bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin‐4 inhibits χ light chain expression and NFχB activation but not IχBα degradation in 70Z/3 murine pre‐B cells
Authors: Clarke Christopher J. P. , Taylor‐Fishwick David A. , Hales Anne , Chernajovsky Yuti , Sugamura Kazuo , Feldmann Marc , Foxwell Brian M. J.
Published: March 2007

The murine pre‐B cell line 70Z/3 responds to lipopolysaccharide by upregulating the surface expression of kappa (χ) light chain through activation of the transcription factor NFχB. Interleukin‐4 (IL‐4), a T cell cytokine, is a known inhibitor of some LPS‐mediated events. We investigated whether IL‐4 could inhibit the up‐regulation of χ light chain and activation of NFχB by LPS in 70Z/3. IL‐4 partially inhibited both the LPS‐induced expression of χ light chain and also the activation of NFχB as judged by an NFχB reporter gene assay. Additionally, electrophoretic mobility shift assays confirmed this effect on LPS‐induced NFχB DNA binding activity in the nucleus. Surprisingly, proteolytic degradation of IχBα (MAD3), a prerequisite for NFχB activation, was unaffected by IL‐4, implying that this cytokine inhibits some subsequent undefined event in the activation of NFχB. IL‐4 was also found partially to inhibit NFχB activity induced by tumor necrosis factor‐alpha (TNF‐α) and interleukin‐1‐beta (IL‐1β). These results indicate that there may be a common mechanism for the well‐documented anti‐inflammatory effects of IL‐4 and that this mechanism involves the transcription factor NFχB.
CD40 ligand‐positive CD8+ T cell clones allow B cell growth and differentiation
Authors: Hermann Patrice , Van‐Kooten Cees , Gaillard Claude , Banchereau Jacques , Blanchard Dominique
Published: March 2007

A fraction of activated CD8+ T cells expresses CD40 ligand (CD40L), a molecule that plays a key role in T cell‐dependent B cell stimulation. CD8+ T cell clones were examined for CD40L expression and for their capacity to allow the growth and differentiation of B cells, upon activation with immobilized anti‐CD3. According to CD40L expression, CD8+ clones could be grouped into three subsets. CD8+ T cell clones expressing high levels of CD40L (≥80% CD40L+ cells) were equivalent to CD4+ T cell clones with regard to induction of tonsil B cell proliferation and immunoglobulin (Ig) production, provided the combination of interleukin (IL)‐2 and IL‐10 was added to cultures. CD8+ T cell clones, with intermediate levels of CD40L expression (10 to 30% CD40L+ cells), also stimulated B cell proliferation and Ig secretion with IL‐2 and IL‐10. B cell responses induced by these CD8+ T cell clones were neutralized by blocking monoclonal antibodies specific for either CD40L or CD40. By contrast, CD40L−− T cell clones (⩽5 % CD40L+ cells), only induced marginal B cell responses even with IL‐2 and IL‐10. All three clone types were able to activate B cells as shown by up‐regulation of CD25, CD80 and CD86 expression. A neutralizing anti‐CD40L antibody indicated that T cell‐dependent B cell activation was only partly dependent on CD40‐CD40L interaction. These CD40L−− clones had no inhibitory effects on B cell proliferation induced by CD40L‐expressing CD8+ T cell clones. Taken together, these results indicate that CD8+ T cells can induce B cell growth and differentiation in a CD40L‐CD40‐dependent fashion.
Differences in the interleukin‐2 (IL‐2) receptor system in human and mouse: α chain is required for formation of the functional mouse IL‐2 receptor
Authors: Nemoto Tadayoshi , Takeshita Toshikazu , Ishii Naoto , Kondo Motonari , Higuchi Masaya , Satomi Susumu , Nakamura Masataka , Mori Shozo , Sugamura Kazuo
Published: March 2007

Reconstitution with mouse interleukin‐2 (IL‐2) receptor subunits demonstrated that the mouse IL‐2 receptor complex was different from the human complex in the α chain requirement for the functional mouse receptor complex. The heterotrimeric complex of the mouse exogenous α and β chains and the endogenous γ chain on mouse lymphoid BW5147 cells showed the ability to bind IL‐2 with high affinity, resulting in IL‐2‐induced tyrosine phosphorylation of a cytosolic tyrosine kinase, JAK3, which is involved in IL‐2‐dependent signals. Exogenous introduction of the β chain with the endogenous γ chain, however, could neither confer appreciable IL‐2 binding nor IL‐2‐induced signal transduction on BW5147 cells, unlike the human βγ heterodimer. Mouse spleen CD8+ cells, not having the α chain initially, showed IL‐2‐dependent cell proliferation only when expression of the α chain was induced. Collectively, these results illustrate that the functional mouse IL‐2 receptor complex necessarily includes the α chain, and that the regulation of CD8+ T cell growth during immune reaction depends upon α chain expression.
Major histocompatibility complex class I‐restricted CD8+ T cells and class II‐restricted CD4+ T cells, respectively, mediate and regulate contact sensitivity to dinitrofluorobenzene
Authors: Bour Hélène , Peyron Eric , Gaucherand Martine , Garrigue Jean‐Luc , Desvignes Cyril , Kaiserlian Dominique , Revillard Jean‐Pierre , Nicolas Jean‐François
Published: March 2007

Contact sensitivity (CS) is a form of delayed‐type hypersensitivity to haptens applied epicutaneously and is thought to be mediated, like classical delayed‐type hypersensitivity responses, by CD4+ T helper‐1 cells. The aim of this study was to identify the effector T cells involved in CS. We studied CS to the strongly sensitizing hapten dinitrofluorobenzene (DNFB) in mice rendered deficient by homologous recombination in either major histocompatibility complex (MHC) class I, MHC class II, or both, and which exhibited deficiencies in, respectively, CD8+, CD4+, or both, T cells. MHC class I single‐deficient and MHC class I/class II double‐deficient mice, both of which have a drastic reduction in the number of CD8+ T cells, were unable to mount a CS response to DNFB. In contrast, both MHC class II‐deficient mice and normal mice treated with an anti‐CD4 monoclonal antibody (mAb) developed exaggerated and persistent responses relative to heterozygous control littermates. Furthermore, anti‐CD8 mAb depletion of class II‐deficient mice totally abolished their ability to mount an inflammatory response to DNFB. Removal of residual CD4+ T cells in class II‐deficient mice by anti‐CD4 mAb treatment did not diminish the intensity of CS. These data clearly demonstrate that class I‐restricted CD8+ T cells are sufficient for the induction of CS to DNFB, and further support the idea that MHC class II‐restricted CD4+ T cells down‐regulate this inflammatory response.
Localization in situ of the co‐stimulatory molecules B7.1, B7.2, CD40 and their ligands in normal human lymphoid tissue
Authors: Vyth‐Dreese Florry A. , Dellemijn Trees A. M. , Majoor Donné , de Jong Daphne
Published: March 2007

Functional interactions between B and T lymphocytes are known to depend on the expression of co‐stimulatory molecules B7.1/CD80, B7.2/CD86 and their counter‐receptors CD28 and CTLA4, as well as CD40 and its ligand CD40L. To study the role of these molecules in situ, an immunohistochemical analysis was carried out on normal human lymphoid tissue. In the germinal centers (GC), B7.1 and B7.2 were differentially expressed. In the dark zone, centroblasts were predominantly B7.1+, while centrocytes in the light zone were B7‐2+, resulting in reversed gradients of both markers in GC. Follicle mantle cells were negative for B7.1 and B7.2. Macrophages and interdigitating dendritic cells (IDC) in T cell zones both expressed B7.1 and B7.2. Moreover, clusters of B7.2+ T cells were demonstrated in interfollicular areas. Intrafollicular CD4+ T cells in GC, predominantly in the apical light zone, expressed CD28 and CTLA4, as did the majority of interfollicular T cells. CTLA4 showed a striking excentric cytoplasmic staining, which was also seen on T cells activated in vitro. CD40 was expressed on all B cells and more strongly on macrophages and IDC. Moreover, small clusters of T cells in a rim outside the GC showed CD40 expression. CD40L was expressed both on intrafollicular CD4+ T cells as well as on T cells in T cell zones. The differential distribution of co‐stimulatory molecules in different compartments of normal human lymphoid tissue in situ indicates that these interactions play a distinctive role in different stages of B cell differentiation and in the immune response.
Interleukin‐7 can induce the activation of Jak 1, Jak 3 and STAT 5 proteins in murine T cells
Authors: Foxwell Brian M. J. , Beadling Carol , Guschin Dimitri , Kerr Ian , Cantrell Doreen
Published: March 2007

The activation of Janus protein tyrosine kinases (Jak) and STAT (signal transducer and activator of transcription) proteins has recently been linked to the signal transduction mechanism of several cytokines. IL‐7 was observed to induce a rapid and dose‐dependent tyrosine phosphorylation of Jak 1 and Jak 3 and concomitantly, the tyrosine phosphorylation and DNA binding activity of multiple STAT proteins. The STAT proteins utilized by IL‐7 were identical to those induced by IL‐2 and could be identified as various STAT 5 isoforms. Moreover, the induction of both Jak 1 and 3, and STAT 5 activity strongly correlated with the growth‐promoting effects of IL‐7, suggesting that this signal transduction mechanism may play a key role in IL‐7‐induced proliferation.
Administration of interleukin ‐5 or ‐10 activates peritoneal B‐1 cells and induces autoimmune hemolytic anemia in anti‐erythrocyte autoantibody‐transgenic mice
Authors: Nisitani Sazuku , Tsubata Takeshi , Murakami Masao , Honjo Tasuku
Published: March 2007

Activation mechanisms of B‐1 (Ly‐1 B) cells have been suggested to be different from those of conventional B cells. To assess the role of various interleukins (IL) in the activation of B‐1 cells, we injected IL‐4, IL‐5 or IL‐10 into nonanemic anti‐red blood cells (RBC) autoantibody‐transgenic mice, in which conventional B cells are clonally deleted but peritoneal B‐1 cells persist without secreting Ig. Intraperitoneal or intramuscular injection of IL‐5 or IL‐10, but not IL‐4, increased the number of antibody‐producing peritoneal B‐1 cells by four‐ to five‐fold, resulting in increased anti‐RBC serum autoantibody and induction of hemolytic anemia. These results suggest that IL‐5 or IL‐10 may play an important role in the terminal differentiation of B‐1 cells into antibody‐producing cells in vivo.
Naive CD4+ T cells confer idiotype‐specific tumor resistance in the absence of antibodies
Authors: Bogen Bjarne , Munthe Ludvig , Sollien Anders , Hofgaard Peter , Omholt Hilde , Dagnaes Frederik , Dembic Zlatko , Lauritzsen Grete F.
Published: March 2007

CD4+ T cells can recognize a processed idiotypic peptide derived from the mouse λ2315 immunoglobulin light chain. The idiotypic peptide is presented on the I‐Ed class II major histocompatibility complex molecule. Mice made transgenic for a λ2315‐specific αβ T cell receptor have been demonstrated to be specifically resistant against a tumor challenge with the MOPC315 (α, λ2315) plasmacytoma (Lauritzsen, G. F., Weiss, S., Dembic, Z. and Bogen, B., Proc. Natl. Acad. Sci. USA 1994. 91: 5700). That study, however, did not rule out a role of either anti‐Id antibodies or T cells expressing nontransgenic specificities due to expression of endogenous T cell receptor (TcR) α chains. Also, the role of different T cell subsets in protection was unclear. To remove these ambiguities, we have now made the transgenic mice homozygous for the scid mutation, known to inhibit both Ig and TcR gene rearrangements. Such transgenic SCID mice lack B cells and antibodies while they still have plenty of CD4+ and CD4−8− cells expressing the transgenic αβ T cell receptor. The number of CD8+ T cell is dramatically reduced. Even so, transgenic SCID mice are protected against a challenge with MOPC315 plasmacytoma cells. Therefore, B cells, as well as novel T cell receptor specificities created by rearrangements of endogenous α‐chain genes, are both dispensable for effective immunosurveillance in our system. Surprisingly, we found that transgenic CD8+ and CD4−8− cells are idiotypespecific and I‐Ed restricted. However, these T cell subsets are not required for resistance because adoptive transfer experiments demonstrated that highly purified transgenic SCID CD4+ cells suffice for tumor protection.
CD28:B7 interactions promote T cell adhesion
Authors: Turcovski‐Corrales Susan M. , Fenton Robert G. , Peltz Gary , Taub Dennis D.
Published: March 2007

CD28 activation by antibody‐mediated ligation has been shown to provide an important co‐stimulatory signal for T cell adhesion to purified protein ligands. However, the effect of CD28 ligation by one of its natural ligands, B7.1, on T cell adhesion to other cells has not been studied. Therefore, in the present manuscript, we characterized the adhesive interactions between human T cells and B7.1‐transfected major histocompatibility complex class II+ and class II− melanoma cells. In our studies, human T cells and T cell clones adhered to B7.1‐transfected melanoma cells, but not to untransfected parental cells. The adhesive reaction in this model was rapid, occurring within 15 min, and was inhibited by anti‐B7.1 antibody and soluble CTLA‐4 immunoglobulin. Antibody inhibition studies demonstrated that adhesion between T cells and B7.1‐transfected melanoma cells was mediated by interactions between LFA‐1:ICAM‐1 and CD2:LFA‐3. Inhibition by pharmacological agents demonstrated that the CD28‐induced adhesion required specific intracellular signaling events. A protein kinase C inhibitor, staurosporin, significantly inhibited T cell binding to transfected melanoma cells, while cyclosporin A and wortmannin, an inhibitor of phosphatidylinositol‐3‐kinase, did not. These results suggest that the presence of B7 on various cell populations may activate lymphocytes to adhere better, thus promoting activation, cytolysis, and migration.
Pertussis toxin‐sensitive GTP‐binding proteins regulate activation‐induced apoptotic cell death of human natural killer cells
Authors: Carracedo Julia , Ramírez Rafael , Marchetti Philippe , Pintado Oscar C. , Baixeras Elena , Martínez Carlos , Kroemer Guido
Published: March 2007

Apoptosis of natural killer (NK) cells can be induced by non‐specific physical damage (UV irradiation, heat shock) or by simultaneous ligation of the CD 16 and the interleukin‐2 receptor (IL‐2R) molecules, but not with either anti‐CD 16 or IL‐2 alone. Whereas blockade of GTP‐binding protein (G protein)‐mediated signal transduction using ADP‐ribosylating bacterial toxins or the GTPase‐resistant GTP analog guanosine 5′‐0‐(3‐thiotriphosphate (GTPγS) does not affect non‐specific induction of NK cell apoptosis, such interventions do inhibit induction of apoptosis by anti‐CD16/IL‐2. The G proteins involved in the regulation of activation‐induced NK apoptosis are sensitive to pertussis toxin (PTX) and to the non‐specific GTP analog GTPγS but not to cholera toxin, Pseudomonas exotoxin A or diphtheria toxin. A pertussis toxin mutant that lacks ADP‐ribosylating activity, but conserves the membrane translocating and T cell‐mitogenic effects of the native molecule, fails to inhibit NK apoptosis. To exert their apoptosis‐inhibitory effect, PTX and GTPγS must be employed before cells are activated. Later addition has no effect, suggesting the implication of G proteins in the transmission of apoptosis‐inducing signals, but not in the effector stage of apoptosis. Pre‐incubation with PTX or GTPγS does not affect the activation of NK cells by CD 16 cross‐linking, IL‐2 stimulation ‐ or both, as assessed by the induction of CD69 expression, protein tyrosine phosphorylation and calcium mobilization. Moreover, neither PTX nor GTPγS compromise the effector function of NK cells or the susceptibility of target cells to NK‐mediated lysis. These data suggest apoptosis as a novel mechanism by which NK responses may be controlled in vivo, as well as an experimental and therapeutical strategy to counteract endogenous down‐regulation of NK responses.
Dendritic cells generated from peripheral blood transfected with human tyrosinase induce specific T cell activation
Authors: Alijagic Selma , Möller Peter , Artuc Metin , Jurgovsky Klaus , Czarnetzki Beate M. , Schadendorf Dirk
Published: March 2007

