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Biomedical subjects

W M Kast

Publications and source records attributed to W M Kast.

At least 109 records · Page 6Linked to original sources
PubMed1993

Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells.

Cytotoxic T lymphocyte (CTL) peptide epitopes can be used for immunization of mice against lethal virus infection. To study whether this approach can be successful against virus-induced tumors we generated a B6 (H-2b) tumorigenic cell line transformed by human papillomavirus (HPV). This virus is detected in over 90% of all human cervical cancers. To identify vaccine candidates, we generated a set of 240 overlapping peptides derived from the HPV type 16 (HPV16) oncogenes E6 and E7. These peptides were tested for their ability to bind H-2Kb and H-2Db MHC class I molecules. Binding peptides were compared with the presently known peptide-binding motifs for H-2Kb and H-2Db and the predictive value of these motifs is shortly discussed. The high-affinity H-2Db-binding peptide and putative CTL epitope E7 49-57 (RAHYNIVTF) was used in vaccination studies against HPV 16-transformed tumor cells. Immunization with peptide E7 49-57 rendered mice insensitive to a subsequent challenge with HPV 16-transformed tumor cells in vivo, and induced a CTL response which lysed the tumor cells in vitro.

Amino Acid Sequence↗
PubMed1993

Differential effect of transporter Tap 2 gene introduction into RMA-S cells on viral antigen processing.

The protein products of the Tap (Transporter associated with antigen processing) 1 and 2 genes are presumed to deliver peptides across the endoplasmic reticulum (ER) for assembly with major histocompatibility complex (MHC) class I molecules. The antigen processing-defective cell line RMA-S (H-2b) has a premature stop in the Tap 2 gene and probably therefore fails to deliver peptides into the ER, which leads to a low level of cell surface MHC class I molecules. Transfection of a Tap 2 gene restores to RMA-S both MHC class I molecule expression and the ability to present influenza viral antigens. We investigated the ability of RMA-S cells transfected with a Tap 2 gene to process and present alloantigens, Sendai and Rauscher viral antigens to allogeneic and virus-specific cytotoxic T lymphocytes. We found that allogeneic peptides as well as Rauscher and Sendai viral peptides can be processed and presented by RMA-S but at reduced levels. Transfection of a Tap 2 gene of mouse (BALB/c, H-2d) or rat origin into RMA-S increased the presentation of Sendai viral antigens and partially restored the presentation of allogeneic antigens. The already low level of Rauscher viral peptides presented by RMA-S is not elevated by transfection of either Tap 2 gene into RMA-S. This indicates a differential effect of transfection of a Tap 2 gene of rat or allogeneic mouse origin into RMA-S on viral antigen processing.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Potential immunogenicity of oncogene and tumor suppressor gene products.

The immunogenicity of viral oncoproteins has been established beyond doubt. Cytotoxic T lymphocytes directed against viral oncogene products can eradicate large established tumor masses. This stage has not yet been reached for cellular oncogene and tumor suppressor gene products, but T cells have been raised against MHC-binding peptides encoded by both mutant and wild-type alleles of the ras oncogene and the p53 tumor suppressor gene. In addition, T cells specific for joining region peptides of abnormal fusion proteins resulting from chromosome translocation in tumor cells have been generated. Some of these peptides are processed in cells infected with, for example, vaccinia-ras, but direct anti-tumor effects of peptide specific T lymphocytes remain to be demonstrated.

Genes, Tumor Suppressor↗
PubMed1993

Human leukocyte antigen-A2.1 restricted candidate cytotoxic T lymphocyte epitopes of human papillomavirus type 16 E6 and E7 proteins identified by using the processing-defective human cell line T2.

