bluuebunny/crossref_metadata_2025 huggingface dataset:
Idiotypic cascade in the human high molecular weight melanoma‐associated antigen system: Fine specificity and idiotypic profile of anti‐anti‐idiotypic monoclonal antibodies
Authors: Yang Hong , Chen Zhi Jian , Kageshita Toshiro , Yamada Masanobu , Ferrone Soldano
Published: March 2007

The mouse anti‐idiotypic (anti‐id) monoclonal antibody (mAb) MK2–23 bears the internal image of the determinant defined by the syngeneic immunizing anti‐human high molecular weight melanoma‐associated antigen (HMW‐MAA) mAb 763.74, since it induces anti‐HMW‐MAA antibodies in syngeneic and xenogeneic hosts. To dissect the humoral immune response induced by mAb MK2–23 at the clonal level, 1351 hybridomas were generated from a BALB/c mouse immunized with mAb MK2–23. Serological and immunochemical assays showed that the anti‐anti‐idiotypic (anti‐anti‐id) mAb GH827, GH1002 and GH1081 react only with the immunizing anti‐id mAb MK2–23 and that the anti‐anti‐id mAb GH149, GH368, GH464, GH518, GH586, GH704, GH786 and GH1151 react with both mAb MK2–23 and HMW‐MAA. The eight HMW‐MAA binding anti‐anti‐id mAb resembled mAb 763.74 in their reactivity patterns with a panel of cell lines, although the extent of reactivity was lower. Staining of surgically removed melanoma lesions detected subtle differences in the reactivity patterns of the eight HMW‐MAA binding anti‐anti‐id mAb. This finding in conjunction with the differential ability of the eight anti‐anti‐id mAb to inhibit the binding of anti‐HMW‐MAA mAb 763.74 to melanoma cells and to mAb MK2–23 and with the different avidity of the binding to mAb MK2–23 suggest diversity in the fine specificity of the eight HMW‐MAA binding anti‐anti‐id mAb. Like mAb 763.74, the eight HMW‐MAA binding anti‐anti‐id mAb express the idiotopes recognized by the anti‐id mAb MK2–23 and MK2‐120. In contrast, the three anti‐anti‐id mAb GH827, GH1002 and GH1081, which do not bind HMW‐MAA binding anti‐anti‐id mAb to the antibodies elicited by the membrane‐bound HMW‐MAA corroborates the validity of the use of anti‐id mAb MK2‐23 as an immunogen to implement active specific immunotherapy in patients with malignant melanoma.
Pre‐exposure of murine macrophages to lipopolysaccharide inhibits the induction of nitric oxide synthase and reduces leishmanicidal activity
Authors: Severn Alison , Xu Damo , Doyle Jacqueline , Leal Ludmila M. C. , O'Donnell Catherine A. , Brett Sara J. , Moss David W. , Liew Foo Y.
Published: March 2007

Murine macrophages produce nitric oxide (NO) from L‐arginine on stimulation with lipopolysaccharide (LPS), alone or with interferon‐γ (IFN‐γ). The effect of incubation of macrophages with low concentrations of LPS on NO synthesis on subsequent stimulation was investigated, using a murine macrophage cell line, J774, and peritoneal macrophages from CBA mice. Cells which had been incubated with LPS produced significantly lower amounts of NO, and expressed lower levels of NO synthase activity, following stimulation with IFN‐γ and LPS, or with a high concentration of LPS. This effect was not reversed by tumor necrosis factor‐α. The ability of CBA macrophages to kill the intracellular parasite Leishmania major was markedly reduced by pre‐incubation with LPS. Reduced NO production by macrophages previously exposed to LPS is a manifestation of endotoxin tolerance, and may represent an important means of regulation of NO synthesis and thus a survival mechanism for intracellular parasites.
Induction of virus‐specific cytotoxic T lymphocytes in vivo by liposome‐entrapped mRNA
Authors: Martinon Frédéric , Krishnan Sivadasan , Lenzen Gerlinde , Magné Rémy , Gomard Elisabeth , Guillet Jean‐Gérard , Lévy Jean‐Paul , Meulien Pierre
Published: March 2007

The induction of anti‐influenza cytotoxic T lymphocytes (CTL) in vivo by immunizing mice with liposomes containing messenger RNA (mRNA) encoding the influenza virus nucleoprotein (NP) is described. NP mRNA, obtained by in vitro transcription, was encapsulated into simple cholesterol/phosphatidylcholine/phosphatidylserine liposomes by the detergent removal technique. The dependance of the route of mRNA‐liposomes delivery on CTL induction was studied. The CTL induced were identical to those obtained in vivo with infectious virus in terms of specificity, lysing both peptide‐sensitized and virus‐infected targets. Furthermore, with the same mRNA‐liposome preparation, virus‐specific CTL responses could be also elicited in mice of three different haplotypes each of them known to present a distinct NP peptide in an MHC‐restricted fashion. The relevance of these results in the context of vaccine development is discussed.
The tum− antigens P91A and P198 derive from proteins located in the cytosolic compartment of cells
Authors: Verlant Vincent , Amar‐Costesec Alain , Godelaine Danièle , Turu Christine , Van Pel Aline , De Plaen Etienne , Dautry‐Varsat Alice , Beaufay Henri
Published: March 2007

To characterize the proteins P91Ap and P198p, of which mutants generate the tum− antigens P91A and P198, respectively, rabbit antisera were raised with ovalbumin‐coupled synthetic peptides that correspond to their respective C terminus. In immunoadsorption tests using immobilized protein A the antisera recognized the translation products synthesized by rabbit reticulocyte lysates programmed with the SP6 polymerase transcripts of the P91A and P198 cDNA. The presence of the two proteins was demonstrated by SDS‐PAGE and immunoblotting in all the mouse cells and organs examined. P91Ap is a constituent of the cytosol; despite a remarkable homology to the Drosophila diphenol oxidase DOX‐A2, it separates from murine catechol oxidase activity in rate zonal sedimentation analysis. P198p is a ribosomal constituent, or a factor firmly linked to both the free and membrane‐bound ribosomes. These subcellular localizations strengthen other evidence that the antigens presented to T lymphocytes by class I products of the major histocompatibility complex derive from proteins of the cytosol, or in direct contact with it.
Methylation status of immunoglobulin x e segments correlates with their recombination potential
Authors: Goodhardt Michele , Cavelier Patricia , Doyen Noëlle , Kallenbach Sacha , Babinet Charles , Rougeon François
Published: March 2007