Peptides of melanosomal proteins have recently been shown to be recognized in an HLA‐restricted mode by specific cytolytic T lymphocytes in melanoma patients. Dendritic antigen‐presenting cells (DC) are considered to be the most effective stimulators of T cell responses, and the use of these cells has therefore been proposed to generate therapeutic responses to tumor antigens in cancer patients. We, therefore, generated DC from peripheral blood of normal donors in the presence of granulocyte/macrophage colony‐stimulating factor and interleukin‐4. Flow cytometric analysis of the cells during a 2‐week culture revealed a loss of CD14 and CD34 expression, a concomittent increase of CD1a, CD11a, b and c, CD44, CD45, CD54, HLA‐class I and II, and intermediate levels of CD26, CD80 and CD86. Cultured DC stimulated proliferation of allogeneic T cells and induced a marked, up to 20‐fold, stimulation of T cell proliferation after pulsing with tetanus toxoid. To achieve independence of already‐identified antigenic peptides presented in HLA class I‐restricted fashion, which limits the general applicability of such peptides for vaccination of melanoma patients, we tested whether DC are transfectable with eukaryotic expression plasmids. DC transfected with two reporter genes (CAT, β‐galactosidase) using a liposome‐based transfection technique, exhibited only low levels of enzymatically active proteins, but were able to degrade rapidly intracellular proteins and to process peptides efficiently. Chloramphenicol acetyltransferase as well as tyrosinase mRNA were detectable after transfection by reverse‐transcriptase‐polymerase chain reaction, and enzyme activities became measurable. Furthermore, DC transfected with the tyrosinase gene were able to induce specific T cell activation in vitro, indicating appropriate peptide processing and presentation in DC after transfection. These data suggest new approaches to future tumor vaccination strategies.
Immature B cells from human and mouse bone marrow can change their surface light chain expression
Authors: Ghia Paolo , Gratwohl Alois , Signer Erich , Winkler Thomas H. , Melchers Fritz , Rolink Antonius G.
Published: March 2007

The capacity of bone marrow‐derived surface immunoglobulin‐positive (sIg+) human and mouse immature B cells, generated either in vitro or in vivo, to change their light (L) chain expression, has been assayed by the number of cells which change in vitro from one type of L chain to the other type, or to no sIg at all. Immature sIg+ B cells were generated in vitro from sIg− precursor cells from human or mouse bone marrow. The immature sIg+ cells expressed RAG‐1. Human sIg+ cells expressed xfr; and λ L chains in ratios between 1:1 and 3:1, whereas in mouse cells, this ratio ranged from 10:1 to 20:1. Upon reculture of the human and mouse xfr;+sIg+ cells, about half of them remained xfr;+, a quarter became λ+, and another quarter became sIg−. Between 1 and 3% expressed both xfr; and λ chains. Of the human λ+ cells, about two‐thirds remained λ+, only 1 to 2% became xfr;+, while the other third became sIg−. Again, between 1 and 3% expressed both xfr; and λ L chains. These results indicate that expression of sIgM in the B cell membrane does not terminate L chain gene rearrangement, and that some order exists in xfr; versus λ gene rearrangements. Hence, human and mouse xfr;+ immature B cells can become λ+, but very few of the λ+ cells can become xfr;+, and both can become sIg−. Further, human CD10+/sIg+ xfr;+ and λ+ cells and mouse B220low/sIglow xfr;+ cells enriched from bone marrow, i.e. immature B cells differentiated in vivo, changed their Ig phenotype upon in vitro culture, but in lower frequencies. By contrast, human and mouse mature B cells did not change their L chain or Ig phenotype. Hence, at least a part of the sIg+ immature B cells in bone marrow retain the capacity to change their L chain and Ig phenotype, and this capacity is lost when they become mature, peripheral B cells.
Efficient immune responses in mice lacking N‐region diversity
Authors: Gilfillan Susan , Bachmann Martin , Trembleau Sylvie , Adorini Luciano , Kalinke Ulrich , Zinkernagel Rolf , Benoist Christophe , Mathis Diane
Published: March 2007

Mice with a null mutation in the terminal deoxynucleotidyl transferase (TdT) gene harbor immunoglobulin and T cell receptor repertoires essentially devoid of N‐region diversity. Consequently, the CDR3 loops important for antigen recognition are shorter and considerably less diverse than those of wild‐type controls. We find surprisingly normal immune responses in TdT° mice, as regards both efficiency and specificity. This provokes a reconsideration of the assumption that N‐region diversity is required for an effective T and B cell repertoire.
Engagement of CD20 suppresses apoptosis in germinal center B cells
Authors: Holder Michelle , Grafton Gillian , Macdonald Ian , Finney Michael , Gordon John
Published: March 2007

In a screen of 67 monoclonal antibodies (mAb) included in the Blind Panel of B cell antibodies for the 5th International Workshop on Human Leukocyte Differentiation Antigens, only the CD20 mAb – included as a positive control for immunophenotyping studies – was found to suppress the spontaneous apoptosis which occurs in human germinal center (GC) B cells when placed in tissue culture at 37°C. Further detailed study using the 1F5 mAb confirmed this observation, showing that rescue from apoptosis via CD20, while not as efficient as that obtained on ligating CD40, was of similar magnitude to that achieved on engagement of surface immunoglobulin (sIg) by immobilized antibody. Also similar to anti‐Ig, the CD20 mAb rescued from apoptosis without priming for the proliferation of GC B cells: this was quite different to its action on resting, non‐GC B cells, where it provides a potent priming signal for cell cycle progression in response to IL‐4 or anti‐CD40. Unlike the survival signal engendered via sIg, CD20 engagement neither mobilized Ca2+ from intracellular stores or opening of a Ca2+ channel with 1F5, nor did it affect the ability of anti‐Ig to open a Ca2+ gate in GC B cells. An unexpected feature of CD20‐mediated rescue of GC B cells from apoptosis was a failure to turn on Bcl‐2 expression.
Are murine marginal‐zone macrophages the splenic white pulp analog of high endothelial venules?
Authors: Lyons A. Bruce , Parish Christopher R.
Published: March 2007

