bluuebunny/crossref_metadata_2025 huggingface dataset:
The influence of positive selection on RAG expression in thymocytes
Authors: Kouskoff Valérie , Vonesch Jean‐Luc , Benoist Christophe , Mathis Diane
Published: March 2007

The expression of recombination activating gene (RAG) products, responsible for T cell receptor (TcR) gene rearrangement, is shut off during positive selection of thymocytes. The precise stage at which this down‐regulation occurs remains somewhat controversial. We have analyzed RAG‐1 expression in thymocytes of TcR transgenic mice carried on selecting versus non‐selecting genetic backgrounds, both by in situ hybridization on thymus sections and by polymerase chain reaction amplification of RNA from sorted cells. The data from several transgenic lines indicate that RAG expression is already reduced in immature, cortical, CD4+CD8+ cells in the presence of positively selecting major histocompatibility complex molecules, although complete shut‐off is not achieved until the mature, medullary, single‐positive stage. This finding has practical and theoretical significance for studies on the mechanism of positive selection.
A cytotoxic CD40/p55 tumor necrosis factor receptor hybrid detects CD40 ligand on herpesvirus saimiri‐transformed T cells
Authors: Hess Sigrun , Kurrle Roland , Lauffer Leander , Riethmüller Gert , Engelmann Hartmut
Published: March 2007

The B cell activation molecule CD40 and the p55 tumor necrosis factor receptor (p55TNFR) belong to the same family of structurally conserved proteins. We constructed a chimeric receptor consisting of the CD40 extracellular and transmembrane domains and the p55TNFR intracellular domain. This receptor hybrid retained the biological activity and the ligand specificity of the respective wild‐type receptor domains. Thus it exerted a marked cytotoxic effect in three different transfected cell lines after activation not only with anti‐CD40 antibody but also with CD40 ligand (CD40L) in soluble and membrane‐bound forms. Using hybrid‐transfected baby hamster kidney cells we demonstrated that herpesvirus saimiri‐transformed human CD4+ T lymphocytes constitutively express bioactive CD40 ligand on their surface. The hybrid receptor‐based assay was highly specific for CD40 activating reagents and more sensitive than an assay measuring CD40‐mediated B cell rescue from apoptosis. Hence CD40/p55TNFR transfectants may be useful for dissecting CD40L‐mediated events in T‐B cell interactions, and also to detect a defective CD40L molecule in putative hyper‐IgM syndrome patients.
Host intestinal intraepithelial γδ T lymphocytes present during acute graft‐versus‐host disease in mice may contribute to the development of enteropathy
Authors: Sakai Tetsu , Ohara‐Inagaki Kyoko , Tsuzuki Toyonori , Yoshikai Yasunobu
Published: March 2007

We reported that T cell receptor (TcR) γδ intestinal intraepithelial lymphocytes (i‐IEL) of host origin increased transiently, then decreased drastically at the early stage of non‐irradiated acute graft‐versus‐host disease (GVHD) in mice. We investigated the role of the TcR γδ i‐IEL of host origin in the pathogenesis of the intestinal lesions that occur during acute GVHD. The acute GVHD was induced in mice which had been depleted of TcR γδ by in vivo administration of hamster monoclonal antibody (mAb) against TcR γδ. Although the degree of splenomegaly after the induction of acute GVHD in mice treated with anti‐TcR γδ mAb was similar to that in control mice treated with hamster anti‐2,4,6‐trinitrophenyl mAb, infiltration of donor‐derived T cells into the epithelium, and mitosis and apoptosis of crypt cells in the intestinal mucosa were dramatically suppressed in these mice. This suggest that host TcR γδ T cells in i‐IEL contribute to the development of enteropathy in acute GVHD in mice.
Clearance of Sendai virus by CD8+ T cells requires direct targeting to virus‐infected epithelium
Authors: Hou Sam , Doherty Peter C.
Published: March 2007

Minimal numbers of CD8+ T cells are found in bronchoalveolar lavage (BAL) populations recovered from Sendai virus‐infected mice that are homozygous (−/−) for β2‐microglobulin (β2‐m) gene disruption. The prevalence of the CD8+ set was substantially increased in the pneumonic lungs of 8−12‐week radiation chimeras made using substantially class I major histocompatibility complex (MHC) glycoprotein‐negative β2‐m (−/−) recipients and normal β2‐m (+/+) bone marrow. Even so, the CD8+ (but not the CD4+) lymphocyte counts were still much lower than in the (+/+)→(+/+) controls. The (+/+)→(+/+) and (+/+)→(−/−) chimeras cleared Sendai virus and potent virus‐immune CD8+ cytotoxic T lymphocytes (CTL) specific for H‐2Kb + viral nucleoprotein peptide were found in the BAL from both groups. However, following in vivo depletion of the CD4+ population, only the (+/+)→(+/+) mice were able to deal with the infection. Similarly, adoptively transferred, H‐2Kb‐restricted CD8+ T cells from previously‐primed (+/+) mice also failed to clear virus from the lungs of (+/+)→(−/−) chimeras infected within 2 weeks of reconstitution with bone marrow, though they were effective in the (+/+)→(+/+) controls. Sendai virus‐immune CD8+ T cells are thus unable to eliminate virus‐infected β2‐m (−/−) lung epithelial cells that might be thought to be expressing very small amounts of either isolated class I heavy chain, or class I MHC glycoprotein that has bound β2‐m derived from β2‐m (+/+) T cells or macrophages present in the pneumonic lung. Furthermore, the CD8+ CTL that are being exposed to β2‐m (+/+) stimulators in the BAL population cannot operate in some bystander mode to clear virus from respiratory epithelium.
Co‐Iigation of ICAM‐1 (CD54) and membrane IgM negatively affects B cell receptor signaling
Authors: van Horssen Martin , Loman Stoffer , Rijkers Ger T. , Boom Saskia E. , Bloem Andries C.
Published: March 2007

