bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific binding of Fyn and phosphatidylinositol 3‐kinase to the B cell surface glycoprotein CD19 through their src homology 2 domains
Authors: Chalupny N. Jan , Aruffo Alejandro , Esselstyn James M. , Chan Po‐Ying , Bajorath Jürgen , Blake James , Gilliland Lisa K. , Ledbetter Jeffrey A. , Tepper Mark A.
Published: March 2007

CD 19 is a B cell surface protein capable of forming non‐covalent molecular complexes with a number of other B cell surface proteins including the CD21/CD81/Leu‐13 complex as well as with surface immunoglobulin. CD19 tyrosine phosphorylation increases after B cell activation, and is proposed to play a role in signal transduction through its cytoplasmic domain, which contains nine tyrosine residues. Several second messenger proteins have been shown to immunoprecipitate with CD 19, including p59 Fyn (Fyn), p59 Lyn (Lyn) and phosphatidylinositol‐3 kinase (PI‐3 kinase). These associations are predicted to occur via the src‐homology 2 (SH2) domains of the second messenger proteins. Two of the cytoplasmic tyrosines in the CD 19 cytoplasmic region contain the consensus binding sequence for the PI‐3 kinase SH2 domain (YPO4‐X‐X‐M). However, the reported consensus binding sequence for the Fyn and Lyn SH2 domains (YPO4‐X‐X‐I/L) is not found in CD 19. We investigated the capacity of CD 19 cytoplasmic tyrosines to bind both Fyn and PI‐3 kinase SH2‐domain fusion proteins. In activated B cells, both Fyn and PI‐3 kinase SH2‐domain fusion proteins precipitate CD 19. Using synthetic tyrosine‐phosphorylated peptides comprising each of the CD 19 cytoplasmic tyrosines and surrounding amino acids, we investigated the ability of the Fyn SH2 and PI‐3 kinase SH2 fusion proteins to bind to the different CD 19 cytoplasmic phosphotyrosine peptides. ELISA revealed that the two CD 19 cytoplasmic tyrosine residues contained within the Y‐X‐X‐M sequences (Y484 and Y515) bound preferentially to the PI‐3 kinase SH2‐domain fusion proteins. Two different tyrosines (Y405 and Y445) bound preferentially to the Fyn SH2‐domain fusion protein via a novel sequence, Y‐E‐N‐D/E, different from that previously reported for the Fyn SH2 domain. In precipitation studies, peptide Y484 was able to compete with tyrosine phosphorylated CD 19 specifically for binding to the PI‐3 kinase SH2 domain fusion proteins, while peptides Y405 and Y445 were able to compete specifically for binding to the Fyn SH2 domain fusion proteins. These results indicate that CD19 may be capable of binding both Fyn and PI‐3 kinase concurrently, suggesting a mechanism for CD 19 signal transduction, in which binding of PI‐3 kinase to the Fyn SH3 domain results in activation of PI‐3 kinase.
Interleukin‐10 inhibits IgE‐mediated nitric oxide synthase induction and cytokine synthesis in normal human keratinocytes
Authors: Bécherel Pierre‐André , Le Goff Liliane , Ktorza Sandra , Ouaaz Fateh , Mencia‐Huerta Jean‐Michel , Dugas Bernard , Debré Patrice , Mossalayi M. Djavad , Arock Michel
Published: March 2007

Human keratinocytes (HK) generate nitric oxide (NO) and proinflammatory mediators following activation with either IgE/anti‐IgE immune complexes or a combination of lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ). Recently, interleukin‐10 (IL‐10) has been shown to down‐regulate various inflammatory responses and to be secreted by lymphocytes and dendritic cells during skin inflammatory reactions. We show here that IL‐10 down‐regulates the production of tumor necrosis factor (TNF)‐α and IL‐6 by activated HK. Also, induction of inducible nitric oxide synthase (iNOS) expression in HK by IgE/anti‐IgE or LPS/IFN‐γ is significantly reduced by the addition of IL‐10. This effect is dose dependent and correlates with reduction of iNOS mRNA production and enzyme level. Therefore, IL‐10 down‐regulates NO‐mediated HK inflammatory responses and may thus participate in the regulation of the skin immune network.
Clonal deletion of major histocompatibility complex class I‐restricted CD4+CD8+ thymocytes in vitro is independent of the CD95 (APO‐1/Fas) ligand
Authors: Müller Klaus‐Peter , Mariani Sara M. , Matiba Bernd , Kyewski Bruno , Krammer Peter H.
Published: March 2007

The CD95 (APO‐1/Fas) ligand (CD95L) mediates apoptosis in sensitive target cells, Ca2+‐independent cytotoxicity of cells from perforin knock‐out mice, and peripheral deletion of activated T cells through engagement of its cognate receptor CD95. Double‐positive thymocytes show a high constitutive expression of CD95. Therefore, we used a model system and investigated whether negative selection through apoptosis might involve CD95/CD95L. We analyzed whether CD95L may induce antigen‐specific deletion of double‐positive thymocytes from mice transgenic for a lymphocytic choriomeningitis virus (LCMV)/H2b‐specific T cell receptor (TCR). These cells are deleted in vitro upon addition of the LCMV‐peptide 33–41 in a major histocompatibility complex‐class I‐restricted fashion. Deletion was not blocked by soluble mouse and human CD95‐Fc receptor decoys. CD95‐Fc receptor decoys, however, were effective in blocking apoptosis induced by mouse CD95L‐transfected L929 cells in sensitive CD95+ target cells and in thymocytes. These results suggest that TCR‐induced deletion of immature thymocytes in vitro is independent of CD95L. Thus, our data argue against a role of CD95L in negative selection of MHC‐class I‐restricted autoreactive thymocytes.
β2‐microglobulin with an endoplasmic reticulum retention signal increases the surface expression of folded class I major histocompatibility complex molecules
Authors: Solheim Joyce C. , Johnson Nancy A. , Carreno Beatriz M. , Lie Wen‐Rong , Hansen Ted H.
Published: March 2007

