Molecular associations required for signalling via dipeptidyl peptidase IV (CD26).
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Animals↗
Biomedical subjects
B Fleischer
Publications and source records attributed to B Fleischer.
Group A streptococcal M protein binds to several human cell types but not via MHC class II molecules.
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Antigens, Bacterial↗
Immune responses to filarial infection in laboratory mice.
Models of filarial infection in laboratory inbred mice are valuable tools for assessing the relevance of anti-filarial immune responses in protection against these parasites. However, laboratory mice are not permissive for those filarial species which are known to infect humans. Therefore, immunity to the different stages of these filariae, i.e. infective third stage larvae (L3), adults and microfilariae, has been analyzed separately, as a surrogate approach. Although much information has been gathered by analysis of immunity and intervention in particular immune responses in these experimental systems, interference of different stage-specific responses as well as modulation of filarial maturation by the immune system cannot be assessed. A newly established infection model of filariasis, namely infection of laboratory mice with Litomosoides sigmodontis, accommodates the full developmental cycle of the parasite and may overcome this deficiency. Although the disadvantage of this latter model is that it deals with a filaria which is not pathogenic to man, it is the only model in which immunity can be analyzed during maturation of infective larvae into adult worms, the period considered most important for vaccination studies.
Animals↗
The T-cell receptor associated zeta-chain is required but not sufficient for CD26 (dipeptidylpeptidase IV) mediated signaling.
CD26 or dipeptidylpeptidase IV (DPP IV) is a cell surface protease involved in T-cell activation. Triggering or costimulation of T-cells via CD26 was shown to be dependent on the expression of the T-cell receptor (TCR) associated zeta-chain with at least one functional immune receptor tyrosine based activation motif (ITAM). Here we tested T-cell lines expressing chimeric proteins (hCD25-zeta) consisting of human IL-2 receptor-alpha chain derived extracellular sequences (hCD25) fused to mouse-specific zeta-chain segments, for their capacity to transfer CD26 mediated signals. Although these 'minimal receptor' expressing T-cell lines were capable of transmitting signals from other costimulatory molecules (e.g. CD2), crosslinking of CD26 did not induce IL-2 secretion. Co-cross-linking of hCD25 and CD26 molecules, however, resulted in the stimulation of the T-cells. Thus, although the zeta-chain is a prerequisite for CD26 mediated signaling events, the sole expression of zeta-protein as a signaling molecule is not sufficient for CD26 mediated triggering but permits CD26 induced costimulation in TCR negative cells.
Cells, Cultured↗
Trypanosoma cruzi induces strong IL-12 and IL-18 gene expression in vivo: correlation with interferon-gamma (IFN-gamma) production.
IFN-gamma, produced after infection with Trypanosoma cruzi, has been shown to be crucial in the determination of resistance or susceptibility. We have performed a detailed study on the expression of IFN-gamma and of the IFN-gamma-inducing cytokines IL-12 and IFN-gamma-inducing factor (IGIF)/IL-18 with regard to time course and tissue localization. IFN-gamma was present in high amounts in the serum and in the supernatants of unseparated spleen cells and isolated CD4+ and CD8+ T cells from the spleens of infected mice which were stimulated ex vivo with T. cruzi. Using the in situ hybridization technique we demonstrate that IL-12 p40 messages were expressed in the spleen and increased during infection, correlating with the expression of IFN-gamma transcripts. Furthermore, we show for the first time that the mRNA for the cytokine IL-18 was induced by a parasitic infection and that this expression increased during infection with T. cruzi. Interestingly, the message for IL-18 was produced earlier during infection and already had declined until day 38, when IFN-gamma and IL-12 p40 transcripts were optimally expressed. Surprisingly, the changes in IL-12 and IL-18 mRNA production were clearly seen only by in situ hybridization, but less clearly by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). This is possibly due to the extensive activation and proliferation of spleen cells observed during infection leading to a dilution of these specific mRNAs.
Animals↗
Accumulation of CTLA-4 expressing T lymphocytes in the germinal centres of human lymphoid tissues.
CTLA-4, a coreceptor with sequence homology to CD28 is expressed on T cells after activation. Mice deficient for CTLA-4 die young from massive infiltration of many organs by activated T cells, which highlights the essential inhibitory role the coreceptor plays in the regulation of the immune response. To study the prevalence and distribution of CTLA-4 in situ immunohistological analyses were carried out on human tonsils and lymph nodes. Expression of CTLA-4 was restricted to alpha beta T cells, and CTLA-4+ B cells were not observed. In T-cell areas, 2-10% of T cells were positive for CTLA-4 with similar percentages in the CD4+ and CD8+ subpopulations. In the germinal centres (GC) the fraction of CTLA-4+ T cells was much higher (70-90%). This was due to frequent expression of CTLA-4 on the CD4+ helper subpopulation. GC CD8+ T cells were rare and mostly did not express the coreceptor. The CTLA-4+ T-cell fraction was also over-represented among intraepithelial tonsillar T cells. Cycling (Ki-67+) and apoptotic (TUNEL+) T cells were never positive for CTLA-4, while a subset of CD25+ cells did express the coreceptor. Since CTLA-4 is essential for the physiological limitation of the immune response, GC T cells, which are mostly CTLA-4 positive, might be important in this process.
Abatacept↗
Production of both IFN-gamma and IL-5 by Onchocerca volvulus S1 antigen-specific CD4+ T cells from putatively immune individuals.
Protective immunity to the parasitic nematode Onchocerca volvulus (Ov) appears to be directed against molecules of invading L3 larvae. In this study, the cellular immune reaction to such an Ov L3 protein (S1) which is protective in an animal model was analyzed using peripheral blood mononuclear cells (PBMC) of individuals from a hyperendemic area in West Africa who were exposed to Ov but remained free from disease ('putatively immune individuals'). Despite seronegativity of these individuals against S1, proliferation of PBMC was inducible, allowing generation of an S1-specific T cell line which produced IFN-gamma upon stimulation with both Ov lysate and S1. However, S1 induced significantly more IL-5 than Ov lysate. S1-specific, DQ6 (DQA1*0103/DQB1*0603)-restricted T cell clones were generated which reacted against synthetic peptides comprising amino acids 99-111 of S1. These clones, which are the first generated against a recombinant fllarial antigen, produced both IFN-gamma and IL-5 as well as little IL-4, suggestive of a Th0-like phenotype. In conclusion, in putative immunity, reactivity against a particular parasite protein can be detectable on the level of T but not B cells. Induction of both IFN-gamma and IL-5 by S1 suggests that it may trigger macrophage plus eosinophil dependent killing of L3 in vivo. The identification of a likely DQ6 (DQA1*0103/DQB1*0603)-restricted T cell epitope may be of more general relevance, given that allele combinations of DQ6, including DQA1*0103/DQB1*0603, are negatively associated with diabetes mellitus.
Amino Acid Sequence↗
Exclusion of bioactive contaminations in Streptococcus pyogenes erythrogenic toxin A preparations by recombinant expression in Escherichia coli.
The streptococcal erythrogenic exotoxin A (SPEA) belongs to the family of bacterial superantigens and has been implicated in the pathogenesis of a toxic shock-like syndrome and scarlet fever. Concerning its biological activity, mainly T-cell-stimulatory properties, conflicting data exist. In this study, we show that most of the SPEA preparations used so far contain biologically active contaminations. Natural SPEA from the culture supernatant of Streptococcus pyogenes NY-5 and recombinant SPEA purified from the culture filtrate of S. sanguis are strongly contaminated with DNases. We show that natural SPEA induces more tumor necrosis factor alpha (TNF-alpha) than recombinant SPEA, but we also show that DNases are able to induce TNF-alpha. In commercial SPEA preparations, we identified a highly active protease, which was shown not to be SPEB. To exclude these contaminations, we overexpressed SPEA cloned in the effective high-level expression vector pIN-III-ompA2 in Escherichia coli. The expressed SPEA shows the same amino acid composition as natural SPEA, whereas functional studies reported so far were carried out with toxins containing an incorrect amino terminus. We describe the rapid purification of lipopolysaccharide-, DNase-, and protease-free SPEA in two steps from the host's periplasm and its structural characterization by circular dichroism. Our results represent for the first time the production in E. coli of recombinant SPEA with the authentic N-terminal sequence and a proven superantigenic activity. Collectively, our results indicate that immunological studies of superantigens require highly purified substances free of biologically active contaminations.
Animals↗
Infection with Trypanosoma cruzi selectively upregulates B7-2 molecules on macrophages and enhances their costimulatory activity.
