Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level

Willkommen in der Schule. Integration neu zugewanderter Schülerinnen und Schüler.

Otto J, Migas K. Willkommen in der Schule. Integration neu zugewanderter Schülerinnen und Schüler. In: Smolka D, ed. Integration als Leitungsaufgabe - Konzepte und Beispiele für Schulen. Köln: Carl Link Verlag/Wolters Kluwer; 2017: 109-113

Integration neu zugewanderter Kinder und Jugendlicher ohne Deutschkenntnisse. Möglichkeiten, Herausforderungen und Perspektiven.

Otto J, Migas K, Austermann N, Bos W. Integration neu zugewanderter Kinder und Jugendlicher ohne Deutschkenntnisse. Möglichkeiten, Herausforderungen und Perspektiven. Münster: Waxmann; 2016

Integration neu zugewanderter Kinder und Jugendlicher ohne Deutschkenntnisse. Möglichkeiten, Herausforderungen und Perspektiven.

Otto J, Migas K, Austermann N, Bos W. Integration neu zugewanderter Kinder und Jugendlicher ohne Deutschkenntnisse. Möglichkeiten, Herausforderungen und Perspektiven. Münster: Waxmann; 2016

Interkulturelle Kompetenz von Lehrkräften. Mythos, Trend oder pädagogische Notwendigkeit?

Otto J, Migas K, Järvinen H, Burghoff M. Interkulturelle Kompetenz von Lehrkräften. Mythos, Trend oder pädagogische Notwendigkeit? In: McElvany N, Bos W, Holtappels HG, Jungermann A, eds. Ankommen in der Schule. Chancen und Herausforderungen bei der Integration von Kindern und Jugendlichen mit Fluchterfahrung. Münster: Waxmann; 2017: 69-85

Microchip screening platform for single cell assessment of NK cell cytotoxicity

Here we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32 400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12 hours long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g. in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy