bluuebunny/crossref_metadata_2025 huggingface dataset:
Amino acid sequence analysis of a mouse interleukin 5 receptor protein reveals homology with a mouse interleukin 3 receptor protein
Authors: Devos René , Vandekerckhove Joel , Rolink Antonius , Plaetinck Geert , van der Heyden José , Fiers Walter , Tavernier Jan
Published: March 2007

A polypeptide chain for the mouse interleukin 5 receptor (IL5R) was purified from detergent‐lysed B13 cells, a mouse IL5‐dependent pre‐B cell line. Purification was by a single immunoaffinity chromatographic step using an anti‐mouse IL 5R monoclonal antibody, R52. Internal amino acid sequence was obtained from four trypsin‐generated peptides. All peptides were found to be present in the published amino acid sequence of a mouse IL 3R and the mouse IL 3R‐like protein deduced from the cDNA. This indicates that the mouse IL 5R and the mouse IL 3R have a homologous polypeptide in common and suggests that the specificity of these lymphokine receptors is mainly generated by association with another ligand‐specific polypeptide chain.
Anti‐T cell receptor antibody treatment of rats with established autologous collagen‐induced arthritis: suppression of arthritis without reduction of anti‐type II collagen autoantibody levels
Authors: Goldschmidt Tom J. , Holmdahl Rikard
Published: March 2007

Activation of T cells is critical for the development of type II collagen (CII)‐induced arthritis (CIA). However, the relative importance of T cells in their delivery of help to B cells, promoting autoantibody formation or acting as inflammatory initiating cells, is unclear. The effect of a monoclonal antibody directed to the α/β T cell receptor (TcR) on the development of autologous CIA was studied. Two weeks after immunization with autologous CII the onset of severe arthritis occurred, followed by a chronic arthritis activity in the peripheral joints. Anti‐TcR treatment before immunization suppressed the incidence of arthritis and the autoantibody response to CII. Treatment given immediately before the expected onset delayed the appearance of arthritis. Treatment given to already arthritic rats reduced the severity. In the latter two groups the serum levels of anti‐CII autoantibodies were not affected. The duration of the ameliorating effect was limited and with the return of arthritis a concomitant antibody response towards the injected mouse anti‐TcR antibody was observed. These results show that the role of T cells in both the induction and perpetuation of CIA is essential and not limited to the triggering of production of pathogenic anti‐CII autoantibodies.
Cross‐protection against influenza A virus infection by passively transferred respiratory tract IgA antibodies to different hemagglutinin molecules
Authors: Tamura Shin‐Ichi , Funato Hirono , Hirabayashi Yoshihiro , Suzuki Vujiro , Nagamine Takashi , Aizawa Chikara , Kurata Takeshi
Published: March 2007

Mice that were intranasally immunized with different influenza A virus hemagglutinins (HA), derived from PR8 (H1N1), A/Yamagata (H1N1) or A/Fukuoka (H3N2) virus, together with cholera toxin B subunit as an adjuvant, were examined for protection against PR8 infection; PR8 HA and A/Yamagata HA immunization conferred complete protection, while A/Fukuoka HA immunization failed to confer protection. In parallel with protection, PR8 HA‐, A/Yamagata HA‐, and A/Fukuoka HA‐immunized mice produced a high, a moderate and a low level of PR8 HA‐reactive IgA in the respiratory tract, respectively. These IgA antibodies were not only higher in content in the nasal secretions, but also more cross‐reactive than IgG. The purified IgA antibodies from respiratory tract washings of PR8 HA‐immunized mice, which contained the HA‐specific IgA corresponding to the amount deteced in the nasal wash, were able to protect mice from PR8 challenge when transferred to the respiratory tract of naive mice. The transfer of IgA from A/Yamagata HA‐immunized mice also afforded cross‐protection against PR8 infection, whereas the IgA from A/Fukuoka HA‐immunized mice failed to provide protection. The ability of transferred IgA to prevent viral infection was dependent on the amount of HA‐reactive IgA remaining in the respiratory tract of the host at the time of infection. These experiments directly demonstrate that IgA antibodies to influenza A virus HA by themselves play a pivotal role in defence not only against homologous virus infection, but also against heterologous drift virus infection at the respiratory mucosa, the portal of entry for the viruses.
Immunization with subunit human immunodeficiency virus vaccine generates stronger T helper cell immunity than natural infection
Authors: Clerici Mario , Tacket Carol O. , Via Charles S. , Lucey Daniel R. , Muluk Satish C. , Zajac Robert A. , Boswell R. Neal , Berzofsky Jay A. , Shearer Gene M.
Published: March 2007

Healthy, human immunodeficiency virus seronegative (HIV−) volunteers were multiply immunized with a recombinant gp160 (rgp160) candidate acquired immunodeficiency syndrome (AIDS) vaccine. Peripheral blood lymphocytes from volunteers immunized with 40 μg or with 80 μg (two volunteers per group) of rgp160, as well as from control donors, were tested for T helper (Th) cell function either prior to immunization, 8 to 12 months after the third immunization, or 2 to 5 months after the fourth immunization. The Th cell functional tests included antigen‐induced in vitro interleukin 2 (IL 2) production and proliferation in response to influenza A virus (FLU) and to four synthetic peptides of HIV gp120 and gp160, previously demonstrated to be recognized by Tcells from HIV naturally infected patients. Our results demonstrate the following: (a) immunization of HIV− individuals with rgp160 results in IL 2 production and T cell proliferation in response to HIV determinants; (b) boosting with rgp160 enhances Th function; (c) HIV‐specific Th function is up to 100‐fold greater in the multiply immunized volunteers than that observed in asymptomatic. HIV‐infected individuals; and (d) multiple immunization with rgp160 does not impair Th function to a non‐HIV antigen such as influenza A virus. These results indicate that immunization of uninfected individuals with an HIV subunit vaccine results in much stronger Th cell immunity than does natural infection and suggests that vaccination against HIV may be possible.
Bispecific F(ab′γ)3 antibody derivatives for redirecting unprimed cytotoxic T cells
Authors: Putt Alison , Greenman John , Stevenson George T. , Glennie Martin J.
Published: March 2007

Bispecific F(ab′γ)3 derivatives have been constructed by selective coupling of monoclonal antibody Fab′γ fragments, using the cross‐linker o‐phenylenedimaleimide attached to hinge‐region sulfhydryl groups. All the derivatives were designed to redirect the cytotoxic activity of circulating human T lymphocytes, via their CD2, CD3 or CD5 molecules, against 51Cr‐labeled chicken red blood cells (CRBC) or the human lymphoblastoid cell line, Namalwa. The composition of F(ab′γ)3 molecules was controlled by the selection of Fab′γ for initial attachment of cross‐linker, allowing production of derivatives with either two anti‐effector and one anti‐target Fab′γ arms or vice versa.

Bispecific F(ab′γ)3 derivatives, recruiting peripheral blood lymphocytes (PBL) via CD3 as effectors against CRBC, gave titers in redirected cellular cytotoxicity assays which were consistently 25–100 times higher than those given by an equivalent F(ab′γ)2 reagent, with full activity being retained down to antibody concentrations of 1 ng/ml. The allocation of valencies – two anti‐target/one anti‐effector or the converse – was not important in determining the increase in titer. No significant lysis was seen when recruiting PBL via CD2 or CD5. It was notable that these CD3‐specific derivatives yielded good tumor‐cell lysis when using fresh, unprimed PBL as effectors. The increased activity over a bispecific F(ab′γ)2 was apparent both in titer and the maximum level of lysis achieved. Blocking studies with excess Fab′γ suggested that the potency of F(ab′γ)3 derivatives lie in the strength of bridging between effector and target cells. This high avidity contact could favor metabolic activation in either the target or effector cell, or could increase the efficiency of lytic machinery already triggered.
The murine heat‐stable antigen: a differentiation antigen expressed in both the hematolymplioid and neural cell lineages
Authors: Rougon Geneviève , Alterman Lesley A. , Dennis Karen , Guo Xia‐Jun , Kinnon Christine
Published: March 2007

The murine heat‐stable antigen (HSA) and the p31 antigen are cell surface glycoconjugates which are transiently expressed during the development and differentiation of the hematolymphoid and neural cell lineages, respectively. We show here that monoclonal antibodies which react with these two species recognize a common antigenic determinant which is expressed on both HSA and p31, and that HSA and p31 share a common protein core. Differences in the molecular weights of the antigens most likely reflect variations in the extent of post‐translational modifications. From these studies we conclude that these antigens are members of the same family of heat‐stable antigens. Our results lead us to speculate on how these molecules are related, their function, and what role they play in cellular differentiation in hematolymphoid and neural cell development.
Class II major histocompatibility complex restriction of human T cell responses to short ragweed allergen, Amb a V
Authors: Huang Shau‐Ku , Zwollo Patty , Marsh David G.
Published: March 2007

Although T cells are known to play a crucial role in the induction of IgE synthesis, the class II major histocompatibility complex (MHC) restriction of aeroallergen‐induced T cell responses in humans is incompletely defined. We have previously shown that, in allergic Caucasoid individuals, HLA‐DR2 and Dw2 (DR2.2) is strongly associated with specific IgE and IgG antibody responses to highly purified Ambrosia (ragweed) allergen, Amb a V, from the artemisiifolia (short) species. For example, 95% of IgE antibody responders to Amb a V were typed as DR2.2. In a novel study of the genetic control of T cell responses to the Amb a V allergen, we have investigated the MHC class II restriction specificity of three CD4, Amb a V‐specific T cell clones derived from a DR2.2+ atopic patient, and a polyclonal Amb a V‐reactive T cell line from another DR2.2+ patient. We observed proliferative responses of all three clones to Amb a V only when either HLA‐DR2.2 or DR2, Dw12 (DR2.12; found on Mongoloid populations) was present on the antigen‐presenting cells, regardless of the HLA‐DQ phenotype of the cells. Moreover, the responses of T cell line and clones were abolished by anti‐DR but not by anti‐DQ nor by anti‐DP monoclonal antibodies, and, significantly, anti‐DRα/β12 (anti‐α/β1w15/w16; anti‐“DR2b”) monoclonal antibody blocked, in a dose‐dependent manner, the cloned T cell responses to Amb a V. These findings demonstrate that DRα/β12.2 (DRα/β11501) and DRα/β12.12 (DRα/β11502) are functional in the restriction of the T cell recognition of Amb a V. These findings also illustrate the power of the allergy model for definitive investigation of the molecular basis of the human immune response.
A highly selected panel of anti‐CD4 antibodies fails to induce anti‐idiotypic antisera mediating human immunodeficiency virus neutralization
Authors: Healey Donald G. , Dianda Lee , Buok David , Schroeder Kurt , Truneh Alemseged , Sattentau Quentin J. , Beverley Peter C. L.
Published: March 2007

Anti‐CD4 antibodies directed to the N terminus of CD4 can inhibit human immunodeficiency virus (HIV) infection. Therefore, it has been proposed that some of these reagents may contain idiotypic determinants which conformationally model the binding site expressed on gp120. In this report, we have selected a panel of anti‐CD4 monoclonal antibodies as idiotypic mimics of gp120 by employing cross‐blocking techniques, and CD4 epitope mapping using site‐directed mutagenesis. These studies suggest that only 4 out of the original panel of 12 would be expected to represent suitable candidates for modelling the gp120 binding site. Nevertheless, anti‐idiotypic antisera raised against these antibodies failed to inhibit gp120 binding to CD4. This negative result may reflect the incomplete modelling of the virus binding site by anti‐CD4, or the lack of internal image antibody in the anti‐idiotypic preparations. Alternatively, the binding site on gp120 may not be accessible to antibody neutralization, excluding the possibility of an idiotypic vaccine to HIV based on anti‐CD4 antibody as surrogate antigen.
Murine epidermal γ/δ T cells express Fcγ receptor II encoded by the FcγRα α gene
Authors: Kuziel William A. , Lewis Julia , Nixon‐Fulton Jackie , Tigelaar Robert E. , Tucker Philip W.
Published: March 2007

