bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the kinetics of antiviral memory T help in vivo: characterization of short‐lived cross‐reactive T help
Authors: Roost Hans‐Peter , Charan Shiv , Zinkernagel Rolf M.
Published: March 2007

To evaluate the kinetics of functional effector and memory T help in vivo the effect of priming with one serotype of vesicular stomatitis virus‐Indiana (VSV‐IND) on the antibody response to a serologically distinct heterologous second serotype (VSV‐New Jersey; VSV‐NJ) was studied. Mice primed with VSV‐IND 4 or 8 days before being given a second infection of VSV‐NJ developed an earlier and enhanced IgG response to neutralizing determinants of the second VSV serotype. However, this enhanced response was not detected in mice primed 15 or more days prior to a second infection. After 15 days, mice challenged with the heterologous VSV‐NJ mounted a strictly normal primary response without evidence of suppression. It was shown by in vivo time‐kinetics experiments that efficient VSV cross‐reactive T help, capable of enhancing the IgG response is short lived and cyclosporin A resistant. Adoptive transfer experiments demonstrated in the absence of experimental evidence for suppression that this short‐lived capacity to enhance neutralizing IgG antibody responses is mediated by T cells. These findings have implications for understanding antiviral protection and immunological memory against related but serologically distinct viruses.
Evidence of IgG‐mediated enhancement of the antibody response in vivo without complement activation via the classical pathway
Authors: Wiersma Erik J. , Nose Masato , Heyman Brigitta
Published: March 2007

The complement (C) dependency of IgG‐mediated enhancement of the antibody response was investigated by immunizing mice with trinitrophenyl‐coupled keyhole limpet hemocyanin (TNP‐KLH) and either a C‐activating TNP‐specific monoclonal IgG2a antibody (Hy‐1.2) or a mutant, non‐C‐activating variant of Hy‐1.2 (M12). Hy‐1.2 as well as M12 efficiently enhanced the anti‐KLH response, although Hy‐1.2 was more active. In addition, also a naturally non‐C‐activating TNP‐specific IgG1 antibody enhanced the response to TNP‐coupled bovine serum albumin. Moreover, C‐activating IgG could enhance the antibody response in mice depleted of C3 by treatment with cobra venom factor. These findings suggest that the classical pathway of C activation is not required for IgG‐mediated enhancement.
A monoclonal antibody directed against the human intercellular adhesion molecule (ICAM‐1) modulates the release of tumor necrosis factor‐α, interferon‐γ and interleukin 1
Authors: Geissler Dietmar , Gaggl Sabine , Möst Johannes , Greil Richard , Herold Manfred , Dierich Manfred
Published: March 2007

ICAM‐1 is a cell surface glycoprotein which is one of the ligands for the leukocyte function‐associated antigen (LFA‐1). It is involved in leukocyte adhesion to endothelial cells as well as in immune functions requiring cell‐cell contact. The quantitative expression of ICAM‐1 in various cell types can be either induced or enhanced by treatment with cytokines, such as interferon‐γ (IFN‐γ), tumor necrosis factor (TNF)‐α or interleukin 1 (IL 1), a phenomenon which results in the augmentation of binding to LFA‐1‐positive cells. In contrast, treatment with anti‐ICAM‐1 antibodies blocks this binding.

A monoclonal antibody (mAb), termed 7F7, which recognizes an epitope on ICAM‐1, was used to investigate the role of ICAM‐1 in cytokine production by T lymphocytes and monocytes. Production of TNF‐α, IFN‐γ and IL 1 was significantly inhibited (p < 0.01) by the incubation of mAb 7F7 with phytohemagglutinin‐activated blood mononuclear cells (MNC) or isolated E rosettepositive T lymphocytes. The maximal level of inhibition was reached with 1 μg/ml of purified antibody. A similar inhibition was obtained using saturating concentrations of 400 μl/ml of mAb 7F7 hybridoma supernatant corresponding to an inhibitory activity of 1 μg of purified mAb. In contrast, granulocyte/macrophage‐colony‐stimulating factor release showed a heterogeneous response over five experiments with an increase found in three experiments and a decrease in two experiments. Addition of increasing concentrations of supernatant or purified mAb to unstimulated MNC or T lymphocyte cultures had no effect on cytokine release.

The observed inhibition of the production of TNF‐α, IFN‐γ and IL 1 by antibody‐mediated blockade of the ICAM‐1 structure probably represents a negative circuit that serves to tune the activation of leukocytes and to avoid an overproduction of cytokines.
Glycosylation of CD45: carbohydrate composition and its role in acquisition of CD45RO and CD45RB T cell maturation‐related antigen specificities during biosynthesis
Authors: Pulido Rafael , Sánchez‐Madrid Francisco
Published: March 2007

The contribution of oligosaccharides to the biochemical composition and antigen heterogeneity of the phosphotyrosine phosphatase CD45 glycoproteins has been studied on the K‐562 erythroleukemic cell line. Treatment of immunoprecipitated CD45 glycoproteins with distinct exo‐ and endoglycosidases revealed the presence of highly sialylated O‐ and N‐linked complex carbohydrates in the composition of mature CD45 glycoproteins. Incubation of K‐562 cells with the N‐glycosylation inhibitor tunicamycin blocked carbohydrate processing during biosynthesis of CD45 proteins, generating unglycosylated polypeptides similar in size to those resulting from digestion of CD45 proteins with a mixture of both N‐and O‐glycanases. Epitopes defining the T cell maturation related CD45RO and CD45RB antigen specificities were present on the mature 180‐ and 190‐kDa K‐562 CD45 proteins, respectively. However, the CD45RO and CD45RB epitopes were not detected on the high mannose biosynthetic CD45 precursors. Furthermore, treatment of CD45 proteins with O‐glycanase or neuraminidase resulted in the loss of both CD45RO and CD45RB epitopes, although reactivity of the anti‐CD45RO and anti‐CD45RB mAb was not affected by mAb preincubation with either sialic acids or sialyllactose in solution. From these results we conclude that the blockade of early steps of N‐glycosylation during carbohydrate processing resulted in the inhibition of subsequent incorporation of O‐linked sugars on CD45 polypeptides, thus preventing the late acquisition of the CD45RO and CD45RB determinants on the 180‐ and 190‐kDa CD45 polypeptides.
Selective lysis of the autologous tumor by δTCS1+ γ/δ+ tumor‐infiltrating lymphocytes from human lung carcinomas
Authors: Raffaella Zocchi M. , Ferrarini Marina , Rugarli Claudio
Published: March 2007

