Kym R Garrod

University of California, Irvine, Irvine, California, United States

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Publications (8)34 Total impact

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    ABSTRACT: We present a simple method for rapid and automatic characterization of lymphocyte migration from time-lapse fluorescence microscopy data. Time-lapse imaging of natural killer (NK) cells in vitro and in situ, both showed that individual cells transiently alter their migration behavior. Typically, NK cells showed periods of high motility, interrupted by transient periods of slow migration or almost complete arrests. Analysis of in vitro data showed that these periods frequently coincided with contacts with target cells, sometimes leading to target cell lysis. However, NK cells were also commonly observed to stop independently of contact with other cells. In order to objectively characterize the migration of NK cells, we implemented a simple method to discriminate when NK cells stop or have low motilities, have periods of directed migration or undergo random movement. This was achieved using a sliding window approach and evaluating the mean squared displacement (MSD) to assess the migration coefficient and MSD curvature along trajectories from individual NK cells over time. The method presented here can be used to quickly and quantitatively assess the dynamics of individual cells as well as heterogeneity within ensembles. Furthermore, it may also be used as a tool to automatically detect transient stops due to the formation of immune synapses, cell division or cell death. We show that this could be particularly useful for analysis of in situ time-lapse fluorescence imaging data where most cells, as well as the extracellular matrix, are usually unlabelled and thus invisible.
    Integrative Biology 06/2011; 3(7):770-8. · 4.32 Impact Factor
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    ABSTRACT: Direct presentation of foreign MHC molecules expressed by donor-derived dendritic cells (DCs) has generally been considered the dominant pathway of allorecognition in acute transplant rejection. However, recent studies implicate preferential activation of the indirect pathway by host DCs. The respective importance of each pathway and the mechanisms that determine their relative contributions remain to be clearly established. In this study, using two-photon microscopy, we visualized host NK cell interactions with syngeneic and allogeneic DCs within intact lymph nodes of mice. Upon contact with allogeneic DCs, NK cells formed prolonged interactions that led directly to target cell lysis. This rapid elimination limited the ability of allogeneic DCs to stimulate primary and recall T cell responses. To discriminate whether donor or host DCs are principally involved in presenting Ag derived from allografts, we used CD11c-diphtheria toxoid receptor mice to conditionally ablate CD11c(+) DCs and to show that direct presentation by donor DCs is alone insufficient to elicit acute allograft rejection. We thus propose that rapid elimination of allogeneic DCs limits direct Ag presentation and thereby favors the indirect pathway of alloreactivity.
    The Journal of Immunology 02/2010; 184(5):2329-36. · 5.36 Impact Factor
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    ABSTRACT: T-cell receptor transgenic (TCR-tg) mouse models with direct CD4 alloreactivity will help elucidate mechanisms of transplant rejection and tolerance in vivo. Although such models exist, they are limited by unusual strain combinations or are based on model antigens. A TCR-tg mouse with direct CD4 specificity in the widely used BALB/c donor --> C57BL/6 host strain combination was created. This TCR-tg mouse, named 4C, was selected for reactivity against BALB/c dendritic cells in order to model early priming events after transplantation. The response of 4C T cells to skin and heart transplants were characterized. The alloantigen is restricted by I-A and appears to be widely distributed in mouse tissues. 4C T cells are able to acutely reject skin but not heart allografts. Paradoxically, heart grafts elicited a stronger proliferation and effector function of TCR-tg T cells than skin grafts. 4C T cells caused cardiac allograft vasculopathy in the absence of other T cells and alloantibodies, suggesting a role for the direct pathway in chronic rejection. Augmentation of priming with an infusion of donor-derived dendritic cells resulted in acute heart allograft rejection by 4C T cells, demonstrating that the level of priming can play a role in determining acute versus chronic rejection by the CD4 direct pathway. Rejection of a graft by the direct CD4 pathway is determined by graft susceptibility to rejection, as well as the degree of T-cell priming caused by the graft. Grafts that are not acutely rejected can develop transplant vasculopathy mediated by the direct CD4 T cells.
