bluuebunny/crossref_metadata_2025 huggingface dataset:
Penicilloyl peptides are recognized as T cell antigenic determinants in penicillin allergy
Authors: Padovan Elisabetta , Bauer Tanja , Tongio Marie Marte , Kalbacher Hubert , Weltzien Hans Ulrich
Published: March 2007

Although hapten immune responses have been intensively studied in the mouse, very little is known about hapten determinants involved in human allergic reactions. Penicillins, as chemically reactive compounds of low molecular weight, constitute typical examples of hapten allergens for humans. Penicillins become immunogenic only after covalent binding to carrier proteins and in this form frequently induce IgE‐mediated allergic reactions in patients subjected to antibiotic treatment. However, our previous data strongly indicated that penicillins also form part of the epitopes contacting the antigen receptors of beta lactam‐specific T cells in allergic individuals. We have therefore investigated the molecular constraints involved in the T cell immune response to penicillin G (Pen G). Designer peptides containing a DRB1*0401‐binding motif and covalently modified with Pen G via a lysine σ‐amino group were found to induce proliferation of Pen G‐specific T cell clones. A precise positioning of the hapten molecule on the peptide backbone was required for optimal T cell recognition. Furthermore, we extended these observations from our designer peptides to show that a peptide sequence derived from a natural DRB1*1101‐binding peptide modified in vitro with Pen G, also acquired antigenic properties. Our data for the first time provide insight into the manner in which allergenic haptens are recognized by human T cells involved in allergic reactions to drugs and suggest possible mechanisms leading to the onset of these adverse immune responses.
TAP‐dependent major histocompatibility complex class I presentation of soluble proteins using listeriolysin
Authors: Darji Ayub , Chakraborty Trinad , Wehland Jürgen , Weiss Siegfried
Published: March 2007

Immunization of mice with mixtures of listeriolysin, a pore‐forming hemolysin secreted by the pathogenic bacterium Listeria monocytogenes, together with soluble ovalbumin, nucleoprotein of influenza virus, or β‐galactosidase of Escherichia coli, resulted in strong cytotoxic CD8 T cell responses to each of the respective passenger proteins in vivo. Also, the concomitant addition of either protein with listeriolysin to target cells elicited efficient sensitization of these cells which could be attributed to the pore‐forming activity of listeriolysin. This response was dependent upon a functional TAP transporter and was inhibitable by brefeldin A, indicating the transfer of the soluble proteins into the cytosol and the classical major histocompatibility (MHC) class I presentation pathway. The treatment of target cells with listeriolysin under our experimental conditions did not affect cell viability and the pores generated by listeriolysin treatment were repaired within 60 min. Introduction of soluble proteins into the MHC class I presentation pathway by listeriolysin provides a powerful system to study the cytotoxic response towards intracellular pathogens and would allow for rapid screening of potential antigens in vaccine formulations.
L‐selectin ligands in rat high endothelium: multivalent sialyl Lewis x glycans are high‐affinity inhibitors of lymphocyte adhesion
Authors: Toppila Sanna , Lauronen Jouni , Mattila Pirkko , Turunen Juha Pekka , Penttilä Leena , Paavonen Timo , Renkonen Ossi , Renkonen Risto
Published: March 2007

Lymphocyte homing is initiated by their tethering to and rolling on the high endothelium and is followed by extravasation into the lymph nodes. We show here that glycosylated cell adhesion molecule‐1 (GlyCAM‐1), CD34, and sialyl Lewis x (sLex) are present on rat lymph node high endothelium analyzed by using monoclonal antibodies. α(1,3)fucosyltransferase VII (Fuc‐TVII), the last enzyme involved in the synthesis of the sLex sequence is also expressed on the rat lymph node high endothelium. We have synthesized a family of sLex‐decorated oligosaccharide structures and used them to inhibit lymphocyte binding to high endothelium in the Stamper‐Woodruff assay. Monovalent sLex, branched di‐ and tetravalent sLex, as well as a linear tetravalent sLex significantly reduce lymphocyte binding to endothelium. The branched and linear forms of tetravalent sLex were clearly superior inhibitors of the L‐selectin‐dependent lymphocyte adhesion, with IC50 values in low nanomolar range. In contrast, the fucose‐free analogs having the same charge and approximately the same size as the corresponding sLex glycans had no effect on lymphocyte binding and served as negative controls. Taken together, these data show the crucial importance of sLex in the endothelial ligands for L‐selectin. Furthermore, we suggest that L‐selectin acts as an oligomer on the lymphocyte surface as it binds multivalent sLex glycans.
Differential response of CD4+ V7+ and CD4+ V7− T cells to T cell receptor‐dependent signals: CD4+ V7+ T cells are co‐stimulation independent and anti‐V7 antibody blocks the induction of anergy by bacterial superantigen
Authors: Soares Luis R. B. , Rivas Alberto , Ruegg Curtis , Engleman Edgar G.
Published: March 2007

V7 is a novel cell surface glycoprotein that is expressed on 25% of circulating T lymphocytes. This molecule appears to play a critical role in T cell activation based on the observation that a monoclonal anti‐V7 antibody inhibits T cell receptor (TCR)‐dependent interleukin‐2 (IL‐2) production and proliferation of T cells. In the current study, CD4+ V7+ and CD4+ V7− T cells were separated from one another and their response to various stimuli analyzed. Although there were only minor differences between the two subsets in the expression of activation/differentiation markers, including CD45RA and R0 isotypes, when exposed to immobilized anti‐CD3 or anti‐TCR antibodies in the absence of APC, CD4+ V7+ T cells alone produced IL‐2 and proliferated vigorously. By contrast, CD4+ V7− cells responded poorly to such stimuli, but they recovered their capacity to respond if antigen‐presenting cells (APC) or anti‐CD28 antibody were added to the cultures. The enhancement of the V7− T cell response by APC appears to be related to augmentation of TCR signals because the effect could be blocked by antibodies against molecules on APC [major histocompatibility (MHC) class II, CD86] that are known to up‐regulate such signals through their interaction with counter‐receptors on T cells. To assess the role of V7 in a system independent of co‐stimulation, CD4+ T cells were stimulated with the bacterial superantigens, staphylococcal enterotoxins A and B. The cells responded by proliferating and then becoming anergic. Addition of anti‐V7 antibody at the initiation of culture with superantigen did not inhibit cellular proliferation but prevented T cells from becoming anergic, while addition of anti‐CD28 antibody had no effect on either proliferation or anergy induction. These results indicate that V7 and CD28 mediate distinct intracellular signals and suggest that V7 functions to preserve T cell reactivity whether the stimulus is mitogenic or anergizing.
Characterization of mouse ALCAM (CD166): the CD6‐binding domain is conserved in different homologs and mediates cross‐species binding
Authors: Bowen Michael A. , Bajorath Jürgen , D'egidio Maurizia , Whitney Gena S. , Palmer Dawn , Kobarg Jörg , Starling Gary C. , Siadak Anthony W. , Aruffo Alejandro
Published: March 2007

Activated leukocyte cell adhesion molecule (ALCAM; CD166) is a member of the immunoglobulin gene superfamily (IgSF) which is expressed by activated leukocytes and thymic epithelial cells and is a ligand for the lymphocyte antigen CD6. Herein, we report on the isolation and characterization of cDNA clones encoding mouse ALCAM (mALCAM). Comparison of the predicted amino acid sequence of mALCAM and human ALCAM (hALCAM) showed an overall identity of 93%. Binding studies with truncated forms of the extracellular region of mALCAM showed that the CD6 binding site is located in the N‐terminal Ig‐like domain and that mALCAM is capable of binding both human and mouse CD6. Mutagenesis studies on hALCAM suggested that residues critical for CD6 binding map to the predicted A‘GFCC’C″ β‐sheet of ALCAM's N‐terminal binding domain. Residue differences in the N‐terminal domains of mALCAM and hALCAM were analyzed with the aid of a molecular model of ALCAM. All residues critical for CD6 binding are conserved in both mALCAM and hALCAM, whereas residue differences map to the predicted BED face which is opposite the CD6 binding site on hALCAM. These findings provide a molecular rationale for the observed cross‐species CD6/ALCAM interaction and the apparent inability to generate monoclonal antibodies (mAb) against the CD6 binding site. RNA blot analysis showed that mRNA transcripts encoding mALCAM are expressed in the brain, lung, liver, and the kidney, as well as by activated leukocytes and a number of cell lines. A rat mAb specific for mALCAM was produced and by two‐color immunofluorescence studies was shown to bind to both activated CD4+ and CD8+ T cells.
Combined structural and immunological refinement of HIV‐1 HLA‐B8‐restricted cytotoxic T lymphocyte epitopes
Authors: Goulder Philip J. R. , Reid Scott W. , Price David A. , O'callaghan Chris A. , McMichael Andrew J. , Phillips Rodney E. , Yvonne Jones E.
Published: March 2007

This study demonstrates that use of structural information improves the definition and optimization of cytotoxic T lymphocyte (CTL) epitopes. Epitope optimization usually requires numerous truncated peptides or a reverse immunogenetic approach, where the peptide binding motif is used to predict epitopes. These binding motifs do not reliably predict all peptides which are CTL epitopes. Comparison of 24 peptides eluted from HLA‐B8 with 10 HLA‐B8‐restricted defined CTL epitopes demonstrated that known epitopes varied considerably at anchor positions. We used structural information based on determination of the crystal structure of the HLA‐B8‐GGKKKYKL complex to reassess previously described CTL epitopes, to predict new epitopes, and to predict the consequences of naturally occurring variation within epitopes. These predictions were confirmed by cytotoxicity and binding assays. Use of combined structural and immunological data more accurately defines the true peptide‐binding motif of a restriction element than eluted peptide data allows.
The C terminus of the human C5a receptor (CD88) is required for normal ligand‐dependent receptor internalization
Authors: Bock Daniel , Martin Ulrich , Gärtner Sören , Rheinheimer Claudia , Raffetseder Ute , Arseniev Lubomir , Barker Mike D. , Monk Peter N. , Bautsch Wilfried , Köhl Jörg , Klos Andreas
Published: March 2007

The biological effects of the potent inflammatory mediator C5a, a complement split product, on human neutrophils and monocytes are limited by the rapid internalization of its specific receptor (C5aR, CD88). The C terminus of the C5aR is phosphorylated after stimulation with C5a of phorbol ester, and this phosphorylation might lead to receptor internalization. In this context, we have studied the effects on C5aR internalization of C5a, phorbol 12‐myristate 13‐acetate (PMA), the protein kinase inhibitor staurosporine, and pertussis toxin on rat basophilic RBL.2H3 cells stably transfected with the human wild‐type or mutant C5aR. C5aR mutants lacked either part of the cytosolic C terminus, including suggested major phosphorylation sites, or a putative phosphorylation motif for protein kinase C in the third cytosolic loop. Additionally, agonist‐induced internalization was analyzed on HEK293 cells co‐transfected with C5aR and the pertussis toxin‐resistant G protein alpha subunit, Gα16. Staurosporine‐sensitive agonist‐dependent C5aR internalization could be detected, suggesting that C5aR phosphorylation, most likely of the C terminus, participates in this type of internalization. In contrast, PMA‐induced C5aR internalization seems to be independent of putative phosphorylation sites in either the truncated section of the C terminus or the third cytosolic loop. The phorbol ester‐induced C5aR internalization may, therefore, be caused by an indirect and less specific effect of protein kinase C on the internalization machinery. Manipulation of the pertussis toxin‐sensitive or ‐resistant G protein‐dependent signal transduction had no effect on ligand‐induced internalization.
Cyclosporin A interferes with the inducible degradation of NF‐xB inhibitors, but not with the processing of p105/NF‐xB1 in T cells
Authors: Marienfeld Ralf , Neumann Manfred , Chuvpilo Sergei , Escher Cornelia , Kneitz Burkhard , Avots Andris , Schimpl Anneliese , Serfling Edgar
Published: March 2007

The transcription factor NF‐xB controls the induction of numerous cytokine promoters during the activation of T lymphocytes. Inhibition of T cell activation by the immunosuppressants cyclosporin A (CsA) and FK506 exerts a suppressive effect on the induction of these NF‐xB‐controlled cytokine promoters. We show for human Jurkat T leukemia cells, as well as human and mouse primary T lymphocytes, that this inhibitory effect is accompanied by an impaired nuclear translocation of the Rel proteins c‐Rel, RelA/p65 and NF‐xB1/p50, whereas the nuclear appearance of RelB remains unaffected. CsA does not interfere with the synthesis of Rel proteins, but prevents the inducible degradation of cytosolic NF‐xB inhibitors IxBα and IxBβ upon T cell activation. CsA neither inhibits the processing of the NF‐xB1 precursor p105 to p50, nor does it “stabilize” the C‐terminal portion of p105, IxBγ, which is degraded during p105 processing to mature p50. These results indicate that CsA interferes with a specific event in the signal‐induced degradation of IxBα and IxBβ, but does not affect the processing of NF‐xB1/p105 to p50.
Germ‐line transcription and methylation status of the TCR‐Jα locus in its accessible configuration
Authors: Villey Isabelle , Quartier Pierre , Selz Françhise , de Villartay Jean‐Pierre
Published: March 2007

We have generated two in vivo mouse models to study the regulation of DNA accessibility to the V(D)J recombinase machinery in the T cell receptor (TCR)‐Jα locus. In recombination activating gene (RAG)‐deficient mice, both injection of a TCR‐β chain transgene (RTB mice) or anti‐CD3‐ε treatment in vivo (RT3 mice) lead to the same phenotype with homogeneous thymocyte populations blocked at the CD4+CD8+ double positive (DP) stage. At this developmental stage, the TCR‐α rearrangements are about to start, and the TCR‐Jα locus is frozen in an accessible but yet unrearranged configuration in these mice. We show high level of TCR‐α germ‐line transcription in thymocytes from RTB and RT3 mice. Transcripts are skewed towards the 5′ end of the TCR‐Jα locus, and the T early alpha (TEA) sterile transcript is predominant and therefore provides a useful marker for the TCR‐Jα locus opening. Analysis of the DNA methylation status reveals a global surmethylation of the TCR‐Jα locus in the thymus in comparison with non‐lymphoid cells in these mice. We propose that hypermethylation of the locus could precede a progressive demethylation, providing a specific protective and regulatory role in the rearrangement events.
B lineage‐restricted rearrangement of a human Igx transgene
Authors: Cavelier Patricia , Nato Faridabano , Coquilleau Isabella , Rolink Antonins , Rougeon François , Goodhardt Michele
Published: March 2007

To study the control of immunoglobulin x light chain gene rearrangement, we generated transgenic mice carrying a germ‐line human x minilocus (HK) containing the Jx‐proximal V gene, VxIV, the V‐J intergenic region, the five Jx segments and the Cx gene. This construct includes the intronic, but not the 3′ x enhancer. Rearrangement of the HK transgene was found to be lymphoid specific and restricted to the B cell lineage. Quantification of x gene rearrangement in pre‐B cell lines established from HK transgenic mice showed that, like endogenous x genes, rearrangement of the transgene is repressed in μ‐negative early B cell precursors. These results indicate that rearrangement of the HK transgene is subjected to the same B/T cell and developmental regulation as Vx‐Jx rearrangement at the endogenous locus. Comparison with an unrearranged x transgenic construct lacking the V‐J intergenic region, suggests that this region, or elements associated with the proximal V gene, may act to restrict x gene rearrangement to the B cell lineage.
Protein disulfide isomerase is the dominant acceptor for peptides translocated into the endoplasmic reticulum
Authors: Lammert Eckhard , Stevanović Stefan , Brunner Josef , Rammensee Hans‐Georg , Schild Hansjörg
Published: March 2007

Peptides derived from cytosolic protein degradation are translocated into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). In the ER, class I molecules bind the peptides fitting to their respective motifs and present them on the cell surface to CD8+ T lymphocytes. However, most TAP‐translocated peptides are not expected to bind to the class I molecules present in a particular cell. Recently, we have demonstrated that TAP‐translocated peptides containing a photoreactive phenylalanine analogue can be cross‐linked to two luminal ER‐resident proteins: with low efficiency to the stress protein gp96 and with high efficiency to a 60‐kDa protein (Lammert, E. et al., Eur. J. Immunol. 1997. 27: 923). Both proteins have also been labeled specifically by TAP‐translocated peptides conjugated to a different photoreactive group (Marusina, K. et al., Biochemistry 1997. 36: 856). Here, we show that the 60‐kDa peptide‐binding protein is identical to the multifunctional protein disulfide isomerase (PDI). Since PDI is the only luminal ER‐resident protein that is labeled by the photoreactive peptides with high efficiency, it might represent the dominant acceptor for TAP‐translocated peptides.
Patterns of lymphokine secretion amongst mouse γδ T cell clones
Authors: Duhindan Nadarajah , Farley Adrian J. , Humphreys Sian , Parker Claire , Rossiter Beth , Brooks Colin G.
Published: March 2007