Human papillomavirus type 16 (HPV-16) is strongly associated with cervical cancer. HPV-16 cytotoxic T lymphocyte (CTL) epitopes may be good candidates for the development of an antitumor peptide vaccine. A set of 240 overlapping peptides nine amino acids in length with an eight amino acid overlap covering the entire sequence of the two viral oncogenes E6 and E7 was synthesized and tested for its ability to bind to the most common human leukocyte antigen class I molecule HLA-A2.1. Binding was measured with the human processing defective cell line T2, which expresses high numbers of empty HLA-A2.1 molecules that are unstable at 37 degrees C. These empty molecules can be stabilized by exogenously added peptides, and the extent of stabilization, measured by cell surface HLA-A2.1-specific staining, can be taken as a measure of the relative HLA-A2.1 binding affinity. Following this analysis, several HLA-A2.1 binding peptides were pinpointed. Preliminary data suggest that at least one of the high-affinity-binding peptides identified is immunogenic even in an in vitro priming protocol, underlining the feasibility of the method described here to identify the immunogenic peptides and potential candidates for CTL peptide-based vaccines.

Amino Acid Sequence↗
PubMed1993

Murine cytomegalovirus interacts with major histocompatibility complex class I molecules to establish cellular infection.

The expression of stable, correctly folded major histocompatibility complex class I molecules conferred susceptibility to murine cytomegalovirus (MCMV) in cells which were previously resistant to infection, demonstrating that these molecules interact critically with MCMV to initiate infection. All class I molecules could potentiate MCMV infection but H-2Dd and Kb molecules were most efficient. Monoclonal antibodies specific for the alpha 1 and/or alpha 2 domains of Dd and Kb inhibited infection. Infection of L cells transfected with hybrid major histocompatibility complex class I molecules demonstrated that allelic control of susceptibility to MCMV mapped to the alpha 1 domain of Dd when in correct configuration with the alpha 2 and alpha 3 domains. In MCMV-resistant RMA-S cells, an improvement in the conformation of class I molecules introduced susceptibility to infection.

Animals↗
PubMed1993

Characterization of cytotoxic T lymphocyte epitopes of a self-protein, p53, and a non-self-protein, influenza matrix: relationship between major histocompatibility complex peptide binding affinity and immune responsiveness to peptides.

We previously described a motif prediction of major histocompatibility complex allele-specific peptides and an in vitro assay for actual measurement of peptide binding to human leukocyte antigen HLA-A2.1 molecules. Using this method we have identified candidate cytotoxic T lymphocyte (CTL) epitopes derived from a non-self-protein (influenza matrix) and self-protein (p53). We now show that results of binding assays performed over a range of peptide concentrations indicate that distinct differences in HLA-A2.1 peptide binding affinities exist between the influenza matrix and p53 protein. The results for the influenza matrix protein indicate that the peptide that shows the highest binding affinity to HLA-A2.1 is identical to the known immunodominant peptide recognized by influenza virus-specific CTLs. The results for p53 indicate that one of the peptides with a low binding affinity is capable of inducing specific CTL responses, but CTLs recognizing the highest affinity binding peptides were not obtained. These findings are discussed in terms of the distinct implications for induction of cellular immune responses directed against peptides with different binding affinities for HLA-A2.1 of proteins that constitute attractive targets for tumor immunotherapy.

Amino Acid Sequence↗
PubMed1992

A rapid isolation procedure for dendritic cells from mouse spleen by centrifugal elutriation.

The standard isolation procedure for antigen presenting dendritic cells (DC) takes 2 days and includes selective adherence to tissue culture plates which may lead to the activation of these cells. This report describes the isolation of DC by centrifugal elutriation (CE). Murine spleen cells were separated on the basis of size and density into 7 CE fractions. This method took 90 min. Cells from each CE fraction were characterized by fluorescence activated cell sorter (FACS) analysis and their antigen presenting cell (APC) activity was determined by a secondary Sendai virus specific T cell proliferation assay. CE fraction 5 contained most of the DC with a concentration of 6-10%, representing an approximately 15-fold enrichment compared to unseparated spleen cells (< 1% DC). This CE fraction also exhibited the highest APC activity, which was almost completely abolished after depletion of DC by treatment with monoclonal antibody 33D1 (DC-marker) and complement. Further enrichment of CE fraction 5 by discontinuous density gradient centrifugation resulted in a cell population containing 35-55% 33D1-positive cells with similar characteristics as DC isolated by the standard procedure, such as the capacity to induce a primary viral peptide specific CTL response. Two-color FACS analysis showed an increase in MHC expression on 33D1-positive cells of CE fraction 5 after 18 h culture involving cell adhesion to a similar level as the MHC expression on DC isolated by the standard procedure. During this same period their morphology changed from a round to a dendritic appearance. In conclusion, our results indicate that CE is well suited for isolating DC more rapidly and without activation of these cells by adherence, a process which readily occurs in the standard isolation procedure.

