Dottorato in INNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Titolo della tesi: The role of the low affinity Fcgamma receptor IIIA (CD16) in the shaping of phenotypic and functional profile of memory NK cells

Human memory NK cells expand and persist in response to human cytomegalovirus (HCMV) infection. They are a partially understood phenotypically and functionally heterogeneous population, characterized by the lack of Fcepsilon Receptor I gamma chain responsible, together with TCRzeta chain, for Fcgamma receptor IIIA (CD16)-mediated signalling. They display the preferential, not fully overlapping, expression of NKG2C (activating receptor for HLA-E) and CD57 maturation marker. Hyperresponsivity to CD16 engagement represents the distinctive functional signature of memory NK cells. Short-term CD16 stimulation was shown to enhance glycolytic and oxidative pathways of human NK cells, but CD16 capability to induce a persisting metabolic reprogramming on NK cells, and the metabolic peculiarities of FcepsilonRIgamma- memory NK cells, are partially studied. We evaluated the capacity of CD16 receptor to regulate the expansion, maintenance, and subset composition of memory NK cell pool in vivo and in vitro in physiological and pathological contexts. We also investigated the impact of CD16 stimulation on CD98, CD71 and GLUT1 nutrient transporter expression of FcepsilonRIgamma- memory and FcepsilonRIgamma+ conventional NK cells, and the role of neutral aminoacids in CD16-induced IFN-gamma secretion. Interindividual differences in memory NK cell pool size and subset composition associated with anti-HCMV IgG levels in vivo, in a large cohort of HCMV+ healthy individuals. HCMV+ Immune Thrombocytopenia (ITP) patients, producing anti-platelet and -megakaryocyte autoantibodies, showed a perturbed composition of the memory population. The relative abundance of NKG2C+CD57+ memory subset was most strongly associated with antibody (or autoantibody) levels in healthy donors and ITP patients, and exhibited the highest in vitro proliferative capacity in response to IgG-opsonized target cells or ITP platelets and the most marked CD16-dependent IFN-gamma production. Peripheral blood memory NK cells displayed higher levels of CD98 neutral aminoacid antiporter and CD71 transferrin receptor than conventional ones. These transporters were upregulated in response to chronic contact with IgG-opsonized target cells (1-3 days), in an mTOR-dependent manner, and with a seemingly different kinetics on memory and conventional NK cells. CD98-mediated neutral aminoacid uptake was higher in memory cells; its blockade decreased CD16-triggered IFN-gamma production in both FcepsilonRIgamma- and FcepsilonRIgamma+ NK cells, without altering memory NK cell relative hyperresponsiveness. Conversely, GLUT1 major glucose transporter was expressed at lower levels in memory NK cells and downregulated by CD16 engagement on both memory and conventional populations. Collectively, we dissected the functional and phenotypical heterogeneity of the HCMV-driven memory NK cell compartment and found that chronic CD16-mediated stimulation promotes the expansion and maintenance of the memory pool, particularly of the most responsive NKG2C+CD57+ subset. Our work also documents that CD16 engagement leads to a persistent metabolic rewiring of human NK cells and suggest that a distinct nutrient transporter expression pattern may contribute to memory NK cell peculiar functional features.