Short‐term bulk cultures and some long‐term clones and lines of murine T cell receptor (TcR) γ/δ‐bearing epidermal T cells (dEC) were found to express an Fcγ receptor II (FcγRII), as revealed by reactivity with the monoclonal antibody 2.4G2. Northern blot analysis showed that the FcγRII expressed on dEC is encoded solely by the FcγR α gene. While all the various cultured dEC cell populations analyzed exhibit lectin‐dependent cellular cytotoxicity, only those which expressed FcγR α were also capable of mediating antibody‐dependent cellular cytotoxicity (ADCC). These results in combination with the previous demonstration of FcγR α on mouse natural killer cells support an essential role for FcγR α in ADCC and extend an analogy with surface CD16 (FcγRIII) expression and ADCC in human natural killer cells and peripheral TcRγ/δ T cells.
Candida albicans‐specific Ly‐2+ lymphocytes with cytolytic activity
Authors: Romani Luigina , Mocci Simonetta , Cenci Elio , Rossi Ruggero , Puccetti Paolo , Bistoni Francesco
Published: March 2007

To determine whether antigen (Ag)‐specific cytotoxic T lymphocytes are generated during experimental Candida albicans infection, purified L3T4+ and Ly‐2+ lymphocytes from immunized mice were cultured in the presence of syngeneic accessory cells, C. albicans Ag, and interleukin 2. Yeast‐infected bone marrow macrophages were used as target cells in a standard 51Cr‐release assay. Freshly isolated L3T4+ and Ly‐2+ lymphocytes failed to lyse either target cell type. However, Ag‐specific, major histocompatibility complex (MHC)‐unre‐stricted lysis of infected macrophages was evident with immune Ly‐2+ cells after 7–10 days in culture. The cultured cells were > 98% Thy‐1+, CD3+, L3T4−, Ly‐2+, T cell receptor α/β+ T cells, and their lytic activity was potentiated by the addition of anti‐CD3 monoclonal antibodies. At limiting effector cell numbers, Ag‐specific MHC‐restricted lymphocytes with cytotoxic activity against infected macrophages could be identified. We suggest that C. albicans infection stimulates multiple cytotoxic cell precursors with varying recognition stringency, which include MHC class I‐restricted, Ag‐specific cytotoxic T lymphocytes.
Dissection of signals controlling T cell function and activation: H7, an inhibitor of protein kinase C, blocks induction of primary T cell proliferation by suppressing interleukin (IL) 2 receptor expression without affecting IL 2 production
Authors: Hengel Hartmut , Allig Birgit , Wagner Hermann , Heeg Klaus
Published: March 2007

T cell activation induced via cross‐linking of the T cell receptor (TcR) stimulates hydrolysis of phosphatidylinositol to the second messengers diacylglycerol (DAG) and inositol 1,4,5‐triphosphate (IP3). DAG is necessary for the activation and function of protein kinase C (PKC) which is suggested to play a key role in the cascade of signal transduction when translocated from the cytosol to the cell membrane. In this report, we investigated responses of resting vs. activated Ly‐2+ and L3T4+ T lymphocytes in the presence of the PKC inhibitor H7 [1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine]. H7 inhibited the induction of primary T cell proliferation, while interleukin 2 (IL 2) production was fully retained. The effect of the PKC inhibitor on primary T cells depended on the type of ligand interacting with the TcR: increasing doses of concanavalin A or of immobilized anti‐CD3 monoclonal antibody (mAb), but not of anti‐Vβ8 or of anti‐TcR α/β mAb, partly overcame the blockade, indicating a differential signaling compared to the former stimuli. The blockade of T cell proliferation by H7 was not due to an inhibition of PKC translocation, but occurred even 4–8 h after T cell induction and correlated with a significant reduction of IL 2 receptor (IL 2R) expression. In contrast, the mRNA levels of IL 2R and the cellular proto‐oncogenes c‐fos and c‐myc were not affected. On activated T cells, H7 neither blocked proliferation nor IL 2R expression. Consequently, H7 dissects the signal resulting in T cell proliferation from those governing the triggering of other T cell functions, i.e. IL 2 production, during primary responses of Ly‐2+ or L3T4+ murine T lymphocytes.
Functional expression of the endogenous Thy‐1 gene and the transfected murine Thy‐1.2 gene in rat basophilic leukemia cells
Authors: Dráberová L'ubica , Dráber Petr
Published: March 2007

An interaction of MRC OX7 monoclonal antibody with Thy‐1.1 antigen of rat peritoneal and pleural mast cells has been previously shown to induce rat mast cell activation (L'. Dráberová, Eur. J. Immunol. 1989. 19: 1715). In the present study we analyzed the expression and function of the Thy‐1 antigen in rat basophilic leukemia cells, clone RBL‐2H3. Two RBL‐2H3‐derived cell lines with stable expression of murine Thy‐1.2 antigen, after transfection of a genomic clone of murine Thy‐1.2 or Thy‐1.2 cDNA under the control of simian virus 40 promoter, were also analyzed. Direct radioantibody binding assays, indirect immuno‐fluorescence studies and flow cytometry analyses revealed that both endogenous Thy‐1.1 and transfected murine Thy‐1.2 gene products were expressed on the surface of the cells under study. Analysis of the distribution of the Thy‐1 antigen in situ in cells grown attached to tissue culture vessels located the Thy‐1 predominantly in regions of cell‐cell contacts. Incubation of RBL‐2H3 cells with Thy‐1.1‐specific antibodies, or of the transfected cells with both Thy‐1.1‐ and Thy‐1.2‐specific antibodies, induced a rapid early increase in the concentration of intracellular free calcium ([Ca2+]i) released from internal stores. Sustained increase of [Ca2+]i required the presence of Ca2+ in the extracellular medium. The increase in [Ca2+]i was followed by histamine release from the target cells. The combined data indicate that RBL‐derived cells can be used as a useful model system for analysis of Thy‐1 antigen‐mediated activation of rat mast cells.
CD3+ T cells in severe combined immunodeficiency (scid) mice. II. Transplantation of dm2 lymphoid cells into semi‐allogeneic scid mice
Authors: Rudolphi Angelika , Spiess Sibylle , Conradt Peter , Claesson Mogens H. , Reimann Jörg
Published: March 2007

