Genetic differentiation for size at first reproduction through male versus female functions in the widespread Mediterranean tree Pinus pinaster.

BACKGROUND AND AIMS: The study of local adaptation in plant reproductive traits has received substantial attention in short-lived species, but studies conducted on forest trees are scarce. This lack of research on long-lived species represents an important gap in our knowledge, because inferences about selection on the reproduction and life history of short-lived species cannot necessarily be extrapolated to trees. This study considers whether the size for first reproduction is locally adapted across a broad geographical range of the Mediterranean conifer species Pinus pinaster. In particular, the study investigates whether this monoecious species varies genetically among populations in terms of whether individuals start to reproduce through their male function, their female function or both sexual functions simultaneously. Whether differences among populations could be attributed to local adaptation across a climatic gradient is then considered. METHODS: Male and female reproduction and growth were measured during early stages of sexual maturity of a P. pinaster common garden comprising 23 populations sampled across the species range. Generalized linear mixed models were used to assess genetic variability of early reproductive life-history traits. Environmental correlations with reproductive life-history traits were tested after controlling for neutral genetic structure provided by 12 nuclear simple sequence repeat markers. KEY RESULTS: Trees tended to reproduce first through their male function, at a size (height) that varied little among source populations. The transition to female reproduction was slower, showed higher levels of variability and was negatively correlated with vegetative growth traits. Several female reproductive traits were correlated with a gradient of growth conditions, even after accounting for neutral genetic structure, with populations from more unfavourable sites tending to commence female reproduction at a lower individual size. CONCLUSIONS: The study represents the first report of genetic variability among populations for differences in the threshold size for first reproduction between male and female sexual functions in a tree species. The relatively uniform size at which individuals begin reproducing through their male function probably represents the fact that pollen dispersal is also relatively invariant among sites. However, the genetic variability in the timing of female reproduction probably reflects environment-dependent costs of cone production. The results also suggest that early sex allocation in this species might evolve under constraints that do not apply to other conifers.

Dexamethasone stimulates the biochemical differentiation of fetal forebrain cells in reaggregating cultures.

The influence of dexamethasone on the development of neurons and oligodendrocytes was studied in serum-free, aggregating rat brain cell cultures. Synaptogenesis and myelination occur in this culture system. The concentration of myelin basic protein and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase were used as oligodendroglia and myelin markers. Choline acetyltransferase and acetylcholinesterase served as neuronal markers, glutamine synthetase reflected astrocyte differentiation, while ornithine decarboxylase served as a general marker for cell growth and maturation. This study showed that dexamethasone stimulated the differentiation of cholinergic neurons and astrocytes. The effect of dexamethasone on oligodendroglial differentiation and myelination depended on the stage of development: during the early phase of myelination dexamethasone had a stimulatory effect, whereas at a later stage it showed a significant inhibition.

Mammalian target of rapamycin complex 1 orchestrates invariant NKT cell differentiation and effector function.

Invariant NKT (iNKT) cells play critical roles in bridging innate and adaptive immunity. The Raptor containing mTOR complex 1 (mTORC1) has been well documented to control peripheral CD4 or CD8 T cell effector or memory differentiation. However, the role of mTORC1 in iNKT cell development and function remains largely unknown. By using mice with T cell-restricted deletion of Raptor, we show that mTORC1 is selectively required for iNKT but not for conventional T cell development. Indeed, Raptor-deficient iNKT cells are mostly blocked at thymic stage 1-2, resulting in a dramatic decrease of terminal differentiation into stage 3 and severe reduction of peripheral iNKT cells. Moreover, residual iNKT cells in Raptor knockout mice are impaired in their rapid cytokine production upon αGalcer challenge. Bone marrow chimera studies demonstrate that mTORC1 controls iNKT differentiation in a cell-intrinsic manner. Collectively, our data provide the genetic evidence that iNKT cell development and effector functions are under the control of mTORC1 signaling.

Strong smooth muscle differentiation is dependent on myocardin gene amplification in most human retroperitoneal leiomyosarcomas.