T-cell-mediated immune responses are essential for protection against infection with the protozoan Trypanosoma cruzi. In this study, we investigated the influence of infection of murine macrophages with T. cruzi on costimulatory signals for T lymphocytes provided by these cells. We demonstrate that bone marrow-derived macrophages (BMMph) selectively and strongly upregulate expression of B7-2 molecules after infection, while the expression of other costimulatory molecules such as B7-1, intercellular adhesion molecule 1, lymphocyte function-associated antigen 3, and heat-stable antigen is not significantly affected. Infection by live trypanosomes was required. As a consequence of the strong B7-2 upregulation, the infected macrophages are able to induce proliferation of splenic CD4+ T cells in the presence of anti-CD3 antibodies with much higher efficiency than uninfected macrophages. Costimulation could be inhibited by an antibody to B7-2. Furthermore, costimulatory activity for established T-cell clones of Th1 and Th2 phenotype was also strongly enhanced in BMMph by infection with T. cruzi. Th1 cells stimulated either via anti-CD3 antibodies or via specific antigen proliferated with higher efficiency in the presence of infected macrophages than in the presence of uninfected cells. BMMph stimulated with gamma interferon (IFN-gamma), expressing elevated levels of B7-2 molecules, are also able to enhance Th1 cell proliferation, which was highest, using macrophages which were infected and in parallel were stimulated with IFN-gamma. Noteworthy, for cloned Th2 cells, the mechanism of costimulation differed, because costimulation of Th2 cells was not dependent on B7-2 upregulation but was due to secretion of interleukin-1alpha. These findings demonstrate that infection of macrophages with T. cruzi transforms the macrophage into a potent costimulatory cell based on the induction of two different costimulatory activities.
Animals↗
Infection of BALB/c mice with the filarial nematode Litomosoides sigmodontis: role of CD4+ T cells in controlling larval development.
Litomosoides sigmodontis is the only filaria which develops from infective larvae into adults in immunocompetent laboratory mice. Depletion of CD4+ T cells from infected BALB/c mice resulted in worm and microfilarial burdens significantly higher than those of infected controls. Th2 cytokines, eosinophilia, and immunoglobulin E, which were strongly induced in infected controls, were diminished in CD4-depleted mice.
Animals↗
Clostridium perfringens enterotoxin lacks superantigenic activity but induces an interleukin-6 response from human peripheral blood mononuclear cells.
We investigated the potential superantigenic properties of Clostridium perfringens enterotoxin (CPE) on human peripheral blood mononuclear cells (PBMC). In contrast to the findings of a previous report (P. Bowness, P. A. H. Moss, H. Tranter, J. I. Bell, and A. J. McMichael, J. Exp. Med. 176:893-896, 1992), two different, biologically active preparations of CPE had no mitogenic effects on PBMC. Furthermore, PBMC incubated with various concentrations of CPE did not elicit interleukin-1, interleukin-2, gamma interferon, or tumor necrosis factor alpha or beta, which are cytokines commonly associated with superantigenic stimulation. However, CPE did cause a dose-related release of interleukin-6 from PBMC cultures.
Clostridium perfringens↗
Functional phenotype of transformed human alphabeta and gammadelta T cells determined by different subgroup C strains of herpesvirus Saimiri.
Based on sequence divergence in the transformation-relevant region, herpesvirus saimiri strains are classified into three subgroups. Only members of subgroup C transform human T lymphocytes to continuous interleukin-2-dependent growth in culture. In this study, human cord blood T cells were immortalized by using different subgroup C strains (C488, C484, and C139). The resulting T-cell lines represented different types of T-cell clones. They were either CD4+ or CD8+ and expressed either the alphabeta or the gammadelta type of T-cell receptors. If transformed by the same virus strain, alphabeta and gammadelta clones were similar with respect to viral persistence, virus gene expression, proliferation, and Th1-type cytokine production. However, major differences were observed in T cells immortalized by different subgroup C strains. Strain C139 persisted at low copy number, compared to the high copy number of prototype C488. The transformation-associated genes stpC and tip of strain C488 were strongly induced after T-cell stimulation. The homologous genes of strain C139 were only weakly expressed and not induced after activation. After CD2 ligation, the C488-transformed T cells produced interleukin-2, whereas the C139-transformed cells did not. Correspondingly, the C139-transformed T cells were less sensitive to cyclosporin A. Sequence comparison from different subgroup C strains revealed a variability of the stpC/tip promoter region and of the Lck-binding viral protein Tip. Thus, closely related subgroup C strains of herpesvirus saimiri cause major differences in the functional phenotype of growth-transformed human T cells.
