bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclosporin depolarizes human lymphocytes: earliest observed effect on cell metabolism
Authors: Damjanovich Sándor , Aszalós Adorján , Mulhern Sally A. , Szöllösi János , Balázs Margit , Trón Lajos , Fulwyler Mack J.
Published: March 2007

Cyclosporin A (CsA) produced dose‐dependent membrane depolarization of human peripheral blood lymphocytes. The phenomenon was investigated applying the membrane potential probe dihexyloxacarbocyanine iodide in a flow cytometer in combination with ionophores, hormones and monoclonal antibodies binding to different subclasses of lymphocytes and the anti‐interleukin 2 receptor antibody. Human interferon‐γ abolished the depolarizing effect of cyclosporin on lymphocytes. Interleukin 2 caused depolarization and also enhanced the effect of CsA. OKT4 and OKT8 monoclonal antibodies slightly hindered depolarization by CsA while OKT3, OKT11 and OKIa1 antibodies had no such effect. Valinomycin decreased CsA's effect on the membrane potential while the ionophore A‐23187 and ionomycin caused depolarizations that were additive with CsA's. CsA treatment released the isotope from 42K‐loaded human lymphocytes in a dose‐dependent fashion. CsA addition increased intracellular calcium content. CsA decreased the motional freedom of a spin probe in the membrane, but did not hinder the binding of fluoresceinated antibodies to the cell surface. These results suggest immediate alteration in membrane structure upon CsA treatment, causing potassium leakage and calcium ion uptake. These are the earliest detected effects of CsA on cells so far.
Interleukin 2 receptor traffic in a murine cytolytic T cell line
Authors: Nabholz Markus , Combe Marie‐Claire , Corthésy Patricia , Lowenthal John , Gabathuler Reinhard
Published: March 2007

We have analyzed different parameters of the interleukin 2 receptor (IL 2R) traffic in a murine IL2‐dependent T cell line, B6.1. These cells express about 105 IL 2R, of which approximately 10% are of high affinity (HAR). About 90% of all mature immunoprecipitable receptor molecules in the cell are on the cell surface. Measurements of the half life and of the rate of receptor synthesis indicate that these cells produce approximately 500 receptor molecules per cell and min. IL2 deprival for less than 6 h does not affect this rate.

B6.1 cells internalize IL2 via their HAR only, with a maximal rate of about 500 molecules per cell and min. Among the receptors present at a given time on the cell surface, about 15% are internalized within 30 min. Receptor internalization is independent of IL2. The data obtained argue against rapid recycling of the HAR. The rate of receptor synthesis can be reconciled with the rate of IL2 internalization and the observation that internalization occurs only via HAR by assuming that cell surface low‐affinity receptors can be transformed into HAR by associating with other molecules.
B cell differentiation and interleukin 2 (IL 2): corticosteroids interact with monocytes to enhance the effect of IL 2
Authors: Emilie Dominique , Karray Saoussen , Crevon Marie‐Claude , Vazquez Aimé , Galanaud Pierre
Published: March 2007

Upon in vitro activation by Staphylococcus aureus Cowan I strain (SAC) human peripheral blood B cells produce only marginal amounts of Ig when cultured in the presence of interleukin 2 (IL2; 10 U/ml). This response is only moderately increased by the addition of monocytes or of IL1. In the presence of dexamethasone (DM; 10−7−10−8 M) microgram amounts of both IgM and IgG are produced in co‐cultures of B cells and monocytes. This response is not modified by inhibitors of cyclooxygenase and is specifically inhibited by a monoclonal antibody interfering with the binding of IL2 to its receptor. This enhancing effect of DM is not observed in the absence of monocytes even if IL 1 is added to the cultures. Moreover, monocytes pretreated with DM stimulate the response of B cells cultures in the absence of DM. Enhancement of Ig production by DM and monocytes could be demonstrated with B cells obtained from a patient suffering from a hyperlymphocytic form of B cell type chronic lymphocytic leukemia, and in this case only IgM was produced. Importantly, DM fully inhibited the IL2‐dependent proliferation of these monoclonal B cells. Thus, physiological concentrations of DM can modulate monocytic function to enhance the differentiative effect of IL2.
A novel 90‐kDa polypeptide (Tp90) possibly involved in an antigen‐independent pathway of T cell activation
Authors: Carrel Stefan , Salvi Suzanne , Giuffrè Laura , Isler Patrick , Cerottini Jean‐Charles
Published: March 2007

A novel surface molecule, Tp90, is described which appears to be involved in an antigen‐independent pathway of human T lymphocyte activation. The Tp90 molecule was identified by a monoclonal antibody (mAb), MX20, obtained from a fusion using spleen cells of a mouse immunized with cells from two T cell leukemia lines, Jurkat and HPB‐ALL. Biochemical data show that Tp90 is distinct and physically independent from the structures already known to be involved in T cell activation, namely T11, T44 or T3/TCR. These results were confirmed by antibody‐induced antigen modulation experiments. Modulation of Tp90 had no effect on the expression of T3 and of the T cell receptor. Conversely, the expression of Tp90 was not affected by modulation of the T3/TCR molecular complex by either anti‐T3 or anti‐TCR antibody.

Functional studies showed that anti‐Tp90 mAb MX20 induced high levels of interleukin 2 production in Jurkat cells. Modulation of the T3/TCR complex significantly decreased the response of Jurkat cells to stimulation by antibody MX20, suggesting that the T3/TCR complex regulates the ability of the Tp90 molecule to induce IL2 synthesis.