Intravenous injection of nonfractionated BALB/c‐H‐2dm2 (dm2) (Ld−) spleen cells into 4‐week‐old, semi‐allogeneic (H‐2d, Ld+) C.B‐17 scid/scid severe combined immunodeficient (scid) mice (2 × 107 cells/mouse) reconstituted T lymphopoiesis in thymi and repopulated the lymphoid white pulp in spleens of these immunodeficient recipients. Transplantation of dm2 thymocytes into young scid mice (5 × 107 cells/mouse) established a donor‐derived CD3+ T cell population in spleens of recipient scid mice, in which CD4+ T cells predominated. This was demonstrated by marker analyses of thymocytes and splenocytes, and determinations of serum immunoglobulin levels in transplanted scid mice. Transfer of splenocytes from young primary scid recipients into young secondary or tertiary recipients (3 × 106 cells/mouse) engrafted preferentially dm2‐derived CD3+CD4+CD8− T cells in spleens of scid mice despite the strong selective Ld‐associated alloantigenic stimulus for CD8+ T cells. Intravenous injections of nonfractionated dm2 spleen cells (2 × 107 cells/mouse) or thymocytes (5 × 107 cells/mouse) into 10‐ to 12‐month‐old, “leaky” scid mice induced severe clinical signs of graft‐vs.‐host disease (GVHD) in all scid recipients. Lymphoid repopulation of spleen and thymus in old scid recipients was incomplete. This GVHD was not transferrable by injecting 3 × 106 spleen cells from old diseased primary scid recipients into secondary or tertiary young scid recipient mice. In these serial transfers, dm2‐derived CD3+CD4+CD8− T cells were again preferentially engrafted in spleens of scid recipients.

Transfer of purified CD4+ dm2 T cells into young scid mice (2 × 105 to 5 × 105 cells/mouse) engrafted this T cell subset into the spleen of semi‐allogeneic scid recipients. This was revealed by histological examinations, surface marker analyses, in vitro isolation of donor‐type CD3+CD4+ T cell lines from spleens of transplanted scid mice, and serial transfer experiments. These data indicated that the CD4+ T cell compartment of scid mice can be selectively repopulated by semi‐allogeneic T cells.

Injections of purified CD8+ dm2 T‐cells into young scid mice (2 × 105 cells/mouse) did not establish a CD8+ T cell graft in spleens of recipients. It was necessary to inject transplanted scid mice biweekly with 104 units recombinant interleukin 2 to establish and/or maintain transferred dm2 CD8+ T cells in spleens of these recipients. dm2 CD8+ T cell‐transplanted and interleukin 2‐treated scid mice did not develop any evidence of GVHD over the 9‐week observation period.
Cloning, expression and cross‐linking analysis of the murine p55 tumor necrosis factor receptor
Authors: Barrett Kathy , Taylor‐Fishwick David A. , Cope Andrew P. , Kissonerghis A. Maija , Gray Patrick W. , Feldmann Marc , Foxwell Brian M. J.
Published: March 2007

Tumor necrosis factor (TNF) mediates its pleiotropic effects via high‐affinity cell surface receptors. In man, molecular cloning has identified two distinct, independent TNF receptors (TNFR) of 55 and 75 kDa. It is unclear, however, whether the multiple effects of TNF are segregated between the receptor types. In the mouse, previous studies had shown functional heterogeneity of TNFR, since the WEHI 164 fibroblast line is sensitive to the cytotoxic effects of both murine and human TNF, whereas the murine T cell line, CT6, proliferates in response to murine but not human TNF. In this study, the cloning of a cDNA encoding the murine homologue of the p55 TNFR is reported. This receptor binds murine and human TNF with equal affinity and is expressed on WEHI 164 and a number of other cell lines, but only low levels of mRNA and no protein is detectable on CT6 cells. CT6 cells, however, express a second TNFR of approximately 75 kDa, identified by cross‐linking analysis, which is also found on WEHI 164 cells, and binds only murine TNF. These studies establish that there are also two TNFR in the mouse, and suggests that there may be segregation of the cytotoxic and proliferative responses between different receptors, at least in these cell lines.
Disorganization and restoration of thymic medullary epithelial cells in T cell receptor‐negative scid mice: Evidence that receptor‐bearing lymphocytes influence maturation of the thymic microenvironment
Authors: Shores Elizabeth W. , Van Ewijk Willem , Singer Alfred
Published: March 2007

In order to assess the possible role of lymphoid cells in the development of thymic epithelium, we compared the organization and maturation of thymic epithelium in scid mice lacking T cell receptor (TcR)‐positive cells with that in scid mice containing TcR+ cells. Immunohistologic examination revealed that thymi from TcR− scid mice were deficient in thymic medullary epithelial cells recognized by the monoclonal antibody ER‐TR5, and that the few thymic medullary epithelial cells present were not organized into discrete medullary areas. In contrast, thymi from scid mice containing TcR+ cells possessed ER‐TR5+ thymic medullary epithelial cells and these cells were organized normally into discrete medullary regions. Thus, normal organization and maturation of thymic medullary epithelial cells did not occur in the absence of TcR+ cells, but did occur upon introduction of TcR+ cells. We conclude that lympho‐stromal cell interactions in the thymus are not unidirectional, and that a symbiotic relationship exists between maturing epithelial cells and developing lymphocytes.
Regulation of lymphokine expression in T cell activation. I. Rapid loss of interleukin‐specific RNA after removal of the stimulating signal
Authors: Swoboda Rolf , Bormmhardt Ursula , Schimpl Anneliese
Published: March 2007