Myocardin (MYOCD), a serum response factor (SRF) transcriptional cofactor, is essential for cardiac and smooth muscle development and differentiation. We show here by array-based comparative genomic hybridization, fluorescence in situ hybridization, and expression analysis approaches that MYOCD gene is highly amplified and overexpressed in human retroperitoneal leiomyosarcomas (LMS), a very aggressive well-differentiated tumor. MYOCD inactivation by shRNA in a human LMS cell line with MYOCD locus amplification leads to a dramatic decrease of smooth muscle differentiation and strongly reduces cell migration. Moreover, forced MYOCD expression in three undifferentiated sarcoma cell lines and in one liposarcoma cell line confers a strong smooth muscle differentiation phenotype and increased migration abilities. Collectively, these results show that human retroperitoneal LMS differentiation is dependent on MYOCD amplification/overexpression, suggesting that in these well-differentiated LMS, differentiation could be a consequence of an acquired genomic alteration. In this hypothesis, these tumors would not necessarily derive from cells initially committed to smooth muscle differentiation. These data also provide new insights on the cellular origin of these sarcomas and on the complex connections between oncogenesis and differentiation in mesenchymal tumors.

Epidermal differentiation and carcinogenesis

Summary Skin, and more precisely the epidermis, plays a crucial role in our survival since it constitutes our first line of defense against our environment. A subtle equilibrium between proliferation and differentiation of keratinocytes, the main epidermal cell type, provides a continous self-renewal of the epidermis, maintaining the integrity of this protective barrier. It is now well established that pertubation of the normal balance between proliferation and differentiation can induce development of several diseases including cancer. The aim of my thesis was first to characterize new genes involved in the differentiation process of keratinocytes and the formation of the epidermis. We show that cornulin, encoded by the c1orf10 gene, is a new marker of epidermal differentiation, mainly expressed in the suprabasal layers of the epidermis. Structurally, cornulin belongs to the "fused genes" protein family and contains a functional calcium-binding domain as well as two repeated sequences of 60 amino acids, the function of which remain unknown. The second part of my work aimed to identify new proteins interacting with CYLD. When mutated, CYLD is responsible for cylindromatosis, a predisposition to benign tumors of skin appendages mainly located on the scalp. CYLD is implicated in the NF-κB signalling pathway. We have identified HBO1 and p30, two nuclear proteins, as potential CYLD partners. Since CYLD was described as a negative regulator of NF-icB-mediated transcription, we have tested the putative effect of HBO1 and p30 on the regulation of this signalling pathway. We have shown that only HBO1 is able to inhibit NF-κB-mediated transactivation. The mechanism of action of HBO1 is still under investigation but our results suggest that an unknown cofactor is involved in this process. Résumé La peau est cruciale à notre survie car elle est notre première ligne de défense contre notre environnement. L'épiderme qui forme cette barrière protectrice entre le corps et l'environnement extérieur est continuellement renouvelé suite aux agressions physiques, chimiques et biologiques répétées qu'il subit. Le but de ce renouvellement étant de garantir l'intégrité de cette barrière. Le keratinocyte est le principal type cellulaire trouvé dans l'épiderme. La formation d'une barrière active dépend essentiellement de la faculté des kératinocytes à proliférer et à se différencier. Il est aujourd'hui admis que tout déséquilibre entre l'activité de prolifération et de différenciation des kératinocytes est la cause du développement de plusieurs maladies, dont certains cancers. Le but de ce travail de thèse était, dans un premier temps d'identifier ou de caractériser de nouveaux gènes impliqués dans le processus de différenciation afin de mieux comprendre la formation de l'épiderme. Noús avons ainsi démontré que la cornulin, produit du gène c1orf10, est un nouveau marqueur de la différenciation épidermique, principalement exprimé dans les couches suprabasales de l'épiderme. D'un point de vue structural, nous avons montré que cette protéine appartient à la famille des « fused gene » et qu'elle possède un domaine de liaison au calcium qui est fonctionnel et deux séquences répétées de 60 acides aminés dont la fonction est encore inconnue. La seconde partie de cette thèse était dédiée à l'étude de la cylindromatose, une prédisposition génétique à la formation de tumeurs bénignes, principalement localisées sur la tête et due à des mutations du gène CYLD. Nous avons cherché de nouvelles protéines qui interagissent avec CYLD afin de mieux caractériser les voies de signalisation impliquées dans le développement de la maladie. Nous avons ainsi identifiés deux nouveaux partenaires potentiels de CYLD ; HBO1 et p30 CYLD ayant été décrit comme un régulateur négatif de la transcription médiée par NF-κB; nous avons testé l'implication de HBO1 et p30 au niveau de cette activité transcriptionnelle. Nous montrons que seul HBO1 est capable d'inhiber la transactivation d'un gène rapporteur régulé par NF-κB. Le mécanisme d'action de HBO1 n'est pas encore connu, néanmoins nos résultats suggèrent l'intervention d'un cofacteur qui reste à déterminer.

