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Biomedical subjects

M Toebes

Publications and source records attributed to M Toebes.

12 recordsLinked to original sources
PubMed2001

Expression of the serpin serine protease inhibitor 6 protects dendritic cells from cytotoxic T lymphocyte-induced apoptosis: differential modulation by T helper type 1 and type 2 cells.

Dendritic cells (DCs) play a central role in the immune system as they drive activation of T lymphocytes by cognate interactions. However, as DCs express high levels of major histocompatibility complex class I, this intimate contact may also result in elimination of DCs by activated cytotoxic T lymphocytes (CTLs) and thereby limit induction of immunity. We show here that immature DCs are indeed susceptible to CTL-induced killing, but become resistant upon maturation with anti-CD40 or lipopolysaccharide. Protection is achieved by expression of serine protease inhibitor (SPI)-6, a member of the serpin family that specifically inactivates granzyme B and thereby blocks CTL-induced apoptosis. Anti-CD40 and LPS-induced SPI-6 expression is sustained for long periods of time, suggesting a role for SPI-6 in the longevity of DCs. Importantly, T helper 1 cells, which mature DCs and boost CTL immunity, induce SPI-6 expression and subsequent DC resistance. In contrast, T helper 2 cells neither induce SPI-6 nor convey protection, despite the fact that they trigger DC maturation with comparable efficiency. Our data identify SPI-6 as a novel marker for DC function, which protects DCs against CTL-induced apoptosis.

Animals↗
PubMed2001

Differentiation of cytomegalovirus-specific CD8(+) T cells in healthy and immunosuppressed virus carriers.

During immunosuppression, cytomegalovirus (CMV) can reactivate and cause serious clinical problems. Normally, abundant virus replication is suppressed by immune effector mechanisms. To study the interaction between CD8(+) T cells and persisting viruses, frequencies and phenotypes of CMV-specific CD8(+) T cells were determined in healthy individuals and compared to those in renal transplant recipients. In healthy donors, function of circulating virus-specific CD8(+) T cells, as measured by peptide-induced interferon gamma (IFN-gamma) production, but not the number of virus-specific T cells enumerated by binding of specific tetrameric peptide/HLA complexes, correlated with the number of CMV-specific IFN-gamma-secreting CD4(+) helper T cells. Circulating CMV- specific CD8(+) T cells did not express CCR7 and may therefore not be able to recirculate through peripheral lymph nodes. Based on coexpression of CD27 and CD45R0 most CMV-specific T cells in healthy donors appeared to be memory-type cells. Remarkably, frequencies of CMV-specific CD8(+) T cells were significantly higher in immunosuppressed individuals than in healthy donors. In these patients CMV-specific cells predominantly had an effector phenotype, that is, CD45R0(+)CD27(-)CCR7(-) or CD45RA(+)CD27(-)CCR7(-) and contained both granzyme B and perforin. Our data show that in response to immunosuppressive medication quantitative and qualitative changes occur in the CD8(+) T-cell compartment. These adaptations may be instrumental to maintain CMV latency. (Blood. 2001;98:754-761)

Adult↗
PubMed1999

Systemic T cell expansion during localized viral infection.

In a local immune response, the priming and expansion of the antigen-specific T cell population has been thought to largely take place in the draining lymphoid tissue. This model was primarily based on indirect enumeration of antigen-specific T cells by limiting dilution analyses. Here, tetrameric MHC class I complexes were used to evaluate the contribution of different secondary lymphoid organs in a local immune response by following the CD8+ T cell responses against the immunodominant epitopes of influenza A virus and herpes simplex virus-1. Mice were either intranasally infected with influenza A virus and developed pneumonia or were intradermally injected with herpes simplex virus-1. Remarkably, even though these viruses cause a local infection, the spleen of infected animals contains approximately 50-fold more antigen-specific cytotoxic T cells than the draining lymph nodes. Although antigen-specific T cells in spleen appear not to have experienced any recent encounter with antigen, this population is actively dividing, and over time, the formation of a memory T cell population is observed. These data reveal that there is a remarkably large and distinct population of antigen-specific T cells in spleen in the course of a local antigenic challenge. This T cell compartment may not only form the foundation of a memory T cell pool but could also provide a safeguard against systemic spreading of an infection.

Animals↗
PubMed1998

Immunization with human papillomavirus type 16 (HPV16) oncoprotein-loaded dendritic cells as well as protein in adjuvant induces MHC class I-restricted protection to HPV16-induced tumor cells.