Amino Acid Sequence↗
TCR V beta families in T cell clones from sarcoid lung parenchyma, BAL, and blood.
The TCR repertoire and the CD4/CD8 ratio of clones from peripheral blood (PB), transbronchial biopsies (TBB), and bronchoalveolar lavage (BAL) of 16 sarcoid patients was analyzed by staining the clones with monoclonal antibodies against nine V beta-families. We observed a striking increase in the CD4/CD8 ratio of the clones from BAL; whereas the CD4/CD8 ratio of the clones from PB was in the normal range. The CD4/CD8 ratio of the TBB-clones had also increased, but this increase did not reach the level of that of the BAL clones. The most prominent changes in the V beta percentages could be detected in the CD4+ subpopulation of the BAL-clones. The most abundant V beta families were V beta 5 in PB and BAL (11.8 and 28.6%, respectively) and V beta 6 in the TBB (12.4%). A similar compartmentalized V beta usage could be demonstrated in one patient with tuberculosis and one patient with HP. The increase in V beta 5, V beta 8, V beta 12, V beta 13.3, and V beta 19 in the BAL and the increase of V beta 5, V beta 6, V beta 13.3, and V beta 19 in the TBB suggest an antigen-driven activation of the T cells in both compartments. Differences in the V beta percentages between BAL and TBB and the lower CD4/CD8 ratio in the TBB, however, demonstrate a relative independence of the two compartments.
Biopsy↗
Synthesis of the CC-chemokines MIP-1alpha, MIP-1beta, and RANTES is associated with a type 1 immune response.
Lymphocytes regulate the immune response by secreting cytokines that control the activity and function of effector cells. Chemokine subsets are ideal candidates for recruitment of specific effector cells to inflammatory sites or to other lesions because of their selective chemoattractant activities. Given the Th1-Th2 model of immune regulation and the particular role of leukocyte recruitment for the outcome of the response, we analyzed whether a subset of human chemokines is associated with a specific type of immune response. Therefore, we have analyzed the human T cell response to Ags prepared from Yersinia enterocolitica and Ascaris suum with respect to cytokine mRNA-synthesis and secretion. For the Gram-negative bacterium Y. enterocolitica, induction of a type 1 response is indicated by IL-2 and IFN-gamma production, and for the nematode A. suum, a type 2 response is based on IL-4 and IL-5 production. Interestingly, expression of three CC-chemokines (i.e., MIP-1alpha, MIP-1beta, and RANTES) correlated with the type 1 response induced by Y. enterocolitica Ag. Chemokine secretion is not restricted to T lymphocytes; therefore, synthesis of MIP-1alpha, MIP-1beta, and RANTES was also characterized in human T cell clones that display a cytokine pattern indicative of the Th2, Th0, or Th1 phenotype. Again CC-chemokine secretion correlated with the Th1-like phenotype. In six analyzed IL-2- and IFN-gamma- secreting Th1 clones and in two Th0 clones, MIP-1alpha, MIP-1beta, and RANTES were detected, while none or only minimal secretion of these CC-chemokines was observed in three IL-4- and IL-5-producing Th2 cell clones.
Animals↗
Selective activation of T cell kinase p56lck by Herpesvirus saimiri protein tip.
Infection with Herpesvirus saimiri, a T lymphotropic virus of non-human primates, immortalizes human T cells in vitro. The cells show a mature activated phenotype and retain their antigen specificity. We have previously shown that in H. saimiri transformed cells a viral gene product termed tyrosine kinase interacting protein (Tip) associates with the T cell-specific tyrosine kinase p56lck and becomes phosphorylated by the enzyme on tyrosine residues. Here we show that p56lck is activated by recombinant and native Tip in cell-free systems. A dramatic increase of Lck activity was also observed in T cell lines transfected with Tip. p60fyn and p53/56lyn, the other Src-related kinases expressed in H. saimiri transformed T cells, did not phosphorylate Tip, and they were not activated by the protein. The selective activation of p56lck by Tip could contribute to the transformed phenotype of H. saimiri infected cells, and it might explain the T cell selectivity of the transformation event.