In addition to its effect on Jurkat cells, anti‐Tp90 mAb was found to be mitogenic for peripheral blood T cells. As the magnitude of the proliferative response elicited by anti‐Tp90 mAb was lower than that induced by anti‐T3 mAb, the possibility was considered that only a subpopulation of T cells is reactive with anti‐Tp90. Indeed as determined by FACS analyses, only 3–14% of E‐rosette‐positive cells were stained with mAb MX20. In addition, multicolor flow cytometry analysis showed that the Tp90+ cells belong preferentially to the CD8 subset.
The murine homologue of the T lymphocyte CD2 antigen: molecular cloning, chromosome assignment and cell surface expression
Authors: Sewell William A. , Brown Marion H. , Owen Michael J. , Fink Pamela J. , Kozak Christine A. , Crumpton Michael J.
Published: March 2007

The human T lymphocyte antigen CD2 (T11, sheep erythrocyte receptor) is expressed on all peripheral T cells and all but the most immature thymocytes. Experiments with monoclonal antibodies against CD2 suggest that CD2 is the cell surface receptor for a natural ligand involved in T cell proliferation. Clarification of the functional role of CD2 would be facilitated by the identification of CD2 in the mouse. However, antibodies that recognize the murine homologue have not been described. An alternative approach to the identification of the murine homologue was to use cross‐species DNA hybridization, employing human CD2 cDNA as a probe. Clones encoding the murine homologue were isolated from a murine T helper cell cDNA library. The murine cDNA sequence encoded a predicted mature polypeptide of 322 amino acids that showed 54% identity with the predicted human sequence. As with the human polypeptide, the cytoplasmic domain was large, and rich in proline and basic residues. CD2 mRNA was expressed in murine thymus and spleen, and in the T cell line EL4. The murine CD2 gene was assigned to chromosome 3 by Southern blot analysis of mouse‐hamster somatic cell hybrids. A rabbit antiserum raised against purified human CD2, precipitated from surface‐labeled mouse thymocytes a glycoprotein of Mr 55000–66000 which decreased to Mr35000 on digestion with endo‐β‐acetyl‐glucosaminidase F. These sizes are consistent with those predicted for the murine CD2 antigen from the cDNA sequence.
The effect on skin allograft survival of a monoclonal antibody specific for a polymorphic CD3‐like cell surface molecule in rhesus monkeys
Authors: Nooij Frans J. M. , Jonker Margreet
Published: March 2007

FN18, a monoclonal antibody specific for the polymorphic rhesus monkey CD3 antigen on peripheral T cells, has been tested for its immunosuppressive effect in a rhesus monkey skin graft model. Animals were injected i.v. daily with antibody and they received an allogeneic skin graft two or three days after the initial antibody treatment. All animals were carefully monitored regarding levels of the major lymphocyte subsets. In animals in which RhT3 (a CD3‐like antigen) is demonstrable (i.e. FN18+ phenotype), all T cells initially disappeared from the circulation. However, T cells without RhT3 on their surface reappeared after several days, indicating that these cells must have been modulated. The survival times of the skin grafts were significantly prolonged in these animals. In monkeys in which RhT3 is not demonstrable (i.e. FN18− phenotype), mainly part of the CD4+ lymphocyte subset was depleted. Although less explicit, skin graft survival was significantly prolonged in these animals as well. In both the FN18+ and FN18− groups a difference in sensitivity between the CD4+ and CD8+ cells for the FN18 antibody could be noticed. From the data it appears that FN18 is immunosuppressive and does not have serious side effects. The data are in agreement with information available for the use of OKT3 in clinical immunosuppression. Thus, extrapolation of data from the rhesus monkey model to the clinical situation seems feasible.
Analysis of T cell responses to poly‐L(GluLys) at the clonal level. I. Presence of responsive clones in nonresponder mice
Authors: De Kruyff Rosemarie H. , Ju Shyr‐Te , Laning Joseph , Dorf Martin E.
Published: March 2007

The synthetic random copolymer of L‐glutamic acid and L‐lysine (GL) is weakly or nonimmunogenic in all inbred strains of mice. Theories proposed to account for nonresponsiveness to GL include a deficient T cell repertoire, failure of antigenpresenting cells to present the antigen and/or the presence of suppressor cells. In this study we examine mechanisms for nonresponsiveness to GL. We demonstrate the existence of GL‐reactive T cells which can be isolated with a relatively high frequency. These clones, which were derived following immunization of H‐2d mice with poly(LGluLLysLTyr), also respond to several GL‐containing polypeptides including the terpolymers of GL with phenylalanine, alanine (GLA) or leucine. Although recognition of GLA by heterogeneous T cell populations usually occurs in association with I‐A determinants, these clones recognize GLA, as well as the other GL‐containing polymers, in association with I‐E determinants. Analysis of the antigen and alloreactivity patterns of these clones indicated that they expressed distinct antigen receptors. These studies imply that the T cell repertoire of “nonresponder” H‐2d mice includes multiple GL‐reactive T cell clones and that the antigen‐presenting cells of these mice are effective in processing and presenting GL.
Defective interleukin 2 receptor expression is associated with the T cell disfunction subsequent to bone marrow transplantation
Authors: López‐Botet Miguel , De Landazuri Manuel O. , Izquierdo Manuel , Ramirez Ana , Figuera Angeia , Cámara Rafael , Fernández‐Rañada José
Published: March 2007

In the present work we have used monoclonal antibodies (mAb) as probes to attempt a dissection of the mechanisms underlying the immunodeficiency subsequent to bone marrow transplantation (BMT). To this end we have studied 19 allogeneic BMT recipients, analyzing the proliferative response of peripheral blood mononuclear cells (PBMC) after activation with either phytohemagglutinin (PHA), anti‐CD3 or anti‐CD 2 mAb. All patients presented normal proportions of CD2+ and CD3+ lymphocytes, as assessed by flow cytometry.

