T‐cell recognition is shaped by epitope sequence conservation in the host proteome and microbiome

Several mechanisms exist to avoid or suppress inflammatory T‐cell immune responses that could prove harmful to the host due to targeting self‐antigens or commensal microbes. We hypothesized that these mechanisms could become evident when comparing the immunogenicity of a peptide from a pathogen or allergen with the conservation of its sequence in the human proteome or the healthy human microbiome. Indeed, performing such comparisons on large sets of validated T‐cell epitopes, we found that epitopes that are similar with self‐antigens above a certain threshold showed lower immunogenicity, presumably as a result of negative selection of T cells capable of recognizing such peptides. Moreover, we also found a reduced level of immune recognition for epitopes conserved in the commensal microbiome, presumably as a result of peripheral tolerance. These findings indicate that the existence (and potentially the polarization) of T‐cell responses to a given epitope is influenced and to some extent predictable based on its similarity to self‐antigens and commensal antigens.     

Computer‐aided design of T‐cell epitope‐based vaccines: addressing population coverage

Epitope‐based vaccines (EVs) make use of short antigen‐derived peptides corresponding to immune epitopes, which are administered to trigger a protective humoral and/or cellular immune response. EVs potentially allow for precise control over the immune response activation by focusing on the most relevant – immunogenic and conserved – antigen regions. Experimental screening of large sets of peptides is time‐consuming and costly; therefore, in silico methods that facilitate T‐cell epitope mapping of protein antigens are paramount for EV development. The prediction of T‐cell epitopes focuses on the peptide presentation process by proteins encoded by the major histocompatibility complex (MHC). Because different MHCs have different specificities and T‐cell epitope repertoires, individuals are likely to respond to a different set of peptides from a given pathogen in genetically heterogeneous human populations. In addition, protective immune responses are only expected if T‐cell epitopes are restricted by MHC proteins expressed at high frequencies in the target population. Therefore, without careful consideration of the specificity and prevalence of the MHC proteins, EVs could fail to adequately cover the target population. This article reviews state‐of‐the‐art algorithms and computational tools to guide EV design through all the stages of the process: epitope prediction, epitope selection and vaccine assembly, while optimizing vaccine immunogenicity and coping with genetic variation in humans and pathogens.     

The amount of self‐antigen determines the effector function of murine T cells escaping negative selection

Autoimmune diseases develop when self‐specific T cells that escaped negative selection initiate a harmful immune response against self. However, factors, which influence the initiation and progression of an autoimmune response remain incompletely understood. By establishing a double‐transgenic BALB/c mouse system in which different amounts of a cell‐surface neo‐self‐antigen are expressed under the CD11c promoter, we demonstrate that antigen dose dramatically influences T‐cell tolerance mechanisms. Moderate antigen expression in both hematopoietic and nonhematopoietic cells favors the development of antigen‐specific Treg cells and the establishment of a tolerogenic environment. In marked contrast, a high dose of antigen expression results in very stringent negative selection, in poor development of antigen‐specific Treg cells and in the early onset of anemia and splenomegaly and the late development of arthritis and high titers of IgG auto Abs. Disease is initiated by autoreactive T cells, which escape negative selection by expressing a second TCR with a different specificity or an altered affinity. Transfer of Ag‐specific Treg cells ameliorates the early onset signs of disease but does not prevent the development of long‐term chronic pathologies. Altogether, our results suggest that Ag dose directly affects Treg‐cell generation and thus, the set‐up of T‐cell tolerance.     

Influenza,but not HIV‐specific CTL epitopes,elicits delayed‐type hypersensitivity (DTH) reactions in HIV‐infected patients

The induction of cytotoxic T lymphocytes (CTLs) is believed to be an important defense mechanism against viral infections. The availability of simple, sensitive, specific and physiologically informative in vivo tests, applicable to humans, would greatly elucidate the nature of protective immune responses and facilitate immune monitoring in large vaccine trials. Here we studied the possibility of using defined HLA‐A*02:01‐restricted CTL epitopes from influenza matrix protein (GL9, GILGFVFTL) and HIV Gag p17 (SL9, SLYNTVATL) to elicit a cutaneous delayed‐type hypersensitivity (DTH) reaction. Our results show that the GL9 but not the SL9 epitope was able to induce a DTH reaction. HIV infection status, HIV RNA level and CD4⁺ T‐cell counts were not predictive of the extent of DTH reactions. However, a markedly reduced expression of skin homing markers CD103 and cutaneous lymphocyte associated Ag (CLA) on epitope‐specific CTL populations was associated with a lack of SL9 DTH reactivity. These data demonstrate that DTH reactions can be elicited by optimally defined CTL epitopes per se and point towards specific homing markers that are required for such reactions. These data may offer new insights into the immune pathogenesis of HIV infection and provide the basis of novel immune monitoring approaches for large‐scale HIV vaccine trials.     

Concurrent cross‐reactivity of microbiota‐derived epitopes to both self and pathogens may underlie the “Hygiene hypothesis”

The current iteration of the “Hygiene hypothesis” proposes precipitous decline in exposure to conserved microbial products and metabolites in individuals in developed countries undermines innate self‐nonself “training” of immune system leading to allergy and autoimmunity. However, lack of innate “training” alone fails to account for the antigen‐driven nature of these immunopathologies. Here, we advance an alternative, antigen‐specific interpretive framework, SPIRAL (Specific ImmunoRegulatory Algorithm) that predicts “loss” of commensal microbiota‐derived epitopes cross‐reactive to both self and pathogens, rather than conserved microbial moieties or metabolites, underlies the “Hygiene hypothesis.” By mechanistically delineating how loss of selective microbiota in predisposed individuals could lead to corresponding “holes” in the epitope‐specific Foxp3⁺ regulatory T cell repertoire and subsequent selective immunopathologies, SPIRAL represents a novel interpretation of cross‐reactivity that could enable targeted discovery of microbiota species and their associated Treg epitopes “missing” in the diseases “Hygiene hypothesis” implicates, and provides a roadmap for a novel unified interpretation of self‐nonself discrimination and T helper phenotype selection.     

Antigens expressed by myelinating glia cells induce peripheral cross‐tolerance of endogenous CD8+ T cells

Auto‐reactivity of T cells is largely prevented by central and peripheral tolerance. Nevertheless, immunization with certain self‐antigens emulsified in CFA induces autoimmunity in rodents, suggesting that tolerance to some self‐antigens is not robust. To investigate the fate of nervous system‐specific CD8⁺ T cells, which only recently came up as being important contributors for MS pathogenesis, we developed a mouse model that allows inducible expression of lymphocytic choriomeningitis virus‐derived CD8⁺ T‐cell epitopes specifically in oligodendrocytes and Schwann cells, the myelinating glia of the nervous system. These transgenic CD8⁺ T‐cell epitopes induced robust tolerance of endogenous auto‐reactive T cells, which proved thymus‐independent and was mediated by cross‐presenting bone‐marrow‐derived cells. Immunohistological staining of secondary lymphoid organs demonstrated the presence of glia‐derived antigens in DC, suggesting that peripheral tolerance of CD8⁺ T cells results from uptake and presentation by steady state DC.     

Genome‐Wide Analysis of Human SNPs at Long Intergenic Noncoding RNAs

Long intergenic noncoding RNAs (lincRNAs) represent a large portion of the noncoding genes in mammals and other eukaryotes but remains among the least well‐understood of genetic factors to date. Here, we systematically analyzed the human SNPs of lincRNAs at a genome level. We found a significantly lower SNP density in lincRNA regions than both their upstream and downstream flanking regions. Functional regions show lower SNP density than other regions in lincRNAs. We revealed that lincRNAs with higher expression levels and broader expression spectrum have significantly lower SNP density. Moreover, we identified lincRNAs that are under recent positive selection and revealed that these lincRNAs show distinct SNP density, expression level, and tissue specificity. Importantly, we identified a genetic variant (rs7990916:T>C) under recent positive selection at a brain‐specific lincRNA that significantly affects the structure of normal brain. Analysis of brain magnetic resonance images showed that individuals with CC genotype have significant bigger regional gray matter volume than individuals with TT genotype. Moreover, the genotype of this SNP shows different distribution in normal elders, mild cognitive impairment, and Alzheimer disease subjects, suggesting that this lincRNA may have a role in physiology and pathophysiology of human brain.     

A high‐throughput shotgun mutagenesis approach to mapping B‐cell antibody epitopes

Characterizing the binding sites of monoclonal antibodies (mAbs) on protein targets, their ‘epitopes’, can aid in the discovery and development of new therapeutics, diagnostics and vaccines. However, the speed of epitope mapping techniques has not kept pace with the increasingly large numbers of mAbs being isolated. Obtaining detailed epitope maps for functionally relevant antibodies can be challenging, particularly for conformational epitopes on structurally complex proteins. To enable rapid epitope mapping, we developed a high‐throughput strategy, shotgun mutagenesis, that enables the identification of both linear and conformational epitopes in a fraction of the time required by conventional approaches. Shotgun mutagenesis epitope mapping is based on large‐scale mutagenesis and rapid cellular testing of natively folded proteins. Hundreds of mutant plasmids are individually cloned, arrayed in 384‐well microplates, expressed within human cells, and tested for mAb reactivity. Residues are identified as a component of a mAb epitope if their mutation (e.g. to alanine) does not support candidate mAb binding but does support that of other conformational mAbs or allows full protein function. Shotgun mutagenesis is particularly suited for studying structurally complex proteins because targets are expressed in their native form directly within human cells. Shotgun mutagenesis has been used to delineate hundreds of epitopes on a variety of proteins, including G protein‐coupled receptor and viral envelope proteins. The epitopes mapped on dengue virus prM/E represent one of the largest collections of epitope information for any viral protein, and results are being used to design better vaccines and drugs.     

Soluble MHC‐II proteins promote suppressive activity in CD4+ T cells

Soluble MHCII (sMHCII) molecules are present in body fluids of healthy individuals and are considered to be involved in the maintenance of self tolerance, and are also related to various diseases. Their concentration increases during in vivo antigen‐specific tolerogenic stimulation and it was recently shown that exosome‐mediated tolerance is MHCII dependent. At the cellular level, sMHCII proteins compete with membrane MHCII for T‐cell receptor binding on CD4⁺ T cells. Immunoaffinity purification techniques isolated sMHCII antigens from the serum of human serum albumin (HSA) ‐tolerant mice as a single highly glycosylated protein of ~ 60 000 molecular weight, specifically interacting with anti‐class II antibodies in Western blotting and ELISA. Mass spectroscopy showed that these sMHCII proteins were loaded with the tolerogenic peptide as well as multiple self peptides. At the cellular level, sMHCII suppressed antigen‐specific, and to a lesser degree antigen‐non‐specific, spleen cell proliferation and induced CD25 in naive T cells. In T cells activated by antigen‐seeded macrophages, sMHCII decreased CD28 and increased CTLA‐4 protein expression, while decreasing interleukin‐2 and increasing interleukin‐10 production. In this case, sMHCII proteins were shown to decrease ZAP‐70 and LAT phosphorylation. The results presented here for the first time provide evidence for the role of sMHCII proteins in immune response suppression and maintenance of tolerance, revealing novel regulatory mechanisms for immune system manipulation.     

Positive selection of self‐antigen‐specific CD8+ T cells by hematopoietic cells

In contrast to thymic epithelial cells, which induce the positive selection of conventional CD8⁺ T cells, hematopoietic cells (HCs) select innate CD8⁺ T cells whose Ag specificity is not fully understood. Here we show that CD8⁺ T cells expressing an H‐Y Ag‐specific Tg TCR were able to develop in mice in which only HCs expressed MHC class I, when HCs also expressed the H‐Y Ag. These HC‐selected self‐specific CD8⁺ T cells resemble innate CD8⁺ T cells in WT mice in terms of the expression of memory markers and effector functions, but are phenotypically distinct from the thymus‐independent CD8⁺ T‐cell population. The peripheral maintenance of H‐Y‐specific CD8⁺ T cells required presentation of the self‐Ag and IL‐15 on HCs. HC‐selected CD8⁺ T cells in mice lacking the Tg TCR also showed these features. Furthermore, by using MHC class I tetramers with a male Ag peptide, we found that self‐Ag‐specific CD8⁺ T cells in TCR non‐Tg mice could develop via HC‐induced positive selection, supporting results obtained from H‐Y TCR Tg mice. These findings indicate the presence of self‐specific CD8⁺ T cells that are positively selected by HCs in the peripheral T‐cell repertoire.     

