bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid induction of tumor necrosis factor cytotoxicity in naive splenic T cells by simultaneous CD80 (B7.1) and CD54 (ICAM‐1) co‐stimulation
Authors: Nishio Makoto , Podack Eckhard R.
Published: March 2007

The co‐expression of B7.1 (CD80) and intercellular adhesion molecule (ICAM)‐1 (CD54) on tumor cells can induce tumor immunity and immunological memory. We show here that the non‐immunogenic tumor lines Lewis lung carcinoma and B16F10 melanoma, co‐transfected with B7.1 and ICAM‐1, induced cytotoxic levels of membrane tumor necrosis factor (TNF) on naive syngeneic T cells within 24 h. Membrane TNF expression, primarily on CD4 cells, was responsible for tumor cell lysis by naive spleen cells and could be completely abolished by anti‐TNF antiserum. It is suggested that the strong induction of TNF cytotoxicity may be important in the establishment of tumor immunity.
The expression of cytokine‐induced neutrophil chemoattractants (CINC‐1 and CINC‐2) in rat peritoneal macrophages is triggered by Fcγ receptor activation: Study of the signaling mechanism
Authors: Alonso Adrés , Bayón Yolanda , Crespo Mariano Sánchez
Published: March 2007

The expression of cytokine‐induced neutrophil chemoattractants (CINC‐1 and CINC‐2) mRNA was studied in rat peritoneal cells stimulated with insoluble IgG/ovalbumin immune complexes. A dose‐ and time‐dependent induction was observed in adherent cells, which was more prominent than that induced by the lipid mediator platelet‐activating factor (PAF), comparable to that observed in response to 10 μg endotoxin in the absence of lipopolysaccharide (LPS)‐binding protein, but lower than that produced by 1 mM dibutyryl cyclic AMP, a compound which stabilizes transiently expressed genes containing AU‐rich sequences in the 3′ untranslated region. Analysis of CINC‐1 protein by specific enzyme‐linked immunosorbent assay confirmed the presence of CINC‐1 in the supernatants at concentrations of ∼4 nM, 4 h after addition of 100 μg/ml immune complexes. CINC‐2β protein was detectable at a lower concentration (∼0.3 nM) under the same conditions. Attempts to relate CINC‐1 induction with the pathways for cytoplasmic signaling showed a dissociation of Ca2+ mobilization and protein kinase C activation as judged from the small effect of thapsigargin and the lack of effect of phorbol ester. In contrast, these agents produced a marked mobilization of arachidonate linked to the MAP kinase‐dependent activation of cytosolic phospholipase A2. The possible dependence of CINC‐1 induction on the autocrine generation of lipid mediators was ruled out by a set of experiments including the use of the PAF receptor antagonist BB823, and the analysis of the effect of free arachidonate and leukotriene B4 on CINC‐1 induction. Surprisingly, the inhibitor of leukotriene synthesis MK‐886 in the range of concentration 1–10 μM inhibited CINC‐1 induction by a mechanism that appears to be independent of its effect on eicosanoid production. Interestingly, CINC‐1 induction appeared to be related to protein tyrosine phosphorylation reactions on the basis of both the appearance of several tyrosine‐phosphorylated protein bands in lysates from adherent peritoneal cells treated with immune complexes and the complete blockade of CINC‐1 induction by treatment with 1 μM herbimycin A, an inhibitor of src protein tyrosine kinases.
Functionally distinct T cells in three compartments of the respiratory tract after influenza virus infection
Authors: Baumgarth Nicole , Kelso Anne
Published: March 2007

This study aimed to resolve, firstly, whether T cell responses induced in one tissue site are similar to those induced by the same antigen in another site and, secondly, whether influenza virus infection induces one predominant type of T cell response locally in the respiratory tract. To address these questions, T cell responses in three compartments of the respiratory tract were compared after infection of mice with a sublethal dose of influenza virus: the draining mediastinal lymph nodes (MLN), the lung parenchyma and the airways. Each compartment harbored a T cell response substantially different from that found at the other sites. A preferential accumulation of ex vivo‐cytolytic CD8+ T cells was found in the airways (CD4/CD8 ratio 1:2) and to a lesser extent in the lung parenchyma (CD4/CD8 ratio 1:1). T cells from both compartments expressed high levels of various cytokine mRNA, but showed differences in their respective expression pattern, with those from lung tissue showing particularly high levels of IFN‐γ mRNA. The response in the draining lymph nodes, on the other hand, was dominated by CD4+ T cells (CD4/CD8 ratio 2:1) with a higher proliferative capacity (after TCR/CD3 cross‐linking) and which provided better B cell help in vitro than CD4+ T cells isolated from lung tissue. T cells from MLN expressed mRNA for a variety of cytokines with only low levels of IFN‐γ mRNA and they showed no CTL activity ex vivo. These functional differences were not due to differences in the kinetics of the response, or to the higher frequencies of activated T cells in lung tissue and airways compared to MLN, since the differences remained when cell‐sorter‐purified activated (CD18hi, CD44hi) T cells from MLN and lung tissue were compared in a time‐course study. Taken together, these findings indicate that pathogens such as influenza virus induce a heterogenous set of T cell responses in different tissue sites affected by the infection.
Glycosylphosphatidylinositol‐anchored H‐2Db molecules are defective in antigen processing and presentation to cytotoxic T lymphocytes
Authors: Cariappa Annaiah , Flyer David C. , Rollins Carl T. , Roopenian Derry C. , Flavell Richard A. , Brown Dennis , Waneck Gerald L.
Published: March 2007

Glycosylphosphatidylinositol‐anchored (GPI)‐Db molecules are defective in mediating cytotoxic T lymphocytes (CTL) lysis of transfected lymphoma cells, compared to their transmembrane (TM) counterpart. This defect is manifest when antigenic peptide must be processed and presented through the endogenous pathway. These same transfectants can be lysed by allospecific CTL, or by antigen‐specific Db‐restricted CTL when pulsed with appropriate exogenous synthetic peptide, demonstrating that they can bind and present peptide for CTL‐mediated lympholysis. The defect apparently results from differences between GPI‐Db and TM‐Db assembly and transport, or from differences in membrane topology that affect CD8+ CTL recognition of major histocompatibility complex/peptide complex.
Differences in Vϰ gene utilization and VH CDR3 sequence among anti‐DNA from C3H‐lpr mice and lupus mice with nephritis
Authors: Wloch Mary Kopke , Alexander Audrey L. , Pippen Anne M. M. , Pisetsky David S. , Gilkeson Gary S.
Published: March 2007

To investigate the molecular properties of anti‐DNA from lpr mice that express high levels of anti‐DNA without immune‐mediated nephritis, the sequences of VH and Vϰ genes encoding 11 monoclonal anti‐DNA antibodies derived from C3H‐lpr/lpr (C3H‐lpr) mice were studied. All of the C3H‐lpr monoclonal anti‐DNA bound single‐stranded DNA while five also bound double‐stranded DNA. Two of the hybridomas were clonally related as determined by Southern analysis and sequencing. Sequence analysis of C3H‐lpr anti‐DNA revealed the use of VH genes that encode anti‐DNA from the MRL‐lpr/lpr and (NZB X NZW)F1 mouse models of lupus, although differences occurred in the VH CDR3 amino acid content. In contrast, the Vϰ genes from C3H‐lpr mice lacked significant identity with previously reported Vϰ genes for anti‐DNA from lupus models. These results indicate that anti‐DNA from C3H‐lpr mice differ from anti‐DNA from lupus mice with nephritis in patterns of V gene expression and suggest a molecular basis for the lack of pathogenicity of anti‐DNA in these mice.
Differential activation of T cell cytokine production by the extracellular signal‐regulated kinase (ERK) signaling pathway
Authors: Egerton Mark , Fitzpatrick David R. , Catling Andrew D. , Kelso Anne
Published: March 2007

Stimulation of T cells via the T cell receptor (TCR) activates a number of signaling pathways that are potentially involved in the elicitation of physiological responses, such as the production of cytokines. The extracellular signal‐regulated kinases (ERK) are a group of molecules activated in response to TCR ligation, whose role in T cell cytokine production is controversial. In this study, we have asked whether ERK activation is coupled to the production of a number of T cell‐derived cytokines, and whether particular cytokines are differentially affected by ERK activation. To address these questions, we have utilized a constitutively active version of the immediate upstream activator of both ERK1 and ERK2, mitogen‐activated/extracellular signal‐regulated kinase 1 (MEK1), to activate ERK signaling selectively in the absence of other TCR‐activated signaling pathways. The effect of constitutive MEK/ERK activation on T cell cytokine production was measured by transiently co‐transfecting newly activated mouse T cells with DNA encoding constitutively active MEK1 (CA‐MEK1) and the human interleukin‐2 (IL‐2) receptor α chain (hCD25), purifying hCD25+ transfectants by flow‐cytometric cell sorting, and measuring the production of IL‐3, IL‐4, interferon (IFN)‐γ and granulocyte/macrophage‐colony‐stimulating factor (GM‐CSF) either in the presence or absence of ionomycin stimulation. Newly activated T cells were used in these experiments as they more closely resemble T cells activated in vivo than do transformed T cells or long‐term established T cell clones. CA‐MEK1 expression led to constitutive ERK activation, which acted synergystically with ionomycin treatment to stimulate cytokine production. Furthermore, these experiments revealed a hierarchy of cytokine responsiveness to MEK/ERK activation, such that the production of IL‐3 was most affected, followed by GM‐CSF, IFN‐γ, and IL‐4.
Noncovalent associations of T lymphocyte surface proteins
Authors: Černý Jan , Stockinger Hannes , Hořejší Václav
Published: March 2007

A number of T cell surface transmembrane molecules such as CD2, CD4, CD8, lymphocyte functional antigen (LFA)‐1 and CD45 are known to interact functionally with the T cell receptor (TCR) complex during T cell activation. Several previous communications have also reported physical associations between some of these molecules. On the other hand, there are indications that signaling through T cell surface molecules anchored via glycosylphosphatidylinositol (GPI), such as Thy‐1, Ly‐6 or CD59, is dependent on the TCR. Therefore, it was of interest to determine in a systematic way which T cell surface molecules are noncovalently associated with the TCR/CD3 complex and with the major intracellular signaling molecules, the protein tyrosine kinases of the Src family. To this aim, membrane proteins of human thymoma HPB‐ALL cells were solubilized in a solution of the mild detergent Brij‐58 and subjected to immunoprecipitation followed by in vitro kinase assays. Two types of large complexes containing protein tyrosine kinases were observed: the first one contained CD3 and the transmembrane proteins CD2, CD4, CD5, CD6, CD7, CD8, CD11a, CD38, CD43, CD45, CD71, CD98 and CD99 and the other contained mainly the GPI‐anchored proteins CD48, CD55, CD59 and CDw108 as well as a fraction of CD4 and CD8. The GPI‐anchored protein complexes were of larger size and lower buoyant density than the CD3 complexes. In agreement with these biochemical data, co‐cross‐linking of CD3 with most of the relevant transmembrane proteins on the surface of another T cell line, Jurkat, markedly enhanced tyrosine phosphorylation of several intracellular proteins. These data indicate the existence of at least two types of membrane microdomains of very different composition in the membranes of T cells which may play a role in signaling through different types of receptors and in functional cooperation between TCR/CD3 and various accessory molecules.
Differential effects of transforming growth factor‐β1 on IgA vs. IgG2b production by lipopolysaccharide‐stimulated lymph node B cells: a comparative study with spleen B cells
Authors: García Belén , Rodríguez Rosa , Angulo Iñigo , Heath Andrew W. , Howard Maureen C. , Subiza José L.
Published: March 2007

Transforming growth factor‐β1 (TGF‐β1) is a multifunctional cytokine that promotes IgA/IgG2b switching and secretion. Here, we show a differential effect of TGF‐β1 on Ig production by lipopolysaccharide‐stimulated spleen and lymph node (LN) B cells. Exogenous TGF‐β1 increased IgA production in B cell cultures and IgG2b production by spleen B cells. In contrast, IgG2b was suppressed by TGF‐β1 in cultures of LN B cells, although endogenous TGF‐β was required for IgG2b production in LN B cell cultures. The suppressor properties of exogenous TGF‐β1 (0.5 ng/ml) on IgG2b production by LN B cells were also seen when testing IgG1 or IgG2a induced by interleukin‐4 or interferon‐γ, respectively. These differences between B cells from each lymphoid tissue appeared to be related to a different TGF‐β antiproliferative effect, since proliferation of LN B cells was extremely sensitive to TGF‐β1 and IgG2b production was more sensitive than IgA to the TGF‐β‐mediated suppression. However, by counteracting the antiproliferative effect of TGF‐β1 with a CD40 agonistic mAb (IC10), the IgG2b response by LN B cells was still lacking. IC10 was nevertheless inhibitory for IgG2b production in most cases, while increasing secretion of IgA in the very same cultures. Taken together, the results suggest that functional differences between spleen and LN B cells do exist, at least with regard to the immunomodulating properties of TGF‐β on both proliferation and Ig production. Moreover, functional differences exist between cells committed for IgA and IgG2b regarding their sensitivity to the antiproliferative activity of TGF‐β1 and the effect of CD40‐derived signals on Ig secretion.
Mapping of the domains required for decay acceleration activity of the human factor H‐like protein 1 and factor H
Authors: Kühn Sabine , Zipfel Peter F.
Published: March 2007

The human factor H‐like protein 1 (FHL‐1) is composed of seven repetitive elements (short consensus repeats; SCR) that are identical in sequence to the seven N‐terminal SCR of complement factor H. We show that the FHL‐1 protein has decay acceleration activity in that it can dissociate C3/C5‐convertases bound to the surface of sheep red blood cells. The same activity was also determined for factor H. However, compared to FHL‐1, factor H was more efficient in decay acceleration, as about 100‐fold less protein was required for a 50% inhibition of activity. The domain required for decay accelerating activity of FHL‐1 and factor H was mapped by the use of recombinant fragments. FHL‐1 and a series of truncated forms of the protein were expressed in the baculovirus system. Recombinant FHL‐1 and all mutants which include SCR 1–4 were functionally active. These four N‐terminal SCR are essential and sufficient for activity, as deletion mutants which lack SCR 1 or SCR 4 showed no activity. These results demonstrate that FHL‐1 and factor H have identical and overlapping regulatory functions in the complement system and that the domain required for this activity is located in the overlapping region of both proteins within the N‐terminal four SCR.
Expression and cleavage of tumor necrosis factor‐α and tumor necrosis factor receptors by human monocytic cell lines upon direct contact with stimulated T cells
Authors: Vey Elisabeth , Burger Danielle , Dayer Jean‐Michel
Published: March 2007

Tumor necrosis factor‐α (TNF‐α) is a potent cytokine in inflammatory processes. A variety of mechanisms that modulate its activity have been described, one being its binding to soluble receptors (sTNFR). In this study, we demonstrate that human monocytic cells such as THP‐1 respond to direct contact with a membrane preparation of stimulated HUT‐78 cells by producing TNF‐α and by releasing sTNFR‐p75, but not sTNFR‐p55, with different kinetics. TNF‐α concentration peaked after 12 h of contact and then decreased, whereas sTNFR‐p75 production increased progressively upon cell/cell contact. The decrease in TNF‐α concentration is not due to trapping of TNF‐α by its soluble receptors or other soluble or cell‐associated molecules, but rather to a proteolytic activity associated to THP‐1 cells. On the other hand, the increase in sTNFR‐p75 release does not result from an increase in the cleavage of pre‐existing cell‐associated sTNFR‐p75 but from an increase in TNFR‐p75 expression, immediately followed by the cleavage of its extracellular domain. Phenylmethylsulfonylfluoride, a serine protease inhibitor, has a negative effect on both TNF‐α degradation and sTNFR‐p75 release by THP‐1 cells. Thus, there may be an enzymatic activity associated to THP‐1 cells that plays an important role in the neutralization of TNF‐α activity both by degrading the molecule and by cleaving its receptors at the cell surface.
Differential modulation of cyclin‐dependent kinase inhibitor p27Kip1 by negative signaling via the antigen receptor of B cells and positive signaling via CD40
Authors: Han Hua , Nomura Takashi , Honjo Tasuku , Tsubata Takeshi
Published: March 2007