Resting T lymphocytes can be induced to express a variety of lymphokines after antigenic or mitogenic stimulation. Expression is regulated both at the transcriptional level, through induction of T cell‐specific DNA‐binding proteins, and at the post‐transcriptional level through alteration of precursors or mRNA stability. To investigate whether lymphokine expression follows a preprogrammed course after activation of T cells or whether it is dependent on the continued monitoring of activating signals, we followed the fate of interleukin (IL) 2, IL 4 and IL 2 receptor mRNA after removal of the stimulating signal concanavalin (Con A) by α‐methylmannoside (αMM). IL 2 and IL 4 needed continued stimulation of the cells for RNA expression, as shown by the rapid disappearance of IL 2 and IL 4 mRNA after addition of αMM to stimulated cells, while IL 2 receptor mRNA which is also induced after Con A stimulation was minimally influenced. In the case of both IL no new mature mRNA was generated after removal of the stimulating signal. The T1/2 of IL 2 mRNA remained unchanged after αMM treatment as compared to actinomycin D treatment, while IL 4 mRNA became labilized after withdrawal of the signal.
Identification of a murine monoclonal antibody specific for an allotypic determinant on mouse CD3
Authors: Coulie Pierre G. , Uyttenhove Catherine , Wauters Pierre , Manolios Nicholas , Klausner Richard D. , Samelson Lawrence E. , Van Snick Jacques
Published: March 2007

A murine monoclonal antibody (mAb; 7D6) that was mitogenic for T cells was derived from 129/Sv animals immunized with a T helper clone from C57BL/6 origin. Fluoresceinated 7D6 labeled T cells from most common mouse strains but not from 129/Sv and LP/J animals, and this labeling was inhibited by the anti‐CD3 ϵ mAb 145‐2C11. The mitogenicity of 7D6 for T cells had a similar strain specificity. The antibody immunoprecipitated the T cell receptor (TcR) complex from a T cell hybridoma. After dissociation of this immunoprecipitate with detergents, the CD3 γ and ϵ chains were retained by the 7D6 antibody. Immunoprecipitation data were also obtained with COS cells transfected with the CD3 γ, δ or ϵ chains alone, in pairs or together. They confirmed that 7D6 bound the CD3 γϵ pair, suggesting that the antibody recognizes a conformational epitope formed by γϵ pairing, whereas 145‐2C11 bound both γϵ and δϵ pairs. These results, therefore, add to current information about TcR structure and subunit stoichiometry. We have demonstrated that the 7D6 mAb specifically binds to a CD3 dimer comprised of γ and ϵ chains. We thus provide additional evidence that indicates that two CD3 ϵ chains are found within the receptor, one linked to CD3 γ and the other to CD3 δ.
In vivo interleukin 6 gene expression in the tumoral environment in multiple myeloma
Authors: Portier Marielle , Rajzbaum Gérald , Zhang Xue Guang , Attal Michel , Rusalen Christiane , Wijdenes John , Mannoni Patrice , Maraninchi Dominique , Piechaczyk Marc , Bataille Régis , Klein Bernard
Published: March 2007

Whether interleukin 6 (IL6) is an autocrine or paracrine myeloma cell growth factor in vivo remains unresolved. To identify which cells are producing IL 6 in vivo, we have studied the IL 6 gene expression in bone marrow mononuclear cells (BMMC) of 19 patients with multiple myeloma (MM) and in peripheral blood mononuclear cells (PBMC) of 9 patients with plasma cell leukemia (PCL). We found that the IL 6 gene was transcribed by BMMC of most patients with MM (79%). Further, IL 6 mRNA was not produced by purified myeloma cells from patients with either MM (5 patients) or PCL, but by the bone marrow environment, mainly by monocytes and myeloid cells (CD13+CD15+ cells). For 2 patients with PCL, for whom PBMC and BMMC samples were available, IL 6 mRNA could be detected in BMMC but not in PBMC. Finally, no IL 6 mRNA was detected in five freshly established IL 6‐dependent myeloma cell lines. The present data give a clear‐cut demonstration of the paracrine origin of IL 6 in vivo in human MM.
Obtention of a human primary humoral response against schistosome protective antigens in severe combined immunodeficiency mice after the transfer of human peripheral blood mononuclear cells
Authors: Mazingue Christine , Cottrez Françoise , Auriault Claude , Cesbron Jean‐Yves , Capron André
Published: March 2007

Peripheral blood mononuclear cells (PBMC) from healthy donors were injected into C.B.‐17 severe combined immunodeficiency (scid) mice which were subsequently immunized with crude Schistosoma mansoni adult worm antigenic preparation (SWAP) or with recombinant S. mansoni 28‐kDa glutathione transferase (r‐Sm‐28‐GST) antigen. PBMC from a S. mansoni‐infected patient were also transfered. The specific human anti‐SWAP and anti‐Sm‐28‐GST antibody responses were monitored. The presence in both cases of human specific antibodies in scid mouse sera was determined by enzyme‐linked immunosorbent assay and Western blotting techniques using anti‐human immunoglobulin reagents. No antibodies were detected in these sera using anti‐mouse immunoglobulin antisera. These antibodies were functional since a cytotoxic activity against schistosomula was observed when monocytes were incubated with scid mouse sera positive for anti‐Sm‐28‐GST antibodies.
Differential regulation of antigen presentation in high‐ and low‐IgE responder mice
Authors: Rizzo Luiz V. , Umetsu Dale T. , Dekruyff Rosemarie H.
Published: March 2007

Strains of mice differ greatly in their capacity to produce interleukin (IL) 4 and high levels of serum IgE. The contribution of macrophages (Mϕ) in enhancing the production of IL 4 by T cells in high‐IgE responder mice was studied. Mϕ from BALB/c mice (high‐IgE responder strain) developed an augmented capacity to present antigen to IL 4‐producing T helper type 2 (Th2) clones, when the Mϕ were pretreated by exposure to the lymphokines IL 4 and IL 5. In contrast, identical treatment of Mϕ from H‐2‐identical DBA/2 mice (low‐IgE responder strain) resulted in inhibition of the capacity to stimulate Th2 clones. The impairment of antigen presentation by DBA/2 Mϕ was closely paralleled by a decrease of cell surface class II major histocompatibility complex (MHC) expression and an abrogation of IL 1 production by these cells, while identical treatment of BALB/c Mϕ enhanced class II MHC expression and did not inhibit secretion of IL 1. Our studies demonstrate that IL 4 and IL 5 can exert different effects on Mϕ from high‐ and low‐IgE responder strains of mice, resulting in down‐regulation of the expansion of IL 4‐producing cells in low‐ but not high‐IgE responder mice.
T cell receptor/CD3 and CD28 use distinct intracellular signaling pathways
Authors: Van Lier René A. W. , Brouwer Miranda , De Groot Els , Kramer Ijsbrand , Aarden Lucien A. , Verhoeven Arthur J.
Published: March 2007

Ligation of the T cell membrane antigen CD28 strongly enhances cytokine secretion in human T lymphocytes that are activated via T cell receptor (TcR)/CD3 or CD2 molecules. This study was undertaken to investigate whether, as has been indicated for activation via TcR/CD3, stimulation via CD28 is dependent on the activation of protein kinase C (PKC). Two inhibitors of PKC, 1‐alkyl 2‐methyl‐glycerol and staurosporine, caused a dose‐dependent inhibition of T cell proliferation induced by anti‐CD3 monoclonal antibodies (mAb). The induction of interleukin (IL)2 secretion was found to be more sensitive to the effects of the PKC inhibitors than the up‐regulation of IL2 receptor expression. In marked contrast, the anti‐CD28 mAb‐mediated enhancement of T cell proliferation and IL 2 secretion were insensitive to the action of either compound. We conclude that two independent signaling pathways may be operational in human T cells. The first used by TcR/CD3 depends on the activation of PKC, whereas the second is employed by CD28 and functions independently of PKC.
Expression of different CD8 isoforms on distinct human lymphocyte subpopulations
Authors: Moebius Ulrich , Kober Gabriela , Griscelli Anne Lise , Hercend Thierry , Meuer Stefan C.
Published: March 2007

Human CD8+ lymphocyte subpopulations were analyzed for their expression of CD8 α and CD8 β subunits. Investigations with uncloned peripheral blood lymphocytes as well as cloned human natural killer and T cell subpopulations demonstrate that CD3− natural killer cells, T cell receptor γ/δ, and CD4+CD8+ T cell clones express exclusively CD8 α gene products. Structural analysis of CD8 molecules demonstrates that CD8 α+/β− T lymphocytes surface express 75‐kDa CD8 α/α homodimers whereas CD8 α/β lymphocytes express concomittantly two CD8 isoforms of different molecular masses (67 kDa and 75 kDa, respectively). Peptide mapping of these latter two isoforms suggests that CD8 is expressed as α/α homodimers and α/β heterodimers on CD8 α/β+ cells. Importantly, we found that the two CD8 isoforms behave functionally different. Thus, in contrast to CD8 α/β+/CD8 α/α+ T lymphocytes, cytolytic activity of CD8α/β−/CD8 α/α+ T cell clones was not inhibited by anti‐CD8 monoclonal antibodies and the latter were not induced to proliferate following CD3/CD8 cross‐linking.
A monoclonal antibody blocking the Schistosoma mansoni 28‐kDa glutathione S‐transferase activity reduces female worm fecundity and egg viability
Authors: Xu Chuan‐Bo , Verwaerde Claudie , Grzych Jean‐Marie , Fontaine Josette , Capron André
Published: March 2007

The protective effects of two different monoclonal antibodies (mAb) raised against the Schistosoma mansoni 28‐kDa glutathione S‐transferase (Sm 28 GST) were investigated. Two mAb of the same isotype (IgM) have been selected according to the blocking effect on Sm 28 GSTenzymatic activity (S13) or the lack of blockade (H12). When passively transfered into Fischer rats, both S13 and H12 significantly reduced the worm burden. In BALB/c mice clear effects on female worm fecundity and egg viability were observed when the S13 mAb was transfered; these effects included significantly reduced loads of intestinal eggs, reduced egg hatching rates and an increased proportion of non‐living eggs. No effect on egg production and egg hatching was observed in H12‐treated mice. In addition, worm pairs recovered from S13‐but not H12‐treated mice laid significantly fewer eggs in vitro, and normal worm pairs incubated in vitro with the S13 mAb produced significantly fewer eggs than those incubated with H12 mAb. The impairment of egg hatching ability was also reproduced in vitro by the S13 mAb. These data suggest the existence of two different effector mechanisms induced by immunization with Sm 28 GST. The effect on the schistosome worm burden appears to be independent of GST activity whereas the effect on S. mansoni female fecundity and egg viability seems to be significantly linked to the inactivation of the enzymatic site.
Preferential development of pre‐B lymphomas with drastically down‐regulated N‐myc in the Eμ‐ret transgenic mice
Authors: Iwamoto Takashi , Pu Meiyi , Ito Masafumi , Takahashi Masahide , Isobe Ken‐Ichi , Nagase Fumihiko , Kawashima Kohei , Ichihara Masatoshi , Nakashima Izumi
Published: March 2007

We established one transgenic mouse line which developed pre‐B leukemic lymphomas by introducing ret cDNA driven by the SV40 promoter and the mouse immunoglobulin (Ig) enhancer. Lymphomas developed not only in the lymph nodes and the spleen but also in the thymus between the ages of 7 and 21 weeks. Analyses of cell surface phenotypes and Ig gene rearrangement revealed that these tumors were surface IgM−B220+ pre‐B lymphomas. The rearrangement pattern of the Ig heavy chain locus indicated that the tumor cells were mono‐ or oligoclonal. Northern blot analysis showed that the ret transgene was expressed at a high level not only in the tumors but also in the prelymphomatous lymphoid tissues. We found that the expression of N‐myc was dramatically down‐regulated in the tumor cells, while the expression of c‐myc was rather stable. Further experiments demonstrated that ret gene product did not directly down‐regulate the expression of N‐myc in transformed pre‐B cell lines by in vitro transfection assay. From these results, we conclude that under the control of Ig enhancer, the ret transgene affected B lymphocytes at the early maturation stage as a prerequisite for transformation, preferentially generating a unique maturation stage of pre‐B lymphomas whose N‐myc expression was developmentally down‐regulated.
The human immunoglobulin x locus. Characterization of the duplicated O regions
Authors: Pargent Walter , Meindl Alfons , Thiebe Rainer , Mitzel Sabine , Zachau Hans G.
Published: March 2007

Two large regions of the human immunoglobulin x locus, the so‐called O regions, have been characterized on cosmid and phage λ clones. The two regions are very similar but not identical duplicates belonging to the Cx proximal (p) and the distal (d) copies of the x locus. The Op and Od regions comprise contigs of 90 and 120 kb, respectively, and contain 20 Vx genes and pseudogenes which have been sequenced. Three pairs of Vx genes were found to be practically identical in the duplicates while allotypic differences, at least for two of the genes, are considerable. The similarities between the duplicate genes may be related to the fact that the two copies of the x locus are arranged in a palindrome‐like fashion with the 5′ sides of the O regions pointing towards each other (CxJx B Lp Ap Op‐Od Ad Ld). This may have contributed to equalizing the sequences. Beyond Op and Od no further Vx genes were found within about 80 kb. Instead, repetitive DNA sequences have been localized there, the structures of which suggest that they may have been involved in the evolution of the Vx gene‐containing regions. The Vx pseudogene containing W regions, that had been transposed in evolution from the short to the long arm of chromosome 2 by a pericentric inversion, may have been derived from the O regions according to structural homologies between defined sections of the O and W regions.
Comparative studies of decay‐accelerating factor and HLA‐DR within the CD8‐brightly positive population
Authors: Tomita Akira , Okada Noriko , Okada Hidechika
Published: March 2007

Decay‐accelerating factor (DAF) is a membrane protein that inhibits C3 convertase activity of autologous complement at the cell surface. We found that DAF+ cells and DAF− (DAFlow, if any) cells are clearly separated from each other among CD8‐brightly positive (CD8bright) cells. Using three‐color fluorescence flow cytometry, we found that whereas the CD8brightDAF− population express HLA‐DR (class II major histocompatibility complex antigens, and an activated T cell marker), the CD8brightDAF+ population does not. Therefore, among the CD8bright T cells, DAF and HLA‐DR are mutually exclusive. In addition, the CD8brightDAF+ population proliferates in the presence of recombinant interleu‐kin 2 (rIL2) while the CD8brightDAF− population does not. After a 4‐day cultivation of peripheral blood lymphocytes in the presence of rIL2, expression of HLA‐DR increased in the CD8brightDAF− population and expression of IL 2Rp55 (α chain, the receptor for IL2, and a marker of T cell activation) occurred in the CD8brightDAF+ population. Furthermore, C3 deposition occurred in the CD8brightHLA‐DR+ population which lacks DAF when lymphocytes, that had been cultured for 3 days in the presence of rIL2, were incubated with fresh autologous serum. This result suggests that the absence of DAF on CD8brightHLA‐DR+ T cells might play a role in permitting complement reaction on the cells in vivo and may be related to the regulation of T cell activation.
Isolation and molecular characterization of the B cells producing the paraprotein in a case of benign monoclonal gammapathy in C57BL mice
Authors: Bos Nicolaas A. , Meeuwsen Cornelia G. , De Glopper‐Van der Veer Elisabeth , Van den Akker Theodoor W. , Radl Jiri , Zwaagstra Kornelisje A. , Benner Robbert
Published: March 2007

Benign monoclonal gammapathy (BMG) is defined as a benign monoclonal B cell proliferative disorder characterized by the presence of a persisting component of homogenous immunoglobulins (H‐Ig) in the serum. A possible role of antigenic stimulation in the development of BMG has been suggested. From a C57BL mouse, a murine model for BMG, we have Isolated clonally related B cells in order to investigate the occurrence of somatic mutations in the variable heavy chain (VH) region of the genes of H‐Ig‐producing B cell clones. Therefore, B cells were immortalized by hybridoma technology. The hybridomas were screened for resemblance of the serum H‐Ig component by Wieme agar electrophoresis, followed by immunoblotting and isoelectrofocusing. Clonal relationship was investigated by Southern blot analysis using a JH probe. In this way we isolated five hybridomas producing an IgG2a,x that was identical to the original serum H‐Ig component according to testing with anti‐idiotypic antisera. mRNA sequencing of four hybridomas showed only one base pair difference in the VH genes. This particular gene belonged to the J558 VH gene family. When compared to the most closely related known VH sequence, three base pair differences were found. The almost complete absence of base pair differences in the VH genes of the four sequenced hybridomas, compared with an independently derived hybridoma, suggests that the same germ‐line VH gene has been used and that somatic mutations were infrequent in our BMG clone.
Inhibition of interleukin 4‐promoted CD23 production in human B lymphocytes by transforming growth factor‐β, interferons or anti‐CD19 antibody is overriden on engaging CD40
Authors: Gordon John , Katira Anita , Strain Alistair J. , Gillis Steven
Published: March 2007

Interleukin 4 (IL4) is an essential component in the sequence of events directing IgE synthesis in uncommitted B lymphocytes. An early consequence of IL4's interaction with the B cell is the induction of CD23, a low‐affinity receptor for IgE (FcϵRII). The present study was designed to explore the detailed regulation of this event. First, we report that transforming growth factor‐β (TGF‐β) is a potent inhibitor of IL4‐promoted CD23 production in human B lymphocytes. The level of inhibition achieved with TGF‐β was greater than that obtained with interferons, or with a monoclonal antibody (mAb) to CD19. Next, we identified three signals, each of which was capable of selectively counteracting the inhibitors of IL 4‐promoted CD23 production: (a) the engagement of surface CD40 antigen with mAb was found to override the influence of all the inhibitors of CD23 expression; (b) mAb to surface IgM overcame the inhibitory actions of TGF‐β and interferons but not that of CD19 ligation; (c) ligation of surface CD72 counteracted the inhibition mediated by TGF‐β but not that generated by interferons or anti‐CD19 antibody. Inhibition of the IL4 signal appeared to be selective for the pathway leading to CD23 induction: none of the inhibitors profoundly altered IL4's ability to enhance surface IgM expression. The study has ramifications for the understanding of events leading to the promotion of IgE synthesis and consolidates the notion of a central role for CD40 in B cell regulation.
Developmental T cell receptor gene rearrangements: Relatedness of the α/β and γ/δ T cell precursor
Authors: Thompson Susan D. , Manzo Andrea R. , Pelkonen Jukka , Larche Mark , Hurwitz Julia L.
Published: March 2007

To examine the relationships between T cell populations at various stages of development, T cell receptor (TcR) gene rearrangements were compared between the four murine populations of (a) early thymocytes, (b) early splenocytes, (c) adult thymocytes and (d) adult splenocytes. TcR α gene rearrangements were shown to progress from 5′ to 3′ regions of the Jα locus and from 3′ to 5′ regions of the Vα locus during the development of T cells in both the thymus and spleen. Thus, the gene rearrangement potentials of proximal genes varied with age, yielding a biased repertoire in the young vs. adult animal. As evidence that γ/δ and α/β gene rearrangements appeared concomitantly in individual precursors, it was found that: (a) multiple adult thymocytes bore α gene rearrangements on one chromosome and δ gene rearrangements on the homologous chromosome, and (b) Vγ3‐Jγ1 rearrangements, prominent joins in the early γ/δ T cell population, were also prominent in the early α/β T cell subset. These data illustrate the non‐random nature of the developmental TcR gene rearrangement and suggest that α/β and γ/δ T cell populations derive from related, if not identical, T cell precursor populations.
Clonal expansion precedes anergy and death of Vβ8+ peripheral T cells responding to staphylococcal enterotoxin B in vivo
Authors: MacDonald H. Robson , Baschieri Selene , Lees Rosemary K.
Published: March 2007

Staphylococcal enterotoxin B (SEB) selectively stimulates T cells bearing T cell receptor Vβ8 domains and hence provides a useful model to study immunity and tolerance in vivo. We show here that Vβ8+ T cells in both CD4+ and CD8+ subsets expand dramatically (fivefold) in lymphoid tissues of mice 2–4 days following injection with SEB. This initial clonal expansion, which is accompanied by a transient hyper‐reactivity to SEB, is followed by a rapid decrease in Vβ8+ cells and a concomitant induction of specific non‐responsiveness which persists for at least 30 days. Selective death of Vβ8+ cells occurs during this latter phase. Taken together, our data indicate that clonal expansion, anergy and death can occur as sequential stages of an immune response in vivo.
A Novel surface molecule expressed by long‐term cultured T and natural killer cells is involved in cell activation
Authors: Ferrini Silvano , Cantoni Claudia , Ciccone Ermanno , Biassoni Roberto , Prigione Ignazia , Bottino Cristina , Venzano Paola , Moretta Lorenzo
Published: March 2007

Two monoclonal antibodies (mAb), termed ED6 and LD6, were obtained by immunizing mice with cytotoxic T cell lines expressing the T cell receptor (TcR)γ/δ. These mAb were selected according to their ability to trigger the cytolytic program of the immunizing cell lines in a redirected killing assay. Both mAb recognized molecule(s) expressed on the surface of most long‐term cultured TcR γ/δ+, TcR α/β+ and CD3−CD16+ lymphocytes, while it was absent on resting peripheral blood lymphocytes. In addition both mAb reacted with neoplastic B cell lines, Epstein‐Barr virus‐transformed B cell lines, small cell lung cancer and glioma cell lines, while no surface reactivity was detected on ovarian, breast, colon and non‐small cell lung cancer lines. The functional activity of these mAb was studied by two cytolytic assays. Both mAb were able to trigger the cytolytic program of CD3+TcR γ/δ+ polyclonal cell lines and of a CD3−CD16+ NK cell clone against the murine mastocytoma target cell line P815 (Fc receptor+) in a 4‐h 51Cr‐release assay. In addition, ED6 and LD6 hybridomas were lysed by TcRγ/δ+ effector cells while other hybridomas (obtained from the same fusion) were not lysed. ED6 and LD6 mAb (in the presence of submitogenic doses of the phorbol 12‐myristate 13‐acetate) also induced the secretion of interleukin 2 by ED6/LD6+ T cell clones expressing TcR γ/δ or α/β. mAb‐induced surface antigen modulation experiments showed that the antigenic determinant recognized by ED6 and LD6 co‐modulated, thus indicating that the two mAb probably recognize the same or closely associated molecules. The molecular characteristics of the antigen recognized by the mAb were investigated by Western blot analysis. The LD6 mAb recognized a major band of approximately 65 kDa, both under nonreducing and reducing conditions. These data indicate that ED6 and LD6 mAb recognize a novel non‐lineage‐specific activation antigen which is involved in the induction of the functional program of long‐term cultured T or natural killer cells.