Final Report Summary - SEQ-CYTOTOX (Next generation sequencing for detection of human primary immunodeficiency syndromes characterized by impaired cytotoxic lymphocyte function)
The project has provided new and unexpected knowledge of the human immune system, contributing to understanding of primary immunodeficiencies in patients. Our findings can briefly be summarized in three main points. First, analyses of healthy volunteers with respect to blood cytotoxic lymphocyte phenotype and function uncovered unprecedented variation in signaling protein expression in NK cell subsets that we associated with CMV infection. This provided entirely new insights to the differentiation and function of so-called adaptive or memory-like NK cells, demonstrating that cells formerly considered as short-lived innate immune cells can also establish long-term forms of immunological memory. A series of papers from our group shows for the first time that lymphocyte subsets can silence expression of key, membrane proximal signaling proteins through DNA methylation, with important functional consequences, including control of malignancies. Thus, some of the insights gained by our studies are of potential interest for rational design of cellular immunotherapy of cancer. Second, we have detailed how non-coding mutations can cause disease and gained insights to novel genes and disease associations. Specifically, insights into regulation of UNC13D, a gene encoding a protein required for the release of granule content from killer cells of the immune system, uncovered new versions of the protein that regulate this process. This study thereby provided first insights to how non-protein coding mutations can cause primary immunodeficiency diseases. Our findings of non-coding aberrations in UNC13D as a common cause of life-threatening familial hyperinflammatory syndromes, particularly in the Swedish population, paved the way for development of neonatal screening methodology that we hope will be implemented in nation-wide newborn screening programs in Sweden enabling rapid, effective, and safe bone marrow transplantation. Furthermore, mutations in RAB27A, another gene encoding a protein required for the release of granule content from killer cells, was shown to be distinctly expressed by different cell types. In the case of associations between IFN-gamma receptor deficiency and hyperinflammatory syndromes, we have identified unexpected disease associations that have important implications for the potential use and efficacy of anti-IFN-g therapy that is current in clinical trials for the treatment of hyperinflammatory syndromes. Remarkably, we also uncovered that gain-of-function mutations in SAMD9L are a cause of a syndrome of bone marrow failure, immunodeficiency, predisposition to cancer, and neurological disease. These findings explain the etiology of a significant proportion of patients with hematological malignancy involving chromosome 7, typically related to myeloid malignancies with poor prognosis. Third, having established live-cell imaging of cytotoxic lymphocytes in the lab, we have gained completely new insights to how cytotoxic lymphocytes release granule contents to kill target cells. Finding that VAMP8-mediated recycling endosome exocytosis is a prerequisite for cytotoxic granule fusion with the plasma membrane, we have posited a new model for the molecular regulation of cytotoxic granule release. These studies raise many questions to the mechanisms of lymphocyte cytotoxicity, which we believe can be answered by further interdisciplinary studies of primary immunodeficiency patients.