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical cancer. In this study, we demonstrate that dendritic cells (DCs) pulsed with HPV16 E7 protein are not only recognized in vitro by E7-specific CTLs but also elicit E7-specific CTL responses in vivo, associated with protection against a challenge with syngeneic HPV16-induced tumor cells. Vaccination with soluble E7 protein in incomplete Freund's adjuvant likewise induces E7-specific CTL responses associated with tumor protection. The presence of HPV16 E7-specific CTLs in vivo and the observation that depletion of CD8+ cells completely abolishes tumor protection demonstrate that CTLs are the major effector cells in mediating antitumor activity. The in vivo involvement of DCs in the activation of protective CTLs is suggested by the surface display of E7 peptide-loaded MHC class I molecules on these cells after E7 protein immunization. These data show that HPV16 E7 protein-pulsed DCs, as well as the administration of E7 protein antigen in adjuvant, can effectively stimulate tumor-specific MHC class I-restricted CD8+ T-cell-mediated protective immunity to HPV16-induced cancers.

Animals↗
PubMed1997

A novel, highly efficient peptide-HLA class I binding assay using unfolded heavy chain molecules: identification of HIV-1 derived peptides that bind to HLA-A*0201 and HLA-A*0301.

A novel cell-free, highly automated peptide-HLA binding assay has been designed during which a mixture of unfolded recombinant HLA heavy chain molecules, beta 2-microglobulin and a fluorescent labeled standard peptide is allowed to form peptide-HLA complexes. The binding of a peptide of interest is monitored as the ability to inhibit the formation of fluorescent peptide-HLA complexes. The assay was validated using published, known HLA-A* 0201 and HLA-A* 0301 binding peptides. In addition a selected set of HIV-1LAI reverse transcriptase derived 10-mer peptides, that had been selected on the basis of HLA-A* 0201 or HLA-A* 0301 binding motifs, were tested for HLA-A* 0201/A* 0301 binding. In that set we identified 8 peptides which bound with high affinity to HLA-A* 0201 and 5 peptides which bound with high affinity to HLA-A* 0301. The major advantage of the use of denatured heavy chain is the improved economy and efficiency, as unfolded protein material is in principle easily accessible by recombinant technology.

HIV Reverse Transcriptase↗
PubMed1997

IkappaB alpha is a target for the mitogen-activated 90 kDa ribosomal S6 kinase.

The activity of transcription factor NFkappaB is regulated by its subcellular localization. In most cell types, NFkappaB is sequestered in the cytoplasm due to binding of the inhibitory protein IkappaB alpha. Stimulation of cells with a wide variety of agents results in degradation of IkappaB alpha which allows translocation of NFkappaB to the nucleus. Degradation of IkappaB alpha is triggered by phosphorylation of two serine residues, i.e. Ser32 and Ser36, by as yet unknown kinases. Here we report that the mitogen-activated 90 kDa ribosomal S6 kinase (p90rsk1) is an IkappaB alpha kinase. p90rsk1 phosphorylates IkappaB alpha at Ser32 and it physically associates with IkappaB alpha in vivo. Moreover, when the function of p90rsk1 is impaired by expression of a dominant-negative mutant, IkappaB alpha degradation in response to mitogenic stimuli, e.g. 12-O-tetradecanoylphorbol 13-acetate (TPA), is inhibited. Finally, NFkappaB cannot be activated by TPA in cell lines that have low levels of p90rsk1. We conclude that p90rsk1 is an essential kinase required for phosphorylation and subsequent degradation of IkappaB alpha in response to mitogens.

Animals↗
PubMed1997

A sensitive fluorometric assay for quantitatively measuring specific peptide binding to HLA class I and class II molecules.

A sensitive, highly reproducible assay was developed for measuring binding of peptides to various HLA class I and II alleles. The assay is based on competition for binding to HLA between a peptide of interest and a fluorescent labelled standard peptide. This mixture is incubated with HLA to obtain equilibrium binding, and subsequently separated on an HPLC size-exclusion column in (i) a protein fraction containing HLA and bound peptide and (ii) a free peptide fraction. Each assay uses only 100 fmol labelled peptide and approximately 10 pmol of HLA. The analytical system contains an autosampler that samples from 96-well microtiter plates. Injections and data recording/evaluation is fully automated. Typical analysis time is 10-12 min per sample. The fluorescence in the HLA-bound peptide and free peptide containing fractions is measured on-line. The ratios of fluorescence signal in protein and peptide fractions at various concentrations of the peptide of interest are determined. IC50 values are calculated from the binding curve as obtained by curve fitting of the data. Here we show results for peptide binding to HLA-DR1 and -DR17 molecules purified from detergent solubilized cell lysates. and for recombinant HLA-A*0201 and HLA-A*0301 expressed in E. coli. The assay reported is sensitive and reproducible. It is non-radioactive and is non-labor intensive due to the high degree of automation.

