A high-density integrated map for grapevine based on three mapping populations genotyped by the Vitis18K SNP chip.

KEY MESSAGE: We present a high-density integrated map for grapevine, allowing refinement and improved understanding of the grapevine genome, while demonstrating the applicability of the Vitis18K SNP chip for linkage mapping. The improvement of grapevine through biotechnology requires identification of the molecular bases of target traits by studying marker-trait associations. The Vitis18K SNP chip provides a useful genotyping tool for genome-wide marker analysis. Most linkage maps are based on single mapping populations, but an integrated map can increase marker density and show order conservation. Here we present an integrated map based on three mapping populations. The parents consist of the well-known wine cultivars 'Cabernet Sauvignon', 'Corvina' and 'Rhine Riesling', the lesser-known wine variety 'Deckrot', and a table grape selection, G1-7720. Three high-density population maps with an average inter-locus gap ranging from 0.74 to 0.99 cM were developed. These maps show high correlations (0.9965-0.9971) with the reference assembly, containing only 93 markers with large order discrepancies compared to expected physical positions, of which a third is consistent across multiple populations. Moreover, the genetic data aid the further refinement of the grapevine genome assembly, by anchoring 104 yet unanchored scaffolds. From these population maps, an integrated map was constructed which includes 6697 molecular markers and reduces the inter-locus gap distance to 0.60 cM, resulting in the densest integrated map for grapevine thus far. A small number of discrepancies, mainly of short distance, involve 88 markers that remain conflictual across maps. The integrated map shows similar collinearity to the reference assembly (0.9974) as the single maps. This high-density map increases our understanding of the grapevine genome and provides a useful tool for its further characterization and the dissection of complex traits.

Genetic Characterization by SSR Markers of a Comprehensive Wine Grape Collection Conserved at Rancho de la Merced (Andalusia, Spain).

The IFAPA research center "Rancho de la Merced" (Jerez, Spain) hosts one of the oldest and most diverse grapevine germplasm repositories in Europe, and is aimed at providing feasible solutions to deal with any agronomic trait by exploring its genetic variability and by means of association and Deoxyribonucleic Acid (DNA) editing studies. In this work, we focused on a wine and dual-use grapevine subcollection that consists of 930 accessions. Genetic analysis allowed to identify 521 unique genotypes. After comparing them with several databases, matches were found for 476 genetic profiles while the remaining 45 have not been previously described. Combination with clustering analysis suggested a total pool of 481 Vitis vinifera accessions that included some table cultivars. Several synonymies, homonymies and mislabeling have also been detected. Structure analysis allowed identifying six clusters according to eco-geographic cultivation areas and one additional group including non-vinifera accessions. Diversity analysis pointed out that Spanish Mediterranean varieties are genetically closer to oriental genotypes than to European varieties typical of oceanic and continental climates. The origin of Spanish varieties is discussed in depth considering our data and previous studies. Analysis of molecular variance partition confirmed a well-structured germplasm, although differentiation among groups had a much lower effect on genetic variability than differences within groups, which are strongly related to a very high heterozygosity. A core collection that covers all allele richness is proposed. It is constituted of about 13% of total accessions, and each cluster inferred by structure analysis is represented.

Somatic variants for seed and fruit set in grapevine.

