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Olofsson, P. E., Brandt, L., Magnusson, K. E. G., Frisk, T., Jaldén, J. & Önfelt, B. (2019). A collagen-based microwell migration assay to study NK-target cell interactions. Scientific Reports, 9, Article ID 10672.
Open this publication in new window or tab >>A collagen-based microwell migration assay to study NK-target cell interactions
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 10672Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-255740 (URN)10.1038/s41598-019-46958-3 (DOI)000476718900058 ()31337806 (PubMedID)2-s2.0-85069667997 (Scopus ID)
Note

QC 20190812

Available from: 2019-08-12 Created: 2019-08-12 Last updated: 2019-08-12Bibliographically approved
Fleming, C. L., Sandoz, P. A., Inghardt, T., Önfelt, B., Grotli, M. & Andreasson, J. (2019). A Fluorescent Kinase Inhibitor that Exhibits Diagnostic Changes in Emission upon Binding. Angewandte Chemie International Edition
Open this publication in new window or tab >>A Fluorescent Kinase Inhibitor that Exhibits Diagnostic Changes in Emission upon Binding
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2019 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773Article in journal (Refereed) Published
Abstract [en]

The development of a fluorescent LCK inhibitor that exhibits favourable solvatochromic properties upon binding the kinase is described. Fluorescent properties were realised through the inclusion of a prodan-derived fluorophore into the pharmacophore of an ATP-competitive kinase inhibitor. Fluorescence titration experiments demonstrate the solvatochromic properties of the inhibitor, in which dramatic increase in emission intensity and hypsochromic shift in emission maxima are clearly observed upon binding LCK. Microscopy experiments in cellular contexts together with flow cytometry show that the fluorescence intensity of the inhibitor correlates with the LCK concentration. Furthermore, multiphoton microscopy experiments demonstrate both the rapid cellular uptake of the inhibitor and that the two-photon cross section of the inhibitor is amenable for excitation at 700 nm.

Place, publisher, year, edition, pages
WILEY-V C H VERLAG GMBH, 2019
Keywords
fluorescence, inhibitors, kinase, solvatochromism
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-261016 (URN)10.1002/anie.201909536 (DOI)000485136100001 ()31411364 (PubMedID)
Note

QC 20191002

Available from: 2019-10-02 Created: 2019-10-02 Last updated: 2019-10-02Bibliographically approved
Radestad, E., Sundin, M., Torlen, J., Thunberg, S., Önfelt, B., Ljungman, P., . . . Uhlin, M. (2019). Individualization of Hematopoietic Stem Cell Transplantation Using Alpha/Beta T-Cell Depletion. Frontiers in Immunology, 10, Article ID 189.
Open this publication in new window or tab >>Individualization of Hematopoietic Stem Cell Transplantation Using Alpha/Beta T-Cell Depletion
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2019 (English)In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 10, article id 189Article in journal (Refereed) Published
Abstract [en]

Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with several potentially lethal complications. Higher levels of CD3+ T-cells in the graft have been associated with increased risk of graft-versus-host disease (GVHD), but also beneficial graft-versus-leukemia effect and reduced infections. To tackle post-transplant complications, donor lymphocyte infusions have been used but with an increased risk of GVHD. To reduce this risk, we performed depletion of alpha beta T-cells and treated 12 patients post-HSCT suffering from infections and/or poor immune reconstitution. The alpha beta T-cell depleted cell products were characterized by flow cytometry. The median log depletion of alpha beta T-cells was -4.3 and the median yield of gamma delta T-cells was 73.5%. The median CD34+ cell dose was 4.4 x 10(6)/kg. All 12 patients were alive 3 months after infusion and after 1 year, two patients had died. No infusion-related side effects were reported and no severe acute GVHD (grade III-IV) developed in any patient post-infusion. Overall, 3 months after infusion 11 out of 12 patients had increased levels of platelets and/or granulocytes. In conclusion, we describe the use of alpha beta T-cell depleted products as stem cell boosters with encouraging results.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2019
Keywords
alpha beta T-cell depletion, gamma delta T-cells, allogeneic hematopoietic stem cell transplantation, stem cell booster, donor lymphocyte infusion, graft manipulation, CliniMACS
National Category
Hematology
Identifiers
urn:nbn:se:kth:diva-244508 (URN)10.3389/fimmu.2019.00189 (DOI)000458248300001 ()2-s2.0-85062186448 (Scopus ID)
Note