A possible role of intercellular adhesion molecule 1 (ICAM‐1, CD54) in transmembrane signaling was investigated in B cells from the Burkitt lymphoma cell line MTLM4. Cross‐linking of membrane IgM (mIgM) induced an increase in intracellular free Ca2+ as a result of the release from intracellular stores and an influx of extracellular Ca2+. When the B cells were incubated with limiting concentrations of anti‐IgM, co‐ligation of mIgM and CD54, but not CD19, resulted in an inhibition of the Ca2+ response. Separate cross‐linking of mIgM and CD54 under these conditions, using isotype mismatched monoclonal antibodies (mAb), did not affect the mobilization of Ca2+. The CD54‐mediated inhibition of the Ca2+ response was also observed in the absence of extracellular Ca2+. All CD54 mAb tested (F10.2, F10.3 and F7.11) interfered with mIgM signaling. The results presented in this report imply that CD54 is linked to intracellular signaling pathways and, via co‐ligation with mIgM, interferes in the release of Ca2+ from intracellular stores.
Hydrogen peroxide as a potent activator of T lymphocyte functions
Authors: Los Marek , Dröge Wulf , Stricker Kirstin , Baeuerle Patrick A. , Schulze‐Osthoff Klaus
Published: March 2007

During inflammatory processes infiltrating cells produce large amounts of reactive oxygen intermediates (ROI). Increasing evidence suggests that ROI besides being cytotoxic may act as important mediators influencing various cellular and immunological processes. In this study, we have investigated the effects of hydrogen peroxide on several aspects of lymphocyte activation. In ESb‐L T lymphoma cells, micromolar concentrations of hydrogen peroxide rapidly induced activation of the transcription factor NF‐χB, whereas DNA‐binding activity of the transcription factor AP‐1 was virtually not affected. In addition, hydrogen peroxide induced early gene expression of interleukin‐2 (IL‐2) and the IL‐2 receptor α chain. The stimulation of IL‐2 expression was found to be conferred by a χB‐like cis‐regulatory region within the IL‐2 gene promoter. In contrast to these activating effects, addition of hydrogen peroxide was largely inhibitory on cell proliferation which is consistent with a general requirement of thiol compounds for lymphocyte proliferation. However, hydrogen peroxide significantly increased T cell proliferation when applied for a short period under reducing conditions. These data indicate that ROI may act as an important competence signal in T lymphocytes inducing early gene expression as well as cell proliferation.
Differential reactivity of residual CD8+ T lymphocytes in TAP1 and β2‐microglobulin mutant mice
Authors: Ljunggren Hans‐Gustaf , van Kaer Luc , Ashton‐Rickardt Philip G. , Tonegawa Susumu , Ploegh Hidde L.
Published: March 2007

TAP1 ‐/‐ and β2‐microglobulin (β2m) ‐/‐ mice (H‐2b background) express very low levels of major histocompatibility complex (MHC) class I molecules on the cell surface. Consequently these mice have low numbers of mature CD8+ T lymphocytes. However, TAP1 ‐/‐ mice have significantly higher numbers of CD8+ T cells than β2m ‐/‐ mice. Alloreactive CD8+ cytotoxic T lymphocyte (CTL) responses were also stronger in TAP1 ‐/‐ mice than in β2m ‐/‐ mice. Alloreactive CTL generated in TAP1 ‐/‐ and β2m ‐/‐ mice cross‐react with H‐2b‐expressing cells. Surprisingly, such cross‐reactivity was stronger with alloreactive CTL from β2m ‐/‐ mice than with similar cells from TAP1 ‐/‐ mice. The β2m ‐/‐ mice also responded more strongly when primed with and tested against cells expressing normal levels of H‐2b MHC class I molecules. Such H‐2b‐reactive CD8+ CTL from β2m ‐/‐ mice but not from TAP1 ‐/‐ mice also reacted with TAP1 ‐/‐ and TAP2‐deficient RMA‐S cells. In contrast, H‐2b‐reactive CD8+ CTL from neither β2m ‐/‐ mice nor TAP1 ‐/‐ mice killed β2m ‐/‐ cells. In line with these results, β2m ‐/‐ mice also responded when primed and tested against TAP1 ‐/‐ cells. We conclude that the reactivity of residual CD8+ T cells differs between TAP1 ‐/‐ and β2m ‐/‐ mice. The MHC class I‐deficient phenotype of TAP1 ‐/‐ and β2m ‐/‐ mice is not equivalent: class I expression differs between the two mouse lines with regard to quality as well as quantity. We propose that the differences observed in numbers of CD8+ T cells, their ability to react with alloantigens and their cross‐reactivity with normal H‐2b class I are caused by differences in the expression of MHC class I ligands on selecting cells in the thymus.
Biphasic control of nuclear factor‐χB activation by the T cell receptor complex: role of tumor necrosis factor α
Authors: Pimentel‐Muiños Felipe X. , Mazana Javier , Fresno Manuel
Published: March 2007

The regulation of nuclear factor (NF)‐χB activation by the T cell receptor (TcR)/CD3 complex in primary human T cells has been studied at various times after activation. Only p50 NF‐χB protein bound the χB element of interleukin‐2 receptor (IL‐2R) α chain promoter on resting T cells. However, immediately after TcR/CD3 cross‐linking (after approximately 1 h; immediate) binding of p50.p65 heterodimers was observed. p50.c‐rel heterodimers were also detected bound to this sequence at early time points (7–16 h; early), and both remained active at later time points (40 h; late) after activation. This regulation takes place mainly at the level of nuclear translocation of p65 and c‐rel, at immediate and early time points. Activation also induced c‐rel and p105/p50 mRNA synthesis, but not p65 mRNA whose expression was constitutive. Interestingly, all those early and late events, but not the immediate ones, were inhibited by a neutralizing anti‐tumor necrosis factor α (TNF‐α) monoclonal antibody. Similarly, cycloheximide prevented the p65 and c‐rel translocation and consequent formation of active binding heterodimers, at early and late times. Cyclosporin A impaired not only early and late, but also immediate events; however, addition of TNF‐α prevented all inhibition. These results indicate that the regulation of NF‐χB activation during T cell activation by TcR/CD3 signals is biphasic: TcR/CD3 triggers its immediate translocation, which is transient if no TNF‐α is present. TNF‐α, therefore, emerges as the main factor responsible for a second phase of NF‐χB regulation, controlling both translocation of p65 and c‐rel, and new mRNA synthesis for c‐rel and p105/p50.
Isotype exclusion in λ1 transgenic mice depends on transgene copy number and diminishes with down‐regulation of transgene transcripts
Authors: Hengstschläger Markus , Maizels Nancy
Published: March 2007

We have compared expression of the endogenous χ and transgenic λ1 light chains in three lines of mice carrying one, four and eight copies of a λ1 transgene. We have found that in very young mice, even a single rearranged transgenic λ allele excludes expression of the endogenous χ loci. As the λ1 transgenic mice age, the proportion of λ‐positive cells decreases, as has been reported by others (Neuberger et al., Nature 1989. 338: 350; Pettersson et al., Nature 1990. 344: 165; Hagman et al., J. Exp. Med. 1989. 169: 1911; Bogen and Weiss, Eur. J. Immunol. 1991. 21: 2391). The decrease in λ‐positive B cells is accompanied by an increase in χ‐positive cells. We show that the decrease in B cells bearing surface λ immunoglobulin depends on transgene copy number and occurs most rapidly in lower copy number lines. The decrease in surface λ expression correlates with a dramatic decrease in the level of λ mRNA in splenic B cells. Transgene down‐regulation cannot be alleviated by stimulation of splenocytes with the mitogen lipopolysaccharide. These are the first data to establish that a single copy transgene can effect isotype exclusion. In addition, these results provide strong evidence that down‐regulation of transgene expression is controlled at the level of transcription, and that it is the level of expressed light chain that regulates isotype exclusion.
Effect of anti‐V3 antibodies on cell‐free and cell‐to‐cell human immunodeficiency virus transmission
Authors: Pantaleo Giuseppe , Demarest James F. , Vaccarezza Mauro , Graziosi Cecilia , Bansal Geetha P. , Koenig Scott , Fauci Anthony S.
Published: March 2007

The present study was undertaken to compare the effects of a type‐specific (HIV‐1 MN) anti‐V3 antibody on in vitro human immunodeficiency virus (HIV) infection of peripheral blood mononuclear cells in systems of cell‐free versus cell‐to‐cell transmission of virus. Anti‐V3 antibody completely prevented HIV‐1 infection when cell‐free virus was the sole mechanism of infection. A significant reduction of the neutralizing activity of the anti‐V3 antibody was observed when infectivity was dependent on both cell‐free and cell‐to‐cell mechanisms of infection. Furthermore, when cell‐to‐cell transfer of virions was the primary mechanism of HIV‐1 infection, inhibition of HIV‐1 infection was not observed. Therefore, a potent neutralizing antibody with a single epitope specificity failed to effectively control dissemination of a persistent HIV‐1 infection in a system characterized predominantly by cell‐to‐cell transfer of virus.
Organ‐specific autoimmunity induced by adult thymectomy and cyclophosphamide‐induced lymphopenia
Authors: Barrett Simon P. , Toh Ban‐Hock , Alderuccio Frank , van Driel Ian R. , Gleeson Paul A.
Published: March 2007

Autoimmune gastritis, a CD4+ T cell‐mediated organ‐specific autoimmune disease, can be induced by thymectomy of neonatal, but not of older, BALB/c mice. Here we have shown that autoimmune gastritis can also be induced in 6–8‐week‐old BALB/c mice by thymectomy combined with a single dose of cyclophosphamide (300 mg/kg). This treatment reduced the numbers of splenic T and B cells approximately 25‐fold. However, by 8 days after treatment, the number of splenic lymphocytes had returned to normal adult levels. Approximately 50% of treated mice developed autoimmune gastritis after 10–12 weeks. These mice had mononuclear cellular infiltrates within the gastric mucosa and serum autoantibodies to the α and β subunits of the gastric H+/K+ ATPase. Transgenic mice, expressing the gastric H+/K+ ATPase β‐subunit in the thymus (Alderuccio, F., Toh, B. H., Tan, S. S., Gleeson, P. A. and van Driel, I. R., J. Exp. Med. 1993. 178: 419), did not develop autoimmune gastritis after the adult thymectomy/cyclophosphamide treatment. Thus a T cell response to the H+/K+ ATPase β‐subunit is likely to be required for the onset of gastritis. These observations suggest that pathogenic autoreactive T cells exist in the periphery of normal adult mice and that autoimmunity can be induced by the activation of these autoreactive T cells following transient lymphopenia. Cyclophosphamide‐treatment of adult mice without thymectomy did not induce autoimmune gastritis, suggesting thymic regulation of these pathogenic T cells.
Fibronectin induces phosphorylation of a 120‐kDa protein and synergizes with the T cell receptor to activate cytotoxic T cell clones
Authors: Ostergaard Hanne L. , Ma Eileen A.
Published: March 2007

