disease
Authors: Schulz Manfred , Aichele Peter , Vollenweider Maja , Bobe Frank W. , Cardinaux Francis , Hengartner Hans , Zinkernagel Rolf M.
Published: March 2007

In mice the immune response to infection with lymphocytic choriomeningitis virus (LCMV), a member of the arenavirus family, is mainly based on the activity of cytotoxic T cells. The immunogenic epitopes of the viral nucleoprotein recognized by cytotoxic T cells in various inbred strains of mice were defined. These epitopes were located in H‐2d and H‐2q mice in the amino‐terminal region and in H‐2b mice in the carboxy‐terminal region of the nucleoprotein. A detailed analysis with synthetic peptides allowed the definition of a common epitope of 9 amino acids in H‐2d and H‐2q mice and of about 15 amino acids in H‐2b mice. These T cell epitopes were all recognized in association with H‐2 D or L transplantation antigen. The protective capacity of recombinant vaccinia viruses expressing these epitopes was documented by assaying prevention of virus replication, protection against LCM and prevention of the local footpad swelling reaction. Thus, distinct T cell epitopes on the same internal viral protein mediate protection in a major histocompatibility complex‐restricted manner.
Interleukin 5 regulation of peritoneal Ly‐1 B lymphocyte proliferation, differentiation and autoantibody secretion
Authors: Wetzel Gayle Delmonte
Published: March 2007

The in vitro effects of recombinant interleukin (IL) 5 on proliferation and maturation of mouse Ly‐1 B cells were studied. Most freshly isolated peritoneal Ly‐1 B cells expressed high levels of IL5 receptor (R). Limiting dilution analyses showed that mitogens could reveal IL5 responsiveness in more than half of low density peritoneal Ly‐1 B cells. IL5 was able not only to increase the proportion of these Ly‐1 B cells induced to proliferate, but it also shifted the clone size distribution of proliferating cells towards larger clone sizes. Splenic Ly‐1 B cells also proliferated in response to mitogens plus IL5. Spontaneous and polyclonal activator‐induced plaque‐forming cell responses of Ly‐1 B cells were increased by IL5. Furthermore, IL 5 increased the frequency of peritoneal Ly‐1 B cells induced to secrete certain autoantibodies. IL5 was certainly the agent responsible since its effects on both proliferation and differentiation were inhibited by either anti‐IL5R monoclonal antibodies or by anti‐IL5 monoclonal antibodies. Hence, Ly‐1 B cells, IL5 and the IL5R appear to constitute a system of cellular proliferation, differentiation and some autoantibody production. Strategies specifically targeting the interleukin and receptor elements of this system might afford external control of these cellular responses.
Elevated temperature regulates tumor necrosis factor‐mediated immune killing
Authors: Gromkowski Stanislaw H. , Yagi Junji , Janeway Charles A.
Published: March 2007

The effect of elevated temperature (heat/fever) on the regulation of inflammatory processes was studied in an in vitro system. Since tumor necrosis factor (TNF), a central mediator in inflammation, is both a pyrogen and a cytokine capable of inducing the death of certain cells, we examined the relationship between heat shock and TNF‐mediated immune killing. Heat shock of WEHI‐164 fibrosarcoma cells significantly (and transiently) decreased the sensitivity of these cells to recombinant TNF‐mediated lysis and to class II major histocompatibility complex‐specific, TNF‐secreting inflammatory T cell‐mediated lysis. Incubating inflammatory T lymphocytes in elevated temperature transiently abolished their lytic potential and their ability to secrete TNF. Our data show that the pyrogen activity of TNF could control cytolytic processes during inflammation both by inducing protective protein(s) synthesis in target cells and by arresting TNF secretion by effector T lymphocytes.
Cyclic AMP can enhance mouse B cell activation by regulating progression into the late G1/S phase
Authors: Li Yue Sheng , Kouassi Edouard , Revillard Jean‐Pierre
Published: March 2007

This study demonstrates that several substances which raise intracellular adenosine 3',5'‐cyclic monophosphate (cAMP) levels in different ways were able to enhance both RNA and DNA synthesis in mouse purified B cells co‐stimulated by the protein kinase C activator phorbol 12‐myristate 13‐acetate and the Ca2+ ionophore ionomycin, while earlier activation events were not modified. These included early changes in cell size and chromatin decondensing demonstrated by light scatter properties at narrow and 90° angles, increase in Ia expression and loss of surface IgD. We concluded that cAMP can up‐regulate mouse B cell activation by controlling progression into the late G1 (G1B)/S phase, but not transition from G0 to early G1 (G1A). Furthermore, because cAMP could synergize with ionomycin but not with phorbol 12‐myristate 13‐acetate to induce RNA and DNA synthesis, we proposed that the cAMP effects in this model may be related to the protein kinase C pathway.
Embryonic neural cell adhesion molecules on human natural killer cells
Authors: Husmann Matthias , Pietsch Torsten , Fleischer Bernhard , Weisgerber Christoph , Bitter‐Suermann Dieter
Published: March 2007

