Abstract
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-h 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.
Highlights
Cytotoxic effector lymphocytes, such as natural killer (NK) cells and T cells, are important for immune defense against cancer and viral infections, the traits that have made these cells valuable in adoptive cell therapy
The microchip platform consisted of a silicon–glass microchip held in place by an aluminum plate, a polydimethylsiloxane (PDMS) gasket, and a plastic lid, and it was designed to fit in a conventional inverted fluorescence or confocal microscope (Figure 1A)
To assess single cell cytotoxicity, NK cells were labeled with the fluorescent viability dye calcein orange and target cells with calcein green and DDAO
Summary
Cytotoxic effector lymphocytes, such as natural killer (NK) cells and T cells, are important for immune defense against cancer and viral infections, the traits that have made these cells valuable in adoptive cell therapy Their activity is associated with detrimental conditions, such as autoimmunity or graft-versus-host disease (GVHD), after allogeneic hematopoietic stem cell transplantation (HSCT). Clinical trials using NK cells from haploidentical donors for cell therapy have shown encouraging results indicating that interindividual differences in NK cell recognition and responsiveness can be used to treat disease [7]. These studies established a link between the number of alloreactive NK cells in the graft and patient survival. New methods to quantify the fraction of alloreactive NK cells and cytolytic potential of individual NK cells could be valuable for the process of selecting donors for therapy
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