Antisense transcripts play an important role in generating regulatory non-coding RNAs but whether these transcripts are also translated to generate functional peptides remains poorly understood. In this study, RNA sequencing and six-frame database generation were combined with mass spectrometry analysis of peptides isolated from polysomes to identify Nascent Pioneer Translation Products (Na-PTPs) originating from alternative reading frames of bi-directional transcripts. Two Na-PTP originating peptides derived from antisense strands stimulated CD8+ T cell proliferation when presented to peripheral blood mononuclear cells (PBMCs) from nine healthy donors. Importantly, an antigenic peptide derived from the reverse strand of two cDNA constructs was presented on MHC-I molecules and induced CD8+ T cell activation. The results demonstrate that three-frame translation of bi-directional transcripts generates antigenic peptide substrates for the immune system. This discovery holds significance for understanding the origin of self-discriminating peptide substrates for the major histocompatibility class I (MHC-I) pathway and for enhancing immune-based therapies against infected or transformed cells.

• Klíčová slova
• MHC-I epitope, Pioneer Translation Products, bi-directional transcripts, bi-directional translation, reverse strand antigenic peptides,
• MeSH
• aktivace lymfocytů imunologie   MeSH
• antisense RNA * genetika   imunologie   MeSH
• CD8-pozitivní T-lymfocyty * imunologie   MeSH
• leukocyty mononukleární imunologie   MeSH
• lidé MeSH
• MHC antigeny I. třídy * imunologie   genetika   MeSH
• peptidy * imunologie   genetika   MeSH
• prezentace antigenu MeSH
• proteosyntéza * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antisense RNA * MeSH
• MHC antigeny I. třídy * MeSH
• peptidy * MeSH

Downregulation of MHC class I expression on the cell surface is a common mechanism by which tumour cells, including cervical carcinoma, can escape the T cell-mediated anti-tumour immunity. This downregulation represents an obstacle for the efficacy of anti-tumour vaccines. In this study, we investigated the efficacy of prophylactic peptide and peptide-pulsed dendritic cell-based vaccines in a murine model of experimental MHC class I-deficient tumours (TC-1/A9), expressing E6/E7 oncogenes derived from HPV16, and compared the efficacy of particular vaccination settings to anti-tumour protection against parental MHC class I-positive TC-1 tumours. Peptide vaccine based on the 'short' peptide E749-57 harbouring solely the CTL epitope and co-administered to the C57BL/6 mice with CpG oligodeoxynucleotide (CpG ODN) 1826 was effective against MHC class I-positive but not -deficient tumours, while the 'longer' peptide E744-62 (peptide 8Q, harbouring CTL and Th epitopes)-based vaccines were also effective against MHC class I-deficient tumours. We have compared the adjuvant efficacies of two CpG ODN, CpG ODN 1826 and CpG ODN 1585. The 8Q peptide immunisation combined with CpG ODN 1585 inhibited growth of the TC-1/A9 tumours more effectively as compared to CpG ODN 1826. Further, we investigated the efficacy of cellular vaccines based on ex vivo cultured dendritic cells pulsed with either E749-57 or E744-62 peptides and matured with CpG ODN 1826. Unlike in the peptide immunisation setting, treatment with dendritic cells pulsed with a 'short' peptide resulted in the tumour growth inhibition, albeit weaker as compared to the immunisation with the longer peptide. Our data demonstrate that peptide and dendritic cell-based vaccines can be designed to elicit protective immunity against MHC class I-deficient tumours.

• MeSH
• adjuvantní radioterapie metody   MeSH
• CpG ostrůvky MeSH
• dendritické buňky cytologie   MeSH
• epitopy chemie   MeSH
• geny MHC třídy I * MeSH
• lidé MeSH
• myši MeSH
• oligonukleotidy genetika   MeSH
• Papillomavirus E7 - proteiny chemie   MeSH
• peptidy chemie   MeSH
• protinádorové vakcíny chemie   MeSH
• průtoková cytometrie MeSH
• regulace genové exprese MeSH
• subjednotkové vakcíny genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• epitopy MeSH
• oligonukleotidy MeSH
• oncogene protein E7, Human papillomavirus type 16 MeSH Prohlížeč
• Papillomavirus E7 - proteiny MeSH
• peptidy MeSH
• protinádorové vakcíny MeSH
• subjednotkové vakcíny MeSH

A broadly used pan-HLA class I-reactive monoclonal antibody W6/32 is believed to recognize a conformational epitope dependent on association between heavy chains and beta2-microglobulin (beta2m). However, in the present study we report that W6/32 does recognize at least some free HLA class I heavy chains under the partially denaturating conditions of nonreducing Western blotting, namely nearly all HLA-B allelic products. Furthermore, we confirm and largely extend our previous observation that complexes of beta2m with heavy chains of a few HLA class I allelic forms (most notably HLA-B27) exhibit unusual resistance to dissociation by SDS, which is reminiscent of MHC class II molecules. In addition, our data indicate the existence of covalent (disulfide-linked) heterodimers of certain HLA class I heavy chains (namely Cw1 and Cw4) and beta2m.

• MeSH
• beta-2-mikroglobulin metabolismus   MeSH
• buněčné linie MeSH
• denaturace proteinů MeSH
• dodecylsíran sodný chemie   MeSH
• epitopy imunologie   MeSH
• HLA-B antigeny imunologie   MeSH
• HLA-B27 antigen imunologie   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• makromolekulární látky MeSH
• MHC antigeny I. třídy imunologie   MeSH
• monoklonální protilátky imunologie   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-2-mikroglobulin MeSH
• dodecylsíran sodný MeSH
• epitopy MeSH
• HLA-B antigeny MeSH
• HLA-B27 antigen MeSH
• makromolekulární látky MeSH
• MHC antigeny I. třídy MeSH
• monoklonální protilátky MeSH

Recombinant adenylate cyclase toxoids are shown to deliver inserted foreign CD4(+)-T-cell epitopes into the major histocompatibility complex class II presentation pathway, inducing a specific CD4(+)-T-cell response in vivo and yielding in vitro stimulation of specific CD4(+) T cells at a 100-times-higher molar efficiency than the free peptide containing the epitope.

• MeSH
• adenylátcyklasový toxin MeSH
• adenylátcyklasy genetika   imunologie   MeSH
• bakteriální proteiny genetika   imunologie   MeSH
• Bordetella pertussis enzymologie   MeSH
• CD4-pozitivní T-lymfocyty cytologie   imunologie   MeSH
• epitopy T-lymfocytární genetika   imunologie   MeSH
• faktory virulence rodu Bordetella genetika   imunologie   MeSH
• H-2 antigeny imunologie   MeSH
• interleukin-2 biosyntéza   MeSH
• kultivované buňky MeSH
• MHC antigeny II. třídy imunologie   MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• prezentace antigenu imunologie   MeSH
• proteinové prekurzory genetika   imunologie   MeSH
• proteiny vázající maltosu MeSH
• sekvence aminokyselin MeSH
• transportní proteiny genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• adenylátcyklasy MeSH
• bakteriální proteiny MeSH
• epitopy T-lymfocytární MeSH
• faktory virulence rodu Bordetella MeSH
• H-2 antigeny MeSH
• H-2K(K) antigen MeSH Prohlížeč
• H-2Kb protein, mouse MeSH Prohlížeč
• interleukin-2 MeSH
• MHC antigeny II. třídy MeSH
• proteinové prekurzory MeSH
• proteiny vázající maltosu MeSH
• transportní proteiny MeSH

Bordetella pertussis secretes an adenylate cyclase toxin (CyaA or ACT) that targets primarily cells expressing the alphaMbeta2 integrin (CD11b/CD18) receptor. This toxin can deliver its N-terminal catalytic AC domain (400 amino acid residues) into the cytosol directly across the cytoplasmic membrane. Various heterologous CD8+, as well as CD4+ T-cell epitopes have been engineered into genetically detoxified CyaA and the resulting toxoids were successfully used as vectors for delivery of inserted epitopes into antigen-presenting cells. Upon processing and presentation, these recombinant CyaAs trigger specific MHC class I and/or class II-restricted T-cell responses both in vitro and in vivo.

• MeSH
• adenylátcyklasový toxin imunologie   MeSH
• antigeny bakteriální imunologie   MeSH
• Bordetella pertussis enzymologie   imunologie   MeSH
• CD4-pozitivní T-lymfocyty imunologie   MeSH
• CD8-pozitivní T-lymfocyty MeSH
• dendritické buňky imunologie   MeSH
• epitopy T-lymfocytární imunologie   MeSH
• infekce bakteriemi rodu Bordetella imunologie   prevence a kontrola   MeSH
• lidé MeSH
• MHC antigeny I. třídy imunologie   MeSH
• MHC antigeny II. třídy imunologie   MeSH
• prezentace antigenu imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• antigeny bakteriální MeSH
• epitopy T-lymfocytární MeSH
• MHC antigeny I. třídy MeSH
• MHC antigeny II. třídy MeSH

