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  • 1. Arruda, L. C. M.
    et al.
    Gaballa, A.
    Uhlin, Michael
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Graft γδ TCR Sequencing Identifies Public Clonotypes Associated with Hematopoietic Stem Cell Transplantation Efficacy in Acute Myeloid Leukemia Patients and Unravels Cytomegalovirus Impact on Repertoire Distribution2019In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 202, no 6, p. 1859-1870Article in journal (Refereed)
    Abstract [en]

    Although the impact of donor graft composition on clinical outcomes after hematopoietic stem cell transplantation (HSCT) has been studied, little is known about the role of intragraft γδ TCR repertoire on clinical outcomes following HSCT. Using a high-throughput sequencing platform, we sought to analyze the TCR γ-chain (TRG) repertoire of γδ T cells within donor stem cell grafts and address its potential impact on clinical response in the corresponding patients. A total of 20 peripheral blood stem cell grafts were analyzed, and donors were classified as CMV+/- The respective acute myeloid leukemia recipients were followed for disease relapse and acute graft-versus-host disease (aGvHD) development post-HSCT. In all samples, TRG repertoire showed a reduced diversity and displayed overrepresented clones. This was more prominent in grafts from CMV+ donors, which presented a more private repertoire, lower diversity, skewed distribution, and reduced usage of the V9-JP pairing. Grafts given to nonrelapse patients presented a more public repertoire and increased presence of long sequence clonotypes. Variable-joining gene segment usage was not associated with aGvHD development, but a higher usage of V2-JP1 pairing and lower usage of V4-J2/V5-J2/V8-JP2 were observed in grafts given to nonrelapse patients. Our work identified five private overrepresented and one public CDR3 sequence (CATWDGPYYKKLF) associated with CMV infection, in addition to 12 highly frequent public sequences present exclusively in grafts given to nonrelapse patients. Our findings show that, despite CMV infection reshaping the TRG repertoire, TRG composition is not associated with aGvHD development, and several public sequences are associated with clinical remission.

  • 2. Enqvist, M.
    et al.
    Ask, E. H.
    Forslund, E.
    Carlsten, M.
    Abrahamsen, G.
    Béziat, V.
    Andersson, S.
    Schaffer, M.
    Spurkland, A.
    Bryceson, Y.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Malmberg, K. -J
    Coordinated expression of DNAM-1 and LFA-1 in educated NK cells2015In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 194, no 9, p. 4518-4527Article in journal (Refereed)
    Abstract [en]

    The functional capacity of NK cells is dynamically tuned by integrated signals from inhibitory and activating cell surface receptors in a process termed NK cell education. However, the understanding of the cellular and molecular mechanisms behind this functional tuning is limited. In this study, we show that the expression of the adhesion molecule and activation receptor DNAX accessory molecule 1 (DNAM-1) correlates with the quantity and quality of the inhibitory input by HLA class I-specific killer cell Ig-like receptors and CD94/NKG2A as well as with the magnitude of functional responses. Upon target cell recognition, the conformational state of LFA-1 changed in educated NK cells, associated with rapid colocalization of both active LFA-1 and DNAM-1 at the immune synapse. Thus, the coordinated expression of LFA-1 and DNAM-1 is a central component of NK cell education and provides a potential mechanism for controlling cytotoxicity by functionally mature NK cells.

  • 3. Forslund, Elin
    et al.
    Sohlberg, Ebba
    Enqvist, Monika
    Olofsson, Per E.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Malmberg, Karl-Johan
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Sci Life Lab, Dept Microbiol Tumor & Cell Biol, S-17165 Stockholm, Sweden.
    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 Cells2015In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 195, no 7, p. 3374-3381Article in journal (Refereed)
    Abstract [en]

    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.