The entry of lymphocytes into the spleen, in contrast to lymph nodes, does not involve high endothelial venule (HEV) interaction. The precise point of entry, as well as the mechanism by which lymphocytes enter the lymphoid areas of the spleen, remains controversial. We examined in detail the effect of two agents, pertussis toxin (PT) and the sulfated polysaccharide fucoidan, on splenic lymphocyte entry and positioning. These have previously been shown to interfere with lymphocyte extravasation across HEV. PT prevents lymphocyte extravasation, but not binding, to HEV, whereas fucoidan prevents binding and thus subsequent extravasation. Studies presented here show that pretreatment of murine lymphocytes with PT does not numerically affect entry into spleen, but profoundly alters lymphocyte positioning within the spleen. When fluorescently labeled, PT‐treated lymphocytes are injected intravenously, they initially accumulate in the marginal zone, in apparent association with the layer of marginal zone macrophages (MZMϕ) which form a shell around the white pulp. They fail to traverse this layer into the white pulp, and subsequently localize in the red pulp. In contrast, untreated cells initially appear in the marginal zone, then continue to migrate into the white pulp after traversing the MZMϕ layer. The localization of PT‐pretreated lymphocytes adjacent to the MZMϕ layer is disrupted by intravenous administration of fucoidan. Using a flow cytometric assay of aggregation between MZMϕ and lymphocytes, we confirmed that fucoidan is also able to inhibit this association in vitro, whereas PT has no effect on this interaction. We propose that MZMϕ in the mouse are the splenic analog of HEV, forming the port of entry of lymphocytes into the white pulp of the spleen.
Cross‐recognition by T cells of an epitope shared by two unrelated mycobacterial antigens
Authors: Harris David P. , Vordermeier Hans‐Martin , Singh Mahavir , Moreno Carlos , Jurcevic Stipo , Ivanyi Juraj
Published: March 2007

The molecular mimicry represented by cross‐recognition of determinants shared by unrelated antigens by antibodies or T cells is of broad immunological interest. In this study, we analyzed the cross‐recognition by CD4+ T cells of a peptide epitope shared by two mycobacterial proteins of diverse sequence, represented by the 19‐kDa antigen of Mycobacterium tuberculosis and the 28‐kDa antigen of Mycobacterium leprae. This epitope was immunodominant with respect to the 19‐kDa antigen, but cryptic in relation to the 28‐kDa antigen. The cross‐reactive epitope cores were identified by Pepscan window analysis and found to be eight residues long in both antigens (residues 69–76 and 127–134). Alignment of these octameric sequences revealed two identical and five conservatively related amino acids. Within the epitope core, two residues (73Asn and 76Ile) were identified as critical for recognition on the basis of inhibition of the cross‐reactive T cell proliferative response using singly substituted analog peptides. These results suggest that T cell cross‐reactive epitopes can exist in proteins with apparently not more than random levels of sequence homology. Their potential for unsuspected cross‐sensitization may play a role in the maintenance of T cell memory, in the pathogenesis of autoimmune diseases and possibly in a wide range of host immune responses to infectious pathogens.
Bacterial superantigens induce T cell unresponsiveness in B cell‐deficient mice
Authors: Miethke Thomas , Wahl Claudia , Heeg Klaus , Wagner Hermann
Published: March 2007

Bacterial superantigens such as staphylococcal enterotoxin B (SEB) cause in vivo a profound and long‐lasting state of unresponsiveness in ligand‐reactive T cells. To test whether presentation of SEB by small resting B cells to ligand‐reactive T cells is essential for the induction of T cell unresponsiveness, we analyzed the effect of SEB in B cell‐deficient mice. We observed T cell deletion and T cell unresponsiveness in both B cell‐deficient mice and control mice. We conclude that presentation of SEB by resting B cells is not a prerequisite for the induction of T cell unresponsiveness in vivo.
Lack of inducible nitric oxide synthase activity in T cell clones and T lymphocytes from naive and Leishmania major‐infected mice
Authors: Thüring Heike , Stenger Steffen , Gmehling Daniela , Röllinghoff Martin , Bogdan Christian
Published: March 2007

Nitric oxide (NO) generated by the inducible isoform of nitric oxide synthase (iNOS) is implicated in a number of immunological processes including killing of intracellular parasites, suppression of T cell proliferation, production of cytokines and destruction of tissue in autoimmune diseases. Considering that cytokine‐activated mouse macrophages, fibroblasts and endothelial cells are potent producers of NO, we investigated whether T cells, as central participants in immune responses, can also be activated for the release of NO. Neither thymocytes nor type 1 or type 2 T helper cell clones generated significant amounts of nitrite (the stable end product of NO in culture supernatants) when stimulated by T cell mitogens, cytokines or antigen in the presence of irradiated antigen‐presenting cells. Similarly, T cells freshly isolated from mice acutely infected with the intracellular pathogen Leishmania major did not produce NO upon restimulation in vitro. The lack of NO production was not due to the expression of enzymatically inactive iNOS, as we were unable to detect any iNOS protein in activated T helper clones or in freshly isolated T cells from infected mice by Western (protein) blot analysis. Finally, we tested whether iNOS expression in T cells might be restricted to a minor subpopulation and therefore only detectable on a single cell level. After immunofluorescence staining of lymph node or spleen cells from infected mice with antibodies against iNOS, F4/80‐ or Thy‐1‐antigen, macrophages, but no T cells, were found to express iNOS. Thus, we have no evidence that activated T helper cell clones or T cells from L. major‐infected mice are high producers of NO.
Processing and presentation of type II collagen, a fibrillar autoantigen, by H‐2q antigen‐presenting cells
Authors: Manoury‐Schwartz Bénédicte , Chiocchia Gilles , Fournier Catherine
Published: March 2007