Reduced immunogenicity of Plasmodium falciparum gamete surface antigen (Pfs48/45) in mice after disruption of disulphide bonds – evaluating effect of interferon‐γ‐inducible lysosomal thiol reductase

Sexual stages of Plasmodium are critical for malaria transmission and stage‐specific antigens are important targets for development of malaria transmission‐blocking vaccines. Plasmodium falciparum gamete surface antigen (Pfs48/45) is important for male gamete fertility and is being pursued as a candidate vaccine antigen. Vaccine‐induced transmission‐blocking antibodies recognize reduction‐sensitive conformational epitopes in Pfs48/45. Processing and presentation of such disulphide‐bond‐constrained epitopes is critical for eliciting the desired immune responses. Mice lacking interferon‐γ‐inducible lysosomal thiol reductase (GILT), an enzyme that mediates reduction of S‐S bonds during antigen processing, were employed to investigate immunogenicity of Pfs48/45. It has been well established that the ability to reduce S‐S bonds in antigens guides effective T‐cell immune responses; however, involvement of GILT in the induction of subsequent B‐cell responses has not been explored. We hypothesized that the ability to reduce S‐S bonds in Pfs48/45 will impact the generation of T‐cell epitopes, and so influence helper T‐cell responses required for specific B‐cell responses. Non‐reduced and reduced and alkylated forms of Pfs48/45 were employed to evaluate immune responses in wild‐type and GILT knockout mice and studies revealed important differences in several immune response parameters, including differences in putative T‐cell epitope recognition, faster kinetics of waning of Pfs48/45‐specific IgG1 antibodies in knockout mice, differential patterns of interferon‐γ and interleukin‐4 secretions by splenocytes, and possible effects of GILT on induction of long‐lived plasma cells and memory B cells responsible for antigen‐recall responses. These studies emphasize the importance of antigen structural features that significantly influence the development of effective immune responses.     

Nanoparticle‐based approaches to immune tolerance for the treatment of autoimmune diseases

Autoimmune diseases are caused by antigenically complex immune responses of the adaptive and innate immune system against specific cells, tissues or organs. Antigen‐specific approaches for induction of immune tolerance in autoimmunity, based on the use of antigenic peptides or proteins, have failed to deliver the desired therapeutic results in clinical trials. These approaches, which are largely relying on triggering clonal anergy and/or deletion of defined autoreactive specificities, do not address the overwhelming antigenic, molecular, and cellular complexity of most autoimmune diseases, which involve various immune cells and ever‐growing repertoires of antigenic epitopes on numerous self‐antigens. Advances in the field of regulatory T‐cell (Treg) biology have suggested that Treg cells might be able to afford dominant tolerance provided they are properly activated and expanded in vivo. More recently, nanotechnology has introduced novel technical advances capable of modulating immune responses. Here, we review nanoparticle‐based approaches designed to induce immune tolerance, ranging from approaches that primarily trigger clonal T‐cell anergy or deletion to approaches that trigger Treg cell formation and expansion from autoreactive T‐cell effectors. We will also highlight the therapeutic potential and positive outcomes in numerous experimental models of autoimmunity.     

Failure of Presented,Non-Dominant Self Epitope to Induce Tolerance: Implications for Autoimmune Diseases

It has been observed that a hierarchy exists among epitopes such that fewer epitopes are actually involved in the induction of T cell response and tolerance than there are epitopes available in a given antigen. Some epitopes which are “cryptic” for immune activity within the protein, are nevetherless able to elicit a response if administered alone and can also be used to by-pass tolerance. We report that tolerance to a self protein shows the same phenomenon seen for non self proteins. In fact, we elicit a proliferative response toward a predicted minor cryptic epitope, to which animals are clearly not self-tolerant. The minor epitope escapes the induction of tolerance to self proteins more easily than the major epitopes, since we cannot elicit proliferative response to the major epitope. A striking feature of our results however is that lack of self tolerance to the minor epitope appears as not being due to the failure of presentation of this epitope in normal, healthy animals.     

