Determination of a predictive cleavage motif for eluted major histocompatibility complex class II ligands

CD4+ T cells have a major role in regulating immune responses. They are activated by recognition of peptides mostly generated from exogenous antigens through the major histocompatibility complex (MHC) class II pathway. Identification of epitopes is important and computational prediction of epitopes is used widely to save time and resources. Although there are algorithms to predict binding affinity of peptides to MHC II molecules, no accurate methods exist to predict which ligands are generated as a result of natural antigen processing. We utilized a dataset of around 14,000 naturally processed ligands identified by mass spectrometry of peptides eluted from MHC class II expressing cells to investigate the existence of sequence signatures potentially related to the cleavage mechanisms that liberate the presented peptides from their source antigens. This analysis revealed preferred amino acids surrounding both N- and C-terminuses of ligands, indicating sequence-specific cleavage preferences. We used these cleavage motifs to develop a method for predicting naturally processed MHC II ligands, and validated that it had predictive power to identify ligands from independent studies. We further confirmed that prediction of ligands based on cleavage motifs could be combined with predictions of MHC binding, and that the combined prediction had superior performance. However, when attempting to predict CD4+ T cell epitopes, either alone or in combination with MHC binding predictions, predictions based on the cleavage motifs did not show predictive power. Given that peptides identified as epitopes based on CD4+ T cell reactivity typically do not have well-defined termini, it is possible that motifs are present but outside of the mapped epitope. Our attempts to take that into account computationally did not show any sign of an increased presence of cleavage motifs around well-characterized CD4+ T cell epitopes. While it is possible that our attempts to translate the cleavage motifs in MHC II ligand elution data into T cell epitope predictions were suboptimal, other possible explanations are that the cleavage signal is too diluted to be detected, or that elution data are enriched for ligands generated through an antigen processing and presentation pathway that is less frequently utilized for T cell epitopes.Fil: Paul, Sinu. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Karosiene, Edita. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Dhanda, Sandeep Kumar. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Jurtz, Vanessa. Technical University of Denmark; DinamarcaFil: Edwards, Lindy. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Nielsen, Morten. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Technical University of Denmark; DinamarcaFil: Sette, Alessandro. University of California at San Diego; Estados Unidos. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Peters, Bjoern. La Jolla Institute for Allergy and Immunology; Estados Unidos. University of California at San Diego; Estados Unido

T-cell dependent immunogenicity of protein therapeutics: Preclinical assessment and mitigation

Protein therapeutics hold a prominent and rapidly expanding place among medicinal products. Purified blood products, recombinant cytokines, growth factors, enzyme replacement factors, monoclonal antibodies, fusion proteins, and chimeric fusion proteins are all examples of therapeutic proteins that have been developed in the past few decades and approved for use in the treatment of human disease. Despite early belief that the fully human nature of these proteins would represent a significant advantage, adverse effects associated with immune responses to some biologic therapies have become a topic of some concern. As a result, drug developers are devising strategies to assess immune responses to protein therapeutics during both the preclinical and the clinical phases of development. While there are many factors that contribute to protein immunogenicity, T cell- (thymus-) dependent (Td) responses appear to play a critical role in the development of antibody responses to biologic therapeutics. A range of methodologies to predict and measure Td immune responses to protein drugs has been developed. This review will focus on the Td contribution to immunogenicity, summarizing current approaches for the prediction and measurement of T cell-dependent immune responses to protein biologics, discussing the advantages and limitations of these technologies, and suggesting a practical approach for assessing and mitigating Td immunogenicity

Identification and characterization of T cell receptors reactive to the E7(11-19) epitope of HPV-16

High-risk human Papilloma viruses (HR-HPVs) are the main causative agents of cervical, anal, vulvar, vaginal and penile cancer as well as head and neck cancer. The most prevalent HR-HPV types are HPV16 and HPV18. HPV16 is responsible for about 50% of HPV-related cancers and thus it is a preferred type to study. The HPV16 E7 (11-19) peptide is a well-characterized epitope presented by HLA-A*02:01 molecules on HPV16+ tumor cells. Therefore, it represents a tumor-specific antigen and is an ideal target for Adoptive T cell therapy (ATC) due to its viral source and being absent in healthy tissues. Recently, a proof-of-concept study in metastatic cervical cancer patients has shown that administration of HPV16-specific tumor infiltrating lymphocytes (TILs) expanded ex vivo and infused back to the patient induced tumor regression. However, selection and expansion of TILs has some important disadvantages compared to genetically modified T cells. This project was aimed at identifying HPV16 E7-specific TCRs that could be applied as an effective immunotherapy to cancers caused by this virus. We believe that tumor samples from patients at developing stages of HPV+ cancers might not be optimal for finding HPV-specific TCRs, since their TILs react weakly against the E6/E7 oncoproteins. Furthermore, HPV16- specific cytotoxic T cells can be frequently found in peripheral blood of healthy donors. Therefore, in vitro stimulation of CD8+ T cells derived from healthy individuals and subsequent identification of their TCR repertoire could be a better way to obtain efficient TCR candidates for adoptive T cell therapy of HPV16+ cancers. For an efficient in vitro stimulation of T cells, we used a previously constructed and purified recombinant fusion protein composed of an N-terminal fragment of E7 (amino acids 1-30, E71- 30) linked to the N terminus of Flt3L. Immature dendritic cells isolated from PBMCs of healthy donors were pre-incubated with the fusion protein and then co-cultured with autologous T cells. E7-reactive CD8+ T cells (IFN- γ + , CD137+ and HLA-A2-E711-19-Tetramer+ ) were isolated. The exact TCR profile of the E7-reactive T cells was determined by single-cell V(D)J sequencing using the 10X Genomics platform. Our results showed that the E7-Flt3L is a functional protein that can efficiently increase the frequency of CD8+ T cells targeting the HLA-A*02:01-restricted iv HPV16 E7(11-19) epitope among peripheral lymphocytes of healthy donors. Moreover, our workflow combined tetramer binding and activation of T cells to improve the selection of truly reactive T cells. Three TCR candidates were screened in vitro, which showed different patterns of specificity, avidity, and reactivity. Further, we were able to identify several E7(11-19)-reactive motifs in the CDR3β region through in silico characterization of 22 selected TCRs. Importantly, most of these motifs were enriched in patients who cleared HPV16 infection compared to patients with cervical intraepithelial neoplasia grade 3 (CIN3) or higher. Taken together, our study has established an efficient workflow for the identification of TCRs targeting tumor viral antigens which can be an important step toward TCR discovery

Deciphering the Landscape of HLA class-I and class-II Phosphopeptidomes leads to Robust Predictions of Phosphorylated HLA ligands

Activation of CD8+ and CD4+ T cells through recognition of antigens presented by class I and class II human leukocyte antigen (HLA-I/HLA-II) molecules is crucial for immune responses against infected or malignant cells. In cancer, neoantigens can arise from cancer-specific genomic or proteomic alterations, including mutations and aberrant post-translational modification, such as phosphorylation. Identifying HLA ligands remains a challenging task that requires either heavy experimental work for in vivo identification or optimized bioinformatics tools for accurate predictions. While much work has been done on unmodified HLA-I and HLA-II ligands, only little is known about the presentation of phosphorylated peptides, in particular by HLA-II molecules. Moreover, none of the existing in silico models for predictions of HLA – ligand interactions are specifically trained on phosphorylated ligands. This thesis presents in-depth analyses of phosphorylated HLA-I and HLA-II ligands and introduces predictors for HLA – phosphorylated ligand interactions. The first part of this thesis comprises the curation of phosphorylated HLA-I ligands from several Mass Spectrometry – based peptidomics studies, identifying more than 2,000 unique phosphorylated peptides covering 72 HLA-I alleles. Furthermore, it was see that phosphorylated HLA-I ligands are shaped by a combination of HLA-I binding motifs, intrinsic HLA-I binding properties of phosphorylated ligands and kinase motifs. Combining phosphorylated HLA-I ligands with unmodified data for training a prediction model resulted in improved predictions of phosphorylated HLA-I ligands. The second part addresses phosphorylated HLA-II ligands presented by professional antigen presenting cells for CD4+ T cell activation. MS – based HLA-II peptidomics data resulted in the identification of binding motifs for more than 30 HLA-II alleles, comprising 2,473 unique phosphorylated ligands. These were used to retrain a predictor for HLA-II - ligand interactions and showed improved accuracy for phosphorylated ligands. The analysis of the phosphorylated HLA-II peptidomes revealed a more diverse repertoire of kinases responsible for the phosphorylation of peptides presented on HLA-II compared to HLA-I. In summary, the current work presents in-depth studies on phosphorylated HLA ligands as well as bioinformatics tools for the predictions of phosphorylated peptide interactions with HLA-I and HLA-II molecules. -- L'activation des cellules T CD8+ et CD4+ suite à la reconnaissance d’antigènes présentés par les antigènes des leucocytes humains de classe I et II (HLA-I/HLA-II) est cruciale pour les réponses immunitaires contre les cellules infectées ou cancéreuses. Dans le cancer, les néoantigènes peuvent provenir d'altérations génomiques ou protéomiques spécifiques au cancer, par exemple des mutations ou des modifications post-traductionnelles aberrantes, telles que la phosphorylation. L'identification des ligands HLA reste une tâche difficile qui nécessite soit un travail expérimental lourd pour l'identification in vivo, soit des outils bio-informatiques optimisés pour des prédictions précises. Si beaucoup de travail a été réalisé sur les ligands HLA-I et HLA-II non modifiés, on ne sait que peu de choses sur la présentation des peptides phosphorylés, en particulier par les molécules HLA-II. De plus, aucun des modèles in silico existants pour la prédiction des interactions HLA - ligands n'est spécifiquement entraîné sur les ligands phosphorylés. Cette thèse présente des analyses détaillées sur les ligands HLA-I et HLA-II phosphorylés et introduit des prédicteurs pour les interactions HLA - ligands phosphorylés. La première partie de cette thèse comprend la curation des ligands HLA-I phosphorylés provenant de plusieurs études peptidiques de spectrométrie de masse, identifiant plus de 2’000 peptides phosphorylés uniques couvrant 72 allèles HLA-I. De plus, il a été constaté que les ligands HLA-I phosphorylés sont obtenus par une combinaison de motifs de liaison aux HLA-I, de propriétés intrinsèques de liaison entre les HLA-I et les ligands phosphorylés et de motifs de kinases. La combinaison de ces ligands HLA-I phosphorylés avec des données de ligands non modifiés pour l’entraînement du prédicteur a permis d'améliorer les prédictions des ligands HLA-I phosphorylés. La deuxième partie de cette thèse porte sur les ligands HLA-II phosphorylés qui sont présentés par des cellules présentatrices d'antigènes professionnelles pour l'activation des lymphocytes T CD4+. Les données peptidiques de HLA-II basées sur la spectrométrie de masse ont permis d'identifier des motifs de liaison pour plus de 30 allèles HLA-II, comprenant 2’473 ligands phosphorylés uniques. Ces motifs ont été utilisés pour re-entraîner un prédicteur des interactions entre les ligands et HLA-II qui a montré une meilleure précision pour les ligands phosphorylés. En outre, l'analyse du peptidome HLA-II phosphorylé a révélé un répertoire plus diversifié de kinases responsables de la phosphorylation des peptides présentés par les HLA-II par rapport aux HLA-I. En résumé, cette thèse présente des études détaillées sur les ligands HLA phosphorylés ainsi que des outils bio-informatiques pour la prédiction des interactions des peptides phosphorylés avec les molécules HLA-I et HLA-II

