TIE-2 and VEGFR Kinase Activities Drive Immunosuppressive Function of TIE-2-Expressing Monocytes in Human Breast Tumors.

PURPOSE: Tumor-associated TIE-2-expressing monocytes (TEM) are highly proangiogenic cells critical for tumor vascularization. We previously showed that, in human breast cancer, TIE-2 and VEGFR pathways control proangiogenic activity of TEMs. Here, we examine the contribution of these pathways to immunosuppressive activity of TEMs. EXPERIMENTAL DESIGN: We investigated the changes in immunosuppressive activity of TEMs and gene expression in response to specific kinase inhibitors of TIE-2 and VEGFR. The ability of tumor TEMs to suppress tumor-specific T-cell response mediated by tumor dendritic cells (DC) was measured in vitro. Characterization of TEM and DC phenotype in addition to their interaction with T cells was done using confocal microscopic images analysis of breast carcinomas. RESULTS: TEMs from breast tumors are able to suppress tumor-specific immune responses. Importantly, proangiogenic and suppressive functions of TEMs are similarly driven by TIE-2 and VEGFR kinase activity. Furthermore, we show that tumor TEMs can function as antigen-presenting cells and elicit a weak proliferation of T cells. Blocking TIE-2 and VEGFR kinase activity induced TEMs to change their phenotype into cells with features of myeloid dendritic cells. We show that immunosuppressive activity of TEMs is associated with high CD86 surface expression and extensive engagement of T regulatory cells in breast tumors. TIE-2 and VEGFR kinase activity was also necessary to maintain high CD86 surface expression levels and to convert T cells into regulatory cells. CONCLUSIONS: These results suggest that TEMs are plastic cells that can be reverted from suppressive, proangiogenic cells into cells that are able to mediate an antitumoral immune response. Clin Cancer Res; 19(13); 3439-49. ©2013 AACR.

Cathepsin B-like and cell death in the unicellular human pathogen Leishmania.

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H(2)O(2)), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.

Unconditioned responses and functional fear networks in human classical conditioning.

Human imaging studies examining fear conditioning have mainly focused on the neural responses to conditioned cues. In contrast, the neural basis of the unconditioned response and the mechanisms by which fear modulates inter-regional functional coupling have received limited attention. We examined the neural responses to an unconditioned stimulus using a partial-reinforcement fear conditioning paradigm and functional MRI. The analysis focused on: (1) the effects of an unconditioned stimulus (an electric shock) that was either expected and actually delivered, or expected but not delivered, and (2) on how related brain activity changed across conditioning trials, and (3) how shock expectation influenced inter-regional coupling within the fear network. We found that: (1) the delivery of the shock engaged the red nucleus, amygdale, dorsal striatum, insula, somatosensory and cingulate cortices, (2) when the shock was expected but not delivered, only the red nucleus, the anterior insular and dorsal anterior cingulate cortices showed activity increases that were sustained across trials, and (3) psycho-physiological interaction analysis demonstrated that fear led to increased red nucleus coupling to insula but decreased hippocampus coupling to the red nucleus, thalamus and cerebellum. The hippocampus and the anterior insula may serve as hubs facilitating the switch between engagement of a defensive immediate fear network and a resting network.

LILRB2 interaction with HLA class I correlates with control of HIV-1 infection.