We have previously shown that unlike endogenous ϰ genes, unrearranged ϰ transgenes undergo Vϰ‐Jϰ recombination in T as well as B cells of transgenic mice. To determine whether the difference in recombination specificity of the transgenic and endogenous ϰ genes is associated with differences in DNA structure, the methylation status of the endogenous genes and three unrearranged ϰ transgenes was compared. The Jϰ‐Cϰ locus of the transgenes was found to be hypomethylated in all tissues of the transgenic mice. In contrast, methylation of the endogenous ϰ genes was tissue and developmentally regulated. Hypomethylation of the endogenous Jϰ‐Cϰ region occurs only in cells of the B lineage undergoing, or having completed ϰ gene recombination. Transfection of fibroblasts from transgenic and control mice with the recombination activating genes, Rag1 and Rag2, led to a high level of rearrangement of the hypomethylated transgenic, but not the endogenous ϰ genes. These results suggest that hypomethylation defines an accessible state of the ϰ locus and that methylation/demethylation could be involved in the control of ϰ gene rearrangement during lymphocyte differentiation.
Thymus‐derived cytokine(s) including interleukin‐7 induce increase of T cell receptor α/β+ CD4−CD8− T cells which are extrathymically differentiated in athymic nude mice
Authors: Kenai Hiroyuki , Matsuzaki Goro , Nakamura Takao , Yoshikai Yasunobu , Nomoto Kikuo
Published: March 2007

Extrathymic T cell differentiation pathways have been reported, although the thymus is the main site of T cell differentiation. The thymus is also known to produce several cytokines that induce proliferation of thymocytes. In the present study, we investigated the influence of thymus‐derived cytokines on extrathymic T cell differentiation by intraperitoneal implantation with a diffusion chamber which encloses fetal thymus (we named it fetal thymus‐enclosed diffusion chamber, FTEDC) in athymic BALB/c nu/nu mice. Increase in number of T cells bearing T cell receptor (TcR) α/β was detected in lymph nodes and spleens of FTEDC‐implanted nude mice 1 week after implantation, whereas no such increase was detected in control nude mice implanted with a diffusion chamber without thymus. The FTEDC‐induced increase of T cells was suppressed by intraperitoneal injection of anti‐interleukin‐7 monoclonal antibody (mAb). The TcR α/β T cells in FTEDC‐inplanted BALB/c nu/nu mice preferentially expressed Vβ11, although Vβ11‐positive T cells are deleted in the thymus of euthymic BALB/c mice by clonal elimination of self‐superantigen Dvb 11‐specific T cells. TcR α/β T cells in FTEDC‐implanted nude mice were of CD4−CD8− phenotype and showed no proliferative response against anti‐TcR monoclonal antibody stimulation. These results suggest that the thymus can induce extrathymic T cell differentiation through the influence of thymus‐derived cytokine(s) including interleukin‐7, and that such extrathymically differentiated T cells have acquired only a little or no ability for proliferation when they recognize antigen by their TcR.
Signaling through the LFA‐1 leucocyte integrin actively regulates intercellular adhesion and tumor necrosis factor‐α production in natural killer cells
Authors: Melero Ignacio , Balboa Maria A. , Aionso Jose L. , Yagüe Enriqueta , Pivel Juan P. , Sanchez‐Madrid Francisco , López‐Botet Miguel
Published: March 2007

The LFA‐1 leucocyte integrin is known to participate in natural killer (NK) cytolytic activity, mediating effector target interactions. The possibility that LFA‐1 may also play an active regulatory role in NK cells has been explored. To this end, we have employed a monoclonal antibody (HP1N) raised against recombinant interleukin‐2 (rIL‐2)‐activated NK cells, which recognizes the α chain of the LFA‐1 heterodimer (CD11a). In contrast to other anti‐CD11a mAb the HP1N and its F(ab)2 fragment did not affect NK cell‐mediated cytotoxicity and triggered a strong homotypic adhesion of NK cells and other LFA‐1+ cells. Cellular aggregation was inhibited by anti‐CD18 mAb, anti‐ICAM‐1 mAb, and other anti‐CD11a mAb. Remarkably, the HP1N mAb was also shown to induce tumor necrosis factor‐α (TNF‐α) production from NK cells upon costimulation with anti‐CD16 mAb. Such an effect appeared to be independent from homotypic adhesion since it took place in Mg2+‐free medium, where NK cell aggregation was inhibited. Moreover, incubation with the HP1N mAb triggered a Ca2+ influx into the cytosol; this effect was clearly observed upon cross‐linking of cell bound HP1N and was also substantiated with other anti LFA‐1 (CD11a and CD18) mAb. Taken together these results indicate that the LFA‐1 molecule is capable of transducing signals in NK cells, which regulate the intercellular interaction with its ligand, and enhance the activation via Fey receptor type III.
Allogeneic recognition of class I molecules: Anti‐H‐2Ld repertoire of H‐2b mice includes T cells recognizing mutant class II H‐2b (Abm12) molecules
Authors: Frangoulis Bernard , Urbero Bruno , Pla Marika
Published: March 2007