The neural cell adhesion molecules (NCAM) are surface glycoproteins that were first described in brain tissue. NCAM mediate adhesion in a variety of cell‐cell interactions. In the present study we show that the so‐called “embryonic” NCAM, i.e., the highly polysialylated forms of these proteins, are expressed on natural killer cells and some CD3+ cells in man. Homotypic binding of NCAM, believed to be of importance for cell‐cell adhesion in neural tissues, appears not to be essential for NK cell‐mediated killing. Yet, NCAM might be involved in NK cell migration, homing or related functions.
Human complement factor H: An additional gene product of 43kDa isolated from human plasma shows cofactor activity for the cleavage of the third component of complement
Authors: Misasi Roberta , Huemer Hartwig P. , Schwaeble Wilhelm , Sölder Elisabeth , Larcher Clara , Dierich Manfred P.
Published: March 2007

In addition to the 150‐kDa factor H protein, we have previously described a 43‐kDa factor H molecule in human plasma, which probably represents a translational product of the additional 1.8‐kb mRNA for factor H. This factor H was isolated from human plasma by means of immunoaffinity chromatography and high‐performance liquid chromatography. When tested for its functional activity, this purified 43‐kDa H protein was shown to act as cofactor for factor I ‐ mediated cleavage of fluid‐phase C3b to iC3b.
Internalization of ige receptors on rat basophilic leukemic cells by phorbol ester. comparison with endocytosis induced by receptor aggregation
Authors: Ra Chisei , Furuichi Kiyoshi , Rivera Juan , Mullins J. Michael , Isersky Chaviva , White Kenneth N.
Published: March 2007

Phorbol myristate acetate (PMA) can induce a rapid and significant decrease in the expression of IgE receptors on RBL‐2H3 cells. Fluorescence microscopy confirmed that the down‐regulation is due to internalization of receptors. The endocytotic response to PMA shares several characteristics with endocytosis induced by immunochemical aggregation of surface‐bound monomeric IgE: the rates of internalization both have a t1/2 of about 5 min, a maximum of 35% of the surface‐bound IgE can be endocytosed by the action of PMA (50% by receptor aggregation), endocytosis is sustained for at least up to 60 min, neither stimulus requires extracellular Ca2+ and endocytosis induced by either stimulus is an active process, i.e., is dependent on temperature and cellular energy. Biochemical studies revealed some differences between the endocytotic responses to the two stimuli. After prolonged treatment of cells with dexamethasone, only endocytosis induced by PMA is inhibited. Cells depleted of protein kinase C by prolonged exposure to PMA can sustain a significant endocytotic response to aggregation of IgE receptors, but become completely desensitized to PMA. These data suggest that different biochemical pathways mediate the signals from the two stimuli and that protein kinase C is directly involved in endocytosis induced by PMA but does not have a major role in endocytosis induced by receptor aggregation.
B cell repertoire in adult antigen‐free and conventional neonatal balb/c mice
Authors: Bos Nicolaas A. , Meeuwsen Cornelia G. , Wijngaarden Peter Van , Benner Robbert
Published: March 2007

Hybridomas were derived from lipopolysaccharide‐reactive splenic B cells of adult germ‐free BALB/c mice fed a chemically defined ultrafiltered “antigen‐free” diet (GF‐CD) and from splenic B cells of 5‐day‐old conventional (CV‐NEO) BALB/c mice. The monoclonal antibodies (mAb) from both collections of hybridomas were tested for reactivity against a large panel of antigens of exogenous and endogenous origin. As a source of natural exogenous antigens 36 different bacteria and 9 different viruses were used, while as endogenous antigens frozen tissue sections of stomach, liver and kidney, the Hep‐2 cell line and the anti‐idiotopic mAb Ac38 and Ac146 were used. In both collections of mAb approximately 70% reacted with one or more bacterial antigens, while no reactivity could be detected against the viral antigens. Of the GF‐CD and CV‐NEO hybridomas, 16% and 19%, respectively, reacted with one or more frozen tissue sections. Overall 56% and 68% of the GF‐CD and CV‐NEO hybridomas, respectively, were producing multireactive antibodies reactive to several exogenous and/or endogenous antigens.