Animals↗
PubMed1992

Cytotoxic T lymphocytes against the antigen-processing-defective RMA-S tumor cell line.

RMA-S is an antigen processing-defective cell line, obtained from a Rauscher virus-induced tumor. The cells express only a low level of cell surface major histocompatibility complex (MHC) class I molecules, which are supposed to be devoid of internally derived antigenic peptides. We investigated Rauscher virus expression and Rauscher peptide presentation to virus-specific cytotoxic T lymphocytes (CTL) by this cell line. Viral proteins are expressed properly, both intracellularly and at the cell surface of RMA-S. Rauscher peptides are presented to virus-specific CTL in the groove of both the class I H-2Kb and Db molecules, but at a low level. Culture of RMA-S cells at room temperature increases their susceptibility to CTL. The RMA-S defect thus affects, but not totally abrogates, Rauscher peptide presentation by MHC class I molecules via the endogenous pathway. This indicates that the RMA-S antigen processing deficit is not absolute.

Animals↗
PubMed1992

Mechanisms of induction of primary virus-specific cytotoxic T lymphocyte responses.

We have investigated the ability of various antigen-presenting cell (APC) types to induce primary anti-viral cytotoxic T lymphocyte (CTL) responses by single in vitro stimulation. Of these APC types, only dendritic cells (DC) and RMA-S lymphoma cells could induce primary CTL responses, but by divergent mechanisms. DC were capable of generating primary virus-specific CTL, either by presenting viral peptide or processed infectious virus. In contrast, RMA-S cells could not present endogenous antigen, e.g. after virus infection, but this cell line very efficiently presented exogenous viral peptides to induce primary virus-specific CTL in vitro. Spleen cells, lipopolysaccharide-induced B cell blasts or the non-mutated RMA cells did not have the ability to trigger unprimed T cells by single in vitro stimulation. We have investigated several characteristics important for primary CTL response induction by DC and RMA-S cells (summarized in Fig. 6). Primary CTL response induction by DC or RMA-S cells was blocked by anti-LFA-1 or anti-CD8 monoclonal antibodies (mAb). DC rapidly aggregated with unprimed T cells, which was independent of LFA-1 and CD8 molecules. RMA-S cells did not form conjugates with unprimed T cells. Despite their abundant major histocompatibility complex (MHC) class I cell-surface expression, DC did not bind much exogenously added viral peptide. In contrast, the MHC class I molecules on RMA-S cells bound a large quantity of exogenously administered peptide. Powerful adhesion by DC and high expression of relevant MHC/peptide complexes on RMA-S cells are important features in the initial contact with unprimed T lymphocytes. In a later stage of contact, both DC and RMA-S cells activate LFA-1 (and CD8) molecules at the T cell surface to strengthen and maintain the contact between T cell and APC.

Amino Acid Sequence↗
PubMed1992

Lessons from T cell responses to virus induced tumours for cancer eradication in general.

Immunotherapy of virus induced tumours by adoptive transfer of virus specific cytotoxic T cells (CTL) is now feasible in experimental murine systems. These CTL recognize viral peptide sequences of defined length presented in the groove of MHC class I molecules. Effective eradication of large tumour masses requires coadministration of IL-2. In essence, T cell immunity against virus induced tumours does not differ from anti-viral T cell immunity in general. Tumour escape strategies are numerous but, in various instances, can be counteracted by defined measures. Initiation of CTL responses against poorly immunogenic non-virus induced tumours (the majority of human cancer) requires novel strategies to overcome T cell inertia. Rather than waiting to see whether tumour specific CTL (against unknown antigens) can be cultured from TIL, we propose an alternative strategy in which CTL are raised against target molecules of choice, including differentiation antigens of restricted tissue distribution (autoantigens) or mutated/overexpressed oncogene products. The various steps proposed include: (a) identification of target molecules of choice; (b) identification in these target molecules of MHC allele specific peptide motifs involved in peptide binding to MHC molecules; (c) evaluation of actual binding of such peptides to specific MHC class I molecules; (d) in vitro CTL response induction by such peptides, presented either by highly efficient antigen presenting cells (such as processing defective cells, which carry empty MHC class I molecules) loaded with a single peptide or by dendritic cells, both cell types being capable of primary CTL response induction in vitro and (e) adoptive transfer of tumour specific CTL generated in vivo or, more conveniently, vaccination with immunodominant peptides. The latter possibility seems to be feasible because peptide vaccination with a single immunodominant viral peptide can install CTL memory and confer protection against lethal virus infection.