Two major histocompatibility complex (MHC) class I‐reactive T cell clones derived from H‐2b mice, generated against the allogeneic Ld molecule, were found to recognize the H‐2b class II mutant Abm12 molecule as well. In addition, these clones also recognize the class II As molecule, and display a class II‐dependent reactivity to staphylococcal enterotoxin B. Neither the class I nor the class II alloreactivities of the clones were found to be dependent on other MHC molecules. Both clones express CD4+CD8− phenotypes. The CD4 molecule appears to be involved in their class II reactivity, while little or no role for CD4 could be detected in the class I reactivity. This is the first report of a class I/class II cross‐reactivity being mediated by CD4+ T cells. The structural basis for this cross‐reactivity is discussed.
The expressed T cell receptor V gene repertoire of rheumatoid arthritis monozygotic twins: Rapid analysis by anchored polymerase chain reaction and enzyme‐linked immunosorbent assay
Authors: Kohsaka Hitoshi , Taniguchi Atsuo , Chen Pojen P. , Ollier William E. R. , Carson Dennis A.
Published: March 2007

Because of heterogeneity in the outbred human population, it has been difficult to determine the genetic factors that influence the expressed T cell receptor (TcR) repertoire in autoimmune diseases. To overcome this problem, we have developed a combination of anchored polymerase chain reaction (APCR) and enzyme‐linked immunosorbent assay (ELISA) that can accurately assess TcR V gene frequencies in numerous clinical samples. The results are independent of amplification efficiency, and Vgene usage can be readily analyzed with an ELISA plate reader and associated software. Using this method, the TcR Vβ gene repertoires in peripheral lymphocytes from nine sets of identical twins, normal, concordant or discordant for rheumatoid arthritis (RA), were studied. The TcR Vγ results were compared with TcR Vβ frequencies in the same specimens as determined by APCR‐ELISA and cDNA sequence analysis. The results showed a marked similarity in the TcR Vβ gene repertoires between identical twins, compared to unrelated subjects (p<0.05) whether or not they were concordant or discordant for RA. In contrast, the TcR Vy gene repertoires in the monozygotic twins differed as much as in controls. The data imply that (a) the human TcR Vβ gene repertoire in peripheral blood is genetically controlled, whereas (b) the TcR Vβ gene repertoire is primarily influenced by environmental stimuli, and (c) RA causes no consistent change in TcR Vβ repertoire of peripheral blood. The APCR‐ELISA method, in the context of large‐scale family and population studies, should facilitate a more precise delineation of the genetic factors that regulate human TcR Vβ expression.
Fcϵ receptor‐positive cells are a major source of antigen‐induced interIeukin‐4 in spleens of mice infected with Schistosoma mansoni
Authors: Williams Megan E. , Kullberg Marika C. , Barbieri Susan , Caspar Patricia , Berzofsky Jay A. , Seder Robert A. , Sher Alan
Published: March 2007

When cultured in vitro with either mitogen or parasite antigens, spleen cells from mice infected with Schistosoma mansoni produce significantly higher levels of IL‐4 than splenocytes from control animals. Previous studies suggested that this increase in IL‐4 production occurs because of a selective expansion of T helper type 2 (Th2) cells in infected mice. However, these experiments employed unfractionated spleen populations rather than purified T lymphocytes. Here we demonstrate that T‐depleted spleen cells from infected animals synthesize high levels of interleukin‐4 (IL‐4), but no IL‐5 when stimulated with parasite antigen in vitro. Nevertheless, when purified by sorting, T cells and non‐B, non‐T (NBNT) populations produced similar amounts of IL‐4 in response to parasite antigen. The IL‐4 producing NBNT cells were found to belong to an Fee receptor (FceR)‐positive population which after sort purification produced high levels of IL‐4 (between 1000 and 2000 U of per 5 × 103 cells). FACS analysis revealed that these FceR+ cells make up 0.53% of splenic NBNT cells in control animals while in 8‐9‐week‐infected animals they increase to 3.8% of that population. In contrast, in mice with 8‐week unisexual worm infections these cells comprise only 1.71% of NBNT cells, indicating that eggs are a major stimulus of the response. The expansion of FceR+ cells and their production of IL‐4 could be an important factor regulating the selection and induction of different CD4+ subsets in schistosome‐infected hosts.
The intronic immunoglobulin ϰ gene enhancer acts independently on rearrangement and on transcription
Authors: Kallenbach Sacha , Babinet Charles , Pournin Sandrine , Cavelier Patricia , And Michele Goodhardt , Rougeon François
Published: March 2007

It is not known which cis‐acting elements regulate ϰ gene rearrangement. However, the onset of rearrangement coincides with the onset of transcription suggesting that common regulatory elements are used. We have investigated the role of the intronic enhancer on rearrangement using mice transgenic for ϰ minigenes. The rabbit ϰ enhancer, which is defective in activation of transcription in mouse cells, allows a high level of rearrangement. However, transgenes which possess no enhancer region at all are rearranged a hundred times less compared to transgenes which possess the rabbit enhancer. Our results suggest that while the enhancer region can activate rearrangement as well as transcription, its action on both phenomena can be uncoupled.
Establishment of apoptosis‐inducing monoclonal antibody 2D1 and 2D1‐resistant variants of human T cell lines
Authors: Takahashi Shuji , Sato Noriyuki , Takayama Shin‐Ichi , Ichimiya Shingo , Satoh Masaaki , Hyakumachi Naomi , Kikuchi Kokichi
Published: March 2007

