bluuebunny/crossref_metadata_2025 huggingface dataset:
Normal thymic selection, normal viability and decreased lymphoproliferation in T cell receptor‐transgenic CTLA‐4‐deficient mice
Authors: Waterhouse Paul , Bachmann Martin F. , Penninger Josef M. , Ohashi Pamela S. , Mak Tak W.
Published: March 2007

CTLA‐4 is a T cell surface receptor essential for the negative regulation of T cell activation. In the CTLA‐4‐deficient mouse, a dramatic accumulation of activated peripheral T cells effects extensive damage to host tissues, resulting in mortality within 5 weeks of age. To determine whether the accumulation of activated T cells in CTLA‐4−/− mice is due to a defect in thymic selection, we examined negative selection in CTLA‐4−/− mice using two transgenic T cell receptor (TCR) models of thymic selection. Neither the H‐Y‐specific TCR nor the lymphocytic choriomeningitis virus (LCMV)‐specific TCR transgenic models revealed a defect in positive or negative selection in CTLA‐4−/− mice in vivo or in vitro. In fact, the negatively selecting phenotype of male H‐YTCR‐transgenic mice greatly mitigated the accumulation of activated peripheral T cells. Further, peripheral CTLA‐4−/− T cells expressing a single LMCV‐specific transgenic TCR did not have an activated phenotype, indicating that CTLA‐4−/− T cells require specific antigen for proliferation. These results demonstrate that specific antigen is required for the lymphoproliferation observed in CTLA‐4−/− mice, and that CTLA‐4 deficiency does not lead to a gross defect in negative selection.
A recombinant single‐chain human class II MHC molecule (HLA‐DR1) as a covalently linked heterotrimer of α chain, β chain, and antigenic peptide, with immunogenicity in vitro and reduced affinity for bacterial superantigens
Authors: Zhu Xiaojie , Bavari Sina , Ulrich Robert , Sadegh‐Nasseri Scheherazade , Ferrone Soldano , McHugh Louise , Mage Michael
Published: March 2007

Major histocompatibility complex (MHC) class II molecules bind to numerous peptides and display these on the cell surface for T cell recognition. In a given immune response, receptors on T cells recognize antigenic peptides that are a minor population of MHC class II‐bound peptides. To control which peptides are presented to T cells, it may be desirable to use recombinant MHC molecules with covalently bound antigenic peptides. To study T cell responses to such homogenous peptide‐MHC complexes, we engineered an HLA‐DR1 cDNA coding for influenza hemagglutinin, influenza matrix, or HIV p24 gag peptides covalently attached via a peptide spacer to the N terminus of the DR1 β chain. Co‐transfection with DR α cDNA into mouse L cells resulted in surface expression of HLA‐DR1 molecules that reacted with monoclonal antibodies (mAb) specific for correctly folded HLA‐DR epitopes. This suggested that the spacer and peptide did not alter expression or folding of the molecule. We then engineered an additional peptide spacer between the C terminus of a truncated β chain (without transmembrane or cytoplasmic domains) and the N terminus of full‐length DR α chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, β chain, and α chain with the expected molecular mass of approximately 66 kDa. These single‐chain HLA‐DR1 molecules reacted with mAb specific for correctly folded HLA‐DR epitopes, and identified one mAb with [MHC + peptide] specificity. Affinity‐purified soluble secreted single‐chain molecules with truncated α chain moved in electrophoresis as compact class II MHC dimers. Cell surface two‐chain or single‐chain HLA‐DR1 molecules with a covalent HA peptide stimulated HLA‐DR1‐restricted HA‐specific T cells. They were immunogenic in vitro for peripheral blood mononuclear cells. The two‐chain and single‐chain HLA‐DR1 molecules with covalent HA peptide had reduced binding for the bacterial superantigens staphylococcal enterotoxin A and B and almost no binding for toxic shock syndrome toxin‐1. The unique properties of these engineered HLA‐DR1 molecules may facilitate our understanding of the complex nature of antigen recognition and aid in the development of novel vaccines with reduced superantigen binding.
Interleukin‐12 acts as an adjuvant for humoral immunity through interferon‐γ‐dependent and ‐independent mechanisms
Authors: Metzger Dennis W. , McNutt Renee M. , Collins John T. , Buchanan J. Matthew , Van Cleave Victor H. , Dunnick Wesley A.
Published: March 2007

Interleukin‐12 (IL‐12) is a pivotal cytokine that has dramatic effects on cellmediated immunity. It is now becoming increasingly recognized that IL‐12 also strongly controls humoral immunity. We have investigated the mechanism by which IL‐12 induces alterations in antibody isotype expression by determining the influence of IL‐12 on in vitro immunoglobulin (Ig) production in polyclonally activated murine spleen cell cultures. Cells exposed to IL‐12 plus lipopolysaccharide or anti‐CD40 monoclonal antibody showed dramatically elevated IgG2a and suppressed IgG1 production compared to cells cultured in the absence of IL‐12. IL‐12 treatment of spleen cell cultures induced expression of γ2a germ‐line transcripts, consistent with initiation of switch recombination to IgG2a. In addition, exposure of limiting dilution cultures to IL‐12 increased IgG2a+ cell precursor frequency. All of the above results were dependent on interferon‐γ (IFN‐γ). However, in the absence of IFN‐γ, IL‐12 still had significant effects on Ig secretion. Specifically, IL‐12 enhanced IgG1 and IgG2b anti‐DNP antibody levels in mice containing specific disruptions in the IFN‐γ gene. Our results suggest that IL‐12 induces T helper type 1 and natural killer cells to secrete large amounts of IFN‐γ which then causes B cells to switch to IgG2a and IgG3 production. In addition, IL‐12 has direct or indirect effects on B cells that are independent of IFN‐γ. The IFN‐γ‐independent effects may include enhancement of Ig expression by post‐switched cells.
The effects of CD3, CD4 and CD28 signaling on lymphocytes during human immunodeficiency virus‐1 infection
Authors: Guntermann Christine , Zheng Richard , Nye Keith E.
Published: March 2007