Fibronectin (FN) has been shown to act as a costimulator in both CD4+ and CD8+ T cell activation through the T cell receptor (TcR). Consistent with previous studies, we found that FN is able to both enhance the maximal amount of TcR‐triggered degranulation and lower the threshold for activation. The density of immobilized anti‐CD3 or anti‐TcR required to induce degranulation and tyrosine phosphorylation of cellular proteins by several cytotoxic T lymphocyte clones is quantitatively about tenfold lower in the presence of FN. We further demonstrate that FN alone stimulates transient tyrosine phosphorylation of a 120‐kDa protein (pp120) in CD8+ T cells and when FN is coimmobilized with substimulatory amounts of anti‐CD3 or anti‐TcR there is a synergistic response, resulting in prolonged and enhanced phosphorylation of pp120. To determine if FN acts as a costimulator in CD8+ cells solely through mediating adhesion events or if it also transduces signals in T cells we conducted remote stimulation experiments. Degranulation was induced when FN and sub‐stimulatory anti‐CD3 were presented on separate surfaces, indicating that FN induces independent transmembrane signals capable of augmenting TcR‐induced signals resulting in a functional response. Both FN plus TcR‐induced tyrosine phosphorylation of pp120 and degranulation are inhibited by RGD‐containing peptides, implying that an RGD‐dependent FN receptor is mediating phosphorylation of pp120 and enhancing TcR‐mediated degranulation.
The alternative splicing of human FcγRII mRNA is regulated by activation of B cells with mIgM cross‐linking, interleukin‐4, or phorbolester
Authors: Sármay Gabriella , Rozsnyay Zoltán , Koncz Gábor , Danilkovich Alla , Gergely János
Published: March 2007

The human type two IgG binding receptors (FcγRII) are encoded by three genes (FcγRIIA, ‐B and C) resulting in at least six protein isoforms generated by alternative mRNA splicing. Surface expression of FcγRII has been shown to be modulated during B cell activation, although data characterizing the isoform(s) expressed are not available. The extracellular as well as the transmembrane domains of various Fcγ;RII are highly homologous. Only the intracellular domains vary between the different FcγRII isoforms, suggesting differences in signal transduction. Using reverse transcriptase and polymerase chain reaction of mRNA obtained from resting tonsil B cells, we show that the majority of FcγRII mRNA species to be of b2 type, although b1 type and a low level of FcγRIIa type are also present. Culturing the cells for 18 h in the presence of 2.5 U/ml interleukin‐4 or 10 μg/ml affinity‐purified anti‐IgM F(ab')2 fragments induced a switch in alternative splicing, resulting in a significant increase of FcγRIIb1 mRNA expression, while the synthesis of FcγRIIb2 mRNA was down‐regulated. Stimulation of B cells with 100 ng/ml phorbol 12‐myristate 13‐acetate induced similar alteration, although only after 48‐h treatment. The accumulation of FcγRIIb1 and the reduction of both FcγRIIb2 and FcγRIIa mRNA in activated cells is accompanied by the enhanced expession of FcγRII on the cell surface, representing most probably the FcγRIIb1 isoform. Heat‐aggregated IgG inhibited the anti‐IgM‐induced proliferation of resting but not that of activated B cells, suggesting that aggregation of FcγRIIb2 constitutively expressed on resting B cells might be responsible for the prevention of inadequate activation of resting B cells.
Evolution of specific antigen recognition: size reduction and restricted length distribution of the CDRH3 regions in the rainbow trout
Authors: Roman Thibaud , De Guerra Arnaud , Charlemagne Jacques
Published: March 2007

The immunoglobulin heavy chain repertoire in fish was investigated by cloning a total of 88 rearranged VDJ junctions from the head kidney B cell mRNA of a salmonid, the rainbow trout (Oncorhynchus mykiss). Trout DH segments are short and cannot be classified into independent DH families. Several of the ten identified putative DH segments had stretches of nucleotide sequence identity with mouse (DQ52, DFL 16.2 and Dsp 2.1), human (DM1) and chicken (DH4) DH. There was a clear preference for one or two of the three putative DH reading frames and a stop codon is often present in the less used reading frame. Four of the six JH segments are preferentially used, and analysis of the VH‐DH and DH‐JH junctions suggest the presence of N‐nucleotides. The absolute size and size heterogeneity of the rainbow trout CDRH3 are smaller than those of the Xenopus, mouse and human CDRH3. About 75% of the 84 in‐frame trout CDRH3 have 8, 9 or 10 residues and none of them have more than 11 residues. This homogeneization of the CDRH3 loop size may partly explain the restricted antibody diversity in lower vertebrates.
The cytoplasmic tail of the T cell receptor ζ chain is required for signaling via CD26
Authors: Mittrücker Hans‐Willi , Steeg Christiane , Malissen Bernard , Fleischer Bernhard
Published: March 2007

The protease dipeptidylpeptidase IV (CD26) provides an alternative activation pathway for T lymphocytes and is involved in several aspects of T cell function. Activation via CD26 requires the expression of the T cell receptor (TcR)/CD3 complex. Here we have investigated the role of the TcR ζ chain for T cell activation via CD26. T cell hybridomas expressing TcR with various deletions in the CD3 ζ chain were transfected with a CD26 cDNA and the response of the transfected cells to anti‐CD26 monoclonal antibodies was tested. Our data show that the ζ chain is essential and that at least one YXXL motif in the cytoplasmic tail of the ζ chain is required for CD26‐mediated signaling. Other TcR components do not replace the ζ chain.
Secretion of the eosinophil‐active cytokines interleukin‐5, granulocyte/macrophage colonystimulating factor and interleukin‐3 by bronchoalveolar lavage CD4+ and CD8+ T cell lines in atopics asthmatics, and atopic and nonatopic controls
Authors: Till Stephen , Li Baiqing , Durham Stephen , Humbert Marc , Assoufi Basil , Huston David , Dickason Richard , Jeannin Pascale , Kay A. Barry , Corrigan Christopher
Published: March 2007

