| 1. |
- Forslund, Elin, et al.
(författare)
-
Microchip-Based Single-Cell Imaging Reveals That CD56(dim) CD57(-)KIR(-)NKG2A(+) NK Cells Have More Dynamic Migration Associated with Increased Target Cell Conjugation and Probability of Killing Compared to CD56(dim)CD57(-)KIR(-)NKG2A(-) NK Cells
- 2015
-
Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 195:7, s. 3374-3381
-
Tidskriftsartikel (refereegranskat)abstract
- NK cells are functionally educated by self-MHC specific receptors, including the inhibitory killer cell Ig-like receptors (KIRs) and the lectin-like CD94/NKG2A heterodimer. Little is known about how NK cell education influences qualitative aspects of cytotoxicity such as migration behavior and efficacy of activation and killing at the single-cell level. In this study, we have compared the behavior of FACS-sorted CD56(dim)CD57(-)KIR(-)NKG2A(+) (NKG2A(+)) and CD56(dim)CD57(-)KIR(-)NKG2A(+) (lacking inhibitory receptors; IR-) human NK cells by quantifying migration, cytotoxicity, and contact dynamics using microchip-based live cell imaging. NKG2A(+) NK cells displayed a more dynamic migration behavior and made more contacts with target cells than IR-NK cells. NKG2A(+) NK cells also more frequently killed the target cells once a conjugate had been formed. NK cells with serial killing capacity were primarily found among NKG2A(+) NK cells. Conjugates involving IR- NK cells were generally more short-lived and IR- NK cells did not become activated to the same extent as NKG2A(+) NK cells when in contact with target cells, as evident by their reduced spreading response. In contrast, NKG2A(+) and IR- NK cells showed similar dynamics in terms of duration of conjugation periods and NK cell spreading response in conjugates that led to killing. Taken together, these observations suggest that the high killing capacity of NKG2A(+) NK cells is linked to processes regulating events in the recognition phase of NK-target cell contact rather than events after cytotoxicity has been triggered.
|
|
| 2. |
- Guldevall, Karolin, et al.
(författare)
-
Microchip screening Platform for single cell assessment of NK cell cytotoxicity
- 2016
-
Ingår i: Frontiers in Immunology. - : FRONTIERS MEDIA SA. - 1664-3224. ; 7
-
Tidskriftsartikel (refereegranskat)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 (approximate to 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.
|
|
| 3. |
- Olofsson, Per E, et al.
(författare)
-
Distinct Migration and Contact Dynamics of Resting and IL-2-Activated Human Natural Killer Cells.
- 2014
-
Ingår i: Frontiers in immunology. - : Frontiers Media SA. - 1664-3224. ; 5, s. 80-
-
Tidskriftsartikel (refereegranskat)abstract
- Natural killer (NK) cells serve as one of the first lines of defense against viral infections and transformed cells. NK cell cytotoxicity is not dependent on antigen presentation by target cells, but is dependent on integration of activating and inhibitory signals triggered by receptor-ligand interactions formed at a tight intercellular contact between the NK and target cell, i.e., the immune synapse. We have studied the single-cell migration behavior and target-cell contact dynamics of resting and interleukin (IL)-2-activated human peripheral blood NK cells. Small populations of NK cells and target cells were confined in microwells and imaged by fluorescence microscopy for >8 h. Only the IL-2-activated population of NK cells showed efficient cytotoxicity against the human embryonic kidney 293T target cells. We found that although the average migration speeds were comparable, activated NK cells showed significantly more dynamic migration behavior, with more frequent transitions between periods of low and high motility. Resting NK cells formed fewer and weaker contacts with target cells, which manifested as shorter conjugation times and in many cases a complete lack of post-conjugation attachment to target cells. Activated NK cells were approximately twice as big as the resting cells, displayed a more migratory phenotype, and were more likely to employ "motile scanning" of the target-cell surface during conjugation. Taken together, our experiments quantify, at the single-cell level, how activation by IL-2 leads to altered NK cell cytotoxicity, migration behavior, and contact dynamics.
|
|
| 4. |
- Verron, Quentin, et al.
(författare)
-
NK cells integrate signals over large areas when building immune synapses but require local stimuli for degranulation
- 2021
-
Ingår i: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 14:684
-
Tidskriftsartikel (refereegranskat)abstract
- Immune synapses are large-scale, transient molecular assemblies that serve as platforms for antigen presentation to B and T cells and for target recognition by cytotoxic T cells and natural killer (NK) cells. The formation of an immune synapse is a tightly regulated, stepwise process in which the cytoskeleton, cell surface receptors, and intracellular signaling proteins rearrange into supramolecular activation clusters (SMACs). We generated artificial immune synapses (AIS) consisting of synthetic and natural ligands for the NK cell-activating receptors LFA-1 and CD16 by microcontact printing the ligands into circular-shaped SMAC structures. Live-cell imaging and analysis of fixed human NK cells in this reductionist system showed that the spatial distribution of activating ligands influenced the formation, stability, and outcome of NK cell synapses. Whereas engagement of LFA-1 alone promoted synapse initiation, combined engagement of LFA-1 and CD16 was required for the formation of mature synapses and degranulation. Organizing LFA-1 and CD16 ligands into donut-shaped AIS resulted in fewer long-lasting, symmetrical synapses compared to dot-shaped AIS. NK cells spreading evenly over either AIS shape exhibited similar arrangements of the lytic machinery. However, degranulation only occurred in regions containing ligands that therefore induced local signaling, suggesting the existence of a late checkpoint for degranulation. Our results demonstrate that the spatial organization of ligands in the synapse can affect its outcome, which could be exploited by target cells as an escape mechanism.
|
|