Our results indicated that in most cases both CD 2 and CD 3‐mediated activation pathways were inefficient to trigger normal T cell proliferation. The addition of exogenous interleukin 2 (IL 2) did not restore in most cases the proliferative response, pointing out that additional defects contribute to the hyporesponsiveness. This was more evident in the group of patients studied during the first 6 months. To further dissect the T cell defect we analyzed the effect of a phorbol ester (phorbol myristate acetate, PMA), which activates protein kinase C, on the anti‐CD 3‐induced response. Our data showed that PMA synergized with anti‐CD 3 similarly to exogenous IL 2, and restored the proliferative response only in certain cases. The expression of IL 2 receptors (CD 25) as assessed by cytofluorimetry, after either PHA or anti‐CD 3 and PMA stimulation, was shown to be depressed, and the addition of IL 2 did not restore it. Finally, we observed that the early increase of intracytoplasmic Ca2+ after anti‐CD 3 stimulation was comparable to that detected in normal PBMC.

Altogether these results indicate that a diminished CD 25 expression is associated with the T cell defect, and cannot apparently be attributed to an inability of the CD 3 molecule to transduce early activation signals thus suggesting that either protein kinase C itself or an as yet undefined metabolic step preceding IL 2 receptor expression is abnormal in variable proportions of T cells after BMT, and constitutes another manifestation of this complex immunodeficiency.
Discrimination of hepatocyte‐stimulating activity from human recombinant tumor necrosis factor α
Authors: Andus Tilo , Heinrich Peter C. , Bauer Joachim , Tran‐Thi Thuy‐Anh , Decker Karl , Männel Daniela , Northoff Hinnak
Published: March 2007

The involvement of tumor necrosis factor α (TNFα) in the regulation of acute‐phase protein synthesis is currently under discussion. In this study the effect of human recombinant TNFα on the regulation of the 4 acute‐phase proteins α2‐macroglobulin, albumin, α1‐proteinase inhibitor and α‐acute‐phase globulin was investigated in rat hepatocyte primary cultures. No changes in synthesis of any of the 4 proteins were observed. However, an acute‐phase response similar to that in vivo could be generated by conditioned media from human monocytes containing natural TNFα. This response remained unchanged after neutralizing TNFα activity by the addition of a specific antibody to TNFα. It is concluded that the hepatocyte‐stimulating activity synthesized by human monocytes is different from TNFα.
Cloned human CD3− lymphocytes with natural killerlike activity do not express nor rearrange T cell receptor gamma genes
Authors: Triebel Frédéric , Graziani Marita , Faure Florence , Jitsukawa Setsuko , Hercend Thierry
Published: March 2007

Here we report the characterization of T cell receptor (TCR) gene activation in 14 CD3− cloned lymphocytes with natural killer‐like activity from different origins. No transcription of either α or β or γ genes is detected in any of these clones. All Jβ, Cβ clusters are in germ‐line configuration. The 5 presently described Jγ loci (JγP1, JγP, Jγ1, JγP2, Jγ2) are also in germ‐line configuration on each chromosome. These results indicate that several distinct populations of CD3− cloned NK cells do not use γ gene products as a recognition structure. Perhaps more importantly, these cells do not appear to be engaged in the earliest presently known stage of T lymphocyte differentiation, namely rearrangement at one of the Jγ loci.
Engineering of immunogenic peptides by co‐linear synthesis of determinants recognized by B and T cells
Authors: Borras‐Cuesta Francisco , Petit‐Camurdan Agnès , Fedon Yann
Published: March 2007

The potential of synthetic peptides as vaccines is restricted by their frequent lack of immunogenicity. As with haptens, coupling to a carrier protein is usually required to provide T cell help to anti‐peptide antibody‐producing B cells. In spite of their short length, a few natural or synthetic peptides are immunogenic: they all include both a determinant recognized by B cells and a proven or putative determinant recognized by T cells. We speculated that it should be possible to induce immunogenicity in peptide haptens by the inclusion of a well characterized determinant recognized by T cells. We thus synthesized two peptides, corresponding to different regions of the major protein VP6 of bovine rotavirus, co‐linearly linked to a peptide of influenza virus hemagglutinin which had been shown to induce T helper cells in BALB/c mice. Both peptides induced anti‐rotavirus antibodies and were more immunogenic than the corresponding bovine serum albumin‐conjugated peptides.
Interleukin‐HP1‐related hybridoma and plasmacytoma growth factors induced by lipopolysaccharide in vivo
Authors: Coulie Pierre G. , Cayphas Sylvie , Vink Anne , Uyttenhove Catherine , Van Snick Jacques
Published: March 2007

Serum of lipopolysaccharide (LPS)‐treated mice was found to support the growth of interleukin‐HP1 (HP1)‐dependent hybridoma and plasmacytoma cell lines. This growth‐promoting activity, which was undetectable in normal serum, rose more than 1000‐fold within 2 h after i.v. injection of the toxin and disappeared in less than 1 day. It could be traced to a single component, which behaved like HP1 in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and reverse phase high performance liquid chromatography, and was completely inhibited by a rabbit anti‐HP1 antiserum. The LPS‐induced factor was apparently not of T cell origin, as indicated by the strong activity found in the serum of LPS‐treated nude mice. In vitro, LPS also considerably enhanced the production by macrophages of a factor with similar characteristics.
Expression of the W6/32 HLA epitope by cells of rat, mouse, human and other species: critical dependence on the interaction of specific MHC heavy chains with human or bovine β2‐microglobulin
Authors: Jefferies Wilfred A. , MacPherson G. Gordon
Published: March 2007