Specific eosinophil accumulation and activation within the asthmatic bronchial mucosa are thought to occur at least partly through the actions of cytokines, including interleukin (IL)‐5, IL‐3 and granulocyte/macrophage colonystimulating factor (GM‐CSF). Although mRNA encoding some of these cytokines has been demonstrated in bronchoalveolar lavage (BAL) fluid cells and bronchial biopsies from asthmatics, it has yet to be established whether these cells produce the translated products and whether expression is associated with CD4+ T helper or CD8+ cytotoxic T cells. We addressed this problem by raising polyclonal CD4+ and CD8+ T cell lines from the BAL fluid of six atopic asthmatics, five atopic non‐asthmatics and seven non‐atopic non‐asthmatic controls. BAL fluid cells obtained at fiberoptic bronchoscopy were depleted of adherent cells, and then T lymphocytes expanded by stimulation with monoclonal anti‐CD3 antibody and recombinant human IL‐2. When lymphocytes had expanded to sufficient numbers, CD4+ and CD8+ cells were separated by positive selection with magnetic beads coated with anti‐CD4 or anti‐CD8 monoclonal antibodies and further expanded. Cytokine secretion by standardized cell numbers was measured by enzyme‐linked immunosorbent assays. BAL CD4+ T cell lines from the asthmatics secreted significantly elevated quantities of both IL‐5 and GM‐CSF as compared with lines from the atopic and non‐atopic controls (p = 0.023–0.003). In contrast, IL‐3 secretion did not significantly differ between the groups. In some subjects, CD8+ T cell lines also secreted significant quantities of these cytokines and there was a trend for IL‐5 secretion by these cells to be higher in asthmatics than non‐atopic controls (p = 0.035). These data are consistent with the hypothesis that activated T lymphocytes from asthmatics, particularly of the CD4+ subset, are predisposed to release elevated quantities of cytokines relevant to the accumulation and activation of eosinophils.
HLA class II‐restricted recognition of common tumor epitopes on human melanoma cells by CD4+ melanoma‐infiltrating lymphocytes
Authors: Dréan Eric Le , Gervois Nadine , Diez Elisabeth , Semana Gilbert , Dreno Brigitte , Jotereau Francine
Published: March 2007

CD4+ T cell clones derived from lymphocytes infiltrating four human melanomas specifically recognized melanoma‐derived tumor epitopes as shown by secretion of tumor necrosis factor (TNF) in vitro upon interaction with autologous melanoma cells, whereas they did not recognize HLA class II‐expressing autologous lymphoblasts or HLA class II mismatched allogeneic melanoma cells. Specificity was further established by demonstrating that TNF responses to tumor cells were inhibited by HLA‐DR or HLA‐DQ monoclonal antibodies. Most of these clones cross‐reacted with allogeneic melanoma cells expressing a potentially restricting HLA allele or a structurally similar one. These data show that shared epitopes of human melanoma cells presented on HLA class II molecules are frequently recognized by autologous CD4+ T lymphocytes.
Polarized Th2 cytokine expression by both mucosal γδ and αβ T cells
Authors: Hiroi Takachika , Fujihashi Kohtaro , McGhee Jerry R. , Kiyono Hiroshi
Published: March 2007

Currently only limited information is available as to why dominant IgA isotype responses are supported by mucosal T cells in effector tissues. To address this issue directly, γδ and αβ T cells were isolated from the submandibular gland (SMG) of mice as an example of mucosal effector tissues. Freshly isolated CD3+ T cells from this tissue contained relatively high numbers of activated cells [approximately 10% interleukin‐2 receptor (IL‐2R)+ cells and 15% of cells in cycle stages S and G2 + M], of which 25% and 75% were γδ and αβ T cells, respectively. The cytokine‐specific quantitative reverse transcriptase‐polymerase chain reaction and enzyme‐linked immunospot analyses revealed that, although both γδ and αβ T cells were capable of producing an array of Th1 or Th2 cytokines following stimulation via the T cell receptor‐CD3 complex, these mucosal T cells were mainly committed to IL‐5 and IL‐6 expression in vivo (Th2 type). Both freshly isolated γδ and αβ T cells expressed mRNA and contained IL‐5 and IL‐6 spot‐forming cells (SFC); however, only the latter exhibited high mRNA levels and SFC for a Th1 cytokine (interferon‐γ). Taken together, the results show that freshly isolated CD3+ T cells from SMG contain activated γδ and αβ T cells which are programmed to produce IL‐5 and IL‐6. Thus, SMG, an example of an IgA effector tissue, can be characterized as a Th2‐dominant site. However, although both γδ and αβ T cells express cytokine profiles consistent with a Th2 phenotype, only the latter subset with a CD4+CD8− phenotype provided effective help for mucosal B cell responses in vitro.
Human CD6 possesses a large, alternatively spliced cytoplasmic domain
Authors: Robinson William H. , de Vegvar Henry E. Neuman , Prohaska Susan S. , Rhee Joon W. , Parnes Jane R.
Published: March 2007

Human CD6 is a monomeric 105/130‐kDa T cell surface glycoprotein that is involved in T cell activation. The apparent discrepancy between the size of the cytoplasmic domain in human (44 amino acids) and mouse (243 amino acids) CD6, led us to use reverse transcriptase‐polymerase chain reaction of human peripheral blood lymphocyte mRNA to isolate cDNA clones that include the carboxyl‐terminal coding region of human CD6. The nucleotide sequence of the longest human cDNA clone, CD6‐PB1, predicts a protein of 668 amino acids with a 244‐amino acid cytoplasmic domain similar in size to and possessing 71.5% amino acid sequence identity with the cytoplasmic domain of mouse CD6. This previously unrecognized 244‐amino acid cytoplasmic domain does not have significant homology to any other known protein (except mouse CD6), but does possess two proline‐rich motifs containing the SH3 domain‐binding consensus sequence, a serine‐threonine‐rich motif repeated three times, three protein kinase C phosphorylation‐site motifs, and 10 casein kinase‐2 phosphorylationsite motifs. These sequences are likely to play a role in the ability of CD6‐specific monoclonal antibodies to stimulate T cell proliferation. Full‐length CD6 cDNA containing this cytoplasmic domain sequence encodes a monomeric 105/130‐kDa protein that can be immunoprecipitated from the surface of transfected cells and comigrates upon SDS‐PAGE with wild‐type CD6 immunoprecipitated from PBL. We also isolated two alternatively spliced forms of human CD6 cDNA lacking sequences encoding membrane‐proximal regions of the cytoplasmic domain which maintain the same reading frame as CD6‐PB1. The short cytoplasmic domain of the previously reported human CD6–15 cDNA clone results from a deletion of a 20‐bp segment through use of an alternative 3' splice site, resulting in a frame shift and premature termination of translation relative to the clones we have isolated. These data demonstrate that human CD6 possesses a large cytoplasmic domain containing sequence motifs that are likely to be involved in signal transduction upon stimulation of T cells through CD6 ligation.
Abundance of H‐2 promiscuous T cells specific for mycobacterial determinants in H‐2b/d F1 hybrid mice
Authors: Vordermeier H. Martin , Arya Anu , Harris David P. , Moreno Carlos , Ivanyi Juraj
Published: March 2007