Natural progression of HIV-1 infection depends on genetic variation in the human major histocompatibility complex (MHC) class I locus, and the CD8+ T cell response is thought to be a primary mechanism of this effect. However, polymorphism within the MHC may also alter innate immune activity against human immunodeficiency virus type 1 (HIV-1) by changing interactions of human leukocyte antigen (HLA) class I molecules with leukocyte immunoglobulin-like receptors (LILR), a group of immunoregulatory receptors mainly expressed on myelomonocytic cells including dendritic cells (DCs). We used previously characterized HLA allotype-specific binding capacities of LILRB1 and LILRB2 as well as data from a large cohort of HIV-1-infected individuals (N = 5126) to test whether LILR-HLA class I interactions influence viral load in HIV-1 infection. Our analyses in persons of European descent, the largest ethnic group examined, show that the effect of HLA-B alleles on HIV-1 control correlates with the binding strength between corresponding HLA-B allotypes and LILRB2 (p = 10(-2)). Moreover, overall binding strength of LILRB2 to classical HLA class I allotypes, defined by the HLA-A/B/C genotypes in each patient, positively associates with viral replication in the absence of therapy in patients of both European (p = 10(-11)-10(-9)) and African (p = 10(-5)-10(-3)) descent. This effect appears to be driven by variations in LILRB2 binding affinities to HLA-B and is independent of individual class I allelic effects that are not related to the LILRB2 function. Correspondingly, in vitro experiments suggest that strong LILRB2-HLA binding negatively affects antigen-presenting properties of DCs. Thus, we propose an impact of LILRB2 on HIV-1 disease outcomes through altered regulation of DCs by LILRB2-HLA engagement.

Biophysical characterization of the interaction of endotoxins with hemoglobins.

Bacterial endotoxin (lipopolysaccharide, LPS) is the major component of the outer leaflet of the outer membrane in gram-negative bacteria. During severe infections, bacteria may reach the blood circuit of humans, and endotoxins may be released from the bacteria due to cell division or cell death. In particular enterobacterial forms of LPS represent extremely strong activator molecules of the human immune system causing a rapid induction of cytokine production in monocytes and macrophages. Various mammalian blood proteins have been documented to display LPS binding activities mediating normally decreasing effects in the biological activity of LPS. In more recent studies, the essential systemic oxygen transportation protein hemoglobin (Hb) has been shown to amplify LPS-induced cytokine production on immune cells. The mechanism responsible for this effect is poorly understood. Here, we characterize the interaction of hemoglobin with LPS by using biophysical methods. The data presented, revealing the changes of the type and size of supramolecular aggregates of LPS in the presence of Hb, allow a better understanding of the hemoglobin-induced increase in bioactivity of LPS.

Electrical neuroimaging reveals intensity-dependent activation of human cortical gustatory and somatosensory areas by electric taste.

To analyze the neural basis of electric taste we performed electrical neuroimaging analyses of event-related potentials (ERPs) recorded while participants received electrical pulses to the tongue. Pulses were presented at individual taste threshold to excite gustatory fibers selectively without concomitant excitation of trigeminal fibers and at high intensity evoking a prickling and, thus, activating trigeminal fibers. Sour, salty and metallic tastes were reported at both intensities while clear prickling was reported at high intensity only. ERPs exhibited augmented amplitudes and shorter latencies for high intensity. First activations of gustatory areas (bilateral anterior insula, medial orbitofrontal cortex) were observed at 70-80ms. Common somatosensory regions were more strongly, but not exclusively, activated at high intensity. Our data provide a comprehensive view on the dynamics of cortical processing of the gustatory and trigeminal portions of electric taste and suggest that gustatory and trigeminal afferents project to overlapping cortical areas.

Regulation of HCF-1 function via proteolytic maturation and HCF-1 subunit interaction

