bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential effects of gangliosides on human IgE and IgG4 production
Authors: Kimata Hajime
Published: March 2007

The effects of gangliosides on human IgE and IgG4 production were studied. Of the various gangliosides tested, only GM2 and GM3 inhibited the IgE and IgG4 production induced by interleukin (IL)‐4 plus hydrocortisone (HC), or that induced by IL‐13 plus HC, in human surface IgE‐ and IgG4‐negative (sIgE−, sIgG4−) B cells without affecting the production of IgG1, IgG2, IgG3, IgM, IgA1 or IgA2. In contrast, GM1, GD1a, GD1b, GD3, GT1b and GQ1b were without effects. The GM2‐ and GM3‐mediated inhibition was specific, since each was blocked by a corresponding antibody. Of the various factors tested, IL‐6, IL‐10, and tumor necrosis factor (TNF)‐α enhanced the IgE and IgG4 production induced by IL‐4 plus HC or by IL‐13 plus HC, while IL‐8 and transforming growth factor (TGF)‐β inhibited these responses. However, only TNF‐α counteracted the GM2‐ and GM3‐mediated inhibition of IgE and IgG4 production, while IL‐6, IL‐10, anti‐IL‐8 monoclonal antibody and anti‐TGF‐β antibody failed to do so. Anti‐TNF‐α monoclonal antibody, but not control IgG1, not only inhibited IgE and IgG4 production in the absence of TNF‐α but also blocked the counteraction of inhibition by TNF‐α. In cultures containing IL‐4 plus HC or IL‐13 plus HC. GM2 and GM3 specifically inhibited TNF‐α production without affecting TNF‐α receptors, IL‐6 production or IL‐6 receptors. These results indicate that GM2 and GM3 inhibit IgE and IgG4 production by inhibiting endogenous TNF‐α production.
Activated T cells enhance nitric oxide production by murine splenic macrophages through gp39 and LFA‐1
Authors: Tian Licheng , Noelle Randolph J. , Lawrence David A.
Published: March 2007

Macrophages can be stimulated to produce a relatively large amount of nitric oxide, which is an important component in macrophage‐mediated defense mechanisms and regulation of T cell activities. It has been known that T helper (Th) cell activation requires intimate physical interaction between T helper cells and macrophages and that cytokines from activated Th cells regulate macrophage activities including nitric oxide production. The current study indicates that surface molecules on activated Th cells also can synergize with cytokines to substantially enhance nitric oxide production by macrophages through cell‐cell contact. The CD40 ligand (gp39) and LFA‐1 appear to be two major contributors for T cell dependent nitric oxide production.
γδ lineage‐specific transcription of human T cell receptor γ genes by a combination of a non‐lineage‐specific enhancer and silencers
Authors: Lefranc Marie‐Paule , Alexandre Daniel
Published: March 2007

The expression of the T cell receptor (TcR) γ genes is restricted to TcR γδ+ T lymphocytes. Transgenic and somatic cell hybrid experiments had suggested that the expression of a functionally rearranged TcR γ gene was extinguished in TcR αP+ T cells, possibly by putative cis‐acting transcriptional silencers. We have identified such negative cis‐acting sequences in the 3′ non‐coding region of the human TcRγ (TRG) locus, upstream of an enhancer located at 6.5 kb of the TcR Cγ2 gene (TRGC2). These silencers were capable of repressing the transcription from a minimal heterologous promoter in a position‐ and orientation‐independent fashion. When analyzed individually, the silencers and the enhancer were equally active in the TcR αβ+ and TcR γδ+ T cell lines studied. In contrast, the association of the enhancer with either silencer was shown to restrict transcription to the TcR γδ+ T cell lines.
Dendritic cells in Leishmania major‐immune mice harbor persistent parasites and mediate an antigen‐specific T cell immune response
Authors: Moll Heidrun , Flohé Stefanie , Röllinghoff Martin
Published: March 2007

Upon infection with Leishmania major, a cause of human cutaneous leishmaniasis, mice of resistant strains are able to control the infection, with lesions resolving spontaneously. A long‐lasting cell‐mediated immunity protects them from reinfection. Nevertheless, small numbers of viable parasites persist in the lymph nodes of these mice. We have recently documented that, in addition to macrophages, epidermal Langerhans cells can ingest L. major. Furthermore, Langerhans cells have the unique ability to transport viable parasites from the infected skin to the draining lymph node for presentation to antigen‐specific T cells and initiation of the cellular immune response. During migration, Langerhans cells develop into dendritic cells. In the present study, we analyzed whether dendritic cells support the persistence of parasites in immune hosts. Immunohistological studies and assays in vitro showed that in the lymph nodes of mice that have recovered from infection with L. major, both macrophages and dendritic cells harbor viable parasites. However, only dendritic cells were able to induce a vigorous T‐cell immune response to L. major in vitro in the absence of exogenous antigen. Tracking experiments conducted in vivo suggested that the infected dendritic cells in the lymph nodes are derived from Langerhans cells that have emigrated from the skin. The data demonstrate that L. major‐infected dendritic cells and macrophages in lymph nodes of immune animals represent long‐term host cells, but only dendritic cells have the ability to present endogenous parasite antigen to T cells. Long‐term infected dendritic cells may thus allow the sustained stimulation of a population of parasite‐specific T cells, protecting the mice from reinfection. Our results favor the hypothesis that the persistence of antigen supports the maintenance of T cell memory and that dendritic cells are critically involved in this process.
Glycoinositolphospholipids of Leishmania major inhibit nitric oxide synthesis and reduce leishmanicidal activity in murine macrophages
Authors: Proudfoot Lorna , O'Donnell Catherine A. , Liew Foo Y.
Published: March 2007