We investigated the effects of early human immunodeficiency virus‐1 infection (HIV‐1) on CD4‐ and CD28‐mediated co‐signaling of the T cell receptor (TCR)/CD3 complex in peripheral blood lymphocytes (PBL). CD4 ligation either alone or in conjunction with TCR occupancy resulted in abrogated signaling shown by impaired co‐association of the tyrosine kinase ZAP‐70 with the CD3‐ζ chains in virally infected PBL. In addition, down‐regulation of CD4‐associated TCR signaling resulted in diminished tyrosine phosphorylation of mitogen‐activated protein kinase (MAPK), a serine threonine kinase which is critically involved in the regulation of transcription factors. Furthermore, these aberrant CD4‐driven signals rendered HIV‐1‐infected PBL susceptible to activation‐induced cell death. By contrast, cross‐linking of the TCR/CD3 complex with the CD28 receptor improved tyrosine phosphorylation of MAPK and salvaged infected PBL from activation‐induced cell death. Our data demonstrate the importance of appropriate CD3, CD4 and CD28 co‐stimulatory function to prevent apoptosis. The CD4‐mediated signaling defects of the TCR could contribute to the loss of immunocompetent cells during HIV‐1 infection via activation‐induced cell death, whereas stimulation through the CD28 pathway could reverse these detrimental effects.
Correction of Fas (CD95) deficiency by haploidentical bone marrow transplantation
Authors: Benkerrou Malika , Le Deist Françoise , de Villartay Jean Pierre , Caillat‐Zucman Sophie , Rieux‐Laucat Frédéric , Jabado Nada , Cavazzana‐Calvo Marina , Fischer Alain
Published: March 2007

Recently, an inherited syndrome characterized by nonmalignant lymphoprolif‐eration with autoimmune manifestations, caused by mutations of the Fas (CD95) receptor gene has been described. Because of disease severity, i.e. unremitting lymphoproliferation in a child with complete Fas deficiency, a haploidentical bone marrow transplantation (BMT) was performed despite the known resistance of Fas‐deficient lpr mice to bone marrow transplantation. Marrow graft was rejected early; however, a second attempt using bone marrow from the mother led to engraftment and to control of lymphoproliferation and of autoimmune thrombocytopenia up to the last follow‐up at 24 months after BMT. This single case shows that resistance to bone marrow engraftment caused by survival of Fas‐deficient cells can be overcome.
Ligation of the T cell antigen receptor induces tyrosine phosphorylation of p105CasL, a member of the p130Cas‐related docking protein family, and its subsequent binding to the Src homology 2 domain of c‐Crk
Authors: Kanda Hiroko , Mimura Toshihide , Morino Noritsugu , Hamasaki Ken , Nakamoto Tetsuya , Hirai Hisamaru , Morimoto Chikao , Yazaki Yoshio , Nojima Yoshihisa
Published: March 2007

p105CasL (CasL) is a recently identified signaling molecule closely related to the p130Cas (Crk‐associated substrate) docking protein. CasL has a single Src homology (SH) 3 domain in its N‐terminal portion followed by multiple consensus motifs for binding to SH2 domains. Like original p130Cas, CasL undergoes tyrosine phosphorylation upon integrin‐mediated cell adhesion. In the present report, we provide direct evidence that CasL is also involved in T cell antigen receptor (TcR)‐mediated signal transduction. In binding studies in vitro using glutathione‐S‐transferase fusion proteins, we have identified 105‐ and 120‐kDa phosphotyrosyl proteins (pp105 and pp120, respectively) tightly bound to the SH2 domain of the Crk adapter protein in the H9 human T cell line after stimulation through the CD3/TcR complex. pp120, but not pp105, also bound to the SH3 of another adapter protein, Ash/Grb2. Immunoblotting with specific antibodies revealed that pp120 and pp105 were identical to the c‐cbl proto‐oncogene product (p120cbl) and CasL, respectively. Association between Crk and tyrosine‐phosphorylated CasL after TcR stimulation was also confirmed in vivo. CasL phosphorylation induced by TcR ligation reached maximal levels within 2 min and rapidly declined thereafter, whereas the integrin‐dependent response occurred slowly and was more prolonged. Finally, we demonstrated that Crk/CasL association occurred in peripheral blood T lymphocytes in response to TcR engagement. Our findings suggest that CasL is involved in T cell activation signals and resides at a point where two distinct receptor‐mediated signaling pathways converge. This provides one mechanism by which integrins may mediate T cell co‐stimulation.
An interleukin‐7 internet for intestinal intraepithelial T cell development: Knockout of ligand or receptor reveal differences in the immunodeficient state
Authors: Fujihashi Kohtaro , McGhee Jerry R. , Yamamoto Masafumi , Peschon Jacques J. , Kiyono Hiroshi
Published: March 2007

Both interleukin‐7 (IL‐7) and IL‐7 receptor (R) gene knockout (IL‐7−/− and IL‐7R−/−) mice were employed in order to directly investigate the importance of the IL‐7 and IL‐7R signaling pathway for the development of intestinal intraepithelial lymphocytes (IEL). Loss of the IL‐7R‐specific gene resulted in complete deficiency of the γδ T cell lineage with lack of Vγ4‐ and Vγ7‐specific messages in the epithelium of the gastrointestinal (GI) tract in comparison to control mice of the same genetic background (∼ 40%). Disruption of the IL‐7‐specific gene resulted in marked, but not complete depletion of γδ T cells (2–3%) in IEL. Furthermore, mRNA for both Vγ4 and Vγ7 genes were detected in the γδ IEL subset of IL‐7−/− mice. The subtle differences between IL‐7−/− and IL‐7R−/− mice suggest that although IL‐7 controls most of the expansion and/or development of γδ IEL, another ligand binding to the IL‐7R also plays a discernable role. Furthermore, αβ IEL developed more slowly in IL‐7R−/− mice when compared with ligand knockouts; however, the frequency of IEL T cells subsequently increased with age and normal levels of CD3+ T cells expressing the αβ TCR were detected by 2 and 3 months of age in IL‐7−/− and IL‐7R−/− mice, respectively. The direct comparison of IL‐7 and IL‐7R−/− mice clearly supports the hypothesis that both IL‐7 and another IL‐7R binding molecule can influence the development of γδ T cells in the intestinal epithelium.
Elevated mutant frequencies in gene lacI in splenic lipopolysaccharide blasts after exposure to activated phagocytes in vitro
Authors: Felix Klaus , Lin Sophia , Janz Siegfried
Published: March 2007