Bordetella pertussis adenylate cyclase (AC) toxin-hemolysin (ACT-Hly) can penetrate a variety of eukaryotic cells. Recombinant AC toxoids have therefore been recently used for delivery of CD8(+) T-cell epitopes into antigen-presenting cells in vivo and for induction of protective antiviral, as well as therapeutic antitumor cytotoxic T-cell responses. We have explored the carrier potential of the ACT molecule by insertional mutagenesis scanning for new permissive sites, at which integration of two- to nine-residue-long peptides does not interfere with membrane interaction and translocation of ACT. A model CD8(+) T-cell epitope of ovalbumin was incorporated at 10 of these permissive sites along the toxin molecule, and the capacity of ACT constructs to penetrate into cell cytosol and deliver the epitope into the major histocompatibility complex (MHC) class I antigen processing and presentation pathway was examined. While all six constructs bearing the epitope within the Hly portion of ACT failed to deliver the epitope to the MHC class I molecules, all four toxoids with inserts within different permissive sites in the AC domain efficiently delivered the epitope into this cytosolic pathway, giving rise to stimulation of a specific CD8(+) T-cell hybridoma. The results suggest that, in contrast to the AC domain, the hemolysin moiety of ACT does not reach the cytosolic entry of the MHC class I pathway.

• MeSH
• adenylátcyklasový toxin MeSH
• adenylátcyklasy genetika   imunologie   metabolismus   MeSH
• bakteriální proteiny genetika   imunologie   metabolismus   MeSH
• Bordetella pertussis enzymologie   genetika   imunologie   MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• DNA primery genetika   MeSH
• epitopy aplikace a dávkování   MeSH
• faktory virulence rodu Bordetella genetika   imunologie   metabolismus   MeSH
• hemolyziny genetika   imunologie   metabolismus   MeSH
• inzerční mutageneze MeSH
• MHC antigeny I. třídy metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• prezentace antigenu * MeSH
• proteinové prekurzory genetika   imunologie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• techniky in vitro MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• adenylátcyklasy MeSH
• bakteriální proteiny MeSH
• DNA primery MeSH
• epitopy MeSH
• faktory virulence rodu Bordetella MeSH
• hemolyziny MeSH
• MHC antigeny I. třídy MeSH
• proteinové prekurzory MeSH

Anti-Iatk monoclonal antibodies (mAbs) were found to inhibit syngeneic mixed lymphocyte reaction (SMLR) of mice with k and a haplotypes (H-2k and H-2a) of the major histocompatibility complex (MHC) by acting on responder T cells but not stimulator cells. Only the early phase of SMLR was inhibited by anti-Iat mAbs. The inhibitory effect was due to the blocking of autoreactive T cells but not induction of suppressor lymphocytes. Cross-linking of Iat epitopes induced T cell proliferation of k haplotype strain. The inhibitory pattern of SMLR by four anti-Iat mAbs varied among different strains of mice. The inhibitory pattern seemed to depend on MHC and unidentified non-MHC background genes possessed by stimulator cells, suggesting that the shape of the MHC recognition site must have different conformation depending on both MHC and non-MHC gene products recognized. However, the inhibitory pattern of SMLR by anti-Iat mAbs of strains which differ at the I-J locus: B10.A(5R) and B10.A(3R) was dependent on the genotype of stimulator cells. The same was observed in B10.S(9R) and B10.HTT strains. The inhibitory activity of anti-Iat mAbs was entirely directed against responder T cells and was not passively carried out by the stimulators. It was concluded that Iat epitopes are I region controlled determinants that are utilized by T cells as receptors for self Ia antigens. It is suggested that anti-Iat mAbs react with an idiotype on the receptor(s) for Ia or I-J, or possibly a "receptor" for these receptors.

• MeSH
• aktivace lymfocytů MeSH
• epitopy imunologie   MeSH
• H-2 antigeny imunologie   MeSH
• imunologická tolerance * MeSH
• inbrední kmeny myší genetika   imunologie   MeSH
• křížení genetické MeSH
• MHC antigeny II. třídy imunologie   MeSH
• monoklonální protilátky imunologie   MeSH
• myši MeSH
• receptory antigenů T-buněk * MeSH
• T-lymfocyty imunologie   MeSH
• test smíšené lymfocytární kultury MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• epitopy MeSH
• H-2 antigeny MeSH
• MHC antigeny II. třídy MeSH
• monoklonální protilátky MeSH
• receptory antigenů T-buněk * MeSH

