The process of Natural Killer (NK) cell adhesion to target cells resulting in NK cell activation and cytotoxicity has been the subject of intense research. However, only very little is known about the detachment of NK cells from target cells after the lytic hit. Since NK cells are able to kill multiple targets in a serial manner, this detachment process could play a pivotal role for an effective cytotoxic NK cell response. We therefore established different assay systems, based on flow cytometry and microscopy, to study NK cell detachment in more detail. Using these assay systems we could demonstrate that recently detached NK cells showed signs of productive conjugates. We found evidence for degranulation and observed down-modulation of activating receptors. This was possibly caused by internalization after receptor engagement, a mechanism suggested to tune down the strength of the interaction between cells. In contrast, plasma membrane-bound metalloproteinases that mediate shedding of surface molecules and thereby also possibly decrease contact between effector and target cell, were not required. In our flow cytometry-based system cultured human NK cells detached from K562 cells with a halflife of about 50 minutes. A similar trend was observed for the half-lives of NK:HeLa cell-contacts analyzed using life cell-microscopy. The maintenance of the NK:K562 conjugates required sustained early activating signaling, e.g. via Src- and Syk-family kinases, and was ATP-dependent. In contrast, MAP kinase and also PKC! activity did not affect the formation nor the decay of conjugates. To investigate the involvement of inhibitory pathways in the detachment process we interfered with phosphatases and found that the serine-threonine phosphatase PP1 was important for the stability of conjugates. In addition to ongoing signaling, the flexibility of the actin cytoskeleton was indispensable to maintain NK:target cell contacts, whereas dynamics of microtubules had only limited impact. Investigating the influence of target cell availability on the detachment revealed that contact to a new target accelerated the separation from a previous one. Therefore, the contact to a target may keep the NK cell in an activated status and thereby may support serial killing. To investigate the role of target cell viability on detachment we interfered with the target cell survival on various levels. Inhibition of myosin IIA, which is involved in exocytosis of lytic granules, led to reduced lysis and moderately decelerated detachment. Depletion of mature perforin by inhibition of the vacuolar H+-ATPase led to a complete loss of NK cell cytotoxicity, while the formation of NK:target cell conjugates remained unaffected. Interestingly, this resulted in a drastic deceleration of NK cell detachment. If the loss of perforin was caused by monensin, a proton ionophor, lysis of target cells was abolished but the half-life of NK:target cell conjugates remained unaffected. Taken together these data indicate that not target cell survival as such, but some NK cell intrinsic mechanisms affect the detachment process.In summary, we established assay systems that allow investigation of the detachment process from target cells and we found first evidences that maintenance of conjugates and the detachment are regulated processes. Thus, understanding the mechanisms leading to detachment may facilitate efforts to increase the efficiency of NK cell-mediated cytotoxicity.
|Supervisor:||Dick, PD Dr. Tobias|
|Date of thesis defense:||8 April 2014|
|Date Deposited:||15 Apr 2014 09:31|
|Faculties / Institutes:||The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences|
|Subjects:||570 Life sciences|