Base Sequence↗
A biological function for the XP motif within the N terminus of major histocompatibility complex class II-associated peptides.
A high proportion (up to 30%) of major histocompatibility complex (MHC) class II-bound peptides in the mouse and humans contains a proline residue at the N-terminal penultimate position (XP motif). We used a set of ovalbumin (OVA)-specific and hen egg lysozyme (HEL)-specific T cell hybridomas and asked whether the XP motif in MHC class II-associated peptides might influence the stimulation of T cells. We created N-terminally substituted variants of OVA323-339, an H2-Ad restricted OVA epitope and of HEL50-63, a dominant epitope in the context of H2-Ak. Our results show that the N-terminal sequence of MHC class II-bound peptides has a strong impact for the overall stimulation of specific T cells. Proline at the N terminus of antigenic peptides, in contrast to other amino acids, is tolerated or even enhances the recognition of MHC class II-bound peptides significantly.
Amino Acid Sequence↗
Trypanosoma cruzi is a potent inducer of interleukin-12 production in macrophages.
Cytokines produced after infection with Trypanosoma cruzi have been shown to be crucial in the determination of resistance or susceptibility. Interferon-gamma (IFN-gamma) is the predominant cytokine produced after infection and has been shown to protect susceptible mice from infection. IFN-gamma production by natural killer cells and T cells is induced by interleukin-12 (IL-12). Therefore, the aim of our study was to analyze the ability of T. cruzi to induce IL-12 production. Spleen cells and bone marrow-derived macrophages incubated with T. cruzi trypomastigotes induced high amounts of IL-12p40 mRNA as shown by reverse transcriptase-polymerase chain reaction. Lipopolysaccharide (LPS) was less efficient in inducing IL-12p40-specific mRNA. Furthermore, biologically active IL-12, detected by the capacity of the supernatant of infected macrophages to induce IFN-gamma production in spleen cells, was produced at very high levels. In comparison, macrophages stimulated with LPS secreted drastically less IL-12. Interestingly, only live, UV- or gamma-irradiated trypanosomes, but not heat-killed parasites or lysates, were functional in this respect. In a kinetic study, in the supernatant obtained from cultures of infected macrophages, IL-12 was already detectable at 2 h after infection, peaked at 32 h and declined after 45 h.
Animals↗
Reactivity of mouse T-cell hybridomas expressing human Vbeta gene segments with staphylococcal and streptococcal superantigens.
A panel of 15 mouse T-cell hybridomas, each expressing a different human Vbeta gene segment (hVbeta) in an otherwise mouse T-cell receptor (i.e., mouse alpha chain and CD3 complex), was constructed by transfection of hVbeta/mouse Cbeta chimeric T-cell receptor (TCR)-beta genes into a mouse T-cell hybridoma recipient lacking the endogenous TCR-beta chain. Several qualities that are conferred by the hVbeta chain of the TCR are retained in the chimeric human-mouse TCR complex: a large panel of hVbeta-specific antibodies specifically stained the hVbeta expressed by the mouse T-cell hybridomas. Moreover, hVbeta-transfected mouse cells could readily produce interleukin 2 when stimulated by superantigens presented by antigen-presenting cells. These characteristics made it possible to refine the reactivity of 17 superantigen preparations with the available transfected Vbetas. Each superantigen gave a characteristic pattern of reactivity on the transfectants. Positive reactivities with some of these transfectants, which differ only by the expressed hVbeta, demonstrate unambiguously the superantigenic character of a protein or fraction and its potential to react with the corresponding Vbetas. Therefore, these hVbeta-transfected cells constituted a valuable tool for determining "specificity fingerprints" of known or putative superantigens. First, commonly used, commercially available superantigens such as staphylococcal enterotoxin B and toxic shock syndrome toxin-1 (TSST-1) showed additional Vbeta reactivities, compared with those of their recombinant counterparts. This stresses the importance of using defined preparations of superantigens for the definition of Vbeta specificities. Second, the stimulatory pattern of a strain of Streptococcus pyogenes demonstrated that this strain, unlike others, produces a potent Vbeta 8-specific superantigen that is an yet undefined at the molecular level.
Animals↗