QC 20190402

Available from: 2019-04-02 Created: 2019-04-02 Last updated: 2019-08-27Bibliographically approved
Prager, I., Liesche, C., van Ooijen, H., Urlaub, D., Verron, Q., Sandström, N., . . . Watzl, C. (2019). NK cells switch from granzyme B to death receptor–mediated cytotoxicity during serial killing. Journal of Experimental Medicine, 7(9), 2113-2127
Open this publication in new window or tab >>NK cells switch from granzyme B to death receptor–mediated cytotoxicity during serial killing
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2019 (English)In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 7, no 9, p. 2113-2127Article in journal (Refereed) Published
Abstract [en]

NK cells eliminate virus-infected and tumor cells by releasing cytotoxic granules containing granzyme B (GrzB) or by engaging death receptors that initiate caspase cascades. The orchestrated interplay between both cell death pathways remains poorly defined. Here we simultaneously measure the activities of GrzB and caspase-8 in tumor cells upon contact with human NK cells. We observed that NK cells switch from inducing a fast GrzB-mediated cell death in their first killing events to a slow death receptor–mediated killing during subsequent tumor cell encounters. Target cell contact reduced intracellular GrzB and perforin and increased surface-CD95L in NK cells over time, showing how the switch in cytotoxicity pathways is controlled. Without perforin, NK cells were unable to perform GrzB-mediated serial killing and only killed once via death receptors. In contrast, the absence of CD95 on tumor targets did not impair GrzB-mediated serial killing. This demonstrates that GrzB and death receptor–mediated cytotoxicity are differentially regulated during NK cell serial killing.

National Category
Immunology in the medical area
Identifiers
urn:nbn:se:kth:diva-256334 (URN)10.1084/jem.20181454 (DOI)000484027100013 ()31270246 (PubMedID)2-s2.0-85071782240 (Scopus ID)
Note

QC 20190822

Available from: 2019-08-22 Created: 2019-08-22 Last updated: 2019-10-28Bibliographically approved
Gaballa, A., Stikvoort, A., Önfelt, B., Mattsson, J., Sundin, M., Watz, E. & Uhlin, M. (2019). T-cell frequencies of CD8(+) gamma delta and CD27(+) gamma delta cells in the stem cell graft predict the outcome after allogeneic hematopoietic cell transplantation. Bone Marrow Transplantation, 54(10), 1562-1574
Open this publication in new window or tab >>T-cell frequencies of CD8(+) gamma delta and CD27(+) gamma delta cells in the stem cell graft predict the outcome after allogeneic hematopoietic cell transplantation
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2019 (English)In: Bone Marrow Transplantation, ISSN 0268-3369, E-ISSN 1476-5365, Vol. 54, no 10, p. 1562-1574Article in journal (Refereed) Published
Abstract [en]

The impact of intra-graft T cells on the clinical outcome after allogeneic hematopoietic cell transplantation has been investigated. Most previous studies have focused on the role of alpha beta cells while gamma delta cells have received less attention. It has been an open question whether gamma delta cells are beneficial or not for patient outcome, especially with regards to graft versus host disease. In this study, graft composition of.d cell subsets was analyzed and correlated to clinical outcome in 105 recipients who underwent allogeneic hematopoietic cell transplantation between 2013 and 2016. We demonstrate for the first time that grafts containing higher T-cell proportions of CD8(+) gamma delta cells were associated with increased cumulative incidence of acute graft versus host disease grade II-III (50% vs 22.6%; P = 0.008). Additionally, graft T-cell frequency of CD27(+) gamma delta cells was inversely correlated with relapse (P = 0.006) and CMV reactivation (P = 0.05). We conclude that clinical outcome after allogeneic hematopoietic cell transplantation is influenced by the proportions of distinct gamma delta cell subsets in the stem cell graft. We also provide evidence that CD8(+) gamma delta cells are potentially alloreactive and may play a role in acute graft versus host disease. This study illustrates the importance of better understanding of the role of distinct subsets of.d cells in allogeneic hematopoietic cell transplantation.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
National Category
Clinical Medicine
Identifiers
urn:nbn:se:kth:diva-262782 (URN)10.1038/s41409-019-0462-z (DOI)000488520400005 ()30723262 (PubMedID)2-s2.0-85061175298 (Scopus ID)
Note