Native type II collagen (CII) is a high molecular‐weight fibrillar molecule which induces a chronic polyarthritis in mice expressing the H‐2q haplotype. The present study was initiated to analyze the processing and the presentation of this nonglobular protein by H‐2q antigen‐presenting cells (APC). Efficiency of presentation was assessed by the ability of antigen‐pulsed APC to activate collagenspecific CD4+ T cell hybridomas. Fixation of APC or the presence of chloroquine completely blocked the reactivity of the T cell hybrids to native, denatured and cyanogen bromide (CB) degraded CII, thus indicating the requirement of intracellular processing for adequate presentation of CII peptides to T cells. In the presence of various processing inhibitors (brefeldin A, leupeptin and Ntosyl‐L‐phenylalanine chloromethylketone) stimulation of T hybrids by CIIpulsed APC was reduced, pointing to the need of newly synthesized class II molecules, the use of several intracellular compartments and the implication of different proteases in the generation of CII peptides. Peritoneal macrophages and, to a lesser extent, total spleen cells, presented native and denatured CII with higher efficiency than purified splenic dendritic cells, naive or even immune B cells from CII‐primed mice. In contrast, these dendritic and B cells were fully competent to present intact ovalbumin to a specific T cell hybrid. The stimulation by dendritic cells and immune B cells was greater when CB peptides of CII were added instead of the native molecule. Similarly, the cleavage of CII was an absolute requirement for its presentation by epidermal cells and B cell lymphomas to the T cell hybridomas. Taken together, these findings emphasize the crucial role of intracellular processing for recognition of soluble CII, similar to most antigens. However, in contrast to ovalbumin, the size and fibrillar nature of the native CII molecule influences its capture by the APC, thus limiting the type of APC able to present this antigen.
In susceptible mice, Leishmania major induce very rapid interleukin‐4 production by CD4+ T cells which are NK1.1−
Authors: Launois Pascal , Ohteki Toshiaki , Swihart Kristin , MacDonald H. Robson , Louis Jacques A.
Published: March 2007

Susceptibility of BALB/c mice to infection with Leishmania major is associated with a T helper type 2 (Th2) response. Since interleukin‐4 (IL‐4) is critically required early for Th2 cell development, the kinetics of IL‐4 mRNA expression was compared in susceptible and resistant mice during the first days of infection. In contrast to resistant mice, susceptible mice exhibited a peak of IL‐4 mRNA in their spleens 90 min after i.v. injection of parasites and in lymph nodes 16 h after s.c. injection. IL‐12 and interferon‐γ (IFN‐γ) down‐regulated this early peak of IL‐4 mRNA; the effect of IL‐12 was IFN‐γ dependent. Treatment of resistant C57BL/6 mice with anti‐IFN‐γ allowed the expression of this early IL‐4 response to L. major. The increased IL‐4 mRNA expression occurred in Vβ8, 7, 2− CD4+ cells in BALB/c mice and NK1.1− CD4+ cells in anti‐IFN‐γ treated C57BL/6 mice. These results show that the NK1.1+ CD4+ cells, responsible for the rapid burst of IL‐4 production after i.v. injection of anti‐CD3, do not contribute to the early IL‐4 response to L. major.
Characterization of processing requirements and metal cross‐reactivities in T cell clones from patients with allergic contact dermatitis to nickel
Authors: Moulon Corinne , Vollmer Jörg , Weltzien Hans‐Ulrich
Published: March 2007

Metal ions such as nickel, cobalt, copper and palladium are known to be potent sensitizers in humans, but the antigenic determinants created by these metals as well as the mechanisms of recognition by specific T cell clones are still not elucidated. In this paper, nickel‐specific T lymphocyte clones were isolated from four patients exhibiting contact dermatitis to this metal. A panel of 42 independent T cell clones was studied. They were shown to recognize nickel in the context of major histocompatibility complex (MHC) class II molecules and to belong to the CD4 subset. Using fixed autologous Epstein‐Barr virus‐transformed B cells as antigen‐presenting cells (APC), we could distinguish two distinct groups of T cell clones on the basis of processing requirements: 40% of the T cell clones were strictly processing dependent, whereas the remaining 60% could proliferate in response to nickel even in the presence of glutaraldehyde‐fixed APC. Furthermore, we present arguments indicating that individual Ni‐specific T cell clones cross‐react with some transition metals (e.g. Cu or Pd), but not with others (e.g. Co, Cr and Pt), presented by identical MHC class II molecules. These results thus provide an explanation for the multiple metal‐reactivities observed in vivo in human patients: they indicate that for Cu and Pd, these co‐reactivities in vivo might be due to cross‐reactivity at the clonal level. Our findings also suggest that this is not the case for cobalt allergy, which might result from co‐sensitization of the patient to cobalt in addition to nickel.
Identification of an ionomycin/cyclosporin A‐responsive element within the human T cell receptor gamma enhancer
Authors: Hettmann Thore , Cohen Amos
Published: March 2007

Activation through the Ca2+/calcineurin pathway is essential to the transcription of many cytokine genes. The conserved cis‐acting sequence, GGAAAA, and transcription factors binding to this sequence are involved in the response to increased intracellular Ca2+ concentrations. Here we report the identification and importance of the same sequence in a non‐cytokine gene, the human T cell receptor gamma (TCRG) enhancer. Results from site‐directed mutations and electrophoretic mobility shift assays strongly suggest that this sequence mediates the ionomycin‐induced activation of the TCRG enhancer. Our studies provide an explanation for a previous observation that TCRG mRNA levels, but not mRNA levels for T cell receptor alpha and ‐beta, are increased by ionomycin treatment.
Carrier‐induced epitope‐specific regulation and its bypass in a protein‐protein conjugate
Authors: Kaliyaperumal Arunan , Chauhan Virender S. , Talwar Gursaran Prasad , Raghupathy Raj
Published: March 2007

