CTLA-4 controls the thymic development of both conventional and regulatory T cells through modulation of the TCR repertoire.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152) is of pivotal importance for self-tolerance, with deficiency or unfavorable polymorphisms leading to autoimmune disease. Tolerance to self-antigens is achieved through thymic deletion of highly autoreactive conventional T (Tconv) cells and generation of FoxP3(+) regulatory T (Treg) cells. The main costimulatory molecule, CD28, augments the negative selection of Tconv cells and promotes the generation of FoxP3(+) Treg cells. The role of its antagonistic homolog CTLA-4, however, remains a topic of debate. To address this topic, we investigated the thymic development of T cells in the presence and absence of CTLA-4 in a T-cell receptor (TCR) transgenic mouse model specific for the myelin basic protein peptide Ac1-9. We reveal that CTLA-4 is expressed in the corticomedullary region of the thymus. Its absence alters the response of CD4(+)CD8(-) thymocytes to self-antigen recognition, which affects the quantity of the Treg cells generated and broadens the repertoire of peripheral Tconv cells. T-cell repertoire alteration after deletion of CTLA-4 results from changes in TCR Vα and Jα segment selection as well as CDR3α composition in Tconv and Treg cells. CTLA-4, therefore, regulates the early development of self-reactive T cells in the thymus and plays a key role in central tolerance.

Pituitary blastoma: a pathognomonic feature of germ-line DICER1 mutations.

Individuals harboring germ-line DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 Syndrome or pleuropulmonary blastoma-familial tumor and dysplasia syndrome [online Mendelian inheritance in man (OMIM) #601200]. In addition, specific somatic mutations in the DICER1 RNase III catalytic domain have been identified in several DICER1-associated tumor types. Pituitary blastoma (PitB) was identified as a distinct entity in 2008, and is a very rare, potentially lethal early childhood tumor of the pituitary gland. Since the discovery by our team of an inherited mutation in DICER1 in a child with PitB in 2011, we have identified 12 additional PitB cases. We aimed to determine the contribution of germ-line and somatic DICER1 mutations to PitB. We hypothesized that PitB is a pathognomonic feature of a germ-line DICER1 mutation and that each PitB will harbor a second somatic mutation in DICER1. Lymphocyte or saliva DNA samples ascertained from ten infants with PitB were screened and nine were found to harbor a heterozygous germ-line DICER1 mutation. We identified additional DICER1 mutations in nine of ten tested PitB tumor samples, eight of which were confirmed to be somatic in origin. Seven of these mutations occurred within the RNase IIIb catalytic domain, a domain essential to the generation of 5p miRNAs from the 5' arm of miRNA-precursors. Germ-line DICER1 mutations are a major contributor to PitB. Second somatic DICER1 "hits" occurring within the RNase IIIb domain also appear to be critical in PitB pathogenesis.

Endoplasmic reticulum stress promotes inflammation through activation of the NLRP3 inflammasome

SummaryMulticellular organisms have evolved an immune system in order to cope with the constant threats they are facing. Foreign pathogens or endogenous danger signals released by injured or dying host cells can be readily detected through a set of germline- encoded pattern-recognition receptors. The NOD-like receptors are a cytoplasmic family of pattern-recognition receptors that have recently attracted considerable attention due to their ability to form inflammasomes, which are molecular complexes responsible for the activation of caspase-1 and the subsequent processing of the pro¬inflammatory cytokines IL-IB and 11-18 into their mature, bioactive form.In this study, we describe a novel pro-inflammatory signaling pathway, whereby the endoplasmic reticulum promotes inflammation through activation of the NLRP3 inflammasome. This was shown to be independent of the classical endoplasmic reticulum stress response pathway constituted by the effectors IREla, PERK and ATF6a. In keeping with other known NLRP3 activators, generation of reactive oxygen species and potassium efflux were required. We also provide evidence that calcium signaling is critical to this pathway, and possibly integrates signaling triggered by various NLRP3 inflammasome activators. Moreover, the mitochondrial channel VDAC1 was instrumental in mediating this response. We thus propose that the endoplasmic reticulum acts as an integrator of stress and is able to activate the mitochondria in a calcium-dependent manner in order to promote NLRP3 inflammasome activation in response to a wide range of activators.Given the role played by inflammation in the pathogenesis of atherosclerosis, we decided to investigate a possible role for the NLRP3 inflammasome in the progression of the disease. Using an ApoE mouse model, we find that deficiency in the NLRP3 inflammasome components NLRP3, ASC or Caspase-1 does not impair atherosclerosis progression, nor does it impact plaque stability. While previous studies have clearly shown a role for the interleukin-1 family of ligands in atherosclerosis, our results suggest that its contribution might be more complex than previously appreciated, and further research is thus warranted in this field.RésuméLes organismes multicellulaires ont développé un système immunitaire pour faire face aux menaces qui les entourent. Des pathogènes étrangers ou des signaux de danger relâchés par des cellules de l'hôte en détresse peuvent être rapidement détectés via un assemblage de récepteurs spécifiques qui sont présents dès la naissance. Certains membres de la famille de récepteurs NOD ont récemment attiré beaucoup d'attention au vu de leur capacité à former des inflammasomes, complexes moléculaires responsables de l'activation de la easpase-1 et de la maturation des cytokines pro-inflammatoires IL- 1β et IL-18 en leur forme bioactive.Dans cette étude, nous décrivons une nouvelle voie de signalisation pro-inflammatoire, par laquelle le réticulum endoplasmique induit l'inflammation via l'activation de l'inflammasome NLRP3. Cette voie est indépendante de la voie classique de réponse au stress du réticulum endoplasmique, qui comprend les effecteurs IRE1, PERK et ATF6. Comme pour d'autres activateurs de NLRP3, la génération de radicaux libres d'oxygène ainsi que Γ efflux de potassium sont requis. Nous montrons également que le calcium joue un rôle critique dans cette voie, et intègre possiblement la signalisation provoquée par divers activateurs de l'inflammasome NLRP3. De plus, le canal mitochondrial VDAC1 est essentiel dans cette réponse. Nous proposons donc que le réticulum endoplasmique agit comme un intégrateur de stress, activant la mitochondrie d'une façon calcium-dépendante pour promouvoir l'activation de l'inflammasome NLRP3 en réponse à divers activateurs.Au vu du rôle joué par l'inflammation dans la pathogenèse de l'athérosclérose, nous avons étudié un possible rôle pour l'inflammasome NLRP3 dans la progression de la maladie. Dans un modèle de souris ApoE, l'absence des composants de l'inflammasome NLRP3 que sont NLRP3, ASC et Caspase-1 n'influence pas la progression des plaques ni leur stabilité. Alors que d'autres études ont démontré un rôle pour les membres de la famille de l'interleukine-1 dans l'athérosclérose, nos résultats suggèrent que leur contribution pourrait être plus complexe que précédemment apprécié, et d'autres recherches dans ce domaine sont donc nécessaires.

Characterization of cytoplasmic antiviral signaling pathways

Summary Multicellular organisms have evolved the immune system to protect from pathogen such as viruses, bacteria, fungi or parasites. Detection of invading pathogens by the host innate immune system is crucial for mounting protective responses and depends on the recognition of microbial components by specific receptors. The results presented in this manuscript focus on the signaling pathways involved in the detection of viral infection by the sensing of viral nucleic acids. First, we describe a new regulatory mechanism controlling RNA-sensing antiviral pathways. Our results indicate that TRIF and Cardif, the crucial adaptor proteins for endosomal and cytoplasmic RNA detection signaling pathway, are processed and inactivated by caspases. The second aspect investigated here involves a signaling pathway triggered upon cytosolic DNA sensing. The interferon inducible protein DAI was recently described as a DNA sensor able to induce the activation of IRFs and NF-κΒ transcription factors leading to type I interferon production. Here we identify two RIP homotypic interaction motifs (RHIMs) in DAI and demonstrate that they mediate the recruitment of RIP1 and RIP3 and the subsequent NF-κΒ activation. Moreover, we observed that the mouse cytomegalovirus RHIM- containing protein M45 has the potential to block this signaling cascade by interfering with the formation of the DAI-RIP1/3 signaling complex. Finally, we report the generation and the initial characterization of NLRX1-deficient mice. NLRX1 is a member of the NOD-like receptor family localized to the mitochondria. The function of NLRX1 is still controversial: one study proposed that NLRX1 acts as an inhibitor of the RIG-like receptor (RLR) antiviral pathway by binding the adaptor protein Cardif, whereas another report implicated NLRX1 in the generation of reactive oxygen species (ROS) and the amplification of NF-κΒ and JNK triggered by TNF-α, poly(I:C) or Shigella infection. Collectively, our results indicate that NLRX1-deficiency does not affect RLR signaling nor TNF-α induced responses. Proteomics analysis identified UQCRC2, a subunit of the complex III of the mitochondrial respiratory chain, as a NLRX1 binding partner. This observation might reveal a possible functional link between NLRX1 and mitochondrial respiration and/or ROS generation. Résumé Au cours de l'évolution, les organismes multicellulaires ont développé le système immunitaire afin de se protéger contre les pathogènes. Une étape cruciale pour le déclenchement des réponses protectrices est la reconnaissance par les cellules du système immunitaire de molécules propres aux microbes grâce à des récepteurs spécifiques. Les résultats présentés dans cette thèse décrivent des nouveaux aspects concernant les voies de signalisation impliquées dans la détection des virus. Le premier projet décrit un mécanisme de régulation des voies activées par la détection d'ARN virale. Nos résultats montrent que TRIF et Cardif, des protéines adaptatrices des voies déclenchées par la reconnaissance de ces acides nucléiques au niveau des endosomes et du cytoplasme, sont clivés et inactivés par les caspases. Le projet suivant de notre recherche concerne une voie de signalisation activée par la détection d'ADN au niveau du cytoplasme. La protéine DAI a été récemment décrite comme un senseur pour cet ADN capable d'activer les facteurs de transcription IRF et NF-κΒ et d'induire ainsi la production des interférons de type I. Ici on démontre que DAI interagit avec RIP1 et RIP3 par le biais de domaines appelés RHIM et que ce complexe est responsable de l'activation de NF-κΒ. On a aussi identifié une protéine du cytomégalovirus de la souris, M45, qui contient ce même domaine et on a pu démontrer qu'elle a la capacité d'interférer avec la formation du complexe entre DAI et RIP1/RIP3 bloquant ainsi l'activation de NF-κΒ. Enfin on décrit ici la génération de souris déficientes pour le gène qui code pour la protéine NLRX1. Cette protéine fait partie de la famille des récepteurs NOD et est localisée dans la mitochondrie. Une étude a suggéré que NLRX1 agit comme un inhibiteur des voies antivirales activées par les récepteurs du type RIG-I (RLR) en interagissant avec la protéine adaptatrice Cardif. Une autre étude propose par contre que NLRX1 participe à la production des dérivés réactifs de l'oxygène et contribue ainsi à augmenter l'activation de NF- κΒ et JNK induite par le TNF-α ou le poly(I:C). Nos résultats montrent que l'absence de NLRX1 ne modifie ni la voie de signalisation RLR ni les réponses induites par le TNF-α. Des analyses ultérieures ont permis d'identifier comme partenaire d'interaction de NLRX1 la protéine UQCRC2, une des sous-unités qui composent le complexe III de la chaîne respiratoire mitochondriale. Cette observation pourrait indiquer un lien fonctionnel entre NLRX1 et la respiration mitochondriale ou la production des dérivés réactifs de l'oxygène au niveau de cette organelle.