The HLA class I epitope W6/32 is conformationally dependent on both heavy chain and β2‐microglobulin (β2M). Previously, the W6/32 epitope has been detected in humans and other primates as well as from bovine sources. Two controversial reports suggest the W6/32 epitope is constitutively expressed by either normal or transformed murine cells expressing the Db allele. Here we show that the appearance of the W6/32 epitope in murine cells results from the association of either the Db or Kd gene products with either bovine or human β2M. We use congenic mouse strains and hybrid H‐2 class I genes between Db and Kb to map the W6/32 epitope to particular amino acid residues in the α2 domain. Subsequently, we show that β2M exchange is not confined to murine or human cells in vitro but can be detected after β2M injection into a mouse. The data presented suggests that β2M exchange takes place at the cell surface under physiological conditions and indicates that MHC class I heavy chains are in an equilibrium between the bound and unbound form of β2M.
Control of prothymocyte proliferation by thymic accessory cells
Authors: Papiernik Martine , Penit Claude , Rouby Soumaya Ei
Published: March 2007

All thymocyte subpopulations derive from intrathymic precursors which are double negative (DN) for Lyt‐2 and L3T4 differentiation antigens. Although nearly half of DN cells express a receptor for interleukin 2 (IL2R), they respond poorly to IL2. DN cell proliferation can be obtained in the presence of various exogenous stimuli, but the in vivo signal for DN cell response to IL2 remains unclear. We show in the present report that phagocytic cells of the thymic reticulum are able to induce the proliferation of DN thymocytes in the presence of recombinant IL2 (rIL2). Cell‐to‐cell contact is needed for this effect. Antibodies directed against class I MHC antigens but not against class II can inhibit DN cell proliferation. DNA‐synthetizing cells were labeled by incubation with 10 μM bromodeoxyuridine either before or at various times during the culture period. Bromodeoxyuridine was then detected in the DNA of proliferating cells and/or their progeny already stained with anti‐Lyt‐2 and L3T4 antibodies. During the initial 16 h and independently of culture conditions, 16–25% of the cells expressed surface antigens and 50–65% of them derived from DN cells which were in S phase just before culture; these differentiated cells had a very short life span.

In the second culture period, the presence of both rIL 2 and thymic accessory cells was necessary for cell survival. In these conditions, DN cell number and proliferation rate were constant and a low number of Lyt‐2+ and/or L3T4+ cells was continuously generated. Thymic accessory cells therefore appear to provide the signal(s) necessary for IL2‐induced proliferation of thymocyte precursors.

Implications of these findings for normal in vivo intrathymic proliferation and differentiation are discussed.
Newly produced virgin B cells migrate to secondary lymphoid organs but their capacity to enter follicles is restricted
Authors: Lortan Jennifer E. , Roobottom Carl A. , Oldfield Susan , MacLennan Ian C. M.
Published: March 2007

The migration of recirculating B cells was compared with that of newly produced virgin B cells following passive cell transfer between congenic strains of rats differing in their kappa immunoglobulin light chain (χ) allotype. The number and location of donor B cells in the secondary lymphoid organs was determined at intervals following transfer by immunohistology using monoclonal antibodies specific for rat χ allotypes. Recirculating B cells were obtained from thoracic duct lymph while bone marrow from rats depleted of recirculating cells was used as a source of newly produced virgin B cells.

B cells from both sources gained immediate access to extrafollicular areas of secondary lymphoid organs rich in interdigitating cells and T cells. However, in lymph nodes extrafollicular B cells were found adjacent to lymphatics and not in the central paracortex. By 8 h after transfer most B cells from thoracic duct lymph were found in follicles. However, the capacity of the bone marrow B cells to enter follicles was very limited. These results are interpreted in relation to previous observations concerning (a) the timing of virgin B cell recruitment into T cell‐dependent antibody responses, and (b) the role of B cells in antigen presentation.
Role of p150,95 in adhesion, migration, chemotaxis and phagocytosis of human monocytes
Authors: Keizer Gerrit D. , Te Velde Anje A. , Schwarting Roland , Figdor Carl G. , De Vries Jan E.
Published: March 2007

The leukocyte function‐associated antigen‐1 (LFA‐1), the C3bi receptor (CR3) and the p150,95 antigen belong to a family of leukocyte surface molecules consisting of bimolecular complexes with ä chains of 170 kDa, 165 kDa and 150 kDa, respectively, and a common β subunit with a mol. mass of 95 kDa. In order to determine the function of the p150,95 antigen on human monocytes and U937 cells, and to study the functional relationship between this antigen and LFA‐1 or CR3, we investigated the influence of monoclonal antibodies (mAb) directed against these cell surface molecules on the adhesive properties of these cells. The observation that anti‐β chain mAb strongly inhibited migration, chemotaxis, adhesion and phagocytosis of monocytic cells indicates a major role for LFA‐1 family antigens in monocyte functions. Detailed analysis with a panel of anti‐α chain antibodies demonstrated that both p150,95 and LFA‐1 mediate random migration whereas in contrast, p150,95 and CR3 were shown to be involved in the directed migration of monocytes to f‐Met‐Leu‐Phe. Furthermore, adhesion of monocytes to plastic surfaces or monolayers of endothelial cells as well as phagocytosis of latex particles was mediated by p150,95.