  • 4.
    Girnyk, Maksym A.
    et al.
    KTH, School of Electrical Engineering (EES), Communication Theory.
    Vehkapera, Mikko
    Rasmussen, Lars Kildehoj
    Christakou, Athanasia
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Onfelt, Bjorn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Orange, Jordan
    Lytic granule convergence is essential for NK cells to promote targeted killing while preventing collateral damage2016In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 196Article in journal (Other academic)
  • 5.
    Kimizuka, Yoshifumi
    et al.
    Massachusetts Gen Hosp, Vaccine & Immunotherapy Ctr, Div Infect Dis, Dept Med, Charlestown, MA 02129 USA..
    Katagiri, Wataru
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). Massachusetts Gen Hosp, Gordon Ctr Med Imaging, Dept Radiol, 149 13th St, Charlestown, MA 02129 USA.;Keio Univ, Grad Sch Fundamental Sci & Technol, Yokohama, Kanagawa 2238522, Japan.
    Locascio, Joseph J.
    Massachusetts Gen Hosp, Alzheimers Dis Res Ctr, Dept Neurol, Boston, MA 02114 USA..
    Shigeta, Ayako
    Massachusetts Gen Hosp, Vaccine & Immunotherapy Ctr, Div Infect Dis, Dept Med, Charlestown, MA 02129 USA..
    Sasaki, Yuri
    Massachusetts Gen Hosp, Vaccine & Immunotherapy Ctr, Div Infect Dis, Dept Med, Charlestown, MA 02129 USA..
    Shibata, Mai
    Massachusetts Gen Hosp, Vaccine & Immunotherapy Ctr, Div Infect Dis, Dept Med, Charlestown, MA 02129 USA..
    Morse, Kaitlyn
    Massachusetts Gen Hosp, Vaccine & Immunotherapy Ctr, Div Infect Dis, Dept Med, Charlestown, MA 02129 USA..
    Sirbulescu, Ruxandra F.
    Massachusetts Gen Hosp, Vaccine & Immunotherapy Ctr, Div Infect Dis, Dept Med, Charlestown, MA 02129 USA..
    Miyatake, Mizuki
    Keio Univ, Fac Sci & Technol, Yokohama, Kanagawa 2238522, Japan..
    Reeves, Patrick
    Massachusetts Gen Hosp, Vaccine & Immunotherapy Ctr, Div Infect Dis, Dept Med, Charlestown, MA 02129 USA..
    Suematsu, Makoto
    Keio Univ, Sch Med, Dept Biochem, Shinjuku Ku, Tokyo 1600016, Japan..
    Gelfand, Jeffrey
    Massachusetts Gen Hosp, Vaccine & Immunotherapy Ctr, Div Infect Dis, Dept Med, Charlestown, MA 02129 USA..
    Brauns, Timothy
    Massachusetts Gen Hosp, Vaccine & Immunotherapy Ctr, Div Infect Dis, Dept Med, Charlestown, MA 02129 USA..
    Poznansky, Mark C.
    Massachusetts Gen Hosp, Vaccine & Immunotherapy Ctr, Div Infect Dis, Dept Med, Charlestown, MA 02129 USA..
    Tsukada, Kosuke
    Keio Univ, Grad Sch Fundamental Sci & Technol, Yokohama, Kanagawa 2238522, Japan.;Keio Univ, Fac Sci & Technol, Yokohama, Kanagawa 2238522, Japan..
    Kashiwagi, Satoshi
    Massachusetts Gen Hosp, Vaccine & Immunotherapy Ctr, Div Infect Dis, Dept Med, Charlestown, MA 02129 USA.;Massachusetts Gen Hosp, Gordon Ctr Med Imaging, Dept Radiol, 149 13th St, Charlestown, MA 02129 USA..
    Brief Exposure of Skin to Near-Infrared Laser Modulates Mast Cell Function and Augments the Immune Response2018In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 201, no 12, p. 3587-3603Article in journal (Refereed)
    Abstract [en]

    The treatment of skin with a low-power continuous-wave (CW) near-infrared (NIR) laser prior to vaccination is an emerging strategy to augment the immune response to intradermal vaccine, potentially substituting for chemical adjuvant, which has been linked to adverse effects of vaccines. This approach proved to be low cost, simple, small, and readily translatable compared with the previously explored pulsed-wave medical lasers. However, little is known on the mode of laser-tissue interaction eliciting the adjuvant effect. In this study, we sought to identify the pathways leading to the immunological events by examining the alteration of responses resulting from genetic ablation of innate subsets including mast cells and specific dendritic cell populations in an established model of intradermal vaccination and analyzing functional changes of skin microcirculation upon the CW NIR laser treatment in mice. We found that a CW NIR laser transiently stimulates mast cells via generation of reactive oxygen species, establishes an immunostimulatory milieu in the exposed tissue, and provides migration cues for dermal CD103(+) dendritic cells without inducing prolonged inflammation, ultimately augmenting the adaptive immune response. These results indicate that use of an NIR laser with distinct wavelength and power is a safe and effective tool to reproducibly modulate innate programs in skin. These mechanistic findings would accelerate the clinical translation of this technology and warrant further explorations into the broader application of NIR lasers to the treatment of immune-related skin diseases.

