bluuebunny/crossref_metadata_2025 huggingface dataset:
Presentation of igg2a antigens to class ii‐restricted t cells by stably transfected b lymphoma cells
Authors: Bikoff Elizabeth K. , Eckhardt Laurel A.
Published: March 2007

Here we describe a panel of BALB/c T cells specific for IgG2a of the b allotype in association with I‐Ad. We used DNA‐mediated gene transfer techniques to localize antigenic determinants recognized by responding T cells. Initially a truncated IgG2aa gene comprising a variable domain and the CH3 domain (not including the membrane exons) from the BALB/c IgG2aa heavy chain was introduced into myeloma cells. The V‐CH3 protein was expressed at high levels under control of the Ig heavy chain enhancer. Secretion of the V‐CH3 protein did not require assembly of H‐H dimers or an association with light chains.

To generate stably transfected B cell lines that would stimulate our class II‐restricted T cells, we replaced most of the BALB/c IgG2aa CH3 exon with CH3 coding sequences from a C57BL/6 IgG2ab cDNA clone and introduced these constructs into Ia+ B lymphoma cells. The IgG2ab CH3‐transfected B cells were recognized by BALB/c Igh‐1b‐specific T cell hybrids in the absence of exogenous antigen. Experiments using glutaraldehyde‐fixed cells as stimulators indicate that presentation of the secreted form of V‐IgG2ab CH3 requires processing. We found that a significant fraction of the endogenously synthesized V‐IgG2ab CH3 protein was, however, present as already processed antigen.
A new cell surface molecule closely related to mouse class i transplantation antigens
Authors: Cochet Madeleine , Casrouge Armanda , Dumont Anne‐Marie , Transy Catherine , Baleux FrançOise , Maloy W. Lee , Coligan John E. , Cazenave Pierre‐André , Kourilsky Philippe
Published: March 2007

The “37” H‐2 class I mouse gene, located in the Tla complex, is poorly polymorphic and is transcribed in a wide variety of cells and tissues. Using antisera directed against peptides deduced from the gene sequence, we demonstrate that the 37 gene encodes a 45.5‐kDa cell surface glycoprotein. This protein, initially identified on the surface of mouse L cells transfected with an overexpressing 37 gene, is present on cells such as splenocytes of various mouse strains except those of A/J mice. Thus, mouse cells may express not only the major transplantation antigens encoded by the K and D region genes, but at least one additional poorly polymorphic class I molecule encoded by the 37 gene.
Camp inhibits the okt3‐ induced increase in cytoplasmic free calcium in the jurkat t cell line: the degree of inhibition correlates inversely with the amount of cd3 binding ligand used*
Authors: Papadogiannakis Nikos , Nordström Tommy E. , Andersson Leif C. , Wolff C. Henrik J.
Published: March 2007

We have investigated the effect of cAMP concentration on the CD3‐mediated rise in intracellular Ca2+ induced by anti‐CD3 monoclonal antibody in the human T cell leukemia line Jurkat. Forskolin, prostaglandin E2 (PGE2) and dibutyryl cAMP (db‐cAMP) were used to increase intracellular cAMP concentrations. Treatment of Jurkat cells with forskolin or db‐cAMP for 3 h inhibited the subsequent rise in intracellular Ca2+ concentration induced by an optimally mitogenic dose of 100 ng/ml of the anti‐CD3 OKT3, whereas PGE2 counteracted the Ca2+ rise only marginally. The inhibitory effect of forskolin and PGE2 on the Ca2+ signal correlated with their abilities to induce increased cAMP levels in Jurkat cells. The suppression of the Ca2+ response was dependent on the concentration of the cAMP‐elevating agent and the time of pre‐incubation with the drug. The cAMP‐mediated inhibition of the Ca2+ response diminished or disappeared when increasing concentrations of OKT3 were used to stimulate the rise in intracellular Ca2+ concentration. The results indicate that cAMP participates in the regulation of T lymphocyte activation at the level of signal transduction. Our findings, which stress the crucial role of the concentration of the ligand used to trigger Ca2+ responses, provide an explanation to previous contradictory reports on the impact of cAMP on the signal transduction in T cells.
Sequence and linkage of the vχ21a and g germ‐line gene segments in the mouse
Authors: Alanen Anna , Weiss Siegfried
Published: March 2007

The germ‐line V gene segments encoding the subgroups A and G of the BALB/c Vχ21 family were cloned and assigned to the previously described 30‐kb cluster of the Vχ21 family. Sequence comparison revealed close homology between the two gene segments at the DNA and the predicted amino acid sequence level, indicating that Vχ21A and Vχ21G originated by a rather recent gene duplication.
Establishment of an anti‐A human monoclonal antibody from a blood group A lung cancer patient: evidence for the occurrence of autoimmune response to difucosylated type‐2 chain A
Authors: Inoue Hiroshi , Hirohashi Setsuo , Shimosato Yukio , Enjoji Munetomo , Clausen Henrik , Hakomori Sen‐Itiroh
Published: March 2007