The results demonstrate that, in spite of its relative low expression, the p150,95 glycoprotein is a major adhesion‐associated molecule expressed by human monocytic cells.
The cytosolic free calcium in anti‐μ‐stimulated human B cells is derived partly from extracellular medium and partly from intracellular stores
Authors: Dugas Bernard , Calenda Alphonse , Delfraissy Jean‐François , Vazquez Aimé , Bach Jean‐François , Galanaud Pierre
Published: March 2007

The inositol phospholipid metabolism and the increase in cytosolic free Ca2+ concentration ([Ca2+]i) into the cell are recognized as two important events in the anti‐μ‐induced B cell activation. The anti‐μ stimulation caused the [3H]inositol incorporation and also a rapid increase in [Ca2+]i from 85 nM to 285 nM. This signal returned to baseline a few minutes after stimulation. By using the fluorescent indicator quin‐2 we demonstrated that this [Ca2+]i uptake was derived part from extracellular medium and part from intracellular stores. Both EGTA (a calcium chelator) and TMB.8 (a drug which interferes with Ca2+ sequestration by smooth endoplasmic retiulum) partially suppressed the intracellular Ca2+ uptake and were fully inhibitory when added together. The role of Ca2+ from intracellular stores may also be evidenced in calcium‐free experiments, or in permeabilized experiments using exogenous inositol 1,4,5‐trisphosphate (IP3, the putative mobilizer of intracellular Ca2+). Preventing the increase in [Ca2+]i also prevents the apparition of early activation markers. These results are consistent with the hypothesis that the Ca2+ increase in B cells stimulated by anti‐μ is caused by the generation of IP3 during the phosphatidyl‐inositol metabolism and also by the entry of extracellular Ca2+ through the plasma membrane.
Immunoglobulin VH region genes of the mouse are organized in overlapping clusters
Authors: Blankenstein Thomas , Krawinkel Ulrich
Published: March 2007

Restriction fragment length polymorphism has been compared between the Igh loci of C57BL/6 and MOLF/EI (Mus musculus molossinus) mice utilizing probes which detect the Cγ2b gene and genes from nine VH‐gene families. Distinct restriction site patterns were found for the CH genes and for VH families PC7183, Q52 and X24. VH families V31 and J558 showed identical patterns. Mixed patterns of identical and distinct bands were detected in VH families S107, J606, V3660 and VGAM3.8. This indicates that a recombination took place involving the Igh loci of a M. m. molossinus and a progenitor of the C57BL/6 strain. The breakpoint of recombination maps to the chromosomal region carrying VH families S107, J606, VGAM3.8 and V3660. VH families PC7183, Q52 and X24 map 3′ to the recombination breakpoint and proximal to the DH‐JH‐CH region, whereas VH families V31 and J558 accordingly map 5′ to the recombination breakpoint and distal to DH‐JH‐CH. This order of VH families was confirmed by deletion mapping utilizing hybridomas which are haploid either for the Ighb or for the Igha locus. The mapping data indicate that the VH families of the mouse are organized in overlapping clusters. This notion is confirmed by demonstration of the physical linkage of VH genes from families V31 and J558 in the Igha locus.
Cyclophilin binds to the region of cyclosporine involved in its immunosuppressive activity
Authors: Quesniaux Valérie F. J. , Schreier Max H. , Wenger Roland M. , Hiestand Peter C. , Harding Matthew W. , Van Regenmortel Marc H. V.
Published: March 2007

Although several cytosolic proteins including calmodulin and cyclophilin have been shown to bind cyclosporine, the direct involvement of these proteins in the immunosuppressive activity of cyclosporine remains to be established. In the present study, a quantitative immunoassay for cyclophilin was developed which made it possible to compare its relative affinity for cyclosporine and any of its analogues. The binding of cyclophilin to cyclosporine coated on a solid phase was revealed by anti‐cyclophilin rabbit antiserum followed by antiglobulin‐enzyme conjugate. This reaction could be inhibited by addition of free cyclosporine or certain cyclosporine analogues. By studying the binding of cyclophilin to more than fifty cyclosporine derivatives modified singly on each of the eleven amino acid residues, it could be shown that cyclophilin binds to the residues of cyclosporine known to be critical for its immunosuppressive activity. These data identify cyclophilin as a highly discriminating stereospecific binding protein for cyclosporine.
Murine and human T11 (CD2) cDNA sequences suggest a common signal transduction mechanism
Authors: Clayton Linda K. , Sayre Peter H. , Novotny Jiri , Reinherz Ellis L.
Published: March 2007

The murine equivalent of the cDNA encoding the human T11 (CD2) sheep erythrocyte‐binding protein has been cloned. It codes for a putative transmembrane protein which is homologous to human T11. In contrast to immunoglobulins whose domains consist of anti‐parallel β sheets, we predict that mouse and human T11 external domains probably belong to the α/β protein folding class. The cytoplasmic region of T11 is a lengthy, proline‐rich semgent; secondary structural analysis predicts it to have a nonglobular conformation. This elongated tail could allow for interaction with multiple other intracellular proteins and may contain a cation‐binding site involved in T lineage activation.
Phorbol ester induces class II gene expression in pre‐B cell lines
Authors: Sarthou Pierre , Jouvain‐Marche Evelyne , Cazenave Pierre‐André , Le Guern Christian
Published: March 2007