Abstract : Host-Cell Factor 1 (HCF-1) was first discovered in the study of the herpes simplex virus (HSV) infection. HCF-1 is one of the two cellular proteins that compose the VP16-induced complex, a key activator of HSV lytic infection. lncleed, when HSV infects human cells, it is able to enter two modes of infection: lytic or latent. The V`P16-induced complex promotes the lytic mode and in so doing the virus targets important cellular regulatory proteins, such as HCF-1, to manipulate the status of the infected cell. Indeed, HCF-1 regulates human cell proliferation and the cell cycle at different steps. In human, HCF-1 is unusual in that it undergoes a process of proteolytic maturation that results from cleavages at six centrally located 26 amino acid repeats called HCF-1pro repeats. This generates a heterodimeric complex of stably associated amino- (HCF-1n) and carboxy- (HCF-1c) terminal subunits. The absence of the HCF-1 N or HCF-1; subunit leads predominantly to either G1 or M phase defects, respectively. We have hypothesized that HCF-1 forms a heterodimeric complex to permit communication between the two subunits of HCF-1 involved in regulating different phases of the cell cycle. Indeed, there is evidence for such inter-subunit communication because a point mutation called P134S in the HCF-1N subunit in the temperature-sensitive hamster cell line tsBN67 causes, addition to G1- phase defects associated with the HCF-1n subunit, M-phase defects similar to the defects seen upon loss of HCF-1 function. Furthermore, inhibition of the proteolytic maturation of HCF-1 by deletion of the six HCF-1pro repeats (HCF-1Aimo) also leads to M-phase defects, specifically cytokinesis defects leading to binucleation, indicating that there is loss of HCF-15 function in the absence of HCF-1 maturation. I demonstrate that individual point mutations in each of the six HCF-1pro repeats that prevent HCF-1 proteolytic maturation also lead to binucleation; however, this defect can be latgely rescued by the presence of just one HCF-1pRO sequence in I-ICF»1. These results argue that processing itself is important for the HCF-1g function. In fact, until now, the hypothesis was that the proteolytic processing per se is more important for HCF-1C function than the proteolytic processing region. But I show that processing per se is not sufticient to rescue multinucleation, but that the HCF-lpm sequence itself is crucial. This discovery leads to the conclusion that the I-ICF-1pRO repeats have an additional function important for HCF-le function. From the studies of others, one potential function of the HCF-lrxo tepeats is as a binding site for O-link NAcetyl glycosamine tansferase (OGT) to glycosylate an HCF-1n-sunbunit region called the Basic region. This new function suggests the Basic region of HCF-1n is also implicated in the communication between the two subunits. This inter-subunit communication was analyzed in more detail with the studies of the Pl34S mutation and the residues 382-450 region of HCF-l that when removed prevents HCF-l subunit association. I demonstrate that the point mutation also leads to a binucleation defect in Hela cells as well as in the tsBN67 cells. In addition, the effect of this mutation on the regulation of HCF-1c activity seems to interfere with that of the HCF-lpgg repeats because the sum of the deletion of the proteolytic processing region and the point mutation surprisingly leads to re-establishment of correct cytokinesis. The study of the 382-450 HCF-lN region also yielded surprising results. This region important for the association of the two subunits is also important for both HCF-1c function in M phase and G1 phase progression. Thus, I have discovered two main functions of this region: its role in the regulation of HCF-lc function in M phase and its involvement in the regulation of G1/S phase ?- an HCF-1n function. These results support the importance of inter-subunit communication in HCF-1 functions. My research illuminates the understanding of the interaction of the two subunits by showing that the whole HCF-1n subunit is involved in the inter-subunit communication in order to regulate HCF-1c function. For this work, I was concentrated on the study of cytokinesis; the first phenotype showing the role of HCF-1c in the M phase. Then, I extended the study of the M phase with analysis of steps earlier to cytokinesis. Because some defects in the chromosome segregation was already described in the absence of HCF-1, I decided to continue the study of M phase by checking effects on the chromosome segregation. I showed that the HCF-1n subunit and HCF-1pro repeats are both important for this key step of M phase. I show that the binucleation phenotype resulting from deletion or mutation in HCF-1pro repeats, Pl34S point mutation or the lack of the region 382-450 are correlated with micronuclei, and chromosome segregation and alignment defects. This suggests that HCF«lç already regulates M phase during an early step and could be involved in the complex regulation of chromosome segregation. Because one of the major roles of HCF-1 is to be a transcription regulator, I also checked the capacity of HCF-1 to bind to the chromatin in my different cell lines. All my recombinant proteins can bind the chromatin, except for, as previously described, the HCF-1 with the P134S point mutation, This suggests that the binding of HCF-1 to the chromatin is not dependant to the Basic and proteolytic regions but more to the Kelch domain. Thus, if the function of HCF-ig in M phase is dependant to its chromatin association, the intercommunication and the proteolytic region are not involved in the ability to bind to the chromatin but more to bind to the right place of the chromatin or to be associated with the co-factors. Résumé : L'étude de l'infection par le virus Herpes Simplex (HSV) a permis la découverte de la protéine HCF-1 (Host-Cell Factor). HCF-1 est une des protéines cellulaires qui font partie du complexe induit par VP16 ; ce