A human monoclonal antibody, NCC‐HAL‐27, has been established by a combination of transformation by Epstein‐Barr virus (EBV) and a newly devised “micro cell fusion” technique. Lymphocytes used were derived from a blood group A lung cancer patient. Cancer cells, as well as normal epithelial cells of lung, stomach and colon (including autologous tissue) from blood group A individuals, fixed in formalin, were immunohistochemically stained by the antibody. A higher concentration (> 140 mg/ml) of the antibody agglutinated only type‐A (not B or 0) erythrocytes. The structural specificity of NCC‐HAL‐27 was determined by particle‐concentrated fluorescence immunoassay and thinlayer chromatography immunostaining using purified, well‐defined glycolipids. The antibody was found to be directed against type‐2 chain A and difucosylated type‐2 chain A antigens with no cross‐reactivity with known A‐like antigens, such as Tn and Forssman antigen, generally expressed irrespective of ABO status. Interestingly, however, the antibody detected the expression of incompatible A antigen in colon cancers of B and 0 individuals with high incidence (30%‐40%). The finding that a monoclonal antibody generated from a blood group A cancer‐bearing host is directed against autologous A antigen is novel because the AB0 blood group antigens are the major alloantigens in man, with strictly controlled immunotolerance. The induction of anti‐A in this patient could, therefore, be directly associated with the lung cancer.
Disparate functions of I‐A and I‐E molecules on B cells as evidenced by the inhibition with anti‐I‐A and anti‐I‐E antibodies of polyclonal B cell activation
Authors: Takahama Yousuke , Ono Shiro , Ishihara Katsuhiko , Muramatsu Minoru , Hamaoka Toshiyuki
Published: March 2007

Polyclonal differentiation of unprimed B cells into IgM‐producing cells induced by lipopolysaccharide (LPS) or T cell‐derived lymphokine B151‐TRF2 has been shown to contain a process of I‐A‐restricted B‐B cell interaction, so that the B cell responses are inhibited by monoclonal antibodies (mAb) specific for I‐A molecules. On the other hand, the B cell responses are also inhibited by anti‐I‐E mAb, although I‐E molecules are not involved in such B‐B cell interaction. In this study, we examined the mechanism underlying the anti‐I‐E‐mediated inhibition of the B cell responses. The B cell responses induced by LPS or B151‐TRF2 were inhibited by either anti‐I‐A or anti‐I‐E mAb added on day 0 over a 5‐day culture period, whereas when added on day 3 the responses were inhibited only by anti‐I‐E mAb and not by anti‐I‐A mAb. To gain insight into the mechanism underlying the anti‐I‐E‐mediated inhibition, we prepared monovalent Fab and divalent F(ab')2 fragments of anti‐I‐A and anti‐I‐E mAb and examined their effects on the B cell responses. We found that the B cell responses were inhibited by the F(ab')2 but not Fab fragment of anti‐I‐E mAb, whereas the Fab fragment of anti‐I‐A mAb still gave effective inhibition. The F(ab')2 but not Fab fragment of anti‐I‐E mAb induced increases in cyclic AMP (cAMP) levels in B cells, whereas the undigested anti‐I‐A mAb did not induce such increases. Furthermore, adenylate cyclase inhibitors, which inhibit cellular cAMP accumulation, circumvented the B cell responses inhibited by anti‐I‐E but not anti‐I‐A mAb. Thus, these results indicate that the anti‐I‐E‐mediated inhibition of the B cell responses requires increases in intracellular cAMP levels induced by cross‐linking of I‐E molecules. In contrast, anti‐I‐A mAb inhibits the B cell responses without cross‐linking of I‐A molecules and cAMP accumulation. These results reinforce a unique function of I‐A molecules as restriction elements in the Ia‐restricted B‐B cell interaction.
Derivation of a T cell hybridoma variant deprived of functional T cell receptor α and β chain transcripts reveals a nonfunctional α‐mRNA of BW5147 origin
Authors: Letourneur FranÇOis , Malissen Bernard
Published: March 2007

We have isolated a variant of the DO‐11.10.7 mouse T cell hybridoma which does not express functional T cell receptor α/β chains. This variant, denoted 58 α−β−, can be used as a recipient for T cell receptor α/β gene transfer experiments to obtain cell lines which express only the products of the transfected α/β genes at their surfaces. In the process of characterizing the defects affecting the 58 α−β− T cell receptor genes, we have found that the parental BW5147 thymoma has undergone a previously unnoticed Vα‐Jα rearrangement. This α rearrangement involves a Vα pseudogene segment and accounts for the high level of α‐mRNA transcripts present in the BW5147 α−β− variant. Knowledge of the existence of this second, albeit nonfunctional, α‐mRNA in BW5147 is of importance, since it could be, and actually already has been, mistakenly identified (due to partial nucleotide sequencing) in T hybrids as a functionally significant message donated by the normal T cell parent.
Surface expression of CD3 in the absence of T cell receptor (TcR): evidence for sorting of partial TcR/CD3 complexes in a post‐endoplasmic reticulum compartment
Authors: Ley Steven C. , Tan Kut‐Nie , Kubo Ralph , Sy Man‐Sun , Terhorst Cox
Published: March 2007

The T cell receptor (TcR) for antigen, on the majority of T cells, is a disulfide‐linked heterodimer composed of the α and β chains, noncovalently associated with the CD3 complex of polypeptides (γ, δ, ϵ and ζ). In this report, two murine thymoma cell lines are described which synthesized incomplete TcR/CD3 complexes and expressed low levels of CD3 on their surface in the absence of the TcR chains. The partial TcR/CD3 complexes were composed primarily of the inherently metabolically stable CD3 γ and ϵ subunits. These results were in contrast to previous studies, which suggested that synthesis of all of the component chains of the TcR/CD3 complex is required for the successful transport of any of the chains to the cell surface. The efficiency of transport of the partial TcR/CD3 complexes from the endoplasmic reticulum (ER) to medial Golgi in the two thymomas was similar to complete complexes. However, the transport of the incomplete receptors was impaired at some point between the medial Golgi and the plasma membrane. Taken together with previous studies, these results suggested that T cells have mechanisms to retain partial TcR/CD3 complexes intracellularly both in the ER and in an undefined post‐ER compartment. However, the transport of low levels of partial TcR/CD3 complexes to the cell surface in some T cell lines implied that the retention mechanisms may not always be completely efficient. Cross‐linking of the surface, partial TcR/CD3 complexes with anti‐CD3 ϵ antibodies did not stimulate interleukin 2 (IL 2) production. It is possible, however, that the partial TcR/CD3 complexes have some function which is unrelated to the stimulation of IL 2 production.
Induction of class II major histocompatibility complex antigens in murine placenta by 5‐azacytidine and interferon‐γ involves different cell populations
Authors: Athanassakis‐Vassiliadis Irene , Thanos Dimitris , Papamatheakis Joseph
Published: March 2007