The cross‐linking of surface immunoglobulins (sIg) of B cells can transmit a negative signal, resulting in cell cycle arrest, apoptosis or both. Signaling via the B cell antigen CD40 reverses the sIg‐mediated negative signaling and induces activation and proliferation of B cells. We investigated the molecular mechanisms for cell cycle regulation by negative and positive signaling via sIg and CD40, respectively, by using the B cell line WEHI‐231. Cross‐linking of sIg almost completely reduced the activity of cyclin‐dependent kinase (Cdk) 2, essential for cell cycle progression in the late G1 phase, although the level of Cdk2 was not reduced. Among the factors that regulate Cdk2 activation, the activity of the Cdk‐activating kinase (CAK) appeared intact and cyclin E was reduced only partially in sIg‐cross‐linked WEHI‐231. In contrast, sIg cross‐linking induced a significant Cdk inhibitor (CKI) activity. Since a 27‐kDa protein was co‐precipitated with Cdk2 in anti‐Ig‐treated, but not untreated WEHI‐231, and the CKI activity in anti‐Ig‐treated WEHI‐231 was neutralized by anti‐p27Kip1, antibodies, it is most likely that p27Kip1 is responsible for the CKI activity induced by sIg cross‐linking. p27Kip1 may thus play a role in growth inhibition of B cells by negative signaling via sIg. In contrast, CD40 signaling enhanced Cdk2 activity and reduced the p27Kip1 level in anti‐Ig‐treated WEHI‐231, suggesting that the reduction of p27Kip1 plays an important role in the abrogation of sIg‐mediated growth arrest by CD40 signaling. Taken together, p27Kip1 is likely to be a crucial target molecule of the negative signaling via sIg and the positive signaling via CD40 essential for T cell‐dependent immune responses.
Biological properties of recombinant chicken interferon‐γ
Authors: Weining Kirsten C. , Schultz Ursula , Münster Uwe , Kaspers Bernd , Staeheli Peter
Published: March 2007

Supernatants of the chicken T cell line 855 contain antiviral and macrophage‐activating factor activity and strongly activate transcription of the guanylate‐binding protein (GBP) gene in chicken cells. To characterize the cytokine responsible for the GBP‐inducing activity, we chose a cDNA expression cloning strategy in COS cells. Sequencing a positive clone revealed that it encodes chicken interferon‐γ (ChIFN‐γ). Histidine‐tagged ChIFN‐γ was expressed in Escherichia coli and purified by nickel chelate affinity chromatography. ChIFN‐γ from COS cells and E. coli both potently induced GBP RNA synthesis but were rather poor antiviral agents. In macrophages, recombinant ChIFN‐γ strongly stimulated secretion of nitric oxide and enhanced expression of major histocompatibility complex class II antigen. A rabbit antiserum to E. coli‐derived ChIFN‐γ effectively neutralized the macrophage‐activating factor activity secreted by concanavalin A‐induced spleen cells and various T cell lines, suggesting that IFN‐γ is the major macrophage‐activating factor of the chicken.
CD28‐deficient mice generate an impaired Th2 response to Schistosoma mansoni infection
Authors: King Christopher L. , Xianli Jia , June Carl H. , Abe Ryo , Lee Kelvin P.
Published: March 2007

Engagement of CD28 on T cells provides a co‐stimulatory signal necessary for T cell activation and differentiation. Recent findings suggest that priming of T helper (Th)2 cells is more dependent on CD28 activation than Th1 cells. The present study examines whether mice that lack expression of CD28 as a result of gene targeting are capable of generating a Th2 response characteristic during infection with the intravascular trematode parasite Schistosoma mansoni. Mutant and control mice were either inoculated in the footpad with S. mansoni eggs (a potent inducer of a Th2 response) or infected percutaneously with the parasite. Draining lymph nodes (after footpad injection) or spleen cells (after natural infection) were harvested at 12 days and 8 weeks, respectively, and examined for cytokine responses to egg antigens. CD28‐deficient mice (−/−) generated diminished egg antigen‐driven interleukin (IL)‐4 and IL‐5 production (by 5‐ to 17‐fold, respectively) compared to CD28‐expressing (+/+) littermates. In contrast, lymphocyte proliferation and interferon (IFN)‐γ production to egg antigens were equivalent for mutant and control mice. Infected CD28−/− mice also had reduced immunoglobulin secretion. Serum levels of parasite antigen‐specific IgG1 and polyclonal IgE were significantly diminished in CD28−/− compared to CD28+/+ mice. Lack of CD28 expression had no effect on granuloma formation around eggs trapped in the liver, but increased susceptibility of mice to primary schistosomiasis infection. These studies indicate that CD28 activation contributes to T cell priming required for generation of a Th2 response to an intravascular dwelling helminth parasite.
Active suppression of the class II transactivator‐encoding AIR‐1 locus is responsible for the lack of major histocompatibility complex class II gene expression observed during differentiation from B cells to plasma cells
Authors: Sartoris Silvia , Tosi Giovanna , De Lerma Barbaro Andrea , Cestari Tiziana , Accolla Roberto S.
Published: March 2007

In this study the genetic control of major histocompatibility complex (MHC) class II gene expression during the transition from B cell to plasma cell has been analyzed. Class II molecules are not expressed in plasma cells because of an active suppression resulting in the abrogation of class II gene transcription. We show here that the plasma cell‐specific repressor function, designated SIR (suppressor of immune response genes), does not act directly on the transcription of class II genes, but instead on the transcription of the AIR‐1 gene, whose product, the class II transactivator (CIITA), is fundamental for the regulation of the constitutive and inducible expression of MHC class II genes. This was unambiguously demonstrated by the fact that plasmacytoma × B cell hybrids carrying an AIR‐1 locus derived from CIITA‐expressing cells do not express CIITA‐specific transcripts. Transfection of a cDNA containing the human CIITA coding sequence under the control of an heterologous promoter restores expression of human MHC class II genes in the hybrids and is responsible for de novo expression of mouse MHC class II genes in both the mouse plasmacytoma cell line and the hybrids. These results confirm and extend the notion of the functional conservation of the AIR‐1 gene product across species barriers. Interestingly, in CIITA‐transfected cell hybrids, cell surface expression of the human HLA‐DQ heterodimer was not observed. This result was not attributable to lack of HLA‐DQα or ‐DQβ transcription, because both transcripts were present in the CIITA‐transfected hybrids, although at reduced levels. These findings further support our previous observations on the distinct regulation of expression of the human HLA‐DQ class II subset, which may be thus controlled at the post‐transcriptional level by a CIITA‐independent mechanism.
Delineation of the minimal encephalitogenic epitope within the immunodominant region of myelin oligodendrocyte glycoprotein: diverse Vβ gene usage by T cells recognizing the core epitope encephalitogenic for T cell receptor Vβb and T cell receptor Vβa H‐2b mice
Authors: Mendel Itzhak , de Rosbo Nicole Kerlero , Ben‐Nun Avraham
Published: March 2007

