Cellular immunity against DNA tumour viruses: possibilities for peptide-based vaccines and immune escape.
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Animals↗
Biomedical subjects
W M Kast
Publications and source records attributed to W M Kast.
The use of Epstein-Barr virus-transformed B lymphocyte cell lines in a peptide-reconstitution assay: identification of CEA-related HLA-A*0301-restricted potential cytotoxic T-lymphocyte epitopes.
In the development of cytotoxic T lymphocyte (CTL)-mediated immunotherapy, the identification of CTL epitopes is of crucial importance. Binding of a peptide to major histocompatibility complex (MHC) class I molecules is one of the prerequisites for its function as a CTL epitope. We describe the technique, validation, and application of a simple cellular assay, intended for the screening of peptides for binding, that can be applied to any human leukocyte antigen (HLA) allele. Reconstitution of peptides in MHC class I molecules after elution by acid treatment was previously shown to be possible in specially engineered cell lines expressing only one type of MHC class I, and was applied for the HLA-A*0201 allele. We now report the optimal conditions for application of this type of binding assay to the HLA-A*0301 allele. The adaptations that were necessary to make the technique operational for HLA-A*0301 are shown in detail. These consisted of lowering the pH during acid treatment to 2.9 and lengthening the duration of elution to 90 s. Furthermore, immediate aspiration of eluted peptides appeared to be essential for this allele. We found also that the use of Epstein-Barr virus (EBV)-transformed B cell lines (B-LCL) yields results similar to those of the use of cell lines expressing only one specific MHC class I allele. Homozygosity for the desired HLA allele improves the sensitivity of the assay, but heterozygous cells can also be employed. Finally, we applied this technique to a search for HLA-A*0301 binding peptides derived from carcinoembryonic antigen (CEA). Of a set of 34 CEA-specific peptides that fit with a specified HLA-A*0301-binding motif, we identified a set of six peptides with high binding affinity to this allele. These peptides can be regarded as potential CTL epitopes.
Amino Acid Sequence↗
Dendritic cells as carriers for a cytotoxic T-lymphocyte epitope-based peptide vaccine in protection against a human papillomavirus type 16-induced tumor.
Previously we have demonstrated that two doses of a cytotoxic T lymphocyte (CTL) epitope-based peptide vaccine of human papillomavirus type 16 (HPV 16) E7 aa 49-57 elicit protection against outgrowth of HPV 16-transformed tumor cells (C3 cells) in B6 mice. Incomplete Freund's adjuvant (IFA), as a carrier, was used to induce this response. To avoid side effects caused by the use of external adjuvants, we have now investigated the effectiveness of highly purified spleen dendritic cells (DC) that efficiently induce primary peptide-specific CTL responses in vitro, as physiological carriers for the HPV 16 E7(49-57) peptide-based vaccine. This is the first report demonstrating that mice immunized once i.v. with syngeneic spleen DCs pulsed with the HPV 16 E7(49-57) peptide in vitro were protected against the outgrowth of C3 tumor cells. In comparison, a single injection of the HPV 16 E7(49-57) peptide in IFA s.c. also resulted in effective induction of tumor-specific immunity in vivo. In both immunization protocols, protective tumor-specific immunity was mediated by CTL that recognized HPV 16 E7(49-57) peptide-pulsed target cells, as well as C3 cells in vitro. Peptide affinity of the CTL induced by both protocols was similar. Thus under the conditions tested, a single injection of spleen DCs pulsed with a CTL epitope-based peptide in vitro elicited tumor-antigen-specific CTL in vivo, which protected mice against a subsequent tumor inoculation. This result indicates that spleen DCs pulsed with a CTL epitope can effectively serve as a tumor-specific vaccine.
Animals↗
T-cell immunotherapy of tumors by adoptive transfer of cytotoxic T lymphocytes and by vaccination with minimal essential epitopes.
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Animals↗
Induction of a primary human cytotoxic T-lymphocyte response against a novel conserved epitope in a functional sequence of HIV-1 reverse transcriptase.
OBJECTIVE: To identify novel major histocompatibility complex (MHC) class I-restricted cytotoxic T-lymphocyte (CTL) epitopes conserved in HIV-1. METHODS: Potential conserved CTL epitopes were selected using a predictive computer algorithm based on a human leukocyte antigen (HLA)-A*0201 peptide-binding motif and tested for actual binding to the human processing defective cell line 174.CEM T2 (T2). Hence, the amino-acid sequences of 14 full-length sequenced HIV-1 strains were analysed. An in vitro primary peptide-specific human CTL response was induced with responding lymphocytes of an HIV-1-seronegative donor. Responding T cells were cloned by limiting dilution and tested for their ability to recognize naturally processed antigen in a 51Cr-release assay using recombinant vaccinia-HIV protein-infected B-lymphoblastoid cells (B-LCL) as target cells. RESULTS: The analysis of peptides bearing the HLA-A*0201 motif for conservation resulted in one peptide of Env, three of Gag and 12 of Pol. Only Gag340-348, Pol83-92, Pol267-277 and Pol960-968 showed binding properties to T2 comparable with those of known CTL epitopes Gag76-84 SLYNTVATL and Pol468-476 ILKEPVHGV. A successful primary MHC class I-restricted CTL response was induced against Pol468-476 and Pol267-277 VLDVGDAYFSV, a peptide in a functional sequence of reverse transcriptase (RT). The resulting CD8+ CTL clones were peptide-specific and able to specifically lyse recombinant vaccinia-HIV-1 RT-infected HLA-A*0201-matched B-LCL. CONCLUSION: The method used to screen proteins sequences for potential CTL epitopes, test selected peptides for binding to MHC class I and induction of an in vitro primary response against optimal binding peptides resulted in the identification of at least one novel conserved CTL epitope. The novel epitope is located in an area crucial for RT activity. This study demonstrates the feasibility of identifying highly conserved HIV-1-derived peptides capable of eliciting novel anti-HIV-1 CTL responses.
Amino Acid Sequence↗
Class I MHC-peptide interactions: structural requirements and functional implications.
In this chapter, we have defined the structural motifs that dictate the capacity of peptides to bind to five different HLA-A alleles that represent some of the most common alleles found in different ethnic populations. In general, these peptide motifs were very specific for the individual HLA-A alleles, with the exception of HLA-A degree 0301 and HLA-A degree 1101, for which the motifs were very similar. When these motifs were tested against an unbiased and complete set of nonamer peptides derived from human papillomavirus E6 and E7 proteins, it was found that the vast majority of high and intermediate binding peptides contained the appropriate motif. Furthermore, using the dominant anchor residues, together with the amino acid positions that interact with secondary anchor residues, it was possible to predict high and intermediate binders. Finally, the finding that there is a direct correlation between binding affinity for MHC and immunogenicity suggests a practical application of being able to predict those peptides that have a high affinity binding for a particular MHC allele--that is, in the design of peptide based vaccines for prophylactic or therapeutic use.
Alleles↗
The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes.
The relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes has been analyzed in two different experimental approaches. In the first approach, the immunogenicity of potential epitopes ranging in MHC binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approximately 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL of acute hepatitis patients. In both cases, it was found that an affinity threshold of approximately 500 nM (preferably 50 nM or less) apparently determines the capacity of a peptide epitope to elicit a CTL response. These data correlate well with class I binding affinity measurements of either naturally processed peptides or previously described T cell epitopes. Taken together, these data have important implications for the selection of epitopes for peptide-based vaccines, and also formally demonstrate the crucial role of determinant selection in the shaping of T cell responses. Because in most (but not all) cases, high affinity peptides seem to be immunogenic, our data also suggest that holes in the functional T cell repertoire, if they exist, may be relatively rare.
Amino Acid Sequence↗
Role of HLA-A motifs in identification of potential CTL epitopes in human papillomavirus type 16 E6 and E7 proteins.
We have measured the binding affinity for five HLA-A alleles: HLA-A1 (A*0101), A2.1 (A*0201), A3 (A*0301), A11 (A*1101), and A24 (A*2401); of a set of all possible nonamer peptides (n = 240) of human papillomavirus type 16 E6 and E7 proteins. High affinity binding peptides were identified for each of the alleles, thus allowing us to select several candidates for CTL-based vaccines. Moreover, this unbiased set of peptides allowed an evaluation of the predictive value of HLA motifs derived either from the analysis of sequencing of pools of naturally processed peptides or from the binding analysis of polyalanine nonameric peptides that differed in the amino acids (aa) present at the anchor positions. Whereas pool sequencing-derived motifs were present in only 27% of high affinity binders, the more expanded motif, based on analysis of different aa substitutions at the anchor positions, was present in 73% of high affinity binders. Furthermore, it was found that the presence of anchor residues in a peptide was in itself not sufficient to determine binding to MHC class I molecules, because the majority of motif-containing peptides failed to bind to the relevant MHC. Finally, specific HLA motifs were used to predict peptide binders of 8, 10, and 11 aa in length. Several high affinity binding peptides were identified for each of the various peptide lengths, indicating a significant size heterogeneity in peptides capable of high affinity binding to HLA-A molecules.
