Faculty Bibliography

Classical alphabeta T cells protect the host by monitoring intracellular and extracellular proteins in a two-step process. The first step is protein degradation and combination with a major histocompatibility complex (MHC) molecule, leading to surface expression of this amalgam (antigen processing). The second step is the interaction of the T cell receptor with the MHC-peptide complex, leading to signaling in the T cells (antigen recognition). The context for this interaction is a T cell-antigen presenting cell junction, known as an immunological synapse if symmetric and stable and as a kinapse if asymmetric and mobile. The physiological recognition of a ligand takes place most efficiently in the F-actin-rich lamellipodium and is F-actin dependent in stages of formation and triggering and myosin II dependent for signal amplification. This review discusses how these concepts emerged from early studies on adhesion, signaling, and cell biology of T cells

The European Molecular Biology Organization (EMBO) meeting Visualizing Immune System Complexity, held in January 2009, covered multiple scales, from imaging single molecules to imaging whole animals. In addition to experimental details, there was an emphasis on modeling both for data analysis and as a predictive tool to support experimental design. Imaging technologies discussed included total internal reflection fluorescence microscopy, fluorescence correlation spectroscopy, two-photon laser scanning microscopy, and magnetic resonance imaging. The biological systems included basic aspects of adaptive and innate immunity. The type 1 diabetes model was used to illustrate how a human disease was dissected at all the scales, from single-molecule analysis of the interactions of T cell receptors with peptide-loaded major histocompatibility complexes to dynamics of immune cell infiltrates by intravital microscopy, as well as the application of imaging diagnostics in humans

The BimEL tumor suppressor is a potent proapoptotic BH3-only protein. We found that, in response to survival signals, BimEL was rapidly phosphorylated on three serine residues in a conserved degron, facilitating binding and degradation via the F box protein betaTrCP. Phosphorylation of the BimEL degron was executed by Rsk1/2 and promoted by the Erk1/2-mediated phosphorylation of BimEL on Ser69. Compared to wild-type BimEL, a BimEL phosphorylation mutant unable to bind betaTrCP was stabilized and consequently potent at inducing apoptosis by the intrinsic mitochondrial pathway. Moreover, although non-small cell lung cancer (NSCLC) cells often become resistant to gefitinib (a clinically relevant tyrosine kinase inhibitor that induces apoptosis through BimEL), silencing of either betaTrCP or Rsk1/2 resulted in BimEL-mediated apoptosis of both gefitinib-sensitive and gefitinib-insensitive NSCLC cells. Our findings reveal that betaTrCP promotes cell survival in cooperation with the ERK-RSK pathway by targeting BimEL for degradation

Lymphocytic choriomeningitis virus infection of the mouse central nervous system (CNS) elicits fatal immunopathology through blood-brain barrier breakdown and convulsive seizures. Although lymphocytic-choriomeningitis-virus-specific cytotoxic T lymphocytes (CTLs) are essential for disease, their mechanism of action is not known. To gain insights into disease pathogenesis, we observed the dynamics of immune cells in the meninges by two-photon microscopy. Here we report visualization of motile CTLs and massive secondary recruitment of pathogenic monocytes and neutrophils that were required for vascular leakage and acute lethality. CTLs expressed multiple chemoattractants capable of recruiting myelomonocytic cells. We conclude that a CD8(+) T-cell-dependent disorder can proceed in the absence of direct T-cell effector mechanisms and rely instead on CTL-recruited myelomonocytic cells

T lymphocytes are a key regulatory component of the adaptive immune system. Understanding how the micro- and nano-scale details of the extracellular environment influence T cell activation may have wide impact on the use of T cells for therapeutic purposes. In this article, we examine how the micro- and nano-scale presentation of ligands to cell surface receptors, including microscale organization and nanoscale mobility, influences the activation of T cells. We extend these studies to include the role of cell-generated forces, and the rigidity of the microenvironment, on T cell activation. These approaches enable delivery of defined signals to T cells, a step toward understanding the cell-cell communication in the immune system, and developing micro/nano- and material- engineered systems for tailoring immune responses for adoptive T cell therapies.

Discovery into the role of renal dendritic cells (rDCs) in health and disease of the kidney is rapidly accelerating. Progress in deciphering DC precursors and the heterogeneity of monocyte subsets in mice and humans is providing insight into the biology of rDCs. Recent findings have extended knowledge of the origins, anatomy and function of the rDC network at steady state and during periods of injury to the renal parenchyma. This brief review highlights these new findings and provides an update on the study of rDCs

Staphylococcus (S.) aureus is a gram-positive bacterium, which causes local and systemic infections in humans and animals. The immune response to this pathogen typically leads to the development of abscesses in the skin and in inner organs. The cavity of the abscess is filled with neutrophils and is surrounded by macrophages, T cells and a fibrinous capsule. Little is known about the cell migration that takes place during abscess formation after S. aureus infection. Here, we describe an experimental system of intravital two-photon microscopy that allows for studying host-pathogen interactions during abscess development in the skin and in the kidney. Different fluorescent proteins (FP) were expressed in S. aureus in order to visualize the microorganisms. The corresponding genes were adapted to the codon usage of gram-positive bacteria and expressed from a plasmid under the control of the agr P3 or the sarA P1 promoter. Furthermore, the genes and the promoters could be stably integrated into the bacterial chromosome at the phage-related pathogenicity island SaPI1 site. The reporter constructs could then be transferred successfully via phage transduction into the commonly used virulent S. aureus strains RN6734 and RN9130. In addition, an ovalbumin expressing S. aureus strain was generated, which will be used to visualize and elucidate the role of antigen-specific T cells during abscess development. LysM-eGFP knock-in mice, CD11c-YFP and OTII transgenic mice will be used to visualize the recruitment of neutrophils, dendritic cells, and T cells to the site of abscess formation. Initial studies in C57BL/6 wild type mice revealed the presence of FP+ S. aureus in the subcapsular blood vessels of the kidney immediately after injection, where some bacteria were able to adhere to the endothelium. Interestingly, accumulation of pathogens during abscess development was also visible predominantly in the capsular and subcapsular area. Altogether, the described system of intravital microscopy using fluorescent S. aureus strains and reporter mice is a powerful tool, which allows us to follow the immune response during abscess formation in real-time and will provide new insights into the pathogenesis of this clinically highly relevant infection

The relationship between intermembrane spacing, adhesion efficiency, and lateral organization of adhesion receptors has not been established for any adhesion system. We have utilized the CD2 ligand CD48 with two (wild type CD48 (CD48-WT)), four (CD48-CD2), or five (CD48-CD22) Ig-like domains. CD48-WT was 10-fold more efficient in mediating adhesion than CD48-CD2 or CD48-CD22. Electron tomography of contact areas with planar bilayers demonstrated average intermembrane spacing of 12.8 nm with CD48-WT, 14.7 nm with CD48-CD2, and 15.6 nm with CD48-CD22. Both CD48-CD2 and CD48-CD22 chimeras segregated completely from CD48-WT in mixed contact areas. In contrast, CD48-CD2 and CD48-CD22 co-localized when mixed contacts were formed. Confocal imaging of immunological synapses formed between primary T lymphocytes and Chinese hamster ovary cells presenting major histocompatibility complex-peptide complexes, and different forms of CD48 demonstrated that CD48-CD2 and CD48-CD22 induce an eccentric CD2/T cell antigen receptor cluster. We propose that this reorganization of the immunological synapse sequesters the T cell antigen receptor in a location where it cannot interact with its ligand and dramatically reduces T cell sensitivity