The nature of the autoimmune T cell response to myelin oligodendrocyte glycoprotein (MOG), recently recognized as a potential target antigen in multiple sclerosis (MS), has not yet been characterized, in contrast to the T cell reactivity to other potential target antigens in MS such as myelin basic protein and proteolipid protein. Here, we show that the encephalitogenicity of the recombinant Ig‐like domain of human MOG is associated, in H‐2b mice, with an immunodominant T cell reactivity against a single region of MOG spanning amino acids 35–55, accounting for the previously reported strong encephalitogenic activity of pMOG 35–55. A single injection of pMOG 35–55 with or without administration of pertussis toxin was sufficient to induce severe clinical experimental autoimmune encephalomyelitis (EAE) in H‐2b mice. Encephalitogenic pMOG 35–55‐specific T cell lines derived from C3H.SW (Vβb) mice were diverse in their TCR Vβ gene usage (Vβ1, Vβ6, Vβ8 and Vβ15), although Vβ8.2 was most predominantly expressed (48%). However, Vβ8+ T cells may only be part of the encephalitogenic MOG‐specific T cell repertoire in H‐2b mice, as demonstrated by the susceptibility of C57L (Vβa) mice to disease induced by pMOG 35–55. Encephalitogenic T cell lines from Vβa mice were also diverse in their TCR Vβ gene usage (Vβ1, Vβ2, Vβ6, Vβ14 and Vβ16). Such a heterogeneous TCR Vβ gene expression by pMOG 35–55/I‐Ab‐reactive T cells from both Vβa and Vβb H‐2b mice suggested multiple epitopes within pMOG 35–55. Analysis of the pattern of reactivity by pMOG 35–55‐reactive T cells to a set of truncated peptides was not commensurate with independent nested epitopes, but revealed a requirement for recognition of a core sequence, YRSPFSRVV (pMOG 40–48). However, optimal stimulation was obtained with longer peptides, with each additional amino acid flanking either the N or the C terminus differentially increasing the stimulatory capacity of pMOG 40–48. Nonetheless, pMOG 40–48 was the minimal encephalitogenic epitope for both Vβa and Vβb mice. Thus, the T cell reactivity against the immunodominant encephalitogenic region of MOG is characterized by a diverse Vβ gene usage and a requirement for the same core epitope. This pattern of reactivity may favor epitope‐directed, rather than TCR‐targeted, approaches to immunospecific therapy for MOG‐related autoimmune disease.
CD94 functions as a natural killer cell inhibitory receptor for different HLA class I alleles: identification of the inhibitory form of CD94 by the use of novel monoclonal antibodies
Authors: Sivori Simona , Vitale Massimo , Bottino Cristina , Marcenaro Emanuela , Sanseverino Lorenza , Parolini Silvia , Moretta Lorenzo , Moretta Alessandro
Published: March 2007

CD94 molecules have been suggested to function as inhibitory natural killer cell (NK) receptors involved in the recognition of HLA‐B alleles sharing the Bw6 supertypic specificity. In this study, we show that CD94 molecules may play a more general role: they are also involved in the recognition of other HLA class I molecules, including HLA‐C and at least some HLA‐A alleles. The inhibitory effect mediated by CD94 molecules on NK cytolytic activity is lower in magnitude than that of bona fide inhibitory receptors such as p58 or p70. Distinct from the other human NK receptors involved in HLA class I recognition, CD94 is expressed on virtually all NK cells. In addition, it has been shown to be functionally heterogeneous since, in different clones, CD94 mediated either cell triggering or inhibition. Although NK cells expressing inhibitory CD94 molecules are usually characterized by a CD94bright phenotype, there is no precise correlation between fluorescence intensity and inhibitory or activating function. Here, we describe two novel monoclonal antibodies (mAb) which selectively recognize inhibitory CD94 molecules and bind to a subset (variable in size among different donors) of CD94bright cells. The use of these mAb allows the direct assessment of NK cells expressing inhibitory CD94 receptors both at the population and at the clonal level.
Physiological activation of the IgH 3′ enhancer in B lineage cells is not blocked by Pax‐5
Authors: Andersson Tove , Neurath Markus F. , Grant Patrick A. , Pettersson Sven
Published: March 2007

The mouse 3′ enhancer contains a high‐affinity binding site for the paired box protein Pax‐5. Here, we demonstrate by genomic footprinting that the rat 3′ enhancer contains a low‐affinity binding site for Pax‐5, which is occupied in activated splenic B cells. Thus, binding of Pax‐5 to the IgH 3′ enhancer appears to be evolutionarily conserved in rodents. Analysis of Pax‐5 expression in primary B cells demonstrates that Pax‐5 remains expressed after 4 days of lipopolysaccharide (LPS) induction, but is down‐regulated in 5‐day stimulated cells. Similarly, the expression of Pax‐5 is down‐regulated in vivo in activated large splenocytes, in contrast to small resting cells. Multimerization of the high‐affinity Pax‐5 binding site linked to a heterologous reporter gene demonstrates that Pax‐5 can function as a transcriptional activator. In contrast, Pax‐5 overexpressed in cell lines represses both the mouse and the rat 3′ enhancer. Surprinsingly, cross‐linking of the IgM receptor in BAL‐17 cells containing a stably integrated 3′ enhancer‐dependent β globin reporter gene demonstrates that induction of 3′ enhancer activity is not blocked by Pax‐5. Moreover, stimulation of 3′ enhancer β globin‐transgenic splenocytes demonstrate that Pax‐5 cannot repress activation of the 3′ enhancer upon LPS induction or CD40 receptor stimulation. Hence, activation of the IgH 3′ enhancer occurs independently of changes in Pax‐5 gene expression. This indicates that previous studies conducted in vitro may be an oversimplification of the function of Pax‐5 and 3′ enhancer activity.
Association of tissue factor with a γ chain homodimer of the IgE receptor type I in cultured human monocytes
Authors: Masuda Midori , Nakamura Shin , Murakami Takashi , Komiyama Yutaka , Takahashi Hakuo
Published: March 2007

Tissue factor (TF) is a high‐affinity receptor for coagulation factors VII (F VII) and VIIa. The F VII/VIIa/TF complex is the major cellular initiator of the extrinsic coagulation cascade. We found that the occupancy of TF by its ligand, F VIIa, is involved with signal transduction and that TF is associated with the γ chain homodimer identified as a component of IgE receptor type I (FcϵRI). When 4‐day cultured human monocytes were incubated with F VIIa, several polypeptides, especially a 70‐kDa polypeptide, were transiently phosphorylated on tyrosine, residues. These phosphorylation events were inhibited by prior binding of anti‐TF monoclonal antibody (mAb) HTF‐K14, but not anti‐TF mAb HTF‐K180 to intact cultured monocytes. HTF‐K14 blocked the binding of FVII/Vila to cell surface TF, whereas HTF‐K180 did not. Anti‐TF immunoprecipitates prepared from 1 % digitonin lysates of cultured human monocytes incorporated phosphate in a γ chain homodimer when incubated with [γ‐32P] ATP. The identity of the TF‐associated structures as γ chains was established by immunoblot analysis of anti‐TF mAb immunoprecipitates with anti‐γ chain mAb. In addition, anti‐TF immunoblot analysis showed that TF co‐precipitated with anti‐γ chain mAb. Our data suggest that γ chains may play an important role in signaling via TF in human monocytes/macrophages.
Autoreactive T cell responses and cytokine patterns reflect resistance to experimental autoimmune myasthenia gravis in Wistar Furth rats
Authors: Zhang Guang‐Xian , Ma Cun‐Gen , Xiao Bao‐Guo , van de Meide Peter H. , Link Hans
Published: March 2007

Various mouse and rat strains show different susceptibilities to experimental autoimmune myasthenia gravis (EAMG) that can be induced by immunization with acetylcholine receptor (AChR) and Freund's complete adjuvant, and represents a model for the antibody‐mediated myasthenia gravis in humans. We examined AChR‐induced B and T cell responses and cytokine mRNA expression to study the mechanisms behind susceptibility to EAMG in Lewis rats and resistance in Wistar Furth (WF) rats. Both strains had similarly elevated concentrations and affinities of serum anti‐AChR antibodies, and no difference between the two strains for frequencies of cells in lymphoid organs expressing mRNA of the B cell stimulating cytokine interleukin‐4 was found. In contrast, T cell responses to AChR measured by proliferation and by enumeration of interferon‐γ‐expressing cells at both mRNA and protein level were lower in the resistant WF rats. This strain showed, instead, an up‐regulation of the anti‐inflammatory transforming growth factor‐β. Strain‐related differences in the susceptibility to actively induced EAMG are thus related to quantitative differences in distribution between pro‐inflammatory and anti‐inflammatory cytokines.
A conditionally immortalized dendritic cell line which differentiates in contact with T cells or T cell‐derived cytokines
Authors: Volkmann Ariane , Neefjes Jacques , Stockinger Brigitta
Published: March 2007

A conditionally immortalized dendritic cell line was established from bone marrow of mice transgenic for a thermolabile mutant of the SV40 large T antigen under the control of the class I Kb promoter. At the permissive temperature of 33°–37°C, the line divides in the absence of granulocyte/macrophage colony stimulating factor. It shares a number of cell surface markers with bone marrow macrophages, but unlike macrophages, is constitutively major histocompatibility complex (MHC) class II+, negative for nonspecific esterase and unable to phagocytose sheep red blood cells. The cells show characteristic dendrites, an abundance of acidic vesicles and are highly active in endocytosis. If maintained at 33°C, the dendritic cell line processes and presents exogenous protein to MHC class II‐restricted T cell hybrids and acts as potent mixed lymphocyte reaction stimulator, but fails to activate naive, resting T cells. Transfer to 39°C arrests growth and results in up‐regulation of surface markers such as B7.1, CD40 and intercellular adhesion molecule‐1. Further up‐regulation of cell surface markers and acquisition of functional maturity occur following contact with T cells and their cognate antigen or in culture with a cytokine mixture derived from activated T cells.
CD43 expression in a B cell lymphoma, WEHI 231, reduces susceptibility to G1 arrest and extends survival in culture upon serum depletion
Authors: Misawa Yukiko , Nagaoka Hitoshi , Kimoto Hiroshi , Ishii Yasunori , Kitamura Kazuyuki , Tsunetsugu‐Yokota Yasuko , Shibuya Masafumi , Takemori Toshitada
Published: March 2007

CD43 is a major surface sialoprotein on hemopoietic cells, whose extracellular domain is heavily O‐glycosylated. The functional role of CD43 in the hemopoietic system is not fully understood; however, it has been suggested that CD43 may have a role in cell‐cell repulsion and in modifying T cell proliferation and activation. CD43 is expressed in immature B cells in the bone marrow, but not by peripheral B cells, except for B‐1 B cells and plasma cells. To analyze the biological effect of CD43 in B‐lineage cells, we transfected mouse CD43 cDNA into a CD43− B cell lymphoma, WEHI 231, and the growth and survival in culture were compared to those of a parental cell line, human CD8 transfectants, and CD43− revertants established from CD43+ clones. We observed that CD43 expression supported cell growth in culture upon serum reduction, whereas growth of CD43− cell lines was barely detected under this condition. CD43− cell lines accumulated in G1 phase of the cell cycle, and the numbers of viable cells were greatly reduced during culture upon serum depletion, whereas expression of CD43 reduced the susceptibility to G1 arrest and temporarily retarded the apoptotic process, which, in turn, resulted in an increase and maintenance of the number of viable cells in culture. The results suggest that CD43 may have some role in the survival and expansion of B‐lineage cells. The biological effect of CD43 was initiated without stimulation by cross‐linking and was significantly impaired by replacement of the extracellular domain by the human CD8 extracellular domain. The basis of these regulatory processes is discussed.
Identification of an immunodominant T cell epitope on cholera toxin
Authors: Cong Yingzi , Bowdon Hazel R. , Elson Charles O.
Published: March 2007