Although the patterns of lymphokine (LK) secretion by CD4 and CD8 αβ T cells have been extensively studied, the question of whether γδ T cells display patterns of restricted LK production and whether these patterns are the same as seen in conventional αδ T cells has not been previously addressed. In this study we generated panels of γδ T cell clones from normal C57BL/6 and BALB/c mice using a lectin‐driven system and compared their patterns of secretion of nine LK with those of CD4 and CD8 αβ T cell clones generated in the same system. The results showed that γδ T cell clones displayed nonrandom patterns of highly restricted LK production with a strong bias towards the production of type 1 LK. The dominant pattern was one of high level secretion of interferon‐γ and tumor necrosis factor (TNF), with variable production of interleukin (IL)‐2, and little or none of the type 2 LK IL‐4, IL‐5, IL‐6, and IL‐10. This pattern differed significantly from that of CD4 Th1 clones in that γδ clones showed a striking deficiency in the production of IL‐3 and granulocyte/macrophage colony‐stimulating factor. A small subset of γδ clones displayed a novel pattern, in which the only LK produced in substantial quantity were TNF and variable amounts of IL‐2. The bias of γδ T cells towards type 1 LK production was not an artefact associated with cloning because bulk populations of splenic γδ T cells behaved in the same way, even when activated in the presence of high concentrations of IL‐4.
Tumor necrosis factor (TNF) receptor type 1 (p55) is a main mediator for TNF‐α‐induced skin inflammation
Authors: Kondo Seiji , Sauder Daniel N.
Published: March 2007

Tumor necrosis factor α (TNF‐α) is a pleiotropic proinflammatory cytokine that elicits a large number of biological effects, including inflammatory and immuno‐regulatory responses. Biological activities of TNF‐α are mediated by two distinct TNF receptors, p55 type 1 receptor (TNFR1) and p75 type 2 receptor (TNFR2). To determine the role of TNF‐α in the induction of inflammatory responses in the skin, gene‐targeted mutant mice lacking either TNFR1 or TNFR2 were painted with irritant chemicals. Both phenol and croton oil painting onto the ears induced less inflammation in TNFR1(−) mice than normal and TNFR2(−) mice. Intradermal injection of TNF‐α (0.2–200 ng for 3 days) into the ear induced less inflammation in TNFR1(−) mice than in normal mice. TNFR2(−) mice developed a normal inflammatory reaction to high doses of TNF‐α (20–200 ng for 3 days), while they showed minimal reactivity to low doses of TNF‐α (0.2–2 ng for 3 days). TNF‐α is known to trigger the release of a series of other cytokines and to induce the expression of cell adhesion molecules, thus contributing to the development of inflammation. The levels of protein and mRNA for interleukin (IL)‐6 were elevated in keratinocytes from normal as well as TNFR2(−) mice after treatment with TNF‐α, while keratinocytes from TNFR1(−) mice did not show any up‐regulation of IL‐6. TNF‐α induced intercellular adhesion molecule (ICAM)‐1 expression in the keratinocytes from normal and TNFR2(−) mice, but not in those from TNFR1(−) mice. These results indicate that TNFR1 is critical for induction of skin inflammation by TNF‐α.
Different cytokine profile and antigen‐specificity repertoire in Helicobacter pylori‐specific T cell clones from the antrum of chronic gastritis patients with or without peptic ulcer
Authors: D'Elios Mario M. , Manghetti Marta , Almerigogna Fabio , Amedei Amedeo , Costa Francesco , Burroni Daniela , Baldari Cosima T. , Romagnani Sergio , Telford John L. , Del Prete Gianfranco
Published: March 2007

Helicobacter pylori (Hp) infection almost invariably results in chronic antral gastritis, but only a proportion of patients develop peptic ulcer. Some Hp strains may be more ulcerogenic than others, but some ulcerogenic mechanisms may also depend on the type of the host immune response. In this study, the antigen specificity and the cytokine profile of 53 Hp‐specific CD4+ T cell clones derived from the antral mucosa of five patients with Hp‐induced uncomplicated chronic gastritis (CG) were assessed and compared with those of 34 Hp‐specific CD4+ T cell clones derived from six Hp‐infected patients with chronic gastritis and peptic ulcer (CG‐PU). The majority (28/34; 82%) of gastric Hp‐specific T cell clones from CG‐PU patients expressed the Th1 profile and 17 (all Th1) of the 34 clones were specific for cytotoxin‐associated protein (CagA). In contrast, 34 (64%) of the 53 Hp‐specific gastric T cell clones derived from CG patients were able to secrete both Th1 and Th2 cytokines (Th0 profile) and only 36% expressed a polarized Th1 profile. The majority (85%) of Hp‐specific clones from CG patients recognized Hp antigens other than CagA, since 13/53 (25%) were specific for urease, 6 (11%) for VacA, 6 (11%) for HSP and 20 (38%) for other undefined Hp antigens. Results provide evidence that the type of T helper cell response against Hp may vary according to the antigen involved and suggest that a polarized Th1 response may play a role in the genesis of peptic ulcer, whereas a local Th0 response, including interleukin‐4 production, may represent an individual host factor which contributes to lower the degree of gastric inflammation and prevent ulcer complication.
Agonist‐induced T cell receptor down‐regulation: molecular requirements and dissociation from T cell activation
Authors: Salio Mariolina , Valitutti Salvatore , Lanzavecchia Antonio
Published: March 2007

T cell receptor (TCR) down‐regulation is a consequence of specific receptor engagement and plays an important role in modulating the T cell response. We have investigated the role of protein kinase C (PKC) and protein tyrosine kinases (PTK) in the induction of TCR down‐regulation. We report that the mutation of S126 in the CD3‐γ chain that is known to inhibit phorbol‐12‐myristate 13‐acetate‐induced TCR down‐regulation does not affect down‐regulation induced by a specific agonist. In addition, agonist‐induced TCR down‐regulation is not affected by blockade or depletion of PKC, neither by blockade or lack of PTK, while the same treatments efficiently interfere with T cell activation. These results demonstrate that TCR down‐regulation is induced by early events which follow specific engagement by an agonist and can be dissociated from those required for full T cell activation.
Independent homeostatic regulation of B cell compartments
Authors: Agenès Fabien , Rosado Maria Manuela , Freitas António A.
Published: March 2007