In the course of clinical trials on a birth control vaccine, it was found that some of the immunized women responded poorly to booster immunizations. This vaccine consists of a dimer of the β chain of human chorionic gonadotropin (βhCG) and the α chain of ovine luteinizing hormone (αoLH), linked to tetanus toxoid (TT) as a carrier. Changing this carrier to diphtheria toxoid resulted in reversion to high anti‐hCG antibody titers, indicating the extent to which the carrier influences anti‐ligand responses in this system. The suppression of anti‐hCG responses after booster immunizations was reminiscent of the phenomenon of carrier‐induced, epitope‐specific regulation. In a mouse model designed to test the effects of preimmunization with TT on anti‐hCG responses, we found that a single preimmunization with TT causes reduced anti‐hCG antibody responses in two out of four mouse strains, while anti‐αoLH antibody responses were not affected by the preimmunization with TT. This is particularly interesting considering that βhCG and αoLH were not presented when linked separately to TT. In an effort to devise a strategy to circumvent this carrier‐induced, ligand‐specific hyporesponsiveness, we investigated the effectiveness of a synthetic T helper epitope from TT as carrier. We show that preimmunization with TT causes a less profound reduction in anti‐hCG titers if the preimmunized mice are subsequently injected with αoLH‐βhCG conjugated to a synthetic tetanus toxin peptide recognized by TT‐induced and peptide‐induced T cells.
Two signaling pathways can lead to Fas ligand expression in CD8+ cytotoxic T lymphocyte clones
Authors: Anel Alberto , Simon Anna Katharina , Auphan Nathalie , Buferne Michel , Boyer Claude , Golstein Pierre , Schmitt‐Verhulst Anne Marie
Published: March 2007

As shown previously, a given cytotoxic T lymphocyte (CTL) clone (KB5.C20) could be induced to express the Fas ligand (FasL) by either T cell receptor (TCR) engagement or phorbol 12‐myristate 13‐acetate (PMA)/ionomycin stimulation. In contrast, another CTL clone (BM3.3) has now been found to exert Fas‐based cytotoxicity only after TCR engagement, but not after PMA/ionomycin stimulation. This suggested the existence of a PMA‐insensitive, antigeninduced pathway leading to FasL expression. The inability of PMA to promote Fas‐based cytotoxicity in BM3.3 cells was correlated with a defect in expression of the classical protein kinase C (PKC) isoforms α and βI. In KB5.C20 cells depleted of PMA‐sensitive PKC isoforms and thus no longer responsive to PMA, Fas‐based cytotoxicity could still be induced via the TCR/CD3 pathway. On the other hand, a requirement for phosphatidylinositol‐3 kinase (PI3K) selectively in this TCR/CD3‐induced pathway was demonstrated by specific inhibition with wortmannin. These results suggest that FasL expression when induced via the TCR/CD3 involves PI3K, and when induced by PMA/ionomycin requires the expression of PMA‐sensitive PKC isoforms absent in clone BM3.3. Additional data suggest that in neither case was NF‐χB activation implicated in FasL expression.
CD14lowCD16high: A cytokine‐producing monocyte subset which expands during human immunodeficiency virus infection
Authors: Thieblemont Nathalie , Weiss Laurence , Sadeghi Hoss M. , Estcourt Claudia , Haeffner‐Cavaillon Nicole
Published: March 2007

Infection with the human immunodeficiency virus HIV‐1 is associated with the expansion of a CD14lowCD16high monocyte subset in peripheral blood. This subset, which represents a minor subpopulation of monocytes in healthy individuals, increases during HIV infection and, in patients with AIDS, may represent up to 40% of the total circulating monocyte cell population. The CD14lowCD16high circulating monocytes co‐express MAX.1, p150,95 and HLADR which are typical of tissue macrophage markers. These cells also express higher levels of intracellular interleukin (IL)‐1α and tumor necrosis factor (TNF)‐α than the CD14highCD16low monocyte population from the same patients. The CD14lowCD16high cells also express low levels of CD35, CD11a and CD4 in common with normal monocytes. When cultured in vitro, monocytes from HIV‐seropositive individuals differentiated within a few hours into an elongated fibroblastoid shape characteristic of migratory cells. Our results suggest that the expansion of the CD14lowCD16high monocyte subset, which produce high amounts of TNF‐α and IL‐1α, may participate in the immune dysfunction observed during HIV infection.
Definition of sites on HLA‐DR1 involved in the T cell response to staphylococcal enterotoxins E and C2
Authors: Hargreaves Roseanna E. G. , Brehm Rossalyn D. , Tranter Howard , Warrens Anthony N. , Lombardi Giovanna , Lechler Robert I.
Published: March 2007

We have exploited the relative inefficiency of interaction between staphylococcal enterotoxins, SEE or SEC2, and H‐2Ek compared to HLA‐DR1 molecules to deduce which regions of the major histocompatibility complex (MHC) class II molecule are involved in the T cell response to these superantigens. Transfectants expressing hybrid DR/H‐2E MHC class II molecules were used to present SEE to the T cell receptor Vβ8.1‐expressing Jurkat cell line, and SEC2 to human peripheral blood T cells. For SEE, the critical region of the class II molecule for T cell reactivity and for binding was the β1 domain α‐helix. The functional data were corroborated by measurements of direct binding. Sequence comparison between DR and H‐2E raised the possibility that the glutamic acid at position 84 in the β chain of H‐2Ek, in place of glycine was responsible for the observed functional effects. This suggestion was supported by the finding that DQw2 (glutamine at 84) transfectants supported the SEE response much more efficiently than DQw6 that has glutamic acid at this position. In addition, amino acid substitutions at either position 36 or 39 in the DRα1 domain abolished T cell reactivity without any obvious alteration in binding. For SEC2, use of transfectants expressing exon‐shuffled α and β chain genes showed that replacement of the α1, α2 and β1 domains with H‐2E sequence inhibited the presentation of SEC2. Similarly, the substitutions at positions 36 and 39 in the α1 domain abolished the Tcell response to SEC2. Taken together, these data may be best explained by a model in which these two toxins have primary binding sites on the β1 domain (SEE) and the α1 and α2 domains (SEC2), but by virtue of a secondary binding site on the opposite surface of the class II molecule, cross‐link two adjacent DR molecules. Such cross‐linking may be important in the induction of T cell reactivity.
Supportive cellular elements for hepatic T cell differentiation: T cells expressing intermediate levels of the T cell receptor are cytotoxic against syngeneic hepatoma, and are lost after hepatocyte damage
Authors: Kawachi Yasuyuki , Arai Katsumitsu , Moroda Tetsuya , Kawamura Toshihiko , Umezu Hajime , Naito Makoto , Ohtsuka Kazuo , Hasegawa Katsuhiko , Takahashi‐Iwanaga Hiromi , Iwanaga Toshihiko , Shultz Leonard D. , Watanabe Hisami , Abo Toru
Published: March 2007