BACKGROUND: Grapevine reproductive development has direct implications on yield. It also impacts on berry and wine quality by affecting traits like seedlessness, berry and bunch size, cluster compactness and berry skin to pulp ratio. Seasonal fluctuations in yield, fruit composition and wine attributes, which are largely driven by climatic factors, are major challenges for worldwide table grape and wine industry. Accordingly, a better understanding of reproductive processes such as gamete development, fertilization, seed and fruit set is of paramount relevance for managing yield and quality. With the aim of providing new insights into this field, we searched for clones with contrasting seed content in two germplasm collections. RESULTS: We identified eight variant pairs that seemingly differ only in seed-related characteristics while showing identical genotype when tested with the GrapeReSeq_Illumina_20K_SNP_chip and several microsatellites. We performed multi-year observations on seed and fruit set deriving from different pollination treatments, with special emphasis on the pair composed by Sangiovese and its seedless variant locally named Corinto Nero. The pollen of Corinto Nero failed to germinate in vitro and gave poor berry set when used to pollinate other varieties. Most berries from both open- and cross-pollinated Corinto Nero inflorescences did not contain seeds. The genetic analysis of seedlings derived from occasional Corinto Nero normal seeds revealed that the few Corinto Nero functional gametes are mostly unreduced. Moreover, three genotypes, including Sangiovese and Corinto Nero, were unexpectedly found to develop fruits without pollen contribution and occasionally showed normal-like seeds. Five missense single nucleotide polymorphisms were identified between Corinto Nero and Sangiovese from transcriptomic data. CONCLUSIONS: Our observations allowed us to attribute a seedlessness type to some variants for which it was not documented in the literature. Interestingly, the VvAGL11 mutation responsible for Sultanina stenospermocarpy was also discovered in a seedless mutant of Gouais Blanc. We suggest that Corinto Nero parthenocarpy is driven by pollen and/or embryo sac defects, and both events likely arise from meiotic anomalies. The single nucleotide polymorphisms identified between Sangiovese and Corinto Nero are suitable for testing as traceability markers for propagated material and as functional candidates for the seedless phenotype.

Candidate genes and SNPs associated with stomatal conductance under drought stress in Vitis.

BACKGROUND: Understanding the complexity of the vine plant's response to water deficit represents a major challenge for sustainable winegrowing. Regulation of water use requires a coordinated action between scions and rootstocks on which cultivars are generally grafted to cope with phylloxera infestations. In this regard, a genome-wide association study (GWAS) approach was applied on an 'ad hoc' association mapping panel including different Vitis species, in order to dissect the genetic basis of transpiration-related traits and to identify genomic regions of grape rootstocks associated with drought tolerance mechanisms. The panel was genotyped with the GrapeReSeq Illumina 20 K SNP array and SSR markers, and infrared thermography was applied to estimate stomatal conductance values during progressive water deficit. RESULTS: In the association panel the level of genetic diversity was substantially lower for SNPs loci (0.32) than for SSR (0.87). GWAS detected 24 significant marker-trait associations along the various stages of drought-stress experiment and 13 candidate genes with a feasible role in drought response were identified. Gene expression analysis proved that three of these genes (VIT_13s0019g03040, VIT_17s0000g08960, VIT_18s0001g15390) were actually induced by drought stress. Genetic variation of VIT_17s0000g08960 coding for a raffinose synthase was further investigated by resequencing the gene of 85 individuals since a SNP located in the region (chr17_10,497,222_C_T) was significantly associated with stomatal conductance. CONCLUSIONS: Our results represent a step forward towards the dissection of genetic basis that modulate the response to water deprivation in grape rootstocks. The knowledge derived from this study may be useful to exploit genotypic and phenotypic diversity in practical applications and to assist further investigations.

Genetic Resources of Olea europaea L. in the Garda Trentino Olive Groves Revealed by Ancient Trees Genotyping and Parentage Analysis of Drupe Embryos.

The area of the Garda Lake within the Trentino province (north of Italy) is the northernmost part of Europe where the Mediterranean species Olea europaea L. is traditionally cultivated. 'Casaliva' is claimed as the main variety traditionally grown in the Garda Trentino area (GT) from which a world renowned niche extra virgin olive oil is produced. Since a dominant presence of 'Casaliva' would link the fruit set success and yield to a self-pollination compatibility system, a deep genetic survey of the olive tree population in the GT has been performed with the aim of establishing the actual varietal composition and of understanding from which pollen donor the 'Casaliva' olives originate. Forty-four different genetic profiles were observed among the 205 leaf samples collected from 106 ancient trees through the analysis of 20 nuclear microsatellite markers. The varietal composition in modern orchards was also explored and the vast majority of the additional 151 trees analyzed showed the same genotype as the ancient accessions of 'Casaliva'. The results support the long historical link of 'Casaliva' with the GT and, besides a high varietal homogeneity, they also revealed the presence of olive genetic resources essential to fruit production. In fact, the parentage analysis of 550 embryos from drupes of 'Casaliva' evidenced that a cross-fertilization system is favored and a list of candidate cultivars most suitable as local pollinizers of 'Casaliva' was identified.