A monoclonal antibody (mAb), 2D1(IgM), was identified for its anti‐proliferative effect on human T leukemia cell line, SUP‐T13. The cells bound with 2D1 showed DNA ladder patterns of oligonucleosomes, demonstrating apoptosis. Peripheral mononuclear cells activated by phytohemagglutinin or OKT3 induced expression of 2D1 antigen and were growth‐inhibited by the antibodies. Amsong the cell lines tested, T cell lines tended to be growth‐inhibited by the antibodies. Epstein‐Barr virus‐transformed B cells were reactive with 2D1, but were not growth‐inhibited by the antibodies. We established stable 2D1‐resistant variants LAC2D1R and JKT2D1R from the original SUP‐T13 and Jurkat T cell lines, respectively. These variant cells demonstrated phenotypes identical to the original cells, including reactivity to 2D1 and expression of cytoplasmic Bc1‐2 protein. The 2D1‐resistant cells were as sensitive as the original cells to the other apoptosis‐inducing stimuli, such as γ‐irradiation or calcium ionophore A23187. However, the 2D1‐resistant variants were also insensitive to anti‐Fas, another apoptosis‐inducing mAb. Binding of 2D1 was blocked by anti‐Fas mAb, suggesting that 2D1 reacts with an epitope of human Fas molecules. The present results demonstrate that a 2D1‐reactive, but not 2D 1‐sensitive, population may exist in highly 2D1‐sensitive human leukemia T cells and that pairs of 2D 1‐sensitive and 2D1‐resistant cells are useful in the biochemical analysis of Fas‐mediated apoptosis in human T cells.
Role of liver endothelial and Kupffer cells in clearance of human Clq in rats
Authors: Coremans Ida E. M. , Rogers Willy M. J. M. , Stad Robert K. , van der Voort Ellen A. M. , Prins Frans A. , van Rooijen Nico , Breedveld Ferdinand C. , Daha Mohamed R.
Published: March 2007

In the present study the contribution of rat liver endothelial cells (EC) and Kupffer cells (KC) in the clearance of human (hu) Clq in rats was investigated. In untreated rats and rats depleted from KC the clearance kinetics and the tissue distribution of hu Clq were measured. In untreated rats, the clearance of hu Clq occurred in a monophasic manner with a half‐life of 66 ± 26.7 min. The clearance of hu Clq in KC‐depleted rats was delayed significantly (p<0.001) and occurred with a half‐life of 217 ± 78.8 min. Fifteen min after injection, 11 ± 3.5% of hu Clq was found in the liver of untreated rats and 8 ± 1.4% was found in the liver of KC‐depleted rats. The percentage non‐trichloroacetic acid precipitable activity in the circulation, as a measure for degradation of Clq, reached a level of 11.6 ± 5.6% at 240 min in untreated rats compared with 4.6 ± 5.8% in KC‐depleted rats. Double immunofluorescence staining 5 min after administration of Clq in untreated rats, revealed that Clq was associated with KC and EC in the liver. Fifteen minutes after i.v. injection of hu Clq, there was an uptake of Clq in the hepatocytes. In KC‐depleted rats, 5 min after administration of hu Clq, Clq was bound to the EC. Fifteen minutes after injection, Clq was also found in the hepatocytes. Electron microscopical studies revealed that Clq binds to EC, and that it is internalized in the hepatocytes and KC. The clearance of hu Clq in untreated rats was inhibited by preadministration of high concentrations of bovine C1q. These data show that rats depleted from KC are able to bind, internalize and degrade Clq, and that EC may play a role in the handling of Clq and Clq bound to immune complexes.
An antigen processing polymorphism revealed by HLA‐B8‐restricted cytotoxic T lymphocytes which does not correlate with TAP gene polymorphism
Authors: Rowland‐Jones Sarah L. , Powis Stephen H. , Sutton Julian , Mockridge Ian , Gotch Frances M. , Murray Nick , Hill Ann B. , Rosenberg William M. , Trowsdale John , McMichael Andrew J.
Published: March 2007

In previous studies of antigen presentation through HLA‐B27, we identified a healthy person whose lymphoblastoid cells do not present three B27‐restricted viral epitopes to specific cytotoxic T lymphocytes (CTL), despite adequate cell surface expression of HLA‐B2702 of normal sequence. Similar findings were observed in all members of his family sharing the HLA‐A3‐B2702 haplotype. The original donor, NW, carries HLA‐B8 on his other class I haplotype, which his daughter, HW, has inherited. We now report a failure to present an HLA‐B8‐restricted epitope from influenza nucleoprotein following viral infection of NW cells, although exogenous added peptide is still presented normally. However, cells from HW, which do not carry the A3‐B2702 haplotype, present the expected epitope after viral infection. Another B8‐restricted epitope, from human immunodeficiency virus‐gag, is presented equally well by both cell lines when infected with gag‐vaccinia. This antigen processing phenotype does not correlate with any of the known human TAP‐1 and TAP‐2 polymorphisms.
Variable binding affinities of listeriolysin O peptides for the H‐2Kd class I molecule
Authors: Wipke Brian T. , Jameson Stephen C. , Bevan Michael J. , Pamer Eric G.
Published: March 2007

Previously we used the peptide‐binding motif for the murine class I major histocompatibility complex molecule H‐2Kd to identify a nonamer peptide of the Listeria monocytogenes listeriolysin (LLO) protein that was recognized by cytotoxic T lymphocytes (CTL) in association with H‐2Kd. Eleven nonamer peptides contained in the LLO sequence were synthesized and one, LLO 91‐99, proved to be a CTL target. Using peptide binding competition assays with H‐2Kd‐restricted CTL, we show that 3 out of the 11 LLO peptides, including the CTL epitope, have a high binding affinity for H‐2Kd; 2 of 11 peptides have approximately 10‐fold lower affinity, while the remaining 6 peptides have no or very low affinity for H‐2Kd. Single residue changes were made in the LLO 91‐99 peptide and two other LLO peptides to identify non‐anchor amino acids that might interfere with peptide binding. In addition, we used the LLO peptides which bound well to H‐2Kd to attempt to restimulate a secondary CTL response from L. monocytogenes‐primed spleen cells. Only LLO 91‐99 was able to induce such a response. Thus only a fraction of nonamer peptides which fit the original binding motif have a high affinity for the H‐2Kd class I molecule, and only a fraction of these serve as CTL epitopes.
Human immunodeficiency virus type 1 gp120 C5 region mimics the HLA class I α1 peptide‐binding domain
Authors: Lopalco Lucia , de Santis Claudio , Meneveri Raffaella , Longhi Renato , Ginelli Enrico , Grassi Fabio , Siccardi Antonio G. , Beretta Alberto
Published: March 2007