complexe est la clef pour l'activation de la phase lytique de HSV. Afin de manipuler les cellules infectées, le complexe induit pas le VPIG devrait donc cibler les protéines importantes pour la régulation cellulaire, telles que la protéine HCF-1. Cette dernière s'avère donc être un senseur pour la cellule et devrait également jouer un rôle de régulation lors des différentes phases du cycle cellulaire. Chez l'humain, HCF-1 a la particularité de devoir passer par une phase de maturation pour devenir active. Lors de cette maturation, la protéine subit une coupure protéolytique au niveau de six répétitions composées de 26 acides aminés, appelé HCF-1pro repeats. Cette coupure engendre la formation d'un complexe formé de deux sous-unités, HCF-1n et HCF-1c, associées l'une à l'autre de façon stable. Enlever la sous-unité HCF-IN ou C entraîne respectivement des défauts dans la phase G1 et M. Nous pensons donc que HCF-1 forme un complexe hétérodimérique afin de permettre la communication entre les molécules impliquées dans la régulation des différentes phases du cycle cellulaire. Cette hypothèse est déduite suite à deux études: l'une réalisée sur la lignée cellulaire tsBN67 et l'autre portant sur l'inhibition de la maturation protéolytique. La lignée cellulaire tsBN67, sensible à la température, porte la mutation Pl 345 dans la sous-unité HCF-1n. Cette mutation, en plus d'occasionner des défauts dans la phase G1 (défauts liés à la sous-unité HCF-1N), a aussi pour conséquence d'entrainer des défauts dans la phase M, défauts similaires à ceux dus a la perte de la sous-unité HCF-1c. Quant à la maturation protéolytique, l'absence de la région de la protéolyse provoque la binucléation, défaut lié à la cytokinèse, indiquant la perte de la fonction de la sous-unité HCF-1c. Au cours de ma thèse, j'ai démontré que des mutations dans les HCF-1=no repeats, qui bloquent la protéolyse, engendrent la binucléation ; cependant ce défaut peut être corrigé pas l'ajout d'un HCF-1pro repeat dans un HCF-1 ne contenant pas la région protéolytique. Ces résultats soutiennent l'idée que la région protéolytique est importante pour le bon fonctionnement de HCF-1c. En réalité jusqu'a maintenant on supposait que le mécanisme de coupure était plus important que la région impliquée pour la régulation de la fonction de HCF-1;. Mais mon étude montre que la protéolyse n'est pas suffisante pour éviter la binucléation ; en effet, les HCF-1pro repeats semblent jouer le rôle essentiel dans le cycle cellulaire. Cette découverte conduit à la conclusion que les HCF-1pro repeats ont sûrement une fonction autre qui serait cruciale pour la foncton de HCF-1c. Une des fonctions possibles est d'être le site de liaison de l'O-linked N-acetylglucosamine transférase (OGT) qui glycosylerait la région Basique de HCF-1n. Cette nouvelle fonction suggère que la région Basique est aussi impliquée dans la communication entre les deux sous- unités. L'intercommunication entre les deux sous-unités ai été d'ailleurs analysée plus en détail dans mon travail à travers l'étude de la mutation Pl34S et de la région 382-450, essentielle pour l'association des deux sous»unités. J'ai ainsi démontré que la mutation P134S entraînait aussi des défauts dans la cytokinése dans la lignée cellulaire Hela, de plus, son influence sur HCF-1c semble interférer avec celle de la région protéolytique. En effet, la superposition de ces deux modifications dans HCF-1 conduit au rétablissement d'une cytokinése correcte. Concernant la région 382 à 450, les résultats ont été assez surprenants, la perte de cette région provoque l'arrêt du cycle en G1 et la binucléation, ce qui tend à prouver son importance pour le bon fonctionnement de HCF-1n et de HCF-1c. Cette découverte appuie par conséquent l'hypotl1èse d'une intercommunicatzion entre les deux sous-unités mettant en jeu les différentes régions de HCF-1n. Grâce à mes recherches, j'ai pu améliorer la compréhension de l'interaction des deux sous-unités de HCF-1 en montrant que toutes les régions de HCF-1n sont engagées dans un processus d'intercommunication, dont le but est de réguler l'action de HCF-1c. J'ai également mis en évidence une nouvelle étape de la maturation de HCF-1 qui représente une phase importante pour l'activation de la fonction de HCF-1c. Afin de mettre à jour cette découverte, je me suis concentrée sur l'étude de l'impact de ces régions au niveau de la cytokinése qui fut le premier phénotype démontrant le rôle de HCF-1c dans la phase M. A ce jour, nous savons que HCF-1c joue un rôle dans la cytokinèse, nous ne connaissons pas encore sa fonction précise. Dans le but de cerner plus précisément cette fonction, j'ai investigué des étapes ultérieures ai la cytokinèse. Des défauts dans la ségrégation des chromosomes avaient déjà été observés, ai donc continué l'étude en prouvant que HCF-1n et les HCF-1pro repeats sont aussi importants pour le bon fonctionnement de cette étape clef également régulée par HCF-1c. J' ai aussi montré que la région 382-450 et la mutation P134S sont associées à un taux élevé de micronoyaux, de défauts dans la ségrégation des chromosomes. L'une des fonctions principales de HCF-1 étant la régulation de la transcription, j'ai aussi contrôlé la capacité de HCF-1 à se lier à la chromatine après insertion de mutations ou délétions dans HCF-1n et dans la région protéolytique. Or, à l'exception des HCF-1 contenant la mutation P134S, la sous-unité HCF-1c des HCF-1 tronquées se lie correctement à la chromatine. Cette constatation suggère que la liaison entre HCF-1c et chromatine n'est pas dépendante de la région Basique ou Protéolytique mais peut-être vraisemblablement de la région Kelch. Donc si le rôle de HCF-1c est dépendant de sa capacité â activer la transcription, l'intercommunication entre les deux sous-unités et la région protéolytique joueraient un rôle important non pas dans son habileté à se lier à la chromatine, mais dans la capacité de HCF-1 à s'associer aux co-facteurs ou à se placer sur les bonnes régions du génome.