Two distinct non‐overlapping populations of TcR1+ (γ/δ) T cells have been described: the first, bearing the disulfide‐linked γ/δ heterodimer, is predominant in the peripheral blood; the second, expressing the non‐disulfide‐linked form of TcR1, is mostly confined to epithelial tissues (lung, gut, skin).TcR1+ lymphocytes may be cytotoxic and could be involved in anti‐tumor immunity, especially against tumors at epithelial sites. Freshly derived tumor‐infiltrating lymphocytes (TIL) obtained from two patients with lung cancer were enriched in CD3+WT31− cells. The percentage of this subset substantially increased upon culture in the presence of interleukin 2. These cells were TcR1+ as demonstrated by immunofluorescence and immunoprecipitation. In one case only 40% of this population reacted with δTCS1 mAb, that recognizes the non‐disulfide‐linked form of TcR1, and co‐expressed the CD8 antigen. Cultured TcR1+ TIL were able to kill fresh autologous tumor cells, K‐562 and, to a lesser extent, some natural killer‐resistant cell lines and allogeneic lung tumor cells in 4‐h 51Cr‐release cytotoxicity assays. The fractionated δTCS1+ TIL lysed only autologous tumor cells and K‐562, whereas the lytic activity against all the other targets was confined to the δTCS1− subset. Moreover, the autotumor cytotoxicity was inhibited by anti‐HLA class I but not by anti‐CD1c or anti‐LFA‐1 mAb, suggesting that killing of the autologous tumor cells and non‐major histocompatibility complex‐restricted cytotoxicity are mediated by different mechanisms.
Use of flanking sequences to study secondary structure‐activity correlations of a Mycobacterium leprae T cell epitope
Authors: Anderson Dave C. , Van Schooten Wim C. A. , Barry Michael E. , Janson Anneke A. M. , Devries René R. P.
Published: March 2007

The 65‐kDa protein of the intracellular pathogen M. leprae is prominent in the immune response to this mycobacterium, and individual T cell epitopes from this protein sequence have been defined. We have tested the stimulatory activity of extended analogs of the minimal peptide representing one such epitope, LQAAPALDKL, with a variety of tetrapeptide extensions added to enhance or destabilize α helix formation. The conformational potential of the peptides was measured by circular dichroism using aqueous trifluoroethanol as a secondary structure inducer. Although analogs with high helical potential activated T cells at low concentrations, a less helical variant was similarly potent. Activity also did not correlate with predicted overall α helical amphipathicity. One analog was found which stimulated T cell proliferation in the 50 pM range. The effect of tetrapeptide extensions on epitope activity is not consistent with the importance in activity of only a single stable secondary structure such as an α helix.
Retroviral interleukin 5 gene transfer into interleukin 5‐dependent growing cell lines results in autocrine growth and tumorigenicity
Authors: Blankenstein Thomas , Li Weiqun , Überla Klaus , Qin Zhihai , Tominaga Akira , Takatsu Kiyoshi , Yamaguchi Naoto , Diamantstein Tibor
Published: March 2007

Two interleukin 5 (IL 5)‐specific retroviral expression vectors have been constructed containing the neomycin gene as selectable marker and either the mouse IL 5 cDNA region or the rat genomic IL 5 gene under the control of the thymidine kinase promoter. High viral titer supernatants derived from the transfected or infected packaging cell line Ψ2 were used to infect the two cell lines B13 and T88M whose growth is dependent on exogenous IL 5. Infection resulted in G418 resistance and IL 5‐independent growth with a high frequency. Clones were established which secrete between 2 and > 1000 U IL 5. The proliferation of the IL 5 autocrine growing cells could be inhibited by an antibody directed against the IL 5 receptor indicating that they grow as a result of the endogenously produced IL 5. Regardless of the amount of IL 5 they produced, all of the clones were highly tumorigenic in nucle mice. The phenotype of the tumors was indistinguishable from that of the injected cells. T88M or B13 cells infected with a control virus neither produced IL 5, nor became factor independent, nor produced tumors. Together, the IL 5 gene transfer and expression into IL 5‐dependent growing cells are in accordance with the “autocrine growth” hypothesis and contrast analogous experiments with IL 4.
cDNA clones encoding immunoglobulin λ chains from rabbit expressing the phenotype c7
Authors: Hayzer David J. , Young‐Cooper Glendowlyn O. , Mage Rose G. , Jaton Jean‐Claude
Published: March 2007

A cDNA library derived from spleen cells of an unimmunized rabbit expressing the c7 phenotype of Igλ chains (c7+, c21−) was screened with Vλ or Cλ probes of a λ light chain bearing c21 epitopes. The nucleotide sequences of three hybridizing clones were found to be identical within the Vλ, Jλ and Cλ regions. The Vλ region was 97% similar to that of the functional germ‐line gene Vλ2, and the Cλ region was identical to that of gene Cλ6, recently identified. Gene Cλ6 exhibited four codon differences when compared with gene Cλ5, the latter encoding c21 epitopes. The data presented here and in the accompanying report (Jaton, J.‐C. et al., Eur. J. Immunol. 1990. 20: 2713) support the view that gene Cλ6 encodes the C region of c7 λ chains and that c7 and c21 markers designate two distinct isotypic forms of λ chains. On the basis of comparative Southern blotting analyses and restriction maps of cloned genomic regions containing Vλ and Cλ genes, a scheme is proposed to account for the c7− and c21− phenotypes.
Isolation and characterization of IgG2a‐reactive autoantibodies from influenza virus‐infected BALB/c mice
Authors: Fazekas György , Rajnavölgyi ÉVa , Kurucz István , Sintár ÉVa , Kiss Katalin , László Glória , Gergely János
Published: March 2007

Repeated influenza virus infection induces the production of dominantly IgG2a‐type virus‐specific antibodies as well as the appearance of IgG2a‐reactive autoantibodies in BALB/c mice characterized by low spontaneous rheumatoid factor‐type autoantibody production. IgG2a‐reactive autoantibody‐producing hybridomas could be isolated from the spleen of influenza virus‐infected BALB/c mice. The mAb produced by these clones represent not only IgM but also IgG and IgA isotypes and show strong isotype or isoallotype specificity. The common functional property of these autoantibodies is their preferential‐ and high‐affinity binding to complexed, solid‐phase‐bound or heat‐aggregated IgG2a when compared to native soluble or cell‐bound IgG2a‐The mechanism of induction and the possible biological function of these autoantibodies are discussed in the light of their fine specificity and functional properties.
Alpha heavy chain disease of patient MAL: structure of the non‐functional rearranged α gene translocated on chromosome 9
Authors: Pellet Pascale , Tsapis Andreas , Brouet Jean‐Claude
Published: March 2007

Alpha heavy chain diseases (HCD) are lymphoproliferative disorders characterized by the production of truncated α immunoglobulin heavy chain without associated light chains, α HCD MAL is featured by multiple structural alterations of the α1 productive gene and on original t(9;14)(p11;q32) translocation involving the other rearranged α allele. We present here the structure of the der(9) chromosome. Sequence analysis provides evidence that the translocation occurred after local pairing of the two chromosomes mediated by an almost perfect nonameric sequence, followed by a staggered double‐strand break of chromosome 14. This translocation occurred on a V(D)J rearranged locus; unexpectedly, there were a deletion of the 3' part of the VH gene, several insertions of non‐immunoglobulin‐related genes and multiple mutations, i.e. alterations reminiscent of those occurring on the HCD productive genes.
The induction of skin graft tolerance in major histocompatibility complex‐mismatched or primed recipients: primed T cells can be tolerized in the periphery with anti‐CD4 and anti‐CD8 antibodies
Authors: Cobbold Stephen P. , Martin Gilly , Waldmann Herman
Published: March 2007

Mice given short courses of anti‐CD4 and anti‐CD8 monoclonal antibodies became tolerant of allogeneic skin grafted at the same time. Tolerance could be obtained without Tcell depletion across multiple minor antigen mismatches, both in naive and primed animals, demonstrating that peripheral T cells could be tolerized, even if they had been previously activated. Where donor and recipient were incompatible across the whole major histocompatibility complex, specific tolerance could be achieved by using a combination of depleting followed by non‐depleting antibodies, where each alone was unsuccessful. Although mice clearly tolerated their original skin grafts, we observed in some strain combinations that a second fresh, but genotypically identical graft, was slowly rejected. Such mice also possessed Tcells which could proliferate to donor‐type stimulator cells in vitro. Whatever the mechanisms, we have demonstrated that operational transplantation tolerance can be achieved with simple, non‐toxic antibody therapy. The introduction of comparable tolerance‐inducing regimens in clinical organ transplantation could obviate the need for long‐term immunosuppression and its unfortunate side effects.
A novel thymic epithelial adhesion molecule
Authors: Couture Clément , Patel Pravin C. , Potworowski Edouard F.
Published: March 2007

Interactions between thymocytes and thymic stromal cells are responsible for positive and negative selection of T cells, their differentiation, maturation and proliferation. The signals required for these events to occur often necessitate close contact, and indeed adhesion, between the cell types involved. The identification of specific adhesion molecules in this context is, therefore, a vital first step in determining the nature of the signal they mediate or facilitate at a given stage of differentiation. In the present work we identify, isolate and partially characterize a ligand present on thymic medullary epithelial cells which selectively binds CD4+CD8+ thymocytes found primarily in the thymic cortex. This adhesion molecule is composed of two non‐covalently associated glycoproteins of 23 kDa and 45 kDa, respectively, both of which are needed to bind to thymocytes. The importance of the finding is that the ligand, in isolated immobilized form, binds the same thymocyte subset as the original epithelial cell line from which it was isolated. The CD4+CD8+ thymocyte subset is the precursor of single‐positive mature Tcells; hence the putative biological activity of the ligand in question takes place at a pivotal stage of Tcell differentiation.
A three‐dimensional model system to study the interactions between human leukocytes and endothelial cells
Authors: Hakkert Betty C. , Rentenaar Jeannette M. , Van Aken Willem G. , Roos Dirk , Van Mourik Jan A.
Published: March 2007

