Profile of a Serial Killer: Cellular and Molecular Approaches to Study Individual Cytotoxic T-Cells following Therapeutic Vaccination

T-cell vaccination may prevent or treat cancer and infectious diseases, but further progress is required to increase clinical efficacy. Step-by-step improvements of T-cell vaccination in phase I/II clinical studies combined with very detailed analysis of T-cell responses at the single cell level are the strategy of choice for the identification of the most promising vaccine candidates for testing in subsequent large-scale phase III clinical trials. Major aims are to fully identify the most efficient T-cells in anticancer therapy, to characterize their TCRs, and to pinpoint the mechanisms of T-cell recruitment and function in well-defined clinical situations. Here we discuss novel strategies for the assessment of human T-cell responses, revealing in part unprecedented insight into T-cell biology and novel structural principles that govern TCR-pMHC recognition. Together, the described approaches advance our knowledge of T-cell mediated-protection from human diseases

Enhanced cytotoxicity and decreased CD8 dependence of human cancer-specific cytotoxic T lymphocytes after vaccination with low peptide dose

In mice, vaccination with high peptide doses generates higher frequencies of specific CD8+ T cells, but with lower avidity compared to vaccination with lower peptide doses. To investigate the impact of peptide dose on CD8+ T cell responses in humans, melanoma patients were vaccinated with 0.1 or 0.5mg Melan-A/MART-1 peptide, mixed with CpG 7909 and Incomplete Freund's adjuvant. Neither the kinetics nor the amplitude of the Melan-A-specific CD8+ T cell responses differed between the two vaccination groups. Also, CD8+ T cell differentiation and cytokine production ex vivo were similar in the two groups. Interestingly, after low peptide dose vaccination, Melan-A-specific CD8+ T cells showed enhanced degranulation upon peptide stimulation, as assessed by CD107a upregulation and perforin release ex vivo. In accordance, CD8+ T cell clones derived from low peptide dose-vaccinated patients showed significantly increased degranulation and stronger cytotoxicity. In parallel, Melan-A-specific CD8+ T cells and clones from low peptide dose-vaccinated patients expressed lower CD8 levels, despite similar or even stronger binding to tetramers. Furthermore, CD8+ T cell clones from low peptide dose-vaccinated patients bound CD8 binding-deficient tetramers more efficiently, suggesting that they may express higher affinity TCRs. We conclude that low peptide dose vaccination generated CD8+ T cell responses with stronger cytotoxicity and lower CD8 dependenc

TCRep 3D: An Automated In Silico Approach to Study the Structural Properties of TCR Repertoires

TCRep 3D is an automated systematic approach for TCR-peptide-MHC class I structure prediction, based on homology and ab initio modeling. It has been considerably generalized from former studies to be applicable to large repertoires of TCR. First, the location of the complementary determining regions of the target sequences are automatically identified by a sequence alignment strategy against a database of TCR Vα and Vβ chains. A structure-based alignment ensures automated identification of CDR3 loops. The CDR are then modeled in the environment of the complex, in an ab initio approach based on a simulated annealing protocol. During this step, dihedral restraints are applied to drive the CDR1 and CDR2 loops towards their canonical conformations, described by Al-Lazikani et. al. We developed a new automated algorithm that determines additional restraints to iteratively converge towards TCR conformations making frequent hydrogen bonds with the pMHC. We demonstrated that our approach outperforms popular scoring methods (Anolea, Dope and Modeller) in predicting relevant CDR conformations. Finally, this modeling approach has been successfully applied to experimentally determined sequences of TCR that recognize the NY-ESO-1 cancer testis antigen. This analysis revealed a mechanism of selection of TCR through the presence of a single conserved amino acid in all CDR3β sequences. The important structural modifications predicted in silico and the associated dramatic loss of experimental binding affinity upon mutation of this amino acid show the good correspondence between the predicted structures and their biological activities. To our knowledge, this is the first systematic approach that was developed for large TCR repertoire structural modeling

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Synthetic CD40L mimetics : biological effects and potential applications in immunotherapy

Le couple CD40/CD40L joue un rôle central dans le système immunitaire. CD40 appartient à la famille des récepteurs au TNF (tumor necrosis factor), et est constitutivement exprimé par les lymphocytes B, les cellules dendritiques et les monocytes/macrophages. Son ligand, CD40L, est un membre de la famille du TNF qui est exprimé transitoirement sur les cellules T activées. Les protéines de la superfamille des TNF-R/TNF s’assemblent selon une symétrie C3, formant des complexes hexavalents importants pour la transduction des signaux intracellulaires. Différents groupes ont utilisé des anticorps dirigés contre CD40 et CD40L à des fins thérapeutiques, dans le but d’inhiber ou d’activer la réponse immunitaire. La recherche de molécules multivalentes synthétiques agissant comme ligands dans des systèmes biologiques complexes représente aujourd’hui un domaine novateur de la chimie médicinale. Nous avons développé des molécules synthétiques, sur la base des données de cristallographie et d’expériences de mutagénèse dirigée, construites sur des plateformes synthétiques de symétrie C3 sur lesquelles, par l'intermédiaire de bras espaceurs de longueur optimale, ont été greffés les résidus de CD40L essentiels à l'interaction avec son récepteur. Les résultats que nous avons obtenus avec les mini-CD40Ls dans divers systèmes moléculaires et cellulaires suggèrent que de petites molécules synthétiques peuvent reproduire les effets biologiques de la protéine CD40L naturelle. Nous avons réalisé une étude complète de structurefonction sur les mini-CD40Ls afin de préciser leurs mécanismes d’action et de les optimiser. En particulier, nous avons généré un mini-CD40L qui a perdu sa capacité d’interagir de manière coopérative avec CD40. Nos données suggèrent l’existence d’une relation entre les propriétés d’interaction au CD40 et les effets biologiques induits par les mini-CD40Ls. Nous avons utilisé les mini-CD40Ls pour décrire les voies de signalisation activées par CD40 dans différents systèmes cellulaires. Enfin, nous avons démontré que les mini-CD40Ls induisent une réponse T efficace dans un modèle d’infection expérimentale par Trypanosoma cruzi chez la souris.The CD40–CD40L interaction plays a central role in development of both humoral and cellular immune responses. CD40 belongs to the tumor necrosis factor receptor (TNF-R) family, and is constitutively expressed on B lymphocytes, dendritic cells and monocytes/macrophages. CD40L belongs to the TNF family, and is mainly expressed transiently on activated T lymphocytes. Proteins from the TNF-R/TNF superfamily assemble in a C3 symmetry, forming hexavalent complexes that are important for transduction of intracellular cell signaling. Many groups have used antibodies directed against CD40 and CD40L as therapeutics, to either inhibit or activate the immune system. Research on synthetic multivalent molecules that could act as ligands in complex biological systems is an innovating field in medicinal chemistry. On the basis of crystallographic data and directed mutagenesis experiments, we have developed synthetic molecules (mini-CD40Ls) built on synthetic C3 platforms linked to the residues essential for interaction with CD40 via spacer arms. The results obtained with mini-CD40Ls in various molecular and cellular systems suggested for the first time that small synthetic molecules could act as functional CD40L mimetics. A complete structure–activity study was performed on mini-CD40Ls to get detailed informations on their mechanisms of action and to optimize them. In particular, we found an interesting mini-CD40L mutant that has lost its cooperative effect in the interaction with CD40. Our data suggested that CD40 binding properties are important for effector functions. We then used mini-CD40Ls as valuable tool to dissect complex signaling pathways activated by CD40 in different cell systems. Finally, we demonstrated that mini-CD40Ls induce an efficient T immune response during experimental infection with Trypanosoma cruzi in mice

Mimes synthétiques de CD40L : effets biologiques et applications potentielles en immunothérapie

Le couple CD40/CD40L'joue un rôle central dans le système immunitaire. CD40 appartient à la famille des récepteurs au TNF (tumor necrosis factor), et est constitutivement exprimé par les lymphocytes B, les cellules dendritiques et les monocytes/macrophages. Son ligand, CD40L, est un membre de la famille du TNF qui est exprimé transitoirement sur les cellules T activées. Les protéines de la superfamille des TNF-R/TNF s'assemblent selon une symétrie C3, formant des complexes hexavalents importants pour la transduction des signaux intracellulaires.Différents groupes ont utilisé des anticorps dirigés contre CD40 et CD40L'à des fins thérapeutiques, dans le but d'inhiber ou d'activer la réponse immunitaire. La recherche de molécules multivalentes synthétiques agissant comme ligands dans des systèmes biologiques complexes représente aujourd hui un domaine novateur de la chimie médicinale. Nous avons développé des molécules synthétiques, sur la base des données de cristallographie et d'expériences de mutagénèse dirigée, construites sur des plateformes synthétiques de symétrie C3 sur lesquelles, par l'intermédiaire de bras espaceurs de longueur optimale, ont été greffés les résidus de CD40L'essentiels à l'interaction avec son récepteur.Les résultats que nous avons obtenus avec les mini-CD40Ls dans divers systèmes moléculaires et cellulaires suggèrent que de petites molécules synthétiques peuvent reproduire les effets biologiques de la protéine CD40L'naturelle. Nous avons réalisé une étude complète de structure-fonction sur les mini-CD40Ls afin de préciser leurs mécanismes d'action et de les optimiser. En particulier, nous avons généré un mini-CD40L'qui a perdu sa capacité d'interagir de manière coopérative avec CD40. Nos données suggèrent l'existence d'une relation entre les propriétés d'interaction au CD40 et les effets biologiques induits par les mini-CD40Ls. Nous avons utilisé les mini-CD40Ls pour décrire les voies de signalisation activées par CD40 dans différents systèmes cellulaires. Enfin, nous avons démontré que les mini-CD40Ls induisent une réponse T efficace dans un modèle d'infection expérimentale par Trypanosoma cruzi chez la souris.The CD40 CD40L interaction plays a central role in development of both humoral and cellular immune responses. CD40 belongs to the tumor necrosis factor receptor (TNF-R) family, and is constitutively expressed on B lymphocytes, dendritic cells and monocytes/macrophages. CD40L belongs to the TNF family, and is mainly expressed transiently on activated T lymphocytes. Proteins from the TNF-R/TNF superfamily assemble in a C3 symmetry, forming hexavalent complexes that are important for transduction of intracellular cell signaling.Many groups have used antibodies directed against CD40 and CD40L as therapeutics, to either inhibit or activate the immune system. Research on synthetic multivalent molecules that could act as ligands in complex biological systems is an innovating field in medicinal chemistry. On the basis of crystallographic data and directed mutagenesis experiments, we have developed synthetic molecules (mini-CD40Ls) built on synthetic C3 platforms linked to the residues essential for interaction with CD40 via spacer arms.The results obtained with mini-CD40Ls in various molecular and cellular systems suggested for the first time that small synthetic molecules could act as functional CD40L mimetics. A complete structure activity study was performed on mini-CD40Ls to get detailed informations on their mechanisms of action and to optimize them. In particular, we found an interesting mini-CD40L mutant that has lost its cooperative effect in the interaction with CD40. Our data suggested that CD40 binding properties are important for effector functions. We then used mini-CD40Ls as valuable tool to dissect complex signaling pathways activated by CD40 in different cell systems. Finally, we demonstrated that mini-CD40Ls induce an efficient T immune response during experimental infection with Trypanosoma cruzi in mice

Mimes synthétiques de CD40L : effets biologiques et applications potentielles en immunothérapie

Le couple CD40/CD40L'joue un rôle central dans le système immunitaire. CD40 appartient à la famille des récepteurs au TNF (tumor necrosis factor), et est constitutivement exprimé par les lymphocytes B, les cellules dendritiques et les monocytes/macrophageThe CD40 CD40L interaction plays a central role in development of both humoral and cellular immune responses. CD40 belongs to the tumor necrosis factor receptor (TNF-R) family, and is constitutively expressed on B lymphocytes, dendritic cells and monocyt

