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R Offringa

Publications and source records attributed to R Offringa.

At least 73 records · Page 4Linked to original sources
PubMed1997

Tumor eradication by wild-type p53-specific cytotoxic T lymphocytes.

The tumor suppressor protein p53 is overexpressed in close to 50% of all human malignancies. The p53 protein is therefore an attractive target for immunotherapy. Cytotoxic T lymphocytes (CTLs) recognizing a murine wild-type p53 peptide, presented by the major histocompatibility complex class I molecule H-2Kb, were generated by immunizing p53 gene deficient (p53 -/-) C57BL/6 mice with syngeneic p53-overexpressing tumor cells. Adoptive transfer of these CTLs into tumor-bearing p53 +/+ nude mice caused complete and permanent tumor eradication. Importantly, this occurred in the absence of any demonstrable damage to normal tissue. When transferred into p53 +/+ immunocompetent C57BL/6 mice, the CTLs persisted for weeks in the absence of immunopathology and were capable of preventing tumor outgrowth. Wild-type p53-specific CTLs can apparently discriminate between p53-overexpressing tumor cells and normal tissue, indicating that widely expressed autologous molecules such as p53 can serve as a target for CTL-mediated immunotherapy of tumors.

Adoptive Transfer↗
PubMed1997

Efficient loading of HLA-DR with a T helper epitope by genetic exchange of CLIP.

The HLA class II-associated invariant chain (Ii)-derived peptide (CLIP) occupies the peptide binding groove during assembly in the endoplasmic reticulum, travels with HLA class II to endosomal compartments, and is subsequently released to allow binding of antigenic peptides. We investigated whether the exchange of CLIP with a known T helper epitope at the DNA level would lead to efficient loading of this helper epitope onto HLA class II. For this purpose, a versatile Ii-encoding expression vector was created in which CLIP can be replaced with a helper epitope of choice. Upon supertransfection of HLA-DR1-transfected 293 cells with an Ii vector encoding a known T helper epitope (HA307-319), predominantly length variants of this epitope were detected in association with the HLA-DR1 molecules of these cells. Moreover, this transfectant was efficiently recognized by a peptide-specific T helper clone (HA1.7). The results suggest that this type of Ii vector can be used to create potent class II+ cellular vaccines in which defined T cell epitopes are continuously synthesized.

Amino Acid Sequence↗
PubMed1997

Cleavage of FLICE (caspase-8) by granzyme B during cytotoxic T lymphocyte-induced apoptosis.

Cytotoxic T lymphocytes induce apoptosis in target cells through the CD95(APO-1/Fas) and the perforin/granzyme B (GrB) pathway. The exact substrate of GrB in vivo is still unknown, but to induce apoptosis GrB requires the activity of caspases in target cells. We show here that in HeLa target cells induction of apoptosis through the perforin/GrB pathway resulted in minor direct cleavage of CPP32 (caspase-3) by GrB. Most caspase-3 cleavage resulted from activation of an upstream caspase. Moreover, target cells derived from caspase-3(-/-) mice displayed GrB-induced poly(ADP-ribose) polymerase (PARP) cleavage with only partially reduced efficiency compared to wild-type target cells. This indicates that other PARP-cleaving caspases can be activated during perforin/GrB-induced cell death. In contrast to caspase-3, FLICE (caspase-8) was directly cleaved by GrB in HeLa cells. We therefore conclude that FLICE not only plays a central role in CD95(APO-1/Fas)-induced apoptosis but can also be directly activated during perforin/GrB-induced apoptosis.

Animals↗
PubMed1997

Activation or frustration of anti-tumor responses by T-cell-based immune modulation.