Molecular mimicry of major histocompatibility (MHC) antigens by viral glycoproteins has been suggested as one of the possible mechanisms of induction of an autoimmune response by human immunodeficiency viruses. A monoclonal antibody (M38) was previously shown to bind to both human immunodeficiency virus type 1 (HIV‐1) gp120 and β‐2 microglobulin‐free HLA class I heavy chains encoded by an HLA C allele. Using HLA C recombinant proteins and synthetic peptides, the M38 class I binding site was mapped to a stretch of 44 amino acids of the al domain. The amino acid residues recognized are clustered in two non‐contiguous regions at positions 66‐69 (KYKR) and 79‐82 (RKLR) shared by almost all HLA C alleles. On HIV‐1 gp120, M38 binds to two non‐contiguous sequences (KYK and KAKR) at positions 490‐492 and 505‐508 located at the edges of a large hydrophobic region that is apparently involved in binding the transmembrane glycoprotein gp41. The C‐terminal gp120 M38‐reactive region (KAKR) lies within the immunodominant sequence APTKAKRRVVQREKR, against which the majority of HIV‐infected individuals produce antibodies. The results indicate that a functionally important region of HIV‐1 gp120 shares similar amino acid sequence motifs with the antigen recognition site of most HLA class I C alleles. The molecular mimicry may be the basis for autoimmune responses in HIV infection.
High IgE secretion capacity of human plasma cells
Authors: Werner‐Favre Christiane , Matthes Thomas , Barnet Marc , Zubler Rudolf H.
Published: March 2007

In accordance with results obtained in another culture system, it has previously been shown that human B cells frequently switch to immunoglobulin E (IgE) when they are co‐cultured with irradiated mutant EL4 thymoma cells (which provide a CD40 ligand‐mediated B cell activation signal), T cell supernatant and recombinant interleukin (IL)‐4. However, because of the potentially severe side effects of IgE, such as anaphylaxis, B cells could have a limited capacity to produce this isotype. The IgE secretion rate of plasma cells is not known. In the present study, we compared the secretion rates for different Ig classes by means of limiting dilution analysis of plasmocytic cells that were harvested after 8 to 9 days from primary EL4/B cell cultures and titrated into secondary cultures in the presence of a cell proliferation‐blocking concentration of hydroxyurea. These cells secreted Ig at constant rates for periods of up to 2 weeks; IgE secretion was IL‐4 independent. The mean cellular secretion rates were similarly high for IgE (150 pg/cell/24 h) and other isotypes (IgM 273 pg, IgG 112 pg, IgA 136 pg/cell/24 h). In terms of molecules per min this represents 3.3 × 105 for IgE versus 1.2 × 105 for IgM, 3.1 × 105 for IgG and 3.6 × 105 for IgA. The relative frequency of IgE‐secreting cells was only 0.3 % of the total number of Ig‐secreting cells, suggesting a small size of IgE‐producing clones in this in vitro system. Whether this is relevant regarding an in vivo response is not known. Clearly, the Ig secretion capacity of plasma cells would not limit an IgE response in the absence of extrinsic control.
Lack of selective Vβ deletion in peripheral CD4+ T cells of human immunodeficiency virus‐infected infants
Authors: Bahadoran Philippe , Rieux‐Laucat Frédéric , Le Deist Françoise , Blanche Stéphane , Fischer Alain , de Villartay Jean‐Pierre
Published: March 2007

To investigate the possibility of superantigen‐mediated deletions of T cells expressing particular T cell receptor Vβ (TcR Vβ) gene segments during human immunodeficiency virus (HIV) infection, TcR Vp usage in CD4+ and CD8+ subsets was analyzed in a cohort of infants maternally infected by HIV and in a group of healthy neonates. We used a semi‐quantitative anchored polymerase chain reaction technique together with cytofluorographic analysis with anti‐Vβ monoclonal antibodies. The representation of the 24 vβ families in CD4+ and CD8+ T cells from normal neonates was very similar to that in adults. Preferential expression of Vβ2 in the CD4+ subset was observed in both the neonates and in healthy adults. The representation of the 24 Vβ families in peripheral CD4+ T cells from the HIV‐infected infants showed no selective vβ deletion, even when the CD4+ subset was globally depleted. Moreover, the main characteristics of the control group (predominance of certain Vβ families and Vβ2 skewing towards the CD4+ subset) were also present in all the HIV‐infected infants.
CD4+ T cell‐mediated rejection of major histocompatibility complex class I‐disparate grafts: A role for alloantibody
Authors: Morton A. Lawrie , Bell Eric B. , Bolton Eleanor M. , Marshall Hilary E. , Roadknight Chris , McDonagh Mark , Bradley J. Andrew
Published: March 2007

Experimental studies of the T cell requirement for rejection of class I major histocompatibility complex (MHC)‐disparate grafts have generated controversy over both the autonomy of CD8+ T cells and the mechanism whereby CD4+ T cells are able to independently mediate rejection. In this study of rejection of RT1Aa class I MHC‐disparate rat cardiac and skin allografts by high‐responder PVG RT1u recipients, we show that elimination of CD8+ T cells [by anti‐CD8 monoclonal antibody (mAb) administration in vivo] fails to prolong graft survival, whereas partial depletion of CD4+ T cells (by anti‐CD4 mAb treatment) markedly delays rejection of class I‐disparate heart grafts, and marginally prolongs survival of skin grafts. Anti‐CD4‐treated PVG‐RT1u athymic nude rats reconstituted with CD8+ T cells failed to reject class I‐disparate skin grafts for several weeks and eventual rejection correlated with re‐emergence of a small number of donor derived CD4+ T cells. Conversely, anti‐CD8‐treated nude rats reconstituted with CD4+ T cells alone rapidly rejected class I‐disparate skin grafts. Passive transfer of anti‐class I immune serum to anti‐CD4‐treated euthymic recipients promptly restored their ability to specifically reject a class I‐disparate heart graft. Similarly, passive transfer of immune serum to PVG‐RT1u nude rats bearing skin allografts caused destruction of class I‐disparate but not third‐party grafts. These results demonstrate that CD4+ T cells are both necessary and sufficient to cause rejection of class I‐disparate heart and skin grafts in this model and that CD4+ T cell‐dependent alloantibody plays a decisive role in effecting rejection.
Leukemia inhibitory factor production by rat mast cells
Authors: Marshall Jean S. , Gauldie Jack , Nielsen Laurie , Bienenstock John
Published: March 2007

