PubMed Health⌕ Search

Biomedical subjects

Johannes B Huppa

Publications and source records attributed to Johannes B Huppa.

9 recordsLinked to original sources
PubMed2005

A hybrid machine-learning approach for segmentation of protein localization data.

MOTIVATION: Subcellular protein localization data are critical to the quantitative understanding of cellular function and regulation. Such data are acquired via observation and quantitative analysis of fluorescently labeled proteins in living cells. Differentiation of labeled protein from cellular artifacts remains an obstacle to accurate quantification. We have developed a novel hybrid machine-learning-based method to differentiate signal from artifact in membrane protein localization data by deriving positional information via surface fitting and combining this with fluorescence-intensity-based data to generate input for a support vector machine. RESULTS: We have employed this classifier to analyze signaling protein localization in T-cell activation. Our classifier displayed increased performance over previously available techniques, exhibiting both flexibility and adaptability: training on heterogeneous data yielded a general classifier with good overall performance; training on more specific data yielded an extremely high-performance specific classifier. We also demonstrate accurate automated learning utilizing additional experimental data.

Animals↗
PubMed2005

Agonist/endogenous peptide-MHC heterodimers drive T cell activation and sensitivity.

Alphabeta T lymphocytes are able to detect even a single peptide-major histocompatibility complex (MHC) on the surface of an antigen-presenting cell. This is despite clear evidence, at least with CD4+ T cells, that monomeric ligands are not stimulatory. In an effort to understand how this remarkable sensitivity is achieved, we constructed soluble peptide-MHC heterodimers in which one peptide is an agonist and the other is one of the large number of endogenous peptide-MHCs displayed by presenting cells. We found that some specific combinations of these heterodimers can stimulate specific T cells in a CD4-dependent manner. This activation is severely impaired if the CD4-binding site on the agonist ligand is ablated, but the same mutation on an endogenous ligand has no effect. These data correlate well with analyses of lipid bilayers and cells presenting these ligands, and indicate that the basic unit of helper T cell activation is a heterodimer of agonist peptide- and endogenous peptide-MHC complexes, stabilized by CD4.

Amino Acid Sequence↗
PubMed2004

Quantitative imaging of lymphocyte membrane protein reorganization and signaling.

Changes in membrane protein localization are critical to establishing cell polarity and regulating cell signaling. Fluorescence microscopy of labeled proteins allows visualization of these changes, but quantitative analysis is needed to study this aspect of cell signaling in full mechanistic detail. We have developed a novel approach for quantitative assessment of membrane protein redistribution based on four-dimensional video microscopy of fluorescently labeled proteins. Our analytic system provides robust automated methods for cell surface reconstruction, cell shape tracking, cell-surface distance measurement, and cluster formation analysis. These methods permit statistical analyses and testing of mechanistic hypotheses regarding cell signaling. We have used this approach to measure antigen-dependent clustering of signaling molecules in CD4+ T lymphocytes, obtaining clustering velocities consistent with single-particle tracking data. Our system captures quantitative differences in clustering between signaling proteins with distinct biological functions. Our methods can be generalized to a range of cell-signaling phenomena and enable novel applications not feasible with single-particle studies, such as analysis of subcellular protein localization in live organ culture.

Algorithms↗
PubMed2004

CD4 enhances T cell sensitivity to antigen by coordinating Lck accumulation at the immunological synapse.

How T cells respond with extraordinary sensitivity to minute amounts of agonist peptide and major histocompatibility complex (pMHC) molecules on the surface of antigen-presenting cells bearing large numbers of endogenous pMHC molecules is not understood. Here we present evidence that CD4 affects the responsiveness of T helper cells by controlling spatial localization of the tyrosine kinase Lck in the synapse. This finding, as well as further in silico and in vitro experiments, led us to develop a molecular model in which endogenous and agonist pMHC molecules act cooperatively to amplify T cell receptor signaling. At the same time, activation due to endogenous pMHC molecules alone is inhibited. A key feature is that the binding of agonist pMHC molecules to the T cell receptor results in CD4-mediated spatial localization of Lck, which in turn enables endogenous pMHC molecules to trigger many T cell receptors. We also discuss broader implications for T cell biology, including thymic selection, diversity of the repertoire of self pMHC molecules and serial triggering.

Animals↗
PubMed2004

T cell killing does not require the formation of a stable mature immunological synapse.

A notable feature of T lymphocyte recognition on other cell surfaces is the formation of a stable mature immunological synapse. Here we use a single-molecule labeling method to directly measure the number of ligands a cytotoxic T cell engages and track the consequences of that interaction by three-dimensional video microscopy. Like helper T cells, cytotoxic T cells were able to detect even a single foreign antigen but required about ten complexes of peptide-major histocompatibility complex (pMHC) to achieve full calcium increase and to form a mature synapse. Thus, cytotoxic T cells and helper T cells are more uniform in their antigen sensitivities than previously thought. Furthermore, only three pMHC complexes were required for killing, showing that stable synapse formation and complete signaling are not required for cytotoxicity.

Animals↗
PubMed2003

Continuous T cell receptor signaling required for synapse maintenance and full effector potential.

Although signals through the T cell receptor (TCR) are essential for the initiation of T helper cell activation, it is unclear what function such signals have during the prolonged T cell-antigen-presenting cell contact. Here we simultaneously tracked TCR-CD3 complex and phosphoinositide 3-kinase activity in single T cells using three-dimensional video microscopy. Despite rapid internalization of most of the TCR-CD3, TCR-dependent signaling was still evident up to 10 h after conjugate formation. Blocking this interaction caused dissolution of the synapse and proportional reductions in interleukin 2 production and cellular proliferation. Thus TCR signaling persists for hours, has a cumulative effect and is necessary for the maintenance of the immunological synapse.

Animals↗
PubMed2003

Linking molecular and cellular events in T-cell activation and synapse formation.

The complex sequence of events in which T cells recognize foreign entities on other cells is not well understood. However, the development of new techniques and approaches in both the molecular and cellular aspects of this problem have provided significant insights into the mechanisms of T-cell recognition and synapse formation. In particular, we have a clearer picture of T-cell sensitivity, the role of co-stimulation in formation of the immunological synapse, and how TCR signaling acts to maintain synapse structure and potentiate the T cells over many hours of engagement. We also are aware of new complexities in the way T-cell receptor molecules bind peptide-MHC (pMHC) ligands and what that may mean for TCR scanning, cross-reactivity, and activation. Ultimately, we want to integrate these cellular aspects of T-cell recognition with key features of the molecular interactions that drive specific events.

Antigen-Presenting Cells↗
PubMed2003

Dynamics of cell surface molecules during T cell recognition.

Recognition of foreign antigens by T lymphocytes is a very important component of vertebrate immunity-vital to the clearance of pathogenic organisms and particular viruses and necessary, indirectly, for the production of high affinity antibodies. T cell recognition is mediated by the systematic scanning of cell surfaces by T cells, which collectively express many antigen receptors. When the appropriate antigenic peptide bound to a molecule of the major histocompatibility complex is found-even in minute quantities-a series of elaborate cell-surface molecule and internal rearrangements take place. The sequence of events and the development of techniques required to observe these events have significantly enhanced our understanding of T cell recognition and may find application in other systems of transient cell:cell interactions as well.

Animals↗