Animals↗
PubMed1991

Pretransplantation blood transfusion revisited.

BACKGROUND: Blood transfusion before organ transplantation has a beneficial effect on allograft survival; the mechanism of this effect has remained a mystery. In murine models, the presence of common histocompatibility antigens in the blood donor and the recipient favors the induction of allograft tolerance. METHODS: To investigate the effect of HLA compatibility between blood donor and recipient on the induction of allograft tolerance, we determined the relative frequency of cytotoxic T-lymphocyte precursors specific for donor cells before and at several times after blood transfusion in 23 patients awaiting a first renal transplant. We correlated the results with the presence of shared HLA antigens. RESULTS: T-cell nonresponsiveness against donor cells developed after blood transfusion in 10 of the 23 patients. Tolerance developed only if the blood donor and the recipient had one HLA haplotype or at least one HLA-B and one HLA-DR antigen in common (as was observed in 9 of these 10 patients). Tolerance developed relatively late after blood transfusion (one to two months) and was long-lasting. No decline in the T-cell response against donor alloantigens was observed in any of the 13 patients who received transfusions without having HLA-antigen compatibility with the donor. CONCLUSIONS: Blood transfusion in which there is a common HLA haplotype or shared HLA-B and HLA-DR antigens induces tolerance to donor antigens. This finding may lead to the development of new strategies with which to induce tolerance for transplantation after blood transfusion. Perhaps transplant donors will be selected not only by HLA-antigen matching, but also on the basis of acceptable HLA-antigen mismatches associated with T-cell non-responsiveness induced by selected blood transfusion.

Blood Transfusion↗
PubMed1991

Peptide selection by MHC class I molecules.

Synthetic peptides have been used to sensitize target cells and thereby screen for epitopes recognized by T cells. Most epitopes of cytotoxic T lymphocytes can be mimicked by synthetic peptides of 12-15 amino acids. Although in specific cases, truncations of peptides improves sensitization of target cells, no optimum length for binding to major histocompatibility complex (MHC) class I molecules has been defined. We have now analysed synthetic peptide captured by empty MHC class I molecules of the mutant cell line RMA-S. We found that class I molecules preferentially bound short peptides (nine amino acids) and selectively bound these peptides even when they were a minor component in a mixture of longer peptides. These results may help to explain the difference in size restriction of T-cell epitopes between experiments with synthetic peptides and those with naturally processed peptides.

Amino Acid Sequence↗
PubMed1991

Protection against lethal Sendai virus infection by in vivo priming of virus-specific cytotoxic T lymphocytes with a free synthetic peptide.

The only peptide of Sendai virus that is recognized by cytotoxic T lymphocytes (CTL) in B6 mice was found with (i) the use of recombinant vaccinia virus constructs containing separate genes of Sendai virus and (ii) a set of overlapping peptides completely spanning the identified nucleoprotein (NP) gene product. This immunodominant NP peptide is recognized by Sendai virus-specific CTL that are known to have therapeutic effects in vivo. By subcutaneous immunization, this peptide induced Sendai virus and NP peptide-specific CTL memory responses in vivo. Most importantly, mice that had been immunized with this peptide were protected against a lethal virus dose, indicating that viral peptides can be used as antiviral T-cell vaccines. The induction of T-cell memory by free peptide immunization potentially has wide applicability in biology and medicine, including protection against infectious disease.

Animals↗
PubMed1991

Peptide loading of empty major histocompatibility complex molecules on RMA-S cells allows the induction of primary cytotoxic T lymphocyte responses.

