Andreas Jansson

Structure and Interactions of the Human Programmed Cell Death 1 Receptor

Background: The inhibitory leukocyte receptor PD-1 binds two ligands, PD-L1 and PD-L2. Results: Nuclear magnetic resonance analysis and rigorous binding and thermodynamic measurements reveal the structure of, and the mode of ligand recognition by, PD-1. Conclusion: PD-L1 and PD-L2 bind differently to PD-1 and much more weakly than expected. Significance: Potent inhibitory signaling can be initiated by weakly interacting receptors. PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC′ sheet, which is flexible and completely lacks a C″ strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC′ sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1·ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The 3–4-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the 3-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T cells with antigen-presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1 and reveal that potent inhibitory signaling can be initiated by weakly interacting receptors.

The Pseudomonas aeruginosa Quorum-Sensing Molecule N-3-(Oxododecanoyl)-L-Homoserine Lactone Inhibits T-Cell Differentiation and Cytokine Production by a Mechanism Involving an Early Step in T-Cell Activation

ABSTRACT The Pseudomonas aeruginosa quorum-sensing molecule N-3-(oxododecanoyl)-l-homoserine lactone (OdDHL) has been reported to have immunomodulatory activity in several systems, although the mechanism of that activity remains to be fully characterized. We demonstrate here, using a defined in vitro model of antigen responses by T-cell receptor (TCR)-transgenic mouse splenic CD4 T cells, that the effect of OdDHL on activation and cytokine production is complete within 4 h of antigen or mitogen stimulation and does not depend on the insertion of OdDHL in the cell membrane, despite a previous report that immunosuppression by homoserine lactones required a minimum acyl chain length of 11 carbons (S. R. Chhabra, C. Harty, D. S. W. Hooi, M. Daykin, B. W. Bycroft, P. Williams, and D. Pritchard, J. Med. Chem. 46:97-104, 2003). We also demonstrate that while OdDHL can have toxic effects on nonlymphoid leukocytes, it does not induce significant cell death in T cells at the concentrations (≤10 μM) used in these experiments. In addition, we show that primary and secondary antigen-specific cytokine responses are equally susceptible to inhibition by OdDHL and that the compound inhibits the differentiation of both Th1 and Th2 cells. However, the precise balance of cytokine production by CD4 T cells stimulated in the presence of OdDHL varies with both the antigen concentration and its affinity for the transgenic TCR. Thus, conflicting reports of the nature of the immunosuppression by OdDHL may be due in part to the differences in antigen affinity and concentration in different models.

Global transcriptional profiling reveals similarities and differences between human stem cell-derived cardiomyocyte clusters and heart tissue

It is now well documented that human embryonic stem cells (hESCs) can differentiate into functional cardiomyocytes. These cells constitute a promising source of material for use in drug development, toxicity testing, and regenerative medicine. To assess their utility as replacement or complement to existing models, extensive phenotypic characterization of the cells is required. In the present study, we used microarrays and analyzed the global transcription of hESC-derived cardiomyocyte clusters (CMCs) and determined similarities as well as differences compared with reference samples from fetal and adult heart tissue. In addition, we performed a focused analysis of the expression of cardiac ion channels and genes involved in the Ca(2+)-handling machinery, which in previous studies have been shown to be immature in stem cell-derived cardiomyocytes. Our results show that hESC-derived CMCs, on a global level, have a highly similar gene expression profile compared with human heart tissue, and their transcriptional phenotype was more similar to fetal than to adult heart. Despite the high similarity to heart tissue, a number of significantly differentially expressed genes were identified, providing some clues toward understanding the molecular difference between in vivo sourced tissue and stem cell derivatives generated in vitro. Interestingly, some of the cardiac-related ion channels and Ca(2+)-handling genes showed differential expression between the CMCs and heart tissues. These genes may represent candidates for future genetic engineering to create hESC-derived CMCs that better mimic the phenotype of the cardiomyocytes present in the adult human heart.

Dynamic modelling of cell death during biofilm development.

Biofilms are currently recognised as the predominant bacterial life-style and it has been suggested that biofilm development is influenced by a number of different processes such as adhesion, detachment, mass transport, quorum sensing, cell death and active dispersal. One of the least understood processes and its effects on biofilm development is cell death. However, experimental studies suggest that bacterial death is an important process during biofilm development and many studies show a relationship between cell death and dispersal in microbial biofilms. We present a model of the process of cell death during biofilm development, with a particular focus on the spatial localisation of cell death or cell damage. Three rules governing cell death or cell damage were evaluated which compared the effects of starvation, damage accumulation, and viability during biofilm development and were also used to design laboratory based experiments to test the model. Results from model simulations show that actively growing biofilms develop steep nutrient gradients within the interior of the biofilm that affect neighbouring microcolonies resulting in cell death and detachment. Two of the rules indicated that high substrate concentrations lead to accelerated cell death, in contrast to the third rule, based on the accumulation of damage, which predicted earlier cell death for biofilms grown with low substrate concentrations. Comparison of the modelling results with experimental results suggests that cell death is favoured under low nutrient conditions and that the accumulation of damage may be the main cause of cell death during biofilm development.

The T Cell Receptor Triggering Apparatus Is Composed of Monovalent or Monomeric Proteins

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.