Class II MHC antigen expression is required for recognition of an alloantigen and generation of immune response. In rodents as well as in humans primary trophoblasts do not express class II MHC antigens. In this study we focused our interest on the mechanism(s) of class II antigen suppression on murine trophoblasts. First, we examined the possibility of gene inactivation by methylation and second the possibility of lymphokine regulation of the class II genes. The first possibility was tested by treatment of placental cells with 5‐azacytidine (5‐AzaC), a cytidine analog which upon incorporation into the DNA inhibits further methylation, thus leading to gene activation. In order to test the second possibility we treated placental cells with interferon‐γ (IFN‐γ) or interleukin 4 (IL 4) which are known to induce class II antigen expression in many systems. We showed that treatment with 5‐AzaC or IFN‐γ but not IL 4 significantly increased class II expression on cytokeratin‐positive and vimentin‐negative adherent placental cells. Following placental cell fractionation we distinguished three cell subsets with different responsiveness to 5‐AzaC and IFN‐γ. The first, characterized as placental macrophages, were induced to express class II MHC antigens only after IFN‐γ treatment. The other two subsets, characterized as trophoblasts, were isolated from the labyrinthine‐ and spongio‐trophoblast layer of the placenta and showed class II inducibility to 5‐AzaC and IFN‐γ, respectively. The results show that depending on the anatomical localization of trophoblasts within the placenta, various regulatory elements control gene expression, so that the placental barrier provides fetal protection at different levels.
The CD3δ gene encodes multiple transcripts regulated by transcriptional and post‐transcriptional mechanisms
Authors: Wilkinson Miles F. , Georgopoulos Katia , Terhorst Cox , Macleod Carol L.
Published: March 2007

CD3 is a multi‐subunit complex of proteins noncovalently associated with the T cell receptor (TcR) for antigen. Considerable evidence indicates a role for CD3 molecules in the transduction of activation signals in T cells. The murine CD3δ gene encodes a 0.7‐kb transcript present in mature T cells. Here we report the characterization of several additional CD3δ transcripts; two nuclear transcripts, 4–4.5 kb in size, and two predominately cytoplasmic transcripts of 1.5 kb and 2.5 kb. Both T lymphoma cell lines and normal thymocytes express the 1.5‐kb and 2.5‐kb CD3δ transcripts. These cytoplasmic transcripts have long 3'‐untranslated sequences which extend beyond the polyadenylation site of the predominant 0.7‐kb transcript. The protein synthesis inhibitor cycloheximide (CHX) increases the expression of all three cytoplasmic CD3δ transcripts, indicating that their level of expression may be regulated by a labile inhibitor protein(s). The CHX elicited increase in CD3δ mRNA appears to result from post‐transcriptional events since the rate of CD3δ gene transcription remains constant. In contrast to CHX, the calcium ionophore A23187 increases the rate of CD3δ gene transcription and, like CHX, also increases the level of cellular CD3δ mRNA. The immunosuppressive agent cyclosporin A inhibits A23187‐mediated stimulation of transcription, but has no effect on the CHX‐mediated induction of CD3δ mRNA. We conclude that both transcriptional and post‐transcriptional mechanisms can regulate the amount of all three cytoplasmic CD3δ transcripts.
Induction of antigen‐specific immunity with monoclonal anti‐idiotypic antibodies in vivo: differences in potency and comparison of immunochemical
Authors: Misplon Julia A. , Reeves James P. , Harvath Liana , Rubinstein Leonard J. , Epstein Suzanne L.
Published: March 2007

Anti‐idiotypic (Id) antibodies provide a means other than antigen of clone‐specific regulation of immune responses, and have been proposed as an alternative form of vaccine. However, the requirements for effective induction of immunity by anti‐Id are not understood. Nine monoclonal anti‐idiotope antibodies (anti‐Id mAb) were derived in the Ia. 7 model system. While all nine anti‐Id mAb induced comparable Ab3 responses in vivo as detected by ELISA, there were dramatic differences in the potency of the antigen‐specific components of the responses induced by the nine anti‐Id mAb. Anti‐Id mAb that were indistinguishable in isotype, combining site relatedness, fine specificity on a panel of mAb, end point binding titers, competitive binding and ability to induce Ab3 differed dramatically in their ability to induce antigen‐specific immunity in vivo, thus ruling out several models for explaining differences in induction.
Glucocorticoid hormones reduce the expression of major histocompatibility class I antigens on human epithelial cells
Authors: von Knebel Doeberitz Magnus , Koch Susanne , Drzonek Heiko , Hausen Harald Zur
Published: March 2007

Expression of a critical level of major histocompatibility complex (MHC) class I antigens on epithelial cells is a prerequisite for the action of specific cytolytic immune response cells. Glucocorticoid hormones have strong immunosuppressive effects. Therefore, we investigated the influence of the synthetic glucocorticoid dexamethasone on the expression level of MHC class I antigens on human epithelial cell lines. Long‐term treatment with dexamethasone leads to reduced MHC class I surface antigen expression and to decreased total membrane‐bound MHC class I protein. The steady‐state mRNA level is significantly decreased and the transcription rate of MHC class I genes is reduced.
Regulation of IgG production by suppressor FcγRII+ T hybridomas
Authors: Brunati Silvana , Moncuit Janine , Fridman Wolf H. , Teillaud Jean‐Luc
Published: March 2007

