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  • 1.
    Brandt, Ludwig
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Pfefferle, Aline
    Goodridge, Jodie
    Malmberg, Karl-Johan
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Cytotoxicity and killing kinetics of KIR educated NK cells2017In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 301-301Article in journal (Other academic)
  • 2.
    Guldevall, Karolin
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brandt, Ludwig
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forslund, Elin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Sweden.
    Olofsson, Karl
    Frisk, Thomas W.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Olofsson, Per E.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gustafsson, Karin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Manneberg, Otto
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Karre, Klas
    Uhlin, Michael
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Microchip screening Platform for single cell assessment of NK cell cytotoxicity2016In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 7, article id 119Article in journal (Refereed)
    Abstract [en]

    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. Meinke, Stephan
    et al.
    Brandt, Ludwig
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Sandgren, Per
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hoglund, Petter
    Platelets become NK cell targets in the presence of anti-platelet antibodies2017In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 257-258Article in journal (Other academic)
  • 4.
    Oei, Vincent Yi Sheng
    et al.
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, KG Jebsen Ctr Canc Immunotherapy, Oslo, Norway..
    Siernicka, Marta
    Med Univ Warsaw, Dept Immunol, Ctr Biostruct Res, Warsaw, Poland.;Med Univ Warsaw, Postgrad Sch Mol Med, Warsaw, Poland..
    Graczyk-Jarzynka, Agnieszka
    Med Univ Warsaw, Dept Immunol, Ctr Biostruct Res, Warsaw, Poland..
    Hoel, Hanna Julie
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway..
    Yang, Weiwen
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway..
    Palacios, Daniel
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, KG Jebsen Ctr Canc Immunotherapy, Oslo, Norway..
    Almasbak, Hilde
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway..
    Bajor, Malgorzata
    Med Univ Warsaw, Dept Immunol, Ctr Biostruct Res, Warsaw, Poland..
    Clement, Dennis
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, KG Jebsen Ctr Canc Immunotherapy, Oslo, Norway..
    Brandt, Ludwig
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst.
    Goodridge, Jodie
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, KG Jebsen Ctr Canc Immunotherapy, Oslo, Norway..
    Winiarska, Magdalena
    Med Univ Warsaw, Dept Immunol, Ctr Biostruct Res, Warsaw, Poland..
    Zagozdzon, Radoslaw
    Med Univ Warsaw, Dept Immunol, Ctr Biostruct Res, Warsaw, Poland.;Med Univ Warsaw, Inst Transplantat, Dept Clin Immunol, Warsaw, Poland..
    Olweus, Johanna
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, KG Jebsen Ctr Canc Immunotherapy, Oslo, Norway..
    Kyte, Jon-Amund
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Oslo Univ Hosp, Dept Oncol, Oslo, Norway..
    Malmberg, Karl-Johan
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, KG Jebsen Ctr Canc Immunotherapy, Oslo, Norway.;Karolinska Inst, Dept Med Huddinge, Ctr Infect Med, Stockholm, Sweden..
    Intrinsic Functional Potential of NK-Cell Subsets Constrains Retargeting Driven by Chimeric Antigen Receptors2018In: CANCER IMMUNOLOGY RESEARCH, ISSN 2326-6066, Vol. 6, no 4, p. 467-480Article in journal (Refereed)
    Abstract [en]

    Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19(+) targets, and maintained their intrinsic degranulation response toward CD19(-) K562 cells. The response of redirected NK-cell subsets against CD19(+) targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA-specific KIR(s), displayed superior ability to kill CD19(+), HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells.

  • 5.
    Olofsson, Per E.
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brandt, Ludwig
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Magnusson, Klas E. G.
    KTH, School of Electrical Engineering (EES), Signal Processing. KTH, School of Electrical Engineering and Computer Science (EECS), Centres, ACCESS Linnaeus Centre.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jaldén, Joakim
    KTH, School of Electrical Engineering (EES), Signal Processing. KTH, School of Electrical Engineering and Computer Science (EECS), Centres, ACCESS Linnaeus Centre.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    A collagen-based microwell migration assay to study NK-target cell interactions2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 10672Article in journal (Refereed)
    Abstract [en]

    Natural killer (NK) cell cytotoxicity in tissue is dependent on the ability of NK cells to migrate through the extracellular matrix (ECM) microenvironment. Traditional imaging studies of NK cell migration and cytotoxicity have utilized 2D surfaces, which do not properly reproduce the structural and mechanical cues that shape the migratory response of NK cells in vivo. Here, we have combined a microwell assay that allows long-term imaging and tracking of small, well-defined populations of NK cells with an interstitial ECM-like matrix. The assay allows for long-term imaging of NK-target cell interactions within a confined 3D volume. We found marked differences in motility between individual cells with a small fraction of the cells moving slowly and being confined to a small volume within the matrix, while other cells moved more freely. A majority of NK cells also exhibited transient variation in their motility, alternating between periods of migration arrest and movement. The assay could be used as a complement to in vivo imaging to study human NK cell heterogeneity in migration and cytotoxicity.