The interaction of B lymphocytes with phagocytes is critical for shaping the humoral immune response, as well as various aspects of normal and malignant B cell development, and has therefore been studied by immunologists in great detail. However, one potential outcome of this confrontation is often neglected, namely the mutagenicity of phagocytes to B lymphocytes. We are interested in phagocyte‐induced B cell mutagenesis and have conducted a feasibility study on the utility of a transgenic reporter assay to evaluate mutant frequencies in B cells that have encountered phagocytes. An in vitro co‐incubation system was designed in which splenic lipopolysaccharide (LPS) blasts carrying a phage Δ‐derived lacI transgene were exposed to pristane‐elicited peritoneal exudate cells (PEC). Mutant frequencies in LPS blasts were significantly increased (up to 6‐fold) when the cells were co‐incubated with PEC that had been stimulated by phorbol myristate acetate to undergo an oxidative burst. The lacI‐based transgenic mutation assay proved also useful for assessing mutagenicity in vivo, as demonstrated by the detection of elevated mutant frequencies in the spleen (3‐fold) and the inflammatory granuloma (4.7‐fold) obtained from pristane‐treated mice. We propose to utilize the lacI‐based transgenic mutagenesis assay as a tool to evaluate mutational levels during normal and aberrant B cell differentiation.
Expression and functions of the high‐affinity IgE receptor on human platelets and megakaryocyte precursors
Authors: Joseph Michel , Gounni Abdelillah Soussi , Kusnierz Jean‐Pierre , Vorng Han , Sarfati Marika , Kinet Jean‐Pierre , Tonnel André‐Bernard , Capron André , Capron Monique
Published: March 2007

Platelets can be activated by IgE and are therefore involved in IgE‐mediated effector mechanisms against parasites and in allergic disorders. Here we show that, besides the low‐affinity IgE receptor (FcεRII/CD23), platelets express the high‐affinity receptor for IgE (FcεRI). Flow cytometry analysis revealed the existence of surface FcεRI on platelets with a large heterogeneity among individual donors, and a low proportion of platelets co‐expressing FcεRI and FcεRII/CD23. Northern hybridization and reverse transcription polymerase chain reaction confirmed the presence of mRNA encoding the α, β and γ chains of FcεRI in platelets and in their megakaryocytic precursors. Cross‐linking of FcεRI with monoclonal antibody (mAb) to α chain using either the whole molecule or F(ab′)2 triggered platelet cytotoxicity for Schistosoma mansoni larvae. Anti‐FcεRII/CD23 mAb significantly inhibited IgE‐ or FcεRI‐mediated cytotoxicity, indicating down‐regulatory effects of FcεRII/CD23 on FcεRI‐dependent functions. These results demonstrate functional properties for FcεRI on platelets and indicate unsuspected interactions between the low‐ and the high‐affinity IgE receptors.
Size‐dependent effect of IgA on the IgA Fc receptor (CD89)
Authors: Reterink Thomas J. F. , van Zandbergen Ger , van Egmond Marjolein , Klar‐Mohamad Ngaisah , Morton Craig H. , van de Winkel Jan G. J. , Daha Mohamed R.
Published: March 2007

The IgA Fc receptor (FcR; CD89) is expressed on several types of cells of the myeloid cell lineage. We investigated whether different sizes of heat‐aggregated IgA (aIgA) bind to CD89 and subsequently induce cellular activation. As a model we used the murine B cell line IIA1.6 transfected with CD89 or IIA1.6 cells transfected with CD89 as well as with the FcR γ chain to study the binding of IgA to CD89. When these cells expressing CD89 were incubated with monomeric IgA, no significant binding of IgA to the cells was detectable by fluorescence‐activated cell sorter analysis; however, incubation of the cells with aggregated IgA resulted in 93 ± 2% positive cells. Incubation of the cells with different sizes of IgA‐containing aggregates revealed optimal binding with aggregates containing five to six molecules of IgA per aggregate. No difference was observed between the binding to CD89 of both IgA1‐ or IgA2‐containing aggregates. Furthermore, the binding of aIgA was found to be CD89‐specific, since the binding of IgA was completely inhibited by the CD89‐specific monoclonal antibody My43 and no detectable binding occurred to the IIA1.6 parent cell line. Activation studies using interleukin‐2 (IL‐2) production as a marker, showed that the FcR γ chain is necessary to induce cellular activation. Only cells transfected with both CD89 and the FcR γ chain (CD89+/γ+) enhance the IL‐2 production 10–12‐fold upon stimulation with aggregates of IgA. Furthermore, triggering of CD89 only results in increase of intracellular calcium concentration ([Ca2+]i) in cells co‐expressing FcR γ chain. Mutation of the tyrosine residues in the FcR γ chain immunoreceptor tyrosine‐based activation motif of the FcR γ chain abolishes this increase in [Ca2+]i, indicating association and involvement of the FcR γ chain in CD89‐mediated signaling.
CD2‐mediated signaling in T cells lacking the ζ‐chain‐specific immune receptor tyrosine‐based activation (ITAM) motif
Authors: Steeg Christiane , Von Benin Arne , Mittrücker Hans‐Willi , Malissen Bernard , Fleischer Bernhard
Published: March 2007

T lymphocytes can be activated via the T cell receptor (TCR) or by triggering through a number of other surface structures, including the CD2 co‐receptor molecule. Signaling through the CD2 molecule was shown previously to be dependent on the TCR‐associated ζ‐chain. Here, we show that CD2‐induced activation also functions in T cells which express ζ‐chains lacking a functional immune‐receptor tyrosine‐based activation motif (ITAM). TCR‐positive T cells that express only the transmembrane part of the ζ‐chain protein and thus lack a functional ζ‐derived ITAM readily produce interleukin (IL)‐2 when cross‐linked with CD2‐specific monoclonal antibodies (mAb). TCR‐negative T cell hybridomas expressing minimal receptors consisting of an extracellular CD25 and an intracellular ζ‐chain‐derived segment were effectively stimulated via CD2‐specific mAb. For CD2‐mediated co‐stimulation of TCR‐negative cells, two ζ‐chain‐derived ITAM were sufficient to induce IL‐2 when the CD2 molecules were co‐cross‐linked with the chimeric CD25‐ζ molecules. Taken together, our results show that CD2‐induced signaling does not necessarily employ the ζ‐chain in TCR‐positive cells and that CD2‐dependent co‐stimulation in TCR‐negative cells can be mediated via two functional ζ‐chain‐derived ITAM.
Mycobacterium bovis Bacillus Calmette Guérin infection prevents apoptosis of resting human monocytes
Authors: Kremer Laurent , Estaquier Jérôme , Brandt Eric , Ameisen Jean‐Claude , Locht Camille
Published: March 2007

Apoptosis plays an essential role in the development and homeostasis of multi‐cellular organisms. Some infectious agents interfere with this programmed cell death to their own benefit. Here, we show that infection of resting human monocytes with Mycobacterium bovis Bacillus Calmette Guérin (BCG) increases monocyte viability by preventing them from undergoing apoptosis. Heat‐killed BCG also prevented apoptosis, indicating that replication of BCG is not required to prevent cell death. Analysis of BCG‐infected monocytes revealed an up‐regulation of the A1 mRNA, whereas the bcl‐2 mRNA was not up‐regulated. Interestingly, preinfection with BCG renders the cells resistant to interleukin (IL)‐10‐induced apoptosis which may be one of the mechanisms mycobacteria use to modulate immune responses. BCG infection was also accompanied by an impairment of the capacity of monocytes to secrete IL‐10 and by an induction of the capacity to secrete tumor necrosis factor‐α, two cytokines known to induce and prevent human monocyte apoptosis, respectively. Since it has been reported that apoptosis is involved in killing of intracellular mycobacteria, the prevention of apoptosis may represent a strategy for mycobacterial survival in the infected host.
Overexpression of the death‐promoting gene bax‐α sensitizes human BL‐41 Burkitt lymphoma cells for surface IgM‐mediated apoptosis
Authors: Weinmann Pamela , Bommert Kurt , Mapara Markus Y. , Dörken Bernd , Bargou Ralf C.
Published: March 2007

Major regulators of programmed cell death, or apoptosis, are the members of the bcl‐2 gene family. Recently, we reported that surface(s) IgM triggering of the human B lymphoma cell line BL‐41 led to strong induction of bax‐α, a death‐promoting member of the bcl‐2 family, and subsequently to induction of apoptosis, suggesting a potential regulatory role of bax‐α in sIgM‐mediated cell death. In contrast, apoptosis‐resistant subclones of this cell line showed only weak bax‐α expression, which was not inducible by sIgM cross‐linking. In this study, we were able to demonstrate the functional significance of this observation. We stably transfected bax‐α into a BL‐41 subline resistant against sIgM‐mediated apoptosis. Several bax‐α overexpressing clones could be selected, which all showed enhanced sensitivity for sIgM‐mediated apoptosis. In contrast, no sensitive clone could be identified in a large number of mock controls. This clearly indicates that induction of bax‐α is a critical regulatory step, which sensitizes B cells for sIgM‐mediated apoptosis.
Chemotactic activity on mononuclear cells in the cerebrospinal fluid of patients with viral meningitis is mediated by interferon‐γ inducible protein‐10 and monocyte chemotactic protein‐1
Authors: Lahrtz Fritz , Piali Luca , Nadal David , Pfister Hans‐Walter , Spanaus Katharina‐Susanne , Baggiolini Marco , Fontana Adriano
Published: March 2007

In viral meningitis the inflammatory response involves activated T cells and monocytes which are recruited into the subarachnoid space. To identify the chemotactic signals attracting the cells to the site of infection in the meninges, we measured the levels of two CXC chemokines, interferon‐γ (IFN‐γ) inducible protein (IP)‐10 and monokine induced by IFN‐γ, four CC chemokines, monocyte chemotactic protein (MCP)‐1, RANTES, macrophage inflammatory protein (MIP)‐1α and MIP‐1β, as well as the cytokines interleukin (IL)‐15 and IL‐16 in the cerebrospinal fluid (CSF) of patients suffering from viral meningitis. The results point to an involvement of two chemokines, MCP‐1 and IP‐10, since (1) unlike the other cytokines, MCP‐1 and IP‐10 were present in 97 % and 79 % of the CSF, respectively, at concentrations sufficient to induce chemotaxis of mononuclear cells; (2) more than 90 % of the CSF of viral meningitis induced chemotaxis of peripheral blood mononuclear cells (PBMC) and all of them induced chemotaxis of activated T cells, and (3) the CSF‐mediated chemotaxis of PBMC was inhibited by anti‐MCP‐1 antibodies and chemotaxis of activated T cells was abolished by the combination of anti‐MCP‐1 and anti‐IP‐10 antibodies. Our data provide evidence that MCP‐1 and IP‐10 lead to accumulation of activated T cells and monocytes in the CSF compartment in viral meningitis.
Normal macrophage functions, but impaired induction of γδ T cells, at the site of bacterial infection in CD45 exon 6‐deficient mice
Authors: Fujise Shigeru , Kishihara Kenji , Lee Kyung‐Yil , Matsuzaki Goro , Nomoto Kikuo
Published: March 2007

