Grants Awarded

Matthias Voss, PhD
Karolinska Institutet
Stockholm, Sweden

Date of Award
December 2015

Amount of Award
$50,000

Layperson Summary
The human immune system is equipped with specialized cells that selectively kill virus-infected cells and thus prevent virus spread within the body. In infant patients with familial hemophagocytic lymphohistiocytosis (FHL), a heritable immunodeficiency syndrome, these specific cells cannot kill their target cells. As consequence, virus infection persists leading to massive systemic inflammation in these patients. This condition is fatal unless patients receive bone marrow transplantation. Through genetic studies, we know that the failure of “killer” cells inFHL patients is largely due to mutations disrupting genes implicated in a process called exocytosis. It is through exocytosis that cellsrelease substances and communicate with other cells. Interestingly, also the killing process exploits this machinery and cytotoxic bloodcells directly secrete toxic molecules into a narrow gap formed between themselves and their respective target cells. While we know now that this process is disturbed in FHL, the finer details of its regulation are not understood and disease in some patients can still not be explained. The outlined research strategy will provide a major technical advancement in the field that can be exploited to further deepen our insights into the processes underlying development of FHL. A recent major breakthrough in molecular biology research was the description of the CRISPR/Cas9 system and its implementation for genome editing. We will adapt this technology, establishing an experimental platform in cytotoxic cells that will enable us to address open questions in FHL research. This platform will be an invaluable tool to systematically evaluatemutations identified in patients. Moreover, this technology at hand, we will specifically dissect the individual contributions of distinctMunc13-4 isoform to lymphocyte cytotoxicity. While the role of Munc13-4 in this process is established, we recently uncovered a novel variant of Munc13-4, but the physiological role of this isoform remains unclear. Moreover, we will use this technology to interrogate the physiological function of LYST. It has been known for 20 years that mutations in LYST underlie Chediak-Higashi syndrome, which also features hemophagocytic lymphohistiocytosis. However, due to technical obstacles, LYST function remains still a mystery. With the new technology, we can investigate LYST function in order to understand how its defects contribute to disease.

Twelve Month Report
Familial hemophagocytic lymphohistiocytosis (FHL) is a life-threatening disease affecting primarily infants or young children. It is caused by inborn gene defects that compromise the function of a certain type of immune cells referred to as cytotoxic lymphocytes, which include cytotoxic T cells and natural killer (NK) cells. These cells have cytotoxic granules, small subcellular vesicles that contain cytotoxic effector molecules, and are specialized to distinguish cancerous or virus-infected from healthy cells in the human body. Upon encounter of the former, the content of the cytotoxic granules is released to kill the malignant or infected target cell. In patients with FHL, the function of cytotoxic granule effector molecules or of factors mediating the granule formation or their release is disrupted due to genetic defects and cytotoxic lymphocytes cannot kill target cells. This causes sustained stimulation of immune responses culminating in a hyperinflammatory state and hemophagocytic lymphohistiocytosis.

While the identification of FHL-causing mutations in genes like UNC13D, STX11 and STXBP2 (causing FHL type 3, 4 and 5, respectively) has revealed the instrumental role of the vesicle fusion machinery during cytotoxic granule release, such insights have at the same time uncovered new key questions relating to cytotoxic granule biology and FHL pathogenesis that have remained unanswered. My research is dedicated to further deepen our molecular understanding of these processes and to characterize new gene defects found in FHL patients.

With the support of the Histiocytosis Assocation, last year’s project aimed to explore the use and application of recently developed gene editing technology to obtain novel cell culture models tailored to experimental dissection of cytotoxic granule exocytosis. To this end, adequate CRISPR/Cas9 reagents were selected and optimized and delivery options were explored. Due to the need for long-term culture of genome-edited cells, NK cell lines were chosen as model system and mRNA transfection techniques proved promising in terms of reagent delivery. With these tools at hand, future efforts can now be directed towards applying this technology to study the individual role of Munc13-4 protein isoforms in granule exocytosis. Moreover, a more intricate genome editing strategy to investigate the still elusive function of LYST was initiated. Mutations in LYST underlie another inborn disorder, Chediak-Higashi syndrome, and affected patients develop life-threatening hemophagocytosis as well. As part of my mentor’s long-term effort to identify novel mutations causing inborn immunodeficiency syndromes in children (like FHL), mutations in a gene relating to the proper function of cytotoxic granule effector molecules were characterized and, finally, I contributed to the characterization of mutations causing a new human syndrome entailing NK cell immunodeficiency.

To conclude, the generous support by the Histiocytosis Association has enabled me to establish line of research and already providing new insights into the biology of cytotoxic lymphocytes to tightly linked to FHL pathogenesis. I will continue to benefit from this initial grant in the future as this grant will be a crucial foundation to new insights in the future.