Provenance and family variation of Pinus caribaea var. hondurensis from Guatemala and Honduras, grown in Brazil, Colombia and Venezuela

Pinus caribaea var. hondurensis (Sénécl) Barr. & Golf. is a tropical pine that naturally occurs in lowland areas of Belize, El Salvador, Guatemala, Honduras, Nicaragua, and eastern Mexico. It has been one of the most studied tropical pines and the one with the most commercial importance in Brazil. The objective of this work was to select the best provenances for plantations and best trees in families for the establishment of seed orchards. For that a trial with five provenances and 47 open-pollinated families was planted near Planaltina, Federal District, in the Cerrado Region of Brazil. The provenances tested were Poptun (Guatemala), Gualjoco, Los Limones, El Porvenir and Santa Cruz de Yojoa (Honduras) and assessed at 12 years of age. Poptun and Gualjoco had larger volume, and Los Limones and El Porvenir the lowest incidence of forks and foxtails. Individual tree heritabilities for volume, stem form and branch diameter were 0.34, 0.06, and 0.26 respectively. More than 90% of the trees had defects, common in unimproved P. caribaea. Selection criteria for quality traits need to be relaxed in the first generation of breeding to allow for larger genetic gains in productivity. Results from this test compared with P. caribaea var. hondurensis trials in other Brazilian, Colombian and Venezuelan sites suggest that provenance x site and family x site interactions are not as strong as in other pine species.

Bioelectrochemical Treatment of Contaminated Groundwater

To remove these pollutants from groundwater, different technologies can be used. Currently, the Environmental Protection Agency (EPA) considers ion exchange, reverse osmosis and reverse electrodialysis to be effective methods for the decrease of their concentrations, below their limit in drinking water. These technologies have some drawbacks, such as low selectivity towards the target pollutant, high energy or chemicals requirements, and the generation of waste brine (pollutants are separated from water, not treated), which require an additional treatment. Bio Electro Chemical Systems (BES) could fill this niche

Comparative analysis of amino acid repeats in rodents and humans

Amino acid tandem repeats, also called homopolymeric tracts, are extremely abundant in eukaryotic proteins. To gain insight into the genome-wide evolution of these regions in mammals, we analyzed the repeat content in a large data set of rat-mouse-human orthologs. Our results show that human proteins contain more amino acid repeats than rodent proteins and that trinucleotide repeats are also more abundant in human coding sequences. Using the human species as an outgroup, we were able to address differences in repeat loss and repeat gain in the rat and mouse lineages. In this data set, mouse proteins contain substantially more repeats than rat proteins, which can be at least partly attributed to a higher repeat loss in the rat lineage. The data are consistent with a role for trinucleotide slippage in the generation of novel amino acid repeats. We confirm the previously observed functional bias of proteins with repeats, with overrepresentation of transcription factors and DNA-binding proteins. We show that genes encoding amino acid repeats tend to have an unusually high GC content, and that differences in coding GC content among orthologs are directly related to the presence/absence of repeats. We propose that the different GC content isochore structure in rodents and humans may result in an increased amino acid repeat prevalence in the human lineage.

Rapid Perturbation in Viremia Levels Drives Increases in Functional Avidity of HIV-specific CD8 T Cells.

The factors determining the functional avidity and its relationship with the broad heterogeneity of antiviral T cell responses remain partially understood. We investigated HIV-specific CD8 T cell responses in 85 patients with primary HIV infection (PHI) or chronic (progressive and non-progressive) infection. The functional avidity of HIV-specific CD8 T cells was not different between patients with progressive and non-progressive chronic infection. However, it was significantly lower in PHI patients at the time of diagnosis of acute infection and after control of virus replication following one year of successful antiretroviral therapy. High-avidity HIV-specific CD8 T cells expressed lower levels of CD27 and CD28 and were enriched in cells with an exhausted phenotype, i.e. co-expressing PD-1/2B4/CD160. Of note, a significant increase in the functional avidity of HIV-specific CD8 T cells occurred in early-treated PHI patients experiencing a virus rebound after spontaneous treatment interruption. This increase in functional avidity was associated with the accumulation of PD-1/2B4/CD160 positive cells, loss of polyfunctionality and increased TCR renewal. The increased TCR renewal may provide the mechanistic basis for the generation of high-avidity HIV-specific CD8 T cells. These results provide insights on the relationships between functional avidity, viremia, T-cell exhaustion and TCR renewal of antiviral CD8 T cell responses.

Detecting the oxidative reactivity of nanoparticles: a new protocol for reducing artifacts

Understanding the oxidative reactivity of nanoparticles (NPs; <100 nm) could substantially contribute to explaining their toxicity. We attempted to refine the use of 2′7-dichlorodihydrofluorescein (DCFH) to characterize NP generation of reactive oxygen species (ROS). Several fluorescent probes have been applied to testing oxidative reactivity, but despite DCFH being one of the most popular for the detection of ROS, when it has been applied to NPs there have been an unexplainably wide variability in results. Without a uniform methodology, validating even robust results is impossible. This study, therefore, identified sources of conflicting results and investigated ways of reducing occurrence of artificial results. Existing techniques were tested and combined (using their most desirable features) to form a more reliable method for the measurement of NP reactivity in aqueous dispersions. We also investigated suitable sample ranges necessary to determine generation of ROS. Specifically, ultrafiltration and time-resolved scan absorbance spectra were used to study possible optical interference when using high sample concentrations. Robust results were achieved at a 5 µM DCFH working solution with 0.5 unit/mL horseradish peroxidase (HRP) dissolved in ethanol. Sonication in DCFH-HRP working solution provided more stable data with a relatively clean background. Optimal particle concentration depends on the type of NP and in general was in the µg/mL range. Major reasons for previously reported conflicting results due to interference were different experimental approaches and NP sample concentrations. The protocol presented here could form the basis of a standardized method for applying DCFH to detect generation of ROS by NPs.

Low TCR avidity and lack of tumor cell recognition in CD8(+) T cells primed with the CEA-analogue CAP1-6D peptide.

The use of "altered peptide ligands" (APL), epitopes designed for exerting increased immunogenicity as compared with native determinants, represents nowadays one of the most utilized strategies for overcoming immune tolerance to self-antigens and boosting anti-tumor T cell-mediated immune responses. However, the actual ability of APL-primed T cells to cross-recognize natural epitopes expressed by tumor cells remains a crucial concern. In the present study, we show that CAP1-6D, a superagonist analogue of a carcinoembriyonic antigen (CEA)-derived HLA-A*0201-restricted epitope widely used in clinical setting, reproducibly promotes the generation of low-affinity CD8(+) T cells lacking the ability to recognized CEA-expressing colorectal carcinoma (CRC) cells. Short-term T cell cultures, obtained by priming peripheral blood mononuclear cells from HLA-A*0201(+) healthy donors or CRC patients with CAP1-6D, were indeed found to heterogeneously cross-react with saturating concentrations of the native peptide CAP1, but to fail constantly lysing or recognizing through IFN- gamma release CEA(+)CRC cells. Characterization of anti-CAP1-6D T cell avidity, gained through peptide titration, CD8-dependency assay, and staining with mutated tetramers (D227K/T228A), revealed that anti-CAP1-6D T cells exerted a differential interaction with the two CEA epitopes, i.e., displaying high affinity/CD8-independency toward the APL and low affinity/CD8-dependency toward the native CAP1 peptide. Our data demonstrate that the efficient detection of self-antigen expressed by tumors could be a feature of high avidity CD8-independent T cells, and underline the need for extensive analysis of tumor cross-recognition prior to any clinical usage of APL as anti-cancer vaccines.

Accuracy profile validation of a new analytical method for propane measurement using headspace-gas chromatography-mass spectrometry

Propane can be responsible for several types of lethal intoxication and explosions. Quantifying it would be very helpful to determine in some cases the cause of death. Some gas chromatography-mass spectrometry (GC-MS) methods of propane measurements do already exist. The main drawback of these GC-MS methods described in the literature is the absence of a specific propane internal standard necessary for accurate quantitative analysis. The main outcome of the following study was to provide an innovative Headspace-GC-MS method (HS-GC-MS) applicable to the routine determination of propane concentration in forensic toxicology laboratories. To date, no stable isotope of propane is commercially available. The development of an in situ generation of standards is thus presented. An internal-labeled standard gas (C3DH7) is generated in situ by the stoichiometric formation of propane by the reaction of deuterated water (D2O) with Grignard reagent propylmagnesium chloride (C3H7MgCl). The method aims to use this internal standard to quantify propane concentrations and, therefore, to obtain precise measurements. Consequently, a complete validation with an accuracy profile according to two different guidelines, the French Society of Pharmaceutical Sciences and Techniques (SFSTP) and the Gesellschaft für toxikologische und Forensische Chemie (GTFCh), is presented.