Mimes synthétiques de CD40L (Effets biologiques et applications potentielles en immunothérapie)

Le couple CD40/CD40L joue un rôle central dans le système immunitaire. CD40 appartient à la famille des récepteurs au TNF (tumor necrosis factor), et est constitutivement exprimé par les lymphocytes B, les cellules dendritiques et les monocytes/macrophages. Son ligand, CD40L, est un membre de la famille du TNF qui est exprimé transitoirement sur les cellules T activées. Les protéines de la superfamille des TNF-R/TNF s assemblent selon une symétrie C3, formant des complexes hexavalents importants pour la transduction des signaux intracellulaires.Différents groupes ont utilisé des anticorps dirigés contre CD40 et CD40L à des fins thérapeutiques, dans le but d inhiber ou d activer la réponse immunitaire. La recherche de molécules multivalentes synthétiques agissant comme ligands dans des systèmes biologiques complexes représente aujourd hui un domaine novateur de la chimie médicinale. Nous avons développé des molécules synthétiques, sur la base des données de cristallographie et d expériences de mutagénèse dirigée, construites sur des plateformes synthétiques de symétrie C3 sur lesquelles, par l'intermédiaire de bras espaceurs de longueur optimale, ont été greffés les résidus de CD40L essentiels à l'interaction avec son récepteur.Les résultats que nous avons obtenus avec les mini-CD40Ls dans divers systèmes moléculaires et cellulaires suggèrent que de petites molécules synthétiques peuvent reproduire les effets biologiques de la protéine CD40L naturelle. Nous avons réalisé une étude complète de structure-fonction sur les mini-CD40Ls afin de préciser leurs mécanismes d action et de les optimiser. En particulier, nous avons généré un mini-CD40L qui a perdu sa capacité d interagir de manière coopérative avec CD40. Nos données suggèrent l existence d une relation entre les propriétés d interaction au CD40 et les effets biologiques induits par les mini-CD40Ls. Nous avons utilisé les mini-CD40Ls pour décrire les voies de signalisation activées par CD40 dans différents systèmes cellulaires. Enfin, nous avons démontré que les mini-CD40Ls induisent une réponse T efficace dans un modèle d infection expérimentale par Trypanosoma cruzi chez la souris.The CD40 CD40L interaction plays a central role in development of both humoral and cellular immune responses. CD40 belongs to the tumor necrosis factor receptor (TNF-R) family, and is constitutively expressed on B lymphocytes, dendritic cells and monocytes/macrophages. CD40L belongs to the TNF family, and is mainly expressed transiently on activated T lymphocytes. Proteins from the TNF-R/TNF superfamily assemble in a C3 symmetry, forming hexavalent complexes that are important for transduction of intracellular cell signaling.Many groups have used antibodies directed against CD40 and CD40L as therapeutics, to either inhibit or activate the immune system. Research on synthetic multivalent molecules that could act as ligands in complex biological systems is an innovating field in medicinal chemistry. On the basis of crystallographic data and directed mutagenesis experiments, we have developed synthetic molecules (mini-CD40Ls) built on synthetic C3 platforms linked to the residues essential for interaction with CD40 via spacer arms.The results obtained with mini-CD40Ls in various molecular and cellular systems suggested for the first time that small synthetic molecules could act as functional CD40L mimetics. A complete structure activity study was performed on mini-CD40Ls to get detailed informations on their mechanisms of action and to optimize them. In particular, we found an interesting mini-CD40L mutant that has lost its cooperative effect in the interaction with CD40. Our data suggested that CD40 binding properties are important for effector functions. We then used mini-CD40Ls as valuable tool to dissect complex signaling pathways activated by CD40 in different cell systems. Finally, we demonstrated that mini-CD40Ls induce an efficient T immune response during experimental infection with Trypanosoma cruzi in mice.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF