42 research outputs found
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
Radial Sizing of Lipid Nanotubes Using Membrane Displacement Analysis
We report a novel method for the measurement of lipid nanotube radii. Membrane translocation is monitored between two nanotube-connected vesicles, during the expansion of a receiving vesicle, by observing a photobleached region of the nanotube. We elucidate nanotube radii, extracted from SPE vesicles, enabling quantification of membrane composition and lamellarity. Variances of nanotube radii were measured, showing a growth of 40-56 nm, upon increasing cholesterol content from 0 to 20%
Trogocytosis-mediated expression of HER2 on immune cells may be associated with a pathological complete response to trastuzumab-based primary systemic therapy in HER2-overexpressing breast cancer patients
Effect of frequently used chemotherapeutic drugs on the cytotoxic activity of human natural killer cells.
Tumors are considered to be possible targets of immunotherapy using stimulated
and expanded autologous or allogeneic natural killer (NK) cells mismatched for
MHC class I molecules and inhibitory NK receptors. NK cell-based immunoadjuvant
therapies are carried out in combination with standard chemotherapeutic
protocols. In the presented study, we characterized the effect of 28 frequently
used chemotherapeutic agents on the capacity of NK cells to kill target cells. We
found that treatment of NK cells with the drugs vinblastine, paclitaxel,
docetaxel, cladribine, chlorambucil, bortezomib, and MG-132 effectively inhibited
NK cell-mediated killing without affecting the viability of NK cells. On the
other hand, the following drugs permitted efficient NK cell-mediated killing even
at concentrations comparable with or higher than the maximally achieved
therapeutic concentration in vivo in humans: asparaginase, bevacizumab,
bleomycin, doxorubicin, epirubicin, etoposide, 5-fluorouracil, hydroxyurea,
streptozocin, and 6-mercaptopurine
The role of the cytoskeleton and protein size in assembly of the human natural killer cell immune synapse
The role of the cytoskeleton and protein size in assembly of the human natural killer cell immune synapse
NK cell-mediated lysis is essential to kill Epstein-Barr virus transformed lymphoblastoid B cells when using rituximab.
Rituximab is a humanized chimeric monoclonal antibody, targeted against the pan B
cell marker CD20. It is frequently used to treat a variety of B cell lymphomas
and immunosuppression associated lymphoproliferations such as posttransplant
lymphoproliferative disorder (PTLD). The response rate of rituximab treatment is
65%, but the exact in vivo mechanism of action is not yet fully understood,
although antibody-dependent cell-mediated cytotoxicity (ADCC),
complement-dependent cytotoxicity (CDC), and direct induction of apoptosis have
been suggested as effector mechanism. Rituximab may affect different types of
lymphomas through different mechanisms. As lymphoblastoid cell lines (LCLs) are
well-established in vitro models of PTLD, we investigated the effect of rituximab
on these cells using a custom built automated laser confocal fluorescent
microscope. We found that rituximab alone was not effective at inducing cell
death of EBV-transformed B cells. The antibody was effective in the
complement-mediated CDC. Rituximab could induce NK cell-mediated ADCC but it was
more effective in the presence of untreated fresh human plasma compared to
heat-inactivated human plasma. Our data suggest that complement-enhanced
NK-mediated ADCC is required for effective rituximab mediated killing of
EBV-transformed B cells. Determining and monitoring of serum complement levels
and in vitro killing efficacy of NK cells of PTLD patients might help to predict
resistant cases to rituximab therapy. On the other hand our results suggest a
possibility that rituximab should be combined only with cytotoxic drugs that
spare NK function when treating PTLD patients
The size of the synaptic cleft and distinct distributions of filamentous actin, ezrin, CD43, and CD45 at activating and inhibitory human NK cell immune synapses.
In this study, we report the organization of cytoskeletal and large transmembrane proteins at the inhibitory and activating NK cell immunological or immune synapse (IS). Filamentous actin accumulates at the activating, but not the inhibitory, NK cell IS. However, surprisingly, ezrin and the associated protein CD43 are excluded from the inhibitory, but not the activating, NK cell IS. This distribution of ezrin and CD43 at the inhibitory NK cell IS is similar to that previously seen at the activating T cell IS. CD45 is also excluded from the inhibitory, but not activating, NK cell IS. In addition, electron microscopy reveals wide and narrow domains across the synaptic cleft. Target cell HLA-C, located by immunogold labeling, clusters where the synaptic cleft spans the size of HLA-C bound to the inhibitory killer Ig-like receptor. These data are consistent with assembly of the NK cell IS involving a combination of cytoskeletal-driven mechanisms and thermodynamics favoring the organization of receptor/ligand pairs according to the size of their extracellular domains
Imaging immune surveillance by T cells and NK cells.
As T cells and natural killer (NK) cells survey the surface of other cells, cognate receptors and ligands are commonly organized into distinct micrometer-scale domains at the intercellular contact, creating an immune or immunological synapse (IS). We aim to address the still unanswered questions of how this organization of proteins aids immune surveillance and how these domains are biophysically constructed. Molecular mechanisms for the formation of the IS include a role for the cytoskeleton, segregation of proteins according to the size of their extracellular domains, and association of proteins with lipid rafts. Towards understanding the function of the IS, it is instructive to compare and contrast the supramolecular organization of proteins at the inhibitory and activating NK cell IS with that at the activating T cell IS. Finally, it is essential to develop new technologies for probing molecular recognition at cell surfaces. Imaging parameters other than fluorescence intensity, such as the lifetime of the fluorophore's excited state, could be used to report on protein environments