Leukocyte adhesion to endothelial cells and migration into the subendothelial matrix was studied with a three‐dimensional model system, consisting of human endothelial cells cultured on a loose collagen matrix. We developed a new method to separate the endothelial cell monolayer and adhering leukocytes from the subendothelial matrix, allowing simultaneous analysis of leukocyte adhesion and transendothelial migration. Monocytes adhered more avidly to untreated endothelial cells than did neutrophils (2.5 ± 0.3 vs. 1.0 ± 0.2 leukocytes per endothelial cell). Only a small fraction (10%–20%) of these leukocytes migrated into the subendothelium. Pretreatment of endothelial cells with interleukin 1 (IL1) enhanced adhesion (20%), but not migration of monocytes. In contrast, neutrophil adhesion was markedly and in a time‐dependent manner increased by IL1 treatment (i.e. 200% after 6h and 110% after 24 h of IL1 treatment). Moreover, IL1 pretreatment enhanced neutrophil migration twofold. Activation of leukocytes with formyl‐methionyl‐leucyl‐phenylalanine (fMLP) enhanced both monocyte and neutrophil adhesion, but did not affect leukocyte migration. Under all conditions, monocyte adhesion was only partly (30%–40%) inhibited by monoclonal antibodies (mAb) against the common β subunit of the leukocyte‐cell adhesion molecules (LeuCAM; CD 18) and 25%–30% by mAb against the α subunit of LFA‐1 (CD11a). In contrast, mAb against the α subunits of Mac‐1 (CD11b) and p150, 95 (CD11c) were hardly effective. fMLP‐mediated neutrophil adhesion was reduced to below baseline levels by anti‐LeuCAM (CD18) mAb, whereas the LeuCAM contribution in IL 1‐mediated neutrophil adhesion was less pronounced and varied in time. IL 1‐mediated neutrophil migration, however, was completely blocked by anti‐LeuCAM mAb. fMLP‐mediated neutrophil adhesion was inhibited by mAb against the α subunits of Mac, while mAb against the α subunits of LFA‐1 and Mac‐1 both reduced IL 1‐mediated adherence. In summary, we describe a novel leukocyte adhesion/migration method and demonstrate that the contribution of the LeuCAM complex in leukocyte‐endothelium interaction varies depending on cell type and stimulus used.
Identification of two distinct phosphoproteins as components of the human B cell antigen receptor complex
Authors: Van Noesel Carel J. M. , Borst Jannie , De Vries Evert F. R. , Van Lier René A. W.
Published: March 2007

In human B cells, the molecules that, upon receptor occupancy, couple membrane immunoglobulin to intracellular signal transduction pathways have never been identified. We here describe two phosphoproteins as integral parts of the B cell antigen receptor complex. Membrane IgM is non‐covalently associated with a disulfide‐linked heterodimer of glycoproteins. These molecules can be demonstrated on B cell lines and freshly isolated polyclonal B cell populations and are subject to phosphorylation at serine residues. Identification of these constituents of the B cell receptor complex opens up the opportunity to study coupling of the B cell antigen receptor to the intracellular signal transduction machinery at the molecular level.
Identification of the genes encoding the IgM‐α and Ig‐β components of the IgM antigen receptor complex by amino‐terminal sequencing
Authors: Hombach Joachim , Lottspeich Friedrich , Reth Michael
Published: March 2007

Beside the immunoglobulin (Ig) heavy and light chains the murine B cell receptor of the IgM class contains a heterodimer of two transmembrane proteins (IgM‐α and Ig‐β). By N‐terminal sequencing of IgM‐α and Ig‐β we have identified the genes encoding these proteins as mb‐1 and B29, respectively. Both genes are B cell specific and have been previously cloned from B minus T cell subtractive cDNA libraries. We have constructed expression vectors of the two genes and demonstrate that expression of the mb‐1 and B29 genes can influence the surface expression of IgM in μm‐transfected myeloma cells. From the known sequences of the IgM‐α and Ig‐β proteins and from the results of previous transfection experiments with various vectors expressing the μ chain we have developed a structural model of the B cell antigen receptor of class IgM which we compare with that of the T cell antigen receptor.
Suppression and prevention of adjuvant arthritis in rats by a monoclonal antibody to the α/β T cell receptor
Authors: Yoshino Shin , Schlipköter Eva , Kinne Raimund , Hünig Thomas , Emmrich Frank
Published: March 2007

Adjuvant arthritis (AA) in rats is an experimentally induced autoimmune disease mediated by T lymphocytes specific for Mycobacterium tuberculosis. We raised the question whether T cells carrying the γ/δ Tcell receptor (TcR), reactive or not to mycobacterial antigens, are involved in the pathogenesis of AA. For this purpose, T cells bearing the TcR α/β were depleted from circulation by treatment with a monoclonal antibody against the rat TcR α/β (R73). This treatment efficiently suppressed existing disease. Even more efficient was pretreatment with R73 from birth, which prevented AA induction completely. In these α/β+ T cell‐depleted animals an elevated level of α/β− T cells (about 15% vs. 1% in normal rats) was evident, which was not significantly increased by Mycobacterium tuberculosis injection. We found no positive evidence that γ/δ+ T cells do contribute to AA induction. Moreover, treatment with an anti‐TcR α/β monoclonal antibody may be very efficient in treatment of Tcell‐mediated autoimmune diseases.
Selective death of T cell receptor γ/δ+ intraepithelial lymphocytes by apoptosis
Authors: Viney Joanne L. , Macdonald Thomas T.
Published: March 2007

In mice, the majority of Tcells expressing the γ/δ Tcell receptor (TcR) are found at mucosal surfaces, especially the intestinal epithelium. Here we show that in vitro, the majority of TcR γ/δ+ intraepithelial lymphocytes, but not TcR α/β+ intraepithelial lymphocytes, undergo rapid and selective programmed cell death by apoptosis.
Fibronectin facilitates the migration of human natural killer cells
Authors: Somersalo Kristina , Saksela Eero
Published: March 2007

The interaction of lymphocytes with the extracellular matrix plays an important role in the immune defence against tumor cells and virus‐infected cells. We have examined the effect of matrix proteins on the migration of large granular lymphocytes (LGL) through 3‐μm pores in Nuclepore filters in a Boyden invasion chamber. Fibronectin bound on the filter surface significantly increased (p < 0.001) the capacity of LGL to migrate, whereas soluble fibronectin did not. In addition, a significantly higher (p < 0.001) percentage of LGL was capable of migration through fibronectin‐coated filters than through untreated filters. With fibronectin‐coated filters, a strong enrichment of CD16+ and CD56+CD3− cells with LGL morphology and reduction of CD3+ cells was found among migrating cells when the incubation time was 4 h or less. Later agranular lymphocytes, mainly CD3+ T lymphocytes, also started to migrate. Laminin coating of filters also facilitated migration, and when filters were coated with both fibronectin and laminin the increase in migration was equal to the sum of the increases induced by each protein alone. Interactions between cell surface and the Arg‐Gly‐Asp (RGD) peptide sequence of many matrix proteins had no role in the LGL migration through untreated flters. However, when filters were coated with either fibronectin or laminin, or with both, peptide containing the RGD sequence reduced migration to the level of untreated filter, whereas an Arg‐Gly‐Glu control peptide had no effect. Our results show that unstimulated LGL/natural killer cells are capable of rapid migration through matrix‐coated porous membranes, and that interactions between cell surface receptors and the RGD sequence of fibronectin and probably laminin are utilized in this process.
Allelic exclusion of membrane but not secreted immunoglobulin in a mature B cell line
Authors: Picarella Dominic , Serunian Leslie A. , Rosenberg Naomi
Published: March 2007

Although many B lymphocytes contain two completely rearranged immunoglobulin (Ig) heavy (H) chain genes, only one of the alleles specifies a protein product. This phenomenon is termed allelic exclusion and is controlled in part by the membrane portion of Ig H chains. We have identified a mature B cell line derived from murine bone marrow that expresses two H chain proteins, γ3 and γ2b. Proteins of appropriate size for the membrane and secreted forms of both H chains are produced and both IgG3 and IgG2b are secreted from the cells. However, only IgG3 is expressed on the membrane. Detailed restriction mapping of the H chain genes indicates that both are rearranged, one containing a VHJ558 gene linked to the γ3 constant region and the other a VHS107 gene linked to the γ2b constant region. Primer extension sequencing of the RNA reveals that the γ2b RNA is transcribed from the allele containing VHS107 sequences while the γ3 RNA is transcribed from the allele containing VHJ558 sequences. Thus, this mature B cell line secretes two distinct antibody molecules but is allelically excluded at the level of surface Ig expression. This cell line provides a model system to study factors, in addition to aberrant rearrangement, that influence allelic exclusion.
Signal transduction by HLA class II antigens expressed on activated T cells
Authors: Ødum Niels , Martin Paul J. , Schieven Gary L. , Hansen John A. , Ledbetter Jeffrey A.
Published: March 2007

Human T cells express HLA class II antigens upon activation. Although activated, class II+ T cells can present alloantigens under certain circumstances, the functional role of class II antigens on activated T cells remains largely unknown. Here, we report that cross‐linking of HLA‐DR molecules expressed on allospecific, CD4+ T clones and cell lines can function as transduction elements that trigger rapid cellular responses including tyrosine phosphorylation of cellular proteins and mobilization of Ca2+ from internal stores. The proteins phosphorylated on tyrosine were distinct from those observed after cross‐linking CD4. Ligation of CD4 and class II molecules generated a synergistic effect of the intracellular free Ca2+ concentration response that required an interaction between the molecules on the cell surface. Since class II is the natural ligand for CD4, the present data suggest that class II is induced on activated T cells to regulate CD4 function, possibly by specific interaction with the CD4‐associated p56lck protein tyrosine kinase.
Non‐mitogenic T cell activation signals are sufficient for induction of human immunodeficiency virus transcription
Authors: Gruters Rob A. , Otto Sigrid A. , Al Bert J. M. , Verhoeven Arthur J. , Verweij Cornelis L. , Van Lier René A. W. , Miedema Frank
Published: March 2007

Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and the Laboratory for Clinical and Experimental Immunology of the University of Amsterdem.

The expression of human immunodeficiency virus type 1 (HIV) is enhanced after T cell activation due to the interaction of cell‐encoded nuclear factors with binding sites in the viral long terminal repeats (LTR). We studied the minimal signal transduction requirements for induction of HIV transcription during T cell activation. Monoclonal antibodies (mAb) against the T cell receptor/CD3 complex induced interleukin (IL) 2 production as well as HIV‐LTR‐directed gene expression in Jurkat T cells. Addition of cyclosporin A or buffering of intracellular Ca2+ changes did not abolish this LTR‐directed gene expression but did block IL 2 production. In contrast, interference with protein kinase C (PKC) activation did inhibit both IL 2 production and LTR‐driven gene expression. Under all conditions HIV‐LTR‐directed gene expression correlated with gene expression induced by the NF‐kB binding enhancer, but not by the NF‐AT or OCT‐1 binding sites. In accordance with observations by Verweij, Geerts and Aarden on the CD28 co‐stimulatory activation of IL2 transcription via an NF‐kB‐like activity, stimulation of the CD2, CD28 and CD44 accessory molecules was tested to mimick physiological activation signals independent of T cell receptor triggering. mAb directed against CD2 and CD44 only marginally induced the LTR. Next, non‐mitogenic stimulation by mAb against CD28 clearly induced the HIV‐LTR‐ and NF‐kB‐ but not NF‐AT‐ and OCT‐1‐driven chloram‐phenicol acetyltransferase CAT expression, showing a direct effect on gene expression via this receptor.