We investigated the protective functions of macrophages and γδ T cells in adult CD45 exon 6‐deficient (CD45−/−) mice against an intraperitoneal (i.p.) infection with Listeria monocytogenes. γδ T cells are preferentially localized in the spleen, liver, and intraperitoneal cavity of the adult CD45−/− mice. Increased numbers of γδ T cells were observed after i.p. infection with L. monocytogenes in the peritoneal cavity of C57BL/6 (CD45+/+) mice but not in CD45−/− mice. The γδ T cells showed predominant usage of Vδ5 and Vδ6 rearranged to Jδ1 in the infected CD45−/− mice which are the same as those used by resident γδ T cells of noninfected CD45+/+ and CD45−/− mice. Furthermore, we analyzed the protective abilities of the CD45−/−, CD45+/+, and γδ T cell‐depleted mice at the early stage of the listerial infection. The numbers of bacteria in the spleens and livers of the CD45−/− mice 5 days after the listerial infection were almost ten times larger than those in the CD45−/− and γδ T cell‐depleted CD45+/+ mice. Macrophages showed normal antigen presentation, nitric oxide production and bactericidal activity for L. monocytogenes despite their lacking CD45 surface expression, suggesting that CD45‐negative macrophages have a minimal influence on the increased bacterial multiplication in the CD45−/− mice. These results suggest that the γδ T cells are induced by the bacterial infection in a CD45‐dependent manner, and that unresponsiveness of the γδ T cells results in only weak protection against L. monocytogenes in CD45−/− mice.
Induction of anti‐carbohydrate antibodies by phage library‐selected peptide mimics
Authors: Phalipon Armelle , Folgori Antonella , Arondel Josette , Sgaramella Giuseppe , Fortugno Paola , Cortese Riccardo , Sansonetti Philippe J. , Felici Franco
Published: March 2007

One of the prerequisites for the development of polysaccharide subunit vaccines is the induction of an efficient immune response to carbohydrate antigens like lipopolysaccharide (LPS) or capsular polysaccharide antigens of pathogens. In an attempt to overcome the problems that arise from the T‐independent immune response induced by such antigens, selecting peptide sequences that mimic protective carbohydrate epitopes has been proposed. In this study, we investigate a new selection strategy for immunogenic peptide mimics using the phage displayed peptide library technology. Two monoclonal antibodies (mAb) of the A isotype (mIgA), mIgA C5 and mIgA I3, specific for the O‐antigen (O‐Ag) part of the human pathogen Shigella flexneri serotype 5a LPS and protective against homologous infection were used to screen two phage‐displayed nona‐peptide libraries in pVIII. Using mIgA C5, 13 different specific clones were selected, and 6 using mIgA I3; 5 of the latter also interacted in enzyme‐linked immunosorbent assay with the first mAb. All of the 19 clones selected were separately used to immunize mice, but only 2 of them, p100c (mIgA I3‐specific) and p115 (interacting with both mIgA) were able to induce anti‐O‐Ag antibodies. The immune response was specific for the O‐Ag of the S. flexneri serotype 5a, and also selectively recognized the corresponding bacterial strain. The amino acid sequences of p100c and p115 immunogenic peptide mimics were YKPL‐GALTH (flanked by two Cys residues) and KVPPWARTA, respectively. These results are the first example of immunogenic mimicry of carbohydrates by phage‐displayed peptides, and indicate a new strategy of selection of immunogens for the development of anti‐polysaccharide vaccines.
A Leishmania protein that modulates interleukin (IL)‐12, IL‐10 and tumor necrosis factor‐α production and expression of B7‐1 in human monocyte‐derived antigen‐presenting cells
Authors: Probst Peter , Skeiky Yasir A. W. , Steeves Michelle , Gervassi Ana , Grabstein Kenneth H. , Reed Steven G.
Published: March 2007

LeIF, a gene homologue of the eukaryotic initiation factor 4A was first described as a leishmanial antigen that induced a Th1‐type T cell response in peripheral blood mononuclear cells (PBMC) from leishmaniasis patients. Moreover, the interferon (IFN)‐γ production by PBMC was found to be interleukin (IL)‐12 dependent. Herein, we characterize the effects of LeIF on cytokine production and expression of surface molecules by normal human monocytes as well as by monocyte‐derived macrophages and dendritic cells (MoDC). LeIF was a strong inducer of IL‐12 and, to a lesser extent, of IL‐10 and tumor necrosis factor (TNF)‐α in macrophages and MoDC. IL‐12 production did not require CD40 triggering, confirming that the ability of LeIF to induce IL‐12 was not mediated through an effect on T cells. However, addition of soluble CD40 ligand (L) synergistically augmented IL‐12 production in macrophages and MoDC. The cytokine‐inducing activity of LeIF is located in the N‐terminal portion of the molecule and was both proteinase K sensitive and polymyxin B resistant. LeIF, lipopolysaccharide and fixed Staphylococcus aureus all induced comparable amounts of IL‐12, validating the potent cytokine‐inducing effects of LeIF. Moreover, of these stimuli, LeIF had the highest IL‐12/IL‐10 and IL‐12/TNF‐α ratio demonstrating the preference of LeIF for IL‐12 induction. Studies investigating the expression of surface molecules showed that LeIF up‐regulated B7‐1 and CD54 (ICAM‐1) on macrophages and MoDC. To our knowledge this is the first report describing IL‐12 production, up‐regulation of co‐stimulatory and intercellular adhesion molecules by monocytic antigen‐presenting cells in response to a protein from a pathogenic microorganism. These immunomodulatory characteristics of LeIF might be excellent properties for a Th1‐type adjuvant.
Anti‐adhesive signals are mediated via major histocompatibility complex class II molecules in normal and neoplastic human B cells: Correlation with B cell differentiation stage
Authors: Ahsmann Els J. M. , Boom Saskia E. , Lokhorst Henk M. , Rijksen Gert , Bloem Andries C.
Published: March 2007