An individual-based network model to evaluate interventions for controlling pneumococcal transmission

BackgroundStreptococcus pneumoniae is a major cause of morbidity and mortality worldwide, but also a common colonizer of the upper respiratory tract. The emergence and spread of antibiotic resistant pneumococcal strains has threatened effective therapy. The long-term effects of measures aiming to limit pneumococcal spread are poorly understood. Computational modeling makes it possible to conduct virtual experiments that are impractical to perform in real life and thereby allows a more full understanding of pneumococcal epidemiology and control efforts.MethodsWe have developed a contact network model to evaluate the efficacy of interventions aiming to control pneumococcal transmission. Demographic data from Sweden during the mid-2000s were employed. Analyses of the models parameters were conducted to elucidate key determinants of pneumococcal spread. Also, scenario simulations were performed to assess candidate control measures.ResultsThe model made good predictions of previous findings where a correlation has been found between age and pneumococcal carriage. Of the parameters tested, group size in day-care centers was shown to be one of the most important factors for pneumococcal transmission. Consistent results were generated from the scenario simulations.ConclusionWe recommend, based on the model predictions, that strategies to control pneumococcal disease and organism transmission should include reducing the group size in day-care centers.

Biochemical modeling with Systems Biology Graphical Notation

The Systems Biology Graphical Notation (SBGN) is an emerging standard for graphical notation developed by an international systems biology community. Standardized graphical notation is crucial for efficient and accurate communication of biological knowledge between researchers with various backgrounds in the expanding field of systems biology. Here, we highlight SBGN from a practical point of view and describe how the user can build and simulate SBGN models from a simple drag-and-drop graphical user interface in PathwayLab.

3D computation modelling of the influence of cytokine secretion on Th-cell development suggests that negative selection (inhibition of Th1 cells) is more effective than positive selection by IL-4 for Th2 cell dominance.

Th‐cell development has been suggested to include selective mechanisms in which certain cytokines select either Th1 or Th2 cells to proliferate and grow. The selective theory is based on the observation that Th2 cells secrete IL‐4, a cytokine that promotes Th2 development, whereas Th1 cells secrete interferon‐γ (IFN‐γ) that favours Th1 development, and both positive and negative selective influences have been suggested to operate. In this study, we investigate the role of autocrine secretion and utilization of IL‐4 by Th2 cells and address the question of whether an activated Th2 cell can be positively selected by IL‐4 secreted from other Th2 cells. We present a spatial three dimensional (3D) modelling approach to simulate the interaction between the IL‐4 ligand and its IL‐4 receptors expressed on discrete IL‐4 secreting cells. The simulations, based on existing experimental data on the IL‐4 receptor–ligand system, illustrate how Th‐cell development is highly dependent on the distance between cells that are communicating. The model suggests that a single Th2 cell is likely to communicate with possible target cells within a range of approximately 100 μm and that an activated Th2 cell manages to fill most of its own IL‐4 receptors, even at a low secretion rate. The predictions made by the model suggest that negative selection against Th1 cells is more effective than positive selection by IL‐4 for promoting Th2 dominance.

A quantitative study of the mechanisms behind thymic atrophy in Gαi2-deficient mice during colitis development.

Mice deficient for the G protein subunit Gαi2 spontaneously develop colitis, a chronic inflammatory disease associated with dysregulated T cell responses. We and others have previously demonstrated a thymic involution in these mice and an aberrant thymocyte dynamics. The Gαi2−/− mice have a dramatically reduced fraction of double positive thymocytes and an increased fraction of single positive (SP) thymocytes. In this study, we quantify a number of critical parameters in order to narrow down the underlying mechanisms that cause the dynamical changes of the thymocyte development in the Gαi2−/− mice. Our data suggest that the increased fraction of SP thymocytes results only from a decreased number of DP thymocytes, since the number of SP thymocytes in the Gαi2−/− mice is comparable to the control littermates. By measuring the frequency of T cell receptor excision circles (TRECs) in the thymocytes, we demonstrate that the number of cell divisions the Gαi2−/− SP thymocytes undergo is comparable to SP thymocytes from control littermates. In addition, our data show that the mature SP CD4+ and CD8+ thymocytes divide to the same extent before they egress from the thymus. By estimating the number of peripheral TREC+ T lymphocytes and their death rate, we could calculate the daily egression of thymocytes. Gαi2−/− mice with no/mild and moderate colitis were found to have a slower export rate in comparison to the control littermates. The quantitative measurements in this study suggest a number of dynamical changes in the thymocyte development during the progression of colitis.

A Mathematical Framework for Analyzing T Cell Receptor Scanning of Peptides

T cells continuously search for antigenic peptides presented on major histocompatibility complexes expressed on nearly all nucleated cells. Because only a few antigenic peptides are presented in a sea of thousands of self-peptides, the T cells have a critical task in discriminating between self- and nonself-peptides. This search process for antigens must be performed with sufficient speed in order to induce a fast response against invading pathogens. This study presents a mathematical framework for analyzing the scanning process of peptides. The framework includes analytic expressions for calculating the sampling rate as well as continuous-systems- and stochastic-agent-based models. The results show that the scanning of self-peptides is a very fast process due to fast off-rates. The simulations also predict the existence of an optimal sampling rate for a certain range of on-rates based on the recently proposed confinement time model. Calculations reveal that most of the self-peptides located within a microdomain are scanned within just a few seconds, and that the T cell receptors have kinetics for self-peptides, facilitating fast scanning. The derived mathematical expressions within this study provide conceptual calculations for further investigations of how the T cell discriminates between self- and nonself-peptides.