[Circadian variation of antibody formation after hepatitis B vaccination].
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Animals↗
Biomedical subjects
B Fleischer
Publications and source records attributed to B Fleischer.
Stimulation of human T cells by streptococcal "superantigen" erythrogenic toxins (scarlet fever toxins).
The pyrogenic (erythrogenic) exotoxins A and C (SPEA and SPEC) of Streptococcus pyogenes belong to the family of mitogenic toxins of which the staphylococcal enterotoxins are the prototypes. The erythrogenic toxin B (SPEB) is a proteinase precursor. All SPE have been reported to be superantigens. Here we have analyzed the human T cell response to these toxins. We used highly purified preparations of SPEA, SPEB, and SPEC from different S. pyogenes strains. These toxins were apparently homogenous in SDS-PAGE, IEF, and HPLC. In addition, recombinant SPEA and SPEC were produced in Escherichia coli. In cultures of PBMC, all three toxins expanded preferentially a fraction of T cells. Using mAb against V beta 2, -5, -6, -8, and -12, we investigated the phenotype of the stimulated cells. Natural SPEA, SPEB, and SPEC strongly stimulated V beta 8+ T cells, whereas recombinant SPEA and SPEC did not. Both natural and recombinant SPEA stimulated V beta 12+ cells and both natural and recombinant SPEC stimulated V beta 2+ cells. In accordance with these findings, a human V beta 8+ line responded to all three toxins derived from S. pyogenes but not to the recombinant proteins. An antiserum against natural SPEC neutralized specifically the V beta 2-stimulating activity of SPEC and the V beta 8-stimulating activity of all three toxins, but had no effect on the response to other superantigens. This shows that trace amounts of a potent novel V beta 8-stimulating activity not identical to SPEA and SPEC are responsible for the stimulation of V beta 8+ T cells by natural SPEA and SPEC reported previously. In a preliminary screening of S. pyogenes strains from patients, we found that this novel superantigen appears to be more widely distributed than SPEA and SPEC. Furthermore, we present evidence that also the superantigenic properties of SPEB are due to contaminations with this V beta 8 stimulator. The response to SPEB usually required 1000 times higher concentrations than to SPEA or SPEC. Antisera to SPEC but not to SPEB inhibited the response of PBMC and V beta 8+ Jurkat cells to SPEB. Furthermore, more stringent purification of SPEB yielded SPEB preparations devoid of mitogenic activity. These results indicate that the mitogenicity that is commonly attributed to SPEB is due to minute contaminations of the V beta 8 stimulator. These results raise two important caveats for the work with these highly potent T cell mitogens.(ABSTRACT TRUNCATED AT 400 WORDS)
Antigens, Bacterial↗
Target sizes of galactosyltransferase, sialyltransferase, and uridine diphosphatase in Golgi apparatus of rat liver.
Target inactivation analysis was used to measure the functional size of uridine diphosphogalactose: N-acetylglucosamine beta(1,4)galactosyltransferase (galactosyltransferase), cytidine monophospho-N-acetyl-neuraminic acid: beta-galactoside alpha(2,6) sialytransferase (sialyltransferase), and uridine diphosphatase (UDPase) in Golgi membranes isolated from rat liver. The size of nucleoside diphosphatase (NDPase), an enzyme similar to UDPase but localized in rat liver endoplasmic reticulum, was also estimated by target inactivation analysis. The related enzymes, UDPase and NDPase, have target sizes of 96 +/- 4 and 77 +/- 3 kDa, while galactosyltransferase and sialyltransferase have target sizes of 97 +/- 10 and 130 +/- 20 kDa, respectively. The target inactivation sizes of galactosyltransferase and of sialyltransferase are about twice the monomer molecular weights of these enzymes obtained from sedimentation studies of the solubilized membranes as well as those predicted from previously reported cDNA sequences. We conclude from our studies that galactosyltransferase and sialyltransferase probably function as dimers in the Golgi membrane.
Animals↗
Dissociation of antigen-presenting capacity of astrocytes for peptide-antigens versus superantigens.
Although superantigens and their molecular interactions with MHC class II molecules have been well characterized recently, little is known concerning the physiological function of different types of APC in inducing superantigen-mediated T cell activation. To evaluate the potential of nonhematopoetic cells to present superantigens to T cells, we have tested astrocytes as a typical "nonprofessional" APC. Although astrocytes can express appropriate levels of MHC class II products and adhesion molecules, they turned out to be unable to mediate superantigen-driven activation of normal T lymphocytes, even in the presence of rather high concentrations of toxins. In contrast, they could properly present equimolar amounts of nominal Ag to various Ag-specific T cell lines under the same experimental conditions. Inability of astrocytes to support T cell responses to superantigens could not be overcome by addition of cytokines IL-1 and IL-6. Binding studies with class II-expressing astrocytes revealed that T cell unresponsiveness was not due to a general failure of astrocytes to bind the superantigen. Moreover, the resulting SA-class II complex was recognizable by TCR, as demonstrated by the capacity to activate IL-2 secretion in T cell hybridomas. Our results extend previous studies demonstrating marked differences of various types of APC to trigger T cell responses to superantigens and describe for the first time a dissociation of the Ag-presenting capacity for peptide-Ag vs superantigen on an accessory cell.
Animals↗
Phosphodiesterase inhibitor pentoxifylline, a selective suppressor of T helper type 1- but not type 2-associated lymphokine production, prevents induction of experimental autoimmune encephalomyelitis in Lewis rats.
The phosphodiesterase inhibitor pentoxifylline (POX), which is known to have pharmacological effects in animal models of multiorgan failure and endotoxin-mediated shock, was tested for its immunosuppressive potential on T lymphocyte activation in vitro and in vivo. POX was found to have a profound inhibitory effect on both mitogen- and antigen-induced proliferation of CD4+ T cells in vitro. This inhibitory activity of the drug could be reproduced by treating T lymphocytes with cAMP analogues during stimulation. Responses of repeatedly in vitro stimulated cells were much more strongly inhibited by the drug and by cAMP analogues than responses of fresh resting lymphocytes. Furthermore, POX could drastically down-regulate tumor necrosis factor regulate production and to a lesser extent interleukin (IL)-2 secretion in activated T cells, but an excess of exogenous IL-2 did not override the antiproliferative effect of the drug. In contrast, the same doses of POX had no inhibitory effect on spontaneous or induced IL-4 and IL-6 production by short-term cultured T lymphocytes, indicating a selective sparing of T helper type 2 (Th2)-associated lymphokine functions by the drug. To test a potential use of POX as an antiinflammatory agent in T cell-mediated autoimmune disease, the influence of POX on myelin basic protein (MBP)-induced experimental autoimmune encephalomyelitis (EAE) was assessed. The onset of EAE in Lewis rats could almost completely be abrogated by oral administration of POX during the induction phase of disease. Lack of clinical symptoms in POX-treated animals coincided with a marked suppression of MBP-specific T cell reactivity in vitro, without any evidence for a generalized impairment of T cell activity. Collectively, our data suggest the potential use of xanthine derivatives of the POX type as a supporting antiinflammatory therapeutic agent in Th1 CD4+ T cell-mediated autoimmune diseases in animal models and possibly in man.
Animals↗
Interleukin-6 production in "normal" and HTLV-1 tax-expressing brain-specific endothelial cells.
Abnormal cytokine production can contribute in many instances to the development of pathology. Our study focuses on the regulation of interleukin (IL)-6 production in vitro in brain-specific endothelial cells (BEC) under physiological conditions and in a model of human T leukemia virus-1 (HTLV-1) infection. IL-6 production was strongly up-regulated in a dose-dependent mode upon exposure to recombinant IL-1 beta, although nearly not detectable in unstimulated BEC. This induction of IL-6 production could be achieved by reagents known to increase intracellular levels of cAMP, such as forskolin, prostaglandin E or pentoxifylline. Furthermore, transcription and production of IL-6 was inducible by addition of dibutyryl cAMP, but not by addition of calcium ionophores or diacylglycerol. To assess a potential role of HTLV-1-infected BEC in the pathogenesis of tropical spastic paraparesis (TSP), the HTLV-1 tax gene was expressed in BEC. Tax gene-expressing BEC produced constitutively very high amounts of IL-6, which were not longer hyperinducible by IL-1 beta or cAMP derivatives. Our results indicate that HTLV-1 tax induces hyperproduction of IL-6 in brain-specific endothelial cells directly by an intracellular mechanism which subsequently renders IL-6 production independent of exogenous stimuli or activators of (cAMP-dependent) second messenger levels. On the basis of these findings we suggest that tax-mediated hyperactivation of IL-6 production in BEC contributes to elevated IL-6 levels found in serum and cerebrospinal fluid of patients with TSP and might have a significance in the immune pathogenesis of the disease.
Animals↗
Function of dipeptidyl peptidase IV (CD26, Tp103) in transfected human T cells.
CD26 (Tp103) is a proteolytic enzyme (dipeptidyl peptidase IV) expressed on the T cell surface that defines an alternative activation signal for human T lymphocytes. It is absent from or present in only low amounts on resting T cells but it is expressed strongly after activation. Crosslinking of CD26/Tp103 via the monoclonal antibody CB.1 triggers functional activities in preactivated T cells. To study the molecular requirements for T cell activation via CD26 we transfected a cDNA encoding CD26 into several CD26-negative cells. In Jurkat T cell leukemia cells that normally do not express the CD26 antigen, the transfected CD26 molecule is functional because the monoclonal antibody CB.1 induces an increase of cytosolic Ca2+ concentration and IL-2 production. For this stimulatory effect a crosslinking of the monoclonal antibody CB.1 is necessary. After modulation of the TCR/CD3 complex the transfected Jurkat cells were insensitive to triggering via CD26. Moreover, a CD26-transfected TCR-negative variant of Jurkat cells did not respond to CD26 triggering despite high levels of expression of the molecule on their surface. These data demonstrate that the function of CD26/Tp103 is dependent on the expression of the T cell receptor complex. In search of a physiological function of CD26 we found a costimulatory effect of mAb CB.1 in combination with the nonstimulatory anti-CD3 antibody BMA030 and an additive effect in the response to the superantigen staphylococcal enterotoxin E. Transfected Jurkat cells, however, did not show a reproducibly enhanced responsiveness to the superantigen compared to that of untransfected cells.
Animals↗
Autoreactive liver-infiltrating T cells in primary biliary cirrhosis recognize inner mitochondrial epitopes and the pyruvate dehydrogenase complex.
Primary biliary cirrhosis (PBC) is characterized by lymphoid infiltrates in the portal tracts of the liver and the occurrence of antimitochondrial autoantibodies in serum directed against components of the pyruvate dehydrogenase complex and the other alpha-keto acid dehydrogenase complexes. These enzymes are located on the inner mitochondrial membrane. The destruction of the biliary tract in PBC is thought to be mediated by autoreactive liver-infiltrating T cells exerting cytotoxic activity or releasing certain lymphokines. In this study the reactivity of liver infiltrating T cells was shown to a bovine pyruvate dehydrogenase complex (PDH), a purified E2 subunit (PDH-E2) and a crude preparation of human liver mitoplasts (HLM), i.e. mitochondria depleted of their outer membranes. Peripheral blood lymphocytes (PBL) from 11 of 15 patients (73.3%) with PBC showed a HLA class II-restricted proliferative response to the PDH complex whereas PBL from patients with chronic viral hepatitis, autoimmune hepatitis or extrahepatic cholestatic icterus (n = 20) and healthy controls (n = 5) did not. In addition 13 of 15 PBL from patients with PBC (86.6%) and three of nine PBL from patients with autoimmune hepatitis (33.3%) reacted with the crude HLM preparation whereas no reactivity was found with PBL from eight patients with chronic viral hepatitis, three patients with extrahepatic cholestasis or five healthy controls. Clonal analysis of 115 liver-infiltrating T cells derived from two diagnostic liver biopsies of patients with PBC revealed a predominance of activated CD4+CD8- T helper cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
Enzymatic activity of CD26 (dipeptidylpeptidase IV) is not required for its signalling function in T cells.
CD26 is a proteolytic enzyme (dipeptidylpeptidase IV) expressed on the T cell surface that defines an alternative activation signal for human T lymphocytes. Crosslinking of CD26 via monoclonal antibodies triggers proliferation and cytotoxicity in preactivated T cells. In this study, we used highly specific competitive and irreversible inhibitors of dipeptidylpeptidase IV to study the role of the enzymatic activity in activation of CD26-transfected T cells as well as of CD26-expressing normal human T cell clones. These inhibitors at concentrations that blocked up to 95% of the enzymatic activity, did not specifically inhibit T cell activation neither via TCR/CD3 nor via CD26 itself. This demonstrates that the enzymatic activity of CD26 is not required for its T cell activating properties.
Animals↗
T cell activation defect in hemodialysis patients: evidence for a role of the B7/CD28 pathway.
The immunosuppressive effect of chronic renal failure is correlated with an impaired proliferation of peripheral blood leukocytes in vitro. This is mainly due to an impaired function of the accessory cells rather than the T cells. Here we tried to define a missing accessory signal for T cell activation in hemodialysis patients. We substituted cell surface bound molecules by adding tumor cell lines to the in vitro assays that express different patterns of accessory molecules. Cell lines that express the costimulatory B7 molecule reconstituted the activation of patients' cells whereas B7 negative cells did not. The reconstitution was also achieved using mouse fibroblasts transfected with human B7 or by monoclonal antibodies that stimulate the B7 ligand CD28 on T cells. These data further emphasize that impaired leukocyte function in renal failure is due to an accessory cell defect, and that T cells of these patients have normal functional capacities when they get the costimulatory signals required. We demonstrate that it is the B7/CD28 pathway that reconstitutes cellular functions in the patients.(ABSTRACT TRUNCATED AT 250 WORDS)
3T3 Cells↗
Multiclonal synovial T cell response to Yersinia enterocolitica in reactive arthritis: the Yersinia 61-kDa heat-shock protein is not the major target antigen.
The T cell response to bacterial antigens plays a major role in the pathogenesis of reactive arthritis (ReA) following enteric infections with Yersinia enterocolitica. To study the antigen specificity of the T cells at the site of inflammation, the response of cloned T cells from the synovial fluid of 2 patients with ReA to partially purified antigens of Yersinia enterocolitica was determined. The clones showed different patterns of response to various fractions, indicating a multiclonal response to Yersinia antigens, and these specificities differed in the 2 patients. Some T cells were specific for Y. enterocolitica; some cross-reacted with other enterobacteria. Proteins of 14 and 19 kDa could be identified as target antigens for the T cell clones, but no clone could be unequivocally found that reacted with the highly purified Yersinia 61-kDa heat shock protein. Thus, the inflammatory T cell response in the synovial fluid in ReA is multiclonal and not predominantly directed against the bacterial heat shock 61-kDa protein.
Adult↗
Low frequency of cytotoxic liver-infiltrating T lymphocytes specific for endogenous processed surface and core proteins in chronic hepatitis B.
To investigate the role of hepatitis B virus (HBV)-specific CD8+ T cells in chronic hepatitis B, the lytic activity of peripheral blood mononuclear cells (PBMC) and liver-infiltrating T cell clones and cytotoxic T cell (CTL) lines stimulated by recombinant vaccinia virus-infected cells were analyzed. Autologous and allogeneic Epstein-Barr virus-transformed B cells infected with vaccinia vectors (VAC) that contain sequences of the surface (S), secretory core (E), cytoplasmatic core (C) VAC antigen of HBV, or the wild-type (WT) VAC served as target cells. ELISA and immunoblotting showed HBV antigen expression in infected cells. Neither PBMC nor C- or E-VAC-stimulated CTL lines showed specific lytic activity. However, S-VAC stimulated blood- and liver-derived CTL from 2 patients with hepatitis B lysed autologous S-VAC but not WT-VAC-infected target cells. One of 158 CD8+ T cell clones from 6 patients with active hepatitis B lysed autologous, but not allogeneic, S-VAC-infected targets. Thus, CTL are present among liver-infiltrating T cells in chronic hepatitis B that recognize endogenously processed hepatitis B surface antigen but not HBc or HBe antigen. The lack of core-specific CTL may contribute to failure in virus elimination in chronic inflammation.
B-Lymphocytes↗
Mutations affecting MHC class II binding of the superantigen streptococcal erythrogenic toxin A.
Streptococcal pyrogenic exotoxin A (SPEA) is an important pathogenicity factor of group A streptococci. It is a member of the family of 'superantigens' produced by Staphylococcus aureus and Streptococcus pyogenes, and its T lymphocyte stimulating activity is involved in the pathogenesis of certain diseases caused by pyogenic streptococci. In this study we have generated nine mutant SPEA molecules by substituting amino acids in the regions of homology between different streptococcal and staphylococcal superantigens. An additional mutant was created by deletion of the 10 N-terminal amino acids. The mutants were expressed as fusion proteins. Several mutations led to a loss of function due to a loss of class II-binding activity. Such loss mutations did not cluster to a certain region of the SPEA molecule. Rather, even a substitution of neighboring amino acids had opposite effects. None of the loss mutations affected the binding of neutralizing mAb and all loss mutants could be precipitated in Ouchterlony tests by a polyclonal anti-SPEA serum. We conclude that the functional activities of SPEA, and probably of other superantigens as well, cannot be attributed to a defined region of the molecule.
Amino Acid Sequence↗
Herpes virus saimiri-transformed human T lymphocytes: normal functional phenotype and preserved T cell receptor signalling.
Herpes virus saimiri (HVS), a primate herpes virus, transforms human CD4+ and CD8+ T lymphocytes to continuous growth in vitro. We have previously shown that HVS-transformed human T cells (HVS-T cells) respond to stimulation via CD2 with autocrine growth. In the present study we have investigated the functional characteristics of HVS-T cells. We describe that these cells can perform all the functions of normal T cells, i.e. cytokine secretion, cytotoxicity, and exocytosis of granule esterases. All these activities can be triggered via CD2 by binding to its natural ligand or via the TCR, e.g. by anti-TCR antibodies, by recognition of a bacterial superantigen and by MHC-restricted recognition of specific antigen. The pattern of tyrosine-phosphorylated proteins after TCR triggering was identical in HVS-T cells and normal T cells. We conclude that HVS-T cells can respond to TCR-mediated signals with the functions of normal T lymphocytes. Furthermore, HVS-T cells are the only transformed human T cells that can be specifically triggered by cytotoxicity and esterase release. The finding that the TCR functions normally in these cells will make HVS a convenient means to immortalize antigen-specific human T lymphocytes.
Antigens↗
Analysis of the in vitro cytokine production by liver-infiltrating T cells of patients with autoimmune hepatitis.
The pathogenic mechanisms underlying the development of autoimmune hepatitis (AIH) are still unclear. Since AIH is associated with the presence of various autoantibodies and certain HLA subtypes, it is likely that T and B cells play a major role in this disease. In this study we have determined the functional capacities of in vivo preactivated liver-infiltrating T cells (LTC) from patients with AIH. As controls we used LTC from patients with non-autoimmune hepatitis (non-AIH). Our results show that preactivated LTC from patients with AIH predominantly (190/255 clones) reside in the CD4+ population, whereas LTC in non-AIH are dominated by the CD8+ phenotype (148/254 clones). In view of this finding we have investigated the cytokine secretion patterns of 102 randomly chosen CD4+ T cell clones from six patients with AIH. As controls we have used 58 CD4+ LTC from 11 patients with non-AIH. All clones were stimulated by lectin and irradiated accessory cells and subsequent cytokine production was evaluated. LTC from patients with AIH have a lower interferon-gamma (IFN-gamma)/IL-4 ratio compared with LTC from non-AIH. Although clones from some patients with AIH produced very high amounts of IL-4 in vitro, this was not a constant finding. These results show that in vivo preactivated LTC from patients with AIH are mostly CD4+ T cells that produce more IL-4 than IFN-gamma. In contrast, LTC from patients with non-AIH are dominated by CD8+ and CD4+ T cells that produce significantly less IL-4 than IFN-gamma. Thus, liver-infiltrating T cells from patients with AIH and non-AIH belong to different functional T cell subsets. This may have implications for the regulation of humoral and cellular immune responses in inflammatory liver disease.