We show that major histocompatibility complex (MHC) class II molecules on B cells transmit signals which regulate adhesion in a negative manner. Engagement of MHC class II molecules with antibodies results in detachment of B cells previously bound to interferon‐γ‐activated human umbilical cord venous endothelial cells. This process depends on metabolic energy, active signaling and protein tyrosine kinase activity. The adhesion pathway influenced by this signaling event is neuraminidase sensitive. The anti‐adhesive signaling program is activated in B cell lines with a mature phenotype, e.g. normal B cells from spleen and tonsil. In contrast, cell lines with a pre‐B cell phenotype and normal B cells from peripheral blood are refractory to MHC class II‐mediated regulation of adhesion. These results extend to neoplastic cells from patients with lymphopro‐liferative diseases representing different stages of B cell maturation. These results suggest that MHC class II‐mediated signals regulate B cell adhesion in a developmentally programmed fashion; this might have implications for clinical behavior of B cell malignancies.
A re‐evaluation of the effects of X‐linked immunodeficiency (xid) mutation on B cell differentiation and function in the mouse
Authors: Klaus Gerry G. B. , Holman Mary , Johnson‐Léger Caroline , Elgueta‐Karstegl Claudio , Atkins Chris
Published: March 2007

CBA/N (xid) mice have a point mutation in Bruton's tyrosine kinase (btk), which results in their failure to respond to T‐independent type 2 (TI‐2) antigens, and to several B cell mitogens [most notably anti‐immunoglobulin (Ig)] in vitro. They have reduced numbers of peripheral (B2) B cells, which are regarded as being phenotypically and functionally immature. We show here that adult CBA/N mice in fact have two distinct B cell populations: some 60% of the cells are CD23+ HSAlo sIgDhi and hence resemble recirculating, follicular (RF) B cells from normal mice, except that they are sIgMhi. The remaining 40% of xid B cells are CD23− HSAhi sIgD−/lo and resemble immature transitional (TR) B cells. TR B cells from xid mice do not synthesize DNA when cultured with lipopolysaccharide (LPS), whereas those from normal mice do so. Only the RF cells from either xid or normal mice proliferate in response to ligation of CD40. In neonatal normal mice the emergence of mitogen responsiveness followed the chronological sequence LPS → anti‐CD40 → anti‐Ig ≈ anti‐CD38. The same developmental sequence was seen with B cells from xid mice (for LPS and anti‐CD40), but it occurred at a significantly slower tempo and this correlated with the later appearance of RF‐type cells. TR xid B cells express very low levels of bcl‐2 and we conclude that these cells resemble very immature (bone marrow) B cells, rather than normal transitional cells. We, therefore, propose that the xid mutation imposes a multistage brake on B cell differentiation in the mouse. The available data suggest that btk is required for the positive selection of B cells throughout their differentiation in the periphery. This in turn implies that low level signaling via surface Ig is needed throughout this process in order for peripheral B cells to become functionally mature.
Rapamycin inhibits proteasome activator expression and proteasome activity
Authors: Wang Xin , Omura Satoshi , Szweda Luc I. , Yang Young , Bérard Jacques , Seminaro Johnny , Wu Jiangping
Published: March 2007

Rapamycin (RAPA) is a potent immunosuppressive drug, and certain of its direct or indirect targets might be of vital importance to the regulation of an immune response. In this study, we used differential hybridization to search for human genes whose expression was sensitive to RAPA. Seven RAPA‐sensitive genes were found and one of them encoded a protein with high homology to the α subunit of a proteasome activator (PA28α). This gene was later found to code for the β subunit of the proteasome activator (PA28β). Activated T and B cells had up‐regulated PA28β expression at the mRNA level. Such up‐regulation could be suppressed by RAPA, FK506, and cyclosporin A. RAPA and FK506 also repressed the up‐regulated PA28α messages in phytohemagglutinin (PHA)‐stimulated T cells. At the protein level, RAPA inhibited PA28α and PA28β in the activated T cells according to immunoblotting and confocal microscopy. Probably as a consequence, there was a fourfold increase of proteasome activities in the peripheral blood mononuclear cell lysate after the PHA activation. RAPA could inhibit the enhanced part of the proteasome activity. Considering the critical role played by the proteasome in degrading regulatory proteins, our data suggest that the proteasome activator is a relevant and important downstream target of rapamycin, and that the immune response could be modulated through the activity of the proteasome.
Expression of the Ly49A gene in murine natural killer cell clones is predominantly but not exclusively mono‐allelic
Authors: Held Werner , Raulet David H.
Published: March 2007