With β2‐microglobulin− (β2m−) cell lines such as R1E/Db, the surface expression of class I major histocompatibility complex molecules is greatly impaired, and class I molecules that are on the surface are generally misfolded. To determine whether β2m must be continually present with the class I heavy chain for the class I molecule to reach the surface in a folded conformation, a sequence encoding an endoplasmic reticulum (ER) retention signal (KDEL) was attached onto the 3′ end of a β2m cDNA. After this chimeric cDNA was transfected into R1E/Db cells, β2m‐KDEL protein was detectable by an anti‐β2m serum within the cells but not at the cell surface. Interestingly, R1E/Db cells transfected with β2m‐KDEL were found to express a high level of conformationally correct Db molecules at the cell surface. This observation implies that β2m has a critical and temporal role in the de novo folding of the class I heavy chain. We propose that the critical time for β2m association is when the class I molecule is docked with the transporter associated with antigen processing (TAP) and first interacts with peptide.
Staphylococcal enterotoxin B and tumor‐necrosis factor‐α‐induced relapses of experimental allergic encephalomyelitis: Protection by transforming growth factor‐β and interleukin‐10
Authors: Crisi Giovanna M. , Santambrogio Laura , Hochwald Gerald M. , Smith Sidney R. , Carlino Joseph A. , Thorbecke G. Jeanette
Published: March 2007

A study was made of the ability of the superantigen staphylococcal enterotoxin B (SEB) to induce relapses of experimental allergic encephalomyelitis (EAE) in SJL mice that had partially or completely recovered from acute EAE. We find that a single injection of 0.05 mg SEB i.v. induces mild relapses in 50% of such mice. In addition, tumor necrosis factor (TNF)‐α (0.2 μg, i.p.) also induces EAE relapses in 43% of SJL mice when injected 1–2 months after recovery. SEB does not induce a second relapse if reinjected when Vβ17a+ T cells are still partially deleted. In these mice, however, TNF‐α is equally effective in inducing relapses as in mice that did not receive SEB previously. We showed earlier that transforming growth factor (TGF)‐β and TNF‐α have antagonistic effects on experimental autoimmune diseases; e.g., in spontaneously relapsing EAE, TGF‐β and anti‐TNF were protective, while anti‐TGF‐β caused disease exacerbation. Interleukin (IL)‐10 is also known to counteract certain TNF effects. We now find that both human IL‐10 and TGF‐β2 lower the incidence of EAE relapses when given simultaneously with SEB or TNF‐α. The protective effect of TGF‐β is significant only against relapses induced by SEB (reduced to 9%), and that of IL‐10 only against relapses induced by TNF (reduced to 0%) with the treatment regimens employed. Neutralizing anti‐TGF‐β does not increase the incidence of SEB‐induced EAE relapses. In contrast, anti‐IL‐10 increases both the incidence and the severity of such relapses. We conclude that TNF production is probably important in causing EAE relapses, but that other aspects of the SEB‐induced reactivation of myelin‐specific T cells also contribute. Furthermore, endogenous IL‐10 rather than TGF‐β production appears to limit the susceptibility to induction of EAE relapses in this model.
Structural and functional analysis of the promoter of the murine Vγ1.1 T cell receptor gene
Authors: Ofir Rivka , Novick Itai , Krup Margalit , Cleveland John L. , Ihle James N. , Weinstein Yacob
Published: March 2007

The expression of the germ‐line gene Vγ1.1‐Cγ4 of the T cell receptor (TcR) γ chain depends on interleukin (IL)‐3 induction in hematopoietic cells, while in T cells, the rearranged gene is expressed constitutively. To understand the mechanism that controls TcRγ gene expression, we cloned and characterized the structure and function of the Vγ1.1‐Cγ4TcR promoter. IL‐3‐dependent cell lines and T cell lines utilized the same transcriptional start sites. In chloramphenicol acetyltransferase (CAT) assays, the minimal 70‐bp promoter confers strong transcriptional activity which is 50–60% of the Moloney long terminal repeat promoter activity. The 500‐bp promoter region linked to the CAT gene exhibits IL‐3 dependency similar to the endogenous TcRγ gene. The immediate 3′ and 5′ flanking sequences inhibit the promoter activity two‐ to fourfold. The promoter lacks an obvious TATA box or CAAT box sequences, but contains a GC box in the untranslated region 3′ to the promoter. The GC box is the core sequence of the element which binds Sp1‐like proteins. Cloning of this Sp1 binding element in front of the thymidine kinase (TK) promoter and mutations generated in this site demonstrate its function as a silencer. Ultraviolet cross‐linking analysis with the Sp1 binding site from the TcRγ promoter revealed binding of a 90–100‐kDa protein in a T cell line (EL‐4) and 40–50 and 90–100‐kDa proteins in FDC‐P1 cells. The possible function of the Sp1‐like protein in silencing the minimal promoter activity is discussed.
Autoreactive T cells in healthy individuals show tolerance in vitro with characteristics similar to but distinct from clonal anergy
Authors: Filion Mario C. , Bradley Amanda J. , Devine Dana V. , Décary Francine , Chartrand Pierre
Published: March 2007