  • 6. McCann, Fiona E.
    et al.
    Eissmann, Philipp
    Önfelt, Björn
    Leung, Rufina
    Davis, Daniel M.
    The activating NKG2D ligand MHC class I-related chain a transfers from target cells to NK cells in a manner that allows functional consequences2007In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 178, no 6, p. 3418-3426Article in journal (Refereed)
    Abstract [en]

    Recently, it has become apparent that surface proteins commonly transfer between immune cells in contact. Inhibitory receptors and ligands exchange between cells during NK cell surveillance and we report here that NK cells also acquire activating ligands from target cells. Specifically, the stress-inducible activating ligand for NKG2D, MHC class I-related chain A (MICA), transferred to NK cells upon conjugation with MICA-expressing target cells. Acquisition of MICA from target cells was dependent on cell contact and occurred after accumulation of MICA at the immunological synapse. Moreover, transfer of MICA was facilitated by specific molecular recognition via NKG2D and augmented by Src kinase signaling. Importantly, MICA associated with its new host NK cell membrane in an orientation that allowed engagement with NKG2D in trans and indeed could down-regulate NKG2D in subsequent homotypic interactions with other NK cells. MICA captured from target cells could subsequently transfer between NK cells and, more importantly, NK cell degranulation was triggered in such NK cell-NK cell interactions. Thus, NK cells can influence other NK cells with proteins acquired from target cells and our data specifically suggest that NK cells could lyse other NK cells upon recognition of activating ligands acquired from target cells. This mechanism could constitute an important function for immunoregulation of NK cell activity.

  • 7.
    Morfeldt, E
    et al.
    Lund University.
    Berggård, K
    Lund University.
    Persson, J
    Lund University.
    Drakenberg, T
    Lund University.
    Johnsson, E
    Lund University.
    Lindahl, E
    KTH, Superseded Departments, Physics.
    Linse, S
    Lund University.
    Lindahl, G
    Lund University.
    Isolated hypervariable regions derived from streptococcal M proteins specifically bind human C4b-binding protein: implications for antigenic variation.2001In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 167, no 7, p. 3870-7Article in journal (Refereed)
    Abstract [en]

    Antigenic variation in microbial surface proteins represents an apparent paradox, because the variable region must retain an important function, while exhibiting extensive immunological variability. We studied this problem for a group of streptococcal M proteins in which the approximately 50-residue hypervariable regions (HVRs) show essentially no residue identity but nevertheless bind the same ligand, the human complement regulator C4b-binding protein (C4BP). Synthetic peptides derived from different HVRs were found to retain the ability to bind C4BP, implying that the HVR corresponds to a distinct ligand-binding domain that can be studied in isolated form. This finding allowed direct characterization of the ligand-binding properties of isolated HVRs and permitted comparisons between different HVRs in the absence of conserved parts of the M proteins. Affinity chromatography of human serum on immobilized peptides showed that they bound C4BP with high specificity and inhibition experiments indicated that different peptides bound to the same site in C4BP. Different C4BP-binding peptides did not exhibit any immunological cross-reactivity, but structural analysis suggested that they have similar folds. These data show that the HVR of streptococcal M protein can exhibit extreme variability in sequence and immunological properties while retaining a highly specific ligand-binding function.