Identification of T-cell epitopes in the Hepatitis C virus genotype 4 proteome: a step towards epitope-driven vaccine development

Hepatitis C is an inflammatory infectious disease of the liver caused by the Hepatitis C Virus (HCV). It is a global pandemic, chronically inflicting 150 million people worldwide, with millions of new infections arising annually. The standard therapy of HCV is expensive, associated with severe side effects, and has variable success rates. Thus far, no HCV vaccine has been developed, owing to the challenges that faced and still face its development. Despite these challenges, several attempts have been taken to develop a vaccine, some of which have progressed to phase II clinical trials. Most of these attempts, however, have focused on HCV genotypes 1 and 2 as vaccine targets, and almost no attention has been given to HCV genotype 4 (HCV-4), the viral genotype most prevalent in the Middle East and Central Africa. In an attempt to fill this gap in HCV-4 vaccine research, this project describes the in silico identification of a group of highly conserved and immunogenic T-cell epitopes from the HCV-4 proteome, using the iVAX immunoinformatics toolkit (EpiVax Inc., RI, USA), as a first step towards the development of an epitope-driven vaccine against the viral genotype. Furthermore, it puts forth a fast and inexpensive method for the validation of the results retrospectively using the repository of empirical HCV immune epitope data on the Immune Epitope Database (IEDB). 90 HLA class I and 14 HLA class II epitopes were identified. From those, 20 HLA class I epitopes were found to be previously uncharacterized, while the in silico HLA binding predictions for 27 others (class I and class II) have been retrospectively validated. The retrospective validation results for 4 of the identified HLA class II epitopes were confirmed by a pilot HLA class II binding assay. Furthermore, an investigation of the conservancy of a selected set of the identified epitopes in newly re-sequenced HCV strains from the Egyptian population was performed. The identified and retrospectively validated set of epitopes constitutes a good target for further immunogenicity testing and epitope-driven vaccine development against HCV-4

