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Natural killer (NK) cells recognize virally infected cells and tumors. NK cell function depends on balanced signaling from activating receptors, recognizing products from tumors or viruses, and inhibitory receptors (such as KIR/Ly49), which recognize major histocompatibility complex class I (MHC-I) molecules. KIR/Ly49 signaling preserves tolerance to self but also conveys reactivity toward MHC-I-low target cells in a process known as NK cell education. Here, we found that NK cell tolerance and education were determined by the subcellular localization of the tyrosine phosphatase SHP-1. In mice lacking MHC-I molecules, uneducated, self-tolerant Ly49A+ NK cells showed accumulation of SHP-1 in the activating immune synapse, where it colocalized with F-actin and the signaling adaptor protein SLP-76. Education of Ly49A+ NK cells by the MHC-I molecule H2Dd led to reduced synaptic accumulation of SHP-1, accompanied by augmented signaling from activating receptors. Education was also linked to reduced transcription of Ptpn6, which encodes SHP-1. Moreover, synaptic SHP-1 accumulation was reduced in NK cells carrying the H2Dd-educated receptor Ly49G2 but not in those carrying the noneducating receptor Ly49I. Colocalization of Ly49A and SHP-1 outside of the synapse was more frequent in educated compared with uneducated NK cells, suggesting a role for Ly49A in preventing synaptic SHP-1 accumulation in NK cell education. Thus, distinct patterning of SHP-1 in the activating NK cell synapse may determine NK cell tolerance., QC 20240829

Studies of patients with inborn errors of immunity provide an important opportunity to understand the human immune system in a natural context. The articles in this thesis are a compilation of such investigations. With our work, we aimed to identify the cause of primary hemophagocytic lymphohistiocytosis (HLH) in an unexplained pediatric patient (paper I), explore the modulation of SAMD9 and SAMD9L gain-of-function (GOF) mutants (paper II), and understand natural killer (NK) cell biology in the context of DEF6 deficiency (paper III). In paper I, we uncovered biallelic loss-of-function variants in RHOG in a 4-monthold patient presenting with HLH and displaying defective lymphocyte exocytosis. Deletion of RHOG in a human NK cell line abrogated exocytosis that could be rescued by constructs expressing wild type RHOG protein. Moreover, we found that MUNC13-4, associated with autosomal recessive familial HLH type 3, required RHOG interactions for recruitment to the plasma membrane during cytotoxic granule exocytosis. Thus, we demonstrated that RHOG is essential in cytotoxic granule exocytosis by human T and NK cells and proposed that biallelic loss-offunction mutations in RHOG are a novel cause for familial HLH. In paper II, we examined a variety of pathogenic SAMD9 and SAMD9L GOF variants associated with syndromes encompassing bone marrow failure, autoinflammation or selective loss of NK cells, B cells, and monocytes. We sought to understand whether viral host range factors, which are known to antagonize wild-type SAMD9 and SAMD9L proteins, could counteract the anti-proliferative and anti-translational activities of these pathogenic variants. SAMD9 or SAMD9L variants and viral factors were overexpressed in a cell line, followed by biochemical and functional analyses. Vaccinia virus K1 exhibited the highest inhibitory capacity but could not antagonize a truncated variant of SAMD9L that lacked the K1 binding site. The other factors (M062, C7 and KI) could interact with al