Abstract Natural killer (NK) cells are an important component of the innate immune system and are capable of killing virally infected cells and malignant cells. Autoimmune NK activity is avoided by inhibitory receptors such as the killer Ig-like receptors (KIR) that recognize self-human leukocyte antigens (HLA) class I molecules present on all autologous tissue cells. NK cells, however, frequently express KIRs for which the individual lacks the appropriate HLA class I ligand, and yet these NK cells do not exhibit autoreactive behavior. This has led to the licensing model where only NK cells expressing KIR for self-HLA ligands are functionally competent and capable of effector activity. How NK cells expressing KIR for self-HLA achieve functional competence while NK cells expressing KIR for non-self HLA achieve tolerance to self is the focus of our proposed studies. Using 6-color flow cytometry for single cell analysis of NK response to study individuals whose functional KIR repertoire is comprised nearly completely by inhibitory receptors, we demonstrate that in the steady state individual, expression of inhibitory KIR for self-HLA class I confers functional competence to the NK cell as assessed by intracellular IFN- production to class I negative targets. Furthermore, there is a correlation between increased effector function and: 1) higher qualitative numbers of KIR specific for self-HLA, and 2) higher NK expression of HLA. We hypothesize that functional competence in part results from HLA and KIR molecules inherent to the cell, whose interaction is necessary for effector function. In Specific Aim 1, using lentiviral constructs to introduce class I shRNA or KIR/HLA cDNA, we will study how altered expression of KIR or HLA in primary NK cells and NK cell lines perturbs the functional capacity of NK cells. Following allogeneic hematopoietic cell transplantation (HCT), we have found that NK cells expressing KIR for non-self HLA are initially endowed with functional capacity, but become hyporesponsive and tolerant to self by day 100. This observation supports the model that the nave NK cell achieves self-tolerance after a period in the circulation of interaction with autologous cells, transitioning from a state where all inhibitory KIR receptors are capable of recognizing lack of ligand (missing ligand) to a mature state where the appropriate inhibitory KIR recognize lack of self- ligand (missing self). Allogeneic HCT offers a unique in vivo environment in which hematopoiesis in the early post-transplant period provides a window to examine NK cell development. We hypothesize that functional competence may be sustained through trans-interaction with HLA- expressing autologous cells, and potentially autoreactive cells expressing KIR for non-self HLA are rendered anergic through lack of class I engagement. Specific Aim 2 seeks to demonstrate that cells that would otherwise adopt a hyporesponsive fate can retain functional competence if cultured during development in an environment expressing non-self class I ligands as presented by allogeneic Langerhans cells or stromal cell transfectants. Elucidating how NK cells achieve functional competence not only has implications for NK alloreactivity in the transplant setting, but also for judicious application of NK cells as adoptive cellular therapy for malignancies.