Clonotype selection and composition of human CD8 T cells specific for persistent herpes viruses varies with differentiation but is stable over time.

Protection from reactivation of persistent herpes virus infection is mediated by Ag-specific CD8 T cell responses, which are highly regulated by still poorly understood mechanisms. In this study, we analyzed differentiation and clonotypic dynamics of EBV- and CMV-specific T cells from healthy adults. Although these T lymphocytes included all subsets, from early-differentiated (EM/CD28(pos)) to late-differentiated (EMRA/CD28(neg)) stages, they varied in the sizes/proportions of these subsets. In-depth clonal composition analyses revealed TCR repertoires, which were highly restricted for CMV- and relatively diverse for EBV-specific cells. Virtually all virus-specific clonotypes identified in the EMRA/CD28(neg) subset were also found within the pool of less differentiated "memory" cells. However, striking differences in the patterns of dominance were observed among these subsets, because some clonotypes were selected with differentiation while others were not. Late-differentiated CMV-specific clonotypes were mostly characterized by TCR with lower dependency on CD8 coreceptor interaction. Yet all clonotypes displayed similar functional avidities, suggesting a compensatory role of CD8 in the clonotypes of lower TCR avidity. Importantly, clonotype selection and composition of each virus-specific subset upon differentiation was highly preserved over time, with the presence of the same dominant clonotypes at specific differentiation stages within a period of 4 years. Remarkably, clonotypic distribution was stable not only in late-differentiated but also in less-differentiated T cell subsets. Thus, T cell clonotypes segregate with differentiation, but the clonal composition once established is kept constant for at least several years. These findings reveal novel features of the highly sophisticated control of steady state protective T cell activity in healthy adults.

Epigenetic features of human mesenchymal stem cells determine their permissiveness for induction of relevant transcriptional changes by SYT-SSX1.

