Grants Awarded

Identification of Molecular Mechanisms Underlying Defective Cytotoxic Lymphocyte Function in Chediak-Higashi Syndrome, a Histiocytic Lysosomal Storage Disorder with Immunodeficiency

Principal Investigator
Konrad Krzewski, PhD
National Institute of Allergy and Infectious Diseases, National Institutes of Health
Rockville, Maryland USA

Date of Award
December 2015

Amount of Award

Layperson Summary
Natural Killer (NK) cells belong to a subset of white blood cells that is very important for our organism defense. NK cells have the abilityto kill abnormal cells, and it has been proposed that inability of NK cells to kill could lead to uncontrolled proliferation of histiocytes andlymphocytes, resulting in a life-threatening condition referred to as lymphohistiocytosis. Indeed, many histiocytic disorders are characterized by the presence of NK cells that cannot kill their targets. One such disorder is Chediak-Higashi syndrome (CHS), a disease caused by mutations in the gene encoding the lysosomal trafficking regulator, LYST. The majority of affected persons develop fatal histiocytosis. We know that NK cells are not efficient in killing their targets in CHS, which implicates LYST as a protein important for normal NK cell function. Very recently, our group has discovered that mutations in different regions of LYST are associated with at least two distinct forms of cellular defects, indicating that CHS is a very complex disease at a molecular level. However, until we get a thorough understanding of the precise stages of NK cell activity that are blocked by LYST mutations, and how LYST fits into the complicated pathways of NK cell signaling, our options to treat the disease and help the patients are greatly restricted. Studying and understanding CHS is limited by the insufficientavailability of patient samples and the lack of means to detect LYST protein. To overcome these difficulties, we propose to (i) generate ahuman cell model of CHS (because animal models are inadequate to fully reproduce cellular defects observed in CHS), and (ii) produceantibodies capable of detecting LYST protein. To this end we will use the most modern gene editing techniques to create NK cell lines mimicking the cellular defects of CHS, and provide a tool for a long-term study of the disease mechanisms. In fact, we have already successfully generated one of the proposed human cell models of CHS, and we are starting to make a second model. Next, we will use state-of-the-art microscopic techniques to examine the size, shape, content and movement of the lysosomal vesicles in the model CHS cells. Using multiple biochemical and flow cytometry methods, we will verify whether the defective killing of target cells by CHS NK cells is due to altered function of lysosomes. We will isolate LYST protein from unaffected NK cells, and investigate what other proteins bind to LYST. Finally, the newlyproduced antibodies against LYST will allow us to examine protein levels in CHS patient samples, providing a useful tool for diagnosisof CHS. The outcome of this study will not only be an in-depth understanding of molecular mechanisms underlying defective NK cell activity inCHS, but also will provide novel insights into NK cell function in histiocytic disorders in general. Our studies will uncover importantaspects of cytotoxic lymphocyte function that will have implications for several diseases with histiocytosis, such as familial hemophagocytic lymphohistiocytosis or Hermansky-Pudlak syndrome. Our proposal of discovering and understanding molecular mechanisms behind the histiocytic diseases will provide the first step in designing new, better and more efficient therapies for the persons suffering from histiocytic disorders.

Twelve Month Report
Defects in lytic granule secretion from cytotoxic lymphocytes are associated with life-threatening conditions, such as hemophagocytic lymphohistiocytosis (HLH). Chediak-Higashi syndrome (CHS) is a rare disorder caused by mutations in the LYST gene, resulting in formation of giant lysosomes or lysosome-related organelles (e.g., lytic granules) in several cell types. The disease is characterized by immunodeficiency and development of a fatal HLH due to impaired function of cytotoxic lymphocytes, mainly Natural Killer (NK) cells. Understanding the mechanism(s) responsible for defective exocytosis and, consequently, cytotoxicity of NK cells could provide a key factor to therapy of CHS, and the syndrome-associated HLH. Therefore, we sought to determine the underlying biochemical cause of the impaired cytotoxicity in CHS NK cells.

The extremely limited availability of patient samples is one of the major obstacles blocking the progress of understanding CHS, or similar disorders. Although few animal models of CHS exist, none of them fully reproduces the human disease. Therefore, we generated a new human cell model of CHS using modern genome editing technology. This model fully mirrors all aspects of the cellular phenotype observed in NK cells isolated from CHS patients, including the lack of LYST expression, the presence of significantly enlarged lytic granules with faulty exocytosis, and defective cytotoxicity. Using multiple biochemical, microscopic, and cytometric methods, we found that NK cell activation is not affected by loss of LYST function, and the large lytic granules in CHS NK cells are functional and have the protein machinery components required for their release. This led us to pursue alternative explanation for their defective secretion in CHS. With the use of super-resolution microscopy, we discovered that the actin cytoskeleton at the cell-cell contact site creates a barrier for release of those large granules, which consequently leads to defective CHS NK cell degranulation and cytotoxicity. Importantly, utilizing combination of gene silencing and small molecules interfering with actin assembly, we demonstrated that decreasing the actin meshwork density, or decreasing the lytic granule size, restores the ability of LYST-deficient NK cells to degranulate and kill target cells, suggesting new possibilities for CHS therapy.

Collectively, our data indicate that the problems resulting from LYST deficiency are related to physical hindrances that affect cell functionality, and that restoration of cytotoxic lymphocyte function is possible in CHS. This not only provides better insights to the pathology of this rare condition, but also offers a new direction for a therapeutic approach to treat this disease. Identification of compounds that could help relaxation of the actin cytoskeleton barrier warrants further investigation, as it could provide a new avenue to restore cytolytic function of CHS cytotoxic lymphocytes. This, in turn, would have a tremendous impact for the patients’ welfare, as it could avert the development of HLH, or extend the period of time CHS patients could wait for a hematopoietic stem cell transplant required for HLH prevention.