Épidémiologie et physiopathologie de la rétinopathie du prématuré [Epidemiology and pathophysiology of retinopathy of prematurity].

Retinopathy of prematurity (ROP) is a major cause of visual impairment in premature infants. It is characterized by an arrest in normal retinal vascular development associated with microvascular degeneration, followed by an abnormal hypoxiainduced neovascularization. Recent studies point out that ROP is a multifactorial disease, implicating both oxygen-dependent and oxygen-independent mechanisms. Oxygen-dependent factors leading to microvascular degeneration include generation of reactive oxygen species and suppression of specific oxygen-regulated vascular survival factors, such as vascular endothelial growth factor (VEGF) and erythropoietin. The other major mechanism for the initial capillary loss is oxygen-independent and implicates a deficit in growth factor IGF-1/IGFBP3. The proliferative, second phase of ROP is triggered by increases in vascular growth factors concentrations, in an attempt to compensate for the hypoxic retina. Novel signaling pathways for vascular repair, implicating both metabolite signaling and inflammatory lipids signaling, represent new therapeutic avenues for ROP.

Storage and uptake of D-serine into astrocytic synaptic-like vesicles specify gliotransmission.

Glial cells are increasingly recognized as active players that profoundly influence neuronal synaptic transmission by specialized signaling pathways. In particular, astrocytes have been shown recently to release small molecules, such as the amino acids l-glutamate and d-serine as "gliotransmitters," which directly control the efficacy of adjacent synapses. However, it is still controversial whether gliotransmitters are released from a cytosolic pool or by Ca(2+)-dependent exocytosis from secretory vesicles, i.e., by a mechanism similar to the release of synaptic vesicles in synapses. Here we report that rat cortical astrocytes contain storage vesicles that display morphological and biochemical features similar to neuronal synaptic vesicles. These vesicles share some, but not all, membrane proteins with synaptic vesicles, including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) synaptobrevin 2, and contain both l-glutamate and d-serine. Furthermore, they show uptake of l-glutamate and d-serine that is driven by a proton electrochemical gradient. d-Serine uptake is associated with vesicle acidification and is dependent on chloride. Whereas l-serine is not transported, serine racemase, the synthesizing enzyme for d-serine, is anchored to the membrane of the vesicles, allowing local generation of d-serine. Finally, we reveal a previously unexpected mutual vesicular synergy between d-serine and l-glutamate filling in glia vesicles. We conclude that astrocytes contain vesicles capable of storing and releasing d-serine, l-glutamate, and most likely other neuromodulators in an activity-dependent manner.

Cilengitide modulates attachment and viability of human glioma cells, but not sensitivity to irradiation or temozolomide in vitro.

Cilengitide is a cyclic peptide antagonist of integrins alphavbeta3 and alphavbeta5 that is currently being evaluated as a novel therapeutic agent for recurrent and newly diagnosed glioblastoma. Its mode of action is thought to be mainly antiangiogenic but may include direct effects on tumor cells, notably on attachment, migration, invasion, and viability. In this study we found that, at clinically relevant concentrations, cilengitide (1-100 microM) induces detachment in some but not all glioma cell lines, while the effect on cell viability is modest. Detachment induced by cilengitide could not be predicted by the level of expression of the cilengitide target molecules, alphavbeta3 and alphavbeta5, at the cell surface. Glioma cell death induced by cilengitide was associated with the generation of caspase activity, but caspase activity was not required for cell death since ectopic expression of cytokine response modifier (crm)-A or coexposure to the broad-spectrum caspase inhibitor zVAD-fmk was not protective. Moreover, forced expression of the antiapoptotic protein marker Bcl-X(L) or altering the p53 status did not modulate cilengitide-induced cell death. No consistent effects of cilengitide on glioma cell migration or invasiveness were observed in vitro. Preliminary clinical results indicate a preferential benefit from cilengitide added to temozolomide-based radiochemotherapy in patients with O(6)-methylguanine DNA methyltransferase (MGMT) gene promoter methylation. Accordingly, we also examined whether the MGMT status determines glioma cell responses to cilengitide alone or in combination with temozolomide. Neither ectopic expression of MGMT in MGMT-negative cells nor silencing the MGMT gene in MGMT-positive cells altered glioma cell responses to cilengitide alone or to cilengitide in combination with temozolomide. These data suggest that the beneficial clinical effects derived from cilengitide in vivo may arise from altered perfusion, which promotes temozolomide delivery to glioma cells.