Murine macrophages express high levels of inducible nitric oxide (NO) synthase and produce large amounts of nitric oxide when activated with interferon‐γ and lipopolysaccharide in vitro. Nitric oxide is a mediator of a variety of biological functions including microbicidal activity against the protozoan parasite Leishmania species. Glycoinositolphospholipids (GIPL) are the predominant surface glycolipids in both developmental stages of Leishmania major. We report here that GIPL can inhibit the synthesis of NO in a time‐ and dose‐dependent manner. In contrast, lipophosphoglycan, which is present in the promastigote stage did not inhibit NO synthesis. GIPL‐treated macrophages also showed markedly reduced leishmanicidal activity. The majority of the inhibitory activity of GIPL was found within the alkylacylglycerol moiety of the GIPL molecule. These data, therefore, suggest that GIPL may contribute towards the survival of the parasite in the immune hosts.
Major histocompatibility complex class I‐restricted presentation of influenza virus nucleoprotein peptide by B lymphoma cells harboring an antibody gene antigenized with the virus peptide
Authors: Billetta Rosario , Filaci Gilberto , Zanetti Maurizio
Published: March 2007

We analyzed the capacity of B cells to process and present a peptide from the variable region of an endogenous immunoglobulin heavy (H) chain to a major histocompatibility complex (MHC) class I‐restricted cytotoxic T lymphocyte (CTL) clone. The H‐chain gene was engineered to express 14‐amino acid peptide from the sequence of the influenza virus nucleoprotein (NP) antigen in the third complementarity‐determining region (CDR3). This NP peptide is presented in association with the Db allele in H−2b mice. We demonstrate that B lymphoma cells (H‐2b) harboring the antigenized H‐chain gene process and present the NP peptide in association with the Db molecule and are lysed by a CTL clone specific for that peptide in an MHC‐restricted way. In contrast, the soluble antigenized antibody failed to mediate lysis of H−2b target cells. The endogenously processed immunoglobulin CDR3 peptide could be eluted from surface Db molecules in transfected cells. This study formally demonstrates that peptides from the hypervariable loops of endogenous immunoglobulin are processed through the endogenous degradative pathway and are presented to CD8+ T cells in the context of MHC class I molecules. The implication of these findings for processing and presentation of endogenous immunoglobulin peptides in B cells and network regulation by idiopeptides is discussed.
Contribution of α/β and γ/δ T lymphocytes to immunity against Mycobacterium bovis Bacillus Calmette Guérin: studies with T cell receptor‐deficient mutant mice
Authors: Ladel Christoph H. , Hess Jürgen , Daugelat Sabine , Mombaerts Peter , Tonegawa Susumu , Kaufmann Stefan H. E.
Published: March 2007

Mutant mice with defined T cell deficiencies were infected with Mycobacterium bovis bacillus Calmette Guérin (BCG) and the relative contribution of α/β T cells and γ/δ T cells to the host immune response was assessed. Recombinase activating gene (RAG‐1)−/− mutants as well as T cell receptor (TcR) β−/−, but not TcR‐δ−/−, mutants succumbed to M. bovis BCG infection and failed to develop granulomatous lesions. Antigen‐induced IFN‐γ production by spleen cells in vitro was abrogated in RAG‐1−/− mutants and markedly diminished in TcR‐β−/− and TcR‐δ−/− mice. Reconstitution experiments suggest that both α/β and γ/δ T cells are essential for antigen‐specific IFN‐γ secretion. Our data formally prove the crucial role of α/β T cells and reveal accessory functions of γ/δ T cells in optimum immunity against M. bovis BCG.
Generation of antigen‐specific CD4+ T cell lines from naive precursors
Authors: Mehta‐Damani Anita , Markowicz Sergiusz , Engleman Edgar G.
Published: March 2007

The conditions required for sensitizing naive T cells to nominal antigen are poorly understood. In this report we describe an in vitro system for generating antigen‐specific CD4+ T cells from previously unprimed individuals. Freshly isolated CD4+ T cells were cultured with keyhole limpet hemocyanin (KLH), sperm whale myoglobin (SWM), or human immunodeficiency virus (HIV) gp 160, antigens to which most persons have not been sensitized, in the presence of either dendritic cells (DC) or macrophages (MΦ). In short‐term (< 8 days) cultures, CD4+ T cells or their CD4+, CD45RA (naive) subpopulation mounted significant proliferative responses to KLH, SWM, and HIV gp160, but only if the antigens were presented by DC. In contrast, CD4+, CD45RO (memory) T cells responded poorly to these antigens, although they responded vigorously to tetanus toxoid, a recall antigen, presented by either DC or MΦ. KLH‐ and SWM‐specific CD4+ T cell lines were established from the starting population that had been sensitized in vitro, following repeated stimulation with antigen and MΦ in medium supplemented with interleukin‐2 and interleukin‐4. Despite the continued presence of these cytokines during T cell expansion, the expanded lines retained their ability to respond to the priming antigen in the absence of exogenous cytokines. When the CD45RA and CD45RO subpopulations were sensitized and expanded separately, the CD45RA cells alone gave rise to antigen‐specific T cell lines, while the CD45RO cells proliferated nonspecifically. These results demonstrate that human naive CD4+ T cells can be sensitized in vitro to nominal antigens presented by DC and that the sensitized cells can be expanded into long‐term lines that retain their antigen specificity.
Studies on the T cell dependence of natural IgM and IgG antibody repertoires in adult mice
Authors: Malanchère Evelyne , Marcos Miguel A. R. , Nobrega Alberto , Coutinho Antonio
Published: March 2007