Many types of tumors (e.g. virus-induced tumors, melanomas, tumors over-expressing certain oncogenes) often express antigens that can induce T-cell-mediated tumor-specific immune responses. Nonetheless, many such tumors manage to circumvent the induction of an effective anti-tumor T-cell response, as is apparent from the many tumor-bearing patients. Therefore, optimally designed vaccination protocols may evoke a more powerful and competent T-cell-mediated anti-tumor response, allowing the host to effectively deal with at least some cancers. These vaccination approaches might include immunization with whole (tumor) cell-based vaccines, entire tumor antigens or selected T-cell epitopes derived from tumor antigens. This survey is an update of several anti-tumor vaccination approaches employed, and on novel possibilities to target the anti-tumor immune response to preselected tumor-derived T-cell epitopes.

Adenoviridae↗
PubMed1997

Identification of potential HLA-A *0201 restricted CTL epitopes derived from the epithelial cell adhesion molecule (Ep-CAM) and the carcinoembryonic antigen (CEA).

The altered expression pattern of the Epithelial Cell Adhesion Molecule (Ep-CAM) and the Carcinoembryonic Antigen (CEA) on tumor cells of epithelial origin as compared to normal epithelia may permit T cells to preferentially recognize and lyse these tumor cells. The binding affinity for human leucocyte antigen A2.1 (HLA-A*0201) and the capacity to form stable peptide-major histocompatibility complex (MHC) interactions with this molecule were tested for 410 Ep-CAM-derived sequences, including an overlapping set of 9 amino-acid-long peptides, and 73 CEA-derived peptides fulfilling the HLA-A*0201 motif. Peptides with a high binding affinity and a low peptide-MHC dissociation rate were subsequently tested for their immunogenicity in HLA-A*0201Kb transgenic mice. One Ep-CAM-derived peptide and 1 CEA-derived peptide were able to reproducibly induce peptide-specific cytotoxic T cells (CTL) in these mice. This indicates that EpCAM and CEA are potential target antigens for CTL-mediated immunotherapy of epithelial cancers.

Animals↗
PubMed1996

Protective antitumor immunity induced by immunization with completely allogeneic tumor cells.

We have shown previously that immunization of B6 mice (H-2b) with tumor cells of B6 origin transformed by the human adenovirus type 5 early region 1 (Ad5E1) induces an H-2Db-restricted CTL response against an E1B-encoded CTL epitope. We now report that immunization of B6 mice with Ad5E1-transformed tumor cells of BALB/c origin (H-2d), apart from inducing a B6 anti-BALB/c allo-response, also induces a strong CTL response against the E1B-encoded H-2Db-presented CTL epitope. BALB/c Ad5E1-transformed tumor cells are not recognized by E1B-specific CTLs, indicating that nontumor cells have processed the E1B-encoded CTL antigen and have presented the E1B peptide to E1B-specific CTLs. These data also show that the B6 anti-BALB/c allo-response does not overwhelm the anti-E1B response induced by the allogeneic tumor cell vaccination. Moreover, B6 mice immunized with allogeneic BALB/c Ad5E1 cells are, in contrast to mice vaccinated with untransformed BALB/c cells, protected against a subsequent challenge with B6 Ad5E1-expressing tumor cells. These data show that immunization with completely allogeneic tumor cells can lead to protective syngeneic antitumor immunity, indicating that completely allogeneic tumor cell vaccines can be used for the induction of antitumor immunity.

Animals↗
PubMed1996

Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance induction.

Vaccination with synthetic peptides representing cytotoxic T lymphocyte (CTL) epitopes can lead to a protective CTL-mediated immunity against tumors or viruses. We now report that vaccination with a CTL epitope derived from the human adenovirus type 5 E1A-region (Ad5E1A234-243), which can serve as a target for tumor-eradicating CTL, enhances rather than inhibits the growth of Ad5E1A-expressing tumors. This adverse effect of peptide vaccination was rapidly evoked, required low doses of peptide (10 micrograms), and was achieved by a mode of peptide delivery that induces protective T-cell-mediated immunity in other models. Ad5E1A-specific CTL activity could no longer be isolated from mice after injection of Ad5E1A-peptide, indicating that tolerization of Ad5E1A-specific CTL activity causes the enhanced tumor outgrowth. In contrast to peptide vaccination, immunization with adenovirus, expressing Ad5E1A, induced Ad5E1A-specific immunity and prevented the outgrowth of Ad5E1A-expressing tumors. These results show that immunization with synthetic peptides can lead to the elimination of anti-tumor CTL responses. These findings are important for the design of safe peptide-based vaccines against tumors, allogeneic organ transplants, and T-cell-mediated autoimmune diseases.

Adenovirus E1A Proteins↗
PubMed1996

Efficient tumor eradication by adoptively transferred cytotoxic T-cell clones in allogeneic hosts.

Tumor-specific cytotoxic T cells (CTLs) can play an important role against cancer as illustrated by the observation that adoptive transfer of tumor-specific CTLs can mediate potent anti-tumor effects. Although such CTLs can be detected at the tumor site, relatively little is known about the mechanisms by which they enter the tumor. In this study, the role of major histocompatibility complex (MHC) class 1 molecules on vascular endothelium in the tumor in entry of, and tumor eradication by, tumor-specific CTL was investigated. Two H-2Db-restricted CTL clones recognizing peptide VNIRNCCYI on human adenovirus type 5 early region 1-(Ad5E1)-induced tumors were used to test whether CTLs were able to cross the vascular endothelium lacking the restricting MHC molecule. One CTL clone recognizes peptide VNIRNCCYI in the context of both H-2Db and H-2Dbm14 molecules. The other CTL clone recognizes this peptide only in the context of H-2Db. Adoptive transfer of these CTLs leads to eradication of Ad5E 1-induced, H-2Db-expressing tumors in B6(H-2Db+) and Bm14(H-2Db-) nude mice. Our data show that presentation of tumor-derived peptides by MHC molecules on endothelial cells of blood vessels in a tumor do not play a major role in eradication of tumors by adoptively transferred CTL in combination with interleukin-2. Moreover, our data show that successful adoptive CTL immunotherapy is possible across allogeneic barriers, without inducing severe side effects, provided the tumor expresses the MHC class 1-peptide complex recognized by the CTLs.

Amino Acid Sequence↗
PubMed1996

Enhanced tumor outgrowth after peptide vaccination. Functional deletion of tumor-specific CTL induced by peptide vaccination can lead to the inability to reject tumors.

CTL can play an important role in the defense against tumors. Protective CTL-mediated immunity can be established in animal tumor models after vaccination with synthetic peptides representing CTL epitopes. We now report that immunization with synthetic peptides can also lead to CTL tolerance associated with the inability to reject tumors. B6 tumor cells transformed by the human adenovirus early region 1 (Ad5E1) present an Ad5E1A- and an Ad5E1B-encoded CTL epitope to the immune system. CTL clones directed against either of these epitopes are able to eradicate established Ad5E1-induced tumors, showing that these CTL epitopes are targets of CTL that can mediate tumor regression. Here, we show that protective immunity against Ad5E1-expressing tumor cells can be established by immunization with Ad5E1-transformed cells and with an adenovirus vector containing the Ad5E1 region. Protective immunity, in either case, is associated with specific CTL memory. To test whether vaccination with synthetic peptides leads to protection against Ad5E1-expressing tumor cells, we vaccinated mice s.c. with a low dose of the Ad5E1B peptide. This peptide was chosen because the CTL response against the Ad5E1B-encoded CTL epitope contributes most to the antitumor response in B6 mice after vaccination with Ad5E1-transformed cells. Ad5E1B peptide-vaccinated mice were not protected against the outgrowth of Ad5E1-expressing tumor cells, but instead were no longer able to reject a tumor inoculum that was rejected by nonvaccinated mice. Moreover, the protection induced by tumor cell vaccination against Ad5E1B-expressing tumors was gone when the Ad5E1B-encoded CTL epitope was injected a few days before tumor challenge. This is associated with peptide-induced tolerance of Ad5E1B-specific CTL activity. These findings are relevant for the design of therapeutic approaches against both malignancies and T cell-mediated autoimmune diseases.