Alleles↗
Prospects for T cell immunotherapy of tumours by vaccination with immunodominant and subdominant peptides.
Immunotherapy of tumours by adoptive transfer of cytotoxic T lymphocytes (CTL) is now feasible in experimental murine systems. These CTL recognize peptide sequences of defined length presented by major histocompatibility complex (MHC) class I molecules. Effective eradication of large tumour masses requires co-administration of interleukin 2. Tumour escape strategies are numerous but in various instances can be counteracted by defined measures. Initiation of CTL responses against poorly immunogenic virally induced tumours and other tumours requires novel strategies to overcome T cell inertia. We propose a 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 steps proposed include: (1) identification of target molecules of choice. (2) Identification in these target molecules of peptides fitting MHC allele-specific peptide motifs involved in peptide binding to MHC molecules. (3) Evaluation of actual binding of such peptides to specific MHC class I molecules. (4) In vitro CTL response induction by such peptides, presented by highly efficient antigen-presenting cells such as antigen processing-defective cells carrying empty MHC class I molecules loaded with a single peptide or dendritic cells. Both types of cells are capable of primary CTL response induction in vitro. (5) Evaluation of proper processing by the demonstration of tumour cell lysis by these CTL. (6) Adoptive transfer of tumour-specific CTL generated in vitro or vaccination with peptides. These various steps have now been taken for several viruses, virally induced tumours and other types of tumours and the first indications that this strategy is useful have been obtained.
Amino Acid Sequence↗
Efficient MHC class I-peptide binding is required but does not ensure MHC class I-restricted immunogenicity.
Cytotoxic T lymphocyte (CTL) epitopes are naturally processed peptides bound and presented by major histocompatibility (MHC) class I molecules. Since they are expressed at the cell surface in sufficient amounts to be recognized by CTL, it is generally believed, and in some cases demonstrated, that they bind efficiently to MHC class I molecules in vivo. Based on this knowledge, candidate CTL epitopes are now searched for by identifying peptides that efficiently bind to MHC class I molecules in vitro. We analysed whether this approach is valid by systematically investigating the relationship between MHC class I-peptide binding and peptide immunogenicity. Fifteen peptides that represent known CTL epitopes were tested for their MHC class I binding ability. In a comparative study with 83 peptides that bear the appropriate MHC class I allele-specific motifs but do not contain known CTL epitopes, the CTL epitope-bearing peptides showed the highest binding affinity for MHC class I. This was true for two MHC class I alleles in two different assay systems that monitor peptide-MHC class I binding. Furthermore, selected motif-bearing Kb binding peptides were used to induce peptide-specific CTL responses in mice. Only a subset of the high affinity Kb binding peptides induced reproducible peptide-specific CTL responses, whereas none of the low affinity Kb binding peptides induced a response. Taken together, these results indicate that efficient peptide-MHC class I binding is required for immunogenicity. Vice versa, immunogenicity is not guaranteed by efficient peptide-MHC class I binding, implying that additional factors are involved. Nevertheless, selection of candidate CTL epitopes on the basis of MHC class I binding seems valid. Our data indicate that, although an excess of peptides might be selected, the chance of missing immunogenic peptides is minimal.
Amino Acid Sequence↗
p53, a potential target for tumor-directed T cells.
Cell lineage-specific cellular proteins, oncogenes from viral or cellular origin and tumor suppressor genes encode tumor-specific/associated antigens. Such antigens can elicit an major compatibility complex (MHC) class I-restricted cytotoxic T lymphocyte (CTL) response, either naturally in cancer patients or following appropriate immunostimulation (in vitro or in vivo). The reported immune responses in humans to the melanoma-associated MAGE gene products, GP100 and tyrosinase, all self-proteins, support the idea to use wild-type p53 products as targets for T cells. An important step towards this goal is identification of potential p53 CTL epitopes. We identified the wild-type p53 peptides with the highest affinity to the HLA-A*0201 molecule using two assays: the previously described MHC peptide-binding assay and the peptide competition assay. We obtained CTL against four p53 peptides with a high affinity for the HLA-A*0201 molecule. These findings are discussed next to a short review concerning the p53 literature.
