Probing the interactions of NK cell receptors with ligand expressed in trans and cis.

Certain receptors on natural killer (NK) cells, which are specific for MHC class I (MHC-I) molecules, do not only interact with ligand expressed on opposing cell membranes (in trans) but also interact with those on the same cell membrane (in cis). Cis interactions have been demonstrated for only a small number of cell surface receptors. However, this has not been tested systematically, raising the possibility that additional receptors may be able to bind ligand expressed in cis. Here we describe a number of approaches to evaluate trans and cis binding of the Ly49A NK cell receptor to its H-2D(d) ligand. These procedures should facilitate the investigation of cis/trans interactions of other receptor-ligand pairs and simplify the analysis of NK cell receptor variants.

Inhibitory receptors specific for MHC class I educate murine NK cells but not CD8αα intestinal intraepithelial T lymphocytes.

The engagement of inhibitory receptors specific for major histocompatibility complex class I (MHC-I) molecules educates natural killer (NK) cells, meaning the improvement of the response of activation receptors to subsequent stimulation. It is not known whether inhibitory MHC-I receptors educate only NK cells or whether they improve the responsiveness of all cell types, which express them. To address this issue, we analyzed the expression of inhibitory MHC-I receptors on intestinal intraepithelial lymphocytes (iIELs) and show that T-cell receptor (TCR)-αβ CD8αα iIELs express multiple inhibitory receptors specific for MHC-I molecules, including CD94/NKG2A, Ly49A, and Ly49G2. However, the presence of MHC-I ligand for these receptors did not improve the response of iIELs to activation via the TCR. The absence of iIEL education by MHC-I receptors was not related to a lack of inhibitory function of these receptors in iIELs and a failure of these receptors to couple to the TCR. Thus, unlike NK cells, iIELs do not undergo an MHC-I-guided education process. These data suggest that education is an NK cell-specific function of inhibitory MHC-I receptors.

Single cell analysis reveals similar functional competence of dominant and nondominant CD8 T-cell clonotypes.

Immune protection from infectious diseases and cancer is mediated by individual T cells of different clonal origin. Their functions are tightly regulated but not yet fully characterized. Understanding the contribution of each T cell will improve the prediction of immune protection based on laboratory assessment of T-cell responses. Here we developed techniques for simultaneous molecular and functional assessment of single CD8 T cells directly ex vivo. We studied two groups of patients with melanoma after vaccination with two closely related tumor antigenic peptides. Vaccination induced T cells with strong memory and effector functions, as found in virtually all T cells of the first patient group, and fractions of T cells in the second group. Interestingly, high functionality was not restricted to dominant clonotypes. Rather, dominant and nondominant clonotypes acquired equal functional competence. In parallel, this was also found for EBV- and CMV-specific T cells. Thus, the nondominant clonotypes may contribute similarly to immunity as their dominant counterparts.

Differential expression of triiodothyronine receptors in schwannoma and neurofibroma: role of Schwann cell-axon interaction.

Regulation of gene expression in Schwann cells may be determined, at least in part, by the interaction of these cells with axons. Two peripheral nerve tumors, neurofibroma and schwannoma, represent good tools for studying Schwann cell activity in the presence or absence of axon action. In the present work we studied the expression of triiodothyronine receptors (T3R) by Schwann cells in these two tumors and also in adult normal sciatic nerve. Confirming the results of the histological examination, immunostaining of the neurofilaments showed the presence of fascicles or scattered axons in all neurofibroma sections studied. In these neurofibromas, Schwann cells did not express T3R immunoreactivity. Furthermore, in adult normal sciatic nerve, Schwann cells which ensheathed axons were devoid of any T3R expression. In contrast, in schwannoma, the complete absence of axons was demonstrated by the lack of neurofilament immunostaining. Here, Schwann cells deprived of axonal interaction displayed clear T3R immunoreactivity. In schwannoma cell cultures, Schwann cells continued to express T3R, even in cultures treated with medium that had been conditioned with rat sensory neurons. On the basis of these results, we suggest that, beside the possible regulatory mechanisms for T3R, the synthesis of T3R is regulated, at least in part, by Schwann cell-axon interaction.

NKT cells: In the beginning...

Invariant natural killer T (iNKT) cells as we know them today are a unique subset of mature T cells co-expressing a semi-invariant Valpha14/Vbeta8 TCR and surface markers characteristic of NK cells. The semi-invariant TCR on iNKT cells recognizes glycolipids bound to monomorphic CD1d molecules, leading to rapid cytokine production. The purpose of this historical perspective is to describe how a series of seemingly unrelated findings in the late 1980s and early 1990s crystallized in the discovery of iNKT cells. The story is told from a personal viewpoint, with a particular effort to place both breakthroughs and misinterpretations in the context of their era.

Integrating receptor interactions and dynamics and Interference with endocrine disruptors in the mechanisms of action of PPAR nuclear receptors

Abstract Peroxisome Proliferator-Activated Receptors (PPARs) form a family of three nuclear receptors regulating important cellular and metabolic functions. PPARs control gene expression by directly binding to target promoters as heterodimers with the Retinoid X Receptor (RXR), and their transcriptional activity is enhanced upon activation by natural or pharmacological ligands. The binding of PPAR/RXR heterodimers on target promoters allows the anchoring of a series of coactivators and corepressors involved in promoter remodeling and the recruitment of the transcription machinery. The transcriptional output finally depends on a complex interplay between (i) the respective expression levels of PPARs, RXRs and of other nuclear receptors competing for DNA binding and RXR recruitment, (ii) the availability and the nature of PPAR and RXR ligands, (iii) the expression levels and the nature of the different coactivators and corepressors and (iv) the sequence and the epigenetic status of the promoter. Understanding how all these factors and signals integrate and fine-tune transcription remains a challenge but is necessary to understand the specificity of the physiological functions regulated by PPARs. The work presented herein focuses on the molecular mechanisms of PPAR action and aims at understanding how the interactions and mobility of the receptor modulate transcription in the physiological context of a living cell: Such observations in vivo rely on the use of engineered fluorescent protein chimeras and require the development and the application of complementary imaging techniques such as Fluorescence Recovery After Photobleaching (FRAP), Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS). Using such techniques, PPARs are shown to reside solely in the nucleus where they are constitutively associated with RXR but transcriptional activation by ligand binding -does not promote the formation of sub-nuclear structures as observed with other nuclear receptors. In addition, the engagement of unliganded PPARs in large complexes of cofactors in living cells provides a molecular basis for their ligand-independent activity. Ligand binding reduces receptor diffusion by promoting the recruitment of coactivators which further enlarge the size of PPAR complexes to acquire full transcriptional competence. Using these molecular approaches, we deciphered the molecular mechanisms through which phthalates, a class of pollutants from the plastic industry, interfere with PPARγ signaling. Mono-ethyl-hexyl-phthalate (MEHP) binding induces the recruitment of a specific subset of cofactors and translates into the expression of a specific subset of target genes, the transcriptional output being strongly conditioned by the differentiation status of the cell. This selective PPARγ modulation induces limited adipogenic effects in cellular models while exposure to phthalates in animal models leads to protective effects on glucose tolerance and diet-induced obesity. These results demonstrate that phthalates influence lipid and carbohydrate metabolism through complex mechanisms which most likely involve PPARγ but also probably PPARα and PPARß, Altogether, the molecular and physiological demonstration of the interference of pollutants with PPAR action outlines an important role of chemical exposure in metabolic regulations. Résumé Les PPARs (Peroxisome Proliferator-Activated Receptors) forment une famille de récepteurs nucléaires qui régulent des fonctions cellulaires et métaboliques importantes. Les PPARs contrôlent l'expression des gènes en se liant directement à leurs promoteurs sous forme d'hétérodimères avec les récepteurs RXR (Retinoid X Receptor), et leur activité transcriptionnelle est stimulée par la liaison de ligands naturels ou pharmacologiques. L'association des hétérodimères PPAR/RXR avec les promoteurs des gènes cibles permet le recrutement de coactivateurs et de corépresseurs qui vont permettre le remodelage de la chromatine et le recrutement de la machinerie transcriptionnelle. Les actions transcriptionnelles du récepteur dépendent toutefois d'interactions complexes qui sont régulées par (i) le niveau d'expression des PPARs, des RXRs et d'autres récepteurs nucléaires entrant en compétition pour la liaison à l'ADN et l'association avec RXR, (ii) la disponibilité et la nature de ligands de PPAR et de RXR, (iii) les niveaux d'expression et la nature des différents coactivateurs et corépresseurs et (iv) la séquence et le marquage épigénétique des promoteurs. La compréhension des mécanismes qui permettent d'intégrer ces aspects pour assurer une régulation fine de l'activité transcriptionnelle est un défi qu'il est nécessaire de relever pour comprendre la spécificité des fonctions physiologiques régulées par les PPARs. Ce travail concerne l'étude des mécanismes d'action moléculaire des PPARs et vise à mieux comprendre comment les interactions du récepteur avec d'autres protéines ainsi que la mobilité de ce dernier régulent son activité transcriptionnelle dans le contexte physiologique des cellules vivantes. De telles observations reposent sur l'emploi de protéines fusionnées à des protéines fluorescentes ainsi que sur le développement et l'utilisation de techniques d'imagerie complémentaires telles que le FRAP (Fluorescence Recovery After Photobleaching), le FRET (Fluorescence Resonance Energy Transfer) ou la FCS (Fluorescence Corrélation Spectroscopy). En appliquant ces méthodes, nous avons pu montrer que les PPARs résident toujours dans le noyau où ils sont associés de manière constitutive à RXR, mais que l'ajout de ligand n'induit pas la formation de structures sub-nucléaires comme cela a pu être décrit pour d'autres récepteurs nucléaires. De plus, les PPARs sont engagés dans de larges complexes protéiques de cofacteurs en absence de ligand, ce qui procure une explication moléculaire à leur activité ligand-indépendante. La liaison du ligand réduit la vitesse de diffusion du récepteur en induisant le recrutement de coactivateurs qui augmente encore plus la taille des complexes afin d'acquérir un potentiel d'activation maximal. En utilisant ces approches moléculaires, nous avons pu caractériser les mécanismes permettant aux phtalates, une classe de polluants provenant de l'industrie plastique, d'interférer avec PPARγ. La liaison du mono-ethyl-hexyl-phtalate (NERF) à PPARγ induit un recrutement sélectif de cofacteurs, se traduisant par l'induction spécifique d'un sous-ensemble de gènes qui varie en fonction du niveau de différentiation cellulaire. La modulation sélective de PPARγ par le MEHP provoque une adipogenèse modérée dans des modèles cellulaires alors que l'exposition de modèles animaux aux phtalates induit des effets bénéfiques sur la tolérance au glucose et sur le développement de l'obésité. Toutefois, les phtalates ont une action complexe sur le métabolisme glucido-lipidique en faisant intervenir PPARγ mais aussi probablement PPARα et PPARß. Cette démonstration moléculaire et physiologique de l'interférence des polluants avec les récepteurs nucléaires PPAR souligne un rôle important de l'exposition à de tels composés dans les régulations métaboliques.