Failure of Presented, Non-Dominant Self Epitope to Induce Tolerance: Implications for Autoimmune Diseases

It has been observed that a hierarchy exists among epitopes such that fewer epitopes are actually involved in the induction of T cell response and tolerance than there are epitopes available in a given antigen. Some epitopes which are “cryptic” for immune activity within the protein, are nevetherless able to elicit a response if administered alone and can also be used to by-pass tolerance. We report that tolerance to a self protein shows the same phenomenon seen for non self proteins. In fact, we elicit a proliferative response toward a predicted minor cryptic epitope, to which animals are clearly not self-tolerant. The minor epitope escapes the induction of tolerance to self proteins more easily than the major epitopes, since we cannot elicit proliferative response to the major epitope. A striking feature of our results however is that lack of self tolerance to the minor epitope appears as not being due to the failure of presentation of this epitope in normal, healthy animals.     

Indirect presentation in the thymus limits naive and regulatory T‐cell differentiation by promoting deletion of self‐reactive thymocytes

Acquisition of T‐cell central tolerance involves distinct pathways of self‐antigen presentation to thymocytes. One pathway termed indirect presentation requires a self‐antigen transfer step from thymic epithelial cells (TECs) to bone marrow‐derived cells before the self‐antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context‐dependent, potentially due to variation in self‐antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane‐bound transgenic self‐antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared with thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHC class II expression in bone marrow‐derived antigen‐presenting cells, insHEL‐mediated thymocyte deletion was abolished, whereas thyroHEL‐mediated deletion occurred at a later stage of thymocyte development and Foxp3⁺ regulatory T‐cell differentiation increased. Indirect presentation increased the strength of T‐cell receptor signalling that both self‐antigens induced in thymocytes, as assessed by Helios expression. Hence, indirect presentation limits the differentiation of naive and regulatory T cells by promoting deletion of self‐reactive thymocytes.     

Co‐expression of HLA‐B7 and HLA‐B27 alleles is associated with B7‐restricted immunodominant responses following influenza infection

It is recognized that host response following viral infection is characterized by immunodominance, but deciphering the different factors contributing to immunodominance has proved a challenge due to concurrent expression of multiple MHC class I alleles. To address this, we generated H2‐K^?/?/D^?/? double‐knockout transgenic mice expressing either one or two human MHC‐I alleles. We hypothesized that co‐expression of different allele combinations figures critically in immunodominance and examined this in influenza‐infected, double Tg MHC‐I mice. In A2/B7 or A2/B27 mice, using ELISpot assays with the A2‐restricted matrix I.58–66, the B7‐restricted NP418–426 or the B27‐restricted NP383–391 influenza A (flu) epitopes, we observed the expected recognition of both peptides for both alleles. In contrast, in flu‐infected B7/B27 mice, a significantly reduced level of B27/NP383‐restricted CTL response was detected while there was no change in the B7/NP418‐restricted CTL response. Flu‐specific tetramer studies revealed a partial deletion of Vβ8.1⁺ NP383/B27‐restricted CD8⁺ T cells, and a diminished Vβ12⁺ CD8⁺ T‐cell expansion in B7/B27 Tg mice. Using HLA Tg chimeric mice, we confirmed these findings. These findings shed light on the immune consequences of co‐dominant expression of MHC‐I alleles for host immune response to pathogens.     

Characterization of cytotoxic T‐lymphocyte epitopes and immune responses to SARS coronavirus spike DNA vaccine expressing the RGD‐integrin‐binding motif

Integrins are critical for initiating T‐cell activation events. The integrin‐binding motif Arg‐Gly‐Asp (RGD) was incorporated into the pcDNA 3.1 mammalian expression vector expressing the codon‐optimized extracellular domain of SARS coronavirus (SARS‐CoV) spike protein, and tested by immunizing C57BL/6 mice. Significant cell‐mediated immune responses were characterized by cytotoxic T‐lymphocyte ⁵¹Cr release assay and interferon‐gamma secretion ELISPOT assay against RMA‐S target cells presenting predicted MHC class I H2‐Kb epitopes, including those spanning residues 884–891 and 1116–1123 within the S2 subunit of SARS‐CoV spike protein. DNA vaccines incorporating the Spike‐RGD/His motif or the Spike‐His construct generated robust cell‐mediated immune responses. Moreover, the Spike‐His DNA vaccine construct generated a significant antibody response. Immunization with these DNA vaccine constructs elicited significant cellular and humoral immune responses. Additional T‐cell epitopes within the SARS‐CoV spike protein that may contribute to cell‐mediated immunity in vivo were also identified. J. Med. Virol. 81:1131–1139, 2009. © 2009 Wiley‐Liss, Inc.     