In the present study we used mice with a developmental arrest of B cell production to study the ability of a limited number of normal B cell precursors to populate peripheral B cell pools. In chimeras reconstituted with mixtures of bone marrow (BM) cells from normal and B cell‐deficient donors, we show that the rate of BM B cell production is a constant function of the number of BM pre‐B cells and is not modified by the peripheral B cell pool size, i.e. there is no feedback regulation of the central pre‐B cell compartment by the number of mature B cells. We also show that the physiological number of peripheral B cells requires a minimum continuous input of newly formed cells, but is not determined by the number of B cell precursors. Chimeras with a threefold reduced rate of BM B cell production have normal numbers of peripheral B cells. Parabiosis between normal and B cell‐deficient mice showed that the BM B cell production of one mouse suffices to replenish the B cell pool of three mice. Finally, we show that the compartment of activated IgM‐secreting B cells is homeostatically autonomous since the number of cells it comprises is regulated independently of the size of the mature B cell pool. The results presented here support a model of the immune system in which the size of the different B cell compartments, i.e. pre‐B, resting B and IgM‐secreting, is autonomously regulated.
Induction of interleukin‐2 unresponsiveness and down‐regulation of the JAK‐STAT system upon activation through the T cell receptor
Authors: Miyatake Shoichiro , Sakuma Machie , Saito Takashi
Published: March 2007

Full activation of T cells with antigen (Ag) and antigen‐presenting cells initiates effector functions and proliferation. When T cells are re‐stimulated through the T cell receptor (TCR) after a primary stimulation with Ag, growth arrest and cell death are induced. Activation of a T cell clone by cross‐linking of TCR induces interleukin (IL)‐2 unresponsiveness and ultimately cell death. While the proliferative signal delivered by IL‐2 induces c‐myc, bcl‐2 and cyclin D3 expression, the expression of bcl‐2 and cyclin D3 is completely suppressed upon TCR stimulation. Furthermore, TCR stimulation induces a decrease in the protein levels of JAK3 and STAT5, suggesting that IL‐2 unresponsiveness and growth arrest of T cells result from down‐regulation of JAK3 and STAT5.
Discrimination between maintenance‐ and differentiation‐inducing signals during initial and intermediate stages of positive selection
Authors: Anderson Graham , Hare Katherine J. , Platt Nick , Jenkinson Eric J.
Published: March 2007

As well as signaling through the αβ T cell receptor complex, positive selection of immature CD4+ 8+ thymocytes involves additional ill‐defined accessory interactions provided by thymic epithelial cells. Here, we have isolated CD4+ 8+ thymocytes at a pre‐positive selection stage of development (TCR− CD69− 4+ 8+ cells), or after initiation of positive selection (CD69+ 4+ 8+ cells), from mice where the normal lifespan of thymocytes is extended by the presence of a bcl‐2 transgene, to allow us to discriminate between requirements for maintenance and differentiation signals during positive selection. We find that MHC class II+ thymic epithelial cells drive positive selection of TCR− CD69− 4+ 8+ bcl‐2 tg thymocytes to the CD4+ and CD8+ stage, while no such mature subsets are observed when thymocytes are cultured alone or with major histocompatibility complex (MHC) class II+ salivary epithelial cells. However, CD4+ 8+ cells remain in such cultures in considerable numbers, and retain the potential for positive selection if re‐cultured with thymic epithelium, suggesting that thymic epithelial cells provide specific differentiation‐inducing signals for positive selection. In contrast, intermediate CD69+ 4+ 8+ thymocytes show some capacity for phenotypic conversion in the absence of thymic stromal cells although strikingly the single‐positive CD4+ and CD8+ cells generated are not functionally competent. Finally, we show that prior culture of thymic epithelial cells under monolayer conditions abrogates their ability to support the initiation of positive selection, suggesting that the epithelial cell molecules necessary for the provision of differentiation signals during positive selection are down‐regulated under such conditions.
Human dendritic cell responses to lipopolysaccharide and CD40 ligation are differentially regulated by interleukin‐10
Authors: Buelens Christel , Verhasselt Valérie , De Groote Donat , Thielemans Kris , Goldman Michel , Willems Fabienne
Published: March 2007

We evaluated the effects of interleukin (IL)‐10 on the maturation of human dendritic cells (DC) induced either by lipopolysaccharide (LPS) or CD40 engagement. For this purpose, DC generated by culturing plastic‐adherent peripheral blood mononuclear cells for 7 days with granulocyte/macrophage‐colony‐stimulating factor and IL‐4 were incubated for 3 days with either LPS (10 ng/ml) or 3T6 fibroblasts transfected with the gene encoding CD40 ligand, in absence or presence of IL‐10. First we found that the membrane expression of CD83, a marker of mature DC, was inhibited by IL‐10 when induced by LPS but not by CD40 engagement. Likewise, IL‐10 inhibited LPS‐induced but not CD40‐dependent CD86 (B7.2) up‐regulation on DC. Furthermore, IL‐10 inhibited the production of IL‐8 and tumor necrosis factor‐α by DC when activated by LPS but not by CD40. In contrast, IL‐10 inhibited IL‐12 production in both activation systems. We conclude that IL‐10 differentially influences LPS‐dependent and CD40‐dependent pathways of DC maturation.
Avian Ikaros gene is expressed early in embryogenesis
Authors: Liippo Jussi , Lassila Olli
Published: March 2007

Ikaros transcription factors dictate the key steps in lymphoid lineage determination. To reveal the evolutionary aspects of regulatory molecules involved in early lymphocyte development, we studied the cDNA of the avian Ikaros homolog. We demonstrate here that the Ikaros gene is highly conserved across avian and mammalian species. Importantly, the Ikaros mRNA is already expressed in early chicken embryos, from embryonic day 2 onwards, thus markedly before the traffic of the first precursor cells to the lymphoid primordia, thymus and the bursa of Fabricius. We suggest that the embryonic expression of the Ikaros transcripts represents an early onset of lymphoid differentiation.
The selective engulfment of apoptotic bodies by dendritic cells is mediated by the αvβ3 integrin and requires intracellular and extracellular calcium
Authors: Rubartelli Anna , Poggi Alessandro , Zocchi M. Raffaella
Published: March 2007

Dendritic cells derived in vitro from monocytes are known to be poor phagocytes. Here we show that, unlike macrophages, monocyte‐derived dendritic cells indeed fail to take up opsonized particles or necrotic cells; however, apoptotic bodies are efficiently engulfed by dendritic cells. The temperature dependence and the sensitivity to cytochalasin D indicate that the apoptotic body engulfment is representative of early stages of phagocytosis. Inhibition studies with ligands for surface molecules involved in recognition of apoptotic bodies, such as vitronectin receptor, CD36 and phosphatidylserine receptor, revealed that apoptotic body engulfment by dendritic cells is mediated preferentially by the vitronectin receptor αvβ3, while all the receptors, with different efficiency, are engaged in phagocytosis of apoptotic bodies by macrophages. The interaction between apoptotic bodies and dendritic cells elicits a rise in intracellular free calcium concentration ([Ca2+]i) which is essential for the process of engulfment. Either intra‐ or extracellular Ca2+ buffering inhibits apoptotic body engulfment by dendritic cells and [Ca2+]i increases, indicating the involvement of both intra‐and extracellular Ca2+. In contrast, Ca2+ mobilization is dispensable for macrophage phagocytosis of apoptotic bodies. The different requirements of Ca2+ in macrophages and dendritic cells is possibly due to the differential usage of phagocytic receptors (CD36 vs. αvβ3) and might reflect different fates of apoptotic bodies in the two cell types.
Differential association of phosphatases with hematopoietic co‐receptors bearing immunoreceptor tyrosine‐based inhibition motifs
Authors: Vély Frédéric , Olivero Sylvain , Olcese Lucia , Moretta Alessandro , Damen Jacqueline E. , Liu Ling , Krystal Gerald , Cambier John C. , Daëron Marc , Vivier Eric
Published: March 2007

A novel family of inhibitory co‐receptors has been recently defined according to the presence in their intracytoplasmic domain of immunoreceptor tyrosine‐based inhibition motifs (ITIM). In particular, this family includes a low‐affinity receptor for IgG, FcγRIIB, which is widely expressed on hematopoietic cells, as well as killer cell inhibitory receptors (KIR) for major histocompatibility complex (MHC) class I proteins, expressed on both T and natural killer (NK) lymphocytes. FcγRIIB and KIR inhibitory function depends upon the tyrosine phosphorylation of their respective ITIM. Phosphorylated FcγRIIB and KIR ITIM bind the tandem SH2 tyrosine phosphatases, SHP‐1 and SHP‐2. Recently, FcγRIIB has been shown to associate with a polyphosphate inositol 5‐phosphatase, SHIP, which appears to be involved in its inhibitory function. Using cell lysate adsorption to phosphorylated ITIM peptides and surface plasmon resonance, we demonstrate here that, in contrast to FcγRIIB, KIR (CD158b: p58.2) do not bind to SHIP, and only recruit SHP‐1 and SHP‐2. In addition, we show that point mutation of the amino acid residue in position tyrosine‐2 of FcγRIIB and KIR ITIM abolihes their binding to SHP‐1 and SHP‐2, but leaves intact the association of SHIP with FcγRIIB ITIM. These data contribute to the structural definition of ITIM and document a differential recruitment of phosphatases by distinct ITIM. These findings also reveal that diverse strategies of inhibition are used by distinct members of the ITIM‐bearing co‐receptor family.
Recent thymic emigrants are distinct from most medullary thymocytes
Authors: Gabor Melinda J. , Godfrey Dale I. , Scollay Roland
Published: March 2007

In the mouse thymus, newly formed single positive (SP) cells spend an average of 14 days in the thymic medulla. During this time, phenotypic and functional maturation occurs with down‐regulation of CD69 and heat stable antigen (HSA), and up‐regulation of Qa‐2. Very little is known about the final steps that allow or direct these T cells to emigrate and join the recirculating peripheral T cell pool. Currently available data suggest that not all recent thymic emigrants (RTE) complete this maturational sequence in the medulla and that emigration may occur at any time during the medullary maturation stage. In this study, we have compared adhesion and activation marker expression on SP thymocytes, RTE and peripheral T cells to determine more precisely which SP medullary thymocytes are exported. Although RTE were heterogeneous for HSA and Qa‐2 expression, they were quite uniform with regard to the expression of other molecules. In contrast to medullary SP thymocytes, most RTE were L‐selectinhigh and CD69−. In addition, CD4+ CD8− and CD4− CD8+ RTE were phenotypically distinct from each other in that the former were β7 integrin−/low, CD45RBintermediate and CD45RC−, while the latter were β7 integrinhigh, CD45RBhigh and CD45RClow. These phenotypes were comparable to only a minor (as little as 6 %) subpopulation of medullary SP thymocytes. Overall, the data indicate that export of cells from the medullary pool of SP thymocytes is not random, but that a series of maturational events within the SP stage are necessary before export can occur.
Stress protein (hsp73)‐mediated, TAP‐independent processing of endogenous, truncated SV 40 large T antigen for Db‐restricted peptide presentation
Authors: Schirmbeck Reinhold , Böhm Waltraud , Reimann Jörg
Published: March 2007

Transporter associated with antigen processing (TAP)‐competent and TAP‐deficient cell lines were transfected with expression plasmids encoding either the wild‐type (wt) large tumor antigen (T‐Ag) of SV40, or a truncated cytoplasmic variant (cT‐Ag) of this viral protein. Stable expression of comparable levels of both forms of the viral protein was observed in different transfectants. The truncated cT‐Ag variant, but not the wtT‐Ag was stably associated with the con‐stitutively expressed, cytosolic heat shock protein (hsp)73 chaperone. Two Db‐binding peptides and one Kb‐binding peptide of T‐Ag were presented to cytotoxic T lymphocyte lines (CTLL) by TAP‐competent transfectants expressing either wtT‐Ag or cT‐Ag. TAP‐deficient transfectants expressing the wtT‐Ag did not present any of these epitopes to CTLL. In contrast, TAP‐deficient transfectants expressing the truncated hsp73‐associated cT‐Ag, presented the two Db‐binding epitopes, but not the Kb‐binding T‐Ag epitope to CTLL. Regurgitation of peptides by transfectants was not detectable. The described data indicate that a pool of post‐Golgi Db molecules is available for 2–3 h in TAP‐deficient transfectants for loading with peptides released during endolysosomal processing of hsp73‐associated, endogenous antigen.
Peptides derived from the whole sequence of BCR‐ABL bind to several class I molecules allowing specific induction of human cytotoxic T lymphocytes
Authors: Buzyn Agnès , Ostankovitch Marina , Zerbib Anne , Kemula Mathilde , Connan Francine , Varet Bruno , Guillet Jean‐Gérard , Choppin Jeannine
Published: March 2007

Chronic myeloid leukemia (CML) is characterized cytogenetically by a t(9;22) translocation which generates a hybrid bcr‐abl gene, encoding a p210bcr‐abl fusion protein. The induction in vitro of leukemia‐specific T cells reactive with p210bcr‐abl is a strategy developed for an immunological therapeutic approach in CML. Peptides from the junction region of this chimeric protein have been considered as potential targets for a cytotoxic response against leukemic cells. However, only a few peptides encompassing the two p210bcr‐abl breakpoints have been shown to bind to the most common HLA class I molecules, which limits the number of patients who could benefit from this approach. We assume that the presence of chimeric BCR‐ABL protein in leukemic cells may affect processing and delivery of peptides, possibly giving rise to new epitopes at the cell surface. We selected 162 peptides from the whole sequence of this protein, including 14 peptides of the b2a2 and b3a2 junctions, which had an anchor motif for a common HLA class I molecule. We tested their ability to bind to eight HLA class I molecules (HLA‐A1, ‐A2, ‐A3, ‐A11, ‐B7, ‐B8, ‐B27, ‐B44). We identified 48 peptides from outside the junction region, with intermediate or strong binding capacities to these HLA class I molecules contrasting with only six junction peptides with a moderate binding capacity to HLA‐A3/A11, ‐B8, or ‐B44 molecules. Moreover, cytotoxic T lymphocyte lines specific for various peptides outside the junction were generated from peripheral blood mononuclear cells of HLA‐A2 or ‐B7 healthy donors and from one CML patient. These results contribute to evaluation of immunity to the BCR‐ABL chimeric protein. Further studies are required to investigate whether such epitopes are correctly processed and presented by leukemic cells.
Molecular characterization of a gene in the rat homologous to human CD94
Authors: Dissen Erik , Berg Siri F. , Westgaard Ingunn H. , Fossum Sigbjørn
Published: March 2007

Three classes of major histocompatibility (MHC) class I binding receptors on natural killer (NK) cells have so far been described: CD94/NKG2 heterodimeric receptors and killer cell inhibitory receptors in the human, and Ly‐49 homodimers in rodents. CD94, NKG2 and Ly‐49 belong to the C‐type lectin super‐family. As yet, CD94 and NKG2 molecules have not been detected in rodents or Ly‐49 in humans. It has therefore been proposed that the two receptors represent functional equivalents in these species. The present study describes the cDNA cloning of a novel rat gene encoding a protein of 179 amino acids, 54.2 % identical to human CD94. The single‐copy Cd94 gene is localized to the rat NK gene complex (NKC), within 50 kb from Nkrp2, between the Nkrp1 and Ly49 gene clusters. By Northern blot analysis, we showed that rat CD94 is selectively expressed by NK cells and a small subset of T cells, similar to the human ortho‐logue. This expression is strain dependent, with high expression in DA NK cells and low in PVG NK cells. Evidence is presented that this difference is not due to receptor repertoire shaping by MHC‐encoded ligands, but is controlled by genetic elements residing within the NKC. The identification of a rat CD94 orthologue suggests that NK cell populations utilize two different C‐type lectin receptors for MHC class I molecules in parallel.
Two separable T cell receptor signals reconstitute positive selection of CD4 lineage T cells in vivo
Authors: Schmitt Steffen , Müller Klaus‐Peter , Kyewski Bruno A.
Published: March 2007

Positive selection is an obligatory step during intrathymic T cell differentiation. It is associated with rescue of short‐lived, self major histocompatibility complex (MHC)‐restricted thymocytes from programmed cell death, CD4/CD8 T cell lineage commitment, and induction of lineage‐specific differentiation programs. T cell receptor (TCR) signaling during positive selection can be closely mimicked by targeting TCR on immature thymocytes to cortical epithelial cells in situ via hybrid antibodies. We show that selection of CD4 T cell lineage cells in mice deficient for MHC class I and MHC class II expression can be reconstituted in vivo by two separable T cell receptor signaling steps, whereas a single TCR signal leads only to induction of short‐lived CD4+CD8la intermediates. These intermediates remain susceptible to a second TCR signal for 12‐48 h providing an estimate for the duration of positive selection in situ. While both TCR signals induce differentiation steps, only the second one confers long‐term survival on immature thymocytes. In further support of the two‐step model of positive selection we provide evidence that CD4 T cell lineage cells rescued by a single hybrid antibody pulse in MHC class II‐deficient mice are pre‐selected by MHC class 1.
Biologically active, alternatively processed interleukin‐1β in psoriatic scales
Authors: Lundqvist Elisabet Nylander , Egelrud Torbjörn
Published: March 2007

The aims of the present work were to elucidate the biochemical properties of interleukin‐1β (IL‐1β) in psoriatic scales to get information on the processing of epidermal IL‐1β in psoriasis, and to elucidate whether the IL‐1β in psoriatic scales possesses biological activity. By means of ion exchange chromatography, IL‐1β in extracts of psoriatic scales was purified to a stage where it could be analyzed with electrophoretic methods and immunoblotting. Compared to mature recombinant human IL‐1β (Ala 117 IL‐1β), IL‐1β in psoriatic scales had a slightly higher apparent molecular mass and a more acidic isoelectric point, as revealed by two‐dimensional electrophoresis under denaturing conditions. Isoelectric focusing under non‐denaturing conditions of IL‐1β partially purified from psoriatic scales, or from non‐inflamed plantar stratum corneum (Nylander Lundqvist, E., Bäck, O. and Egelrud, T., J. Immunol. 1996. 157: 1699), and of mature IL‐1β, followed by immunoblotting with IL‐1β‐specific antibodies, showed that psoriatic scales contained two components with IL‐1β‐like immuno‐reactivity which were isoelectric at pH 6.1 and 6.3, respectively. Theses components could also be detected in extracts of plantar stratum corneum, which also contained small amounts of an IL‐1β‐like component isoelectric at pH 6.9. Mature IL‐1β was isoelectric at pH 6.9. No IL‐1β‐like biological activity could be detected in crude extracts of psoriatic scales. These extracts also contained high amounts of IL‐1 receptor antagonist. Partially purified preparations of IL‐1β from psoriatic scales, in which an apparently total separation of IL‐1β and IL‐1 receptor antagonist had been achieved, could induce expression of E‐selectin in human umbilical vein endothelial cells. This activity was inhibited by antibodies specific for IL‐1β, but not by antibodies specific for IL‐1α. It is concluded that psoriatic scales contain biologically active IL‐1β, which has been processed by a mechanism which may be similar to that present in non‐inflamed plantar stratum corneum, and which does not involve IL‐1β converting enzyme.
Interleukin‐2 receptor β and γ chain dysregulation during the inhibition of CD4 T cell activation by human immunodeficiency virus‐1 gp120
Authors: Bani Lynda , David Denis , Février Michèle , Pialoux Gilles , Dupont Bertrand , Sugamura Kazuo , Thèze Jacques
Published: March 2007

We have observed that CD4 T lymphocytes from human immunodeficiency virus (HIV)‐infected patients marginally express interleukin‐2 receptor (IL‐2R)β and IL‐2Rγ chains which are essential for IL‐2 signal transduction. To analyze this observation further, we studied the influence of gp120 on the cell surface expression of IL‐2Rβ and IL‐2Rγ by purified CD4 lymphocytes in vitro. Cross‐linking of the T cell receptors of these lymphocytes initiates entry into the cell cycle as measured by CD69 and CD71 cell surface expression and [3H]thymidine incorporation. It also induces the cell surface expression of IL‐2Rβ and IL‐2Rγ. We have shown that treatment of the CD4 T lymphocytes with HIV‐1 gp120 before anti‐CD3 stimulation impedes cell cycle progression as measured by reduced CD71 expression and inhibition of [3H]thymidine incorporation. Furthermore, cell surface expression of IL‐2Rβ and IL‐2Rγ subunits, which form the functional intermediate‐affinity IL‐2R, are significantly inhibited. More importantly, addition of exogenous IL‐2 does not restore the proliferation of the CD4 T cells treated with gp120, suggesting that cells are anergic and/or that the remaining IL‐2R are not functional. This is the first study of IL‐2Rβ and IL‐2Rγ dysregulation in the context of HIV infection and shows that CD4 is also involved in IL‐2R expression.
Enhanced sensitivity of P‐glycoprotein‐positive multidrug resistant tumor cells to complement‐mediated lysis
Authors: Bomstein Yonit , Fishelson Zvi
Published: March 2007

The interaction of KB‐V1, a multidrug resistant (MDR) variant of the KB‐3–1 human oral carcinoma, with human complement was investigated. KB‐V1 cells were found to be more sensitive than KB‐3–1 cells to complement‐mediated lysis. Detailed analysis of the capacity of KB cells to activate human complement demonstrated that both C3b deposition and formation of the membrane attack complex (MAC) are higher on KB‐V1 than on KB‐3–1 cells. Furthermore, the MAC formed on KB‐V1 cells, but not on KB‐3–1 cells, was found to be resistant to trypsin treatment, i.e. more stably inserted into the plasma membrane. Immunofluorescence analysis by flow cytometry showed that KB‐V1 cells express less decay‐accelerating factor (DAF, CD55) than KB‐3–1 cells. Two other complement regulatory proteins, membrane cofactor protein (MCP, CD46) and CD59 are expressed to a similar extent on both KB‐V1 and KB‐3–1 cells. Treatment of KB‐V1 cells with neutralizing anti‐P‐glycoprotein (P‐gp) monoclonal antibodies reduced their sensitivity to complement. In addition, KB‐V1 revertants which cease to express P‐gp become more resistant to complement. These results indicate that multiple factors, such as reduced expression of DAF, enhanced deposition of C3b and increased binding and stability of the MAC may contribute to the increased complement sensitivity of KB‐V1 cells. It is suggested that P‐gp is responsible for the complement‐sensitive phenotype of KB‐V1 cells.