In this work, we analyzed the immunoglobulin heavy (H) and light (L) chain production by two variant B hybridomas, UN2.C3 and UN2.C17.K1 co‐cultured with cells from a FcγRII, IgG‐binding factor (IgG‐BF)‐producer T hybridoma (T2D4.C1) or with cells of a FcγRII−, IgG‐BF‐nonproducer variant (D10C5). We showed that only the FcγRII hybridoma directly inhibits the IgG secretion by UN2.C3 through a soluble mediator. This inhibition affects the H and L chain synthesis as well as the H and L chain‐encoding mRNA steady state. No apparent cytotoxic effect could be detected. In contrast, the production of ϰ chain by an H chain‐negative variant (UN2.C17.K1) was unaffected. This indicates that a complete IgG molecule is required to observe the inhibitory effect induced by T2D4.C1. The pattern of effector/target cell interactions observed in our work suggests that the soluble factor involved in the suppression of IgG production is IgG‐BF, able to transiently modify the IgG gene expression in target cells.
T cell receptor‐negative thymocytes from SCID mice can be induced to enter the CD4/CD8 differentiation pathway
Authors: Shores Elizabeth W. , Sharrow Susan O. , Uppenkamp Ingeborg , Singer Alfred
Published: March 2007

In order to investigate the role of T cell receptor (TcR) expression in thymocyte maturation, we have analyzed thymocytes from C.B‐17/SCID mice, which are unable to productively rearrange their antigen receptor genes and fail to express TcR. Despite this defect, SCID thymocytes are functional as they produce lymphokines and proliferate in response to a variety of stimuli. Phenotypic analysis revealed that thymocyte populations from young adult SCID mice resemble thymocyte populations from normal embryonic mice in that they are large, Thy‐1.2+, CD4−, CD8−, TcR− and enriched in CD5lo, IL2R+ and Pgp1+ cells. However, other TcR− populations normally present in adult mice (i.e., CD4−CD8+ cells and CD4+CD8+ cells) are absent from the thymus of TcR− adult SCID mice. To understand the basis of the developmental arrest of TcR− SCID thymocytes at the CD4−CD8− stage of differentiation, we analyzed thymi from the occasional “leaky” SCID mouse which possesses small numbers of TcR+ thymocytes. We found that the presence of TcR+ cells within a SCID thymus was invariably associated with the presence of CD4+ and/or CD8+ SCID thymocytes. Interestingly, however, the CD4+/CD8+ SCID thymocytes were not themselves necessarily TcR+. That is, emergence of SCID thymocytes expressing CD4/CD8 was tightly linked to the presence of TcR cells within that SCID thymus, but the SCID thymocytes that expressed CD4/CD8 were not necessarily the same cells that expressed TcR. Finally, we found that the introduction into TcR− SCID mice of normal bone marrow cells that give rise to TcR+ cells within the SCID thymus promoted the differentiation of SCID thymocytes into CD4−CD8+ and CD4+CD8+ TcR− cells.

These data indicate that TcR+ cells within the thymic milieu provide critical signals which promote entry of CD4−CD8−TcR− precursor T cells into the CD4/CD8 differentiation pathway. When applied to differentiation of normal thymocytes, these findings may imply a critical role for early appearing CD4−CD8− TcR (γ/δ)+ cells in initiating normal thymic ontogeny.
Antigen density on target cells determines the immunosuppressive potential of rat IgG2b monoclonal antibodies
Authors: Kummer Udo , Thierfelder Stefan , Mysliwietz Josef
Published: March 2007

The current studies were designed to determine the relevance of T cell antigen density, besides antibody isotype, with regard to the success of antibody serotherapy. We compared the immunosuppressive effects of two rat IgG2b monoclonal anti‐Thy‐1 antibodies, RmT1 and 30‐H12, with distinct binding sites in a graft‐vs.‐host disease (GVHD) model of fully H‐2 and I‐A regionmismatched bone marrow transplantation, making use of the difference in Thy‐1.2 antigen density between homozygous (BALB/c) and heterozygous (BALB/c×AKR/J)F1 GVHD‐promoting donor cells. Antibodies RmT1 (directed against a monomorphic determinant on mouse Thy‐1) and 30‐H12 (reactive with the Thy‐1.2 allele‐specific determinant) did not differ in their anti‐GVHD activity with regard to Thy‐1.2 homozygous grafts. However, in the region of a critical number of binding sites a small difference in the amounts of the two antibodies bound (about 8×103 IgG molecules/cell) obviously accounts for a great difference in anti‐GVHD activity. This is shown in a two haplotype host‐graft disparity between C57BL/6 recipients treated with either RmT1 or 30‐H12 before challenging them with (BALB/c×AKR/J)F1 grafts, where the Thy‐1.2 antigen concentration is approximately 50% compared to the density on BALB/c lymphocytes. Here, mAb 30‐H12 loses its remarkable in vivo immunosuppressive quality, whereas RmT1 treatment protects mice against lethal GVHD. Binding sites were quantitated using a computerized approach for the analysis of data from ligand binding experiments of the respective mAb, RmT1 and 30‐H12, coated to LN cells of BALB/c and F1 hybrid origin. Furthermore, the in vivo immunosuppressive activity of rat IgG2b antibodies directed against Thy‐1 was found to correlate with their ability to generate stable antibody‐Clq complexes on the cell surface of immunocompetent T cells.
Inhibition of human interleukin 4‐induced IgE synthesis by a subset of anti‐CD23/FcϵRII monoclonal antibodies
Authors: Bonnefoy Jean‐Yves , Shields John , Mermod Jean‐Jacques
Published: March 2007

