Chronic NK cell activation results in a dysfunctional, tissue resident-like state mediated by KLF2 deficiency

Adoptive transfer of NK cells can induce complete remissions in 30-50% of patients with refractory acute myeloid leukemia and lymphoma. While blood chimerism occurs, attaining functional homing to the site of tumor without exhaustion has been elusive. During chronic infections and tumorigenesis, exposure to activating stimuli weakens the effector activity of NK cells. Despite this knowledge, there is little known about the mechanisms that govern this dysregulation and whether these disparate activating stimuli use distinct pathways to downregulate effector immunity. In this study, we reveal that chronic NK cell activating receptor (NKAR) stimulation and chronic IL-15 exposure impart distinct modes of dysregulation, with NKAR stimulation inducing a tissue resident-like state that resembles that of tumor-infiltrating NK cells in cancer patients. Using loss- and gain-of-function studies, we identify the transcription factor KLF2 as a master regulator of the NK cell response to chronic activation and provide evidence that KLF2 overexpression promotes NK cell cytotoxicity, cytokine production, and chemotaxis, and inhibits the development of dysfunctional, tissue resident-like features. Using KLF2 reporter mice, we show that in certain tissues, tissue resident NK cells are predominantly KLF2-negative while circulating NK cells in these tissues are overwhelmingly KLF2 + . Lastly, using mixed bone marrow chimeras, we demonstrate that conditional KLF2 deficiency in NK cells leads to altered homing and the acquisition of tissue resident-like features in vivo. Together, these findings highlight the profound changes NK cells undergo during prolonged activation and advance our understanding of how some NK cell therapies fail during malignant relapse. Overall design: Details of experimental design can be found within the associated publication. There are datasets from two separate figures in this directory. The following is a summary of design for the data in Figure 2: in brief, NK cells from healthy human donor peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with either NK cell activating receptor (NKAR) agonists (i.e. anti-NKp46 and MICA/B) or IL-15 for 4 or 10 days. 10 day stimulations were performed with either continuous IL-15 or with a 2-day (days 5 and 6) reduction in the concentration of IL-15. The following is a summary of design for the data in Figure 4: in brief, human NK cells were isolated as previously described and expanded using K562 leukemia cells expressing membrane-bound IL-21 and 4-1BB-L. Following expansion, NK cells were transduced with AAV vectors carrying either a control insert (mNG) or KLF2-overexpression vector (KLF2-mNG). Following transduction, cells were stimulated for 4 days on control or NKAR-agonist plates as previously described. Cell samples were also collected post-transduction, but prior to plate-bound activation.