Giraudo Research

Cytotoxic T-lymphocytes (CTL) and Natural Killer (NK) cells are critical components of the innate and adaptive immune system responsible for destroying virus-infected and cancer cells. CTLs and NK cells perform this fundamental function by releasing apoptotic proteins contained within lytic granules at the contact area with a target cell known as Immunological Synapse. Patients with defects in these processes most commonly manifest with Familial Hemophagocytic Lymphohistiocytosis (f-HLH), a fulminant Primary Immunodeficiency characterized by a disproportionate dysregulation of the immune system.  In particular, my lab is interested in determining how gene mutations affect the molecular machinery that controls the membrane fusion steps required for the cytotoxic immune response. By providing a better understanding of WT and mutant FHL-associated proteins, our studies will shed new light on novel pathways and proteins that control cell-killing by CTL and NK cells. To study how these processes are achieved we utilize multidisciplinary approaches including biochemical reconstitution, proteomics, biophysical analysis, live cell imaging, Super-resolution Microscopy (STED) and Total Internal Reflection Microscopy. Also, the lab specialized in developing novel simplified fusion assays that will help us to investigate the biological significance of novel mutations associated with f-HLH and more generally with PIDs. In summary, our studies will contribute more broadly to our understanding of the membrane trafficking steps involved during cell-mediated immune responses, establish common and gene-specific pathways affected in a wide range of Primary Immune Deficiencies (PID).

Very early onset inflammatory bowel disease (VEO-IBD) is characterized by severe intestinal inflammation affecting infants and children less than six years of age.  A subset of patients has been identified to have highly penetrant homozygous recessive or dominant mutations in genes affecting a myriad of immune-related pathways and intestinal barrier function. Strikingly, there is a significant number or VEO-IBD patient with abnormal cytotoxic immune responses that manifest with Immune Dysregulation Disorder such as FHL, Primary Immune Deficiency, and Autoimmunity. However, the underlying pathophysiological mechanisms of VEO-IBD is still poorly understood. The Giraudo Lab, in collaboration with Dr. Judith Kelsen and Kathleen Sullivan (Directors of VEO-IBD Program of The Children's Hospital of Philadelphia), is currently interested in understanding how genetic mutations found in this cohort of patients affect basic cellular and molecular functions of both immune and intestine epithelial cells.  To do this, we apply cell biological and biochemical test, as well as functional assays and cell profiling on patient samples. Our goal is to gain insights into novel pathways shared by CTLs and Intestine cells that could be used for diagnostic purposes and eventually, be manipulated for potential therapeutic approaches.

The Giraudo lab host the Center for Immune Disorders and Human Translational Immunology (CIDHTI). The CIDHTI harnesses the expertise of a team of basic immunologists with cutting-edge technologies to provide support on diagnostic, genetic testing, and identification of molecular defects in patients with rare Immune Dysregulation Disorders. The Center has an approved IRB protocol to serve as a repository of patient samples, perform immune cell profiling, run an array of biochemical, genetics and functional assays to test various cellular functions. Our goal is to discover novel defective pathways that could be exploited for targeted therapies for these conditions as well as to explore innovative approaches to fight cancer cells. The CIDHTI is open to establishing collaborations with doctors from the TJU-network and from different Institutes around the World to assist on the care of patients with rare immunological disorders. Among the disorders that we study are Primary Immune Deficiencies, Immune Disorders of Hyperinflammation and Genetics autoimmune/inflammatory disorders.

Regulation of membrane fusion is a fundamental biological process, which is central to many areas of endocrine and exocrine physiology, and imbalances in these processes give rise to important diseases, such as diabetes, brain disorders and immunodeficiency. All these processes rely upon the assembly between vesicular (v-) SNARE and target (t) SNARE proteins into a single SNARE complex, which brings the bilayers into close proximity and triggers their fusion. In regulated exocytosis, there are additional proteins such as Sec1/Munc18 (SM) family proteins and calcium-sensing proteins that control membrane fusion through their interactions with a specific set of SNARE proteins in different cell types. How such proteins accomplish this, or potentially contribute directly to the bilayer fusion event itself, has been difficult to discern with any precision. The goal of this project is to bridge this gap in knowledge by focusing on the exocytosis of lytic granules (LG) by Cytotoxic T-Lymphocytes (CTLs) as a model of regulated exocytosis. LG exocytosis provides a unique angle to investigate the functions of SM- and calcium-sensing proteins on membrane fusion because of its intrinsic slow kinetics, the well-defined step-wise process and the additional energetic barrier for regulation posed by the atypical lipid-anchored SNARE involved in the fusion reaction. To achieve this we perform detailed protein:protein interaction analyses using recombinant proteins and immunoprecipitation from cell lysates, functional analyses using fusion assays, optical tweezers to assess the strength of forces on single SNARE complex. Additionally, we use TIRF and super-resolution microscopy to analyze genetically-modified human CTLs for trafficking and study dynamics of single LGs at distinct fusion steps at the IS, formed with either target cells or artificial surfaces mimicking physiological condition. Our studies will contribute to a new, in-depth understanding of the perforin-dependent cell-killing pathway as a model of regulated exocytosis and begin to dissect the roles for f-HLH-associated proteins and other factors in LG exocytosis. Our studies will also contribute more broadly to our understanding of the regulation of granule exocytosis in general and will establish common and cell-type specific ways by which SM and calcium-sensing proteins control the SNARE fusion machinery.