The present experiments address the thymic dependence of IgM and IgG natural antibody repertoires in adult euthymic and athymic BALB/c mice, as well as in athymic animals reconstituted with a fixed number of syngeneic T cells. Within 3 weeks of the transfer of 107 syngeneic splenic T lymphocytes to athymic mice, the T cell compartment is essentially reconstituted in the peritoneal cavity (up to 80% of the numbers in euthymic animals), but is only 10–20% of controls in the spleen and lymph nodes. Early after transfer, there is an increase in the numbers of activated B cells and of immunoglobulin‐secreting cells in the spleen, and within 1–2 weeks, the serum concentrations of IgG1 and IgG2a are fully reconstituted to control levels (30–40‐fold increased). Multiparametric analyses of serum IgM and IgG repertoires revealed that euthymic and athymic mice share essentially all natural antibody reactivities towards syngeneic extracts of liver and muscle. When tested at the same immunoglobulin concentrations, however, nude sera consistently show higher values of reactivity in all detectable bands. The transfer of 107 splenic T cells into athymic mice results in a general decrease of serum IgM reactivities, some of which become undetectable, and in alterations of the serum IgG repertoire as early as 1 week, and for at least 4 weeks after transfer. T cell transfer, however, fails to restore the euthymic IgM and IgG repertoires within 4 weeks. The present observations demonstrate that, after limited T cell reconstitution of nude mice, there is a rapid and quantitatively important increase of serum IgG1 and IgG2a production; the serum IgM reactivity repertoire is qualitatively similar in euthymic and athymic animals, but is generally decreased by T cell activity; and the serum IgG repertoire, which is qualitatively similar in euthymic and athymic animals, is amplified by T cell activity and partially altered by T cell transfer into athymic animals. These results raise questions on the mechanisms of B cell activation and natural antibody repertoire selection in T cell‐deficient adult individuals.
Early parasite containment is decisive for resistance to Leishmania major infection
Authors: Laskay Tamás , Diefenbach Andreas , Röllinghoff Martin , Solbach Werner
Published: March 2007

We investigated the early spread of Leishmania major in various mouse strains. In BALB/c mice, which are extremely vulnerable to L. major infection, the parasites disseminated within 10‐24 h from the site of subcutaneous footpad infection in to the popliteal lymph node, spleen, lung, liver and bone marrow. Application of recombinant (r)IL‐12 prior to infection prevented the early dissemination of parasites into visceral organs and the animals healed the infection. In three mouse strains tested, C57BL/6, CBA/J and C3H/HeJ, which are all resistant to L. major infection, the parasites remained localized in the footpad and in the draining LN for 3 days without evidence of dissemination. In C57BL/6 mice, depletion of NK1.1+ cells or neutralization of interferon (IFN)‐γ prior to infection led to rapid parasite spreading with kinetics similar to those seen in susceptible animals. Depletion of either CD4+ or CD8+ T cells in vivo prior to infection did not alter the kinetics of dissemination in any mouse strain tested. Experiments with severe‐combined immunodeficient mice provided further evidence that parasite containment depends on natural killer cells and IFN‐γ, but is independent of T cells. The finding that all resistant mouse strains restrict the spread of the parasites within the first 24 h after infection strongly suggests that early parasite containment is closely associated with a resistant phenotype. The data show that local restriction of parasites in the pre‐T cell phase of the infection is mediated by the innate immune system and suggest that this function plays an important role in the development of a protective T cell response.
A B7‐1‐transfected human melanoma line stimulates proliferation and cytotoxicity of autologous and allogeneic lymphocytes
Authors: Sulé‐Suso Josep , Arienti Flavio , Melani Cecilia , Colombo Mario Paolo , Parmiani Giorgio
Published: March 2007

B7 co‐stimulation is necessary to activate resting T cells upon antigen recognition by the T cell receptor. To see whether expression of B7 may render human melanoma cells able to stimulate T cells, a cloned melanoma line (Me1B6), which did not express B7‐1, was transfected with the human B7‐1 gene. In proliferation assays, B7‐1 transfected cells (Me1B6/B7) showed greater stimulatory activity of allogeneic and autologous peripheral blood lymphocytes (PBL) compared to parental, non‐transfected tumor cells. This effect was also seen when allogeneic CD8+ and CD4+ subpopulations were used as effectors. In these studies, activation of lymphocytes was B7‐1‐dependent and HLA classes I and II mediated. The higher proliferation correlated with an increased lytic activity by PBL stimulated with B7‐1+ tumor cells against the untransfected Me1B6. Furthermore, PBL from a metastatic melanoma patient stimulated by Me1B6/B7 developed an higher lytic activity not only against Me1B6 but also against their autologous, B7‐1− tumor. Finally, after Me1B6/B7 stimulation, PBL released interleukin (IL)‐2 and interferon‐γ, but not IL‐4, suggesting a Th1‐mediated response. These data support the use of B7‐1 transfected melanoma cells in the therapeutic vaccination of melanoma patients.
Internalization of B cell and pre‐B cell receptors is regulated by tyrosine kinase and phosphatase activities
Authors: Salamero Jean , Fougereau Michel , Seckinger Philippe
Published: March 2007

Prior to the expression of the B cell antigen receptor, the μ heavy chain associates with two non‐polymorphic polypeptides, λ like and VpreB, which form a pseudo‐light chain complex in pre‐B cells and pre‐B cell lines. Surface expression of the so‐called pre‐B cell receptor (pre‐BCR) occurs only in the presence of Igα and Igβ, known to be involved both in B cell antigen receptor (BCR) signaling and trafficking. Although the pre‐BCR organization is consistent with an efficient transport to the cell surface, most of the newly synthesized receptor remains within the cells, and so far, no data are available concerning the rate of exit from the endoplasmic reticulum. Using the human pre‐B cell line Nalm‐6, we found that only a small fraction (2%) of newly synthesized pre‐BCR is transported to the cell surface within 4‐6 h after synthesis, where it is constitutively re‐internalized. Membrane Ig‐heavy chain cross‐linking induced internalization of surface pre‐BCR within a few minutes, and the mechanisms underlying endocytosis were analyzed by immunofluorescence and confocal microscopy. Prein‐cubation of the cells with either genistein or orthovanadate, which inhibit, respectively, tyrosine kinases and tyrosine phosphatases, blocked pre‐BCR internalization in a dose‐dependent manner, indicating that both activities are required for endocytosis. BCR internalization was also inhibited in a reversible manner by the drugs. In contrast, neither drug affected the size of the steadystate pool of internalized transferrin receptors. Thus, our data show that tyrosine phosphorylation and dephosphorylation are both required for cross‐linkinginduced pre‐BCR and BCR internalization.
Carrier‐reactive hapten‐specific cytotoxic T lymphocyte clones originate from a highly preselected T cell repertoire: implications for chemical‐induced self‐reactivity
Authors: Martin Stefan , Ruh Helga , Hebbelmann Sabine , Pflugfelder Ulrike , Rüde Barbara , Weltzien Hans Ulrich
Published: March 2007

We have recently described trinitrophenyl (TNP)‐specific cytotoxic T lymphocyte (CTL) clones from C57BL/6 mice specific for hapten‐modified peptides bearing a TNP‐lysine in a peripheral position, i.e. in position 7 of H‐2Kb‐bound octapeptides. CTL recognition of such determinants is always sequencedependent due to co‐recognition of TNP as well as amino acid side chains of the carrier peptide. By the use of glycine‐based designer peptides for primary induction of CTL in vitro, we have identified two sub‐epitopes on individual position 7‐haptenated peptides that form two TcR contact points and which can be independently recognized by cloned CTL. One of these sub‐epitopes is represented by the hapten itself, the other by the amino acids tyrosine and lysine in positions 3 and 4 of the carrier peptide, respectively. Immunization with such TNP‐modified peptides frequently results in the specific induction of CTL also reacting with the unmodified carrier peptides. DNA sequence analyses of the TcR revealed an extraordinary similarity of several independent TcR of CTL from individual mice and induced with different TNP‐peptides. These receptor similarities clearly correlate with structural elements common to the immunizing peptides and suggest their origin from positive thymic selection of TcR on Kb‐associated self‐peptides bearing Tyr in position 3. Our data provide additional information concerning the topology of TcR binding to peptide/MHC complexes with, but also without, TNP. They also indicate a mechanism which might explain the potential of chemicals or drugs to induce autoimmune phenomena.
Different forms of human vascular adhesion protein‐1 (VAP‐1) in blood vessels in vivo and in cultured endothelial cells: implications for lymphocyte‐endothelial cell adhesion models
Authors: Salmi Marko , Jalkanen Sirpa
Published: March 2007

Vascular endothelium plays a pivotal role in controlling leukocyte extravasation from the blood into the tissues. Vascular adhesion protein‐1 (VAP‐1) is a novel endothelial cell molecule which mediates lymphocyte binding to the vascular lining (Salmi, M., and Jalkanen, S., Science 1992. 257: 1407). In this study, we analyzed endothelial cell type‐specific differences of VAP‐1. In vivo, VAP‐1 is a 90/170‐kDa molecule which is mainly expressed on the lumenal surface and in cytoplasmic granules of peripheral lymph node‐type postcapillary venules (high endothelial venules, HEV). In tonsil HEV, VAP‐1 is modified with abundant sialic acids. VAP‐1 is also detectable in the cytoplasm of human umbilical vein endothelial cells (HUVEC) and in an endothelial cell hybrid EaHy‐926, although both cell types lack detectable surface VAP‐1. Cultured endothelial cells do not express MECA‐79‐defined peripheral lymph node addressins either. VAP‐1 was not translocated onto the endothelial cell surface after stimulation with multiple cytokines, mitogens or secretagogues which induced expression of other known endothelial adhesion molecules. Biochemical analyses revealed that VAP‐1 is a ∼ 180‐kDa protein in these endothelial cell types. Digestions with neuraminidase, O‐glycanase and N‐glycanase, as well as treatment of cells with tunicamycin and benzyl‐N‐acetylgalactosaminide, did not alter the molecular mass of VAP‐1 in EaHy‐926. Pulse‐chase experiments showed that VAP‐1 is directly synthesized as a 180‐kDa molecule without any detectable precursors. Thus, in cultured endothelial cells, VAP‐1 is a 180‐kDa protein which is devoid of post‐translational modifications, and in particular, lacks the sialic acids crucial for the function of VAP‐1 in tonsil vessels. Notably, the endothelial cell types commonly used as a model in studying lymphocyte‐endothelial cell interactions lack surface expression of VAP‐1 and peripheral node addressins, and hence are inherently of limited use in analyses of the initial adhesion of lymphocytes.
Glucocorticoids down‐regulate dendritic cell function in vitro and in vivo
Authors: Moser Muriel , De Smedt Thibaut , Sornasse Thierry , Tielemans Françoise , Chentoufi Aziz Alami , Muraille Eric , Van Mechelen Marcelle , Urbain Jacques , Leo Oberdan
Published: March 2007