Peripheral tolerance to self antigens has been suspected to play an important role in the regulation of the immune response in humans since autoreactive T cells can be isolated from the peripheral blood of healthy individuals. The mechanism of this tolerance is not known, but a number of groups have shown that autoreactive T cells can be induced to proliferate in vitro by the addition of their specific antigen and exogenous interleukin (IL)‐2. In this report, we present the analysis of autoreactive T cells, isolated from healthy individuals, to the autoantigen GpIIb‐IIIa present on circulating bone‐marrow‐derived cells and on thymic epithelial cells. We found that the response of GpIIb‐IIIa autoreactive T cells in vitro, when stimulated with GpIIb‐IIIa, shares characteristics with the response found for anergic T cells. In response to GpIIb‐IIIa, the GpIIb‐IIIa‐autoreactive T cells are neither able to proliferate nor produce IL‐2 on their own, but do express IL‐2 receptors α on their cell surface and produce IFN‐γ. This state of unresponsiveness can be broken by the addition of exogenous IL‐2 and IL‐7, as in the case of anergic T cells. However, GpIIb‐IIIa‐autoreactive T cells differ from anergic T cells in their capacity to be stimulated by IL‐12 and by their production of IL‐2 mRNA. Interestingly, once the unresponsive state to GpIIb‐IIIa has been broken by the addition of IL‐2, GpIIb‐IIIa autoreactive T cells can produce IL‐2 and proliferate when restimulated by GpIIb‐IIIa alone. Altogether, these results suggest that the tolerance of GpIIb‐IIIa autoreactive T cells from healthy individuals could involve post‐transcriptional regulation of IL‐2 expression.
Fas receptor expression on B‐lineage cells
Authors: Mandik Laura , Nguyen Kim‐Anh T. , Erikson Jan
Published: March 2007

Mice homozygous for the lpr mutation have B and T cell defects and develop autoantibodies, suggesting that lpr plays a role in their genesis. The lpr defect has been identified as a mutation in the apoptosis‐associated Fas receptor (FasR) gene. To begin to define the role of FasR in B cells, we have surveyed FasR expression on B‐lineage cells from early progenitors in the bone marrow through their maturation in the periphery. Contrary to some reports, we found that FasR is expressed on B cells at all stages of their development and is highest on germinal center B cells. FasR is not expressed on lpr/lpr‐derived cells. These data are consistent with the idea that lpr/lpr mice have an intrinsic B cell defect that may be manifested in developing as well as peripheral B cells. An unexpected finding is that B‐1 (CD5) B cells do not constitutively express FasR: FasR becomes detectable on B‐1 B cells only after activation.
Der p I, a major allergen of the house dust mite, proteolytically cleaves the low‐affinity receptor for human IgE (CD23)
Authors: Schulz Oliver , Laing Peter , Sewell Herb F. , Shakib Farouk
Published: March 2007

The nature of the proteases that cleave CD23 in vivo is of considerable interest, but remains unknown. Here, we demonstrate that Der p I, a major allergen of the house dust mite Dermatophagoides pteronyssinus, cleaves CD23 from the surface of cultured human B cells (RPMI 8866 B cell line). The cleavage of the receptor from the B cell surface was associated with a parallel increase in soluble CD23 (sCD23) in the culture supernatant. Furthermore, the proteolytic effect of Der p I was specific for CD23, since none of the other B cell markers tested (CD20, HLA‐DR, CD71 and CD49d) were affected. Labeled antibody experiments and protease inhibition assays clearly demonstrate that Der p I is a cysteine protease that directly cleaves a 25‐kDa fragment of CD23. These data suggest that the cysteine protease Der p I, in addition to being highly immunogenic, may up‐regulate IgE synthesis by virtue of its ability to cleave CD23.
Differential interactions between the immunoglobulin heavy chain μ intron and 3′ enhancer
Authors: Mocikat Ralph , Kardinal Christian , Klobeck Hans‐Gustav
Published: March 2007

The immunoglobulin heavy chain 3′ enhancer may be a novel type of a transcriptional regulation element in as much as its function is position dependent. We show that there are interactions between the μ intron and 3′ enhancer which are differentially regulated depending on the distance between the two elements. Thus, a transcriptional repression is exerted by the 3′ enhancer when juxtaposed to the intron enhancer. Whereas no or only modest synergism between the immunoglobulin μ intron and 3′ enhancer has been reported to date, we show here that the stimulatory effect is substantially increased by extending the distance between the two enhancers. In our stable expression system, the μ intron enhancer insulated the test gene from neighboring chromatin.
Relevance of residue 116 of HLA‐B27 in determining susceptibility to ankylosing spondylitis
Authors: D'amato Mauro , Fiorillo Maria Teresa , Carcassi Carlo , Mathieu Alessandro , Zuccarelli Angelo , Bitti Pier Paolo , Tosi Roberto , Sorrentino Rosa
Published: March 2007

Ankylosing spondylitis (AS) is an autoimmune disorder strongly associated with HLA‐B27. A direct role of B27 molecules in the disease pathogenesis has been postulated, possibly by presenting to T cells an as‐yet unidentified arthritogenic peptide that triggers the autoimmune response. There are nine HLA‐B27 alleles differing from each other at one or more amino acid positions. It is important, for the identification of the arthritogenic peptide, to define which alleles, and therefore which polymorphic positions, predispose to the disease. Here, we report that HLA‐B★2709 is not associated with AS, as it was not found in patients. HLA‐B★2709 differs from the most frequent and disease‐associated HLA‐B★2705 allele for a single substitution (His vs. Asp) at position 116. Amino acid 116 is located at the bottom of the groove where the antigenic peptide sits, and it has been proven to influence the peptide‐binding specificity of HLA class I molecules. The most likely interpretation of these data is that the differences in charge and size that accompany the His‐to‐Asp substitution exclude the acceptance of the arthritogenic peptide.
Epitopes for human CD4+ cells on diphtheria toxin: Structural features of sequence segments forming epitopes recognized by most subjects
Authors: Raju Raghavanpillai , Navaneetham Duraiswamy , Okita David , Diethelm‐Okita Brenda , McCormick Daniel , Conti‐Fine Bianca M.
Published: March 2007