Amino Acid Sequence↗
Production of interleukin-2 by EL4 tumor cells induces natural killer cell- and T-cell-mediated immunity.
Systemic administration of recombinant interleukin (rIL)-2 to cancer patients has met with limited clinical success since, despite significant antitumor effects, its use is associated with severe toxicity. Local production of IL-2 by IL-2 gene transfected tumor cells in murine model systems has been reported to induce specific immunity--devoid of toxicity--to the parental non-IL-2-producing tumor cells. We now report enhanced resistance in nonimmunized mice to murine EL4 thymoma cells, producing murine IL-2 following gene transfer (EL4pIL-2). This effect is mediated by activated natural killer (NK) cells, since we observed the same effect in nude mice but not in NK-depleted mice. Additionally, in mice repeatedly vaccinated with irradiated EL4pIL-2 cells, we observed immunity to challenge with a tumorigenic dose of EL4 cells transfected with a control vector, EL4p. EL4-specific cytotoxic T-lymphocytes (CTLs) were detected in these mice. Mice vaccinated with irradiated EL4p cells were less protected against challenge with a tumorigenic dose of EL4p cells. This study indicates that although some IL-2-producing autologous tumor cells elicit NK-mediated responses and not CTL responses upon inoculation, tumor-specific CTL responses are generated upon repeated vaccinations with these cells. This strategy has potential application for treating a wide variety of cancer patients with autologous IL-2 producing tumor cells.
Animals↗
Identification of an H-2 Kb-presented Moloney murine leukemia virus cytotoxic T-lymphocyte epitope that displays enhanced recognition in H-2 Db mutant bm13 mice.
Upon infection with the Moloney murine sarcoma virus-murine leukemia virus (MuLV) complex, H-2b C57BL/6 (B6) mice respond with a class I Db-restricted cytotoxic T-lymphocyte (CTL) response, which protects against virus-induced tumorigenesis. In the B6-derived Db mutant B6.CH-2bm13 (bm13) strain, part of the class I Db antigen-presenting groove is shaped by a class I Kb-encoded sequence. Like B6 mice, bm13 mice reject Moloney virus-induced tumors, but the protective CTL response is Kb restricted. In this study we show enhanced levels of Moloney MuLV-specific CTLp with a restriction for Kb in bm13 mice. Through the use of CTL clones from Moloney virus-immunized bm13 mice, the class I Kb-presented CTL epitope was identified. The epitope is located in the Moloney virus gp70 envelope protein region (Moloney envelope, amino acids 189 to 196 [Mol env (189-196)]), SSWDFITV and has the Kb allele-specific binding motif. The Dbm13 molecule does not present the env(189 to 196) epitope to Kb-restricted bm13 CTL. In B6 mice, Mol env(189-196)-specific CTL could be induced by peptide vaccination. B6 mice thus have CTL precursors specific for this epitope but at considerably lower levels than do bm13 mice. We hypothesize that additional positive selection of Kb-restricted CTL on the Dbm13 molecule in bm13 mice explains this difference in precursor frequencies. We examined related strains of MuLV for the presence of Mol env(189-196) sequence equivalents. Rauscher, Friend, and AKV MuLV-encoded Mol env(189-196) epitope equivalents were properly recognized in cytotoxicity assays, both as synthetic and as endogenously expressed (Rauscher MuLV) peptides. In contrast, the mink cell focus-forming virus MuLV-encoded epitope equivalent, lacking a Kb anchor residue, was not presented for CTL recognition and hence can be excluded as an important CTL epitope for mink cell focus-forming viruses.
Amino Acid Sequence↗
Tumor rejection antigens and tumor specific cytotoxic T lymphocytes.
In several tumor models and in certain types of human malignancies, T cell mediated immune responses can be involved in the host's defences against cancer. Adoptively transferred tumor-specific T cells can mediate complete tumor regression in several animal models and the first effective therapeutic interventions with adoptively transferred (virus-specific) CTL in man have been reported. With the identification of tumor rejection antigens new antigen specific therapeutic approaches come into sight. The development of anti-tumor vaccines based upon known tumor rejection antigens might take off rapidly in the near future, since only the first few tumor rejection antigens of an enormous potential have been reported. This development will go hand in hand with the utilization of cytokines in these vaccines. Cytokines might promote T cell activity or antigen presentation, but can also be used to counteract tumor immune escape mechanisms. Escape from immune surveillance and T cell mediated elimination by tumor cells might account for the failure of the immune system to eradicate or prevent the outgrowth of many types of tumors. The identification of tumor rejection antigens and tumor-specific immune responses, together with a better understanding of the mechanisms by which tumors escape from T cell mediated immunity brings fresh encouragement in the field of tumor immunology.