Specific monoclonal antibodies (mAb) directed against the CD23 antigen were used to study human interleukin 4 (hIL 4)‐induced IgE production by blood and tonsillar mononuclear cells. Both peripheral blood and tonsillar mononuclear cells stimulated by hIL 4 expressed membrane CD23 as detected by the binding of all anti‐CD23 mAb. Nevertheless, two sets of anti‐CD23 mAb could be distinguished. The first set, including mAb25, was able to decrease significantly hIL 4‐induced IgE synthesis by mononuclear cells. The second set, including EBVCS#1, did not affect hIL 4‐induced IgE synthesis. All the anti‐CD23 mAb were able to bind specifically to a human B cell line expressing recombinant CD23. Inhibition experiments revealed that the two sets of anti‐CD23 mAb did not recognize the same epitope on the CD23 antigen. In fact, all the anti‐CD23 mAb, except EBVCS#1, were able to inhibit IgE binding to CD23 on RPMI 8866 cells. Moreover, the first set of antibodies, which decreased IgE production, was able to up‐regulate membrane CD23 expression on hIL4‐stimulated tonsillar mononuclear cells. Conversely, EBVCS#1, which had no effect on IgE production, did not affect hIL 4‐induced CD23 expression. These results indicate that CD23 plays a key role in human IgE synthesis.
Chemical residues of ganglioside molecules involved in interactions with lymphocyte surface targets leading to CD4 masking and inhibition of mitogenic proliferation
Authors: Grassi Fabio , Lopalco Lucia , Lanza Paola , Ciccomascolo Franca , Cazzola Flavia , Martino Alessandro Di , Kirschner Gunther , Callegaro Lanfranco , Chieco‐Bianchi Luigi , Siccardi Antonio G.
Published: March 2007

Human lymphocyte CD4 becomes undetectable in the presence of exogenous gangliosides (CD4 masking), as originally described by Offner et al. (J. Immunol. 1987. 139: 3295). CD4 masking is apparently due to in situ rearrangement of the glycoprotein; since no direct binding of ganglioside to CD4 could be demonstrated, it was suggested that the effect could be mediated by interactions with other, as yet unidentified, surface structures. To gain insight into the structural requirements of the interaction(s) that leads to CD4 masking, we assayed the effects of a battery of gangliosides and of ganglioside derivatives on (a) CD4 masking; (b) cholera toxin binding (as a well known ganglioside‐protein interaction) and (c) inhibition of lymphocyte mitogenic proliferation (as a second ganglioside interaction with a lymphocyte surface target). Our results indicate that the three interactions are distinctly different, since ganglioside chemical groups which are essential for one of the interactions are irrelevant for the others, and lead to the conclusion that gangliosides can interact with lymphocyte surface targets in a number of ways, causing a number of independent biological effects.
Interleukin 4 activates human B lymphocytes via transient inositol lipid hydrolysis and delayed cyclic adenosine monophosphate generation
Authors: Finney Michael , Guy Graeme R. , Michell Robert H. , Gordon John , Dugas Bernard , Rigley Kevin P. , Callard Robin E.
Published: March 2007

We report from three independent centers that, in human tonsillar B lymphocytes, human IL4 switches on a series of second messenger changes, the precise sequence of which constitutes a novel signal transduction cascade. It involves an immediate and transient elevation of inositol 1,4,5‐trisphosphate and Ca2+ levels. This is followed several minutes later by a sustained rise in cellular cyclic adenosine monophosphate concentration, the triggering of which involves both the Ca2+ rise and an additional, as yet unidentified, IL4‐generated signal. Both the products of the initial inositol lipid hydrolysis and the delayed cyclic adenosine monophosphate accumulation are essential for the later induction of CD23 expression, a major phenotypic change promoted in these cells by IL4. The striking contrast between these findings and those that have been observed for the IL4 triggering of murine B cells is discussed.
Immunodeficient mice recover from infection with vaccinia virus expressing interferon‐γ
Authors: Kohonen‐Corish Maija R. J. , King Nicholas J. C. , Woodhams Cathy E. , Ramshaw Ian A.
Published: March 2007

Recombinant vaccinia viruses (VV)‐encoding murine interferon‐γ (IFN‐γ) were constructed and the effect of virus‐encoded IFN‐γ on the immune response towards VV in vivo investigated. In athymic nude mice and sublethally irradiated euthymic mice, IFN‐γ expression by VV enabled the mice to recover from the infection, whereas mice infected with the control virus died. In normal CBA/H mice also, the growth of VV was greatly reduced and it was cleared faster from mouse organs than the control virus. Natural killer (NK) cell responses in these mice were not enhanced suggesting that this recovery is not NK cell mediated. Other possible mechanisms and implications of this observation are discussed.
In vivo cytokine gene expression in T cell subsets of the autoimmune MRL/Mp‐lpr/lpr mouse
Authors: Murray Lesley J. , Lee Rozanne , Martens Christine
Published: March 2007

Expression of cytokine genes in freshly isolated T cell subsets in the autoimmune lpr mouse has been studied to determine what factors may be produced by these cells in vivo. RNA prepared from T cell subsets from diseased lpr mice and from the normal congenic strain, MRL/n, was tested for the presence of cytokine‐specific message using the polymerase chain reaction. Cells of the expanded abnormal T cell subset were shown to express genes encoding interferon (IFN)‐γ, tumor necrosis factor (TNF)‐β,TNF‐α and interleukin (IL) 6, cytokines which are associated with inflammatory immune responses. These cells may thus play an important role in exacerbation of the pathological symptoms of the systemic autoimmune disease. These cells expressed no detectable IL 1, IL 2, IL 3, IL 4 or IL 5. Phenotypically normal CD4+ and CD8+T cells from both lpr and MRL/n also contained transcripts for IFN‐γ, TNF‐α, TNF‐β and IL 6. IL 2 mRNA was found almost exclusively in the CD4+ subset, indicating that the CD8+ T cells in the lpr mouse are not highly activated through their class I major histocompatibility complex molecules to produce IL 2, as could occur if a virus infection was inducing autoimmunity in these mice. Similar levels of IL 2 mRNA were present in the CD4+ T cells of lpr and MRL/n mice, demonstrating that these cells are not defective in IL 2 production in vivo.
Metabolic inhibitors distinguish cytolytic activity of CD4 and CD8 clones
Authors: Strack Peggy , Martin Carla , Saito Saburu , Dekruyff Rosemarie H. , Ju Shyr‐Te
Published: March 2007