The sequence regions of diphtheria toxin (DTX) recognized by CD4+ T cells of seven healthy humans of different major histocompatibility complex haplotypes were identified. Overlapping synthetic peptides, screening the DTX sequence, were used to test in proliferation assays unselected blood CD4+ cells, or DTX‐specific CD4+ lines propagated by stimulation with DTX of blood mononuclear cells. Blood CD4+ cells and DTX‐specific CD4+ lines gave consistent results. Although each subject had an individual pattern of peptide recognition, six peptide sequences (residues 271–290, 321–340, 331–350, 351–370, 411–430 and 431–450) were recognized by all subjects. In the native DTX molecule, these sequence regions are flanked by sequence loops exposed on the DTX surface. They overlap uncharged segments of the DTX sequence. These structural properties may be general requirements for immunodominance in CD4+ cell sensitization in humans.
Raf‐1 provides a dominant but not exclusive signal for the induction of CD69 expression on T cells
Authors: Taylor‐Fishwick David A. , Siegel Jeffrey N.
Published: March 2007

Stimulation of the T cell antigen receptor (TCR) induces a number of intracellular signaling pathways which lead to the transcription of a variety of new genes. Of the newly synthesized proteins, the earliest to be detected on the cell surface is the type II integral membrane protein CD69. Cross‐linking of this activation antigen induces signaling events related to T cell activation. The proto‐oncogene product Ras has been reported to up‐regulate CD69. However, which of the potential effectors of Ras induces the expression of CD69 has remained unclear. Using transient transfection, we have shown a constitutively active form of the serine/threonine kinase Raf‐1 to be sufficient to induce CD69 expression in human Jurkat T cells. Raf‐1 was further shown to be necessary for PMA‐induced CD69 expression, since transfection of a dominant inhibitory form of Raf‐1 blocked the up‐regulation of CD69 by PMA. In addition, studies with the calcium ionophore ionomycin identified a previously uncharacterized pathway regulating the expression of CD69 in T cells. Elevation of intracellular calcium induced the expression of CD69 in both Jurkat cells and peripheral blood T cells. This effect was sensitive to the immunosuppressive drug cyclosporin A, indicating that calcium‐induced CD69 expression is mediated by the protein phosphatase calcineurin. Taken together, these results define Raf‐1 as the major signaling mediator of CD69 expression in T cells and suggest that multiple mechanisms exist to regulate the level of CD69 expression following TCR stimulation.
The roles of perforin‐ and Fas‐dependent cytotoxicity in protection against cytopathic and noncytopathic viruses
Authors: Kägi David , Seiler Peter , Pavlovic Jovan , Ledermann Birgit , Bürki Kurt , Zinkernagel Rolf M. , Hengartner Hans
Published: March 2007

In vitro, T cell‐dependent cytotoxicity is mediated by two distinct mechanisms, one being perforin‐, the other Fas‐dependent. The contribution of both of these mechanisms to clearance of viral infections was investigated in mice for the noncytopathic lymphocytic choriomeningitis virus (LCMV) and the cytopathic vaccinia, vesicular stomatitis (VSV) and Semliki forest (SFV) viruses. Clearance of an acute LCMV infection was mediated by the perforin‐dependent mechanism without measurable involvement of the Fas‐dependent pathway. For the resolution of vaccinia virus infection and for resistance against VSV and SFV, however, neither of the two pathways was required. These data suggest that perforin‐dependent cytotoxicity mediated by T cells is crucial for protection against noncytopathic viruses, whereas infections with cytopathic viruses are controlled by nonlytic T cell‐dependent soluble mediators such as cytokines (IFN‐γ against vaccinia virus) and neutralizing antibodies (against VSV and SFV).
Peptide anchor residue glycosylation: effect on class I major histocompatibility complex binding and cytotoxic T lymphocyte recognition
Authors: Haurum John S. , Tan Linda , Arsequell Gemma , Frodsham Penny , Lellouch Annemarie C. , Moss Paul A. H. , Dwek Raymond A. , McMichael Andrew J. , Elliott Tim
Published: March 2007

This study extends our previous observation that glycopeptides bind to class I major histocompatibility complex (MHC) molecules and elicit carbohydrate‐specific CTL responses. The Sendai virus nucleoprotein wild‐type (WT) peptide (FAPGNYPAL) binds H‐2Db using the P5‐Asn as an anchor. The peptide K2 carrying a P5 serine substitution did not bind Db. Surprisingly, glycosylation of the serine (K2‐O‐GlcNAc) with N‐acetylglucosamine (GlcNAc), a novel cytosolic O‐linked glycosylation, partially restored peptide binding to Db. We argue that the N‐acetyl group of GlcNAc may fulfil the hydrogen bonding requirements of the Db pocket which normally accomodates P5‐Asn. Glycosylation of the P5‐Asn residue itself abrogated binding similar to K2, probably for steric reasons. The peptide K2‐O‐GlcNAc readily elicited Db‐restricted cytotoxic T lymphocytes (CTL), which did not cross‐react with K2 or WT. However, all Db‐restricted CTL raised against K2‐O‐GlcNAc cross‐reacted strongly with another glycopeptide, K3‐O‐GlcNAc, where the GlcNAc substitution is on a neighboring P4‐Ser. Furthermore, Db‐restricted CTL clones raised against K2‐O‐GlcNAc or K3‐O‐GlcNAc displayed a striking TCR conservation. Our interpretation is that the carbohydrate of K2‐O‐GlcNAc not only mediates binding to Db, but also interacts with the TCR in such a way as to mimic K3‐O‐GlcNAc. This unusual example of molecular mimicry extends the known effects of peptide glycosylation from what we and others have previously reported: glycosylation may create a T cell neo‐epitope, or, conversely, abrogate recognition. Alternatively, glycosylation may block peptide binding to MHC class I and finally, as reported here, restore binding, presumably through direct interaction of the carbohydrate with the MHC molecule.
Interleukin‐4‐producing CD4+ NK1.1+ TCRα/βintermediate liver lymphocytes are down‐regulated by Listeria monocytogenes
Authors: Emoto Masashi , Emoto Yoshiko , Kaufmann Stefan H. E.
Published: March 2007

