EpiToolKit—a web server for computational immunomics

Predicting the T-cell-mediated immune response is an important task in vaccine design and thus one of the key problems in computational immunomics. Various methods have been developed during the last decade and are available online. We present EpiToolKit, a web server that has been specifically designed to offer a problem-solving environment for computational immunomics. EpiToolKit offers a variety of different prediction methods for major histocompatibility complex class I and II ligands as well as minor histocompatibility antigens. These predictions are embedded in a user-friendly interface allowing refining, editing and constraining the searches conveniently. We illustrate the value of the approach with a set of novel tumor-associated peptides. EpiToolKit is available online at www.epitoolkit.org

Proof of a pronounced individuality of HLA ligandomes by mass spectrometric analyses

Die HLA-Ligandomik, die systematische Untersuchung von HLA-Peptidrepertoires im großen Stil, ist Grundvoraussetzung für zwei wichtige Forschungsfelder. Zum einen gewährt sie Einblicke in die fundamentalen Mechanismen und Prinzipien der Antigenprozessierung und -präsentation. Zum anderen erlaubt sie die Übertragung dieses Wissens auf klinische Anwendungen wie TZellepitopvorhersagen, Immundiagnostik oder peptidbasierte Immuntherapie. Zunächst wurden die Peptidmotive von 18 HLA-Allelen – basierend auf knapp 2000 natürlichen Liganden – entweder neu aufgestellt oder aber überarbeitet und verfeinert. Diese Motive, die durch Anker, Hilfsanker und Längenverteilungen charakterisiert sind, zeigen, dass sich verschiedene Allele meist durch viele einzigartige Merkmale voneinander abheben. Dies spiegelt sich auch in der geringen Überlappung der HLA-Peptidrepertoires verschiedener Allotypen wider. Diese wurde insbesondere für den HLA-B44-Supertyp eingehend mit Hilfe verschiedenster massenspektrometrischer Methoden untersucht. Dabei zeigte sich, dass die Mitglieder des Supertyps weniger als 3% ihrer Peptide gemeinsam haben. Diese HLA-LigandomÜberschneidungen können allerdings im Einzelfall für kleine Gruppen von Subtypen oder HLA-Antigenen sowie für andere Supertypen auch größer sein. Die Proteomik und die HLA-Ligandomik sind durch gemeinsame Methoden sowie durch analog anwendbare Konzepte und Forschungsfragen eng verwandt. Dies wurde an den Beispielen Gewebsspezifität, allotypische Peptide und Quellproteinanalysen näher betrachtet. So konnte etwa der HLA-A*02-Ligand YLLPAIVHI bislang auf 77% aller HLA-A*0+ Sources detektiert werden und stellt somit ein „Markerpeptid“ für HLA-A*02 dar. Schließlich wurde die Bedeutung der HLA-Ligandomik für die klinische Anwendung an zwei Beispielen verdeutlicht. Zum einen wurden Peptide aus potentiell tumor-assoziierten Antigenen zur in vitro-Stimulation von CD8+ T-Zellen verwendet. Zum anderen konnte anhand von vier HLA-B*44- bzw. HLA-B*40-restingierten EBV-T-Zellepitopen bei 130 Spendern gezeigt werden, dass es innerhalb des HLA-B44-Supertyps keine promiskuitive T-Zell-Epitoperkennung gibt. Peptidvakzinierungen werden also patienten- und allotypspezifisch erfolgen müssen.HLA ligandomics – the systematic large-scale analysis of HLA peptide repertoires – provides a basis for two important fields of research. On the one hand it contributes to our understanding of fundamental mechanisms and principles of antigen processing and presentation. On the other hand it enables transfer of this knowledge into clinical applications such as T cell epitope predictions and the formulation of immunodiagnostic and immunotherapeutic strategies. First, peptide motifs of 18 HLA allotypes were build up or refined based on nearly 2000 natural HLA ligands. Such motifs are characterised by primary and auxiliary anchors as well as peptide length distributions and reveal the uniqueness of different HLA allotypes. This is reflected by an only marginal overlap of their peptide repertoires. Particularly the HLA-B44 supertype was investigated in detail by different mass spectrometric methods. These analyses showed an overlap of HLA ligandomes of below 3%. Nevertheless, HLA ligandome overlaps might be more pronounced for small groups of subtypes or other HLA supertypes. Proteomics and HLA ligandomics share not only mass spectrometry as core technology but also concepts and research questions. This was exemplified for tissue specificity, allotypic peptides and source proteins analyses. For example, YLLPAIVHI – an HLA-A*02-presented natural ligand – was detected in 77% of all HLA-A*02+ sources and can therefore be considered as marker peptide for HLA-A*02. Finally the importance of HLA ligandomics for clinical applications was pointed out by two examples. First, peptides derived from potential tumor-associated antigens were used for in vitro stimulations of CD8+ T cells. Second, T cell responses to four HLA-B*44- or HLA-B*40-restricted EBV-derived epitopes were analysed in 130 blood donors. No promiscuous T cell recognition was observed among allotypes of the HLA-B44 supertype. Thus, patient-specific approaches are now called for