Peroxisome proliferator-activated receptors (PPARs) in skin health, repair and disease.

Peroxisome proliferator-activated receptors, PPARalpha, PPARbeta/delta and PPARgamma, are fatty acid activated transcription factors that belong to the nuclear hormone receptor family. While they are best known as transcriptional regulators of lipid and glucose metabolism, evidence has also accumulated for their importance in skin homeostasis. The three PPAR isotypes are expressed in rodent and human skin. Various cell culture and in vivo approaches suggest that PPARalpha contributes to fetal skin development, to epidermal barrier maturation and to sebocyte activity. PPARbeta/delta regulates sebocyte differentiation, promotes hair follicle growth and has pro-differentiating effects in keratinocytes in normal and inflammatory conditions. In contrast, the role of PPARgamma appears to be rather minor in keratinocytes, whereas its activity is required for sebaceous gland differentiation. Importantly, PPARalpha and beta/delta are instrumental in skin repair after an injury, each of them playing specific roles. Due to their collective diverse functions in skin biology, PPARs represent a major research target for the understanding and treatment of many skin diseases, such as benign epidermal tumors, papillomas, acne vulgaris and psoriasis.

The N-terminal DNA-binding 'zinc finger' of the oestrogen and glucocorticoid receptors determines target gene specificity.

Steroid hormone receptors activate specific gene transcription by binding as hormone-receptor complexes to short DNA enhancer-like elements termed hormone response elements (HREs). We have shown previously that a highly conserved 66 amino acid region of the oestrogen (ER) and glucocorticoid (GR) receptors, which corresponds to part of the receptor DNA binding domain (region C) is responsible for determining the specificity of target gene activation. This region contains two sub-regions (CI and CII) analogous to the 'zinc-fingers' of the transcription factor TFIIIA. We show here that CI and CII appear to be separate domains both involved in DNA binding. Furthermore, using chimaeric ERs in which either the first (N-terminal) (CI) or second (CII) 'zinc finger' region has been exchanged with that of the GR, indicates that it is the first 'zinc finger' which largely determines target gene specificity. We suggest that receptor recognition of the HRE is analogous to that of the helix-turn-helix DNA binding motif in that the receptor binds to DNA as a dimer with the first 'zinc finger' lying in the major groove recognizing one half of the palindromic HRE, and that protein-DNA interaction is stabilized through non-specific DNA binding and dimer interactions contributed by the second 'zinc finger'.

High level expression of human neuropeptide Y receptors in mammalian cells infected with a recombinant vaccinia virus.

Neuropeptide Y (NPY) is a 36 amino acid peptide present in the central and peripheral nervous system. Numerous studies point to a role of NPY in cardiovascular regulation. NPY effects are mediated through stimulation of specific cell surface G protein-coupled receptors. To allow biochemical studies of the receptor and of its interaction with the ligand, we have developed a potent expression system for NPY receptors using a recombinant vaccinia virus. A human NPY receptor cDNA was fused to a strong vaccinia virus promoter and inserted into the viral genome by homologous recombination. Recombinant viruses were isolated and tested for their ability to induce NPY binding site expression following infection of mammalian cell lines. Using saturation and competition binding experiments we measured a Bmax of 5-10 x 10(6) NPY binding sites per cell. The Kd for the binding of NPY is about 20 nM. Labelling of infected cells with a fluorochrome-labelled NPY indicated that the recombinant protein integrates into the cell membrane.

No implication of thromboxane prostanoid receptors in reactive hyperemia of skin and skeletal muscle in human forearm.

There is evidence that reactive hyperemia (ie, the transient increase of blood flow above resting level after a short period of ischemia) could be negatively modulated by vasoconstrictor prostanoids. The present study tested whether pharmacological blockade of the thromboxane prostanoid receptors with the specific antagonist S18886 (terutroban) would amplify reactive hyperemia in human skin and skeletal muscle. Twenty healthy young male volunteers were enrolled in a randomized, blinded, crossover trial of oral S18886 30 mg/d for 5 days versus placebo. Reactive hyperemia was evaluated in forearm skin and skeletal muscle, after occlusion of the brachial artery with a pneumatic cuff inflated at suprasystolic pressure. Blood flow was measured with laser Doppler imaging (skin) and strain gauge venous occlusion plethysmography (muscle). On the first and last day of each treatment period, recordings of reactive hyperemia were obtained immediately before and 2 hours after drug intake. Whether in forearm muscle or skin, S18886 had no discernible effect on peak postocclusion blood flow, nor on the global hyperemic response as quantified by the area under curve. These results do not support that thromboxane prostanoid receptor activation could exert a moderating influence on reactive hyperemia in human skin and skeletal muscle, at least in young subjects.

Molecular and physiological characterisation of lonotropic receptors in the sacculus in drosophila

Chemosensation is the detection of chemical signals in the environment that enable an animal to make informed decisions about food choice, mate preference or predator detection. Dissecting the molecular and neural mechanisms by which animals detect chemical cues is an important goal towards understanding how they interact with the environment. An attractive system to dissect the mechanisms of chemosensation is the olfactory system. One of the most-investigated olfactory systems is that of Drosophila melanogaster, a model organism that is amenable to a powerful combination of genetic and physiological analyses. Embedded within the antennal olfactory organ of Drosophila is an unusual sensory structure called the sacculus. The sacculus is comprised of three distinct chambers, each lined with several sensilla housing two to three neurons. Previous morphological, anatomical and surgical studies of sacculus neurons have implicated sacculus neurons in chemosensation, hygrosensation and/or thermosensation. While a subset of sacculus neurons have been physiologically characterised as temperature sensors, the role of this organ has remained largely mysterious, due to its inaccessibility to peripheral electrophysiological analysis. Recently a new family of olfactory receptors, the lonotropic Receptors (IRs), was identified. Five IRs are expressed in sacculus neurons providing the first selective molecular markers for these cells. In this thesis I describe the molecular, physiological and anatomical characterisation of these neurons. Genetic labelling of specific populations of sacculus neurons with anatomical (CD8:GFP) reporters has identified neurons in sacculus chambers I and II express IR40a+IR93a together with their co- receptor IR25a, while neurons in chamber III express IR64a with its co-receptor IR8a. Both these sets of neurons project to two distinct glomeruli in the antennal lobe; IR40a neurons project to the column and arm, IR64a neurons project to DC4 and DP1m. Through a live optical imaging screen I showed that these neurons are indeed olfactory and IR64a neurons recognise acidic ligands, while IR40a neurons recognise amine ligands. IR40a and IR64a neurons are in fact composed of anatomically and physiologically distinct subpopulations, strongly implying the existence of other factors that define their functional properties. My thesis identifies the sacculus as a specialised olfactory organ capable of detecting acids and bases, which are of widespread importance to insects. The data from my thesis along with data from other labs show the sacculus is composed of different populations of olfactory sensory neurons and thermosensory neurons. Comparative genomic analysis of sacculus IRs across insects reveals them to be among the most conserved of this receptor repertoire, suggesting that the sacculus represents an evolutionarily ancient insect olfactory acid-base sensor. - La détection des produits chimiques se trouvant dans l'environnement (perception chimiosensorielle) permet à un animal de choisir sa nourriture, son partenaire ou encore d'identifier ses prédateurs. Décortiquer les mécanismes moléculaires et neuronaux grâce auxquels les animaux détectent ces signaux chimiques permet de comprendre comment ces animaux interagissent avec leur environnement. Un système intéressant pour décortiquer ces mécanismes de perception chimiosensorielle est le système olfactif, de la drosophile (Drosophila melanogaster), aussi appelée mouche du vinaigre. C'est un animal modèle très utile grâce à la combinaison d'outils génétiques puissants et d'analyses physiologiques facilement réalisables. Dans l'antenne de la drosophile, qui est l'organe olfactif principal de cet animal, se trouve une structure appelée sacculus. Celui-ci est composé de trois chambres distinctes, chacune comprenant plusieurs sensilles à l'intérieur desquelles se trouvent deux à trois neurones. De précédentes études morphologiques et anatomiques des ces neurones ont déterminé qu'ils sont impliqués dans la perception des odeurs, de l'humidité et de la température. Malgré ceci, la fonction principale de cet organe reste largement inconnue, principalement car il est inaccessible aux analyses électrophysiologiques. Récemment, une nouvelle famille de soixante-six récepteurs olfactifs, nommés Récepteurs lonotropiques (IRs), a été découverte chez la drosophile. Cinq IRs sont exprimés dans les neurones du sacculus. Pour la première fois, une sélection de marqueurs moléculaires est disponible pour l'étude de ces cellules. Dans cette thèse, les caractéristiques moléculaires, physiologiques et anatomiques des neurones du sacculus sont décrites. Ces populations de neurones situés dans le sacculus ont été marquées avec des gènes rapporteurs (CD8:GFP). Ceci a montré que les récepteurs IR40a et IR93a sont exprimés ensemble avec le co-récepteur IR25a dans les chambres I et II, tandis que les neurones de la chambre III expriment IR64a avec son co-récepteur IR8a. Ces deux groupes de neurones projettent vers deux glomérules distincts du lobe antennaire : les neurones IR40a projettent vers la column et le arm, alors que les neurones IR64a projettent vers DC4 et DP1m. Un screen d'imagerie optique a démontré que ces neurones sont en effet des neurones olfactifs, et que les neurones IR64a reconnaissent des ligands acides, tandis que les neurones IR40a reconnaissent des ligands aminés. De plus, les neurones IR40a et IR64a sont séparés en sous-populations distinctes anatomiquement et physiologiquement, et d'autres facteurs permettant de définir leurs propriétés fonctionnelles sont probablement impliqués. Cette thèse identifie ainsi le sacculus comme un organe olfactif spécialisé capable de détecter des acides et amines, lesquels sont très importants pour les insectes. Toutes les données collectées durant cette thèse, combinées aux données d'autres laboratoires, montrent que le sacculus est composé de différentes populations de neurones olfactifs et thermosenseurs. Ces IRs sont très conservés parmi les insectes, suggérant que le sacculus représente révolution d'un ancien détecteur olfactif d'acides et de bases chez l'insecte. - Tous les animaux sont capables de percevoir les signaux chimiques dans leur environnement, comme les odeurs ou le goût, via différents organes. L'odorat est le sens qui permet de percevoir les odeurs, et il est implique des neurones olfactifs qui se trouvent dans le nez des mammifères ou les antennes des insectes. La capacité d'un neurone olfactif à détecter une molécule odorante dépend des types de récepteurs olfactifs qu'il exprime. Il existe deux grandes familles de récepteurs qui perçoivent les odeurs : les Récepteurs Olfactifs, ORs, et Récepteurs lonotropiques IRs, qui détectent différents types d'odeurs avec différents mécanismes. Lorsqu'un récepteur reconnaît une molécule odorante, il convertit ce signal en un signal électrique qui est ensuite transmis au centre olfactif dans le cerveau. La drosophile (Drosophila melanogaster), aussi appelée mouche du vinaigre, est utilisée comme animal modèle pour étudier l'odorat, parce que son génome entier a été séquencé et que ses gènes sont facilement manipulables. De plus, l'anatomie du système olfactif de la mouche est similaire à celui des mammifères, malgré qu'il possède moins de neurones, ce qui le rend moins complexe. Ma thèse a pour objectif d'étudier les Récepteurs lonotropiques dans un organe spécifique, appelé le sacculus, situé dans les antennes. Les neurones du sacculus exprimant des IRs envoient leurs projections au centre olfactif du cerveau, suggérant que ces neurones perçoivent les odeurs. Une technique d'imagerie optique a été utilisée sur le cerveau de mouches vivantes afin de mesurer la réponse des neurones du le sacculus à différentes odeurs. J'ai démontré que ces récepteurs détectent des acides et des amines, qui sont très importants pour les insectes. Par exemple, les acides se retrouvent dans les fruits mûrs sur lesquels les mouches vont se nourrir, s'accoupler et poser leurs oeufs, et les amines sont souvent produites par des bactéries pouvant être nuisible pour la mouche. La principale découverte de ma thèse est donc l'identification du sacculus comme un organe capable de détecter deux des principales odeurs importantes pour la mouche. Ces récepteurs sont aussi présents dans d'autres insectes où ils jouent peut-être des rôles différents. Les acides et les amines se retrouvent aussi dans les excrétions (comme la sueur ou l'urine) de beaucoup de mammifères, qui pourraient potentiellement être dangereux pour la mouche, mais qui attirent les moustiques se nourrissant de leur sang.

