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M Guttinger

Publications and source records attributed to M Guttinger.

At least 19 recordsLinked to original sources
PubMed2000

Adoptive immunotherapy by avidin-driven cytotoxic T lymphocyte-tumor bridging.

We have shown previously that T cells, tagged with biotinylated anti-CD3 antibody fragments, can exert avidin-dependent cytolytic activity on suitably biotinylated tumor cells in vitro. In this study, we demonstrate that avidin-driven CTL-tumor bridging in vivo leads to growth inhibition of murine tumors WEHI-164 fibrosarcoma and RMA lymphoma. The biodistribution of biotin-tagged 111In-labeled T cells demonstrated a selective avidin-dependent and time-dependent accumulation of radioactivity at tumor sites. The specificity of lymphocyte tumor localization was demonstrated by the concurrent time-dependent decrease of radioactivity in the blood and in all other organs. Furthermore, we documented a therapeutic effect of the adoptively transferred T cells, i.e., a significant delay of tumor growth at early stages. All of the experiments included a control group of mice, which received all of the reagents, except avidin. These avidin-minus mice showed no specific localization and no delay in tumor growth, indicating that avidin bridging was essential for T-cell activity at tumor sites.

Animals↗
PubMed1999

GKLF in thymus epithelium as a developmentally regulated element of thymocyte-stroma cross-talk.

Gut-enriched Krüppel-like factor (GKLF) is a transcriptional regulator expressed in differentiated epithelia. We identified GKLF transcript as a regulated element in thymic epithelium of recombinase-deficient mice during thymus development induced by anti-CD3 antibody injection. This treatment recapitulates the organogenetic process depending on productive rearrangement of T cell receptor (TCR) beta gene with thymocytes expansion and acquisition of the CD4+8+ double positive phenotype. In wildtype mice, GKLF is expressed very early in embryogenesis and becomes intensely up-regulated in thymus epithelium at day 18 of gestation when TCR beta expressing cells have selectively expanded and express both CD4 and CD8. The results presented here suggest that thymocytes may regulate GKLF transcriptionally in the cortical epithelium at the developmental check-point controlled by TCR beta gene rearrangement. Furthermore, GKLF expression in hematopoietic stroma might suggest the thus far uncharacterised participation of this factor in hematopoiesis.

3T3 Cells↗
PubMed1998

Epithelial V-like antigen (EVA), a novel member of the immunoglobulin superfamily, expressed in embryonic epithelia with a potential role as homotypic adhesion molecule in thymus histogenesis.

Thymus development depends on a complex series of interactions between thymocytes and the stromal component of the organ. To identify regulated genes during this codependent developmental relationship, we have applied an RNA fingerprinting technique to the analysis of thymus expansion and maturation induced in recombinase-deficient mice injected with anti-CD3 antibodies. This approach led us to the identification of a gene encoding a new member of the immunoglobulin superfamily, named epithelial V-like antigen (EVA), which is expressed in thymus epithelium and strongly downregulated by thymocyte developmental progression. This gene is expressed in the thymus and in several epithelial structures early in embryogenesis. EVA is highly homologous to the myelin protein zero and, in thymus-derived epithelial cell lines, is poorly soluble in nonionic detergents, strongly suggesting an association to the cytoskeleton. Its capacity to mediate cell adhesion through a homophilic interaction and its selective regulation by T cell maturation might imply the participation of EVA in the earliest phases of thymus organogenesis.

Amino Acid Sequence↗
PubMed1998

Expression pattern of the epithelial v-like antigen (Eva) transcript suggests a possible role in placental morphogenesis.

Adhesive mechanisms are considered to be of crucial importance for blastocyst adherence to the uterine wall, as well as for the interactions between embryonal and decidual tissues during hemochorial placenta formation. Epithelial V-like Antigen (Eva) is a novel homophilic adhesion molecule of the immunoglobulin superfamily, which during mouse embryonic development is expressed by various differentiating epithelia. In the present paper we describe Eva expression during mouse trophoblast invasion and placental morphogenesis, analysing day 5.5 to 18.5 postcoitum (p.c.) placentas and deciduomas by in situ hybridization. Eva transcripts were detected in spongiotrophoblast cells from 7.5 to 18.5 days p.c. Expression was uniform at early stages, but after day 11.5, p.c. became limited to the invasive subpopulation of spongiotrophoblasts (known as glycogen cells). Trophoblast giant cells did not express Eva in any of the stages analysed. Besides trophoblasts, also early postimplantation decidua was positive for Eva transcripts. In decidual tissue, Eva expression was present at day 5.5 p.c., peaked at day 7.5 p.c., and declined on successive days. The expression pattern of Eva transcripts suggests that during mouse placenta formation, its protein product may play a role in the processes of trophoblast invasion, decidual response, and trophoblast-decidual interaction.