Leukemia inhibitory factor (LIF) is a pluripotent cytokine of importance in the regulation of immune and inflammatory responses. This cytokine may play an important role in neuronal development and bone metabolism. We have examined the ability of freshly isolated rat mast cells and mast cell lines to produce LIF at both the mRNA and bioactivity levels. Initial experiments demonstrated that two mucosal mast cell‐like cell lines RBL.2H3 and RCMC9 endogenously produced low levels of LIF bioactivity. The production of this cytokine was examined using a hepatocyte‐stimulating factor activity assay and confirmed by the use of neutralizing antibodies specific for LIF. This production was enhanced by treatment with the calcium ionophore A23187. No interleukin‐6 production was observed by these cells either endogenously or following ionophore activation. Freshly isolated highly purified rat peritoneal mast cells also expressed mRNA for LIF. These results could have important implications for the role of mast cells in neuronal development, hematopoiesis bone metabolism and the acute‐phase response.
A novel immunosuppressant, IR‐1116, which has a different biological mechanism from that of cyclosporine A
Authors: Matsumoto Sumie , Marui Shogo , Shiho Osamu , Tsukamoto Kyozo , Yoshimura Norio , Oka Takahiro
Published: March 2007

The effects of an epifumagillol derivative, IR‐1116, on human lymphocytes in vitro, mouse antibody production in vivo and rat renal and cardiac transplantation were investigated. IR‐1116 suppressed the human primary mixed lymphocyte reaction (MLR) in a dose‐dependent manner with a value of the dose required for 50% suppression of 0.32 μM. IR‐1116 suppressed the antibody production by human B lymphocytes activated with pokeweed mitogen or Epstein‐Barr virus, but did not affect the interleukin‐2 production by human lymphocytes stimulated with phytohemagglutinin. Furthermore, IR‐1116 did not have any effect on the proliferation of B or T lymphocytes. IR‐1116 is thought to have a different mechanism from cyclosporine A (CsA). BALB/c mice were immunized by s.c. injection of bovine γ‐globulin (BGG) and then received IR‐1116 or CsA by i.p. injection at a dose of 20 mg/kg every other day for 2 weeks. IR‐1116 suppressed the production of serum antibodies against BGG as strongly as CsA. In this experiment, IR‐1116 apparently showed no adverse effects, while CsA‐treated mice suffered from diarrhea and appeared to be irritated. Histological studies showed that IR‐1116 suppressed the formation of germinal centers in the spleen of immunized mice. Flow cytometric analysis showed that IR‐1116 caused a reduction of B lymphocyte population in splenocytes. On the other hand, CsA caused a marked reduction of helper T lymphocyte population in splenocytes and thymocytes. Furthermore, the i.p. administration of IR‐1116 prolonged the survival time in a rat renal allograft model and the vital period of grafted hearts in rats. Based on the above, IR‐1116 seems to be a new type of immunosuppressant acting via novel mechanisms different from those of immunosuppressants such as CsA, a potent T lymphocyte suppressant.
Characterization of rat LECAM‐1 (L‐selectin) by the use of monoclonal antibodies and evidence for the presence of soluble LECAM‐1 in rat sera
Authors: Tamatani Takuya , Kitamura Fujiko , Kuida Keisuke , Shirao Makoto , Mochiznki Manabu , Suematsu Makoto , Schmid‐Schönbein Geert W. , Watanabe Kazuyoshi , Tsurufuji Susumu , Miyasaka Masayuki
Published: March 2007

We have characterized the rat LECAM‐1 (L‐selectin) by the use of newly generated hamster anti‐rat LECAM‐1 monoclonal antibodies (mAb) (HRL1, HRL2, HRL3, HRL4), with respect to the biochemistry, cellular distribution and function, and developed an ELISA system to detect the soluble form of rat LECAM‐1. In the rat, lymphocyte and neutrophil LECAM‐1 have apparent molecular masses of 65 and 62 kDa, respectively, and differential glycosylation may account for the molecular heterogeneity. Readily detectable levels of LECAM‐1 are expressed on peripheral blood lymphocytes and neutrophils, but not on thymocytes. Lymphocyte LECAM‐1 is rapidly shed from the cell surface upon cell activation with PMA, but not with interleukin (IL)‐8. In contrast, neutrophil LECAM‐1 showed rapid shedding upon stimulation with phorbol 12‐myristate 13‐acetate (PMA) or IL‐8. Concomitantly there is up‐regulated expression of Mac‐1 in PMA‐ and IL‐8‐stimulated neutrophils. Neutrophil rolling in mesenteric venules was significantly inhibited by administration of function‐blocking anti‐rat LECAM‐1 mAb HRL3, but not by non‐blocking HRL4, indicating that LECAM‐1 plays a significant role in leukocyte rolling. Given that LECAM‐1 is rapidly shed from the cell surface, we attempted to develop an ELISA system for detecting LECAM‐1 is soluble form, and measured the levels in experimental autoimmune uveitis. The circulating levels of LECAM‐1 increased from day 4, which preceded the appearance of clinical signs of uveitis and remained high until uveitis subsided, suggesting that soluble LECAM‐1 is potentially a useful parameter to monitor certain types of inflammatory or immune disorders.
Mycobacteria induce CD4+ T cells that are cytotoxic and display Th1‐like cytokine secretion profile: Heterogeneity in cytotoxic activity and cytokine secretion levels
Authors: Mutis Tuna , Cornelisse Yolanthe E. , Ottenhoff Tom H. M.
Published: March 2007

Protective immunity against mycobacteria is dependent on antigen‐specific T cells. Current evidence suggests that not only helper T cells that activate infected macrophages but also cytotoxic T cells (CTL) that lyse infected macrophages are involved in protection. Mycobacterium‐specific CD4+ CTL are readily detectable among primary peripheral T cells but what proportion of CD4+ T cells display cytotoxic activity is not known. Whether the cytotoxic CD4+ T cells are identical to or distinct from those that produce interferon (IFN)‐γ is also unknown. In addition, studies on CTL in mycobacterial infections have focused primarily on selected antigens like hsp65 but have not analyzed systematically whether other mycobacterial antigens can activate CTL as well. These issues are relevant not only to a further understanding of protective immunity and immunopathology but also may have implications for the design of effective vaccines. To start addressing these issues, we have studied a large panel of CD4+ T cell clones specific for a broad range of mycobacterial antigens, and analyzed their ability to lyse mycobacterium‐pulsed target cells and to release IFN‐γ and interleukin (IL)‐4. Our results show that the vast majority of CD4+ T cell clones are able to lyse mycobacterial antigen‐pulsed target cells, and that those CTL can be triggered by a wide variety of mycobacterial antigens. CD4+ CTL released high levels of IFN‐γ, but low or nondetectable levels of IL‐4. In contrast, control tetanus toxoid‐specific T cell clones or lines displayed poor or weak cytotoxic activity and released high levels of IL‐4. The antimycobacterial clones appeared to be heterogeneous in their levels of cytotoxic activity and IFN‐γ release. Interestingly one T cell clone was able to lyse only mycobacterium‐pulsed macrophages but not B cells suggesting possible selectivity in target cell recognition for some CTL. These in vitro data have to be interpreted with some caution. Nevertheless they confirm and significantly extend previous observations and suggest that mycobacteria preferentially induce CD4+ T helper type 1 (Th1)‐like cells that display cytotoxic activity, and release high levels of IFN‐γ but no or little IL‐4. The induction of such Th1 like cells is specific for mycobacteria since tetanus toxoid induced T cells that were poorly or not cytolytic and secreted high levels of IL‐4.
Natural killer cells participate in the early defense against Leishmania major infection in mice
Authors: Laskay Tamás , Röllinghoff Martin , Solbach Werner
Published: March 2007

In this study the role of natural killer (NK) cells in the course of experimental Leishmania major infection was investigated. NK cells in genetically resistant C57BL/6 mice were depleted by in vivo administration of anti‐asialo‐GM1 or anti‐NK1.1 antibodies. A marked exacerbation of the infection was found in the NK‐depleted mice within the first two weeks of infection. Both the local tissue swelling and the number of parasites in the lesions were significantly higher than in normal animals. Lymph node cells taken from infected NK‐depleted mice released less interferon‐γ (IFN‐γ) when cultured in vitro. As an alternate approach we have used poly I: C treatment in order to activate NK cell activity in vivo in BALB/c mice, which are genetically susceptible to L. major infection. Poly I: C treatment led to milder symptoms and to a significantly lower parasite burden in the early course of infection. Lymph node cells from infected and poly I: C‐treated BALB/c mice released higher amount of IFN‐γ in vitro than cells from control mice. These data show that NK cells are active participants in the non‐specific phase of anti‐leishmanial activity in the control of parasite multiplication early in the course of L. major infection in mice.
The cytoplasmic tail of the T cell receptor ζ chain is dispensable for antigen‐mediated T cell activation
Authors: Hermans Mirjam H. A. , Malissen Bernard
Published: March 2007

The T cell antigen receptor consists of an antigen‐binding αβ heterodimer and a group of invariant polypeptides denoted CD3‐γ, CD3‐δ, CD3‐εand CD3‐ζ. Whether antigen responsiveness is dependent on the expression of functional CD3‐ζ subunit remains controversial. Forinstance, transfection of a ζ− / n− variant of the 2B4.11. T cell hybridoma with mutated ζ cDNA that encoded a ζ protein truncated at residue 108, restored the surface expression of T cell antigen receptor complexes with, however, impaired antigen responsiveness [Frank, S. J., Niklinska, B. B., Orloff, D. G., Mercep, M., Ashwell, J. D. and Klausner, R. D., Science 1990. 249: 174.]. In marked contrast, BW5147 transfectants that expressed T cell antigen receptors devoid of functional ζ subunits were still able to trigger the production of interleukin‐2 in response to antigen [Wegener, A.‐M. K., Letourneur, E, Hoeveler, A., Brocker, T., Luton, F. and Malissen, B., Cell 1992. 68: 83.].

To assess if the above discrepancies may have resulted from the use of different recipient T cells, we transfected a ζ/n‐deficient variant of 2B4.11 (MA5.8) with the very same truncated ζ cDNA we previously used in BW5147. Consistent with our initial observations in BW5147, the cytoplasmic tail of the ζ polypeptide was found dispensable for antigenic responsiveness. Furthermore, a difference between the two recipient T cells was detected when cells were challenged via the Thy‐1 and Ly‐6 molecules. Once expressed in MA5.8, but not in BW5147, T cell antigen receptor complexes devoid of functional ζ subunits were able to sustain activation initiated via Thy‐1 and Ly‐6 molecules.
Plasmodium berghei‐specific T cells respond to non‐processed sporozoites presented by B cells
Authors: Link Heidi T. , White Katherine , Krzych Urszula
Published: March 2007

The mechanism of malaria protective immunity induced by immunization with radiation‐attenuated Plasmodium sporozoites (SPZ) is only partially understood. For example, B and T cell responses specific for the circumsporozoite (CS) protein, a 46 kDa SPZ surface protein, have been characterized; however, events leading to SPZ‐specific T cell activation, i.e., processing and presentation of SPZ by antigen‐presenting cells have not been investigated. In the present study we describe the in vitro analysis of requirements for accessory cell function in the presentation of SPZ to SPZ‐immune T cells. The results establish that SPZ‐induced proliferative T cells are reactive to non‐processed SPZ presented by activated B cells and, thus, imply that the non‐processed form of the SPZ‐associated CS protein restricts the induction of the potential CS protein T cell repertoire.
Collaboration of helper and cytotoxic T lymphocytes
Authors: Stuhler Gemot , Walden Peter
Published: March 2007