A majority of immunodominant epitopes of mycobacterial antigens are known to be recognized by murine T cells in the context of several H‐2 haplotypes. In this study, we established the frequency of T cells able to recognize these peptides promiscuously, i.e. in the context of allogeneic antigen‐presenting cells, using hybridomas from peptide‐immunized H‐2 homologous and heterologous mice. The degree of promiscuity in homozygous mice varied between 4–27% between different specificities and genetic backgrounds. In particular, the results showed that promiscuity between Ab and Ad in respect to a peptide from the Mycobacterium tuberculosis 38‐kDa protein (residues 350–369) was displayed by 22% of BALB/c and 4% of C57BL/10‐derived hybrids, but by 42% of [BALB/c x C57BL/10] F1‐derived clones. This represents a significant increase (p < 0.001) of T cell promiscuity compared to the parental haplotypes. It is noteworthy that considerably lower peptide concentrations were able to stimulate the promiscuous hybridomas compared to the H‐2‐restricted hybrids. This finding suggests a functional advantage of promiscuous T cells which enables them to expand preferentially in the initial stages of infections with M. tuberculosis and thus enables the host to mount a rapid protective immune response.
The B lymphocyte in rheumatoid arthritis: analysis of rearranged Vx genes from B cells infiltrating the synovial membrane
Authors: Gause Angela , Gundlach Karen , Zdichavsky Marty , Jacobs Georg , Koch Bernhard , Hopf Thomas , Pfreundschuh Michael
Published: March 2007

The participation of the humoral immune system in rheumatoid arthritis (RA) is characterized by the production of rheumatoid factors (RF). RF are autoantibodies against the Fc part of IgG which are encoded by diverse germ‐line genes. Most of the RF‐encoding genes are unmutated, but in RA, a substantial quantity is encoded by somatically mutated genes. In addition, the synovial membranes (SM) of the diseased joints of RA patients are infiltrated by B lymphocytes which form germinal center‐like aggregates. To analyze the local immune response, B cell foci from two RA SM were isolated by micromanipulation. From DNA of these foci, the rearranged kappa light chain variable region (Vx) genes were amplified by polymerase chain reaction (PCR), cloned and sequenced. The amplification of different Vx‐Jx combinations of different foci suggested oligoclonal expansion of B lymphocytes, which was confirmed by sequence analysis: each PCR product contained members of a single B cell clone. The sequence analysis of 29 different clones revealed rearrangements of diverse Vx genes. Both frequent representatives of the Vx3 and the Vx1 family, as well as rarely used genes such as the L10 and B2 genes of the Vx2 and Vx5 families were found. Of the eleven potentially functional gene rearrangements, eight were significantly mutated, indicating their derivation from antigen‐selected B cells. Intraclonal diversity in one of these clones may suggest ongoing mutation in the diseased synovial membrane of patients with RA.
Role of splenic B cells in the immune privilege of the anterior chamber of the eye
Authors: Niederkorn Jerry Y. , Mayhew Elizabeth
Published: March 2007

The immune privilege of the anterior chamber (AC) of the eye is largely due to the active down‐regulation of systemic delayed‐type hypersensitivity (DTH) that is evoked when antigens are introduced into this ocular compartment. This antigen‐specific suppression of DTH has been termed anterior chamberassociated immune deviation (ACAID) and has been demonstrated with a wide variety of antigens. Previous studies have shown that antigens introduced into the AC are processed by resident antigen‐presenting cells which then migrate to the spleen where they transmit a signal that culminates in the generation of regulatory cells that prevent the development of DTH. Although considerable effort has focused on the nature of the ocular phase of ACAID, the role of the spleen has been largely ignored. The present study tested the hypothesis that B cells are the essential cell population responsible for the splenic phase of ACAID. Splenectomy prevented the induction of ACAID; however, introduction of B cell‐enriched spleen cells into the AC of splenectomized mice restored the hosts' capacity to develop ACAID. The same effect, however, could not be produced with B cell‐depleted spleen cells. B cell depletion of eusplenic mice by chronic administration of anti‐μ antiserum prevented the development of ACAID and thus, had the same of effect as splenectomy. The results indicate that an intact B cell population is necessary for the induction of ACAID. These findings also support the hypothesis that antigens arising in the AC and subsequently delivered to spleen are captured by B cells and presented to T cells in a manner that promotes the development of down‐regulatory T cells.
CD8+ cell activation to a major mastocytoma rejection antigen, P815AB: requirement for tum− or helper peptides in priming for skin test reactivity to a P815AB‐related peptide
Authors: Grohmann Ursula , Bianchi Roberta , Fioretti Maria C. , Fallarino Francesca , Binaglia Lucia , Uyttenhove Catherine , van Pel Aline , Boon Thierry , Puccetti Paolo
Published: March 2007

Delayed‐type hypersensitivity (DTH) responses, mediated by CD8+ cells and detected by skin test assay, occur in sensitized mice in response to challenge with class I‐restricted antigenic peptides of mutagenized (tum−) P815 mastocytoma cells. In contrast, a nonapeptide related to a tumor rejection antigen, P815AB, failed in this study to elicit DTH after sensitization of mice with irradiated tumor cells or adoptive transfer of P815AB‐pulsed dendritic cells. Unresponsiveness, however, could be overcome by immunization with tumor cells co‐expressing P815AB and tum− antigens. When used for cell pulsing in vitro, a mixture of P815AB and tum− peptides was also highly effective in inducing anti‐P815AB reactivity, as was the combined use of P815AB and class II‐restricted peptides of tetanus toxin or Plasmodium berghei circumsporozoite protein. While the effector phase of the CD8+ cell‐mediated DTH to P815AB was unaffected by the ablation of CD4+ cells, the same treatment, or neutralization of IFN‐γ, negated the induction of reactivity if it occurred at the time of sensitization. Thus, defective activation of CD4+ cells may contribute to the poor immunogenicity of P815AB. Besides providing an insight into the mechanisms of anti‐tumor protection induced by tum− cells, these data offer useful information for the design of vaccination strategies against identified tumor antigens.
CD27/CD70 interaction directly drives B cell IgG and IgM synthesis
Authors: Agematsu Kazunaga , Kobata Tetsuji , Yang Feng‐Chun , Nakazawa Takayuki , Fukushima Keitaro , Kitahara Masashi , Mori Tetsuo , Sugita Kanji , Morimoto Chikao , Komiyama Atsushi
Published: March 2007

CD27 is a T cell activation antigen expressed on a majority of peripheral blood T cells. CD27 is also expressed on a subpopulation of human B cells, and it is reported that CD27+ B cells secrete both IgG and IgM. CD70, a ligand for CD27, is expressed on activated T and B cells, suggesting an interaction between T and B cells via CD27/CD70 ligation. Here, we analyze B cell immunoglobulin synthesis using a CD70 transfectant and present functional data showing that B cells secrete large amounts of IgG and IgM as a result of the CD27/CD70 interaction. A flow cytometric analysis showed that CD27 expression was increased and CD70 was expressed on tonsillar and peripheral blood B cells after activation with Staphylococcus aureus Cowan strain (SAC) plus interleukin (IL‐2). In addition, the proliferation of B cells was enhanced mildly by the addition of CD70 transfectant, and its proliferation was blocked by anti‐CD70 mAb. More importantly, the CD70 transfectant enhanced IgG and IgM production by purified B cells greatly in the presence of SAC plus IL‐2. The enhancement was completely blocked by the addition of either anti‐CD70 mAb or anti‐CD27 mAb. Strongly suggesting that the interaction of CD27 with its ligand, CD70, on B cells plays an important role in B cell growth and differentiation to produce IgG and IgM.
Low‐dose UVB radiation perturbs the functional expression of B7.1 and B7.2 co‐stimulatory molecules on human Langerhans cells
Authors: Weiss Johannes M. , Renkl Andreas C. , Denfeld Ralf W. , de Roche Roland , Spitzlei Michael , Schöpf Erwin , Simon Jan C.
Published: March 2007

In previous studies, we have shown that ultraviolet (UV) B radiation perturbs the APC function of Langerhans cells (LC) by interfering with as‐yet unidentified co‐stimulatory signals. Recently, B7.1 and B7.2 on APC were shown to deliver important co‐stimulatory signals through interaction with their counterreceptors CD28 and CTLA‐4 on T cells. To determine whether UVB affects the functional expression of B7.1 or B7.2 on LC, B7.1 and B7.2 expression was studied on human LC by multiparameter flow cytometry. Little, if any, B7.1 or B7.2 was detected on LC freshly isolated from skin. However, following 48 h of tissue culture, expression of both B7.1 and B7.2 were markedly up‐regulated. To test whether these molecules were functional, primary mixed epidermal cell leukocyte reactions (MECLR) were performed. Blocking monoclonal antibody (mAb) to B7.1 or B7.2 both inhibited the MECLR, with anti‐B7.2 being much more effective than anti‐B7.1. UVB radiation dose‐dependently (100–200 J/m2) suppressed the culture‐induced up‐regulation of B7.1 and B7.2 on LC. Since LC exposed to the same UVB flux (UVB‐LC) failed to stimulate alloreactive T cells in a MECLR, we questioned whether this was related to their inability to provide B7 co‐stimulation. Indeed, when effective B7‐CD28 signaling was ascertained by adding submitogenic doses of exogenous anti‐CD28 mAb to UVB‐LC, the proliferative response of alloreactive T cells was restored. We conclude that the suppressive effects of low‐dose UVB radiation on the APC function of LC are, at least in part, due to an inhibition of functional B7.1 and B7.2 expression.
Major histocompatibility complex class II‐associated peptides determine the binding of the superantigen toxic shock syndrome toxin‐1
Authors: von Bonin Arne , Ehrlich Svenja , Malcherek Georg , Fleischer Bernhard
Published: March 2007

Superantigens bind to major histocompatibility complex (MHC) class II proteins and interact with variable parts of the T cell antigen receptor (TCR) β‐chain. Cross‐linking the TCR with MHC class II molecules on the antigen‐presenting cell by the superantigen leads to T cell activation that plays an essential role in pathogenesis. Recent crystallographic data have resolved the structure of the complexes between HLA‐DR1 and staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin‐1 (TSST‐1), respectively. For TSST‐1, these studies have revealed possible contact sites between the superantigen and the HLA‐DR1‐bound peptide. Here, we show that TSST‐1 binding is dependent on the MHC‐II‐associated peptides by employing variants of T2 mutant cells deficient in loading of peptides to MHC class II molecules as superantigen‐presenting cells. On HLA‐DR3‐transfected T2 cells, presentation of TSST‐1, but not SEB, was dependent on HLA‐DR3‐associated peptides. Thus, although these superantigens can be recognized in the context of multiple MHC class II alleles and isotypes, they clearly bind to specific subsets of MHC molecules displaying appropriate peptides.
Differential susceptibility to monomeric HIV gp120‐mediated apoptosis in antigen‐activated CD4+ T cell populations
Authors: Tuosto Loretta , Montani Maria Saveria Gilardini , Lorenzetti Serena , Cundari Enrico , Moretti Simona , Lombardi Giovanna , Piccolella Enza
Published: March 2007

To support the hypothesis that indirect mechanisms mediated by viral products like the HIV envelope glycoprotein gp120 could be responsible for T lymphocyte depletion in HIV infection, we developed a system in which the impairment of T cell functions could be investigated in vitro. In particular, we characterized the conditions that allow T lymphocytes repeatedly stimulated with an antigen to be sensitive or resistant to gp120‐mediated apoptotic signals. To achieve this goal, a panel of antigen‐specific CD4+ T cell clones and primary CD4+ T lymphocytes were treated for 2 and 18 h with saturating amounts of monomeric gp120 (without cross‐linking with specific antibodies) and antigen‐driven T cell proliferation and apoptosis were analyzed. We show that monomeric gp120 induces apoptosis only in T lymphocytes repeatedly stimulated with the antigen, that primary T lymphocytes are resistant to programmed cell death mediated by monomeric gp120, but are sensitive to anti‐CD4 antibodies, and that gp120‐mediated apoptosis is dependent on the period of time between the binding of gp120 to CD4 and the encounter with antigen. To investigate the different susceptibility to gp120 induced apoptosis of primary CD4+ and T cell clones further, the number of membrane CD4 molecules and their affinity for gp120, together with Bcl‐2 and Fas expression, were studied. Our data suggest that a down‐modulation of membrane CD4 together with high expression of the Bcl‐2 gene and protein characterizes the susceptibility to apoptosis of gp120‐treated cells. In conclusion, our results define the phenotypic features of T cells susceptible to HIV gp120‐induced apoptosis and demonstrate that the same clonotype, depending on the activation state, may present a differential sensitivity to apoptosis induction.
Involvement of CD44 variant isoforms in hyaluronate adhesion by human activated T cells
Authors: Galluzzo Edi , Albi Nicola , Fiorucci Stefano , Merigiola Carla , Ruggeri Loredana , Tosti Antonella , Grossi Carlo E. , Velardi Andrea
Published: March 2007

The standard, 85–95‐kDa form of the hyaluronic acid (HA) receptor CD44 and a number of CD44 mRNA splice variants play important roles in immune responses and tumor metastasis. Variants carrying exon 6 (v6), or 9 (v9) products are transiently expressed on activated human T cells. Here, modulation experiments with specific monoclonal antibodies (mAb) indicate that v6 and v9 are expressed independently on distinct sets of CD44 molecules, and that their combined expression is necessary for HA adhesion. Moreover, the finding that mAb‐mediated cross‐linking of v6 and v9 promoted cytosolic free Ca2+ mobilization and co‐stimulated CD3‐triggered T cell proliferation indicates that v6 and v9 possess signaling and effector function activation ability. Finally, HA‐mediated signaling appears to be required for variant‐dependent adhesion to HA. The observation that soluble HA promoted cytosolic free Ca2+ mobilization indicates that HA‐induced Ca2+ mobilization can occur during T cell‐HA interaction. Since Ca2+ mobilization was inhibited by pretreatment of cells with an anti‐CD44 mAb directed against the HA‐binding domain of CD44, CD44 receptors appear to be involved in HA‐mediated signal transduction. The requirement of cytosolic free Ca2+ for adhesion is shown by the fact that ionomycin (a Ca2+ ionophore) stimulated, and EGTA (a Ca2+ chelator), inhibited HA adhesion. In addition, cytoskeletal functional activation is required for cell adhesion to HA, since drugs that block actin polymerization, such as cytochalasin B, or actomyosin contraction, such as the calmodulin antagonist W‐7, inhibited cell adhesion to HA. As this adhesion is also ADP ribosylation‐sensitive, it may involve a GTP‐dependent function of CD44v, i.e. ankyrin binding. Our data indicate that there is a functional hierarchy among the CD44 molecules expressed on human peripheral blood T cells and that the splice variants, as compared to the standard form, exhibit a greater HA binding ability which involves CD44‐mediated signaling and effector function activation.
Interleukin‐7 activates p56lck and p59fyn, two tyrosine kinases associated with the p90 interleukin‐7 receptor in primary human T cells
Authors: Page Theresa H. , Lali Ferdinand V. , Foxwell Brian M. J.
Published: March 2007

We have investigated signaling events associated with the cloned 90‐kDa (p90) interleukin‐7 receptor (IL‐7R) to determine whether changes in the signaling pathways initiated by this molecule can explain the ability of T cells to proliferate to IL‐7 following activation. Using in vitro kinase assays we find that the p90 IL‐7R in both unstimulated and activated human T cells is physically associated with two molecules with intrinsic kinase activity. Western blotting analysis reveals these proteins to be the src kinase enzymes, p59fyn and p56lck. Binding of human recombinant IL‐7 to the p90 IL‐7R results in increased activity of both receptor‐associated kinases in both resting and activated mature T cells. Thus, the signaling pathways initiated via the p90 IL‐7R‐associated src kinases are unlikely to be solely responsible for the proliferation of only activated T cells in response to IL‐7. Additional signals, which may derive from other IL‐7R‐associated molecules such as the γc, are clearly required for IL‐7‐driven proliferation of activated primary T cells.