Cholera toxin (CT), the enterotoxin of Vibrio cholerae, is a potent mucosal immunogen as well as a strong mucosal adjuvant to related and unrelated antigens. The mucosal immune response to CT is T cell dependent and MHC class II restricted. The epitopes on CT recognized by T cells have not been identified. The purpose of this study was to determine the fine specificity of T cell recognition of both the CT A subunit (CT‐A) and the CT B subunit (CT‐B) by using a range of synthetic peptides. After immunization with CT‐B or CT‐A in CFA subcutaneously, the peripheral lymph node T cells were stimulated with different synthetic peptides in vitro The peptide specificity of T cell recognition was identified by assaying T cell proliferation and interleukin‐3 production. T cells from C57BL/6 (H‐2b) high responder mice recognized one immunodominant epitope (peptide 89–100) and one weak epitope (peptide 31–50) on CT‐B and two epitopes (peptide 21–39 and 180–194) on CT‐A. The immunization of C57BL/6 mice with synthetic immunodominant CT‐B peptide 89–100 induced T cell immunity to the pentameric CT‐B. Induction of tolerance to CTB peptide 89–100 by i.v. injection in high responder C57BL/6 mice induced unresponsiveness to mucosal immunization with CT, compatible with an immunodominant role for this T cell epitope.
Definition of the mitogenic factor (MF) as a novel streptococcal superantigen that is different from streptococcal pyrogenic exotoxins A, B, and C
Authors: Toyosaki Tomoko , Yoshioka Takeshi , Tsuruta Yuji , Yutsudo Takashi , Iwasaki Makoto , Suzuki Ryuji
Published: March 2007

Human T cell activation by recombinant mitogenic factor (rMF) was investigated in comparison with that by recombinant streptococcal pyrogenic exotoxins (rSPE) A, B, and C. Recombinant MF, rSPEA, and rSPEC were mitogenic for peripheral blood mononuclear cells (PBMC), whereas rSPEB was not. Recombinant MF required only HLA‐DR for the stimulation of PBMC, as determined using monoclonal antibodies (mAb) to HLA class II molecules and the mouse L cells transfected with HLA class II molecules. Recombinant SPEA and rSPEC required HLA‐DR or HLA‐DQ molecule. Recombinant MF selectively stimulated Vβ2, Vβ7, Vβ8, Vβ18 and Vβ21‐bearing T cells, whereas rSPEA and rSPEC activated Vβ2 and Vβ6‐bearing T cells as evaluated by the quantitative T cell receptor (TCR) analytical method. No clonality was observed in the nucleotide sequences of complementarity determining region 3 of TCR Vβ in T cells responding to rMF. The profiles of cytokine production by PBMC in response to rMF, rSPEA, and rSPEC were quite similar. In summary, these results demonstrate that both HLA class II molecules and the TCR Vβ required for rMF‐mediated T cell activation are distinct from those required for rSPEA or rSPEC‐mediated activation. Therefore, the MF is a novel streptococcal superantigen which is different from SPEA, SPEB, and SPEC.
Identification of a novel surface molecule, RM3/1, that contributes to the adhesion of glucocorticoid‐induced human monocytes to endothelial cells
Authors: Wenzel Ileana , Roth Johannes , Sorg Clemens
Published: March 2007

The functions of different populations of peripheral blood monocytes in the course of an inflammatory reaction are presently not fully understood. In particular, the mechanisms for their specific recruitment to an inflammatory site are not yet known. We investigated a dexamethasone (Dex)‐inducible monocyte subtype and its adhesion to either unstimulated or lipopolysaccharide (LPS)‐ or interferon (IFN)‐γ‐stimulated human umbilical vein endothelial cells (HUVEC). The Dex‐induced monocytes were characterized by the expression of the surface glycoprotein RM3/1. It was found that pretreatment of monocytes with Dex increased their adhesion to unstimulated and stimulated HUVEC. This increase in adhesion was paralleled by the expression of the RM3/1 surface molecule. Treatment with cyclosporin A (CsA) caused a down‐regulation of the RM3/1 density per cell by 67% and also decreased adhesion to HUVEC. In contrast, the expression of other adhesion molecules remained unaffected by Dex or CsA treatment. Treatment of Dex‐induced monocytes with antibodies against RM3/1, CD11a, CD11b, CD11c, CD14 and CD18 resulted in suppression of adhesion to stimulated HUVEC by 46%, 18%, 17%, 12%, 25% and 15%, respectively. Antibodies to the known adhesion molecules on endothelial cells (vascular cell adhesion molecule‐1, E‐selectin) did not block adhesion of Dex‐induced monocytes. However, the combination of antibodies to RM3/1 and CD14 inhibited adhesion to LPS‐stimulated HUVEC by 74%. These effects were also seen on IFN‐γ‐stimulated HUVEC, where adhesion of Dex‐induced monocytes was blocked with antibodies to RM3/1 + CD14 by 63%. From this, it is concluded that the RM3/1‐molecule is a novel surface molecule that contributes to the adhesion of cortisone‐induced monocytes to LPS or cytokine‐stimulated endothelial cells.
The peptide binding motif of the disease associated HLA‐DQ (α 1* 0501, β 1* 0201) molecule
Authors: Vartdal Frode , Johansen Bente H. , Friede Thomas , Thorpe Christopher J. , Stevanović Stefan , Eriksen Jon E. , Sletten Knut , Thorsby Erik , Rammensee Hans‐Georg , Sollid Ludvig M.
Published: March 2007