BACKGROUND: A characteristic SYT-SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas, a malignant soft tissue tumor widely believed to originate from as yet unidentified pluripotent stem cells. The resulting fusion protein has no DNA binding motifs but possesses protein-protein interaction domains that are believed to mediate association with chromatin remodeling complexes. Despite recent advances in the identification of molecules that interact with SYT-SSX and with the corresponding wild type SYT and SSX proteins, the mechanisms whereby the SYT-SSX might contribute to neoplastic transformation remain unclear. Epigenetic deregulation has been suggested to be one possible mechanism. METHODOLOGY/PRINCIPAL FINDINGS: We addressed the effect of SYT/SSX expression on the transcriptome of four independent isolates of primary human bone marrow mesenchymal stem cells (hMSC). We observed transcriptional changes similar to the gene expression signature of synovial sarcoma, principally involving genes whose regulation is linked to epigenetic factors, including imprinted genes, genes with transcription start sites within a CpG island and chromatin related genes. Single population analysis revealed hMSC isolate-specific transcriptional changes involving genes that are important for biological functions of stem cells as well as genes that are considered to be molecular markers of synovial sarcoma including IGF2, EPHRINS, and BCL2. Methylation status analysis of sequences at the H19/IGF2 imprinted locus indicated that distinct epigenetic features characterize hMSC populations and condition the transcriptional effects of SYT-SSX expression. CONCLUSIONS/SIGNIFICANCE: Our observations suggest that epigenetic features may define the cellular microenvironment in which SYT-SSX displays its functional effects.

Quantification of nicotine, cotinine, trans-3'-hydroxycotinine and varenicline in human plasma by a sensitive and specific UPLC-tandem mass-spectrometry procedure for a clinical study on smoking cessation.

A sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, its metabolites cotinine and trans-3'-hydroxycotinine and varenicline in human plasma was developed and validated. Sample preparation was realized by solid phase extraction of the target compounds and of the internal standards (nicotine-d4, cotinine-d3, trans-3'-hydroxycotinine-d3 and CP-533,633, a structural analog of varenicline) from 0.5mL of plasma, using a mixed-mode cation exchange support. Chromatographic separations were performed on a hydrophilic interaction liquid chromatography column (HILIC BEH 2.1×100mm, 1.7μm). A gradient program was used, with a 10mM ammonium formate buffer pH 3/acetonitrile mobile phase at a flow of 0.4mL/min. The compounds were detected on a triple quadrupole mass spectrometer, operated with an electrospray interface in positive ionization mode and quantification was performed using multiple reaction monitoring. Matrix effects were quantitatively evaluated with success, with coefficients of variation inferior to 8%. The procedure was fully validated according to Food and Drug Administration guidelines and to Société Française des Sciences et Techniques Pharmaceutiques. The concentration range was 2-500ng/mL for nicotine, 1-1000ng/mL for cotinine, 2-1000ng/mL for trans-3'-hydroxycotinine and 1-500ng/mL for varenicline, according to levels usually measured in plasma. Trueness (86.2-113.6%), repeatability (1.9-12.3%) and intermediate precision (4.4-15.9%) were found to be satisfactory, as well as stability in plasma. The procedure was successfully used to quantify nicotine, its metabolites and varenicline in more than 400 plasma samples from participants in a clinical study on smoking cessation.

Early processing in the human lateral occipital complex is highly responsive to illusory contours but not to salient regions.

Human electrophysiological studies support a model whereby sensitivity to so-called illusory contour stimuli is first seen within the lateral occipital complex. A challenge to this model posits that the lateral occipital complex is a general site for crude region-based segmentation, based on findings of equivalent hemodynamic activations in the lateral occipital complex to illusory contour and so-called salient region stimuli, a stimulus class that lacks the classic bounding contours of illusory contours. Using high-density electrical mapping of visual evoked potentials, we show that early lateral occipital cortex activity is substantially stronger to illusory contour than to salient region stimuli, whereas later lateral occipital complex activity is stronger to salient region than to illusory contour stimuli. Our results suggest that equivalent hemodynamic activity to illusory contour and salient region stimuli probably reflects temporally integrated responses, a result of the poor temporal resolution of hemodynamic imaging. The temporal precision of visual evoked potentials is critical for establishing viable models of completion processes and visual scene analysis. We propose that crude spatial segmentation analyses, which are insensitive to illusory contours, occur first within dorsal visual regions, not the lateral occipital complex, and that initial illusory contour sensitivity is a function of the lateral occipital complex.