The effect of various metabolic inhibitors on the expression of cytolytic activity of CD4 (TH1) and CD8 (CTL) clones was studied. The cytolytic activity of CD4 clones, but not CD8 clones, was sensitive to the RNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide. Conversely, cholera toxin (CT) inhibited cytolytic activity of CD8, but not CD4 clones. Both mitomycin C, a DNA synthesis inhibitor, and cyclosporin A (CsA) failed to inhibit the cytolytic activity of either CD4 or CD8 clones.

Although pretreatment with CsA or CT did not inhibit the cytolytic activity of CD4 clones, lymphokine (interleukin 2, IL 2, interferon‐γ, IFN‐γ, and tumor necrosis factor, TNF) production was strongly inhibited. Similarly, pretreatment of a CD8 clone with actinomycin D or CsA inhibited lymphokine production without affecting cytolytic activity. The production of mRNA for TNF and IFN‐γ by concanavalin A‐activated CD4 clones was also inhibited by CsA and CT. Moreover, perforin‐specific mRNA was not detected in activated CD4 clones. Collectively, these observations demonstrated that de novo synthesis of RNA and protein is required for expression of cytolytic activity of CD4 clones, yet production of TNF, INF‐γ, IL 2 and perforin is not involved. In contrast, the cytolytic machinery of CD8 clones is present prior to activation and is quickly expressed following activation even when de novo synthesis of RNA, protein and lymphokines is blocked.
Monoclonal antibodies reacting with the interleukin 1 receptor define a multi‐molecular complex
Authors: Lewis Cynthia , Mazzei Gonzalo , Shaw Alan
Published: March 2007

By sequential immunization with a variety of different interleukin (IL) 1 receptor (IL 1R)‐bearing cells and by generating a large number of clones from a single fusion experiment, we have managed to produce two monoclonal antibodies which react with the lymphocyte IL 1R. The antibodies also react with the mouse fibroblast line, 3T3, but we have not yet defined their reactivity with cells carrying low‐affinity receptors. Immunoprecipitation allows the IL 1R to be isolated from EL4 6.1 cell membrane preparations and shows that the IL 1R exists as a complex. The antibodies clearly interfere with IL 1 responses both in vitro and in vivo. These are the first anti‐mouse IL 1R antibodies to be described which clearly and profoundly affect the immune response.
Detection of primary cytotoxic T lymphocytes specific for the envelope glycoprotein of HIV‐1 by deletion of the env amino‐terminal signal sequence
Authors: McChesney Michael , Tanneau Françoise , Regnault Armelle , Sansonetti Philippe , Montagnier Luc , Kieny Marie Paule , Rivière Yves
Published: March 2007

A heterogenous population of envelope glycoprotein‐specific cytotoxic effector cells are found in the peripheral blood of individuals infected with HIV‐1, and in many cases env‐specific lysis is not restricted by MHC molecules and is not blocked by antibody to CD3 (Rivière, Y. et al., J. Virol. 1989. 63: 2270). In order to detect env‐specific cytotoxic T lymphocytes (CTL) in fresh peripheral blood mononuclear cells of HIV‐1‐infected donors, a mutant env gene with deletion of the amino‐terminal signal sequence was inserted into vaccinia virus. This deletion of the amino‐terminal signal sequence was inserted into vaccinia virus. This deletion results in synthesis of an envelope protein that is not glycosylated and not expressed at the surface of infected cells. Target cells infected with this recombinant vaccinia virus are not lysed by antibody‐mediated cellular cytotoxicity, but they are recognized by secondary CTL. Comparing lysis of target cells expressing gp160 of HIV‐1 and the signal peptide deletion mutant, primary env‐specific CTL were detected in some individuals infected with HIV‐1.
Inhibition of antibody formation by receptor cross‐linking: the molecular characteristics of inhibitory haptenated polymers
Authors: Dintzis Renée Z. , Okajima Masako , Middleton Marjorie H. , Dintzis Howard M.
Published: March 2007

Previous studies from this laboratory have reported on the characteristics of a molecule which make it inhibitory. These findings were based on studies using the T cell‐independent haptenated polymer, 2,4‐dinitrophenyl‐polyacrylamide (DNP‐PA). The present study was undertaken to determine whether the same molecular properties which defined the inhibitory potentials of DNP‐PA were characteristic of other haptenated polymers as well. The molecules studied here consisted of a series of five diverse fluoresceinated polymers with varying molecular mass and hapten valence. In agreement with the previous findings on DNP‐PA, definable molecular forms of fluoresceinated dextrans, Ficolls, polyacrylamides, carboxymethyl celluloses and polyvinyl alcohols were each found to be capable of inhibiting the anti‐fluorescein response to immunogenic forms of haptenated polymers of either the same or different carrier chemistry. This inhibitory ability was relatively independent of the chemical composition and conformation of the carrier polymer. These studies allow some logical generalizations to be made as to which quantitative molecular properties of haptenated type 2 T cell‐independent antigens determine whether they will be stimulatory or inhibitory of an anti‐hapten immune response.
Interleukin 6 production in the central nervous system during experimental autoimmune encephalomyelitis
Authors: Gijbels Koen , van Damme Jo , Proost Paul , Put Willy , Carton Herwig , Billiau Alfons
Published: March 2007