Adult↗
Identification of the Yersinia enterocolitica urease beta subunit as a target antigen for human synovial T lymphocytes in reactive arthritis.
The local T-cell response to bacterial antigens is involved in the pathogenesis of reactive arthritis (ReA). Here, we have identified a 19-kDa antigen of Yersinia enterocolitica O:9 recognized by Yersinia-specific synovial fluid CD4+ T cells in two patients with Yersinia-induced ReA. N-terminal amino acid sequencing of this protein revealed that it was identical to the 19-kDa urease beta subunit of Y. enterocolitica O:9. This protein has previously been shown to be arthritogenic in preimmunized rats after intra-articular injection. Analysis of the T-cell response to this protein showed that it contains several T-cell epitopes, one of which cross-reacts with other enterobacteria not able to induce ReA. This indicates that the arthritogenicity of the 19-kDa antigen is not a property of the 19-kDa protein alone but is dependent on its expression in bacteria able to induce ReA.
Amino Acid Sequence↗
Concomitant loss of conformation and superantigenic activity of staphylococcal enterotoxin B deletion mutant proteins.
The T-cell-stimulating activity of staphylococcal enterotoxin B (SEB) is an important factor in the pathogenesis of certain staphylococcal diseases. To investigate the immunologically active domains of the SEB molecule, we have produced truncated fragments of recombinant SEB by C-terminal and N-terminal deletions. The fragments were expressed as fusion proteins with protein A, including a cleavage site to remove the protein A part. Mutant proteins were tested for the ability to stimulate human resting T cells and SEB-reactive T-cell clones. Deletion of only 9 amino acids from the C terminus leads to complete loss of T-cell-stimulating activity. Removing further amino acids from the SEB molecule did not lead to a reexpression of T-cell-mitogenic activity. A mutant protein, however, in which the 9 C-terminal amino acids were replaced with a tail of 68 amino acids encoded by the vector was fully active. Two mutant proteins with N-terminal deletions of 60 and 81 amino acids were inactive as well. A neutralizing monoclonal antibody against a conformational epitope lost binding with all the inactive mutant proteins only, whereas a monoclonal antibody recognizing an epitope involved in emetic activity reacted with all mutant proteins. These results suggest that even small deletions at the C terminus affect the three-dimensional conformation of the SEB molecule.
Amino Acid Sequence↗
Hepatitis A: hepatotropism and influence on myelopoiesis.
Immunopathologic mechanisms leading to liver tissue injury in hepatitis caused by hepatitis A virus (HAV) were studied in an autologous in vitro model. Data show virus-specific killing by liver-infiltrating T lymphocytes in man and support the hypothesis that hepatocellular damage as well as efficient elimination of virus-infected hepatocytes is mediated by HLA-restricted, HAV-specific CD8+ T lymphocytes. Furthermore, experimental results demonstrate that human interferon-gamma produced by HAV-specific T cells may act as a key factor in T-cell-promoted clearance of HAV-infected hepatocytes. Besides the well-known hepatotropism, the myelotropic properties of HAV have some important clinical implications. Perturbations of hematopoietic regulation, ranging from transient granulocytopenia to rare cases of bone marrow failure, are associated with HAV infection. In an attempt to elucidate the pathogenetic mechanisms, we could show a direct suppressive effect of HAV on human bone marrow progenitors and a significant progressive decline in these cells in HAV-infected long-term bone marrow cultures.
Bone Marrow↗