Molecular design of the Calphabeta interface favors specific pairing of introduced TCRalphabeta in human T cells.

A promising approach to adoptive transfer therapy of tumors is to reprogram autologous T lymphocytes by TCR gene transfer of defined Ag specificity. An obstacle, however, is the undesired pairing of introduced TCRalpha- and TCRbeta-chains with the endogenous TCR chains. These events vary depending on the individual endogenous TCR and they not only may reduce the levels of cell surface-introduced TCR but also may generate hybrid TCR with unknown Ag specificities. We show that such hybrid heterodimers can be generated even by the pairing of human and mouse TCRalpha- and TCRbeta-chains. To overcome this hurdle, we have identified a pair of amino acid residues in the crystal structure of a TCR that lie at the interface of associated TCR Calpha and Cbeta domains and are related to each other by both a complementary steric interaction analogous to a "knob-into-hole" configuration and the electrostatic environment. We mutated the two residues so as to invert the sense of this interaction analogous to a charged "hole-into-knob" configuration. We show that this inversion in the CalphaCbeta interface promotes selective assembly of the introduced TCR while preserving its specificity and avidity for Ag ligand. Noteworthily, this TCR modification was equally efficient on both a Mu and a Hu TCR. Our data suggest that this approach is generally applicable to TCR independently of their Ag specificity and affinity, subset distribution, and species of origin. Thus, this strategy may optimize TCR gene transfer to efficiently and safely reprogram random T cells into tumor-reactive T cells.

Prevalent role of TCR alpha-chain in the selection of the preimmune repertoire specific for a human tumor-associated self-antigen.

The specificity of recognition of pMHC complexes by T lymphocytes is determined by the V regions of the TCR alpha- and beta-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more V alpha- than V beta-restricted usage. Whether V alpha usage is narrowed during immune responses to Ag or if, on the contrary, restricted V alpha usage is already defined at the early stages of TCR repertoire selection, however, has remained unexplored. Here, we analyzed V and CDR3 TCR regions of single circulating naive T cells specifically detected ex vivo and isolated with HLA-A2/melan-A peptide multimers. Similarly to what was previously observed for melan-A-specific Ag-experienced T cells, we found a relatively wide V beta usage, but a preferential V alpha 2.1 usage. Restricted V alpha 2.1 usage was also found among single CD8(+) A2/melan-A multimer(+) thymocytes, indicating that V alpha-restricted selection takes place in the thymus. V alpha 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected V alpha-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to V alpha 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR.

Trachea, lung, and tracheobronchial lymph nodes are the major sites where antigen-presenting cells are detected after nasal vaccination of mice with human papillomavirus type 16 virus-like particles.

Vaccination by the nasal route has been successfully used for the induction of immune responses. Either the nasal-associated lymphoid tissue (NALT), the bronchus-associated lymphoid tissue, or lung dendritic cells have been mainly involved. Following nasal vaccination of mice with human papillomavirus type 16 (HPV16) virus-like-particles (VLPs), we have previously shown that interaction of the antigen with the lower respiratory tract was necessary to induce high titers of neutralizing antibodies in genital secretions. However, following a parenteral priming, nasal vaccination with HPV16 VLPs did not require interaction with the lung to induce a mucosal immune response. To evaluate the contribution of the upper and lower respiratory tissues and associated lymph nodes (LN) in the induction of humoral responses against HPV16 VLPs after nasal vaccination, we localized the immune inductive sites and identified the antigen-presenting cells involved using a specific CD4(+) T-cell hybridoma. Our results show that the trachea, the lung, and the tracheobronchial LN were the major sites responsible for the induction of the immune response against HPV16 VLP, while the NALT only played a minor role. Altogether, our data suggest that vaccination strategies aiming to induce efficient immune responses against HPV16 VLP in the female genital tract should target the lower respiratory tract.

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches.

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.