Cell differentiation to "mating bodies" induced by an integrating and conjugative element in free-living bacteria.

Lateral gene transfer (LGT) is one of the most important processes leading to prokaryotic genome innovation. LGT is typically associated with conjugative plasmids and bacteriophages, but recently, a new class of mobile DNA known as integrating and conjugative elements (ICE) was discovered, which is abundant and widespread among bacterial genomes. By studying at the single-cell level the behavior of a prevalent ICE type in the genus Pseudomonas, we uncover the remarkable way in which the ICE orchestrates host cell differentiation to ensure horizontal transmission. We find that the ICE induces a state of transfer competence (tc) in 3%-5% of cells in a population under nongrowing conditions. ICE factors control the development of tc cells into specific assemblies that we name "mating bodies." Interestingly, cells in mating bodies undergo fewer and slower division than non-tc cells and eventually lyse. Mutations in ICE genes disrupting mating-body formation lead to 5-fold decreased ICE transfer rates. Hence, by confining the tc state to a small proportion of the population, ICE horizontal transmission is achieved with little cost in terms of vertical transmission. Given the low transfer frequencies of most ICE, we anticipate regulation by subpopulation differentiation to be widespread.

Growth and differentiation of aggregating fetal brain cells in a serum-free defined medium.

Aggregating cultures of mechanically dissociated fetal brain cells provide an excellent system for neurobiological studies of cellular growth and differentiation, but, in common with almost all culture systems, they have the disadvantage that crude serum is required in the medium. Although several cell lines have either been adapted to serum-free conditions or grown normally in serum-free media supplemented with hormones, trace elements and defined serum components, this approach has never been applied to differentiating primary cells of the central nervous system. We now describe the successful cultivation of aggregating fetal rat brain cells in a chemically defined, serum-free medium.

Mult-class differentiation of cannabis seedlings in a forensic context

This article presents an experimental study about the classification ability of several classifiers for multi-classclassification of cannabis seedlings. As the cultivation of drug type cannabis is forbidden in Switzerland lawenforcement authorities regularly ask forensic laboratories to determinate the chemotype of a seized cannabisplant and then to conclude if the plantation is legal or not. This classification is mainly performed when theplant is mature as required by the EU official protocol and then the classification of cannabis seedlings is a timeconsuming and costly procedure. A previous study made by the authors has investigated this problematic [1]and showed that it is possible to differentiate between drug type (illegal) and fibre type (legal) cannabis at anearly stage of growth using gas chromatography interfaced with mass spectrometry (GC-MS) based on therelative proportions of eight major leaf compounds. The aims of the present work are on one hand to continueformer work and to optimize the methodology for the discrimination of drug- and fibre type cannabisdeveloped in the previous study and on the other hand to investigate the possibility to predict illegal cannabisvarieties. Seven classifiers for differentiating between cannabis seedlings are evaluated in this paper, namelyLinear Discriminant Analysis (LDA), Partial Least Squares Discriminant Analysis (PLS-DA), Nearest NeighbourClassification (NNC), Learning Vector Quantization (LVQ), Radial Basis Function Support Vector Machines(RBF SVMs), Random Forest (RF) and Artificial Neural Networks (ANN). The performance of each method wasassessed using the same analytical dataset that consists of 861 samples split into drug- and fibre type cannabiswith drug type cannabis being made up of 12 varieties (i.e. 12 classes). The results show that linear classifiersare not able to manage the distribution of classes in which some overlap areas exist for both classificationproblems. Unlike linear classifiers, NNC and RBF SVMs best differentiate cannabis samples both for 2-class and12-class classifications with average classification results up to 99% and 98%, respectively. Furthermore, RBFSVMs correctly classified into drug type cannabis the independent validation set, which consists of cannabisplants coming from police seizures. In forensic case work this study shows that the discrimination betweencannabis samples at an early stage of growth is possible with fairly high classification performance fordiscriminating between cannabis chemotypes or between drug type cannabis varieties.

Plac8 is an inducer of C/EBPβ required for brown fat differentiation, thermoregulation, and control of body weight.

Brown adipocytes oxidize fatty acids to produce heat in response to cold or to excessive energy intake; stimulation of brown fat development and function may thus counteract obesity. Brown adipogenesis requires activation of the transcription factor C/EBPβ and recruitment of the zinc finger protein Prdm16, but upstream inducers of these proteins are incompletely defined. Here, we show that genetic inactivation of Plac8, a gene encoding an evolutionarily conserved protein, induces cold intolerance, and late-onset obesity, as well as abnormal morphology and impaired function of brown adipocytes. Using brown preadipocyte lines we show that Plac8 is required for brown fat differentiation, that its overexpression induces C/EBPβ and Prdm16, and that upon induction of differentiation Plac8 associates with C/EBPβ and binds to the C/EBPβ promoter to induce its transcription. Thus, Plac8 is a critical upstream regulator of brown fat differentiation and function that acts, at least in part, by inducing C/EBPβ expression.

Smooth muscle differentiation identifies two classes of poorly differentiated pleomorphic sarcomas with distinct outcome.

The clinical relevance of accurately diagnosing pleomorphic sarcomas has been shown, especially in cases of undifferentiated pleomorphic sarcomas with myogenic differentiation, which appear significantly more aggressive. To establish a new smooth muscle differentiation classification and to test its prognostic value, 412 sarcomas with complex genetics were examined by immunohistochemistry using four smooth muscle markers (calponin, h-caldesmon, transgelin and smooth muscle actin). Two tumor categories were first defined: tumors with positivity for all four markers and tumors with no or incomplete phenotypes. Multivariate analysis demonstrated that this classification method exhibited the strongest prognostic value compared with other prognostic factors, including histological classification. Secondly, incomplete or absent smooth muscle phenotype tumor group was then divided into subgroups by summing for each tumor the labeling intensities of all four markers for each tumors. A subgroup of tumors with an incomplete but strong smooth muscle differentiation phenotype presenting an intermediate metastatic risk was thus identified. Collectively, our results show that the smooth muscle differentiation classification method may be a useful diagnostic tool as well as a relevant prognostic tool for undifferentiated pleomorphic sarcomas.

The anti-apoptotic factor Bcl-2 can functionally substitute for the B cell survival but not for the marginal zone B cell differentiation activity of BAFF.

The TNF family ligand B cell-activating factor (BAFF, BLyS, TALL-1) is an essential factor for B cell development. BAFF binds to three receptors, BAFF-R, transmembrane activator and CAML interactor (TACI), and B cell maturation antigen (BCMA), but only BAFF-R is required for successful survival and maturation of splenic B cells. To test whether the effect of BAFF is due to the up-regulation of anti-apoptotic factors, TACI-Ig-transgenic mice, in which BAFF function is inhibited, were crossed with transgenic mice expressing FLICE-inhibitory protein (FLIP) or Bcl-2 in the B cell compartment. FLIP expression did not rescue B cells, while enforced Bcl-2 expression restored peripheral B cells and the ability to mount T-dependent antibody responses. However, many B cells retained immaturity markers and failed to express normal amounts of CD21. Marginal zone B cells were not restored and the T-independent IgG3, but not IgM, response was impaired in the TACI-IgxBcl-2 mice. These results suggest that BAFF is required not only to inhibit apoptosis of maturating B cells, but also to promote differentiation events, in particular those leading to the generation of marginal zone B cells.

Population differentiation and its consequences in the white campion "Silene latifolia" (Caryophyllaceae): a functional analysis