Insight in the factors associated with the presence of protective antiviral T-cell response.

T cells play a primordial role in antiviral immunity. Virus-specific T-cell responses can be characterized by a number of independent variables. These include the magnitude of the response; the functional quality of the response, i.e. the types of cytokines secreted after stimulation and the proliferative or lytic potential; the tissue distribution of the T cells; the breadth of the response; and the avidity of the response. All of these together constitute the T-cell response to antigen (Ag) and comprise potential variables that may correlate with antiviral protective immunity. Substantial advances have recently been obtained in the characterization of virus-specific T-cell responses. These studies have shown that the quality (in term of functional profile) rather than the quantity of Ag-specific T cells was associated with protection. Recently, the term polyfunctional has been used to define T-cell responses that, in addition to typical effector functions such as secretion of IFN-g, TNF-a and MIP-1b and cytotoxic activity, comprise distinct T-cell populations, also able to secrete IL-2 and retaining Ag-specific proliferation capacity. The term \only effector" defines T-cell responses/ populations able to secrete cytokines such as IFN-g, TNF-a and MIP-1b and endowed with cytotoxic activity but lacking IL-2 and proliferation capacity. Several models of virus infections (HIV-1, cytomegalovirus [CMV], Epstein Barr virus [EBV], influenza [Flu] and Herpes Simplex virus) exclusively differentiated on the basis of Ag exposure and persistence, were investigated: 1) antigen clearance, 2) protracted Ag exposure and persistence and low Ag levels, 3) Ag persistence and high Ag levels, and 4) acute Ag exposure/re-exposure. These analyses have demonstrated that polyfunctional and not \only effector" T-cell responses were associated with protective antiviral immunity. However, the factors and mechanisms governing the generation of functionally distinct T-cell populations remain to be elucidated. Recently, several studies have shown a major influence of HLA genotype in the evolution of HIV and the progression of HIV-associated disease. In particular, certain HLA-B alleles were most closely associated with non-progressive disease and low viral load or disease and had a dominant involvement on the clinical course of HIV-associated diseases. In this study, we have investigated the relationship between HLA restriction and the functional profile of Tcell responses in order to determine whether HLA-B influenced the generation of polyfunctional CD8 T-cell responses. To be able to address this issue, we studied CD8 T-cell responses against HIV-1, CMV, EBV and Flu in 128 subjects. These analyses enabled us to demonstrate that HLA-Arestricted epitopes were mostly associated with \only effector" T-cell responses while, in contrast, polyfunctional CD8 T-cell responses were predominantly driven by virus epitopes restricted by HLA-B alleles. We then characterized eventual differences in the responsiveness of CD8 T-cell populations restricted by different HLA-A and HLA-B alleles. For this purpose, we investigated the T-cell receptor (TCR) avidity for the cognate epitope of polyfunctional and \only effector" CD8 T-cell populations. Our results indicated that overall virus-specific CD8 T-cell populations recognizing virus epitopes restricted by HLA-B alleles were equipped with lower avidity TCR for the cognate epitopes when compared to those recognizing epitopes restricted by HLA-A alleles. In conclusion, these results provide the rationale for the observed protective role of HLA-B genotypes in HIV-1- infection and new insights into the relationship between TCR avidity and functional profile of virus-specific CD8 Tcells.