The Ly49 family of natural killer (NK) cell receptors are major histocompatibility complex (MHC) class I‐specific inhibitory receptors that are distributed to overlapping NK cell subsets. Extending earlier studies of polyclonal NK cell populations, we have employed an analysis of short‐term NK cell clones from Ly49A heterozygous mice, to demonstrate that the Ly49A gene is usually expressed from one or the other allele in each Ly49A+ cell. However, we also detected a small percentage of clones that expressed both Ly49A alleles. The possibility that the colonies exhibiting bi‐allelic Ly49A gene expression had been inoculated with more than one cell was ruled out by parallel analysis of clones isolated from mixtures of NK cells from Ly49A homozygous mice. The frequency of bi‐allelic Ly49A+ clones suggested that the two Ly49A alleles in an NK cell are chosen for expression independently. These data are consistent with the proposal that mono‐allelic Ly49A gene expression may arise as a consequence of a stochastic Ly49 gene activation mechanism. Analysis of Ly49A+ clones from MHC‐different mice demonstrated that class I‐deficient mice harbored a greater number of bi‐allelic Ly49A+ cells than did H‐2d mice, which express a Ly49A ligand. Although the numbers were insufficiently large for a clear assignment, H‐2b mice may harbor an intermediate number of bi‐allelic Ly49A+ NK cells. The effects of MHC expression on the prevalence of bi‐allelic Ly49A+ cells suggest that an MHC‐dependent education process modifies the Ly49 repertoire.
The roles of complement receptors type 1 (CR1, CD35) and type 3 (CR3, CD11b/CD18) in the regulation of the immune complex‐elicited respiratory burst of polymorphonuclear leukocytes in whole blood
Authors: Nielsen Claus H. , Antonsen Steen , Matthiesen Steen H. , Leslie R. Graham Q.
Published: March 2007

The binding of immune complexes (1C) to polymorphonuclear leukocytes (PMN) and the consequent respiratory burst (RB) were investigated in whole blood cell preparations suspended in 75% human serum, using flow cytometry. Blockade of the complement receptor (CR)1 receptor sites for C3b on whole blood cells using the monoclonal antibody (mAb) 3D9 resulted in a 1.9‐fold increase in the IC‐elicited PMN RB after 5 min of incubation, rising to 3.1‐fold after 40 min. This enhancement was not due to increased IC deposition on PMN. Blockade of CR3 abrogated the mAb 3D9‐induced rise in RB activity and inhibited the IC binding to PMN in a whole blood cell preparation, with or without mAb 3D9, by approximately 40% from 15–40 min while reducing their RB over 40 min to approximately one third. Blockade of CR1 on either erythrocytes (E) or leukocytes, before mixing the populations, revealed that the potentiation of the RB by mAb 3D9 was associated with abrogation of E‐CR1 function, whereas blockade of leukocyte‐CR1 had a diminishing effect. Exposure to IC at high concentrations induced release of both specific and azurophilic granule contents from PMN. The latter was CR3 dependent in that blockade of the receptor inhibited the lactoferrin release by one third during 40 min of incubation. In conclusion, CR3 plays a significant role in the IC‐mediated generation of an RB and release of specific granules by PMN, while CR1 on whole blood cells, primarily E CR1, restricts the IC‐elicited RB in PMN. We propose that CR1 in whole blood promotes the degradation of IC‐bound iC3b to C3dg, thereby rendering the IC inaccessible for binding to CR3.
In vivo rolling and endothelial selectin binding of mononuclear leukocytes is distinct from that of polymorphonuclear cells
Authors: Xie Xun , Raud Johan , Hedqvist Per , Lindbom Lennart
Published: March 2007

In inflammation, rolling of leukocytes along the microvascular endothelium is a precondition for subsequent integrin‐mediated firm adhesion and extravasation. Rolling characteristics of polymorphonuclear leukocytes (PMNL) and mononuclear leukocytes (MNL) in small venules (15–25 μm) of the rat mesentery were studied by intravital fluorescence microscopy under basal conditions and after intravenous treatment with an anti‐rat neutrophil serum (ANS). The baseline rolling fraction of the venular total leukocyte flux was 36 ± 15% (mean ± SD). The PMNL fraction of the systemic leukocyte count was 27 ± 9%. Treatment with ANS resulted in total depletion of circulating PMNL and reduced the leukocyte rolling fraction to 12 ± 5%, in this situation represented only by MNL. In rats treated intraperitoneally with interleukin (IL)‐1β for 4 h, the leukocyte rolling fraction was 53 ± 13% and was reduced to 33 ± 11% after ANS treatment. These data indicated that most, if not all, circulating PMNL rolled along the venular endothelial lining in the rat mesentery prepared for intravital microscopy, whereas MNL rolling was minor (∼ 10%) under the same basal condition. In cytokine‐activated tissue, on the other hand, the number of rolling MNL was greatly increased. While PMNL rolling is known to be entirely selectin dependent, the increased MNL rolling after IL‐1 stimulation was likely mediated by α4 integrins, inasmuch as the rolling fraction of isolated peripheral blood lymphocytes injected into the microcirculation of the cytokine‐stimulated mesentery was reduced from 31 ± 14% to 6 ± 2% by pre‐treatment of the cells with a monoclonal antibody against the rat integrin α4 chain. In accordance with the in vivo rolling characteristics of the two cell populations, binding of soluble P‐ or E‐selectin (selectin/IgG chimeras) was less intense for blood lymphocytes than for granulocytes, as determined by flow cytometric analyses of rat and human leukocytes. Taken together, our findings in vivo indicate that the adhesive interactions responsible for rolling of PMNL and MNL, respectively, are distinct in terms of receptor occupancy, and may help explain the temporal selectivity in recruitment of different leukocyte subpopulations in inflammatory or immune reactions.
αβ Lineage‐committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain
Authors: Livák Ferenc , Wilson Anne , MacDonald H. Robson , Schatz David G.
Published: March 2007

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)‐transgenic and TCR mutant murine strains. TCRγδ‐transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ‐positive T cells, develop phenotypically αβ‐like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ‐like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR‐mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.
Hypermutation, diversity and dissemination of human intestinal lamina propria plasma cells
Authors: Dunn‐Walters Deborah K. , Boursier Laurent , Spencer Jo
Published: March 2007

In this work we have microdissected lamina propria plasma cells and used polymerase chain reaction and sequencing to investigate immunoglobulin (Ig) gene rearrangements and mutations in human intestine. In addition, specific primers were designed for individual Ig gene rearrangements to analyze the distribution of related B cell and plasma cell clones at different sites along the bowel. Confirming our earlier work, intestinal IgVH genes were highly mutated in plasma cells from older individuals (> 30 years). IgVH genes were significantly less mutated in samples taken from patients aged 11–30 years, and there were fewer mutations again in samples from young children (< 11 years). In age‐matched specimens the number of mutations was equivalent in the duodenum and colon. Using complementarity‐determining region 3 primers to amplify specific Ig gene rearrangements, evidence was also found for the existence of related lamina propria plasma cells along the small bowel and colon, although these were quite scarse. In addition, analysis of the numbers of related clones in a random sampling from discrete areas of lamina propria indicates that the local population is diverse. These results suggest that the highly mutated IgVH genes in adult intestinal plasma cells are a consequence of chronic antigen exposure with age. Duodenal plasma cells are as highly mutated as colonic plasma cells, despite the fact that the upper bowel has no indigenous microbial flora (the stimulus for intestinal plasma cells). They also show that the plasma cell population is diverse and can be widely disseminated along the bowel.
Analysis of immunoreceptor tyrosine‐based activation motif (ITAM) binding to ZAP‐70 by surface plasmon resonance
Authors: Vély Frédéric , Nunès Jacques A. , Malissen Bernard , Hedgecock Charles J. R.
Published: March 2007

The signaling function of the T cell antigen receptor (TCR) is mediated via CD3 polypeptides, the cytoplasmic sequences of which bear conserved immunoreceptor tyrosine‐based activation motifs (ITAM). ITAM are defined by two YxxL/I sequences separated by a six–eight amino acid long spacer. Upon antigen recognition, ITAM become phosphorylated on both tyrosine residues, creating a high affinity binding site for the tandem SH2 domains found in the protein tyrosine kinase ZAP‐70. Using surface plasmon resonance, we further dissected the sequences required for the binding of ZAP‐70 to each TCR‐associated ITAM. First, we generated protein tyrosine phosphatase‐resistant ITAM peptide analogs, in which difluorophosphonomethyl phenylalanyl (F2P) replaced both phosphotyrosines, and showed that those protein tyrosine phosphatase‐resistant analogs bind ZAP‐70 with high affinity, establishing a rational strategy for the design of novel pharmacological tools capable of interfering with TCR signaling function. Second, we substituted the five amino acids separating the two YxxL/I sequences of the CD3ζ1 ITAM with a non‐peptidic linker made up of γ‐amino butyric acid units and demonstrated that the length of this intervening sequence rather than its chemical composition is essential for high affinity binding of phosphorylated ITAM to the ZAP‐70 SH2 domains.
Molecular mechanisms regulating induction of interleukin‐6 gene transcription by interferon‐γ
Authors: Faggioli Laura , Merola Marcello , Hiscott John , Furia Adriana , Monese Rossana , Tovey Michael , Palmieri Marta
Published: March 2007

The multifunctional cytokine interleukin‐6 (IL‐6) plays a central role in host defence mechanisms and hematopoiesis. Furthermore, dysregulation of IL‐6 gene expression is associated with the pathogenesis of various immunologically related diseases such as myeloma, systemic lupus erythematosus, rheumatoid arthritis, psoriasis and Kaposi's sarcoma. The regulation of IL‐6 gene expression occurs mainly at transcriptional level, although mechanisms of post‐transcriptional regulation have also been described. In the present study we demonstrate that in HeLa cells, induction of IL‐6 by interferon‐γ (IFN‐γ) is transcriptionally controlled, as shown by run on assays and analysis of the IL‐6 mRNA stability. Gel‐retardation experiments using antibodies specific for factors of the IRF family identified four protein‐DNA complexes, which bind to the interferon regulatory factor (IRF) binding site at position −267 to −254, in nuclear extracts from IFN‐γ treated cells. Furthermore, transient transfection analyses of the 5′‐flanking region of IL‐6 gene linked to the chloramphenicol acetyltransferase (CAT) reporter gene demonstrated that the −267 to −254 IRF site is necessary for IL‐6 induction by IFN‐γ. However, transfection experiments in which IRF‐1 and IxBα were overexpressed show that full‐scale transcriptional activation of the IL‐6 promoter directing CAT expression requires the co‐operation between IRF‐1 and NF‐xB at a low constitutive level.
Human immunodeficiency virus type‐1‐specific immune responses induced by DNA vaccination are greatly enhanced by mannan‐coated diC14‐amidine
Authors: Sasaki Shin , Fukushima Jun , Arai Hiroyuki , Kusakabe Ken‐Ichi , Hamajima Kenji , Ishii Norihisa , Hirahara Fumiki , Okuda Katsuji , Kawamoto Susumu , Ruysschaert Jean‐Marie , Vandenbranden Michel , Wahren Britta , Okuda Kenji
Published: March 2007

Use of mannan‐coated N‐t‐butyl‐N′‐tetradecyl‐3‐tetradecylamino‐propion‐amidine (diC14‐amidine) as an adjuvant for a DNA vaccine encoding glycoprotein 160 of human immunodeficiency virus type‐1 (HIV‐1) enhanced the antigen‐specific immune responses. The role of interferon‐γ (IFN‐γ) and interleukin‐12 in the mechanism of adjuvant action was also evaluated. Coating of diC14‐amidine with mannan significantly augmented the HIV‐specific delayed‐type hypersensitivity reaction induced by the immunogenic DNA. HIV‐1‐specific cytotoxic T lymphocyte activity was also markedly enhanced by the mannan‐diC14‐amidine cocktail. An immunomodulatory effect of this cocktail was inhibited by treatment with anti‐IFN‐γ monoclonal antibody in vivo, which suggests that IFN‐γ plays an important role in inducing cell‐mediated immunity by the DNA vaccine containing this adjuvant. The results of both antigen‐specific immunoglobulin isotype analysis and cytokine measurement showed that the immunogenic DNA incorporated into mannan‐coated diC14‐amidine elicits Th1‐biased immune responses.