Protein-protein interactions at the adrenergic receptors.

The adrenergic receptors are among the best characterized G protein-coupled receptors (GPCRs) and knowledge on this receptor family has provided several important paradigms about GPCR function and regulation. One of the most recent paradigms initially supported by studies on adrenergic receptors is that both βarrestins and G proteincoupled receptors themselves can act as scaffolds binding a variety of proteins and this can result in growing complexity of the receptor-mediated cellular effects. In this review we will briefly summarize the main features of βarrestin binding to the adrenergic receptor subtypes and we will review more in detail the main proteins found to selectively interact with distinct AR subtype. At the end, we will review the main findings on oligomerization of the AR subtypes.

The efficiency of antigen recognition by CD8+ CTL clones is determined by the frequency of serial TCR engagement.

Using H-2Kd-restricted CTL clones, which are specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS(252-260) (SYIPSAEKI) and permit assessment of TCR-ligand interactions by TCR photoaffinity labeling, we have previously identified several peptide derivative variants for which TCR-ligand binding and the efficiency of Ag recognition deviated by fivefold or more. Here we report that the functional CTL response (cytotoxicity and IFN-gamma production) correlated with the rate of TCR-ligand complex dissociation, but not the avidity of TCR-ligand binding. While peptide antagonists exhibited very rapid TCR-ligand complex dissociation, slightly slower dissociation was observed for strong agonists. Conversely and surprisingly, weak agonists typically displayed slower dissociation than the wild-type agonists. Acceleration of TCR-ligand complex dissociation by blocking CD8 participation in TCR-ligand binding increased the efficiency of Ag recognition in cases where dissociation was slow. In addition, permanent TCR engagement by TCR-ligand photocross-linking completely abolished sustained intracellular calcium mobilization, which is required for T cell activation. These results indicate that the functional CTL response depends on the frequency of serial TCR engagement, which, in turn, is determined by the rate of TCR-ligand complex dissociation.

Distinct conformations of Ly49 natural killer cell receptors mediate MHC class I recognition in trans and cis.

Certain cell-surface receptors engage ligands expressed on juxtaposed cells and ligands on the same cell. The structural basis for trans versus cis binding is not known. Here, we showed that Ly49 natural killer (NK) cell receptors bound two MHC class I (MHC-I) molecules in trans when the two ligand-binding domains were backfolded onto the long stalk region. In contrast, dissociation of the ligand-binding domains from the stalk and their reorientation relative to the NK cell membrane allowed monovalent binding of MHC-I in cis. The distinct conformations (backfolded and extended) define the structural basis for cis-trans binding by Ly49 receptors and explain the divergent functional consequences of cis versus trans interactions. Further analyses identified specific stalk segments that were not required for MHC-I binding in trans but were essential for inhibitory receptor function. These data identify multiple distinct roles of stalk regions for receptor function.