Experimental infection of mice with the intracellular bacterium, Listeria monocytogenes, provides a paragon model for immune defence dominated by T helper type 1 (Th1) responses. Potent production of interleukin (IL)‐12 by infected macrophages is considered the determining factor in Th1 cell development. In contrast, it is assumed that IL‐4 producers remain virtually unstimulated in listeriosis. In the liver, the major target organ of listeriosis, an unusual T lymphocyte population exists with the intriguing phenotype CD4+NK1.1+ TCRα/βintermediate (TCRα/βint). Here we show that IL‐4‐producing CD4+NK1.1+TCRα/βint liver lymphocytes are down‐regulated early in listeriosis. We assume that curtailment of IL‐4‐producing CD4+NK1.1+TCRα/βint liver lymphocytes promotes unconstrained development of Th1 cells which are central to protection against intracellular bacteria.
Malignant B lymphocytes from non‐Hodgkin's lymphoma induce allogeneic proliferative and cytotoxic T cell responses in primary mixed lymphocyte cultures: An important role of co‐stimulatory molecules CD80 (B7‐1) and CD86 (B7‐2) in stimulation by tumor cells
Authors: Plumas Joël , Chaperot Laurence , Jacob Marie‐Christine , Molens Jean‐Paul , Giroux Claudine , Sotto Jean‐Jacques , Bensa Jean‐Claude
Published: March 2007

We analyzed the stimulating capacities of malignant B cells from non‐Hodgkin's lymphomas (NHL) to induce an allogeneic response in primary mixed lymphocyte reaction (MLR). T cells purified from a single healthy donor (KS) were used to compare the responses induced by either malignant or hyperplastic cells. Malignant B cells induced strong proliferation of KS cells independently of their level of expression of adhesion molecules. The KS cells after MLR were predominantly CD3+, CD25+, HLA‐DR+, Ki67+ and CD45RO+ T cells, and the CD4/CD8 ratio was heterogeneous (from 0.8 to 2.7). To investigate the role of co‐stimulatory molecules CD80 and CD86 for the stimulatory capacities of B cells, the expression of both molecules was analyzed before and during the MLR. Most fresh malignant B cells were negative for CD80 and CD86, whereas co‐cultured B cells expressed high levels of both molecules. This expression was crucial for T cell proliferation, since monoclonal antibodies directed against CD80 and CD86 completely abrogated the MLR. We also report that KS responding cells at the end of co‐culture were able to lyse fresh B cells used as stimulator cells to different extents (from 10 to 51%), and the level of lysis was enhanced after PMA activation of the target cells. Inhibition experiments using CD8 and CD4 mAb showed that effector cells were mainly CD8+. This report is the first to describe the accessory function of human malignant B cells from NHL and their sensitivity to lysis mediated by CD8+ T cells, and suggests new strategies for the development of antitumor immunity in NHL.
Down‐regulation of class II major histocompatibility complex molecules on antigen‐presenting cells by antibody fragments
Authors: Vidović Damir , Falcioni Fiorenza , Siklodi Botond , Belunis Charles J. , Bolin David R. , Ito Kouichi , Nagy Zoltan A.
Published: March 2007

Certain HLA class II‐specific monoclonal antibodies (mAb) cause up to 90% decrease in the cell surface expression of class II molecules. This down‐regulation is isotype‐specific, i.e. DR‐specific mAb do not affect the expression of DP and DQ molecules. However, antibodies binding to one DR allotype down‐regulate both allotypes in heterozygous antigen‐presenting cells (APC), indicating that the phenomenon is not a direct consequence of ligation. All down‐regulating mAb identified recognize the first (peptide binding) domains of class II heterodimers, and strongly inhibit the activation of class II‐restricted human T cells in vitro. Conversely, non‐down‐regulating mAb fail to inhibit T cell activation, and most of them (four out of five) recognize class II second domains. Down‐regulating antibodies are cytotoxic for B lymphoblastoid cell lines and for a small proportion of normal activated B cells. Their F(ab′)2 fragments mediate both down‐regulation and cytotoxicity, whereas the monovalent Fab fragments are not cytotoxic, but retain the down‐regulatory and T cell inhibitory properties. These findings raise the possibility of a class II major histocompatibility complex‐specific, antibody‐based immunosuppressive therapy without cytotoxic side effects.
Precursor cells to CD3‐intermediate (CD3int) liver mononuclear cells in the adult liver: Further evidence for the extrathymic development of CD3int liver mononuclear cells
Authors: Kenai Hiroyuki , Matsuzaki Goro , Lin Tesu , Yoshida Hiroki , Nomoto Kikuo
Published: March 2007

Liver mononuclear cells (LMNC) can be divided into CD3‐high positive (CD3hi), CD3‐intermediate positive (CD3int) and CD3− populations. CD3int LMNC, but not CD3hi LMNC, are considered to be extrathymically derived because they are found in the liver of nude mice and adult thymectomized lethally irradiated bone marrow chimeras. CD3int LMNC express NK1.1 and show a skewed T cell receptor (TCR) V region repertoire with relatively high levels of Vβ8 and Vα14 expression, further suggesting that they belong to a different lineage from conventional T cells, which lack NK1.1 expression and V region skewing. Since the liver has been proposed to be a site of extrathymic T cell differentiation in adult mice, we analyzed LMNC for the presence of T cell precursors. CD3− LMNC contained CD4−CD8− cells and CD4lo CD8− cells. Reverse transcription‐polymerase chain reaction analysis showed both CD3− populations express the recombination‐activating gene −1, which is indispensable in gene rearrangement of TCR and expressed by thymic T cell precursors. Furthermore, when electronically sorted CD3− LMNC were cultured in medium without any feeder cells or exogenously added cytokines for 24 h, CD3int cells, but not CD3hi cells, appeared. These results suggest that the adult liver contains T cell precursors that lack the expression of the CD3/TCR complex, but are strongly committed to differentiate into extrathymic liver CD3int T cells.
B1 and B2 cells differ in their potential to switch immunoglobulin isotype
Authors: Tarlinton David M. , McLean Michelle , Nossal Gustav J. V.
Published: March 2007