Genomic signatures of different adaptations to environmental stimuli between wild and cultivated Vitis vinifera L.

The application of population genetic methods in combination with gene mapping strategies can help to identify genes and mutations selected during the evolution from wild plants to crops and to explore the considerable genetic variation still maintained in natural populations. We genotyped a grapevine germplasm collection of 44 wild (Vitis vinifera subsp. sylvestris) and 48 cultivated (V. vinifera subsp. sativa) accessions at 54 K single-nucleotide polymorphisms (SNPs) to perform a whole-genome comparison of the main population genetic statistics. The analysis of Wright Fixation Index (FST) along the whole genome allowed us to identify several putative "signatures of selection" spanning over two thousand SNPs significantly differentiated between sativa and sylvestris. Many of these genomic regions included genes involved in the adaptation to environmental changes. An overall reduction of nucleotide diversity was observed across the whole genome within sylvestris, supporting a small effective population size of the wild grapevine. Tajima's D resulted positive in both wild and cultivated subgroups, which may indicate an ongoing balancing selection. Association mapping for six domestication-related traits was performed in combination with population genetics, providing further evidence of different perception and response to environmental stresses between sativa and sylvestris.

SNP-Discovery by RAD-Sequencing in a Germplasm Collection of Wild and Cultivated Grapevines (V. vinifera L.).

Whole-genome comparisons of Vitis vinifera subsp. sativa and V. vinifera subsp. sylvestris are expected to provide a better estimate of the valuable genetic diversity still present in grapevine, and help to reconstruct the evolutionary history of a major crop worldwide. To this aim, the increase of molecular marker density across the grapevine genome is fundamental. Here we describe the SNP discovery in a grapevine germplasm collection of 51 cultivars and 44 wild accessions through a novel protocol of restriction-site associated DNA (RAD) sequencing. By resequencing 1.1% of the grapevine genome at a high coverage, we recovered 34K BamHI unique restriction sites, of which 6.8% were absent in the 'PN40024' reference genome. Moreover, we identified 37,748 single nucleotide polymorphisms (SNPs), 93% of which belonged to the 19 assembled chromosomes with an average of 1.8K SNPs per chromosome. Nearly half of the SNPs fell in genic regions mostly assigned to the functional categories of metabolism and regulation, whereas some nonsynonymous variants were identified in genes related with the detection and response to environmental stimuli. SNP validation was carried-out, showing the ability of RAD-seq to accurately determine genotypes in a highly heterozygous species. To test the usefulness of our SNP panel, the main diversity statistics were evaluated, highlighting how the wild grapevine retained less genetic variability than the cultivated form. Furthermore, the analysis of Linkage Disequilibrium (LD) in the two subspecies separately revealed how the LD decays faster within the domesticated grapevine compared to its wild relative. Being the first application of RAD-seq in a diverse grapevine germplasm collection, our approach holds great promise for exploiting the genetic resources available in one of the most economically important fruit crops.

Induction of Terpene Biosynthesis in Berries of Microvine Transformed with VvDXS1 Alleles.

Terpenoids, especially monoterpenes, are major aroma-impact compounds in grape and wine. Previous studies highlighted a key regulatory role for grapevine 1-deoxy-D-xylulose 5-phosphate synthase 1 (VvDXS1), the first enzyme of the methylerythritol phosphate pathway for isoprenoid precursor biosynthesis. Here, the parallel analysis of VvDXS1 genotype and terpene concentration in a germplasm collection demonstrated that VvDXS1 sequence has a very high predictive value for the accumulation of monoterpenes and also has an influence on sesquiterpene levels. A metabolic engineering approach was applied by expressing distinct VvDXS1 alleles in the grapevine model system "microvine" and assessing the effects on downstream pathways at transcriptional and metabolic level in different organs and fruit developmental stages. The underlying goal was to investigate two potential perturbation mechanisms, the former based on a significant over-expression of the wild-type (neutral) VvDXS1 allele and the latter on the ex-novo expression of an enzyme with increased catalytic efficiency from the mutated (muscat) VvDXS1 allele. The integration of the two VvDXS1 alleles in distinct microvine lines was found to alter the expression of several terpenoid biosynthetic genes, as assayed through an ad hoc developed TaqMan array based on cDNA libraries of four aromatic cultivars. In particular, enhanced transcription of monoterpene, sesquiterpene and carotenoid pathway genes was observed. The accumulation of monoterpenes in ripe berries was higher in the transformed microvines compared to control plants. This effect is predominantly attributed to the improved activity of the VvDXS1 enzyme coded by the muscat allele, whereas the up-regulation of VvDXS1 plays a secondary role in the increase of monoterpenes.

Experimental Review of DNA-Based Methods for Wine Traceability and Development of a Single-Nucleotide Polymorphism (SNP) Genotyping Assay for Quantitative Varietal Authentication.

The genetic varietal authentication of wine was investigated according to DNA isolation procedures reported for enological matrices and also by testing 11 commercial extraction kits and various protocol modifications. Samples were collected at different stages of the winemaking process of renowned Italian wines Brunello di Montalcino, Lambruschi Modenesi, and Trento DOC. Results demonstrated not only that grape DNA loss is produced by the fermentation process but also that clarification and stabilization operations contribute to the reduction of double-stranded DNA content on wine. Despite the presence of inhibitors, downstream PCR genotyping yielded reliable nuclear and chloroplast SSR markers for must samples, whereas no amplification or inconsistent results were obtained at later stages of the vinification. In addition, a TaqMan genotyping assay based on cultivar-specific single-nucleotide polymorphisms (SNPs) was designed, which allowed assessment of grapevine DNA mixtures. Once the wine matrix limitations are overcome, this sensitive tool may be implemented for the relative quantification of cultivars used for blend wines or frauds.

New candidate genes for the fine regulation of the colour of grapes.

In the last decade, great progress has been made in clarifying the main determinants of anthocyanin accumulation in grape berry skin. However, the molecular details of the fine variation among cultivars, which ultimately contributes to wine typicity, are still not completely understood. To shed light on this issue, the grapes of 170 F1 progeny from the cross 'Syrah'×'Pinot Noir' were characterized at the mature stage for the content of 15 anthocyanins during four growing seasons. This huge data set was used in combination with a dense genetic map to detect genomic regions controlling the anthocyanin pathway both at key enzymatic points and at particular branches. Genes putatively involved in fine tuning the global regulation of anthocyanin biosynthesis were identified by exploring the gene predictions in the QTL (quantitative trait locus) confidence intervals and their expression profile during berry development in offspring with contrasting anthocyanin accumulation. New information on some aspects which had scarcely been investigated so far, such as anthocyanin transport into the vacuole, or completely neglected, such as acylation, is provided. These genes represent a valuable resource in grapevine molecular-based breeding programmes to improve both fruit and wine quality and to tailor wine sensory properties according to consumer demand.

Omental infarction and its mimics: imaging features of acute abdominal conditions presenting with fat stranding greater than the degree of bowel wall thickening.

The segmental omental infarction is a rare self-limited disorder presenting with aspecific clinical symptoms that may mimic several acute abdominal conditions. Therefore, a correct noninvasive diagnosis is important because treatment approaches range from monitoring to surgery. As omental infarction results in an important fat stranding that is much greater than the degree of bowel wall thickening, it suggests a narrower differential diagnosis: appendicitis, diverticulitis, epiploic appendagitis, and mesenteric panniculitis. In this pictorial essay, we point out the importance of imaging in identifying this typical sign allowing alternate diagnoses such as segmental omental infarction that can be conservatively managed.

ERAP1 regulates natural killer cell function by controlling the engagement of inhibitory receptors.

The endoplasmic reticulum aminopeptidase ERAP1 regulates innate and adaptive immune responses by trimming peptides for presentation by MHC class I (MHC-I) molecules. Herein, we demonstrate that genetic or pharmacological inhibition of ERAP1 on human tumor cell lines perturbs their ability to engage several classes of inhibitory receptors by their specific ligands, including killer cell Ig-like receptors (KIR) by classical MHC-I-peptide (pMHC-I) complexes and the lectin-like receptor CD94-NKG2A by nonclassical pMHC-I complexes, in each case leading to natural killer (NK) cell killing. The protective effect of pMHC-I complexes could be restored in ERAP1-deficient settings by the addition of known high-affinity peptides, suggesting that ERAP1 was needed to positively modify the affinity of natural ligands. Notably, ERAP1 inhibition enhanced the ability of NK cells to kill freshly established human lymphoblastoid cell lines from autologous or allogeneic sources, thereby promoting NK cytotoxic activity against target cells that would not be expected because of KIR-KIR ligand matching. Overall, our results identify ERAP1 as a modifier to leverage immune functions that may improve the efficacy of NK cell-based approaches for cancer immunotherapy.

Molecular identification and genetic relationships of Palestinian grapevine cultivars.

Palestine has a wide range of agro-ecological concerns and hosts a large variety of plants. Grapes are part of the cultural heritage and provide an indispensable food ingredient. Local cultivars have been traditionally identified on the basis of morphological traits, geographical origin, or names of the vineyard owner; therefore, the occurrence of homonymy, synonymy, and misnaming significantly prevents their valorization. DNA profiling by 22 common SSR markers was used to characterize 43 putative cultivars grown mainly for local table grape consumption at the southern highland regions of West-Bank, to further evaluate genetic diversity and relationships of the population. Consistent matching of SSR markers with grapevines cultivated in neighboring countries or maintained in European germplasm collections was found for 8 of the 21 different non-redundant genotypes discovered, suggesting possible synonyms as well as the occurrence of breeding selections formerly developed in the USA. Genetic relationships inferred from SSR markers clearly assigned Palestinian cultivars to the Proles orientalis subpr. Antasiatica ancestral population, and they even remarked the connection between local resources and cultivars generated from international table grape breeding. This study supports the value of collection and conservation of vines endemic to a region of immense historical importance for viticulture.

Candidate loci for phenology and fruitfulness contributing to the phenotypic variability observed in grapevine.

KEY MESSAGE: In this study, we identified several genes, which potentially contribute to phenological variation in the grapevine. This may help to maintain consistent yield and suitability of particular varieties in future climatic conditions. The timing of major developmental events in fruit crops differs with cultivar, weather conditions and ecological site. This plasticity results also in diverse levels of fruitfulness. Identifying the genetic factors responsible for phenology and fertility variation may help to improve these traits to better match future climates. Two Vitis vinifera populations, an F1 progeny of Syrah × Pinot Noir and a phenological core collection composed of 163 cultivars, were evaluated for phenology and fertility subtraits during three to six growing seasons in the same geographical location. The phenotypic variability in the core collection mostly overlapped with that observed in the F1 progeny and several accessions had exceeding values of phenological response. The progeny population was used together with SSR and SNP markers to map quantitative trait loci (QTLs). This allowed us to detect nine QTLs related to budburst, flowering beginning, the onset of ripening (véraison) and total fertility, explaining from 8 to 44 % of phenotypic variation. A genomic region on chromosome 15 was associated with budburst and véraison and two QTLs for fruitfulness were located on chromosomes 3 and 18. Several genes potentially affecting fertility and the timing of fruit development were proposed, based on their position and putative function. Allelic variation at these candidate loci may be explored by sampling accessions from the core collection.

Linkage mapping and molecular diversity at the flower sex locus in wild and cultivated grapevine reveal a prominent SSR haplotype in hermaphrodite plants.

Cultivars used for wine and table grape have self-fertile hermaphrodite flowers whereas wild European vines and American and Asian species are dioecious, having either male or female flowers. Consistent with previous studies, the flower sex trait was mapped as a single major locus on chromosome 2 based on a pure Vitis vinifera population segregating for hermaphrodite and female progeny, and a hybrid population producing all three flower sex types. The sex locus was placed between the same SSR and SNP markers on both genetic maps, although abnormal segregation hampered to fine map the genomic region. From a total of 55 possible haplotypes inferred for three SSR markers around the sex locus, in a population of 132 V. sylvestris accessions and 171 V. vinifera cultivars, one of them accounted for 66 % of the hermaphrodite individuals and may be the result of domestication. Specific size variants of the VVIB23 microsatellite sequence within the 3'-UTR of a putative YABBY1 gene were found to be statistically significantly associated with the sex alleles M, H and f; these markers can provide assistance in defining the status of wild grapevine germplasm.

Genetic diversity and population structure assessed by SSR and SNP markers in a large germplasm collection of grape.

BACKGROUND: The economic importance of grapevine has driven significant efforts in genomics to accelerate the exploitation of Vitis resources for development of new cultivars. However, although a large number of clonally propagated accessions are maintained in grape germplasm collections worldwide, their use for crop improvement is limited by the scarcity of information on genetic diversity, population structure and proper phenotypic assessment. The identification of representative and manageable subset of accessions would facilitate access to the diversity available in large collections. A genome-wide germplasm characterization using molecular markers can offer reliable tools for adjusting the quality and representativeness of such core samples. RESULTS: We investigated patterns of molecular diversity at 22 common microsatellite loci and 384 single nucleotide polymorphisms (SNPs) in 2273 accessions of domesticated grapevine V. vinifera ssp. sativa, its wild relative V. vinifera ssp. sylvestris, interspecific hybrid cultivars and rootstocks. Despite the large number of putative duplicates and extensive clonal relationships among the accessions, we observed high level of genetic variation. In the total germplasm collection the average genetic diversity, as quantified by the expected heterozygosity, was higher for SSR loci (0.81) than for SNPs (0.34). The analysis of the genetic structure in the grape germplasm collection revealed several levels of stratification. The primary division was between accessions of V. vinifera and non-vinifera, followed by the distinction between wild and domesticated grapevine. Intra-specific subgroups were detected within cultivated grapevine representing different eco-geographic groups. The comparison of a phenological core collection and genetic core collections showed that the latter retained more genetic diversity, while maintaining a similar phenotypic variability. CONCLUSIONS: The comprehensive molecular characterization of our grape germplasm collection contributes to the knowledge about levels and distribution of genetic diversity in the existing resources of Vitis and provides insights into genetic subdivision within the European germplasm. Genotypic and phenotypic information compared in this study may efficiently guide further exploration of this diversity for facilitating its practical use.

IRF1 and NF-kB restore MHC class I-restricted tumor antigen processing and presentation to cytotoxic T cells in aggressive neuroblastoma.

Neuroblastoma (NB), the most common solid extracranial cancer of childhood, displays a remarkable low expression of Major Histocompatibility Complex class I (MHC-I) and Antigen Processing Machinery (APM) molecules, including Endoplasmic Reticulum (ER) Aminopeptidases, and poorly presents tumor antigens to Cytotoxic T Lymphocytes (CTL). We have previously shown that this is due to low expression of the transcription factor NF-kB p65. Herein, we show that not only NF-kB p65, but also the Interferon Regulatory Factor 1 (IRF1) and certain APM components are low in a subset of NB cell lines with aggressive features. Whereas single transfection with either IRF1, or NF-kB p65 is ineffective, co-transfection results in strong synergy and substantial reversion of the MHC-I/APM-low phenotype in all NB cell lines tested. Accordingly, linked immunohistochemistry expression patterns between nuclear IRF1 and p65 on the one hand, and MHC-I on the other hand, were observed in vivo. Absence and presence of the three molecules neatly segregated between high-grade and low-grade NB, respectively. Finally, APM reconstitution by double IRF1/p65 transfection rendered a NB cell line susceptible to killing by anti MAGE-A3 CTLs, lytic efficiency comparable to those seen upon IFN-γ treatment. This is the first demonstration that a complex immune escape phenotype can be rescued by reconstitution of a limited number of master regulatory genes. These findings provide molecular insight into defective MHC-I expression in NB cells and provide the rational for T cell-based immunotherapy in NB variants refractory to conventional therapy.