The antigen processing-defective mutant cell line RMA-S expresses at the cell surface major histocompatibility complex (MHC) class I molecules devoid of peptide that can be efficiently loaded with exogenous immunogenic peptides. We now report that viral peptide-loaded RMA-S cells, unlike parental RMA cells, can induce primary cytotoxic T lymphocyte (CTL) responses in vitro, in a T helper cell-independent fashion. This was shown for an H-2Kb-binding peptide of Sendai virus nucleoprotein and an H-2Db-binding peptide of adenovirus type 5 E1A protein with responding spleen cells of C57BL/6 mice, the strain of origin of RMA and RMA-S cells. Primary Sendai peptide-induced CTL lyse both peptide-loaded and virus-infected cells. Pre-culture of RMA-S cells at low temperature (22 degrees - 26 degrees C), which increases the amount of empty MHC class I molecules at the cell surface, decreases the peptide concentrations required for the induction of primary CTL responses. Primary peptide-specific CTL responses induced by peptide-loaded RMA-S cells are CD4+ cell- and MHC class II+ cell-independent. CTL response induction is blocked by the presence of anti-CD8 monoclonal antibody during culture. Direct peptide binding studies confirm the efficient loading of empty MHC molecules on RMA-S cells with peptide and show 2.5-fold more peptide bound per RMA-S cell compared to RMA cells. An additional factor explaining the difference in primary response induction between RMA and RMA-S cells is related to the CD8 dependence of these responses. MHC class I molecules occupied with irrelevant peptides (a majority present on RMA, largely absent on RMA-S) may interfere in the interaction of the CD8 molecule with relevant MHC/peptide complexes. The results delineate a novel strategy of peptide based in vitro immunization to elicit CD8+ cytotoxic T cell responses.

Adenoviridae↗
PubMed1991

Fine peptide specificity of cytotoxic T lymphocytes directed against adenovirus-induced tumours and peptide-MHC binding.

A peptide encoded by the adenovirus type 5 early region I (Ad5 EI) is the target structure for H-2Db-restricted cytotoxic T lymphocytes (CTL) that are capable of tumour eradication in vivo. With the use of a set of peptides in which each individual amino acid (aa) was deleted out of the sequence, we analyzed to what extent these deletion mutant peptides were still recognized by an Ad5-specific CTL clone and which deletion mutant peptides still bound to major histocompatibility-complex (MHC) class-I molecules. Binding was analyzed with RMA-S cells that express largely empty and unstable MHC-class-I molecules which are stabilized by peptide binding. We show here that flanking an 8 mer aa sequence, originally described by us as the minimal epitope recognized by CTL, 2 additional aa are important for MHC binding. This leads to the conclusion that this 10-mer peptide is optimal for MHC binding and T-cell recognition. Areas of the peptide primarily involved in binding to MHC or in T-cell recognition are delineated.

Adenoviruses, Human↗
PubMed1991

In vivo efficacy of virus-derived peptides and virus-specific cytotoxic T lymphocytes.

The in vivo efficacy of virus- and tumor-specific cytotoxic T lymphocytes (CTL) is discussed as well as ways to activate these cells in vivo with lymphokines, monoclonal antibodies and immunization. In vivo and in vitro peptide immunization can induce such virus- and tumor-specific CTL but several questions have to be answered before peptide vaccination can be implemented as a novel immunotherapeutic or preventive approach in man.

Animals↗
PubMed1991

Cytotoxic T lymphocyte therapy of cancer and tumor escape mechanisms.

In animal models complete and permanent eradication of tumors can be achieved by adoptive transfer of tumor specific T cells, combined with interleukin 2. The most active cells are CD8+ cytotoxic T lymphocytes (CTL), which recognize peptides of 8-10 amino acids in length, bound to the antigen presenting groove of MHC class I molecules. In the case of virus-induced tumors these peptides are processed from viral proteins. Potentially immunogenic human tumors include melanoma and renal cell carcinoma in addition to the virus-associated cancers Burkitt's lymphoma and cervical carcinoma. The potential of CTL therapy of human cancer needs to be tested with cloned tumor cells. Remedies to over-come poor immunogenicity and evasion by tumor cells of CTL mediated-destruction are discussed.

Amino Acid Sequence↗