Adenovirus E1A Proteins↗
PubMed1996

Peptide-based cancer vaccines.

The application of molecularly defined vaccines composed of a limited number of tumor-specific T cell epitopes has resulted in protective antitumor T cell immunity in several mouse tumor models. The first encouraging results with such vaccines have been obtained in human beings. The development of the next generation of rationally designed vaccines that are both effective and safe for application in a clinical setting requires comparison of different modes of delivery of tumor-associated T cell epitopes in multiple epitope constructs.

Animals↗
PubMed1996

Immunotherapy of cancer by peptide-based vaccines for the induction of tumor-specific T cell immunity.

Recent progress in defining the molecular nature of antigens and in finding ways to manipulate T cell-mediated immune responses may provide new modalities for cancer treatment. In this report, we review preclinical studies as well as the first clinical trials with vaccination strategies aiming at the induction of anti-tumor immunity. In particular, we focus on the development of a vaccine against human papillomavirus-induced cervical carcinoma.

Animals↗
PubMed1995

Purification and functional analysis of the Mycobacterium leprae thioredoxin/thioredoxin reductase hybrid protein.

In Mycobacterium leprae, thioredoxin and thioredoxin reductase are expressed from a single gene. This results in the expression of a hybrid protein with subunits attached to each other by a hydrophilic peptide linker. In all other organisms studied so far, thioredoxin (Trx) and thioredoxin reductase (TR) are expressed as two separate proteins. This raises the question of whether the hybrid protein is enzymatically active and, if so, whether TR reduces its own Trx partner or alternatively a heterologous Trx subunit. To address this question, the hybrid TR/Trx protein of M. leprae as well as the individual parts of the hybrid gene coding for either TR or Trx were overexpressed in Escherichia coli and purified. The purified proteins were tested for their ability to catalyze NADPH-dependent insulin disulfide reduction. Here we show that the M. leprae hybrid protein is indeed enzymatically active. Compared with the enzymatic activity of the separately expressed Trx and TR proteins, the hybrid protein is shown to be more efficient, particularly at low equimolar concentrations. This suggests that the hybrid protein of M. leprae is active by itself and that its activity involves intramolecular interactions between the TR and Trx domains. The activity of the hybrid protein increases when exogenous TR or Trx is added, indicating an additional role for intermolecular interactions.

Bacterial Proteins↗
PubMed1995

An adenovirus type 5 early region 1B-encoded CTL epitope-mediating tumor eradication by CTL clones is down-modulated by an activated ras oncogene.

Mouse embryo cells (C57BL/6, H-2b) transformed by the E1A and E1B genes of adenovirus type 5 (Ad5E1 MEC) are highly immunogenic. Previously, CTL were cloned from mice immunized with Ad5E1 MEC. These CTL clones were capable of tumor eradication in nude mice, and were directed against the Ad5E1A-encoded decapeptide SGPSNTPPEI, presented by the H-2Db MHC molecule. We have now generated Ad5E1 MEC containing a mutated Ad5E1A-encoded epitope. The mutant Ad5E1 MEC induce a strong CTL response when injected into immunocompetent mice. CTL clones generated against mutant Ad5E1-transformed tumor cells recognize an Ad5E1B-encoded epitope (VNIRNCCYI) in the context of H-2Db. Because this epitope is also present on wild-type Ad5E1 MEC, it is concluded that Ad5E1-transformed tumor cells express at least two CTL epitopes. Interestingly, the lysis of Ad5E1 MEC by the Ad5E1B-specific, but not by the Ad5E1A-specific, CTL clones was strongly diminished by the action of the activated ras oncogene. CTL directed against the Ad5E1B-encoded epitope were, like Ad5E1A-specific CTL, able to eradicate large established Ad5E1-induced tumors in B6 nude mice, demonstrating that CTL activity directed against different CTL epitopes expressed by the same tumor can be exploited for immunotherapy of cancer.