Amino Acid Sequence↗
PubMed1995

Phosphorylation of a specific cdk site in E2F-1 affects its electrophoretic mobility and promotes pRB-binding in vitro.

The E2F transcription factor family participates in growth control presumably through transcriptional activation of genes that promote entry into S phase. E2F activity is believed to be controlled across the cell cycle by association with various cellular proteins, including the product of the retinoblastoma gene (pRB). We find that E2F-1 proteins are heterogeneously phosphorylated in insect cells, as a result of which they migrate as a doublet on SDS-polyacrylamide gels. This electrophoretic shift is shown to be dependent upon specific phosphorylation of E2F-1 on serine-375 (S375), near the pRB-binding site. Phosphorylation on S375 also occurs in human cells. E2F-1 was most efficiently phosphorylated on this residue by cyclin A/cdk2 kinase, and to a lesser extent by cyclin A/cdk2, irrespective of the presence of the pRB-related p107 protein. Phosphorylation of E2F-1 on S375 greatly enhanced its affinity of pRB in vitro. These results suggest a novel way of regulating E2F-1 activity, namely by cell-cycle-dependent phosphorylation of this transcription factor.

Animals↗
PubMed1995

Induction of differentiation-regulated transcription factor Oct-6 specifically accompanies major histocompatibility complex class I down-regulation by E1A of oncogenic adenovirus type 12.

The E1A genes from adenovirus (Ad) types 5 and 12 share the capacity to cooperate with a second oncogene to transform primary rodent cells in vitro. However, only Ad12-transformed cells are oncogenic in immunocompetent rodents, an event that requires conserved region 3 (CR3) of E1A to be intact. Ad12-induced tumorigenicity correlates with the E1A-CR3-dependent down-modulation of MHC class I transcription, contributing to escape from CTL-mediated immune surveillance. Expression of MHC class I antigens is also lacking in undifferentiated embryonal carcinoma cells. In these cells, MHC class I expression increases during differentiation in a process possibly involving octamer-binding proteins. We found that both nononcogenic and oncogenic Ad-transformed cells contained the ubiquitously expressed factor Oct-1. In contrast, only oncogenic Ad12-transformed cells that are derived from primary cell cultures expressed an additional octamer-binding factor, which we identified as Oct-6. The induction of Oct-6 expression was at the RNA level and was found to require an intact CR3 domain in Ad12 E1A. Like MHC class I expression, Oct-6 expression was not affected in already established cell lines expressing Ad12 E1A. The presence of Oct-6 in Ad12-transformed cells correlated with an increase in octamer-dependent transcription of a reporter gene, relative to Ad5-transformed cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenovirus E1A Proteins↗
PubMed1993

A- and B-type cyclins differentially modulate substrate specificity of cyclin-cdk complexes.

Both cyclins A and B associate with and thereby activate cyclin-dependent protein kinases (cdks). We have investigated which component in the cyclin-cdk complex determines its substrate specificity. The A- and B-type cyclin-cdk complexes phosphorylated histone H1 and their cyclin subunits in an indistinguishable manner, irrespective of the catalytic subunit, p33cdk2 or p34cdc2. In contrast, only the cyclin A-cdk complexes phosphorylated the Rb-related p107 protein in vitro. Likewise, binding studies revealed that cyclin A-cdk complexes bound stably to p107 in vitro, whereas cyclin B-cdk complexes did not detectably associate with p107, under identical assay conditions. Binding to p107 required both cyclin A and a cdk as neither subunit alone bound to p107. These results demonstrate that although the kinase subunit provides a necessary component for binding, it is the cyclin subunit that plays the critical role in targeting the complex to p107. Finally, we show that the cyclin A-p33cdk2 complex phosphorylated p107 in vitro at most of its sites that are also phosphorylated in human cells, suggesting that the cyclin A-p33cdk2 complex is a major kinase for p107 in vivo.

Animals↗