Summary In his theory On the Origin of Species by Means of Natural Selection (1859), Darwin describes evolution as a gradual change in population over time and that natural selection is a process that caused evolution. Because quantitative variation in species is partly influenced by several genes and thus heritable, association between levels of genetic variation at neutral markers and at quantitative traits and their partitioning within and among populations are important to study mechanisms that drive evolution in populations. Most studies addressing quantitative variation in plants focused on morphological and life history traits but not in traits affecting reproductive success. The aim of this thesis is to better understand how patterns of variation for neutral molecular markers and phenotypic traits drive the evolution of reproduction and defensive mechanisms in six European populations of Silene latifolia, a dioecious plant species. We found evidence for extremely high within and between population variation at six microsatellite loci and at most quantitative traits studied in plants grown under standardized conditions (morphology, life history and reproductive traits). Interestingly, there was clinal variation between age at first flowering and latitude. This pattern is likely due to natural selection since differentiation of this trait was high, heritable and probably higher than differentiation at neutral markers. Our study focused on sex specific selective pressures: mechanisms of intersexual coadaptation and defence mechanism against the seed predator Hadena bicruris. To address divergence at reproductive traits, we studied male and female population of origin effects and in particular pollen competitive ability on male post-pollination success in the study populations with within and between populations crosses. We crossed the same female plant with pollen from a male within the same population of origin and pollen from two males from two distinct populations, using a fixed tester male as a competitor. Additionally, we conducted control crosses with pollen from each male as a single donor. We analysed paternity success of each competitor with two microsatellite loci, seed set and offspring fitness. Male population of origin showed significant among-population variation for siring success at pollen competition. In vitro pollen germination rate showed heritable variation among populations and was positively correlated to siring success. Local or foreign pollen did not have a consistent advantage. Furthermore, female population of origin affected the outcome of pollen competition in some populations. There was no difference of seed set or offspring fitness in within/ between population crosses. This suggests that reproductive divergence may occur via pollen competition in Silene latifolia. The specialist seed predator Hadena bicruris may also induce divergence between populations. We tested potential constitutive and induced defence mechanisms against the specialist predator Hadena bicruris. Because fruit wall thickness is smaller in the invasive range (Northern America) were the moth is absent, this suggests that a thicker fruit wall is a potentially defensive trait against larval attack, and that relaxed selection in the absence of the seed predator has resulted in an evolutionary loss of this defence in the invasive range. Fruit wall thickness was different among three populations. Experimental exposure to moth eggs increased fruit abortion. Fruits built after attack on exposed plants did not have thicker fruit walls compared to fruits on non-exposed plants. Furthermore, fruits with thicker fruit walls were not less profitable, nor did they require longer handling time when exposed to larvae, suggesting no defensive role of fruit wall thickness. Our results show that there is high molecular and phenotypic variation in Silene latifolia and that traits potentially involved in reproductive success both for intra-specific (between sexes) and inter-specific interactions are heritable. Different selective forces may thus interact and cause differential evolution of geographically separated Silene latifolia populations in Europe, leading to the observed differentiation. Résumé Dans sa théorie de l'évolution, L'origine des espèces, ch. 4 (1859), Darwin décrit l'évolution comme un processus continu au cours du temps à l'intérieur de populations et que la sélection naturelle en est le moteur. La variation quantitative est en partie déterminée par plusieurs gènes, donc transmissible à la descendance. Associer le niveau de variation génétique à des marqueurs neutres au niveau de la variation à des traits quantitatifs, ainsi que la répartition à l'intérieur et entre les populations d'une espèce donnée de cette variation, sont importants dans la compréhension des forces évolutives. La plupart des études scientifiques sur la variation quantitative chez les plantes se sont intéressées à la morphologie et à la phénologie mais pas aux caractères impliqués dans le succès reproducteur. L'objectif de cette thèse est de mieux comprendre comment la répartition de la variation à des marqueurs neutres et des caractères quantitatifs influence l'évolution de la reproduction et des mécanismes de défense dans six populations Européennes de l'espèce dioïque Silene latifolia. Nous avons mis en évidence une grande diversité intra et inter-population à six loci microsatellites ainsi qu'à la plupart des caractères quantitatifs mesurés (morphologie, phénologie et traits reproducteurs) sur des plantes cultivées dans des conditions standardisées. Un résultat intéressant est la présence d'un cline latitudinal pour l'âge à la floraison. Ceci est probablement une conséquence de la sélection naturelle, puisque ce caractère est différencié entre les populations étudiées, héritable et que la différenciation de ce trait est supérieure à la différenciation des marqueurs neutres étudiés. Notre étude a ensuite porté plus précisément sur les pressions de sélection spécifiques aux sexes : la coadaptation entre les sexes et les mécanismes de défense contre l'insecte granivore Hadena bicruris. Afin d'évaluer la divergence sur les traits reproducteurs, nous avons étudié les effets des populations d'origine des mâles et des femelles et en particulier le succès reproducteur des mâles après pollinisation à l'aide de croisements inter et intra-population. Nous avons pollinisé la même femelle avec du pollen provenant d'un mâle de la même population ainsi qu'avec le pollen de deux mâles provenant de deux autres populations en situation de compétition avec un pollen provenant d'une population test. Des croisements contrôle ont été réalisés avec les mêmes mâles en pollinisation pure. Nous avons évalué le succès reproducteur de chaque mâle à l'aide d'analyses de paternité ainsi que la production de graines et la fitness de la descendance. L'origine du mâle avait un effet sur la paternité. Le taux de croissance in vitro du pollen est un caractère héritable et a eu un effet positif sur le succès reproducteur. De plus, l'origine de la femelle avait un effet sur le succès des mâles en compétition dans certaines populations. Nos résultats suggèrent qu'une divergence reproductive chez Silene latifolia pourrait apparaître suite à la compétition pollinique. Nous avons ensuite testé des mécanismes potentiels de défense constitutive et induite contre l'herbivore spécialiste Hadena bicruris, un papillon nocturne qui pourrait aussi jouer un rôle dans la différenciation des populations. L'épaisseur des fruits étant plus faible dans les régions où la plante est invasive (Amérique du Nord) et où l'insecte est absent, ce trait pourrait jouer un rôle défensif. Une pression de sélection plus faible causée par l'absence de l'herbivore aurait abouti à une perte de cette défense dans ces régions. Nous avons montré que l'épaisseur du fruit est variable selon les populations. L'infestation artificielle de fruit par l'insecte induit l'abscission sélective des fruits. Les fruits produits après une infestation n'étaient pas plus épais que les fruits issus de plantes non infestées. De plus, les fruits épais n'étaient pas moins nutritifs et ne causaient pas de perte de temps pour la prédation pour les larves, ce qui suggère que l'épaisseur des fruits ne joue pas un rôle défensif. Nos résultats montrent que plusieurs pressions de sélection interviennent et interagissent dans l'évolution de populations distantes, provoquant la divergence des populations Européennes de l'espèce Silene latifolia.