    Transplantation 02/2008; 85(2):247-55. · 3.78 Impact Factor
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    Kym R Garrod, Michael D Cahalan
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    ABSTRACT: As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take <10 min to perform.
    Journal of Visualized Experiments 02/2008;
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    ABSTRACT: T cell activation by APC requires cytosolic Ca(2+) ([Ca(2+)](i)) elevation. Using two-photon microscopy, we visualized Ca(2+) signaling and motility of murine CD4(+) T cells within lymph node (LN) explants under control, inflammatory, and immunizing conditions. Without Ag under basal noninflammatory conditions, T cells showed infrequent Ca(2+) spikes associated with sustained slowing. Inflammation reduced velocities and Ca(2+) spiking in the absence of specific Ag. During early Ag encounter, most T cells engaged Ag-presenting dendritic cells in clusters, and showed increased Ca(2+) spike frequency and elevated basal [Ca(2+)](i). These Ca(2+) signals persisted for hours, irrespective of whether T cells were in contact with visualized dendritic cells. We propose that sustained increases in basal [Ca(2+)](i) and spiking frequency constitute a Ca(2+) signaling modality that, integrated over hours, distinguishes immunogenic from basal state in the native lymphoid environment.
    The Journal of Immunology 09/2007; 179(3):1586-94. · 5.36 Impact Factor
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    ABSTRACT: Natural killer (NK) cells are known to reject MHC-mismatched targets within blood organs, yet their role in peripheral lymphoid tissue remains unresolved. Here we address the capacity of NK cells to migrate within lymph nodes (LN) using two-photon microscopy to characterize cell velocities and interaction dynamics within the native lymphoid-tissue environment. Adoptively transferred unmanipulated NK cells were highly motile (6-7 microm/min) and capable of forming transient contacts with both syngeneic and allogeneic B cells. Stable conjugate interactions (lasting >5 min) formed preferentially with allogeneic cells, resulting in diminished motility and subsequent elimination of the target cell. In marked contrast to unmanipulated cells, NK cells purified by CD49b-positive selection exhibited only limited motility (2-3 microm/min). This velocity impairment arose largely because CD49b cross-linking enhanced NK cell adhesion to collagen fibers within the node. Moreover, CD49b cross-linking prevented NK cells from reconstituting effector cytolytic function in vivo, inhibited target cell lysis in vitro, and augmented IFN-gamma responses to IL-2 activation in vitro. Taken together our data demonstrate that NK cells are a functionally important component of the LN microenvironment, and that cell motility and effector function are strongly modulated via CD49b manipulation.
    Proceedings of the National Academy of Sciences 07/2007; 104(29):12081-6. · 9.81 Impact Factor
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    ABSTRACT: Accumulating evidence that dendritic cells (DC) are important regulators of peripheral immune tolerance has led to the concept that donor-derived DC may be useful for inducing donor-specific transplantation tolerance. Although in vitro studies in this field have been encouraging, in vivo results have been inconsistent. Recent evidence has suggested a critical role of lymphoid organs in tolerance induction. In this study, we use a novel gene transduction technique to show that engineered expression of CCR7 on immature DC can markedly increase DC homing to lymphoid organs, leading to increased interaction with Ag-specific T cells. Moreover, we show that a single infusion of DC coexpressing CCR7 and the immunomodulatory molecule viral IL-10 (vIL-10) markedly prolongs cardiac allograft survival (mean survival time >100 days); importantly, DC expressing either vIL-10 alone or CCR7 alone was not effective. These results demonstrate an important paradigm for immune modulation using DC.
    The Journal of Immunology 08/2006; 177(2):863-8. · 5.36 Impact Factor
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Publication Stats

144 Citations
34.00 Total Impact Points

Institutions

  • 2008–2011
    • University of California, Irvine
      • Department of Physiology & Biophysics
      Irvine, California, United States
  • 2006
    • University of California, San Francisco
      • Department of Surgery
      San Francisco, CA, United States