The ability of purified B1a (Ly‐1B) and B2 cells to switch immunoglobulin isotype was assessed by limiting dilution analysis in two in vitro culture systems. When stimulated in the presence of interleukins‐4 and −5 by either lipopolysaccharide or CD40 ligand, the frequency of IgG1 precursors in the B1a population was at most one third that of IgM precursors. In B2 cells, however, the frequency of IgG1 precursors was up to seven times that of IgM precursors. B1a cells were shown to respond to interleukin‐4 by virtue of up‐regulating major histocompatibility complex class II expression when exposed to the cytokine, precluding non‐responsiveness as a reason for not switching to IgG1. Indeed, interleukin‐4 was found to specifically induce transcription of the germ‐line IgG1 constant region locus in B1a cells as it did in B2 cells. Collectively these results suggest that the ability of B1 cells to respond to isotype switch commitment factors such as interleukin‐4 may be secondary to the production of IgM by these cells.
Interleukin‐2 down‐modulates memory T helper lymphocyte development during antigenic stimulation in vitro
Authors: Bemer Véronique , Motta Iris , Perret Raymond , Truffa‐Bachi Paolo
Published: March 2007

Using an in vitro antigenic stimulation model of murine spleen cells in the presence of the immunosuppressor cyclosporin A (CSA) we have previously reported that not only does this drug not interfere with the differentiation of T lymphocytes into memory cells it appears to favor this differentiation (Motta, I. et al., Eur. J. Immunol. 1991. 21: 551). Because CSA blocks interleukin‐2 (IL‐2) gene expression, we have analyzed the effect of this cytokine on memory T helper cell development. Murine splenic cells were primed for 6 days with sheep red blood cells (SRBC) in protocols in which either IL‐2 was not produced or its biological activity was neutralized by anti‐IL‐2 receptor (R) antibodies. The helper function of the recovered T cells was revealed by their capacity to help virgin B splenocytes produce anti‐SRBC antibodies upon challenge in vitro. We found that CD4+ cells primed in the absence of IL‐2, provoked either by IL‐2 gene transcription blockade by CSA or by treatment with anti‐IL‐2R antibodies, afford the best helper functions. These cells exhibit a memory‐type phenotype characterized by the low expression of the MEL‐14 marker and the high expression of the CD44 marker. Evidence is also presented that memory T helper cells originate in part from naive subset displaying the MEL‐14hi phenotype. The pattern of expression of the genes encoding different cytokines (IL‐2, IL‐4, IL‐5 and interferon‐γ) following a secondary antigenic stimulation shows that the helper function of the cells primed in the absence of IL‐2 correlates with the upregulation of the IL‐2 and the IL‐5 genes. From these data, we conclude that IL‐2 plays a major role in the control of memory T helper cell induction.
The Yaa gene‐mediated acceleration of murine lupus: Yaa− T cells from non‐autoimmune mice collaborate with Yaa+ B cells to produce lupus autoantibodies in vivo
Authors: Fossati Liliane , Sobel Eric S. , Iwamoto Masahiro , Cohen Philip L. , Eisenberg Robert A. , Izui Shozo
Published: March 2007

The BXSB Y chromosome‐linked mutant gene, Yaa, promotes autoimmune responses in mice predisposed to a lupus‐like autoimmune disease. We have previously shown that a cognate interaction of T cells with B cells expressing the Yaa gene appears to be responsible for the accelerated production of autoantibodies. To investigate whether T cells that provide help for autoantibody production by Yaa+ B cells need to express the Yaa gene, we have made radiation bone marrow chimeras containing two sets of T and B cells from mice with or without the Yaa gene and differing by the Thy‐1 and Igh allotypes. We then determined autoantibody production following the selective elimination of T cells of Yaa+ origin by treating mice with allele‐specific anti‐Thy‐1 monoclonal antibody. Our results demonstrated that the selective production of autoantibodies by Yaa+ B cells in Yaa+‐Yaa− double bone marrow chimeras can be mediated as efficiently by T cells from non‐autoimmune mice lacking the Yaa gene as by T cells from autoimmune mice bearing the Yaa gene. This indicates that T cells from non‐autoimmune Yaa− mice are capable of providing help for autoimmune responses by collaborating with Yaa+ B cells. These data thus strongly suggest that the Yaa gene defect is not functionally expressed in T cells, but only in B cells, and contrast with parallel experiments in the lpr model, in which defects of the Fas antigen in both T and B cells are crucial for the lpr gene‐mediated promotion of autoantibody production.
Bacterial superantigen specificities of mouse T cell receptor Vβ20
Authors: de Alba Yolanda Bravo , Cazenave Pierre‐André , Marche Patrice N.
Published: March 2007

The study of the mouse T cell receptor (TcR) β chain repertoire in BALB/c thymocytes led to the identification of the Vβ20 gene segment. The expression of Vβ20 estimated at the transcriptional level differs among mouse strains, suggesting clonal deletion. In the present study, we reconstituted by transfection functional TcR using the Vβ20 segment with different Vα segments and studied the action of superantigen toxins. The Vβ20‐transfectant T cells are activated by staphylococcal enterotoxins A and E (SEA and SEE) but not by the other tested toxins. The activation is dependent on the presence of cells expressing major histocompatibility complex class II molecules. Different HLA DR alleles can present the bacterial toxins, establishing that they interact with TcRVβ20 as super‐antigens. Moreover, the Vα domain associated with the Vβ20 domain has an influence on the response to these toxins. The fact that Vβ20 is recognized by SEA and SEE, although both toxins are known to interact with different sets of Vβ, suggests the presence of different TcR binding sites on the toxins.
Long‐term humoral and cellular immunity induced by a single immunization with replication‐defective adenovirus recombinant vector
Authors: Juillard Véronique , Villefroy Pascale , Godfrin Dominique , Pavirani Andrea , Venet Alain , Guillet Jean‐Gerard
Published: March 2007

