Precursors of functional MHC class I- or class II-restricted CD8alphaalpha(+) T cells are positively selected in the thymus by agonist self-peptides.

The origin and specificity of alphabeta TCR(+) T cells that express CD8alphaalpha have been controversial issues. Here we provide direct evidence that precursors of functional CD8alphaalpha T cells are positively selected in the thymus in the presence of agonist self-peptides. Like conventional positive selection, this agonist selection process requires functional TCR alpha-CPM, whereas it is independent of CD8beta expression. Furthermore, CD8alphaalpha expression on mature, agonist-selected T cells does not imply selection by MHC class I, and CD8alphaalpha(+) T cells can be either class I or class II restricted. Our data define a distinct agonist-dependent, positive selection process in the thymus, and they suggest a function for CD8alphaalpha distinct from the conventional TCR coreceptor function of CD8alphabeta or CD4.

Molecular modeling study of the binding and signaling properties of the TCRpMHC complex

The activation of the specific immune response against tumor cells is based on the recognition by the CD8+ Cytotoxic Τ Lymphocytes (CTL), of antigenic peptides (p) presented at the surface of the cell by the class I major histocompatibility complex (MHC). The ability of the so-called T-Cell Receptors (TCR) to discriminate between self and non-self peptides constitutes the most important specific control mechanism against infected cells. The TCR/pMHC interaction has been the subject of much attention in cancer therapy since the design of the adoptive transfer approach, in which Τ lymphocytes presenting an interesting response against tumor cells are extracted from the patient, expanded in vitro, and reinfused after immunodepletion, possibly leading to cancer regression. In the last decade, major progress has been achieved by the introduction of engineered lypmhocytes. In the meantime, the understanding of the molecular aspects of the TCRpMHC interaction has become essential to guide in vitro and in vivo studies. In 1996, the determination of the first structure of a TCRpMHC complex by X-ray crystallography revealed the molecular basis of the interaction. Since then, molecular modeling techniques have taken advantage of crystal structures to study the conformational space of the complex, and understand the specificity of the recognition of the pMHC by the TCR. In the meantime, experimental techniques used to determine the sequences of TCR that bind to a pMHC complex have been used intensively, leading to the collection of large repertoires of TCR sequences that are specific for a given pMHC. There is a growing need for computational approaches capable of predicting the molecular interactions that occur upon TCR/pMHC binding without relying on the time consuming resolution of a crystal structure. This work presents new approaches to analyze the molecular principles that govern the recognition of the pMHC by the TCR and the subsequent activation of the T-cell. We first introduce TCRep 3D, a new method to model and study the structural properties of TCR repertoires, based on homology and ab initio modeling. We discuss the methodology in details, and demonstrate that it outperforms state of the art modeling methods in predicting relevant TCR conformations. Two successful applications of TCRep 3D that supported experimental studies on TCR repertoires are presented. Second, we present a rigid body study of TCRpMHC complexes that gives a fair insight on the TCR approach towards pMHC. We show that the binding mode of the TCR is correctly described by long-distance interactions. Finally, the last section is dedicated to a detailed analysis of an experimental hydrogen exchange study, which suggests that some regions of the constant domain of the TCR are subject to conformational changes upon binding to the pMHC. We propose a hypothesis of the structural signaling of TCR molecules leading to the activation of the T-cell. It is based on the analysis of correlated motions in the TCRpMHC structure. - L'activation de la réponse immunitaire spécifique dirigée contre les cellules tumorales est basée sur la reconnaissance par les Lymphocytes Τ Cytotoxiques (CTL), d'un peptide antigénique (p) présenté à la suface de la cellule par le complexe majeur d'histocompatibilité de classe I (MHC). La capacité des récepteurs des lymphocytes (TCR) à distinguer les peptides endogènes des peptides étrangers constitue le mécanisme de contrôle le plus important dirigé contre les cellules infectées. L'interaction entre le TCR et le pMHC est le sujet de beaucoup d'attention dans la thérapie du cancer, depuis la conception de la méthode de transfer adoptif: les lymphocytes capables d'une réponse importante contre les cellules tumorales sont extraits du patient, amplifiés in vitro, et réintroduits après immunosuppression. Il peut en résulter une régression du cancer. Ces dix dernières années, d'importants progrès ont été réalisés grâce à l'introduction de lymphocytes modifiés par génie génétique. En parallèle, la compréhension du TCRpMHC au niveau moléculaire est donc devenue essentielle pour soutenir les études in vitro et in vivo. En 1996, l'obtention de la première structure du complexe TCRpMHC à l'aide de la cristallographie par rayons X a révélé les bases moléculaires de l'interaction. Depuis lors, les techniques de modélisation moléculaire ont exploité les structures expérimentales pour comprendre la spécificité de la reconnaissance du pMHC par le TCR. Dans le même temps, de nouvelles techniques expérimentales permettant de déterminer la séquence de TCR spécifiques envers un pMHC donné, ont été largement exploitées. Ainsi, d'importants répertoires de TCR sont devenus disponibles, et il est plus que jamais nécessaire de développer des approches informatiques capables de prédire les interactions moléculaires qui ont lieu lors de la liaison du TCR au pMHC, et ce sans dépendre systématiquement de la résolution d'une structure cristalline. Ce mémoire présente une nouvelle approche pour analyser les principes moléculaires régissant la reconnaissance du pMHC par le TCR, et l'activation du lymphocyte qui en résulte. Dans un premier temps, nous présentons TCRep 3D, une nouvelle méthode basée sur les modélisations par homologie et ab initio, pour l'étude de propriétés structurales des répertoires de TCR. Le procédé est discuté en détails et comparé à des approches standard. Nous démontrons ainsi que TCRep 3D est le plus performant pour prédire des conformations pertinentes du TCR. Deux applications à des études expérimentales des répertoires TCR sont ensuite présentées. Dans la seconde partie de ce travail nous présentons une étude de complexes TCRpMHC qui donne un aperçu intéressant du mécanisme d'approche du pMHC par le TCR. Finalement, la dernière section se concentre sur l'analyse détaillée d'une étude expérimentale basée sur les échanges deuterium/hydrogène, dont les résultats révèlent que certaines régions clés du domaine constant du TCR sont sujettes à un changement conformationnel lors de la liaison au pMHC. Nous proposons une hypothèse pour la signalisation structurelle des TCR, menant à l'activation du lymphocyte. Celle-ci est basée sur l'analyse des mouvements corrélés observés dans la structure du TCRpMHC.

Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen

Human cancer vaccines are often prepared with altered "analog" or "heteroclitic" antigens that have been optimized for HLA class I binding, resulting in enhanced immunogenicity. Here, we take advantage of CpG oligodeoxynucleotides as powerful vaccine adjuvants and demonstrate the induction of high T cell frequencies in melanoma patients, despite the use of natural (unmodified) tumor antigenic peptide. Compared with vaccination with analog peptide, natural peptide induced T cell frequencies that were approximately twofold lower. However, T cells showed superior tumor reactivity because of (i) increased functional avidity for natural antigen and (ii) enhancement of T cell activation and effector function. Thus, novel vaccine formulations comprising potent immune stimulators may allow to circumvent the need for modified antigens and can induce highly functional T cells with precise antigen specificity

Switch-peptides: From conformational studies to Alzheimer's disease

Studies on designed peptides that exhibit high tendencies for medium-induced conformational transitions have recently attracted much attention because structural changes are considered as molecular key processes in degenerative diseases. The experimental access to these events has been limited so far mainly due to the intrinsic tendency of the involved polypeptides for self-association and aggregation, e.g. amyloid P plaque formation, thought to be at the origin of Alzheimer's disease. We have developed a new concept termed 'switch-peptides' which allows the controlled onset of polypeptide folding and misfolding in vitro and in vivo, starting from a soluble, non-toxic precursor molecule. As a major feature, the folding process is initiated by enzyme-triggered N,O-acyl migrations restoring the native peptide backbone in situ. As the folding is set off in the moment of creating the bioactive molecule ('in statu nascendi', ISN), our concept allows for the first time the investigation of the early steps of protein misfolding as relevant in degenerative diseases, opening new perspectives for the rational design of therapeutically relevant compounds.

Positional scanning-synthetic peptide library-based analysis of self- and pathogen-derived peptide cross-reactivity with tumor-reactive Melan-A-specific CTL.

Synthetic combinatorial peptide libraries in positional scanning format (PS-SCL) have recently emerged as a useful tool for the analysis of T cell recognition. This includes identification of potentially cross-reactive sequences of self or pathogen origin that could be relevant for the understanding of TCR repertoire selection and maintenance, as well as of the cross-reactive potential of Ag-specific immune responses. In this study, we have analyzed the recognition of sequences retrieved by using a biometric analysis of the data generated by screening a PS-SCL with a tumor-reactive CTL clone specific for an immunodominant peptide from the melanocyte differentiation and tumor-associated Ag Melan-A. We found that 39% of the retrieved peptides were recognized by the CTL clone used for PS-SCL screening. The proportion of peptides recognized was higher among those with both high predicted affinity for the HLA-A2 molecule and high predicted stimulatory score. Interestingly, up to 94% of the retrieved peptides were cross-recognized by other Melan-A-specific CTL. Cross-recognition was at least partially focused, as some peptides were cross-recognized by the majority of CTL. Importantly, stimulation of PBMC from melanoma patients with the most frequently recognized peptides elicited the expansion of heterogeneous CD8(+) T cell populations, one fraction of which cross-recognized Melan-A. Together, these results underline the high predictive value of PS-SCL for the identification of sequences cross-recognized by Ag-specific T cells.

Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a severe and incurable autoimmune disease characterized by chronic activation of plasmacytoid dendritic cells (pDCs) and production of autoantibodies against nuclear self-antigens by hyperreactive B cells. Neutrophils are also implicated in disease pathogenesis; however, the mechanisms involved are unknown. Here, we identified in the sera of SLE patients immunogenic complexes composed of neutrophil-derived antimicrobial peptides and self-DNA. These complexes were produced by activated neutrophils in the form of web-like structures known as neutrophil extracellular traps (NETs) and efficiently triggered innate pDC activation via Toll-like receptor 9 (TLR9). SLE patients were found to develop autoantibodies to both the self-DNA and antimicrobial peptides in NETs, indicating that these complexes could also serve as autoantigens to trigger B cell activation. Circulating neutrophils from SLE patients released more NETs than those from healthy donors; this was further stimulated by the antimicrobial autoantibodies, suggesting a mechanism for the chronic release of immunogenic complexes in SLE. Our data establish a link between neutrophils, pDC activation, and autoimmunity in SLE, providing new potential targets for the treatment of this devastating disease.

Homologous DNA pairing domain peptides of RecA protein: intrinsic propensity to form beta-structures and filaments.

The 20 amino acid residue peptides derived from RecA loop L2 have been shown to be the pairing domain of RecA. The peptides bind to ss- and dsDNA, unstack ssDNA, and pair the ssDNA to its homologous target in a duplex DNA. As shown by circular dichroism, upon binding to DNA the disordered peptides adopt a beta-structure conformation. Here we show that the conformational change of the peptide from random coil to beta-structure is important in binding ss- and dsDNA. The beta-structure in the DNA pairing peptides can be induced by many environmental conditions such as high pH, high concentration, and non-micellar sodium dodecyl sulfate (6 mM). This behavior indicates an intrinsic property of these peptides to form a beta-structure. A beta-structure model for the loop L2 of RecA protein when bound to DNA is thus proposed. The fact that aromatic residues at the central position 203 strongly modulate the peptide binding to DNA and subsequent biochemical activities can be accounted for by the direct effect of the aromatic amino acids on the peptide conformational change. The DNA-pairing domain of RecA visualized by electron microscopy self-assembles into a filamentous structure like RecA. The relevance of such a peptide filamentous structure to the structure of RecA when bound to DNA is discussed.