Animals↗
PubMed1996

Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect.

Physiological concentrations of urokinase plasminogen activator (uPA) stimulated a chemotactic response in human monocytic THP-1 through binding to the urokinase receptor (uPAR). The effect did not require the protease moiety of uPA, as stimulation was achieved also with the N-terminal fragment (ATF), while the 33 kDa low molecular weight uPA was ineffective. Co-immunoprecipitation experiments showed association of uPAR with intracellular kinase(s), as demonstrated by in vitro kinase assays. Use of specific antibodies identified p56/p59hck as a kinase associated with uPAR in THP-1 cell extracts. Upon addition of ATF, p56/p59hck activity was stimulated within 2 min and returned to normal after 30 min. Since uPAR lacks an intracellular domain capable of interacting with intracellular kinase, activation of p56/p59hck must require a transmembrane adaptor. Evidence for this was strongly supported by the finding that a soluble form of uPAR (suPAR) was capable of inducing chemotaxis not only in THP-1 cells but also in cells lacking endogenous uPAR (IC50, 5 pM). However, activity of suPAR require chymotrypsin cleavage between the N-terminal domain D1 and D2 + D3. Chymotrypsin-cleaved suPAR also induced activation of p56/p59hck in THP-1 cells, with a time course comparable with ATF. Our data show that uPA-induced signal transduction takes place via uPAR, involves activation of intracellular tyrosine kinase(s) and requires an as yet undefined adaptor capable of connecting the extracellular ligand binding uPAR to intracellular transducer(s).

Animals↗
PubMed1995

Syk and ZAP-70 mediate recruitment of p56lck/CD4 to the activated T cell receptor/CD3/zeta complex.

During antigen recognition by T cells, CD4 and the T-cell receptor (TCR)/CD3/zeta complex are thought to interact with the same major histocompatibility complex II molecule in a stable ternary complex. Evidence has suggested that the association of CD4 with TCR/CD3/zeta requires the interaction of the protein tyrosine kinase p56lck with CD4. We have taken a biochemical approach to understand the mechanism by which p56lck and, in particular, its src homology (SH) 2 domain contributes to the association of CD4 with TCR/CD3/zeta during activation. We have previously shown that the p56lck SH2 domain binds directly to tyrosine-phosphorylated ZAP-70. Here we formally demonstrate the in vivo association of p56lck with the homologous protein tyrosine kinases Syk and ZAP-70 after CD3 stimulation of Jurkat cells. A tyrosine-phosphorylated peptide containing the sequence predicted to be optimal for binding to the SH2 domain of src family kinases specifically competes for this association, indicating that tyrosine-phosphorylated ZAP-70 and Syk bind to p56lck by an SH2-mediated interaction. We also show that the same peptide is able to compete for the activation-dependent TCR/CD4 association in Jurkat cells. Moreover, ZAP-70 and CD4 cocap only after CD3 stimulation in human T lymphoblasts. We propose that the interaction of the p56lck SH2 domain with zeta-associated tyrosine-phosphorylated ZAP-70 and/or Syk enables CD4 to associate with antigen-stimulated TCR/CD3/zeta complexes.

Antigens, CD↗
PubMed1992

Protein tyrosine kinase p59fyn is associated with the T cell receptor-CD3 complex in functional human lymphocytes.

The binding of antigen to the multicomponent T cell antigen receptor (TcR)-CD3 complex leads to the activation of several signal transduction pathways which result in T lymphocyte proliferation and lymphokine secretion by molecular mechanisms and catalytic molecules as yet poorly defined. One of the earliest events that follows the triggering of the antigen-specific TcR-CD3 complex is a rapid tyrosine phosphorylation of several intracellular substrates, suggesting stimulation of at least one protein tyrosine kinase (PTK). Since none of the seven TcR-CD3 subunits exhibits a recognizable kinase domain, it seems likely that the receptor complex is associated with an intracellular PTK. p59fyn and the T lymphocyte-specific p56lck are two intracellular, non-receptor, cell membrane-associated PTK of the src family expressed in T lymphocytes. Here, we show by double immunofluorescence microscopy a specific co-distribution of p59fyn, but not p56lck, with antibody-induced TcR or CD3 caps in intact human T lymphocytes. These findings provide direct evidence for a significant association of p59fyn with the TcR-CD3 complex under physiologically relevant conditions in functional T lymphocytes. They suggest that p59fyn is a crucial component of the TcR signal transduction machinery and that one of the earliest consequences of antigen recognition by the TcR is p59fyn-mediated phosphorylation of intracellular substrates on tyrosine residues.

Animals↗
PubMed1992

CD45 phosphotyrosine phosphatase and p56lck protein tyrosine kinase: a functional complex crucial in T cell signal transduction.

Tyrosine phosphorylation of several intracellular proteins is observed very early during T cell activation. p56lck, a non-receptor src-like protein tyrosine kinase (PTK) which is associated with the intracellular domains of CD4 and CD8 co-receptors, has been implicated in these early signal transduction events. Furthermore, recent experiments indicate that the receptor phosphotyrosine phosphatase, CD45, might be important in the regulation of p56lck PTK activity and that its expression is required for the generation of second messenger molecules following TCR triggering. Here, using co-capping experiments and double indirect immunofluorescence microscopy in functional human T lymphocytes, a specific co-distribution of a significant fraction of p56lck with CD45, but not with several other cell surface proteins, has been revealed. This is the first demonstration of a physical interaction between a receptor phosphotyrosine phosphatase and a PTK under physiologically relevant conditions. In addition, after antibody-induced capping of CD4, both a co-localization of p56lck and CD4, and concomitantly a significant increase in intracellular phosphotyrosine at the sites of CD4 caps were observed. In strong contrast to these results, co-clustering of CD4 with CD45 did not result in any detectable intracellular phosphotyrosine at the cap sites. These data indicate that CD45 can act on CD4-associated phosphoproteins in viable human T lymphocytes. Further, this provides evidence that p56lck PTK is a substrate of CD45 phosphotyrosine phosphatase in vivo and thereby supports the idea that CD45 is an early regulator of T cell activation involved in the modulation of the coupling of receptor-triggered events to intracellular signalling pathways.

CD4 Antigens↗
PubMed1991

HLA polymorphism and T cell recognition of a conserved region of p190, a malaria vaccine candidate.

We have examined T cell recognition of a recombinant polypeptide (190L), corresponding to a 175-amino-acid-long conserved region of the major surface antigen (p190) of Plasmodium falciparum merozoites. We show that 190L contains a variety of T cell epitopes, and can be recognized in association with many different MHC class II molecules, including HLA-DR, DP, and DQ antigens. Most of the epitope-containing peptides are able to bind to more than one DR, and a single DR molecule can bind to different peptides. These findings, together with the fact that humans are generally heterozygous at the DR, DQ, and DP beta chain loci, suggest that MHC restriction should not be a major constraint in the development of malaria subunit vaccines.

Amino Acid Sequence↗
PubMed1991

Cloned antigens from Plasmodium falciparum as malaria vaccine candidates.

The problems of genetic polymorphism and poor immunogenicity of malaria vaccine candidates are discussed, with emphasis on the Circumsporozoite (CS) and p190 proteins of Plasmodium falciparum. It may be possible to use conserved regions of these proteins to raise protective immune responses against non-polymorphic determinants. Better adjuvants or delivery systems will be a critical factor in development of an effective vaccine.

Amino Acid Sequence↗
PubMed1990

Malaria antigens and MHC restriction.

In the case of the malaria CS protein we have shown that there is at least one T cell determinant which is able to bind to and be recognized by most human MHC class II molecules, while for the 190L polypeptide, derived from a conserved region of the p190 merozoite surface protein, we have identified several epitopes recognized by T cell clones in association with different HLA-class II isotypes and alleles. In addition, binding analysis of these epitopes indicated that most of the peptides are able to bind to multiple allelic forms of class II molecules. Although there are important obstacles to malaria vaccine development we believe that, in the light of these results, unresponsiveness in humans, caused by MHC restriction, might not be a major constraint in development of a subunit vaccine.

Amino Acid Sequence↗
PubMed1990

An invariant, "universal" T-cell epitope in the P. falciparum circumsporozoite protein.