Taken together, this report shows that non‐mitogenic T cell activation signals are sufficient to induce HIV transcription. The finding that these signals may be delivered by receptors that are not dependent on antigen‐specific activation may have important implications for our understanding of HIV pathogenesis.
Morphological features of cloned lymphocytes expressing γ/δ T cell receptors
Authors: Arancia Giuseppe , Malorni Walter , Iosi Francesca , Zarcone Daniela , Cerruti Giannamaria , Favre Anna , Zeromski Jan , Grossi Carlo E. , Moretta Alessandro
Published: March 2007

We have analyzed the morphological characteristics of human T lymphocytes bearing CD3‐associated T cell receptor (TcR) γ and δ chains. BB3 and δ‐TCS1 monoclonal antibodies (mAb) were used to identify two distinct, nonoverlapping populations of TcR γ/δ+ cells which express the products of Vδ2 and Vδ1 gene segments, respectively. In the peripheral blood, most Vδ1+ (δTCS‐1+) lymphocytes express the non‐disulfide‐linked form of receptor whereas Vδ2+ (BB3+) cells express the disulfide‐linked form. The majority of cloned TcR γ/δ+ cells exhibit a growth pattern different from that of conventional TcR α/β+ cells as they adhere promptly to surfaces and undergo morphological changes which can be summarized as follows: cells spread on the surface, form a distinct uropod and, in the final phase of adherence, emit long filopodia ending with adhesion plaques. Immunofluorescence studies of TcR γ/δ+ clones demonstrated the presence of submembranous actin microfilaments and actin‐binding protein confirming that these cells are capable of active motility which is related to the propensity of TcR γ/δ+ cells to home to epithelia.

Scanning electron microscope analyses of effector/target cell conjugates showed that in TcR γ/δ+ cells the region of the uropodia next to the cell body is responsible for the binding to tumor target cells. Interestingly, immunofluorescence analyses revealed that LFA‐1 molecules are predominantly distributed in the uropodium whereas they are virtually absent in the cell bodies. These morphological characteristics of TcR γ/δ+ cells may pertain to defensive mechanisms at the mucosal level.
Evidence that the CD45 phosphatase regulates the activity of the phospholipase C in mouse T lymphocytes
Authors: Marvel Jacqueline , Rimon Galia , Tatham Peter , Cockcroft Shamshad
Published: March 2007

The importance of the tyrosine phosphatase CD45 in the regulation of lymphocyte activation was first demonstrated using antibodies against the extracellular domain of CD45 in functional assays. More recently it was reported that CD45‐negative mutants were nonresponsive to stimulation through the T cell receptor‐CD3 complex. We have studied the effect of CD45 cross‐linking on the early signals induced by CD3 in mouse T cells. We show that CD45 cross‐linking inhibits the increase in inositol phosphates and cytoplasmic Ca2+ induced by cross‐linking of CD3. This indicates that CD45 is involved in the regulation of phospholipase C.
L‐Arginine‐dependent destruction of intrahepatic malaria parasites in response to tumor necrosis factor and/or interleukin 6 stimulation
Authors: Nüssler Andreas , Drapier Jean‐Claude , Rénia Laurent , Pied Sylviane , Miltgen François , Gentilini Marc , Mazier Dominique
Published: March 2007

There is growing evidence that cytokines (interleukin [IL] 1, IL 6, interferon‐γ, tumor necrosis factor [TNF]) directly or indirectly interfere with the intrahepatic development of malaria parasites. Recent work in our laboratory clearly showed that TNF can affect the hepatic development of parasites via IL 6 secreted by liver nonparenchymal cells. The possible participation of an L‐arginine‐dependent effector mechanism has been studied to explain the TNF/IL 6‐induced inhibition. We thus investigated if NGmonomethyl‐L‐arginine and Nω‐nitro‐L‐arginine, two specific inhibitors of inorganic nitrogen oxide synthesis from L‐arginine, were able to affect the inhibitory effect of TNF and/or IL 6 in co‐cultures. At 0.1 and 0.5 mM both L‐arginine analogues reversed the inhibitory effect of these cytokines. An interesting observation is that L‐arginine analogues enhance schizont development in the absence of prior cytokine contact. This result indicates an hepatic basal L‐arginine‐dependent anti‐parasitic activity which might explain the existence of self‐degenerating hepatic forms as previously reported.
Soluble Fcγ receptors II (FcγRII) are generated by cleavage of membrane FcγRII
Authors: Sautès Catherine , Varin Nadine , Teillaud Christophe , Daëron Marc , Even Jos , Hogarth P. Mark , Fridman Wolf H.
Published: March 2007

This study describes the production of soluble FcγRII by a cell line, D1B1, obtained by transfection of mouse L cells with a murine β1 FcγRII cDNA. Upon incubation at 37 °C, radioiodinated D1B1 cells release a 39‐kDa soluble FcγRII, reacting with the rat anti‐mouse FcγRII monoclonal antibody 2.4G2, and binding to mouse IgG2a, IgG2b and IgG1 but not IgG3. In contrast to the transmembrane 50‐ to 70‐kDa receptor, this soluble FcγRII does not react with antibodies directed against a peptide corresponding to the 15 carboxy‐terminal intracytoplasmic amino acids of β FcγRII. N‐Glycosidase F treatment generates a 18‐kDa polypeptide. A 32‐ to 40‐kDa soluble FcγRII, which resolves into 18.5‐ and 20‐kDa polypeptides after deglycosylation, was also isolated from the culture medium of unlabeled D1B1 cells. Therefore, this study indicates that soluble FcγRII corresponding to the two extracellular domains of FcγRII are generated by cleavage of membrane FcγRII. Proteolysis occurs most probably at the vicinity of the transmembrane region of the receptor, around amino acids 165 to 180.
T cell receptor δ diversity of freshly isolated T lymphocytes in rheumatoid synovitis
Authors: Sioud Mouldy , Førre Øystein , Natvig Jacob B.
Published: March 2007

