ABSTRACT
Natural killer cells are lymphocytes specialized to participate in host defense through their innate ability to mediate cytotoxicity by secreting the contents of preformed secretory lysosomes (lytic granules) directly onto a target cell. This form of directed secretion requires the formation of an immunological synapse and occurs stepwise with actin reorganization preceding microtubule-organizing center (MTOC) polarization to the synapse. Because MTOC polarization to the synapse is required for polarization of lytic granules, we attempted to define their interrelationship. We found that compared with the time required for MTOC polarization, lytic granules converged to the MTOC rapidly. The MTOC-directed movement of lytic granules was independent of actin and microtubule reorganization, dependent on dynein motor function, occurred before MTOC polarization, and did not require a commitment to cytotoxicity. This defines a novel paradigm for rapid MTOC-directed transport as a prerequisite for directed secretion, one that may prepare, but not commit cells for precision secretory function.
Subject(s)
Cytoplasmic Granules/metabolism , Cytotoxicity, Immunologic , Dyneins/metabolism , Killer Cells, Natural/cytology , Killer Cells, Natural/metabolism , Microtubule-Organizing Center/metabolism , Actins/metabolism , Antigens, CD/genetics , Antigens, CD/metabolism , Cell Line , Humans , Microscopy, Confocal , Microtubules/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tubulin/genetics , Tubulin/metabolismABSTRACT
Since NK cells specialize in contact-dependent functions including cytotoxicity, interest has focused on the direct study of the interface between the NK cell and the cell with which it is interacting. This interface is also known as the immunological synapse and is characterized by an extraordinary number of dynamic molecular events that have the potential to result in NK cell function. Here we describe microscopy-based methods for evaluating and quantifying the NK cell immunological synapse that can be useful in enabling experimental studies.
Subject(s)
Immunological Synapses/immunology , Killer Cells, Natural/immunology , Actins/metabolism , Algorithms , Animals , Cell Line , Cell Survival , Humans , Immunological Synapses/metabolism , Killer Cells, Natural/cytology , Killer Cells, Natural/metabolism , Microscopy, Confocal , Microscopy, Fluorescence , Microtubule-Organizing Center/metabolism , Molecular Imaging , Protein Transport , Recombinant Fusion Proteins/metabolism , Software , Transduction, Genetic , TransfectionABSTRACT
Adeno-associated virus (AAV) vectors are effective gene delivery vehicles mediating long-lasting transgene expression. Data from a clinical trial of AAV2-mediated hepatic transfer of the Factor IX gene (F9) into hemophilia B subjects suggests that CTL responses against AAV capsid can eliminate transduced hepatocytes and prevent long-term F9 expression. However, the capacity of hepatocytes to present AAV capsid-derived antigens has not been formally demonstrated, nor whether transduction by AAV sensitizes hepatocytes for CTL-mediated destruction. To investigate the fate of capsids after transduction, we engineered a soluble TCR for the detection of capsid-derived peptide:MHC I (pMHC) complexes. TCR multimers exhibited antigen and HLA specificity and possessed high binding affinity for cognate pMHC complexes. With this reagent, capsid pMHC complexes were detectable by confocal microscopy following AAV-mediated transduction of human hepatocytes. Although antigen presentation was modest, it was sufficient to flag transduced cells for CTL-mediated lysis in an in vitro killing assay. Destruction of hepatocytes was inhibited by soluble TCR, demonstrating a possible application for this reagent in blocking undesirable CTL responses. Together, these studies provide a mechanism for the loss of transgene expression and transient elevations in aminotransferases following AAV-mediated hepatic gene transfer in humans and a potential therapeutic intervention to abrogate these limitations imposed by the host T cell response.
