Abstract Natural killer (NK) cell tolerance to self is incompletely understood despite wide-spread acceptance of the now familiar “missing-self” hypothesis. Serving as a guiding principle for several decades, it proposed that NK cells survey tissues for ubiquitously expressed major histocompatibility complex class I (MHCI) molecules as self. Normal levels of MHCI do not allow NK cell attack but if MHCI is down-regulated in a pathologic event, NK cells attack. The applicant and his laboratory discovered the Ly49 family of receptors specific for target cell MHCI molecules that inhibit NK cell activation receptor function, providing a basis for understanding the missing-self hypothesis. However, some predictions of the missing-self hypothesis were not observed, such as hyper-reactive NK cells in MHCI-deficient hosts, rather hypo-responsive NK cells were found. This can now be explained by other findings from the applicant's laboratory that the inhibitory Ly49 receptors have a second function to license or educate NK cells to self-MHCI, such that licensed NK cells have functionally competent activation receptors. Meanwhile, other issues. For example, in prior studies, the applicant's lab showed that different MHCI alleles appear to have graduated effects on Ly49 functions, suggesting signaling strength accounts for these functions, possibly due to Ly49 affinities for self-MHCI, in part related to the rheostat model for receptor function that has not been well studied. Moreover, MHCI affects the repertoire of Ly49s that are expressed in a variegated manner with more than one Ly49 per NK cell. Data from the applicant's laboratory suggest that signaling by a self-MHCI-specific Ly49 influences expression of another Ly49 that is self-MHCI- specific, potentially providing an explanation for how MHCI alleles affect the Ly49 repertoire. Finally, it is not known how the Ly49s confer licensing, such as the possibility that the inhibitory receptors may directly signal the licensing phenotype. Herein the applicant proposes to study NK cell tolerance utilizing novel mice recently generated in his laboratory, including knockout mice lacking all Ly49 expression on conventional NK cells and knockin mice with essentially monoclonal expression of a single Ly49 on all NK cells. Therefore, the Specific Aims of this proposal are to study: 1) Ly49 affinity for self-MHCI in licensing, effector inhibition and missing-self; 2) Establishment of the Ly49 repertoire; and 3) Inhibitory Ly49 signaling. Thus, these studies will markedly enhance our understanding of NK cell tolerance.