In some patients with rheumatoid synovitis the distribution of Vδ gene families used by freshly isolated T lymphocytes from the synovial fluid and peripheral blood is different: peripheral blood T lymphocytes preferentially use the Vδ2 gene segment, whereas the majority of synovial fluid T lymphocytes use the Vδ1. The antigenic diversity of T cell receptor γ/δ is mainly dependent upon base sequences in the V‐J junction. In the present study we investigated whether the synovial fluid T lymphocytes expressing the Vδ1 gene segment were derived from a polyclonal or monoclonal expansion. The sequences encoding the V‐J junctions of 11 cDNA clones derived from the synovium of two patients with chronic arthritis were determined. Our data demonstrate that the Vδ1 cDNA clones have different V‐J junction sequences. This indicates that the Vδ1‐expressing T cells found in the synovial compartment are polyclonally expanded.
Influence of I‐E expression on induction of neonatal transplantation tolerance
Authors: Streilein J. Wayne , Socarras Sandra , Powell Thomas J.
Published: March 2007

Neonatal transplantation tolerance was one of the first experimental systems to reveal that tolerance could be achieved to non‐self antigens in living animals. Functional and direct evidence (obtained by the use of monoclonal antibodies directed at T cell receptors specifically reactive with I‐E molecules) confirm that tolerance is achieved, at least in part, via clonal elimination of developing thymocytes. In this report, we show that induction of tolerance of class I alloantigens in neonatal mice is governed by expression of I‐E molecules. Neonatal I‐E non‐expressor mice proved to be highly resistant to the acquisition of class I tolerance if the donor inoculum expressed disparate class I antigens as well as I‐E molecules. The spleens of the few class I‐tolerant, I‐E non‐expressor mice that were generated were found to be depleted of I‐E‐reactive (RR315+) T cells, whereas no such depletion was observed in their neonatally injected, but non‐tolerant littermates. By contrast we found no resistance to tolerance of I‐A alloantigens when neonatal I‐E non‐expressor mice received injections of I‐A‐disparate, I‐E‐bearing donor cells. In these tolerant mice, splenic I‐E‐reactive T cells were readily detected in apparently normal amounts. These results indicate that lack of I‐E expression in newborn mice confers resistance to tolerance induction to class I alloantigens, especially when the latter are expressed on donor cells that also display I‐E molecules. The possible mechanisms operating to produce resistance to tolerance induction in neonatal mice are discussed, including the possibilities that (a) I‐E may act as a restricting element during tolerance induction (an ontogenic process), and (b) the expression of I‐E on H‐2‐disparate, I‐E‐expressing test skin allografts may provide a source of “help” for CD8+ cytotoxic T cell precursors, leading to graft rejection.
Analysis of a positive feedback mechanism in the anti‐Sm autoantibody response of MRL/MPJ‐lpr/lpr mice
Authors: Stocks Martin Robert , Williams David Gareth , Maini Ravinder Nath
Published: March 2007

The mechanism of induction of anti‐Sm antibodies by passive transfer of anti‐Sm mAb in MRL/lpr mice was investigated. No idiotypic relationship was detected between the inducing monoclonal antibody KSm2 and either the induced circulating anti‐Sm antibodies or the products of anti‐Sm‐producing hybridomas derived from spleen cell fusion of treated mice. Treatment of mice with ribonucleoprotein Sm antigen, alone or as an immune complex, induced anti‐Sm and anti‐ribonucleoprotein antibodies similarly to treatment with KSm2. This suggests that autoantigen contributes to the development of the anti‐Sm response in MRL mice.
Age‐related changes in lymphokine production related to a decreased number of CD45RBhi CD4+ T cells
Authors: Nagelkerken Lex , Hertogh‐Huijbregts Anita , Dobber Ruud , Dräger Angelika
Published: March 2007

The ability of CD4+ T cells from CBA/Rij mice to produce interleukin (IL) 2 after stimulation with anti‐CD3, concanavalin A, or the combination of phorbol 12‐myristate 13‐acetate and ionomycin declines during aging. This phenomenon was accompanied by an increased production of IL 4 and interferon‐γ. These age‐related changes in lymphokine production correlated with the decrease in the percentage of CD45RBhi CD4+ T cells from about 80% in 2‐month‐old to about 40% in 27‐month‐old mice. This phenotypic shift was responsible for the decline in IL 2 production, because in young and in old mice CD45RBhi CD4+ T cells were more potent IL 2 producers than CD45RBlo cells. Moreover, old CD45RBhi CD4+ T cells produced less IL 2 than their young counterparts. Proliferative responses by T cells from old mice were lower than those of young mice, regardless whether the cultures were supplemented with IL 2, IL 4 or both lymphokines. As far as CD4+ T cells were concerned, this hyporesponsiveness was found in the CD45RBlo as well as in the CD45RBhi CD4+ T cell population.
Elevated levels of intracellular cAMP sensitize resting B lymphocytes to immune complex‐induced unresponsiveness
Authors: Stein Sidney H. , Phipps Richard P.
Published: March 2007

The ability of immune complexes (IC) to regulate B lymphocyte differentiation was investigated. Using an in vitro model, we previously demonstrated that macrophages (MΦ) or lymphoid dendritic cells pulsed with IC differentially regulated B cell function, inducing unresponsiveness or stimulation, respectively. The capacity of MΦ to induce unresponsiveness was dependent upon two signals, an antigen‐specific one supplied by the IC and MΦ‐secreted prostaglandin (PG)E2. Total inhibition of antibody production was never achieved as a small percentage of B lymphocytes were resistant to IC‐induced unresponsiveness. In this study, utilizing an accessory cell‐free system, we demonstrate that splenic B cell fractions separated on Percoll density gradients are heterogeneous in their sensitivity to IC‐mediated unresponsiveness. Small resting B lymphocytes are exquisitely sensitive to IC‐mediated negative signaling and exhibit virtual total ablation of antibody responses. Conversely, large activated B cells are more refractory to this inhibitory pathway. PGE2 and other agents which elevate cAMP potentiate IC‐induced unresponsiveness in resting, but not activated B lymphocytes. In addition treatment of resting B cells with PGF2α, which did not elevate cAMP, failed to sensitize these cells to IC‐mediated negative signaling. Unresponsiveness induced by IC is selective for specific aspects of B lymphocyte activation, since B cell differentiation but not proliferation is affected. Furthermore, pre‐treatment of resting B lymphocytes with interleukin 4 prevents the IC‐induced ablation of IgM antibody responses. Overall, our results indicate that the binding of IC by resting B lymphocytes provides a potent mechanism for inhibiting differentiation without affecting proliferation. These observations suggest that in vivo, IC play an important role in regulating the memory B lymphocyte pathway.
Formation of covalently linked C3‐C3 dimers on IgG immune aggregates
Authors: Barrio Elena , Antón Luis C. , Marqués Guillermo , Sánchez Angel , Vivanco Fernando
Published: March 2007