Interleukin 6 (IL6) is one of the major inflammation‐associated cytokines. Elevated serum or tissue levels of IL 6 have been reported to occur in several human diseases, including infections of the central nervous system (CNS), but not in non‐infectious CNS inflammation, e.g. multiple sclerosis. While studying experimental autoimmune encephalomyelitis (EAE) as an animal model for autoimmune inflammation of the CNS, we found increased IL 6 levels in the CNS of mice suffering from a lethal form of the disease. IL 6 levels in the spleens and sera were not significantly increased. These findings are indicative of local production of IL6 in the CNS during EAE, and represent the first demonstration of IL6 production in non‐infectious CNS inflammatory disease.
Recombinant C5a enhances interleukin 1 and tumor necrosis factor release by lipopolysaccharide‐stimulated monocytes and macrophages
Authors: Cavaillon Jean‐Marc , Fitting Catherine , Haeffner‐Cavaillon Nicole
Published: March 2007

Lipopolysaccharide (LPS) is a potent inducer of interleukin 1 (IL 1) synthesis and release, and of tumor necrosis factor (TNF) secretion. Many signals can enhance the LPS‐induced production of these cytokines. We have previously observed that addition of low amounts of normal human serum to the culture medium enhances IL 1 production. Among serum factors, anaphylatoxins C3a and C5a and/or their desArg derivatives have been shown to enhance LPS‐induced IL 1 and TNF production. However, the capacity of natural anaphylatoxins to induce by themselves the production of cytokines remains a controversal issue. We have investigated the capacity of human recombinant C5a (hrC5a) to induce IL 1 and TNF production. Despite its lack of direct triggering, hrC5a was able to act synergistically with LPS, leading to higher IL 1 and TNF release by human monocytes and mouse peritoneal macrophages. As assessed by the comitogenic assay, hrC5a increased IL 1 release, whereas cell‐associated IL 1 activity was not significantly modified. Measurement by enzyme‐linked immunosorbent assay of human IL 1β led to similar conclusions, whereas measurement of IL 1α by radioimmunoassay indicated, in addition, an increase in intracellular IL 1α.
Difference in the clustering of complement receptor type 1 (CR1) on polymorphonuclear leukocytes and erythrocytes: Effect on immune adherence
Authors: Paccaud Jean‐Pierre , Carpentier Jean‐Louis , Schifferli Jürg Alfred
Published: March 2007

Complement receptor type 1 (CR1) mediates the adherence of complement‐reacted immune complexes (IC) to various blood cells. On the erythrocyte, CR1 are clustered, a distribution which favors efficient multivalent binding of C3b‐coated IC. IC can also bind to CR1 expressed on polymorphonuclear (PMN) leukocytes. To evaluate the respective importance of these two cell types in immune adherence reactions, functional analysis of IC binding, as well as morphological studies of CR1 distribution at their surface were undertaken. At equal cell concentrations, resting PMN leukocytes bound the same percentage of IC as erythrocytes, despite expressing four times more CR1 at their surface. At equal CR1 concentrations, IC binding to resting or formyl‐methionyl‐leucyl‐phenylalanine (fMLP)‐stimulated PMN leukocytes was always lower than to erythrocytes. The morphological counterpart of these differences was studied by label‐fracture immunoelectron microscopy. On erythrocytes, almost 50% of the CR1 were distributed in clusters of ⩾ 3 units, while < 15% were grouped in such clusters on the surface of PMN leukocytes. Activation of PMN leukocytes by fMLP increased the surface density of CR1, but the proportion of clustered CR1 remained unchanged.

These observations suggest that the low responsiveness of PMN leukocytes towards C3b‐coated IC may be due to the unaggregated state of CR1. In the circulation, erythrocytes might function as a “buffer” for PMN leukocytes, which would otherwise engage too swiftly in reactions with IC.
Activated but not resting T cells or thymocytes express colony‐stimulating factor 1 mRNA without co‐expressing c‐fms mRNA
Authors: Cerdan Chantal , Courcoul Marianne , Razanajaona Diane , Pierrès Anne , Maroc Nicolas , Lopez Marc , Mannoni Patrice , Mawas Claude , Olive Daniel , Birg Françoise
Published: March 2007

Following the observation that, besides acute myeloid leukemia cells, acute lymphoid leukemia cells of either B or T phenotype could express the transcript for the colony‐stimulating factor 1 (CSF‐1), a growth factor known to be restricted to the monocytic‐macrophage lineage, various sources of resting and/or activated T cells and thymocytes were screened for expression of this hemopoietic growth factor. We report here that the CSF‐1 transcript was rapidly (7 h) induced in T cells by a variety of stimuli, but was not detectable in either resting T cells or thymocytes. In addition, secretion of CSF‐1 was detectable in the supernatants of activated T cells by 72 h, with a peak around 92‐120 h. In contrast to activated monocytes, the transcript of the c‐fms proto‐oncogene, the product of which is the receptor for CSF‐1, was not detectable in either resting or activated T cells. This observation could be relevant to the intimate relationships between T cells and antigen‐presenting cells during immune responses.
Identification and characterization of rat T cell subpopulations expressing T cell receptors α/β and γ/δ
Authors: Lawetzky Alexandra , Tiefenthaler Georg , Kubo Ralph , Hünig Thomas
Published: March 2007