Animals↗
In vivo detection of fluorescent tumor-specific cytotoxic T cell clones.
Cytotoxic T lymphocytes (CTL) can be very effective mediators of tumor-specific immunity in vivo. Since little is known about the in vivo behaviour of cultured tumor-specific CTL, a fast and simple method has been developed utilizing a lipophilic carbocyanine, 1,1'-dioctadecyl 3,3,3',3'-tetramethylin-docarbocyanine perchlorate (DiI), for the in vivo detection of tumor-specific CTL clones in (tumor-bearing) mice. The two CTL clones used in this study are directed against human papillomavirus type 16- or human adenovirus type 5 early region 1 (Ad5E1)-transformed mouse embryo cells. Growth ability, cytotoxic capacity and tumor-eradicating potential remained unaltered when the CTL were labeled with this dye. Thus, in neither in vitro nor in vivo testing was the biological function of the CTL clones affected. The in vivo localization in the spleen of an adoptively transferred DiI-labeled Ad5E1-specific CTL clone is described. This adoptively transferred CTL clone was also detectable at the site of a subcutaneously growing human Ad5E1-induced tumor within 1 day after intravenous injection.
Adenovirus Infections, Human↗
Strict peptide length is not required for the induction of cytotoxic T lymphocyte-mediated antiviral protection by peptide vaccination.
Sendai virus nuclear protein peptides of different lengths were titrated in peptide vaccination experiments. We observed that peptide length was not important in inducing cytotoxic T lymphocyte-mediated protective immunity in vivo against a challenge with a lethal dose of virus. These results suggest that long peptides are trimmed in vivo to peptides that fit into the groove of major histocompatibility complex class I molecules. In addition several adjuvants were screened for their effectiveness in peptide vaccination protocols. Incomplete Freund's adjuvant and Titermax turned out to be useful, whereas alum was much less effective.
Adjuvants, Immunologic↗
Identification of peptide sequences that potentially trigger HLA-A2.1-restricted cytotoxic T lymphocytes.
We used the human processing defective cell line 174CEM.T2 (T2) to identify potential cytotoxic T lymphocyte (CTL) epitopes of human proteins. Exogenously added peptides can increase the number of properly folded HLA-A2.1 molecules on the cell surface of T2 cells, as shown by immunofluorescence measurements using the mouse monoclonal antibody BB7.2 (anti-HLA-A2.1) and fluorescein isothiocyanate-labeled goat anti-mouse F(ab')2 antibody. The peptides were selected on the basis of a computer score derived from the recently described HLA-A2.1 specific motif. Analysis of the influenza matrix protein showed that 15 out of 35 high-scoring peptides up-regulate the expression of HLA-A2.1 molecules on the T2 cell surface. The combination of the computer scoring program and an immunofluorescence-based peptide binding assay allows rapid detection of potential CTL target peptides.
Amino Acid Sequence↗
In vitro induction of human cytotoxic T lymphocyte responses against peptides of mutant and wild-type p53.
The central role of the p53 tumor suppressor gene product in oncogenesis is gradually being clarified. Point mutations in the p53 tumor suppressor gene are common in most human cancers and are often associated with p53 protein overexpression. Overexpressed wild-type or mutant determinants of the p53 protein thus represent an attractive target for immunotherapy of cancer directed against a structure involved in malignant transformation. An important step towards this goal is identification of epitopes of p53 that can be recognized by human cytotoxic T lymphocytes. We identified peptides of (mutant) p53 capable of binding to HLA-A2.1 in an in vitro assay. These HLA-A2.1-binding peptides were utilized for in vitro induction of primary cytotoxic T lymphocyte responses using a human processing-defective cell line (174CEM.T2) as antigen-presenting cell. These cells display "empty" HLA class I surface molecules, that can efficiently be loaded with a single peptide. We obtained CD8+ cytotoxic T lymphocyte clones capable of specifically lysing target cells loaded with wild-type or tumor-specific mutant p53 peptides. This strategy allows the in vitro initiation of human cytotoxic T lymphocyte responses against target molecules of choice.
Amino Acid Sequence↗