Tolerance toward tumor antigens, which are shared by normal tissues, have often limited the efficacy of cancer vaccines. However, wild type epitopes can be tweaked to activate cross-reactive T-cell clones, resulting in antitumor activity. The design of these analogs (i.e., heteroclitic peptides) can be difficult and time-consuming since no automated in silico tools are available. Hereby we describe the development of an in silico framework to improve the selection of heteroclitic peptides. The Epitope Discovery and Improvement System (EDIS) was first validated by studying the model antigen SIINFEKL. Based on artificial neural network (ANN) predictions, we selected two mutant analogs that are characterized by an increased MHC-I binding affinity (SIINFAKL) or increased TCR stimulation (SIIWFEKL). Therapeutic vaccination using optimized peptides resulted in enhanced antitumor activity and against B16.OVA melanomas in vivo. The translational potential of the EDIS platform was further demonstrated by studying the melanoma-associated antigen tyrosinase related protein 2 (TRP2). Following therapeutic immunization with the EDIS-derived epitope SVYDFFAWL, a significant reduction in the growth of established B16.F10 tumors was observed, suggesting a break in the tolerance toward the wild type epitope. Finally, we tested a multi vaccine approach, demonstrating that combination of wild type and mutant epitopes targeting both TRP2 and OVA antigens increases the antitumor response. In conclusion, by taking advantage of available prediction servers and molecular dynamics simulations, we generated an innovative platform for studying the initial sequences and selecting lead candidates with improved immunological features. Taken together, EDIS is the first automated algorithm-driven platform to speed up the design of heteroclitic peptides that can be publicly queried online.

• Klíčová slova
• Cancer vaccine, heteroclitic peptides, immunotherapy, in silico, prediction servers, tumor antigens,
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cellular immune reactions against non-self-epitopes require activation of cytotoxic CD8+ T-cells via cross-presentation of MHC class I-restricted peptides by professional antigen presenting cells (pAPCs), with the consequent detection and elimination of cells expressing the same antigens via the endogenous (direct) pathway. The source of peptides for the endogenous pathway is constituted of alternative mRNA translation products; however, it is still unclear which source of peptides is used for cross-presentation. Furthermore, the presentation of non-canonical translation products, produced during a non-conventional translation event, on class I molecules of tumor cells has been reported but how these peptides are generated, presented to pAPCs, and their capacity to stimulate CD8+ T cells is still not known. Here, we report that pioneer translation peptides (PTPs) derived from intron or exon pre-mRNAs can serve as tumor-associated antigens (TA-PTPs) and are delivered from the producing tumor cells to pAPCs via exosomes where they are processed by the cytosolic pathway. Injection of TA-PTPs and tumor-derived exosomes efficiently induce CD8+ T-cell proliferation and prevent tumor growth in mice. Our results show that TA-PTPs represent an efficient source of antigenic peptides for CD8+ T cell activation and that full-length proteins are not required for cross-presentation. These findings can have interesting implications for generating tolerance and for designing vectors to generate vaccines.

• Klíčová slova
• Exosomes, MHC-I antigen cross presentation, pioneer translation products, tumor rejection, vaccines,
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Adaptive immune responses to newly encountered pathogens depend on the mobilization of antigen-specific clonotypes from a vastly diverse pool of naive T cells. Using recent advances in immune repertoire sequencing technologies, models of the immune receptor rearrangement process, and a database of annotated T cell receptor (TCR) sequences with known specificities, we explored the baseline frequencies of T cells specific for defined human leukocyte antigen (HLA) class I-restricted epitopes in healthy individuals. METHODS: We used a database of TCR sequences with known antigen specificities and a probabilistic TCR rearrangement model to estimate the baseline frequencies of TCRs specific to distinct antigens epitopespecificT-cells. We verified our estimates using a publicly available collection of TCR repertoires from healthy individuals. We also interrogated a database of immunogenic and non-immunogenic peptides is used to link baseline T-cell frequencies with epitope immunogenicity. RESULTS: Our findings revealed a high degree of variability in the prevalence of T cells specific for different antigens that could be explained by the physicochemical properties of the corresponding HLA class I-bound peptides. The occurrence of certain rearrangements was influenced by ancestry and HLA class I restriction, and umbilical cord blood samples contained higher frequencies of common pathogen-specific TCRs. We also identified a quantitative link between specific T cell frequencies and the immunogenicity of cognate epitopes presented by defined HLA class I molecules. CONCLUSIONS: Our results suggest that the population frequencies of specific T cells are strikingly non-uniform across epitopes that are known to elicit immune responses. This inference leads to a new definition of epitope immunogenicity based on specific TCR frequencies, which can be estimated with a high degree of accuracy in silico, thereby providing a novel framework to integrate computational and experimental genomics with basic and translational research efforts in the field of T cell immunology.

• Klíčová slova
• Antigen, Immune repertoire, Immunogenicity, T cell receptor,
• MeSH
• epitopy imunologie   MeSH
• lidé MeSH
• MHC antigeny I. třídy imunologie   MeSH
• peptidy imunologie   MeSH
• receptory antigenů T-buněk imunologie   MeSH
• statistické modely MeSH
• T-lymfocyty imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• epitopy MeSH
• MHC antigeny I. třídy MeSH
• peptidy MeSH
• receptory antigenů T-buněk MeSH