The effect of phorbol myristate acetate on the induction of major histocompatibility complex class II gene expression was studied in four Abelson murine leukemia virus‐transformed pre‐B cell lines. In three cell lines, low concentrations of PMA (0.1–10 ng/ml) induced the expression of high levels of surface la molecules, and this effect was mediated at the transcriptional level. PMA induced a program of coordinated transcription of all four genes involved in the biosynthesis of Ia molecules. A spontaneous Ia‐positive variant pre‐B cell line was derived from an Ia‐negative parental cell line. This variant was highly sensitive to the toxic effects of PMA, and highly responsive to the Ia‐inductive effects of phorbol ester. Our findings suggest that in pre‐B cells the appearance of Ia molecules and the regulation of their expression are controlled, at least partially, by variations in the activity of protein kinase C, which is the cellular receptor for phorbol esters.
Functional interleukin 2 receptors on B cells lacking Tac antigens
Authors: Tanaka Toshio , Saiki Osamu , Doi Satoru , Hatakeyama Masanori , Doi Takeshi , Kono Takeshi , Mori Hisashi , Fujii Masahiro , Sugamura Kazuo , Negoro Shigeru , Taniguchi Tadatsugu , Kishimoto Susumu
Published: March 2007

Human B lymphoblastoid line, SKW 6‐4, cells were induced to IgM‐secreting cells by high concentrations of interleukin 2 (IL2). These cells were found to be unreactive with anti‐Tac antibody and did not express mRNA detectable for Tac antigen. In Scatchard plot analysis, low‐affinity IL 2‐binding sites were found on SKW 6‐4 cells. Moreover, analysis of the IL2‐binding molecules revealed ones (molecular weight 70000 and 75000) distinct from Tac antigen. It is conceivable that IL2 exerts its effect through its interaction with these novel IL2‐binding molecules in SKW 6‐4 cells.
A common idiotope on T cell receptors and antibodies expressed in the absence of network selection
Authors: Martinez‐A. Carlos , Pereira Pablo , De La Hera Antõnio , Toribio Marisa , Marcos Miguel R. A. , Marquez Carlos , Coutinho Antõnio
Published: March 2007

The monoclonal antibody F6(51), directed to an idiotope of MOPC 460‐like anti‐1,4,6‐trinitrophenl (TNP) antibodies produced in IgHa mouse strains, identifies in helper T cells from C57BL/6 (IgHb) mice with (4‐hydroxy‐3‐nitrophenyl)acetyl (NP)‐self specificity, a clonotypic determinant functionally and biochemically associated with the specific T cell receptor. The expression of this antibody‐related T cell clonotype in C57BL/6 mice, although “recurrent”, is independent of network selection, as shown by its presence in B mice suppressed from birth with anti‐μ antibodies, and in IgH‐congenic mouse strains. These results indicate aleatory cross‐reactivity between T cell receptors and antibodies and command caution in network interpretations for “idiotype sharing”.
Biological properties of suppressive E‐receptor factor on lymphokine function
Authors: Oh Se‐Kyung , Leung Mun‐Fai , Knee Treyce , Williams John M.
Published: March 2007

A potent immunosuppressive factor isolated from malignant ascites fluids showed serological cross‐reactivity with the E‐receptor of human peripheral blood T lymphocytes. Thus, this factor was named suppressive E‐receptor (SER) factor. In this study, we examined the effect of this immunosuppressor, SER, on lymphokine functions of human mononuclear cells participating in polyclonal T cell activation.

SER is active at nanomolar concentrations in vitro and the inhibitory effect of SER was most pronounced when added at the initiation of stimulation with phytohemag‐glutinin or anti‐T3 antibody. Concomitant with the inhibition on PHA‐induced DNA synthesis, lymphocytes that were treated with SER failed to progress beyond G1 phase of cell cycle. These growth‐arrested cells did expire after 7 days of culture in vitro. This anti‐proliferative effect of SER was more easily demonstrated with normal lymphoid cells in culture than transformed cells or fibroblast cells. SER effectively interfered with the lympho‐proliferative properties of interleukin 2 (IL2) on human peripheral blood mononuclear cells and an IL2‐dependent murine cytotoxic T cell line. However, excess quantities of exogenous IL2, especially when added in conjunction with IL1, were able to partially overcome the ability of SER to inhibit T cell proliferation.

In contrast to the inhibition on DNA synthesis of human lymphoblasts, expression of IL2 receptor was only minimally inhibited by SER during the first 24 h of culture (24% inhibition at 12 h and 34% inhibition at 24 h) but it was followed by full expression of IL2 receptor by 48 h. Thus, SER merely reduced the rate of expression of IL2 receptor and was not able to inhibit the transcription of new message from activated T lymphocytes. Taken together, these studies indicate that SER acts as a noncytolytic anti‐proliferative factor on immune responses that are mediated by T cells. SER appears to act on a relatively late event during T cell activation, perhaps on some portion of the DNA replication pathway.
A second signal for T cell mitogenesis provided by monoclonal antibodies CD45 (T200)
Authors: Martorell Jaime , Vilella Ramón , Borche Luis , Rojo Isabel , Vives Jordi
Published: March 2007

The induction of T cell mitogenesis through CD3 is a complex process that requires at least two signals. The first one can be provided by Sepharose‐bound CD3. The second one is normally provided by monocytes. The signal provided by Sepharose‐bound CD3 is unable by itself to induce mitogenesis in monocyte highly depleted cells (MHDC).

We describe here that the monoclonal antibody (mAb) 72‐5D3 belonging to CD45 (T200), which was not mitogenic by itself, could replace monocytes when MHDC were activated by Sepharose‐bound CD3. That is to say, in the absence of monocytes, mAb 72‐5D3 gave a second signal necessary for T cell proliferation. Using eleven anti‐CD45 mAb from other investigators we show that this effect is not a peculiar characteristic of 72‐5D3 mAb. The effect of the mAb 72‐5D3 was only effective in CD4‐positive cells and was not observed when MHDC were activated with either soluble CD3 or concanavalin A.

As both phorbol myristate acetate and mAb 72‐5D3 can replace monocytes, a comparative study of their effects was undertaken. Phorbol myristate acetate but not mAb 72‐5D3 induced proliferation of MHDC when recombinant interleukin 2 (rIL2) was added. On the other hand mAb 72‐5D3 induced IL2 production in MHDC activated by Sepharose‐bound CD3 and increased the IL2 production in Sepharose‐bound CD3‐activated peripheral blood mononuclear cells.

In conclusion, data presented in this report indicate that the T200 molecule could be involved in T cell proliferation by giving a signal that induces the production of IL 2 and bypasses the necessity of monocytes.
The human mononuclear phagocyte high‐affinity Fc receptor, FcRI, defined by a monoclonal antibody, 10.1
Authors: Dougherty Graeme J. , Selvendran Yogi , Murdoch Sarah , Palmer David G. , Hogg Nancy
Published: March 2007

The properties of the mononuclear phagocyte (Mph) high‐affinity Fc receptor, FcRI, were investigated using a novel monoclonal antibody (mAb) designated 10.1. This receptor was shown to be a protein of 71 kDa, presented chiefly on monocytes and the myeloid cell lines U937 and HL60. mAb 10.1 inhibited the binding to Mph of erythrocytes opsonized with rabbit IgG or human IgG3. It also blocked T cell proliferation induced by murine CD3 mAb of the IgG2a but not the IgG1 subclass. These results suggest that rabbit IgG, human IgG3 and murine IgG2a all bind to FcRI in a similar manner and that mAb 10.1 reacts with an epitope on FcRI near to the binding site for the Fc region of IgG. In addition, although it is well known that FcRI has a high affinity for both monomeric human IgG1 and IgG3, we show in this study that while erythrocytes opsonized with human IgG3 bind to Mph, equivalent cells opsonized with IgG1 surprisingly do not. These results define further the nature of the constraints on the interaction between Mph FcRI and particular IgGs.
Relationships between B‐lineage lymphocytes and stromal cells in long‐term bone marrow cultures
Authors: Witte Pamela L. , Robinson Margaret , Henley Anna , Low Martin G. , Stiers David L. , Perkins Steve , Fleischman Roger A. , Kincade Paul W.
Published: March 2007

In long‐term culture of mouse bone marrow, the growth and differentiation of B‐lineage lymphocytes depends on interaction with adherent cells or their products. The objectives of these studies were to characterize the types of cells present in the supporting adherent layer as well as the physical relationships of these cells with lymphocytes. With an extensive panel of antibodies against hemopoietic and lymphocyte antigens, two discrete nonlymphoid populations were identified: macrophages and undefined, large cells which we termed “stromal cells”. Lymphocyte clusters grew in actual contact with the latter cells only. Stromal cells lacked expression of most hemopoietic antigens, including the common leukocyte antigen, J11d, heat stable antigen (M1/69), Thy‐1 and BP1. Antigens expressed by stromal cells were detected by AA4.1, our 94.2 antibody, and antibody to the Forsmann antigen, but the most distinguishing characteristics of the lymphocyte‐binding stromal cells were production of basement membrane components, laminin and collagen IV, and the extremely low uptake of acetylated low density lipoprotein (LDL). Using acetylated LDL uptake as a sorting criterion, the lymphocyte‐binding stromal cells were separated from the macrophages, recultured and shown to support lymphocyte proliferation. We found the binding between stromal cells and lymphocytes to be highly selective and dependent on divalent cations; hence, specialized adhesion mechanisms may have a role in B cell development. Moreover, our studies suggest that phosphatidylinositolanchored cell surface molecules may be involved in this adhesion. Our findings demonstrate the possibility that a single cell type provides physical support and proliferation stimuli for early B‐lineage cells. This accessory cell is not a macrophage; rather, it has features of an endothelial or epithelial cell.
Two monoclonal anti‐CD3 antibodies can induce different events in human T lymphocyte activation
Authors: Roosnek Eddy E. , van Lier René A. , Aarden Lucien A.
Published: March 2007

Two monoclonal antibodies, WT32 and CLB‐T3/4.2a, directed against the CD3 complex were used to study the mechanism of activation of human peripheral T lymphocytes. WT32, a mouse monoclonal IgG2a antibody with a low avidity (≫ OKT3) for the CD3 complex, effectively induces mitogenesis of purified T lymphocytes when used in the 1 ng‐10 μg range in the presence of monocytes or recombinant interleukin 2 (IL 2). In contrast, CLB‐T3/4.2a, a mouse monoclonal antibody of the same isotype with a high avidity (< OKT3) for the CD3 complex, induces IL 2 receptor expression and IL 2 responsiveness only at very low concentrations (< 5 ng/ml), yet in the presence of monocytes this antibody induces proliferation within a similar dose range as WT32. Apparently, in the absence of accessory cells which can cross‐link the antibody CD3 complexes, the binding properties (avidity) of an antibody and thereby the number of receptors that are occupied are important parameters for induction of IL 2 responsiveness. Furthermore, we show that Ca2+ mobilization only occurs when the cells are stimulated by saturating amounts of antibody, so that, when the conditions are optimal for the induction of IL 2 responsiveness, no Ca2+ mobilization will be detected.
Recognition by major histocompatibility complex class I‐restricted cytolytic T lymphocytes of cells expressing vaccinia‐encoded viral and class I proteins
Authors: Andrew Marion E. , Coupar Barbara E. H. , Boyle David B. , Blanden Robert V.
Published: March 2007

