Role of chemokine/chemokine receptor axes in the regulation of bone marrow NK cell localization in physiological and pathological conditions

Natural Killer (NK) cells represent a lymphocyte population of the innate immunity, involved in the control of viral infections and tumor cells. Among the wide number of functions that NK cells can mediate, the most important are the ability to lyse target cells via the release of cytotoxic granules, and the production of pro-inflammatory mediators, such as cytokines and chemokines. In mouse, according to the expression of the surface markers CD11b and KLRG1, within the NK cell population 4 discrete subsets can be distinguished, which represent different developmental stages and show heterogeneous functional capacities. Moreover, their tissue localization is dissimilar in primary and secondary lymphoid organs, and in non-lymphoid organs: the most immature populations are mainly represented in the bone marrow (BM), thymus and lymph nodes (LN), while the most mature ones preferentially distribute in blood, spleen, liver and lungs. Chemokines are chemotactic molecules of proteic nature, which bind specific GPCR receptors, and direct the migration of several cell types through the formation of a concentration gradient. NK cells express a great variety of chemokine receptors, which regulate their organ distribution and their trafficking to inflamed tissues. The first part of this work dealt with the study of the function of CX3CR1 receptor, specific for the chemokine CX3CL1, in the NK cell positioning in the BM microenvironment. To this aim, a murine knock-in model, in which one or both copies of cx3cr1 gene were replaced by GFP cDNA, was used. Through the analysis of GFP expression it was possible to track CX3CR1+ NK cells in heterozygous mice, or the cells that should express CX3CR1, but express only GFP, in the homozygous mice. Data obtained by adoptive transfer experimental approaches, single or mixed BM chimeras and the phenotyping of NK cells in BM compartments, demonstrated that CX3CR1 is expressed by a terminally differentiated subset, which mainly resides in the vascular compartment of the BM, and displays reduced effector functions. Moreover, CX3CR1 was associated to a positive regulation in the exit of most mature NK cells from the BM towards the periphery, similarly to what was previously demonstrated for S1P5. Data regarding these studies have been published in two international peer-reviewed journals. The second part of this thesis work was centered on the influence of multiple myeloma (MM) growth on the functional and trafficking capacities of BM NK cells, in a murine model of this pathology. MM was followed at different growth stages, and we observed the selective exclusion from BM of the most functional NK cell subpopulation (KLRG1-), at an intermediate stage of tumor progression. Adoptive transfer experiments on healthy controls and MM-bearing mice have elucidated that the KLRG1- NK cell reduction was attributable to a trafficking alteration within BM. We analysed the mechanism of this phenomenon, by studying the production of several chemokines and the expression of many chemokine receptors on BM NK cells in presence or absence of MM. At the same stage of MM growth we observed a decrease of CXCR3 expression, while its ligands CXCL9 and CXCL10 were augmented in tumor-bearing mice. Furthermore, CXCL9 concentration was higher as compared to healthy controls also in the sera in MM-bearing mice.In parallel, we highlighted that CXCR4/CXCL12 axis was altered too, as CXCL12 level was significantly lower in the BM of tumor-bearing mice. With competitive adoptive transfer experiments, we demonstrated that CXCR3+ NK cells are selectively excluded from BM, and accumulate in liver and spleen. Globally these data suggest a strategy for the tumor cells to evade the NK-cell mediated effect in the tumor microenvironment, by altering directly or indirectly different chemokine/receptor axes, important for BM NK cell migration.