Human monoclonal Fab and human plasma antibodies to carbamyl‐epitopes cross‐react with malondialdehyde‐adducts

Oxidized low‐density lipoprotein (OxLDL) plays a crucial role in the development of atherosclerosis. Carbamylated LDL has been suggested to promote atherogenesis in patients with chronic kidney disease. Here we observed that plasma IgG and IgM antibodies to carbamylated epitopes were associated with IgG and IgM antibodies to oxidation‐specific epitopes (ρ = 0·65–0·86, P < 0·001) in healthy adults, suggesting a cross‐reaction between antibodies recognizing carbamyl‐epitopes and malondialdehyde (MDA)/malondialdehyde acetaldehyde (MAA) ‐adducts. We used a phage display technique to clone a human Fab antibody that bound to carbamylated LDL and other carbamylated proteins. Anti‐carbamyl‐Fab (Fab106) cross‐reacted with oxidation‐specific epitopes, especially with MDA‐LDL and MAA‐LDL. We showed that Fab106 bound to apoptotic Jurkat cells known to contain these oxidation‐specific epitopes, and the binding was competed with soluble carbamylated and MDA‐/MAA‐modified LDL and BSA. In addition, Fab106 was able to block the uptake of carbamyl‐LDL and MDA‐LDL by macrophages and stained mouse atherosclerotic lesions. The observed cross‐reaction between carbamylated and MDA‐/MAA‐modified LDL and its contribution to enhanced atherogenesis in uraemic patients require further investigation.     

Synergistic inhibition of PARP‐1 and NF‐κB signaling downregulates immune response against recombinant AAV2 vectors during hepatic gene therapy

Host immune response remains a key obstacle to widespread application of adeno‐associated virus (AAV) based gene therapy. Thus, targeted inhibition of the signaling pathways that trigger such immune responses will be beneficial. Previous studies have reported that DNA damage response proteins such as poly(ADP‐ribose) polymerase‐1 (PARP‐1) negatively affect the integration of AAV in the host genome. However, the role of PARP‐1 in regulating AAV transduction and the immune response against these vectors has not been elucidated. In this study, we demonstrate that repression of PARP‐1 improves the transduction of single‐stranded AAV vectors both in vitro (～174%) and in vivo (two‐ to 3.4‐fold). Inhibition of PARP‐1, also significantly downregulated the expression of several proinflammatory and cytokine markers such as TLRs, ILs, NF‐κB subunit proteins associated with the host innate response against self‐complementary AAV2 vectors. The suppression of the inflammatory response targeted against these vectors was more effective upon combined inhibition of PARP‐1 and NF‐κB signaling. This strategy also effectively attenuated the AAV capsid‐specific cytotoxic T‐cell response, with minimal effect on vector transduction, as demonstrated in normal C57BL/6 and hemophilia B mice. These data suggest that targeting specific host cellular proteins could be useful to attenuate the immune barriers to AAV‐mediated gene therapy.     

Mature proteins derived from Epstein–Barr virus fail to feed into the MHC class I antigenic pool

The immediate presentation of peptide epitopes on MHC class I (MHC I) after antigen expression has led to the concept that MHC I ligands are mostly derived from defective ribosomal products (DRiPs), a subset of newly synthesized proteins that are rapidly degraded by the proteasome. Whether and to what extent mature proteins contribute to the antigenic pool, however, has remained elusive. Here, we developed a conditional antigen expression system that allows studying antigen presentation from mature proteins by inducing their rapid proteasomal degradation in the absence of further antigen synthesis. Target cells in which expression of two Epstein–Barr virus (EBV) antigens was induced were rapidly recognized by antigen‐specific CD8⁺ T cells in a time‐ and dosage‐dependent manner, demonstrating that antigen presentation was linked to antigen synthesis. By contrast, T cells failed to recognize target cells containing large amounts of mature protein even after induction of their rapid proteasomal degradation. Thus, the presentation of these antigens proved to be strictly dependent on protein synthesis whereas mature proteins failed to furnish the antigenic pool. These results have implications for the design of immunotherapeutic strategies that aim at targeting proteins with increased half‐lives and are hence overexpressed in tumors.