PPARbeta/delta regulates paneth cell differentiation via controlling the hedgehog signaling pathway.

BACKGROUND & AIMS: All 4 differentiated epithelial cell types found in the intestinal epithelium derive from the intestinal epithelial stem cells present in the crypt unit, in a process whose molecular clues are intensely scrutinized. Peroxisome proliferator-activated receptor beta (PPARbeta) is a nuclear hormone receptor activated by fatty acids and is highly expressed in the digestive tract. However, its function in intestinal epithelium homeostasis is understood poorly. METHODS: To assess the role of PPARbeta in the small intestinal epithelium, we combined various cellular and molecular approaches in wild-type and PPARbeta-mutant mice. RESULTS: We show that the expression of PPARbeta is particularly remarkable at the bottom of the crypt of the small intestine where Paneth cells reside. These cells, which have an important role in the innate immunity, are strikingly affected in PPARbeta-null mice. We then show that Indian hedgehog (Ihh) is a signal sent by mature Paneth cells to their precursors, negatively regulating their differentiation. Importantly, PPARbeta acts on Paneth cell homeostasis by down-regulating the expression of Ihh, an effect that can be mimicked by cyclopamine, a known inhibitor of the hedgehog signaling pathway. CONCLUSIONS: We unraveled the Ihh-dependent regulatory loop that controls mature Paneth cell homeostasis and its modulation by PPARbeta. PPARbeta currently is being assessed as a drug target for metabolic diseases; these results reveal some important clues with respect to the signals controlling epithelial cell fate in the small intestine.

Simultaneous coexpression of memory-related and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation.

Phenotypic and functional cell properties are usually analyzed at the level of defined cell populations but not single cells. Yet, large differences between individual cells may have important functional consequences. It is likely that T-cell-mediated immunity depends on the polyfunctionality of individual T cells, rather than the sum of functions of responding T-cell subpopulations. We performed highly sensitive single-cell gene expression profiling, allowing the direct ex vivo characterization of individual virus-specific and tumor-specific T cells from healthy donors and melanoma patients. We have previously shown that vaccination with the natural tumor peptide Melan-A-induced T cells with superior effector functions as compared with vaccination with the analog peptide optimized for enhanced HLA-A*0201 binding. Here we found that natural peptide vaccination induced tumor-reactive CD8 T cells with frequent coexpression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3, and CCR5) and effector-related genes (IFNG, KLRD1, PRF1, and GZMB), comparable with protective Epstein-Barr virus-specific and cytomegalovirus-specific T cells. In contrast, memory/homing-associated and effector-associated genes were less frequently coexpressed after vaccination with the analog peptide. Remarkably, these findings reveal a previously unknown level of gene expression diversity among vaccine-specific and virus-specific T cells with the simultaneous coexpression of multiple memory/homing-related and effector-related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor-specific and virus-specific T cells.