Exogenous glucocorticoid hormones are widely used as therapeutical agents, whereas endogenous glucocorticoids may act as physiological immunosuppressants involved in the control of immune and inflammatory responses. The optimal activation of T lymphocytes requires two distinct signals: the major histocompatibility complex‐restricted presentation of the antigen and an additional co‐stimulatory signal provided by the antigen‐presenting cells. There is ample evidence that, among the cells able to present the antigen, the dendritic cells (DC) have the unique property to activate antigen‐specific, naive T cells in vitro and in vivo, and are therefore required for the induction of primary immune responses. In this work, we tested whether glucocorticoids affected the capacity of DC to sensitize naive T cells. Our data show that, in vitro, the steroid hormone analog dexamethasone (Dex) affects the viability of DC, selectively downregulates the expression of co‐stimulatory molecules on viable DC, and strongly reduces their immunostimulatory properties. In vivo, a single injection of Dex results in impaired antigen presenting function, a finding which correlates with reduced numbers of splenic DC. These results show that glucocorticoids regulate DC maturation and immune function in vitro and in vivo and suggest that this mechanism may play a role in preventing overstimulation of the immune system.
Induction of Th1 and Th2 CD4+ T cell responses by oral or parenteral immunization with ISCOMS
Authors: Maloy Kevin J. , Donachie Anne M. , Mowat Allan M.
Published: March 2007

We examined the ability of oral or parenteral immunization with immune stimulating complexes containing ovalbumin (ISCOMS‐OVA) to prime T cell proliferative and cytokine responses. A single subcutaneous immunization with ISCOMS‐OVA primed potent antigen‐specific proliferative responses in the draining popliteal lymph node, which were entirely dependent on the presence of CD4+ T cells. CD8+ T cells did not proliferate in vitro even in the presence of the appropriate peptide epitope and exogenous interleukin (IL)‐2. Primed popliteal lymph node cells produced IL‐2, IL‐5 and interferon (IFN)‐γ, but not IL‐4 when restimulated with OVA in vitro. Serum antigen‐specific IgG1 and IgG2a antibody responses were also primed by subcutaneous immunization with ISCOMS‐OVA, confirming the stimulation of both Th1 and Th2 cells in vivo. Spleen cells from subcutaneously primed mice produced a similar pattern of cytokines, indicating that disseminated priming had occurred. Oral immunization with ISCOMS‐OVA also primed local antigen‐specific proliferative responses in the mesenteric lymph node and primed an identical pattern of systemic cytokine responses in the spleen. The ability of ISCOMS to prime both Th1 and Th2 CD4+ T cell responses may be central to their potent adjuvant activities and confirm the potential of ISCOMS as future oral vaccine vectors.
Induction of diabetes in standard mice by immunization with the p277 peptide of a 60‐kDa heat shock protein
Authors: Elias Dana , Marcus Hadar , Reshef Tamara , Ablamunits Vitaly , Cohen Irun R.
Published: March 2007

We previously reported that immunity to the p277 peptide of the human 60‐kDa heat shock protein (hsp60) was a causal factor in the diabetes of non‐obese diabetic (NOD) mice, which are genetically prone to develop spontaneous autoimmune diabetes. The present study was done to test whether immunization with the p277 peptide could cause diabetes in standard strains of mice. We now report that a single administration of the p277 peptide conjugated to carrier molecules such as bovine serum albumin or ovalbumin can induce diabetes in C57BL/6 mice and in other strains not genetically prone to develop diabetes. The diabetes was marked by hyperglycemia, insulitis, insulin autoantibodies, glucose intolerance and low blood levels of insulin. The diabetes could be transferred to naive recipients by anti‐p277 T cell lines. Similar to other experimentally induced autoimmune diseases, the autoimmune diabetes remitted spontaneously. After recovery, the mice were found to have acquired resistance to a second induction of diabetes. Susceptibility to induced diabetes in C57BL/6 mice was influenced by sex (males were much more susceptible than were females) and by class II genes in the major histocompatibility complex (B6.H‐2bm12 mice with a mutation in the MHC‐II molecule were relatively resistant). Other strains of mice susceptible to induced diabetes were C57BL/KSJ, C3HeB/FeJ, and NON/Lt. BALB/c and C3H/HeJ strains were relatively resistant. Immunization to p277‐carrier conjugates could also induce transient hyperglycemia in young NOD mice, but upon recovery from the induced diabetes, the NOD mice were found to have acquired resistance to later development of spontaneous diabetes. Thus, T cell immunity to the p277 peptide can suffice to induce diabetes in standard mice, and a short bout of induced diabetes can affect the chronic process that would otherwise lead to spontaneous diabetes in diabetes‐prone NOD mice.
The role of tyrosine phosphorylation in the interaction of cellular tyrosine kinases with the T cell receptor ζ chain tyrosine‐based activation motif
Authors: Osman Narin , Lucas Susan , Cantrell Doreen
Published: March 2007

Immunoglobulin receptor family tyrosine‐based activation motifs (ITAM) define a conserved signaling sequence, EX2 YX2L/IX7YX2L/I, that mediates coupling of the T cell antigen receptor (TCR) to protein tyrosine kinases (PTK). In the present study, we explored the role of phosphorylation of the two ITAM tyrosine residues in the interactions of the motif with the PTK ZAP‐70 and p59fyn. The data show that the phosphorylation of a single tyrosine within the motif enables binding of p59fyn, whereas phosphorylation of both tyrosines within the motif is required for maximal binding of the PTK ZAP‐70. Quantitative binding experiments show that nanomolar concentrations of the doubly phosphorylated ζ1‐ITAM are sufficient for ZAP‐70 recruitment, whereas micromolar levels of singly phosphorylated ITAM are necessary for p59fyn binding. ZAP‐70 binds with low efficiency to a singly phosphorylated ITAM, but shows preferential binding to the C‐terminal phosphotyrosine in the ITAM, whereas p59fyn binds selectively to the N‐terminal phosphotyrosine. The present data thus show that there is the potential for a singly phosphorylated ITAM to couple to cellular PTK. Moreover, the data suggest a mechanism for heterogeneity in signal transduction responses by the TCR, since ITAM could differentially couple the TCR to downstream signaling events depending on their phosphorylation state.
CD30 ligation induces nuclear factor‐ϰB activation in human T cell lines
Authors: McDonald Patrick P. , Cassatella Marco A. , Bald Anette , Maggi Enrico , Romagnani Sergio , Gruss Hans‐Jürgen , Pizzolo Giovanni
Published: March 2007