This study examines the suitability of replication‐defective adenovirus vectors for engineering recombinant vaccines. The immunological abilities and limitations of E1‐deleted adenoviruses containing the lac Z gene (Ad‐β‐gal) were investigated by examining the humoral and cellular immune responses to the β‐galactosidase protein. BALB/c mice (H‐2d) were given in a single injection of recombinant adenovirus. The cytotoxic T lymphocyte (CTL) response of spleen cells was evaluated. Recognized target cells were H‐2d‐derived tumor cells transfected by the lac Z gene, or incubated with the 876–884 β‐galactosidase peptide known to be restricted by the Ld molecule of the major histocompatibility complex. A long‐lasting β‐galactosidase‐specific cytotoxic T cell response was obtained. By contrast, CTL from mice immunized with the Ld‐restricted peptide were less specific for the endogenous epitope presented by the transfectants expressing β‐galactosidase. Ad‐β‐gal‐immunized mice were also protected against an intra‐cerebral challenge with a recombinant vaccinia virus expressing the lac‐Z gene. These results suggest that Ad‐β‐gal‐induced CTL have protective abilities in vivo. The induction of β‐galactosidase‐specific T helper lymphocytes and humoral IgG responses were also examined. A proliferative response occurred only late after immunization and the primed T lymphocytes produced interleukin‐2, but no interleukin‐4. A humoral IgG response to the β‐galactosidase protein was detected 15–30 days after a single immunization and remained stable for 6 months without boosting. Lastly, we followed the evolution of the immune response over the course of successive immunizations. The magnitude and kinetics of the cellular and humoral responses were similar to those obtained after a single immunization. Consistent with these observations, an adenovirus‐specific neutralizing antibody response was detected as early as the second immunization. Thus, a single immunization with a replication‐defective adenovirus recombinant vector induces long‐lasting humoral and cellular immune responses specific to the transgene product.
IgD class switching: Identification of a novel recombination site in neoplastic and normal B cells
Authors: Kluin Philip M. , Kayano Hidekazu , Zani Valter J. , Kluin‐Nelemans Hanneke C. , Tucker Philip W. , Satterwhite Ed , Dyer Martin J. S.
Published: March 2007

IgD on normal B lymphocytes usually is co‐expressed with IgM. A minority of normal plasma cells and rare B cell malignancies express exclusively IgD (IgM−‐IgD+). The low frequency has been explained by the lack of a recognizable switch region within the Cμ‐Cδ intron. We analyzed four cases of IgM−IgD+ hairy cell leukemia (HCL) by Southern (DNA) blot analysis and identified two cases with a recombinatorial event within the Cμ‐Cδ intron and deletion of Cμ. DNA sequence analysis of junctional regions showed that Sμ or the immediate upstream region was used as a donor site and that the Cμ‐Cδ intronic σδ region was used as acceptor site. Using polymerase chain reaction, we subsequently analyzed whether similar Sμ‐σδ recombinations occur in normal tonsils containing IgM−IgD+ plasma cells. Multiple products with a size range of 200–800 base pairs were detected in all four individuals, suggesting clustering of acceptor sites within σδ. Sequence analysis of three cloned products showed Sμ‐σδ recombinations similar those observed in HCL. The σδ region contains a relatively high content of pentameric repeats with an extremely G‐rich area and appears to function as a vestigial switch recombination site in normal and neoplastic IgM−‐IgD+ B cells.
Regulation of Fas(Apo‐1/CD95)‐ and perforinmediated lytic pathways of primary cytotoxic T lymphocytes by the protooncogene bcl‐2
Authors: Schröter Michael , Lowin Bente , Borner Christoph , Tschopp Jürg
Published: March 2007

Cytotoxic T cells (CTL) induce cell death of their target cells either by the surface interaction between Fas ligand and Fas or by the release of perforin and granzymes. Both lytic pathways induce apoptosis yet it is not known whether identical or distinct apoptotic pathways are activated. The protooncogene bcl‐2 is known to protect various hematopoietic cells from apoptosis induced by diverse agents. Here we show that overexpression of the Bcl‐2 protein in the murine mastocytoma line P815 or in concanavalin A‐activated splenocytes suppresses apoptotic cell death induced by allospecific primary cytotoxic T lymphocytes (CTL) in which only the Fas lytic pathway was functional. Bcl‐2 also reduced target cell killing induced by CTL whose lytic activity was dependent on the perforin/granzyme pathway only. These data provide evidence that, in the target cells studied here, both perforin/granzyme and Fas apoptotic pathways are modulated by Bcl‐2 and suggest that these two pathways converge at a step prior to Bcl‐2 inhibition.
Transdermal immunization with large proteins by means of ultradeformable drug carriers
Authors: Paul Amla , Cevc Gregor , Bachhawat Bemal K.
Published: March 2007

By means of novel, ultradeformable and self‐optimizing agent carriers called transfersomes, large molecules can be brought into the body through intact permeability barriers. This permits non‐invasive immunization through normal skin and gives rise to a similar or even slightly higher antibody titer than subcutaneous injections of the same immunogen formulation. The former type of immunization also results in a higher IgA/IgG ratio in the blood than the repeated immunogen injections, as shown here for a soluble protein, human serum albumin, as well as for an integral membrane protein, gap junction protein, in mice.
Enhanced inflammatory responses of chronic granulomatous disease leukocytes involve ROS‐independent activation of NF‐κB
Authors: Bylund Johan , MacDonald Kelly L. , Brown Kelly L. , Mydel Piotr , Collins L. Vincent , Hancock Robert E. W. , Speert David P.
Published: March 2007