Although there are important obstacles to malaria vaccine development, we believe they might be overcome by a strategy of searching for conserved regions of a vaccine candidate that are recognized in association with many different HLA molecules and, if necessary, deliberately modifying the conserved sequences to improve their immunogenicity for T cells. This approach is illustrated by work on the circumsporozoite (CS) protein of Plasmodium falciparum, which covers the surface of the malaria sporozoite and is the best characterized of current vaccine candidates.

Animals↗
PubMed1989

Requirements for screening recombinant DNA libraries for T cell epitope expression.

The possibility of screening cDNA expression libraries with T cell clones was investigated. The model system was based on human T cell clones specific for the recombinant malaria protein 190L, which was expressed fused to beta-galactosidase in lambda gt11. Several membranes were tested for their capacity to bind antigen and stimulate T cell proliferation. Pretreatment of membranes with DMSO and/or sonication to release the antigen improved the sensitivity of the assay. Under optimal conditions, T cell proliferation in response to antigen bound to a low protein binder membrane was comparable to that observed with the antigen in solution. A dot-blot type apparatus was designed for screening large numbers of plaques with T cells. The technical problems of this approach, its requirements and possible applications are discussed.

Clone Cells↗
PubMed1989

Vaccine T-cell epitope selection by a peptide competition assay.

The binding of several peptides derived from the Plasmodium falciparum circumsporozoite protein (CS protein) to the human major histocompatibility complex class II proteins HLA-DR5 and -DRw6 was examined in a competition assay. Fixed antigen-presenting cells (APCs) were incubated with various concentrations of each peptide and suboptimal concentrations of stimulator peptides. The binding of the CS peptides to DR5 or DRw6 proteins was then determined in a proliferation assay using two established DR5 or DRw6-restricted T-cell clones with specificity for the stimulator peptides as responder cells. One of five CS peptides, comprising together about 50% of the CS protein sequence, was found to compete with the binding of the stimulator peptides to DR5 and DRw6. The CS peptide CS-(378-398), binding to DR5 and DRw6, was then shown to be able to induce primary in vitro responses of T cells from donors with DR5 and DRw6 haplotypes. CS-(378-398)-induced T-cell clones responded not only to the homologous peptide but also to the native CS protein in the presence of appropriate APCs. The strategy we have applied is of considerable general interest for the engineering of vaccines against any pathogen, since it greatly facilitates the selection of appropriate T-cell epitopes to be incorporated in the vaccine.

Amino Acid Sequence↗
PubMed1988

Epitopes recognized by human T lymphocytes on malaria circumsporozoite protein.

The circumsporozoite protein of the malaria parasite Plasmodium falciparum contains regions of nonrepetitive sequences which are predicted to be T cell recognition sites. We synthesized peptides corresponding to three of these regions, and tested their ability to stimulate proliferation of peripheral blood lymphocytes from donors living in a malaria-endemic area, or from nonimmune donors. Cells from 15 out of 22 donors (including 4 of 6 nonimmune individuals) were stimulated by one or more of the peptides. T cell clones specific for one of the peptides were obtained and shown to recognize the native protein purified from sporozoites. These data help to identify T cell epitopes which could be incorporated into a malaria vaccine.

Animals↗
PubMed1988

Human T cells recognize polymorphic and non-polymorphic regions of the Plasmodium falciparum circumsporozoite protein.

In order to characterize T cell epitopes in the Plasmodium falciparum circumsporozoite (CS) protein sequence, we isolated T cell clones, from non-immune donors, which reacted with synthetic peptides corresponding to two predicted CS protein T cell epitopes. Peptide CS.T3 (corresponding to a non-polymorphic region of the CS protein, residues 378-398) was recognized in association with either DR2 or DRw9 restriction elements. T cell clones recognizing CS.T3 also reacted with the sporozoite-derived CS protein. Peptide CS.T2 corresponds to a polymorphic region (residues 325-341) of the CS protein. Unlike the CS.T3-specific clones, the CS.T2-specific clones did not recognize the CS protein. Since the CS.T2 peptide includes residues which are polymorphic in different P. falciparum isolates, we investigated whether these residues were critical for recognition of the peptide. We show here that a single amino acid substitution at a position of the CS protein which shows genetic polymorphism affects recognition of the sequence by human T cells. The implications of these data for malaria vaccine development are discussed.

Animals↗