Adenovirus E1 Proteins↗
PubMed1995

Targeted recombination in plants using Agrobacterium coincides with additional rearrangements at the target locus.

The use of Agrobacterium for gene targeting in plants has been investigated. Leaf protoplasts of five transgenic tobacco lines, containing a T-DNA insertion with a defective npt-II gene at different positions in the plant genome, were transformed via Agrobacterium with a T-DNA containing a npt-II repair gene. After selection for kanamycin resistance and PCR analysis six recombinants were derived from four of the target lines. The recombination frequencies were similar for the different target lines with one recombinant from approximately 3 x 10(5) transformants. Apparently gene targeting is more or less independent of the location of the target construct in the plant genome. Molecular analysis revealed that gene targeting had occurred in five of the six recombinant lines. However precise recombination had occurred in only one line, while in the other four lines restoration of the npt-II gene was accompanied by a deletion of part of the target locus. The sixth recombinant line showed restoration of the npt-II gene of the incoming T-DNA construct which was inserted in the plant genome at a position closely linked to the target locus. The different recombination products favour a model in which recombination is via gene conversion followed by reintegration of the synthesized DNA via homologous or illegitimate recombination rather than a reciprocal exchange of DNA between two cross-overs.

Agrobacterium tumefaciens↗
PubMed1995

Unique gene organization of thioredoxin and thioredoxin reductase in Mycobacterium leprae.

The thioredoxin system comprising thioredoxin (Trx), thioredoxin reductase (TR) and NADPH operates via redox-active disulphides and provides electrons for a wide variety of different metabolic processes in prokaryotic and eukaryotic cells. Thioredoxin is also a general protein disulphide reductase involved in redox regulation. In bacteria, the Trx and TR proteins previously identified were encoded by separate genes (trxA and trxB). In this study, we report a novel genomic organization of TR and Trx in mycobacteria and show that at least three modes of organization of TR and Trx genes can exist within a single bacterial genus: (i) in the majority of mycobacterial strains the genes coding for TR and Trx are located on separate sites of the genome; (ii) interestingly, in all pathogenic Mycobacterium tuberculosis complex mycobacteria both genes are found on the same locus, overlapping in one nucleotide; (iii) in the pathogen Mycobacterium leprae, TR and Trx are encoded by a single gene. Sequence analysis of the M. leprae gene demonstrated that the N-terminal part of the protein corresponds to TR and the C-terminal part to Trx. A corresponding single protein product of approximately 49 kDa was detected in cell extracts of M. leprae. These findings demonstrate the very unusual phenomenon of a single gene coding for both the substrate (thioredoxin) and the enzyme (thioredoxin reductase), which seems to be unique to M. leprae.

Amino Acid Sequence↗
PubMed1995

Molecular characterization and T-cell-stimulatory capacity of Mycobacterium leprae antigen T5.

A Mycobacterium leprae lambda gt11 clone designated T5 has previously been selected with sera from tuberculoid leprosy patients. Sequence analysis of this clone revealed the presence of two overlapping open reading frames (ORFs) present on the two cDNA strands. The first ORF codes for the serologically recognized antigen, which was fused with the lacZ gene in the lambda gt11 clone. The second ORF, present on the complementary strand, displays strong sequence homology with the aspartyl-tRNA synthetase genes of Escherichia coli and Thermus thermophilus. Here we show that the purified T5-derived product, overexpressed in E. coli, is recognized by T cells of the majority of the leprosy patients tested, including lepromatous leprosy patients who do not respond to whole M. leprae bacilli.

Amino Acid Sequence↗