QC 20191022

Available from: 2019-10-22 Created: 2019-10-22 Last updated: 2019-10-22Bibliographically approved
Sarha, D., Brandt, L., Felices, M., Guldevall, K., Lenvik, T., Hinderlie, P., . . . Miller, J. S. (2018). 161533 TriKE stimulates NK-cell function to overcome myeloid-derived suppressor cells in MDS. BLOOD ADVANCES, 2(12), 1459-1469
Open this publication in new window or tab >>161533 TriKE stimulates NK-cell function to overcome myeloid-derived suppressor cells in MDS
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2018 (English)In: BLOOD ADVANCES, ISSN 2473-9529, Vol. 2, no 12, p. 1459-1469Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
AMER SOC HEMATOLOGY, 2018
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-232250 (URN)10.1182/bloodadvances.2017012369 (DOI)000436548300013 ()29941459 (PubMedID)2-s2.0-85060540460 (Scopus ID)
Funder
Swedish Foundation for Strategic Research , SBE13-0092Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20180720

Available from: 2018-07-20 Created: 2018-07-20 Last updated: 2019-08-20Bibliographically approved
Olofsson, K., Carannante, V., Ohlin, M., Frisk, T., Kushiro, K., Takai, M., . . . Wiklund, M. (2018). Acoustic formation of multicellular tumor spheroids enabling on-chip functional and structural imaging. Lab on a Chip, 18(16), 2466-2476
Open this publication in new window or tab >>Acoustic formation of multicellular tumor spheroids enabling on-chip functional and structural imaging
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2018 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 18, no 16, p. 2466-2476Article in journal (Refereed) Published
Abstract [en]

Understanding the complex 3D tumor microenvironment is important in cancer research. This microenvironment can be modelled in vitro by culturing multicellular tumor spheroids (MCTS). Key challenges when using MCTS in applications such as high-throughput drug screening are overcoming imaging and analytical issues encountered during functional and structural investigations. To address these challenges, we use an ultrasonic standing wave (USW) based MCTS culture platform for parallel formation, staining and imaging of 100 whole MCTS. A protein repellent amphiphilic polymer coating enables flexible production of high quality and unanchored MCTS. This enables high-content multimode analysis based on flow cytometry and in situ optical microscopy. We use HepG2 hepatocellular carcinoma, A498 and ACHN renal carcinoma, and LUTC-2 thyroid carcinoma cell lines to demonstrate (i) the importance of the ultrasound-coating combination, (ii) bright field image based automatic characterization of MTCS, (iii) detailed deep tissue confocal imaging of whole MCTS mounted in a refractive index matching solution, and (iv) single cell functional analysis through flow cytometry of single cell suspensions of disintegrated MTCS. The USW MCTS culture platform is customizable and holds great potential for detailed multimode MCTS analysis in a high-content manner.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2018
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-234606 (URN)10.1039/c8lc00537k (DOI)000442265100012 ()30033460 (PubMedID)2-s2.0-85051357097 (Scopus ID)
Funder
Stockholm County CouncilSwedish Cancer SocietySwedish Childhood Cancer FoundationSwedish Research CouncilSwedish Foundation for Strategic Research
Note

