Previous studies of NK cell inhibitory Ly49 receptors suggested their expression is stochastic. However, relatively few studies have examined this stochasticity in conjunction with activating Ly49 receptors. We hypothesized that the expression of activating Ly49 receptors is not stochastic and is influenced by inhibitory Ly49 receptors. We analyzed NK cell "clusters" defined by combinatorial expression of activating (Ly49H, Ly49D) and inhibitory (Ly49I, Ly49G2) receptors in C57BL/6 mice. Using the product rule to evaluate the interdependencies of the Ly49 receptors, we found evidence for a tightly regulated expression at the immature NK cell stage, with the highest interdependencies between clusters that express at least one activating receptor. Further analysis demonstrated that certain NK clusters predominated at the immature (CD27+CD11b-), transitional (CD27+CD11b+) and mature (CD27-CD11b-) NK cell stages. Using parallel in vitro culture and in vivo transplantation of sorted NK clusters, we discovered nonrandom upregulation of Ly49 receptors, suggesting that prescribed pathways of NK cluster differentiation exist. Our data infer that upregulation of Ly49I is an important step in NK cell maturation. Ki-67 expression and cell counts confirmed that immature NK cells proliferate more than mature NK cells. We found that MHC-I is particularly important for regulation of Ly49D and Ly49G2, even though no known MHC-I ligand for these receptors is present in B6 mice. Our data indicate that the regulatory systems controlling the expression of both activating and inhibitory Ly49 receptors are non-stochastic and support the idea that NK cell clusters develop in a non-random process correlated to their maturation stage.

16Previous studies of NK cell inhibitory Ly49 receptors suggested their expression is 17 stochastic. However, relatively few studies have examined this stochasticity in 18 conjunction with activating Ly49 receptors. We hypothesized that the expression of 19activating Ly49 receptors is not stochastic and is influenced by inhibitory Ly49 receptors. 20We analyzed NK cell "clusters" defined by combinatorial expression of activating (Ly49H, 21Ly49D) and inhibitory (Ly49I, Ly49G2) receptors in C57BL/6 mice. Using the product rule 22to evaluate the interdependencies of the Ly49 receptors, we found evidence for a tightly 23 regulated expression at the immature NK cell stage, with the highest interdependencies 24 between clusters that express at least one activating receptor. Further analysis 25 demonstrated that certain NK clusters predominated at the immature (CD27+CD11b-), 26 transitional (CD27+CD11b+) and mature (CD27-CD11b-) NK cell stages. Using parallel 27in vitro culture and in vivo transplantation of sorted NK clusters, we discovered non-28 random upregulation of Ly49 receptors, suggesting that prescribed pathways of NK 29 cluster differentiation exist. Our data infer that upregulation of Ly49I is an important step 30in NK cell maturation. Ki-67 expression and cell counts confirmed that immature NK cells 31 proliferate more than mature NK cells. We found that MHC-I is particularly important for 32 regulation of Ly49D and Ly49G2, even though no known MHC-I ligand for these receptors 33is present in B6 mice. Our data indicate that the regulatory systems controlling the 34 expression of both activating and inhibitory Ly49 receptors are non-stochastic and 35 support the idea that NK cell clusters develop in a non-random process correlated to their 36 maturation stage. 37 38 39 42 surveillance by the recognition and elimination of cellular targets. Unlike their T and B 43 lymphocyte counterparts, which acquire antigen-specific diversity through genetic 44 recombination events, NK cells generate germline-encoded receptors that can recognize 45 and lyse cellular targets by releasing perforin, granzymes, and secrete regulatory and 46 proinflammatory cytokines. Activating and inhibitory Ly49 receptors employ NK cell 47 signaling pathways, which dictate NK cell effector functions through cytoplasmic 48immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-49based activating motifs (ITAM) (1-4). NK cell subsets are thought to utilize a "rheostat" 50 process in which NK cell subsets are tuned quantitatively by self-MHC class I ligands 51corresponding to specific Ly49 inhibitory receptors, which in turn provides a diversity of 52 responsiveness towards cellular targets (5, 6). Through this mechanism, NK cells 53 distinguish healthy from unhealthy cells. However, what regulates the acquisition of 54 specific NK cell Ly49 receptors during NK cell development and maturation is still an 55 unanswered and complex question. In addition, how models derived from the biology of 56Ly49 inhibitory receptors ...