  • 6.
    Sarha, Dhifaf
    et al.
    Univ Minnesota, Mason Canc Ctr, Dept Med, Div Hematol Oncol & Transplantat, Minneapolis, MN 55455 USA..
    Brandt, Ludwig
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Felices, Martin
    Univ Minnesota, Mason Canc Ctr, Dept Med, Div Hematol Oncol & Transplantat, Minneapolis, MN 55455 USA..
    Guldevall, Karolin
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Lenvik, Todd
    Univ Minnesota, Mason Canc Ctr, Dept Med, Div Hematol Oncol & Transplantat, Minneapolis, MN 55455 USA..
    Hinderlie, Peter
    Univ Minnesota, Mason Canc Ctr, Dept Med, Div Hematol Oncol & Transplantat, Minneapolis, MN 55455 USA..
    Curtsinger, Julie
    Univ Minnesota, Mason Canc Ctr, Translat Therapy Lab, Minneapolis, MN 55455 USA..
    Warlick, Erica
    Univ Minnesota, Mason Canc Ctr, Dept Med, Div Hematol Oncol & Transplantat, Minneapolis, MN 55455 USA..
    Spellma, Stephen R.
    Ctr Int Blood & Marrow Transplant Res, Minneapolis, MN USA..
    Blazar, Bruce R.
    Univ Minnesota, Mason Canc Ctr, Div Blood & Marrow Transplantat, Dept Pediat, Minneapolis, MN 55455 USA..
    Weisdorf, Daniel J.
    Univ Minnesota, Mason Canc Ctr, Dept Med, Div Hematol Oncol & Transplantat, Minneapolis, MN 55455 USA..
    Cooley, Sarah
    Univ Minnesota, Mason Canc Ctr, Dept Med, Div Hematol Oncol & Transplantat, Minneapolis, MN 55455 USA..
    Vallera, Daniel A.
    Univ Minnesota, Mason Canc Ctr, Lab Mol Canc Therapeut, Dept Therapeut Radiol Radiat Oncol, Minneapolis, MN 55455 USA..
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden.
    Miller, Jeffrey S.
    Univ Minnesota, Mason Canc Ctr, Dept Med, Div Hematol Oncol & Transplantat, Minneapolis, MN 55455 USA..
    161533 TriKE stimulates NK-cell function to overcome myeloid-derived suppressor cells in MDS2018In: BLOOD ADVANCES, ISSN 2473-9529, Vol. 2, no 12, p. 1459-1469Article in journal (Refereed)
    Abstract [en]

    Myelodysplastic syndrome (MDS) is a clonal heterogeneous stem cell disorder driven by multiple genetic and epigenetic alterations resulting in ineffective hematopoiesis. MDS has a high frequency of immune suppressors, including myeloid-derived suppressor cells (MDSCs), that collectively result in a poor immune response. MDSCs in MDS patients express CD155 that ligates the T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) and delivers an inhibitory signal to natural killer (NK) cells. To mediate a productive immune response against MDS, negative regulatory checkpoints, like TIGIT, expressed on MDS NK cells must be overcome. NK cells can be directed to lyse MDS cells by bispecific killer engagers (BiKEs) that ligate CD16 on NK cells and CD33 on MDS cells. However, such CD16 x CD33 (1633) BiKEs do not induce the proliferative response in MDS NK cells needed to sustain their function. Here, we show that the addition of an NK stimulatory cytokine, interleukin-15 (IL-15), into the BiKE platform leads to productive IL-15 signaling without TIGIT upregulation on NK cells from MDS patients. Lower TIGIT expression allowed NK cells to resist MDSC inhibition. When compared with 1633 BiKE, 161533 trispecific killer engager (TriKE)-treated NK cells demonstrated superior killing kinetics associated with increased STAT5 phosphorylation. Furthermore, 161533 TriKE-treated MDS NK cells had higher proliferation and enhanced NK-cell function than 1633 BiKE-treated cells without the IL-15 linker. Collectively, our data demonstrate novel characteristics of the 161533 TriKE that support its application as an immunotherapeutic agent for MDS patients.

  • 7.
    Verron, Quentin
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Guldevall, Karolin
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brandt, Ludwig
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Olofsson, Per E.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden.
    Microchip screening for single cell assessment and isolation of serial killing NK cells2017In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 347-347Article in journal (Other academic)
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