Extrathymic T cells exist in the liver and are often seen in close contact with Kupffer cells in the hepatic sinusoids. Since selective depletion of Kupffer cells has become possible by using liposome‐encapsulated clodronate, it was investigated whether elimination of Kupffer cells influences the level of extrathymic T cells in the liver. Extrathymic T cells were identified as interleukin‐2 receptor β‐chain (IL‐2Rβ) intermediate TCR (TCRint) cells by two‐color staining for CD3 or T cell receptor (TCR) and IL‐2Rβ. The elimination of Kupffer cells did not significantly affect levels of TCRint cells up to 7 days after treatment. We then examined monocyte colony stimulating factor (M‐CSF)‐deficient op/op mice (low levels of Kupffer cells). Extrathymic T cells both in the liver and spleen of these mice were detected at a level comparable to that of control mice. Since extrathymic T cells in the liver are sometimes located in the parenchymal space, the relationship between extrathymic T cells and hepatocytes was then examined. Electron microscopy revealed that some hepatic T cells adhered directly to hepatocytes. When hepatocytes were damaged by a single injection of CCl4, hepatocyte death and subsequent hepatic fibrosis were induced. Beginning 3 days after injection, CD3int cells, but not other type of cells, decreased prominently. Purified CD3int cells, as well as whole lymphocytes in the liver, were cytotoxic against syngeneic hepatoma. In parallel with the above‐mentioned hepatic damage, the cytotoxic activity of lymphocytes against such targets was impaired in the liver. These results suggest that extrathymic generation of TCRint cells and their acquisition of cytotoxic function are relatively independent of Kupffer cells, but are dependent on hepatocytes.
Expression of the complement alternative pathway by human myoblasts in vitro: Biosynthesis of C3, factor B, factor H and factor I
Authors: Legoedec Jocelyne , Gasque Philippe , Jeanne Jean‐François , Fontaine Marc
Published: March 2007

In this study, we demonstrate expression in vitro of complement alternative pathway components C3, factor B, factor H and factor I by normal human myoblasts and human rhabdomyosarcoma cell lines CRL1558 and HTB153. Proteins in culture supernatants were detected by Western (protein) blot analysis and biosynthetic labeling followed by immunoprecipitation experiments, and quantified by ELISA. Newly secreted proteins were structurally and functionally similar to their serum counterparts. An additional polypeptide of 43 kDa with factor H immunoreactivity was detected, which could correspond to the N‐terminal truncated form found in plasma. Protein expression was correlated with mRNA expression by reverse transcriptase‐polymerase chain reaction analysis. The major proteins of complement alternative pathway C3, factor B and factor H were produced constitutively by skeletal muscle cells at a rate of 50 to 150 ng/106 cells/ml and factor I was expressed 20 ng/106 cells/ml. These syntheses in vitro were regulated by inflammatory cytokines. Interferon‐γ significantly upregulated C3, factor B and factor H expression, but had no effect on factor I production. Interleukin‐1β strongly enhanced C3 and factor B production and had a weak enhancing or no effect on factor I and factor H secretion. Human myoblast cell lines constitute an interesting model to analyze complement biosynthesis by human skeletal muscle cells. Local complement expression by skeletal muscle in vivo may be implicated in some muscular inflammatory or pathological processes.
Interleukin‐6 signal transducer gp130 has specific binding sites for different cytokines as determined by antagonistic and agonistic anti‐gp130 monoclonal antibodies
Authors: Wijdenes John , Heinrich Peter C. , Müller‐Newen Gerhard , Roche Catherine , Gu Zong‐Jiang , Clément Claude , Klein Bernard
Published: March 2007

The cytokines interleukin (IL)‐6, IL‐11, ciliary neurotrophic factor (CNTF), leukemia inhibitor factor (LIF), oncostatin M (OSM) and probably the recently cloned cytokine cardiotrophin‐1, signal, in combination with their specific receptors, through the common signal transducer gp130. Here, we report that the signaling activities of IL‐6, IL‐11, CNTF and OSM/LIF can be specifically blocked by different anti‐gp130 monoclonal antibodies (mAb). Furthermore, we found two mAb, B‐P8 and B‐S12, which directly activate gp130 independently of the presence of cytokines or their receptors. This agonistic activity includes induction of cytokine‐dependent cell proliferation and stimulation of acute‐phase protein synthesis in liver cells. Compared to B‐P8 mAb, the B‐S12 mAb exhibited the strongest agonistic activity, while both mAb are synergistic in their action. This activity could not be blocked by inhibiting mAb against IL‐6 and the IL‐6 receptor. In contrast to F(ab′)2 of B‐S12 which still could activate gp130, Fab fragments completely lost their agonistic activity. Activation by tyrosine phosphorylation of the transcription factors Stat1 and APRF/Stat3 was also induced by B‐S12 and B‐P8, suggesting that both mAb induce homodimerization of gp130. Since hematopoietic stem cells express gp130 on their plasma membrane, it was anticipated that the agonistic anti‐gp130 mAb could stimulate the proliferation of these stem cells. Indeed, B‐S12 and B‐P8 were able to stimulate CD34+ cells. In summary, our data show for the first time that mAb against gp130 can specifically block the action of distinct IL‐6‐type cytokines that signal through gp130. Such mAb might be of great value for therapeutic applications in diseases where a single cytokine action needs to be inhibited. In addition, the agonistic gp130 mAb may be used as growth factors for maintenance and expansion of stem cells prior to grafting.