Role of endoplasmic reticulum aminopeptidases in health and disease: from infection to cancer.

Endoplasmic reticulum (ER) aminopeptidases ERAP1 and ERAP2 (ERAPs) are essential for the maturation of a wide spectrum of proteins involved in various biological processes. In the ER, these enzymes work in concert to trim peptides for presentation on MHC class I molecules. Loss of ERAPs function substantially alters the repertoire of peptides presented by MHC class I molecules, critically affecting recognition of both NK and CD8(+) T cells. In addition, these enzymes are involved in the modulation of inflammatory responses by promoting the shedding of several cytokine receptors, and in the regulation of both blood pressure and angiogenesis. Recent genome-wide association studies have identified common variants of ERAP1 and ERAP2 linked to several human diseases, ranging from viral infections to autoimmunity and cancer. More recently, inhibition of ER peptide trimming has been shown to play a key role in stimulating innate and adaptive anti-tumor immune responses, suggesting that inhibition of ERAPs might be exploited for the establishment of innovative therapeutic approaches against cancer. This review summarizes data currently available for ERAP enzymes in ER peptide trimming and in other immunological and non-immunological functions, paying attention to the emerging role played by these enzymes in human diseases.

Type I IFNs control antigen retention and survival of CD8α(+) dendritic cells after uptake of tumor apoptotic cells leading to cross-priming.

Cross-presentation is a crucial mechanism for generating CD8 T cell responses against exogenous Ags, such as dead cell-derived Ag, and is mainly fulfilled by CD8α(+) dendritic cells (DC). Apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector CD8 T cell responses. Type I IFNs (IFN-I) have been shown to promote cross-priming of CD8 T cells against soluble or viral Ags, partly through stimulation of DC. By using UV-irradiated OVA-expressing mouse EG7 thymoma cells, we show that IFN-I promote intracellular Ag persistence in CD8α(+) DC that have engulfed apoptotic EG7 cells, regulating intracellular pH, thus enhancing cross-presentation of apoptotic EG7-derived OVA Ag by CD8α(+) DC. Notably, IFN-I also sustain the survival of Ag-bearing CD8α(+) DC by selective upmodulation of antiapoptotic genes and stimulate the activation of cross-presenting DC. The ensemble of these effects results in the induction of CD8 T cell effector response in vitro and in vivo. Overall, our data indicate that IFN-I cross-prime CD8 T cells against apoptotic cell-derived Ag both by licensing DC and by enhancing cross-presentation.

Natural killer cells efficiently reject lymphoma silenced for the endoplasmic reticulum aminopeptidase associated with antigen processing.

The endoplasmic reticulum aminopeptidase ERAAP is involved in the final trimming of peptides for presentation by MHC class I (MHC-I) molecules. Herein, we show that ERAAP silencing results in MHC-I peptide-loading defects eliciting rejection of the murine T-cell lymphoma RMA in syngeneic mice. Although CD4 and CD8 T cells are also involved, rejection is mainly due to an immediate natural killer (NK) cell response and depends on the MHC-I-peptide repertoire because replacement of endogenous peptides with correctly trimmed, high-affinity peptides is sufficient to restore an NK-protective effect of MHC-I molecules through the Ly49C/I NK inhibitory receptors. At the crossroad between innate and adaptive immunity, ERAAP is therefore unique in its two-tiered ability to control tumor immunogenicity. Because a large fraction of human tumors express high levels of the homologous ERAP1 and/or ERAP2, the present findings highlight a convenient, novel target for cancer immunotherapy.