Jagged1-dependent Notch signaling is dispensable for hematopoietic stem cell self-renewal and differentiation.

Jagged1-mediated Notch signaling has been suggested to be critically involved in hematopoietic stem cell (HSC) self-renewal. Unexpectedly, we report here that inducible Cre-loxP-mediated inactivation of the Jagged1 gene in bone marrow progenitors and/or bone marrow (BM) stromal cells does not impair HSC self-renewal or differentiation in all blood lineages. Mice with simultaneous inactivation of Jagged1 and Notch1 in the BM compartment survived normally following a 5FU-based in vivo challenge. In addition, Notch1-deficient HSCs were able to reconstitute mice with inactivated Jagged1 in the BM stroma even under competitive conditions. In contrast to earlier reports, these data exclude an essential role for Jagged1-mediated Notch signaling during hematopoiesis.

Combination of lentivector immunization and low-dose chemotherapy or PD-1/PD-L1 blocking primes self-reactive T cells and induces anti-tumor immunity.

In the last two decades, anti-cancer vaccines have yielded disappointing clinical results despite the fact that high numbers of self/tumor-specific T cells can be elicited in immunized patients. Understanding the reasons behind this lack of efficacy is critical in order to design better treatment regimes. Recombinant lentivectors (rLVs) have been successfully used to induce antigen-specific T cells to foreign or mutated tumor antigens. Here, we show that rLV expressing a murine nonmutated self/tumor antigen efficiently primes large numbers of self/tumor-specific CD8(+) T cells. In spite of the large number of tumor-specific T cells, however, no anti-tumor activity could be measured in a therapeutic setting, in mice vaccinated with rLV. Accumulating evidence shows that, in the presence of malignancies, inhibition of T-cell activity may predominate overstimulation. Analysis of tumor-infiltrating lymphocytes revealed that specific anti-tumor CD8(+) T cells fail to produce cytokines and express high levels of inhibitory receptors such as programmed death (PD)-1. Association of active immunization with chemotherapy or antibodies that block inhibitory pathways often leads to better anti-tumor effects. We show here that combining rLV vaccination with either cyclophosphamide or PD-1 and PD-L1 blocking antibodies enhances rLV vaccination efficacy and improves anti-tumor immunity.

Cytosolic sensing of extracellular self-DNA transported into monocytes by the antimicrobial peptide LL37.

The intracellular location of nucleic acid sensors prevents recognition of extracellular self-DNA released by dying cells. However, on forming a complex with the endogenous antimicrobial peptide LL37, extracellular DNA is transported into endosomal compartments of plasmacytoid dendritic cells, leading to activation of Toll-like receptor-9 and induction of type I IFNs. Whether LL37 also transports self-DNA into nonplasmacytoid dendritic cells, leading to type I IFN production via other intracellular DNA receptors is unknown. Here we found that LL37 very efficiently transports self-DNA into monocytes, leading the production of type I IFNs in a Toll-like receptor-independent manner. This type I IFN induction was mediated by double-stranded B form DNA, regardless of its sequence, CpG content, or methylation status, and required signaling through the adaptor protein STING and TBK1 kinase, indicating the involvement of cytosolic DNA sensors. Thus, our study identifies a novel link between the antimicrobial peptides and type I IFN responses involving DNA-dependent activation of cytosolic sensors in monocytes.

3D culture of postnatal retinal stem cells using self assembling polymers

PURPOSE We have previously shown that retinal stem cells (RSCs) can be isolated from the radial glia population of the newborn mouse retina (Angénieux et al., 2006). These RSCs have a great capacity to renew and to generate a large number of neurons including cells differentiated towards the photoreceptor lineage (Mehri-Soussi et al., 2006). However, recent published results from our lab revealed that such cells have a poor integration and survival rate after grafting. The uncontrolled environment of a retina seems to prevent good integration and survival after grafting in vivo. To bypass this problem, we are evaluating the possibility of generating in vitro a hemi-retinal tissue before transplantation. METHODS RSC were expanded and cells passaged <10 were seeded in a solution containing poly-ethylene-glycol (PEG) polymer based hydrogels crosslinked with peptides that are chosen to be substrates for matrix metalloproteinases. Various doses of cross linkers peptides allowing connections between PEG polymers were tested. Different growth factors were studied to stimulate cell proliferation and differentiation. RESULTS Cells survived only in the presence of EGF and FGF-2 and generated colonies with a sphere shape. No cells migrated within the gel. To improve the migration and the repartition of the cells in the gels, the integrin ligand RGDSP was added into the gel. In the presence of FGF-2 and EGF, newly formed cell clusters appeared by cell proliferation within several days, but again no outspreading of cells was observed. No difference was even seen when the stiffness of the hydrogels or the concentration of the integrin ligand RGDSP were changed. However, our preliminary results show that RSCs still form spheres when laminin is entrapped in the gel, but they started to spread out having a neuronal morphology after around 2 weeks. The neuronal population was assessed by the presence of the neuronal marker b-tubulin-III. This differentiation was achieved after successive steps of stimulations including FGF-2 and EGF, and then only FGF-2. Glial cells were also present. Further characterizations are under process. CONCLUSIONS RSC can be grown in 3D. Preliminary results show that neuronal cell phenotype acquisition can be instructed by exogenous stimulations and factors linked to the gel. Further developments are necessary to form a homogenous tissue containing retinal cells.

Impact of orthologous melan-A peptide immunizations on the anti-self melan-A/HLA-A2 T cell cross-reactivity.

In HLA-A2 individuals, the CD8 T cell response against the differentiation Ag Melan-A is mainly directed toward the peptide Melan-A26-35. The murine Melan-A24-33 sequence encodes a peptide that is identical with the human Melan-A26-35 decamer, except for a Thr-to-Ile substitution at the penultimate position. Here, we show that the murine Melan-A24-33 is naturally processed and presented by HLA-A2 molecules. Based on these findings, we compared the CD8 T cell response to human and murine Melan-A peptide by immunizing HLA-A2 transgenic mice. Even though the magnitude of the CTL response elicited by the murine Melan-A peptide was lower than the one elicited by the human Melan-A peptide, both populations of CTL recognized the corresponding immunizing peptide with the same functional avidity. Interestingly, CTL specific for the murine Melan-A peptide were completely cross-reactive against the orthologous human peptide, whereas anti-human Melan-A CTL recognized the murine Melan-A peptide with lower avidity. Structurally, this discrepancy could be explained by the fact that Ile32 of murine Melan-A24-33 created a larger TCR contact area than Thr34 of human Melan-A26-35. These data indicate that, even if immunizations with orthologous peptides can induce strong specific T cell responses, the quality of this response against syngeneic targets might be suboptimal due to the structure of the peptide-TCR contact surface.

Melanoma patients respond to a cytotoxic T lymphocyte-defined self-peptide with diverse and nonoverlapping T-cell receptor repertoires.

HLA-A2+ melanoma patients develop naturally a strong CD8+ T cell response to a self-peptide derived from Melan-A. Here, we have used HLA-A2/peptide tetramers to isolate Melan-A-specific T cells from tumor-infiltrated lymph nodes of two HLA-A2+ melanoma patients and analyzed their TCR beta chain V segment and complementarity determining region 3 length and sequence. We found a broad diversity in Melan-A-specific immune T-cell receptor (TCR) repertoires in terms of both TCR beta chain variable gene segment usage and clonal composition. In addition, immune TCR repertoires selected in the patients were not overlapping. In contrast to previously characterized CD8+ T-cell responses to viral infections, this study provides evidence against usage of highly restricted TCR repertoire in the natural response to a self-differentiation tumor antigen.