To identify the binding motifs of peptides which bind to the celiac disease and insulin‐dependent‐diabetes‐mellitus (IDDM)‐associated DQ2 molecule, peptides were eluted from affinity‐purified DQ2 molecules. The eluted peptides were separated by reverse‐phase HPLC. Prominent peptide peaks and the remaining pool of peptides were sequenced by Edman degradation. Truncated variants of eight different peptides with a length of 9–19 amino acids were identified; among them class II‐associated invariant chain peptides (CLIP) and peptides that stem from HLA class I α, HLA‐DQα1*0501, Ig and CD20 molecules. Data from the pool sequencing and the biochemical binding analyses of synthetic variants of an eluted high‐affinity ligand (HLA class I α 46–60), indicate that the side chains of amino acid residues at relative position P1 (bulky hydrophobic), P4 (negatively charged or aliphatic), P6 (Pro or negatively charged), P7 (negatively charged) and P9 (bulky hydrophobic) are important for binding of peptides to DQ2. Computer modeling of the DQ2 with variants of the high‐affinity ligand in the groove suggests that peptides bind to DQ2 through the primary anchors P1, P7 and P9 and making additional advantageous interactions using the P4 and P6 positions.
Characterization of H4: a mouse T lymphocyte activation molecule functionally associated with the CD3/T cell receptor
Authors: Redoglia Valter , Dianzani Umberto , Rojo Josè M. , Portolés Pilàr , Bragardo Manuela , Wolff Henrik , Buonfiglio Donatella , Bonissoni Sara , Janeway Charles A.
Published: March 2007

The monoclonal antibody C398.4A was produced by immunizing Armenian hamsters with the mouse T cell clone D10.G4.1. It recognizes a molecule selectively expressed by activated mouse T cells and was named H4. H4 is expressed on the T cell surface about 24 h after activation and peaks at day 7. By contrast, it is not expressed by resting or activated B cells, macrophages, or fibroblasts. It is also expressed by CD4 or CD8 single‐positive mature thymocytes. Immunoprecipitation showed that H4 is a disulfide‐linked dimer, precipitating as a broad band at about 50–65 kDa under nonreducing conditions and at 25 and 29 kDa under reducing conditions. Deglycosylation of the reduced H4 by N‐glycanase gave rise to a single band of about 21 kDa, suggesting that the two chains may be differentially glycosylated forms of the same protein. The H4 expression pattern and biochemical features, together with cross‐blocking, co‐capping, co‐modulation, and immunoprecipitation preclearing experiments showed that H4 is different from other known co‐stimulatory molecules such as CD69, CD2, Ly‐6, CD25, OX‐40, Mac‐1 and LFA‐1. By in vitro kinase assay, H4 was found to co‐precipitate a tyrosine kinase activity that phosphorylated substrates of about 29 and 25 kDa. Co‐modulation and co‐capping experiments showed that H4 is physically associated with the CD3/T cell receptor. These data suggest that H4 may function as a T cell‐specific co‐stimulatory molecule and play a role in the T cell response when the activation stimulus is limited either because the antigen is only available in low concentration or has a low agonistic activity.
High‐level expression of the ER‐MP58 antigen on mouse bone marrow hematopoietic progenitor cells marks commitment to the myeloid lineage
Authors: De Bruijn Marella F. T. R. , Ploemacher Rob E. , Mayen Angelique E. M. , Voerman Jane S. A. , Slieker Walentina A. T. , van Ewijk Willem , Leenen Pieter J. M.
Published: March 2007

Studies on the early events in the differentiation of the nonspecific immune system require the identification and isolation of myeloid‐committed progenitor cells. Using the monoclonal antibodies (mAb) ER‐MP12 and ER‐MP20, generated against immortalized macrophage precursors, we have shown previously that the earliest macrophage colony‐stimulating factor (M‐CSF)‐responsive cells in the bone marrow have the ER‐MP12hi20− phenotype. In addition, we found that the ER‐MP12hi20− subset (comprising about 2 % of total nucleated marrow) contains progenitor cells of all hematopoietic lineages. Aiming at the identification and purification of the myeloid progenitor cells within the ER‐MP12hi20− subset, we used ER‐MP58, a marker expressed at high level by all M‐CSF‐responsive bone marrow progenitors. With this marker the ER‐MP12hi20− cell population could be divided into three subfractions: one with absent or low level ER‐MP58 expression, one with intermediate, and one with high ER‐MP58 expression. These subfractions were isolated by fluorescence‐activated cell sorting and tested in vitro and in vivo for their differentiation capacities. In addition, the expression of ER‐MP58 on stem cell subsets was examined in the cobblestone area‐forming cell (CAFC) assay. Our data indicate that in the ER‐MP12hi20− subpopulation myeloid‐committed progenitors are characterized by high‐level expression of the ER‐MP58 antigen, whereas cells with other or broader differentiation capacities have an ER‐MP58 negative/low or intermediate phenotype. These myeloid‐committed progenitors have no significant repopulating ability in vivo, in contrast to the ER‐MP58 intermediate cells. Primitive CAFC‐28/35, corresponding to cells providing long‐term hematopoietic engraftment in vivo, also did not express the ER‐MP58 Ag at a high level. Thus, cells committed to the myeloid lineage can be separated from progenitor cells with other differentiation capacities by means of multiparameter cell sorting using ER‐MP58 in combination with ER‐MP12 and ER‐MP20.