Cytotoxic T lymphocytes are major effector cells in responses to viral infections and in allograft rejection and are implicated in many other immunological reactions. Efficient induction of cytotoxic activity in these cells in many but not all cases depends upon helper Tand antigen‐presenting cells so that at least three different cell types have to work together. Here we present an in vitro model for the helper T cell‐dependent induction of cytotoxic T lymphocytes which allows the investigation of the collaboration of helper and cytotoxic T cells. First results demonstrate that linkage of helper and killer epitopes on the surface of one antigen‐presenting cell is a prerequisite for productive interaction between the two T cells that results in induction of cytolytic activity. T helper 1 and T helper 2 cells are equally efficient. The crucial roles of interleukin‐2 and interferon‐y in this process were confirmed. Activated CD4 cells can influence cytotoxic T lymphocytes in such a way that they produce interferon‐y independent from recognition of cognate peptide. The possibility of direct T‐T contacts mediated by adhesion molecules that promote collaboration of the two cells is discussed.
B cells are anergic in transgenic mice that express IgM anti‐DNA antibodies
Authors: Tsao Betty P. , Chow Anne , Cheroutre Hilde , Song Yeong W. , McGrath Mary E. , Kronenberg Mitchell
Published: March 2007

B lymphocytes in individuals with systemic lupus erythematosus (SLE) secrete pathogenic auto antibodies to DNA which cause clinical nephritis. (NZB x NZW) Fl (BW) female mice also secrete pathogenic anti‐DNA auto antibodies, and therefore are considered to be an animal model of SLE. The rearranged immunoglobulin (Ig) genes that encode an anti‐DNA antibody from a diseased BW mouse have been cloned, and transgenic (Tg) mice have been created by microinjection of these constructs into fertilized eggs from normal mice. As we reported previously, when the construct contains the Cγ2a heavy chain constant (CH) region, the mice spontaneously secrete anti‐DNA IgG and they develop mild nephritis. This demonstrated that the Ig encoded by the transgene is pathogenic. In contrast, here we report that when the construct contains the same anti‐DNA Ig variable (V) regions used previously, along with the Cμ region, the auto reactive B cells are rendered tolerant. Most B cells in the Tg mice express the μ transgene product on their surface, and rearrangement of endogenous light chain genes is partially suppressed. Furthermore, most hybridomas made from Tg B cells secrete IgM anti‐DNA. Despite this, the Tg mice have reduced levels of total serum Ig and they do not secrete anti‐DNA IgM either spontaneously or following immunization with DNA. We conclude that most B cells in the Tg mice have been rendered anergic. Anergy is however reversiblein vitro;lipopolysaccharide stimulation of Tg B cells leads to the production of a significant amount of IgM anti‐DNA antibody. The studies demonstrate that in this line of Tg mice on a normal mouse genetic background potentially pathogenic B cells that express a high‐affinity Ig specific for a natural autoantigen are subject to tolerance by induction of anergy.
Leukotriene B4 induces interleukin 5 generation from human T lymphocytes
Authors: Yamaoka Kunio A. , Kolb Jean‐Pierre
Published: March 2007

Leukotriene B4 (LTB4) has been shown to affect several interleukin (IL)‐linked functions of human lymphocytes. In this study, we investigated whether LTB4 regulates IL‐5 generation from human T cells and subsequently modulates eosinophil functions. Preincubation of T cells with very low concentrations (10−12 to 10−8 M) of LTB4 induced concentration‐dependent IL‐5 production, the event occurring after the first 24 h of cultivation. However, direct action of LTB4 to IL‐5 generation is strictly dependent on a preincubation with appropriate concentration of LTB4. In contrast, the stereoisomer of LTB4,5S,12S‐dihydroxy‐6,8,10,14‐eicosatetraenoic acid showed no enhancement of IL‐5 production. IL‐5 released from LTB4‐primed T cells elicited sustained viability of mature eosinophils and reduced the content of eosinophil cationic protein in their crystalloid matrix by degranulation. These data suggest that LTB4 induces bioactive IL‐5 production from T cells and that the released IL‐5 modulates eosinophil functions which might play a crucial role in eosinophil‐linked allergic inflammatory process.
Elevation of cytosolic calcium is sufficient to induce growth inhibition in a B cell lymphoma
Authors: Muthukkumar Subramanian , Udhayakumar Venkatachalam , Bondada Subbarao
Published: March 2007

Recently, we have described that anti‐IgM antibodies profoundly inhibited the growth of BKS‐2, an immature B cell lymphoma. In this report, we demonstrated that ionomycin alone at very low concentrations (20 nM) inhibited the growth of BKS‐2 cells completely. The levels of intracellular Ca2+ induced by the inhibitory concentrations of ionomycin were comparable to those in anti‐IgM‐treated cells. The growth inhibition caused by ionomycin was reversed by phorbol 12‐myristate 13‐acetate and lipopolysaccharide. In addition, the immunosuppressants, cyclosporin A and FK506 conferred significant protection from the negative signal induced by ionomycin. However, either cyclosporin A, FK506 or lipopolysaccharide was not found to have direct effect on ionomycin‐induced Ca2+ mobilization in BKS‐2 cells. Also, ionomycin augmented the anti‐IgM‐induced growth arrest in these cells. Furthermore, BKS‐2 cells that were exposed to anti‐IgM or ionomycin underwent apoptosis as characterized by DNA fragmentation. Thus, the characteristics of growth inhibition induced by ionomycin and anti‐IgM appeared to be similar in that phorbol 12‐myristate 13‐acetate, lipopolysaccharide, cyclosporin A and FK506 caused significant reversal from such negative signals and both ionomycin and anti‐IgM induced apoptosis in these cells. Altogether, these results showed that the elevation of intracellular Ca2+ alone was sufficient to inhibit the growth of some B lymphoma cells.