Among the GF‐CD hybridomas a correlation was found between multireactivity and the usage of the VH gene family PC7183. In CV‐NEO hybridomas, however, the preferential utilization of the VH gene family PC7183 was found among both mono‐and multireactive hybridomas. The results suggest (a) that the actual B cell repertoire of neonatal mice consists of a large proportion of multireactive B cells which are reactive with autoantigens and bacterial antigens, but not viral antigens and (b) that in antigen‐deprived mice the neonatal repertoire is largely preserved during maturation of the mice.
Detection of soluble class i molecules (non hla‐a or hla‐b) in serum, spleen membranes and lymphocytes in culture
Authors: Villar Luisa M. , Roy Garbine , Lázaro Iciar , Álvarez‐Cermeno José C. , González Miguel , Brieva José A. , Del La Sen María L. , Leoro Galo , Bootello Alfredo , González‐Porqué Pedro
Published: March 2007

Soluble major histocompatibility complex class I molecules (sHLA) present in human serum can be resolved by gel filtration into two different peaks with an apparent molecular mass of about 200 kDa (30% of the total) and 50–60 kDa (60%–70%). The serological analysis of the peaks shows that A or B specificities can only be detected in the 200 kDa peak while both are recognized by the monomorphic W6/32 monoclonal antibody (mAb) and anti‐β2‐microglobulin mAb. Such sHLA (non HLA‐A or ‐B) molecules are released from human spleen membranes upon incubation at 37 °C and have been purified by affinity chromatography with mAb W6/32 bound to Sepharose. The molecular mass analysis by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the sHLA (non HLA‐A or ‐B) and of the classical HLA‐A or ‐B antigens still bound to the membranes and purified from the same membranes after detergent solubilization does not show a significant difference, indicating that sHLA do not represent proteolytic fragments of the classical HLA‐A or ‐B antigens. The presence of sHLA (non HLA‐A or ‐B) has also been detected in the supernatants of lymphocyte cultures and increases dramatically upon stimulation by mitogens. The effect of pokeweed mitogen, phytohemagglutinin, Staphylococcus aureus Cowan strain and phorbol 12‐myristate 13‐acetate on the secretion of sHLA has been studied. The molecular mass of the secreted sHLA (detected using [14C]leucine) is compared with the classical transmembrane proteins.
Only dfl16, dsp2, and dq52 gene families exist in mouse immunoglobulin heavy chain diversity gene loci, of which dfl16 and dsp2 originate from the same primordial dh gene
Authors: Ichihara Yoshikazu , Hayashida Hidenori , Miyazawa Sanzo , Kurosawa Yoshikazu
Published: March 2007

In mice, 12 germ‐line DH genes belonging to three different families (DQ52, DSP2 and DFL16) have been identified. The DH genes other than DQ52 are clustered in the 60 kb‐long region located between VH and JH genes. Since there are seven DH gene families (DHQ52, DXP, DA, DK, DN, DM and DLR) in humans, we tried to identify new DH gene families in the 60 kb‐long region using human DH gene probes. Mouse and human DH genes showing the highest similarity were mouse DFL16 genes and human DA genes. Southern hybridization of the mouse clones covering the 60‐kb region with human DH probes did not detect any other DH genes. Nucleotide sequence analysis of the 4.0‐kb fragment containing the DFL16.1 gene confirmed this conclusion. Comparison of the 12 germ‐line DH genes and more than 150 somatic DH sequences also indicated that there are not more germ‐line DH genes in the mouse genome. Moreover, comparison of nucleotide sequences of DFL16.1 and DSP2.2 genes and their surrounding regions suggests that both DH gene families originate from the same primordial DH gene. Using the flanking sequences of both DH genes, the divergence date between DFL16 and DSP2 genes was estimated at around 37 million years ago.
Allo‐class i‐reactive cd4‐cd8‐ t cell hybridomas recognize the conformational change of class i molecule resulting from the exchange of the α3 domain
Authors: Yamamoto Junji , Tanabe Manabu , Minami Mutsuhiko , Kano Kyoichi , Takiguchi Masafumi
Published: March 2007

H‐2Kb‐reactive, interleukin (IL) 2‐producing T cell hybridomas possessing neither CD8 nor CD4 molecules were employed for the study of allorecognition on interspecies hybrid antigens by T cells in the absence of an influence of these accessory molecules. Both HTB176.10 and HTB177.2 T cell hybridomas reacted with KbKbB7 hybrid antigens as well as Kb antigens and they produced more IL2 in response to Kb antigens than KbKbB7 hybrid antigens. IL2 production of these T cell hybridomas was dependent on the surface expression level of Kb molecules on stimulators. Therefore, L cells expressing almost equivalent levels of Kb or hybrid antigens were selected for further functional studies by these T cell hybridomas. They apparently produced less IL2 in response not only to interspecies hybrid antigens but also to interspecies hybrid antigens in response to Kb antigens. These results indicated that T cell hybridomas recognized the conformational change of class I molecule resulting from the exchange of the α3 domain via their T cell receptors.