A new infectious mammary tumor virus in the milk of mice implanted with C4 hyperplastic alveolar nodules.

We have previously characterized an infectious mouse mammary tumor virus [(MMTV(SW)] which induces a strong superantigen response in vivo. Here we describe the isolation and characterization of MMTV(C4) which was derived from milk of mice implanted with hyperplastic alveolar nodules. MMTV(C4) stimulates V beta 2 expressing T cells after local injection in vivo. Comparison with known open reading frame (orf) sequences revealed high homology to Mtv-6, an endogenous virus interacting with V beta 3-expressing T cells. The carboxyl-terminal amino acids were, however, altered. High homology including the carboxyl-terminal orf amino acids were found with MMTV(C3H-K). We show here that MMTV(C3H-K) has lost its superantigen function. Sequence comparisons permitted the characterization of few key amino acids which could be important for T cell receptor interaction and superantigen processing.

Rôle des molécules de co-stimulations et de CD93 dans la différenciation en plasmocytes

Le développement des cellules B est constitué d'une première phase qui se déroule dans la moelle en absence d'antigène et d'une deuxième phase qui se déroule dans les organes lymphoïdes secondaires et qui débute uniquement en présence d'antigène. Cette deuxième partie est extrêmement importante et doit être très bien régulée pour lutter efficacement contre les pathogènes, ainsi que pour éviter de nombreuses maladies de type auto-immunes. Ce travail est basé à l'origine sur l'étude de souris mutantes dans lesquelles une protéine des cellules T est modifiée, impliquant une très forte activation des cellules B en absence d'antigène et de manière non spécifique. Ces souris constituent donc un outil de travail très intéressant pour étudier tout d'abord le mécanisme aboutissant à l'activation des cellules B dans ce contexte particulier. De plus comme ces souris contiennent énormément de cellules sécrétant des anticorps, à savoir les plasmocytes, il est facile d'étudier leur phénotype. Cela nous a permis de démontrer qu'un récepteur membranaire, CD93 est exprimé à leur surface. Cette observation a ensuite été confirmée dans des souris normales, de type sauvage. L'utilisation de ce marqueur de surface nous a permis de caractériser plus en détail les étapes du développement des plasmocytes. De plus nous avons tenté de trouver la fonction jouée par cette molécule à la surface de ces cellules, en utilisant des souris dans lesquelles ce récepteur a été supprimé. Si les premières étapes de l'activation des cellules B étaient normales, ces souris n'étaient par contre pas capables de produire des anticorps à long-terme dans le sang. Nous avons pu montrer que la survie des plasmocytes en l'absence de CD93 est moins efficace dans la moelle, probablement du au fait qu'en absence de cette molécule, les plasmocytes ont plus de difficultés à adhérer dans ce que l'on appelle des niches de survie. Nous avons essayé ensuite de déterminer si CD93 peut être utilisé comme cible thérapeutique dans le cadre de maladies auto-immunes ou de lymphomes. Bien que CD93 soit exprimé à la surface des cellules d'intérêt dans les souris souffrant de lupus, il n'a pas été possible de les éliminer avec un anticorps dirigé contre CD93. De plus nous n'avons pas pu mettre en évidence l'expression de CD93 à la surface des plasmocytes humains induits in vitro. SUMMARY : Antigen dependent B cell activation is a key aspect of the adaptive immunity which is involved in the efficient response against pathogens, but also in vaccination and in numerous pathologies. The aim of this project was to investigate two key aspects of the late B cell development, namely the role of costimulatory molecules in the immunological synapse between T and B cells and the characterization of a new plasma cell marker, CD93. This work was initially based on the study of the LatY136F mutant mouse. The latter harbors a point mutation in the LAT adaptor protein which is involved in T cell receptor signaling. As a consequence of this mutation, CD4 T cells in the periphery expand strongly and are polarized in a TH2 manner leading to a normal but exaggerated B cell response. For this reason, these mice provide a useful tool to investigate different aspects of the late B cell development. The first part of the project was focused on the role played by costimulatory molecules in LotY136F CD4 T cell mediated B cell activation. In vitro studies showed that CD80/CD86, IL-4 and LFA-1 were required for LatY136FT cells to activate B cells whereas CD40 and IcosL were not necessary. In vivo we showed that CD80/CD86 was required for initial T cell expansion whereas CD40 and IcosL deficiency led to a less efficient B cell activation. The large amount of plasma cells present in LatY136F mice allows investigating in more details their phenotype and CD93 was found to be expressed on their surface, This observation was confirmed in wild type B cells activated either in vivo or in vitro with T-independent or T-dependent antigens. Moreover we found that CD93 expression can occur either before CD138/Blimp-1 induction or after, showing that two independent pathways can lead to the formation of CD93/CD138 double positive population, which was shown to be the more mature. Indeed, their phenotype correlated with modified transcriptional network, high isotype switched antibody secretion and cell cycle arrest. Analysis of CD93 deficient mice demonstrated that the initial B cell activation after immunization was normal, but also showed that these mice failed to maintain a high antibody secretion level at later time points both after primary and boost immunization. This was shown to be due to a less efficient survival of the long-lived plasma cells in the bone marrow niches, most likely related with a defective adhesion process in absence of CD93. We investigated the possibility to use CD93 as a target to treat plasma cell pathologies, but even if this molecule is expressed on cells of interest in the bone marrow of lupus mice, it was not possible to deplete them using anti-CD93 antibodies. Moreover we were not able to show its expression on the surface of in vitro activated B cells and multiple myeloma cell lines of human origin. In conclusion, our data helped understand both the mechanisms leading to the polyclonal B cell activation occurring in the LatY136F KI mouse and the role played by CD93 on the surface of plasma cells, which could potentially open the way to therapeutic application.

Role of CD8 in aberrant function of cytotoxic T lymphocytes.

Using H-2Kd-restricted photoprobe-specific cytotoxic T lymphocyte (CTL) clones, which permit assessment of T cell receptor (TCR)-ligand interactions by TCR photoaffinity labeling, we observed that the efficiency of antigen recognition by CTL was critically dependent on the half-life of TCR-ligand complexes. We show here that antigen recognition by CTL is essentially determined by the frequency of serial TCR engagement, except for very rapid dissociations, which resulted in aberrant TCR signaling and antagonism. Thus agonists that were efficiently recognized exhibited rapid TCR-ligand complex dissociation, and hence a high frequency of serial TCR engagement, whereas the opposite was true for weak agonists. Surprisingly, these differences were largely accounted for by the coreceptor CD8. While it was known that CD8 substantially decreases TCR-ligand complex dissociation, we observed in this study that this effect varied considerably among ligand variants, indicating that epitope modifications can alter the CD8 contribution to TCR-ligand binding, and hence the efficiency of antigen recognition by CTL.

Transformed and nontransformed human T lymphocytes migrate to skin in a chimeric human skin/SCID mouse model.

To study human T cell migration to human skin in vivo, we grafted severe combined immunodeficient mice with 500-microm thick human skin. Two weeks after grafting, epidermal and dermal structures in the grafts were of human origin. When we intraperitoneally injected grafted mice with clones of the human HUT-78 T cell line derived from a patient with cutaneous T cell lymphoma and Sézary syndrome, we detected in the grafts the rare Vbeta23-Jbeta1.2 T cell receptor transcripts characteristic for the HUT-78 clones. These signals were found 2-6 d after cell injection in about 40% of the grafted and HUT-78 cell injected mice but not in grafts from mice that received no exogenous T cells. In contrast to HUT-78 cells, which only accumulate in low number, grafts topically challenged with nickel sufate in vaseline from mice that were injected with autologous nickel-reactive T cell lines led to massive accumulation of T cells within 3 d. Only scattered T cells accumulated in the skin when grafted mice received vaseline plus T cells, nickel sulfate alone, T cells alone, or nickel sulfate plus an allogeneic nickel-nonreactive T cell clone. When the T cell lines were labeled with the fluorochrome PKH-26 before cell injection, spots of fluorescent label in the size and shape of cells were found in the grafts challenged with nickel. Together, these results clearly demonstrate that human T cells can migrate to human skin in this chimeric human/mouse model.

Ly49E-dependent inhibition of natural killer cells by urokinase plasminogen activator.

The Ly49 natural killer (NK)-cell receptor family comprises both activating and inhibitory members, which recognize major histocompatibility complex (MHC) class I or MHC class I-related molecules and are involved in target recognition. As previously shown, the Ly49E receptor fails to bind to a variety of soluble or cell-bound MHC class I molecules, indicating that its ligand is not an MHC class I molecule. Using BWZ.36 reporter cells, we demonstrate triggering of Ly49E by the completely distinct, non-MHC-related protein urokinase plasminogen activator (uPA). uPA is known to be secreted by a variety of cells, including epithelial and hematopoietic cells, and levels are up-regulated during tissue remodeling, infections, and tumorigenesis. Here we show that addition of uPA to Ly49E-positive adult and fetal NK cells inhibits interferon-gamma secretion and reduces their cytotoxic potential, respectively. These uPA-mediated effects are Ly49E-dependent, as they are reversed by addition of anti-Ly49E monoclonal antibody and by down-regulation of Ly49E expression using RNA interference. Our results suggest that uPA, besides its established role in fibrinolysis, tissue remodeling, and tumor metastasis, could be involved in NK cell-mediated immune surveillance and tumor escape.

Molecular modeling of peptide-MHC class I complexes : applications to peptide based immunotherapy

Summary The specific CD8+ T cell immune response against tumors relies on the recognition by the T cell receptor (TCR) on cytotoxic T lymphocytes (CTL) of antigenic peptides bound to the class I major histocompatibility complex (MHC) molecule. Such tumor associated antigenic peptides are the focus of tumor immunotherapy with peptide vaccines. The strategy for obtaining an improved immune response often involves the design of modified tumor associated antigenic peptides. Such modifications aim at creating higher affinity and/or degradation resistant peptides and require precise structures of the peptide-MHC class I complex. In addition, the modified peptide must be cross-recognized by CTLs specific for the parental peptide, i.e. preserve the structure of the epitope. Detailed structural information on the modified peptide in complex with MHC is necessary for such predictions. In this thesis, the main focus is the development of theoretical in silico methods for prediction of both structure and cross-reactivity of peptide-MHC class I complexes. Applications of these methods in the context of immunotherapy are also presented. First, a theoretical method for structure prediction of peptide-MHC class I complexes is developed and validated. The approach is based on a molecular dynamics protocol to sample the conformational space of the peptide in its MHC environment. The sampled conformers are evaluated using conformational free energy calculations. The method, which is evaluated for its ability to reproduce 41 X-ray crystallographic structures of different peptide-MHC class I complexes, shows an overall prediction success of 83%. Importantly, in the clinically highly relevant subset of peptide-HLAA*0201 complexes, the prediction success is 100%. Based on these structure predictions, a theoretical approach for prediction of cross-reactivity is developed and validated. This method involves the generation of quantitative structure-activity relationships using three-dimensional molecular descriptors and a genetic neural network. The generated relationships are highly predictive as proved by high cross-validated correlation coefficients (0.78-0.79). Together, the here developed theoretical methods open the door for efficient rational design of improved peptides to be used in immunotherapy. Résumé La réponse immunitaire spécifique contre des tumeurs dépend de la reconnaissance par les récepteurs des cellules T CD8+ de peptides antigéniques présentés par les complexes majeurs d'histocompatibilité (CMH) de classe I. Ces peptides sont utilisés comme cible dans l'immunothérapie par vaccins peptidiques. Afin d'augmenter la réponse immunitaire, les peptides sont modifiés de façon à améliorer l'affinité et/ou la résistance à la dégradation. Ceci nécessite de connaître la structure tridimensionnelle des complexes peptide-CMH. De plus, les peptides modifiés doivent être reconnus par des cellules T spécifiques du peptide natif. La structure de l'épitope doit donc être préservée et des structures détaillées des complexes peptide-CMH sont nécessaires. Dans cette thèse, le thème central est le développement des méthodes computationnelles de prédiction des structures des complexes peptide-CMH classe I et de la reconnaissance croisée. Des applications de ces méthodes de prédiction à l'immunothérapie sont également présentées. Premièrement, une méthode théorique de prédiction des structures des complexes peptide-CMH classe I est développée et validée. Cette méthode est basée sur un échantillonnage de l'espace conformationnel du peptide dans le contexte du récepteur CMH classe I par dynamique moléculaire. Les conformations sont évaluées par leurs énergies libres conformationnelles. La méthode est validée par sa capacité à reproduire 41 structures des complexes peptide-CMH classe I obtenues par cristallographie aux rayons X. Le succès prédictif général est de 83%. Pour le sous-groupe HLA-A*0201 de complexes de grande importance pour l'immunothérapie, ce succès est de 100%. Deuxièmement, à partir de ces structures prédites in silico, une méthode théorique de prédiction de la reconnaissance croisée est développée et validée. Celle-ci consiste à générer des relations structure-activité quantitatives en utilisant des descripteurs moléculaires tridimensionnels et un réseau de neurones couplé à un algorithme génétique. Les relations générées montrent une capacité de prédiction remarquable avec des valeurs de coefficients de corrélation de validation croisée élevées (0.78-0.79). Les méthodes théoriques développées dans le cadre de cette thèse ouvrent la voie du design de vaccins peptidiques améliorés.

Limited heterogeneity of autoantigens and T cells in autoimmune diseases?

For many induced and spontaneous autoimmune diseases, a predominant role for T cells in the organ-specific destruction process has been shown. In one of the induced models of autoimmunity, experimental allergic encephalomyelitis (EAE), a very small heterogeneity of T-cell receptor (TcR) molecules is expressed by the pathogenic T cells in both rats and mice. Contrary to induced autoimmune diseases, little is known about the autoantigens recognized by these autoimmune T cells and the heterogeneity of their TcR in spontaneous autoimmune diseases. The aim of this work was to establish a system which allows characterization of relevant autoantigens in spontaneous insulin-dependent diabetes mellitus (IDDM) in non-obese diabetic (NOD) mice. A completely different approach was taken to characterize the gene products of the minor lymphocyte stimulatory (Mls) loci. These gene products are responsible for the clonal elimination or the clonal stimulation of T cells expressing particular TcR V beta genes and therefore could be implicated in induction of autoimmune diseases by oligoclonal T-cell populations. The finding that Mls antigens are encoded by retroviral sequences leads to the hypothesis that viruses could be the inducing agents of autoimmune diseases.

Superantigen-induced immune stimulation amplifies mouse mammary tumor virus infection and allows virus transmission.

Endogenous and infectious mouse mammary tumor viruses (MMTVs) encode in their 3' long terminal repeat a protein that exerts superantigen activity; that is, it is able to interact with T cells via the variable domain of the T cell receptor (TCR) beta chain. We show here that transmission of an infectious MMTV is prevented when superantigen-reactive cells are absent through either clonal deletion due to the expression of an endogenous MTV with identical superantigen specificity or exclusion due to expression of a transgenic TCR beta chain that does not interact with the viral superantigen. A strict requirement for superantigen-reactive T cells is also seen for a local immune response following MMTV infection. This immune response locally amplifies the number of MMTV-infected B cells, most likely owing to their clonal expansion. Collectively, our data indicate that a superantigen-induced immune response is critical for the MMTV life cycle.

Distinct sets of alphabeta TCRs confer similar recognition of tumor antigen NY-ESO-1157-165 by interacting with its central Met/Trp residues.

Naturally acquired immune responses against human cancers often include CD8(+) T cells specific for the cancer testis antigen NY-ESO-1. Here, we studied T cell receptor (TCR) primary structure and function of 605 HLA-A*0201/NY-ESO-1(157-165)-specific CD8 T cell clones derived from five melanoma patients. We show that an important proportion of tumor-reactive T cells preferentially use TCR AV3S1/BV8S2 chains, with remarkably conserved CDR3 amino acid motifs and lengths in both chains. All remaining T cell clones belong to two additional sets expressing BV1 or BV13 TCRs, associated with alpha-chains with highly diverse VJ usage, CDR3 amino acid sequence, and length. Yet, all T cell clonotypes recognize tumor antigen with similar functional avidity. Two residues, Met-160 and Trp-161, located in the middle region of the NY-ESO-1(157-165) peptide, are critical for recognition by most of the T cell clonotypes. Collectively, our data show that a large number of alphabeta TCRs, belonging to three distinct sets (AVx/BV1, AV3/BV8, AVx/BV13) bind pMHC with equal antigen sensitivity and recognize the same peptide motif. Finally, this in-depth study of recognition of a self-antigen suggests that in part similar biophysical mechanisms shape TCR repertoires toward foreign and self-antigens.

In vivo T-lymphocyte tolerance in the absence of thymic clonal deletion mediated by hematopoietic cells.

Thymic negative selection renders the developing T-cell repertoire tolerant to self-major histocompatability complex (MHC)/peptide ligands. The major mechanism of induction of self-tolerance is thought to be thymic clonal deletion, ie, the induction of apoptotic cell death in thymocytes expressing a self-reactive T-cell receptor. Consistent with this hypothesis, in mice deficient in thymic clonal deletion mediated by cells of hematopoietic origin, a twofold to threefold increased generation of mature thymocytes has been observed. Here we describe the analysis of the specificity of T lymphocytes developing in the absence of clonal deletion mediated by hematopoietic cells. In vitro, targets expressing syngeneic MHC were readily lysed by activated CD8(+) T cells from deletion-deficient mice. However, proliferative responses of T cells from these mice on activation with syngeneic antigen presenting cells were rather poor. In vivo, deletion-deficient T cells were incapable of induction of lethal graft-versus-host disease in syngeneic hosts. These data indicate that in the absence of thymic deletion mediated by hematopoietic cells functional T-cell tolerance can be induced by nonhematopoietic cells in the thymus. Moreover, our results emphasize the redundancy in thymic negative selection mechanisms.

A V beta 4-specific superantigen encoded by a new exogenous mouse mammary tumor virus.

The superantigen (SAg) expressed by mouse mammary tumor virus (MMTV) has been shown to play an essential role in the course of the viral life cycle. In the present study, we describe a V beta 4-specific SAg encoded by a new exogenous MMTV carried by the SIM mouse strain. This is the first report of a viral or bacterial SAg reacting with mouse V beta 4+ T cells. Injection of MMTV(SIM) into adult BALB/c mice leads to a rapid and strong stimulation of V beta 4+ CD4+ T cells, followed by a slow deletion of these cells. Neonatal exposure to the virus also leads to a progressive deletion of V beta 4+ T cells. In contrast to other strong MMTV SAg, this new SAg requires the presence of major histocompatibility complex class II I-E molecules to be presented efficiently to T cells. Sequence analysis revealed a new predicted amino acid sequence in the C-terminal polymorphic region of this SAg. Furthermore, sequence comparisons to the most closely related SAg with different V beta specificities hint at the specific residues involved in the interaction with the T cell receptor.

Viral superantigen-induced negative selection of TCR transgenic CD4+ CD8+ thymocytes depends on activation, but not proliferation.

T-cell negative selection, a process by which intrathymic immunological tolerance is induced, involves the apoptosis-mediated clonal deletion of potentially autoreactive T cells. Although different experimental approaches suggest that this process is triggered as the result of activation-mediated cell death, the signal transduction pathways underlying this process is not fully understood. In the present report we have used an in vitro system to analyze the cell activation and proliferation requirements for the deletion of viral superantigen (SAg)-reactive Vbeta8.1 T-cell receptor (TCR) transgenic (TG) thymocytes. Our results indicate that in vitro negative selection of viral SAg-reactive CD4+ CD8+ thymocytes is dependent on thymocyte activation but does not require the proliferation of the negatively signaled thymocytes.

Interactions of Ly49 family receptors with MHC class I ligands in trans and cis.

The Ly49A NK cell receptor interacts with MHC class I (MHC-I) molecules on target cells and negatively regulates NK cell-mediated target cell lysis. We have recently shown that the MHC-I ligand-binding capacity of the Ly49A NK cell receptor is controlled by the NK cells' own MHC-I. To see whether this property was unique to Ly49A, we have investigated the binding of soluble MHC-I multimers to the Ly49 family receptors expressed in MHC-I-deficient and -sufficient C57BL/6 mice. In this study, we confirm the binding of classical MHC-I to the inhibitory Ly49A, C and I receptors, and demonstrate that detectable MHC-I binding to MHC-I-deficient NK cells is exclusively mediated by these three receptors. We did not detect significant multimer binding to stably transfected or NK cell-expressed Ly49D, E, F, G, and H receptors. Yet, we identified the more distantly related Ly49B and Ly49Q, which are not expressed by NK cells, as two novel MHC-I receptors in mice. Furthermore, we show using MHC-I-sufficient mice that the NK cells' own MHC-I significantly masks the Ly49A and Ly49C, but not the Ly49I receptor. Nevertheless, Ly49I was partly masked on transfected tumor cells, suggesting that the structure of Ly49I is compatible in principal with cis binding of MHC-I. Finally, masking of Ly49Q by cis MHC-I was minor, whereas masking of Ly49B was not detected. These data significantly extend the MHC-I specificity of Ly49 family receptors and show that the accessibility of most, but not all, MHC-I-binding Ly49 receptors is modulated by the expression of MHC-I in cis.

Superantigens and retroviral infection: insights from mouse mammary tumor virus.

Superantigens induce a vigorous immune response by stimulating T cells that express particular T-cell receptor V beta chains. Mouse mammary tumor virus is a milk-transmitted retrovirus that encodes such a superantigen. Paradoxically, as discussed by Werner Held and colleagues, the strong superantigen-induced immune response permits the survival of the virus via T-cell dependent clonal expansion of infected B cells.

Autoimmunity-Associated LYP-W620 Does Not Impair Thymic Negative Selection of Autoreactive T Cells.

A C1858T (R620W) variation in the PTPN22 gene encoding the tyrosine phosphatase LYP is a major risk factor for human autoimmunity. LYP is a known negative regulator of signaling through the T cell receptor (TCR), and murine Ptpn22 plays a role in thymic selection. However, the mechanism of action of the R620W variant in autoimmunity remains unclear. One model holds that LYP-W620 is a gain-of-function phosphatase that causes alterations in thymic negative selection and/or thymic output of regulatory T cells (Treg) through inhibition of thymic TCR signaling. To test this model, we generated mice in which the human LYP-W620 variant or its phosphatase-inactive mutant are expressed in developing thymocytes under control of the proximal Lck promoter. We found that LYP-W620 expression results in diminished thymocyte TCR signaling, thus modeling a "gain-of-function" of LYP at the signaling level. However, LYP-W620 transgenic mice display no alterations of thymic negative selection and no anomalies in thymic output of CD4(+)Foxp3(+) Treg were detected in these mice. Lck promoter-directed expression of the human transgene also causes no alteration in thymic repertoire or increase in disease severity in a model of rheumatoid arthritis, which depends on skewed thymic selection of CD4(+) T cells. Our data suggest that a gain-of-function of LYP is unlikely to increase risk of autoimmunity through alterations of thymic selection and that LYP likely acts in the periphery perhaps selectively in regulatory T cells or in another cell type to increase risk of autoimmunity.