Peripheral lymphoid organs of the rat were investigated for the presence of lymphocytes that expressed the pan‐T cell markers CD5 and OX‐52 but not the T cell receptor (TcR) α/β. Two such populations were identified: 2% to 4% of lymphocytes in adult spleen, lymph nodes and peripheral blood are CD5+ TcR α/β− and express the OX‐52 antigen at the same density as TcR α/β+ T‐cells. About 90% of these cells are CD8+. A second population is CD5−, CD8+ and OX‐52low. Radioimmunoprecipitation from digitonin lysates of surface‐labeled cells with an anti‐CD3 antiserum showed that the CD5+, but not the CD5− population of TcR α/β− cells expresses a CD3‐associated disulfide‐linked cell surface molecule of about 100 kDa apparent mol. mass. Upon reduction, one major band, migrating with 48 kDa was observed. A band of the same size was obtained with an anti‐human δ chain peptide antiserum, indicating that the CD3‐associated non‐TcR α/β molecule is the rat TcR α/δ. Functional assays showed that most, if not all natural killer (NK) cell activity is present in the CD5−‐OX‐52low population. Reactivity to foreign major histocompatibility complex (MHC) antigens in mixed lymphocyte reaction was exclusively found in TcR α/β+ splenic T cells. It is concluded that rat α/δ T cells in the spleen do not contain a high frequency of cells with specificity for foreign MHC antigens. The seeding of the periphery with α/β and the presumptive α/δ T cells was followed from birth. Most prominently in the spleen, α/β T cells reached adult levels much later than α/δ T cells. Taken together, these findings demonstrate the expression of the TcR α/δ on a minor population of peripheral rat T cells with the predominant phenotype CD4−CD8+ that has no NK cell activity when freshly isolated and does not contain a high frequency of alloreactive cells.
Reversal of autoimmune encephalomyelitis by membranes presenting myelin basic protein‐associated class II MHC molecule as an approach to immunotherapy of organ‐specific autoimmune diseases
Authors: Ben‐Nun Avraham , Yossefi Sara
Published: March 2007

Experimental autoimmune encephalomyelitis (EAE), a well‐accepted experimental model for multiple sclerosis in humans, is a paralytic disease mediated by CD4+ T cells specific for myelin basic protein (MBP). Several approaches to immune‐specific therapy of EAE as a model for other organ‐specific autoimmune diseases have previously been reported. We now show that macrophages (MΦ) or B cells, as antigen‐presenting cells, when pulsed with MBP and intraperitoneally (but not intravenously) inoculated after the encephalitogenic challenge, are highly effective in blocking the development of EAE. Moreover, MΦ pulsed with an organ tissue homogenate, mouse spinal cord homogenate, can also present the relevant target antigen, MBP, and are as effective as MBP‐pulsed MΦ in blocking the development of EAE. This capacity of the MΦ to identify and present the relevant target antigen indicates that this approach is also applicable to organ‐specific autoimmune diseases other than EAE, regardless of how much is known about their etiological agent or specific target antigen. Nonviable glutaraldehyde‐fixed MBP‐pulsed MΦ or membranes derived from MBP‐pulsed MΦ retain their capacity to block the development of EAE.
Restricted utilization of VH and DH genes in leukemic rabbit B cells
Authors: Becker Robert S. , Suter Mark , Knight Katherine L.
Published: March 2007

Polyclonal B cell leukemias have been generated in 17‐ to 20‐day old Eμ‐myc transgenic rabbits. To analyze the repertoire of VH genes utilized in early B cells, eight VDJ genes were cloned from these leukemic cells. The nucleotide sequences of these genes indicated that seven of the eight VDJ genes encoded prototype VHal, VHa2 or VHa3 allotypes. The two VDJ genes encoding VHa1 molecules had VH segments with identical nucleotide sequences; similarly, the VH segments of the four VDJ genes encoding VHa2 molecules were identical, with the exception of a single base pair. These data suggest that a limited repertoire of VH genes were utilized in the generation of these VDJ genes. The DH segments of these genes were limited to two DH families, D1 and D2, indicating that a restricted repertoire of DH genes also had been utilized. Since these leukemic cells probably developed early in ontogeny, we suggest that this restricted utilization of VH and DH genes is representative of B cells from developmentally immature rabbits.
Cholera holotoxin and its B subunit enhance Peyer's patch B cell responses induced by orally administered influenza virus: Disproportionate cholera toxin enhancement of the IgA B cell response
Authors: Chen Ker‐Sang , Strober Warren
Published: March 2007

In these studies we analyzed the adjuvant effect of cholera holotoxin or cholera toxin (CT) B subunit on the B cell response to mucosal antigens. Purified Peyer's patch B cells obtained from mice at varying periods of time after oral administration of inactivated influenza virus, with or without a CT preparation, were stimulated in vitro in the absence or presence of various lymphokines. Responses were measured by an antigen‐and isotype‐specific ELISPOT assay. In this system cultures containing a combination of lymphokines [interleukin 5 (IL 5), interferon‐γ (IFN‐γ), IL 4] gave comparable responses to those containing T cells from immunized mice or supernatant of concanavalin A‐stimulated T cells and therefore were assumed to express optimum or near optimum B cell responses. Administration of a CT preparation along with influenza virus increased the number of B cells producing anti‐influenza antibodies of both the IgM and IgA isotypes, with the effect on the IgA response at least threefold greater than the effect on the IgM response. These results thus indicate that CT preparations enhance the memory B cells response in Peyer's patches and, in addition, suggest that CT enhances isotype switching. In this antigen‐specific B cell system IL 4 augmented responses in cultures containing IL 5 but not IFN‐γ; in addition, IL 5 and IFN‐γ acted in an additive fashion. Thus, these findings suggest that the effects of IL 5 and IFN‐γ are at least in part, mediated via different cellular differentiation pathways.