QC 20180914

Available from: 2018-09-14 Created: 2018-09-14 Last updated: 2018-09-14Bibliographically approved
Sohlberg, E., Haroun-Izquierdo, A., Bjorklund, A. T., Cooley, S., Wiiger, M. T., Goodridge, P., . . . Malmberg, K.-J. (2018). Efficient Scale-up and Pre-Clinical Evaluation of NKG2C+Adaptive NK Cell Expansion for Therapy Against High-Risk AML/MDS. Paper presented at 60th Annual Meeting of the American-Society-of-Hematology (ASH), DEC 01-04, 2018, San Diego, CA. Blood, 132
Open this publication in new window or tab >>Efficient Scale-up and Pre-Clinical Evaluation of NKG2C+Adaptive NK Cell Expansion for Therapy Against High-Risk AML/MDS
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2018 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 132Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
American Society of Hematology, 2018
National Category
Hematology
Identifiers
urn:nbn:se:kth:diva-245961 (URN)10.1182/blood-2018-195 (DOI)000454837600249 ()
Conference
60th Annual Meeting of the American-Society-of-Hematology (ASH), DEC 01-04, 2018, San Diego, CA
Note

QC 20190313

Available from: 2019-03-13 Created: 2019-03-13 Last updated: 2019-03-13Bibliographically approved
Oei, V. Y., Siernicka, M., Graczyk-Jarzynka, A., Hoel, H. J., Yang, W., Palacios, D., . . . Malmberg, K.-J. (2018). Intrinsic Functional Potential of NK-Cell Subsets Constrains Retargeting Driven by Chimeric Antigen Receptors. CANCER IMMUNOLOGY RESEARCH, 6(4), 467-480
Open this publication in new window or tab >>Intrinsic Functional Potential of NK-Cell Subsets Constrains Retargeting Driven by Chimeric Antigen Receptors
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2018 (English)In: CANCER IMMUNOLOGY RESEARCH, ISSN 2326-6066, Vol. 6, no 4, p. 467-480Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
AMER ASSOC CANCER RESEARCH, 2018
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-228141 (URN)10.1158/2326-6066.CIR-17-0207 (DOI)000429044000010 ()29459477 (PubMedID)2-s2.0-85048084731 (Scopus ID)
Note

QC 20180518

Available from: 2018-05-18 Created: 2018-05-18 Last updated: 2018-10-16Bibliographically approved
Stikvoort, A., Gaballa, A., Solders, M., Nederlof, I., Önfelt, B., Sundberg, B., . . . Uhlin, M. (2018). Risk Factors for Severe Acute Graft-versus-Host Disease in Donor Graft Composition. Biology of blood and marrow transplantation, 24(3), 467-477
Open this publication in new window or tab >>Risk Factors for Severe Acute Graft-versus-Host Disease in Donor Graft Composition
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2018 (English)In: Biology of blood and marrow transplantation, ISSN 1083-8791, E-ISSN 1523-6536, Vol. 24, no 3, p. 467-477Article in journal (Refereed) Published
Abstract [en]

Acute graft-versus-host disease (aGVHD) is 1 of the main major complications of post-hematopoietic stem cell transplantation (HSCT). Identifying patients at risk of severe aGVHD may lead to earlier intervention and treatment, resulting in increased survival and a better quality of life. We aimed to identify biomarkers in donor grafts and patient plasma around the time of transplantation that might be predictive of aGVHD development. We build on our previously published methods by using multiplex assays and multicolor flow cytometry. We identified 5 easily assessable cellular markers in donor grafts that combined could potentially be used to calculate risk for severe aGVHD development. Most noteworthy are the T cell subsets expressing IL-7 receptor-a (CD127) and PD-1. Additionally, we identified a potential role for elevated tumor necrosis factor-a levels in both graft and patient before HSCT in development of aGVHD. 

Place, publisher, year, edition, pages
ELSEVIER SCIENCE INC, 2018
Keywords
Stem cell transplantation, GVHD, T cells, Donor grafts, Risk factors
National Category
Basic Medicine
Identifiers
urn:nbn:se:kth:diva-225738 (URN)10.1016/j.bbmt.2017.11.026 (DOI)000427663000008 ()29197674 (PubMedID)
Funder
Swedish Research CouncilStockholm County CouncilSwedish Foundation for Strategic Research
Note

QC 20180410

Available from: 2018-04-10 Created: 2018-04-10 Last updated: 2018-04-10Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-5178-7593

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