CD30 is a recently described member of the tumor necrosis factor/nerve growth factor receptor superfamily. In this report, we show that following incubation of L540 cells (Hodgkin's disease‐derived, T cell‐like, CD30+ cells) with the agonistic anti‐CD30 monoclonal antibodies (mAb) M44 and M67, two nuclear factor (NF)‐ϰB DNA binding activities were induced in nuclear extracts, as determined in gel retardation assays. The effect of the mAb towards NF‐ϰB activation was rapid, as it occurred within 20 min, and was sustained for up to 6h. By comparison, an isotype‐matched antibody had no effect on NF‐ϰB activation. Moreover, in human T helper (Th) clones functionally characterized as being of the type 0, type 1 and type 2 (28%, < 1% und 93% CD30+, respectively), the extent of CD30‐mediated NF‐ϰB activation correlated with the proportion of CD30+ cells. In all cell lines investigated, the NF‐ϰB complexes induced following CD30 engagement were shown to contain p50 NF‐ϰB1, p65 RelA, and possibly other transcription factors. Collectively, our results demonstrate that nuclear translocation and activation of NF‐ϰB rank among the short‐term cellular responses elicited following CD30 ligation.
Enhancement of endotoxin‐induced interleukin‐10 production by SR 31747A, a sigma ligand
Authors: Bourrie Bernard , Bouaboula Monsif , Benoit Jean‐Marc , Derocq Jean‐Marie , Esclangon Martine , Le Fur Gérard , Casellas Pierre
Published: March 2007

SR 31747A is a new sigma ligand eliciting immunosuppressive and anti‐inflammatory properties. Here, we show that SR 31747A greatly enhances lipopolysaccharide (LPS)‐induced systemic release of interleukin (IL)‐10, while it inhibits the secretion of tumor necrosis factor (TNF)‐α and interferon (IFN)‐γ. In line with this finding, we also show by using quantitative reverse transcription‐polymerase chain reaction analysis that SR 31747A increased LPS‐induced IL‐10 mRNA accumulation in spleen cells, whereas the level of both TNF‐α and IFN‐γ mRNA was dramatically decreased. The enhancement of IL‐10 production by SR 31747A treatment was also apparent in nude and severe‐combined immunodeficient mice treated with LPS, clearly indicating that T and B cells were not involved. Finally, SR 31747A conferred protection against the lethal effect of LPS. The finding that SR 31747A strongly stimulates the synthesis of the natural anti‐inflammatory cytokine IL‐10, a property not observed with dexamethasone, provides new insights for the clinical use of this original compound, particularly in chronic inflammatory diseases where IL‐10 is believed to be a pivotal regulatory component.
NKG2‐C is a receptor on human natural killer cells that recognizes structures on K562 target cells
Authors: Düchler Markus , Offterdinger Martin , Holzmüller Harry , Lipp Joachim , Chu Chien‐Ts , Aschauer Barbara , Bach Fritz H. , Hofer Erhard
Published: March 2007

NKG2‐C is a member of the recently discovered NKG2 family of genes and proteins, which are preferentially expressed on human natural killer (NK) cells. These potential NK cell receptors belong to a larger class of type II transmembrane proteins with a C‐type lectin domain. We show here that NKG2‐C is expressed as a 36‐kDa glycoprotein by translation in vitro, recombinant expression and immunoprecipitation from a human NK cell clone. Further, a recombinant soluble NKG2‐C‐receptor binds specifically to K562 cells, which are target cells for NK cell killing, and to RPMI 8866 cells, which are feeder cells for NK cells; several other hematopoietic cell lines tested do not show any binding. The binding structures on the surface of K562 cells disappear, concomitant with a loss in susceptibility to killing when the cells are induced to differentiate with phorbol ester and Ca2+ ionophore. Our data suggest the presence of specific target molecules for NKG2‐C on K562 cells, since overall glycosylation, Lewis X and Lewis Y structures, as well as the mucin‐like CD43 molecule, do not change following induction of the cells. We propose that NKG2‐C mediates a specific interaction of NK cells and their target cells with functional importance for NK cell killing.
Modulation of immunity to Borrelia burgdorferi by ultraviolet irradiation: Differential effect on Th1 and Th2 immune responses
Authors: Brown Eric L. , Rivas Jorge M. , Ullrich Stephen E. , Young Colin R. , Norris Steven J. , Kripke Margaret L.
Published: March 2007

Ultraviolet B (UVB) radiation suppresses the delayed‐type hypersensitivity (DTH) response to alloantigen by a mechanism involving interleukin (IL)‐10. It has been hypothesized, based on this result, that UV irradiation shifts the immune response from a Th1 to a Th2 response. We tested this hypothesis using Borrelia burgdorferi (Bb) as an antigen under conditions where both DTH and antibody responses could be assessed. Mice were irradiated with a single dose of UV and then immunized with Bb in complete Freund's adjuvant (CFA). DTH was assessed by footpad challenge. At various time points thereafter, mice were bled, and the serum antibodies to Bb were quantitiated. Only IgG1, IgG2a, and IgG2b were produced in response to Bb. The IgG2a and IgG2b antibody responses, as well as the DTH response to Bb, showed UV dose‐dependent reductions after UV irradiation. The primary IgG1 response to Bb was very low and was unaffected by UV irradiation; however, the IgG1 secondary response was elevated in UV‐irradiated mice. Injection of anti‐IL‐10 antibody into UV‐irradiated mice within 24 h after UV exposure restored the DTH response, as well as the IgG2a and IgG2b antibody responses. In addition, injecting recombinant murine IL‐10 mimicked some of the effects of UV radiation. Our results support the hypothesis that in vivo, UV irradiation down‐regulates Th1 immune responses, while leaving Th2 responses intact, and suggest that IL‐10 is an important mediator of this effect.
The proto‐oncogene Vav product is constitutively tyrosine‐phosphorylated in normal human immature T cells
Authors: Gouy Hélène , Debré Patrice , Bismuth Georges
Published: March 2007