Exploring the MHC-peptide matrix of central tolerance in the human thymus

Ever since it was discovered that central tolerance to self is imposed on developing T cells in the thymus through their interaction with self-peptide major histocompatibility complexes on thymic antigen-presenting cells, immunologists have speculated about the nature of these peptides, particularly in humans. Here, to shed light on the so-far unknown human thymic peptide repertoire, we analyse peptides eluted from isolated thymic dendritic cells, dendritic cell-depleted antigen-presenting cells and whole thymus. Bioinformatic analysis of the 842 identified natural major histocompatibility complex I and II ligands reveals significant cross-talk between major histocompatibility complex-class I and II pathways and differences in source protein representation between individuals as well as different antigen-presenting cells. Furthermore, several autoimmune- and tumour-related peptides, from enolase and vimentin for example, are presented in the healthy thymus. 302 peptides are directly derived from negatively selecting dendritic cells, thus providing the first global view of the peptide matrix in the human thymus that imposes self-tolerance in vivo

All eyes on the patient: the influence of oncologists' nonverbal communication on breast cancer patients' trust

Trust in the oncologist is crucial for breast cancer patients. It reduces worry, enhances decision making, and stimulates adherence. Optimal nonverbal communication by the oncologist, particularly eye contact, body posture, and smiling, presumably benefits patients' trust. We were the first to experimentally examine (1) how the oncologist's nonverbal behavior influences trust, and (2) individual differences in breast cancer patients' trust. Analogue patients (APs) viewed one out of eight versions of a video vignette displaying a consultation about chemotherapy treatment. All eight versions varied only in the oncologist's amount of eye contact (consistent vs. inconsistent), body posture (forward leaning vs. varying), and smiling (occasional smiling vs. no smiling). Primary outcome was trust in the observed oncologist (Trust in Oncologist Scale). 214 APs participated. Consistent eye contact led to stronger trust (β = -.13, p = .04). This effect was largely explained by lower educated patients, for whom the effect of consistent eye contact was stronger than for higher educated patients (β = .18, p = .01). A forward leaning body posture did not influence trust, nor did smiling. However, if the oncologist smiled more, he was perceived as more friendly (rs = .31, p < .001) and caring (rs = .18, p = .01). Older (β = .17, p = .01) and lower educated APs (β = -.25, p < .001) were more trusting. Trust was weaker for more avoidantly attached APs (β = -.16, p = .03). We experimentally demonstrated the importance of maintaining consistent eye contact for breast cancer patients' trust, especially among lower educated patients. These findings need to be translated into training for oncologists in how to optimize their nonverbal communication with breast cancer patients while simultaneously managing increased time pressure and computer use during the consultatio

WARS1 and SARS1: Two tRNA synthetases implicated in autosomal recessive microcephaly

Aminoacylation of transfer RNA (tRNA) is a key step in protein biosynthesis, carried out by highly specific aminoacyl-tRNA synthetases (ARSs). ARSs have been implicated in autosomal dominant and autosomal recessive human disorders. Autosomal dominant variants in tryptophanyl-tRNA synthetase 1 (WARS1) are known to cause distal hereditary motor neuropathy and Charcot-Marie-Tooth disease, but a recessively inherited phenotype is yet to be clearly defined. Seryl-tRNA synthetase 1 (SARS1) has rarely been implicated in an autosomal recessive developmental disorder. Here, we report five individuals with biallelic missense variants in WARS1 or SARS1, who presented with an overlapping phenotype of microcephaly, developmental delay, intellectual disability, and brain anomalies. Structural mapping showed that the SARS1 variant is located directly within the enzyme's active site, most likely diminishing activity, while the WARS1 variant is located in the N-terminal domain. We further characterize the identified WARS1 variant by showing that it negatively impacts protein abundance and is unable to rescue the phenotype of a CRISPR/Cas9 wars1 knockout zebrafish model. In summary, we describe two overlapping autosomal recessive syndromes caused by variants in WARS1 and SARS1, present functional insights into the pathogenesis of the WARS1-related syndrome and define an emerging disease spectrum: ARS-related developmental disorders with or without microcephaly