Studies of MHC class I antigen presentation & the origins of the immunopeptidome

La présentation d'antigène par les molécules d'histocompatibilité majeure de classe I (CMHI) permet au système immunitaire adaptatif de détecter et éliminer les agents pathogènes intracellulaires et des cellules anormales. La surveillance immunitaire est effectuée par les lymphocytes T CD8 qui interagissent avec le répertoire de peptides associés au CMHI présentés à la surface de toutes cellules nucléées. Les principaux gènes humains de CMHI, HLA-A et HLA-B, sont très polymorphes et par conséquent montrent des différences dans la présentation des antigènes. Nous avons étudié les différences qualitatives et quantitatives dans l'expression et la liaison peptidique de plusieurs allotypes HLA. Utilisant la technique de cytométrie de flux quantitative nous avons établi une hiérarchie d'expression pour les quatre HLA-A, B allotypes enquête. Nos résultats sont compatibles avec une corrélation inverse entre l'expression allotypique et la diversité des peptides bien que d'autres études soient nécessaires pour consolider cette hypothèse. Les origines mondiales du répertoire de peptides associés au CMHI restent une question centrale à la fois fondamentalement et dans la recherche de cibles immunothérapeutiques. Utilisant des techniques protéogénomiques, nous avons identifié et analysé 25,172 peptides CMHI isolées à partir des lymphocytes B de 18 personnes qui exprime collectivement 27 allotypes HLA-A,B. Alors que 58% des gènes ont été la source de 1-64 peptides CMHI par gène, 42% des gènes ne sont pas représentés dans l'immunopeptidome. Dans l'ensemble, l’immunopeptidome présenté par 27 allotypes HLA-A,B ne couvrent que 17% des séquences exomiques exprimées dans les cellules des sujets. Nous avons identifié plusieurs caractéristiques des transcrits et des protéines qui améliorent la production des peptides CMHI. Avec ces données, nous avons construit un modèle de régression logistique qui prédit avec une grande précision si un gène de notre ensemble de données ou à partir d'ensembles de données indépendants génèrerait des peptides CMHI. Nos résultats montrent la sélection préférentielle des peptides CMHI à partir d'un répertoire limité de produits de gènes avec des caractéristiques distinctes. L'idée que le système immunitaire peut surveiller des peptides CMHI couvrant seulement une fraction du génome codant des protéines a des implications profondes dans l'auto-immunité et l'immunologie du cancer.Antigen presentation by major histocompatibility complex class I (MHCI) molecules allows the adaptive immune system to detect and eliminate intracellular pathogens or abnormal cells. Immune surveillance is executed by CD8 T cells that monitor the repertoire of MHCI-associated peptides (MAPs) presented at the surface of all nucleated cells. The primary human MHCI genes, HLA-A and HLA-B, are highly polymorphic and consequentially demonstrate differences in antigen presentation. We investigated qualitative and quantitative differences in expression and peptide binding. Using quantitative flow cytometry we establish clear hierarchy of expression for the four HLA-A,B allotypes investigated. Our results are consistent with an inverse correlation between expression and peptide diversity although further work is necessary to solidify this hypothesis. The global origins of the MAP repertoire remains a central question both fundamentally and in the search for immunotherapeutic targets. Using proteogenomics, we identified and analyzed 25,172 MAPs isolated from B lymphocytes of 18 individuals who collectively expressed 27 HLA-A,B allotypes. While 58% of genes were the source of 1-64 MAPs per gene, 42% of genes were not represented in the immunopeptidome. Overall, we estimate the immunopeptidome presented by 27 HLA-A,B allotypes covered only 17% of exomic sequences expressed in subjects’ cells. We identified several features of transcripts and proteins that enhance MAP production. From these data we built a logistic regression model that predicts with high accuracy whether a gene from our dataset or from independent datasets would generate MAPs. Our results show preferential selection of MAPs from a limited repertoire of gene products with distinct features. The notion that the immune system can monitor MAPs covering only a fraction of the protein coding genome has profound implications in autoimmunity and cancer immunology

Effect of HLA DR epitope de-immunization of Factor VIII \u3ci\u3ein vitro\u3c/i\u3e and \u3ci\u3ein vivo\u3c/i\u3e

T cell-dependent development of anti-Factor VIII (FVIII) antibodies that neutralize FVIII activity is a major obstacle to replacement therapy in hemophilia A. To create a less immunogenic therapeutic protein, recombinant FVIII can be modified to reduce HLA binding of epitopes based on predicted anchoring residues. Here, we used immunoinformatic tools to identify C2 domain HLA DR epitopes and predict site-specific mutations that reduce immunogenicity. Epitope peptides corresponding to original and modified sequences were validated in HLA binding assays and in immunizations of hemophilic E16 mice, DR3 and DR4 mice and DR3 × E16 mice. Consistent with immunoinformatic predictions, original epitopes are immunogenic. Immunization with selected modified sequences lowered immunogenicity for particular peptides and revealed residual immunogenicity of incompletely de-immunized modified peptides. The stepwise approach to reduce protein immunogenicity by epitope modification illustrated here is being used to design and produce a functional full-length modified FVIII for clinical use