The proto‐oncogene Vav product is markedly tyrosine‐phosphorylated after the recruitment of various receptors of cells of hematopoietic origin, including mature T cells. Recent studies on Vav‐deficient mice have clearly implicated the product of the proto‐oncogene Vav in intrathymic T cell development. Vav tyrosine phosphorylation is probably crucial to connect early tyrosine kinase(s) to downstream molecular events leading to cell division and maturation that occur in the thymus. We investigated the tyrosine phosphorylation of Vav in human thymocytes. Immunoblotting experiments demonstrate that, as in mature T cells, tyrosine phosphorylation of Vav is induced following thymocyte stimulation through the T cell receptor. The main observation, however, is that an important fraction of cellular Vav is constitutively tyrosine‐phosphorylated in freshly isolated cells. This phenomenon takes place apparently both in the CD4+CD8+ and the more mature CD4+CD8− and CD4−CD8+ thymocyte cell subsets. Co‐immunoprecipitation experiments showed, moreover, that a small amount of Vav is engaged in the multimolecular complex that includes elements of the T cell receptor and the T cell specific ZAP‐70 tyrosine kinase. Altogether, these data suggest that a critical pathway for T cell development in the human thymus likely involves the permanent activation of Vav in vivo.
Generalized autoimmune disease in interleukin‐2‐deficient mice is triggered by an uncontrolled activation and proliferation of CD4+ T cells
Authors: Sadlack Benjamin , Löhler Jürgen , Schorle Hubert , Klebb Gabriele , Haber Hildegard , Sickel Eckart , Noelle Randolph J. , Horak Ivan
Published: March 2007

Interleukin‐2‐deficient mice (IL‐2−/−) crossed to a BALB/c genetic background develop a lymphoproliferative syndrome with severe hemolytic anemia and die within 5 weeks of age. The presence of autoantibodies of various specificities and inflammatory lesions in several organs are indicative of a generalized autoimmune disease. No alterations of the immune system were observed in 6‐day‐old animals, but 10‐day‐old mice already showed an increased proliferation and polyclonal activation of lymphocytes. The treatment of IL‐2−/− mice with anti‐gp39(CD40L) antibody prevented the disease and indicated that the appearance of activated CD4+ T cells (CD44high, CD69+) represents the first alteration of the immune system in IL‐2−/− mice. Collectively, our results suggest that an essential role of IL‐2 in vivo, which is not compensated by other cytokines, is the maintenance of self tolerance.
Preferential dependence of autoantibody production in murine lupus on CD86 costimulatory molecule
Authors: Nakajima Atsuo , Azuma Miyuki , Kodera Sanki , Nuriya Satoru , Terashi Akiro , Abe Masaaki , Hirose Sachiko , Shirai Toshikazu , Yagita Hideo , Okumura Ko
Published: March 2007

Blockade of the interactions between CD28/CTLA‐4 and their ligands, CD80 (B7, B7.1)/CD86 (B70, B7.2), seems an attractive means to induce antigenspecific peripheral tolerance in organ transplantation and autoimmune disease. Recently, diversities between CD80 and CD86 in expression, regulation, and function have been reported in certain cell populations and murine experimental disease models. To investigate the possible differential role of CD80 and CD86 in the development of lupus, we treated lupus‐prone NZB/WF1 mice with specific monoclonal antibodies (mAb) against CD80, CD86, or both. The treatment with a combination of anti‐CD80 and CD86 mAb before the onset of lupus completely prevented autoantibody production and nephritis, and prolonged survival. Interestingly, we found that anti‐CD86 mAb alone, but not anti‐CD80 mAb, efficiently inhibited autoantibody production. Subclass study on IgG antidouble‐stranded (ds) DNA antibody revealed that the treatment with anti‐CD86 mAb almost completely inhibited both IgG1 and IgG2b, but not IgG2a production. The incomplete reduction of IgG2a anti‐dsDNA antibody by anti‐CD86 mAb was compensated by the addition of anti‐CD80 mAb. A significant reduction of mRNA for interleukin (IL)‐2, interferon‐γ, IL‐4 and IL‐6 was observed in mice treated with a combination of anti‐CD80 and CD86 mAb or anti‐CD86 mAb alone. Treatment with both mAb after the onset of lupus resulted in a significantly prolonged survival with reduction of autoantibody production. These results suggest that CD86 plays a more critical role in autoantibody production, and CD86, but not CD80, contributes to Th2‐mediated Ig production. However, the blockade of both CD80 and CD86 are required for preventing the development and progression of lupus.
Allo‐skin graft rejection, tumor rejection and natural killer activity in mice lacking p56lck
Authors: Wen Tao , Zhang Li , Kung Sam K. P. , Molina Thierry J. , Miller Richard G. , Mak Tak W.
Published: March 2007

Mice lacking the p56lck molecule (lck−/−) have a profound block in the maturation of thymocytes and a greatly reduced number of peripheral mature T cells. To analyze further the functions of the T cells developed in lck −/− mice in vivo, we evaluated the ability of lck−/− mice to reject allo‐skin grafts and methylcholanthrene (MCA)‐induced syngeneic fibrosarcoma, and also examined the biological activity of lck −/− natural killer (NK) cells. Mice lacking p56lck failed to reject skin grafts from either MHC‐disparate or minor‐histocompatibility‐different donors, even after they had been primed with donor spleen cells. They also failed to reject the MCA‐induced immunogenic syngeneic fibrosarcoma, MC57X. NK activity in mice lacking p56lck was normal, and there were no differences in the NK cell activation induced by poly(I) · poly(C) stimulation or interleukin‐2 stimulation (lymphokine‐activated killer induction) between mice lacking p56lck and their immunocompetent heterozygous littermates. NK cells lacking p56lck mediated a normal antibody‐dependent cell‐mediated cytotoxicity (ADCC) response. The results of this study indicate that the loss of p56lck severely impairs the effectors of the immune system which mediate the rejection of allo‐skin grafts and syngeneic tumors. The normal NK activity in lck −/− mice suggests that p56lck is not required for the development and activation of NK cells.
A sensitive assay for detecting low‐affinity interactions at the cell surface reveals no additional ligands for the adhesion pair rat CD2 and CD48
Authors: Brown Marion H. , Preston Sandy , Barclay A. Neil
Published: March 2007

The ligand for the T cell antigen CD2 is CD48 in rodents, but CD58 in humans. The extracelluar parts of these three antigens are structurally related in that all contain two immunoglobulin superfamily (IgSF) domains. There have been reports of alternative ligands for CD2 in the human, but not so far in rodents. We describe the analysis of ligands for rat CD2 and CD48 using fluorescent beads capable of displaying a high ligand density and detecting low‐affinity interactions like that of CD2 with CD48 (Kd = 60 − 90 μM). Monovalent chimeric proteins containing the two IgSF domains of rat CD48 or CD2 and domains 3 and 4 of rat CD4 (CD4d3+4) were anchored to fluorescent covaspheresTMvia a CD4 monoclonal antibody (mAb) with the CD48 or CD2 domains available for ligand binding. Multivalent CD48‐CD4d3 + 4 covaspheresTMgave strong specific binding to rat CD2 expressed on the surface of transfected Jurkat cells. CD48‐CD4d3+4 was compared with CD48‐IgG and CD48‐IgM as tools for detecting binding at the cell surface. Soluble divalent CD48‐IgG and decavalent CD48‐IgM bound to soluble CD2 with a Koffof around 10−3s−1as determined using a BIAcoreTMbiosensor. However, binding to cells by CD48‐IgG and CD48‐IgM was only detectable when they were immobilized on covaspheresTMand represented no increase in sensitivity over CD48‐CD4 covaspheresTMwhen tested for binding to cells expressing high and low levels of CD2. CD48‐CD4d3 + 4 covaspheresTMonly bound to rat cells expressing CD2. In the reverse orientation, binding of CD2‐CD4d3 + 4 covaspheresTMwas dependent on expression of CD48. Pre‐incubation of cells with CD2 or CD48 mAb abolished all binding of CD48‐CD4d3 + 4 or CD2‐CD4d3 + 4, respectively. The data provide no evidence for an alternative ligand for rat CD2 or CD48.
Role of tumor necrosis factor‐α in the regulation of activated synovial T cell growth: Downregulation of synovial T cells in rheumatoid arthritis patients
Authors: Lai Ning‐Sheng , Lan Joung‐Liang , Yu Chia‐Li , Lin Rong‐Hwa
Published: March 2007

To characterize the role of tumor necrosis factor (TNF)‐α in regulating synovial T cell growth, cell cycle progression associated with TNF‐α in mitogen‐activated synovial T cells of patients with rheumatoid arthritis (RA) were analyzed. After mitogen stimulation, the majority of synovial T cells in RA patients accumulated in S‐phase. Anti‐human TNF‐α monoclonal antibody and soluble recombinant human TNF receptor (rhTNFR) can block S‐phase accumulation. Furthermore, synovial fluid (SF) from RA patients was able to inhibit the proliferation of these S‐phase‐accumulated T cells. These data indicate that TNF‐α could regulate activated synovial T cell growth by driving them into S‐phase. Combined with the activities of other components of SF, TNF‐α seems to play an important role in down‐regulating activated synovial T cells in RA patients. In addition, the elevated level of soluble TNFR in the SF of disease‐active RA patients is believed to be associated with the promotion of synovial T cell responses.
A human follicular lymphoma B cell line hypermutates its functional immunoglobulin genes in vitro
Authors: Wu Hongyan , Pelkonen Eila , Knuutila Sakari , Kaartinen Matti
Published: March 2007

The functional immunoglobulin (Ig) genes of B lymphocytes undergo somatic mutations during immune responses. These mutations modify the antigen binding site of the immunoglobulins, thereby enhancing the average affinity of the antibodies produced. The molecular mechanism underlying these B cell hypermutations remains unresolved, partly because it is difficult to grow normal B cells in long‐term cell cultures and because there is no suitable transformed or malignant B cell line which generates mutations in its immunoglobulin genes in vitro. Here, we show that the recently established follicular lymphoma line HF‐1.3.4 generates somatic hypermutations in vitro at a high frequency of 0.7 × 10−6 mutations per base pair per generation in standard cell cultures (RPMI 1640 + 5% fetal calf serum). This shows for the first time that B cell hypermutation can occur without T cells or T cell factors. The mutation frequency increased approximately tenfold to 1 × 10−5 mutations/base pair/generation with B cell‐specific growth factors (interleukins −2 and −4 and three antibodies stimulatory to HF‐1.3.4 cells). This HF‐1.3.4 lymphoma line may help to elucidate the molecular mechanism of Ig gene hypermutation.