Upon activation of the complement system by IgG immune aggregates several components become tightly bound to the aggregates. The covalent interaction of C3 with immune complexes is essential for the solubilization and inhibition of immune precipitation of the complexes. It has recently been reported that on erythrocytes that have a fixed complement, activated C3 can become involved in the formation of C3b‐C3b covalent dimers, which act as high‐affinity binding sites for C5 (Kinoshita, T., Takata, Y, Kozono, H., Takeda, J., Hong, K. and Inoue, K., J. Immunol. 1988. 141: 3895). To characterize the molecular composition of immune aggregates that have fixed complement by the alternative pathway, we have investigated whether such C3b‐C3b dimers are formed in IgG immune complexes. For this purpose immune aggregates bearing covalently bound C3 were analyzed by two‐dimensional gel electrophoresis and the resolved bands transferred to polyvinylidene difluoride membranes and sequenced. When immune aggregates were incubated with serum for 15 min at 37 °C, the major high‐molecular mass bands detected by gel electrophoresis corresponded to heavy chain‐C3α65 and C3α65‐C3α43 (derived from iC3b‐iC3b‐IgG) covalent complexes. If K76COONa, an inhibitor of factor I, was added to the serum, before incubation with the immune complexes, then the major C3α fragment detected on the complexes corresponded to the C3α′ chain (105 kDa) and not C3α65. Hence C3b‐C3b covalent dimers are readily formed on the immune aggregates incubated with normal human serum, and are degraded to iC3b‐iC3b by factor I. The second C3b molecule was shown to be bound to the C3α43 region (C‐terminal portion of the C3α′ chain) of the first C3b molecule, which was itself covalently bound to the heavy, chain of IgG. Covalent complexes of heavy chain‐(C3α65)2 molecular composition were also detected, but their precise bonding pattern has not been established.
Anti‐CD3‐induced cell death in T cell hybridomas: mitochondrial failure and DNA fragmentation are distinct events
Authors: Vukmanović Stanislav , Zamoyska Rose
Published: March 2007

Triggering of the T cell receptor of T cell hybridomas leads to interleukin (IL) 2 secretion, inhibition of spontaneous growth, degradation of genomic DNA and cell death. We have investigated the relationship between the ability of mitochondria to convert 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), DNA fragmentation and growth arrest in hybridomas stimulated with anti‐CD3/T cell receptor antibodies. We describe a variant T hybridoma whose mitochondrial function remains unaffected upon stimulation with anti‐CD3 antibody, although it does undergo DNA fragmentation. By contrast, treatment of another anti‐CD3‐stimulated T hybridoma with endonuclease inhibitor completely inhibits the DNA fragmentation response but not mitochondrial failure induced by anti‐CD3 antibody. Thus, we have been able to dissociate anti‐CD3‐induced mitochondrial failure and DNA fragmentation, suggesting that they are separate events. Although both undoubtedly contribute to cell death induced by activation the primary cause of death may be mitochondrial failure rather than DNA fragmentation.
Genetic reconstitution of the T cell receptor (TcR) α/β heterodimer restores the association of CD3 ζ2 with the TcR/CD3 complex
Authors: Blumberg Richard S. , Sancho Jaime , Ley Steven C. , McDermott Francis V. , Tan Kut‐Nie , Breitmeyer James , Terhorst Cox
Published: March 2007

The cell surface expression of the T cell receptor (TcR)/CD3 complex and, consequently, the functional competence of the cell is partly dependent on CD3ζ. In its absence, a pentameric complex (TcR α/β/CD3γδϵ) is formed which is inefficiently transported to the cell surface. Reconstitution of CD3ζ by transfection, in turn, restores the cell surface expression and function of the complex. Through the use of transfection experiments, we here provide direct evidence that the association of CD3 ζ2 with the TcR/CD3 complex is dependent on the presence of both the TcR α and β polypeptide chains. Despite wild‐type levels of the CD3ζ protein in a TcR α‐negative mutant human T cell line, a complex was formed intracellularly which lacked CD3 ζ2 and consisted of βγδϵ and β2γδϵ. Upon transfection of the mutant with a TcR α cDNA, a TcR/CD3 complex which contained CD3 ζ2 was observed intracellularly. In contrast to the partial subcomplex on the cell surface of the untransfected cell line, the TcR/CD3 complex on the transfectant was functional as demonstrated by its ability to mobilize intracellular calcium after stimulation with a mitogenic CD3 ζ‐specific monoclonal antibody. Transient transfection studies performed in COS cell fibroblasts indicated that CD3 ζ2 was not interacting with the TcR α protein alone, implying that a conformation provided by either the TcR α/β heterodimer or the TcR α/β/CD3 γδϵ complex was necessary for the association of CD3 ζ2. Transfection studies performed in a TcR α/β‐negative murine T‐T hybridoma confirmed the requirement of both the TcR α and β proteins in CD3 ζ2 binding. We conclude that the TcR α and β chains harbor polypeptide sequences essential for the association of CD3 ζ2 with the TcR/CD3 complex.