Reactive oxygen species (ROS) generated by the cellular NADPH‐oxidase are crucial for phagocytic killing of ingested microbes and have been implicated as signaling molecules in various processes. For example, ROS are thought to be involved in activation of the transcription factor NF‐κB, central for mediating production of proinflammatory cytokines in response to inflammatory stimuli. Several studies have demonstrated that inhibitors of the NADPH‐oxidase interfere with NF‐κB activation and production of proinflammatory cytokines. Curiously, patients with chronic granulomatous disease (CGD), an immunodeficiency characterized by an inability to produce ROS, are not only predisposed to severe infections, but also frequently develop various inflammatory complications indicative of exaggerated inflammatory responses. Here, we show that human CGD leukocytes display a hyperinflammatory phenotype with increased production of proinflammatory cytokines in response to stimulation with Toll‐like receptor agonists. The hyperinflammatory phenotype was also evident in mononuclear cells from CGD mice (gp91phox–/–), but not in control cells in the presence of NADPH‐oxidase inhibitor diphenyleneiodonium, probably reflecting NADPH‐oxidase‐independent effects of the inhibitor. Furthermore, we show that the major steps involved in NF‐κB activation were intact in human CGD cells. These data indicate that ROS were nonessential for activation of NF‐κB and their production may even attenuate inflammation.
Regulated expression of the inhibitory receptor LAIR‐1 on human peripheral T cells during T cell activation and differentiation
Authors: Jansen Christine A. , Cruijsen Carin W. A. , de Ruiter Talitha , Nanlohy Nening , Willems Neeltje , Janssens‐Korpela Pirkko‐Liisa , Meyaard Linde
Published: March 2007

The leukocyte‐associated Ig‐like receptor‐1 (LAIR‐1) is capable of inhibiting immune cell function through interaction with collagens. LAIR is expressed on the majority of peripheral blood mononuclear cells. The abundant expression of both receptor and ligand calls for regulatory mechanisms to relieve the continuous interaction between collagens and LAIR‐1. This regulation may occur at the expression level of the receptor. Here, we report that LAIR‐1 is indeed differentially expressed during human T cell differentiation. Naive CD4+ and CD8+ T cells as well as CD8+ T cells of the effector phenotype express higher levels of LAIR‐1 compared to memory T cells. In vitro stimulation revealed a decrease in LAIR‐1 expression upon activation, and the lower LAIR‐1 expression on CD127– T cells suggests that activation‐induced down‐modulation of LAIR‐1 may also occur in vivo. Furthermore, crosslinking of LAIR‐1 on primary T cells results in an inhibition of T cell function. Our data suggest that regulated expression of LAIR‐1 and the subsequent change in the threshold for activation may be a mechanism to modulate inhibition of the immune system.
Nanocauliflowers: A Nanostructured Polyaniline‐Modified Screen‐Printed Electrode with a Self‐Assembled Polystyrene Template and Its Application in an Amperometric Enzyme Biosensor
Authors: Luo Xiliang , Vidal Gloria Domínguez , Killard Anthony J. , Morrin Aoife , Smyth Malcolm R.
Published: March 2007

A simple method for the nanostructuring of polyaniline (PANI)‐modified screen‐printed electrodes (SPE) was developed. Polystyrene (PS) nanoparticles were self‐assembled onto a PANI‐modified SPE with the help of the negatively charged polyelectrolyte poly(sodium 4‐styrenesulfonate) (PSS) and positively charged poly(diallyldimethylammonium chloride) (PDDA). The self‐assembled PS nanoparticles could then act as a template for the electropolymerization of an additional layer of PANI. It was found that the additional layer of PANI preferred to grow around the PS nanoparticles, and a highly nodular, ‘cauliflower–like’ nanostructure of PANI was thus formed on the electrode surface, which was characterized by scanning electron microscopy. The SPE modified with the nanostructured PANI (nano‐PANI) was further used for the immobilization of horseradish peroxidase (HRP) and developed into a biosensor for hydrogen peroxide. Under optimized conditions, the proposed biosensor exhibited a fast response time of about 5 s, a linear range from 2.5×10−6 to 1.9×10−4 μM with a detection limit of 0.36 μM, and a high sensitivity of 41.0 μA mM−1 due to the benefits of the nanostructured PANI.
Evaluation of Accuracy of Electrochemical Isoflavonoid Index for the Determination of Total Isoflavones in Soy Samples
Authors: Escarpa Alberto , González María Cristina , Blasco Antonio Javier , del Carmen Rogerio Maria , Hervás Miriam
Published: March 2007

The accuracy of a novel electroanalytical route to determine total isoflavones using a secondary standard from Drug Master File (SW/1211/03) as metrological reference with well‐known traceability and its applicability using representative soy samples is demonstrated. Calibration protocols were used i) for choosing a suitable isoflavone standard to determine the total isoflavone content, ii) to evaluate matrix effects and, iii) to evaluate the overall reliability and performance of the method in analytical operations, extraction and analysis. The inherent electroactivity of both, aglycones and glycoside structures and the similar analytical sensitivity exhibited by the prominent soy isoflavones was relevant to determine the total amount with reliability in terms of accuracy (E<10%) and precision (RSDs<7%) in soy samples. In consequence, the introduction of the term isoflavonoid index as the total amount of isoflavones obtained when they are amperometrically monitorized at +1.0 V as a particular case of the electrochemical index concept is proposed.
Amperometric Glucose Biosensing of Gold Nanoparticles and Carbon Nanotube Multilayer Membranes
Authors: Liu Ying , Wu Shuo , Ju Huangxian , Xu Li
Published: March 2007

A novel multilayer gold nanoparticles/multiwalled carbon nanotubes/glucose oxidase membrane was prepared by electrostatic assembly using positively charged poly(dimethyldiallylammonium chloride) to connect them layer by layer. The modification process and membrane structures were characterized by atomic force microscopy, scanning electron microscopy and electrochemical methods. This membrane showed excellent electrocatalytic character for glucose biosensing at a relatively low potential (−0.2 V). The Km value of the immobilized glucose oxidase was 10.6 mM. This resulting sensor could detect glucose up to 9.0 mM with a detection limit of 128 μM and showed excellent analytical performance.