Target cells expressing influenza virus hemagglutinin (HA) could be recognized by cytotoxic T lymphocytes (CTL) in conjunction with the murine major histocompatibility complex class I antigen, H‐2Kd, when both antigens were encoded by recombinant vaccinia virus. This recognition occurred if HA and H‐2Kd were encoded by separate vaccinia viruses following dual infection of target cells or if HA and H‐2Kd were encoded by a single recombinant virus. In contrast, target cells expressing nucleoprotein (NP) were only recognized by H‐2Kd‐restricted CTL if both NP and H‐2Kd were encoded by the same vaccinia virus. These results show that the requirements for association of H‐2Kd with different viral antigens derived from HA or NP can vary. Possible factors contributing to this difference are discussed.
CD43 monoclonal antibodies recognize the large sialoglycoprotein of human leukocytes
Authors: Borche Luis , Lozano Francisco , Vilella Ramon , Vives Jordi
Published: March 2007

In this study data are presented indicating that the molecule identified by the recently described CD43 cluster of monoclonal antibodies (mAb; Oxford, 1986) is the human analogue to the one originally described in rat as leukocyte sialoglycoprotein (LSGP). This conclusion is based on several criteria. Both molecular mass (105 kDa) and cellular distribution are similar to the antigens previously described in the rat with the mAb W3/13 and subsequently in humans with L10 mAb. Sialidase treatment of the molecule immunoprecipitated by 84‐3C1 mAb (CD43) resulted in a decreased electrophoretic mobility on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. L10 mAb inhibited the binding of 84‐3C1 mAb to the cell membrane suggesting that both mAb recognized the same molecule.

Moreover, the presence in the CD3−4−8− thymic populations of the sialidase‐sensitive epitope recognized by mAb 84‐3C1 suggests that there is no simple correlation between the thymic maturation and the degree of sialylation.
In vitro production of human interleukin lα and interleukin 1β by peripheral blood mononuclear cells examined by sensitive sandwich enzyme immunoassay
Authors: Tanaka Koichiro , Ishikawa Eiji , Ohmoto Yasukazu , Hirai Yoshikatsu
Published: March 2007

The in vitro production of human interleukin 1α (hIL 1α) and interleukin 1β (hIL 1β) by peripheral blood mononuclear cells was examined by sensitive sandwich enzyme immunoassays which could discriminate hIL 1α and hIL 1β without cross‐reaction with human IL2. In culture supernatants of mononuclear cells, two components were detected by sandwich enzyme immunoassay for hIL 1α or hIL 1β. The molecular weight of one component was shown to be equal to that of recombinant hIL 1α or hIL 1β by gel filtration. The elution volume of the other component corresponded to a molecular weight of about 30000. The sum of the two components for both hIL 1α and hIL 1β in culture supernatants of peripheral blood mononuclear cells from healthy subjects increased 1.7 to 38‐fold by Escherichia coli lipopolysaccharide. The sum of the two components for hIL 1β was 13 to 97‐fold larger than that for hIL 1α.
Idiotype switching of CD4‐specific monoclonal antibodies can prolong the therapeutic effectiveness in spite of host anti‐mouse IgG antibodies
Authors: Jonker Margreet , Den Brok Jan H. A. M.
Published: March 2007

Immunotherapy using murine monoclonal antibodies (mAb) is limited by the host anti‐mouse IgG response. Previous investigations demonstrated that a large proportion of the anti‐mouse response was specific for idiotypic determinants of the mAb. This study demonstrates the feasibility of idiotype switching of therapeutic mAb to evade this anti‐idiotype response. In this way prolongation of the therapeutic effectiveness of mAb treatment can be achieved. Using different CD4‐specific mAb consecutively in rhesus monkeys it was possible to obtain therapeutic effectiveness in spite of host anti‐mouse IgG antibodies, provided that no anti‐idiotypic antibodies were present. Anti‐constant part antibodies may even enhance therapeutic effectiveness.
Relationship of B cell Fc receptors to T cell recognition of Mls antigen
Authors: Nicolas Jean‐Francois , Wegmann Dale , Lebrun Philippe , Kaiserlian Dominique , Tovey Julie , Glasebrook Andy L.
Published: March 2007

The Mls locus on chromosome 1 controls the expression of cellular determinants that are responsible for stimulating mixed lymphocyte reactions between H‐2‐identical strains. However, the biochemical nature of Mls antigenic determinants remains undefined. It has been proposed that Ly‐17 lymphoid cell surface antigens (also known as Ly.m.20.2 and LyM‐1) and Mls antigens could be identical because they are both encoded by loci on chromosome 1 and display similar tissue distribution. The Ly‐17 locus encodes polymorphic alleles of the IgG Fc receptor (FcγR). In the present study, two approaches were used to address the question of whether FcγR are involved in T cell recognition of Mls antigen. In the first approach we tested the effect of FcγR blockade by heat‐aggregated mouse IgG or anti‐FcγR monoclonal antibodies (2.4.G2) on the ability of an Ia+, FcγR+ Mlsa‐expressing B cell hybrid (LBB.3.4.16) to stimulate interleukin 2 secretion by an anti‐Mlsa‐specific T cell hybrid. We show that blockade of FcγR does not inhibit the Mlsa‐specific stimulation of T cells during a 24‐h culture period in which FcγR remain blocked. In the second approach, we derived irradiation‐induced variants of LBB.3.4.16 to dissociate FcγR expression and Mls antigen expression. We describe 2 LBB variants which no longer stimulate Mlsa‐reactive T cells but do express FcγR. Compared to parental LBB cells, the capacity of variant LBB cells to present soluble antigen to Ia‐restricted T cells is unaffected. Collectively, these results indicate that FcγR expression and Mlsa antigen stimulation can be dissociated. We conclude that FcγR expression may be necessary, but not sufficient for T cell recognition of Mls antigen.