  • 8.
    Walwyn-Brown, Katherine
    et al.
    Univ Manchester, Fac Biol Med & Hlth, Manchester Collaborat Ctr Inflammat Res, Core Technol Facil Bldg,46 Grafton St, Manchester M13 9NT, Lancs, England..
    Guldevall, Karolin
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Saeed, Mezida
    Univ Manchester, Fac Biol Med & Hlth, Manchester Collaborat Ctr Inflammat Res, Core Technol Facil Bldg,46 Grafton St, Manchester M13 9NT, Lancs, England..
    Pende, Daniela
    Osped Policlin San Martino, Ist Ricovero & Cura Carattere Sci, Lab Immunol, I-16132 Genoa, Italy..
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Dept Microbiol Tumor & Cell Biol, S-17177 Stockholm, Sweden..
    MacDonald, Andrew S.
    Univ Manchester, Fac Biol Med & Hlth, Manchester Collaborat Ctr Inflammat Res, Core Technol Facil Bldg,46 Grafton St, Manchester M13 9NT, Lancs, England..
    Davis, Daniel M.
    Univ Manchester, Fac Biol Med & Hlth, Manchester Collaborat Ctr Inflammat Res, Core Technol Facil Bldg,46 Grafton St, Manchester M13 9NT, Lancs, England..
    Human NK Cells Lyse Th2-Polarizing Dendritic Cells via NKp30 and DNAM-12018In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 201, no 7, p. 2028-2041Article in journal (Refereed)
    Abstract [en]

    Cross-talk between NK cells and dendritic cells (DCs) is important in Th1 immune responses, including antitumor immunity and responses to infections. DCs also play a crucial role in polarizing Th2 immunity, but the impact of NK cell-DC interactions in this context remains unknown. In this study, we stimulated human monocyte-derived DCs in vitro with different pathogen-associated molecules: LPS or polyinosinic-polycytidylic acid, which polarize a Th1 response, or soluble egg Ag from the helminth worm Schistosoma mansoni, a potent Th2-inducing Ag. Th2-polarizing DCs were functionally distinguishable from Th1-polarizing DCs, and both showed distinct morphology and dynamics from immature DCs. We then assessed the outcome of autologous NK cells interacting with these differently stimulated DCs. Confocal microscopy showed polarization of the NK cell microtubule organizing center and accumulation of LFA-1 at contacts between NK cells and immature or Th2-polarizing DCs but not Th1-polarizing DCs, indicative of the assembly of an activating immune synapse. Autologous NK cells lysed immature DCs but not DCs treated with LPS or polyinosinic-polycytidylic acid as reported previously. In this study, we demonstrated that NK cells also degranulated in the presence of Th2-polarizing DCs. Moreover, time-lapse live-cell microscopy showed that DCs that had internalized fluorescently labeled soluble egg Ag were efficiently lysed. Ab blockade of NK cell-activating receptors NKp30 or DNAM-1 abrogated NK cell lysis of Th2-polarizing DCs. Thus, these data indicate a previously unrecognized role of NK cell cytotoxicity and NK cell-activating receptors NKp30 and DNAM-1 in restricting the pool of DCs involved in Th2 immune responses.

  • 9. Önfelt, Björn
    et al.
    Nedvetzki, S.
    Yanagi, K.
    Davis, D. M.
    Cutting edge: Membrane nanotubes connect immune cells2004In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 173, no 3, p. 1511-1513Article in journal (Refereed)
    Abstract [en]

    We present evidence that nanotubular highways, or membrane nanotubes, facilitate a novel mechanism for intercellular communication in the immune system. Nanotubes were seen to connect multiple cells together and were readily formed between a variety of cell types, including human peripheral blood NK cells, macrophages, and EBV-transformed B cells. Nanotubes could be created upon disassembly of the immunological synapse, as cells move apart. Thus, nanotubular networks could be assembled from transient immunological synapses. Nanotubes were seen to contain GFP-tagged cell surface class I MHC protein expressed in one of the connected cells. Moreover, GPI-conjugated to GFP originating from one cell was transferred onto the surface of another at the connection with a nanotube. Thus, nanotubes can traffic cell surface proteins between immune cells over many tens of microns. Determining whether there are physiological functions for nanotubes if an intriguing new goal for cellular immunology.

  • 10. Önfelt, Björn
    et al.
    Nedvetzki, Shlomo
    Benninger, Richard K. P.
    Purbhoo, Marco A.
    Sowinski, Stefanie
    Hume, Alistair N.
    Seabra, Miguel C.
    Neil, Mark A. A.
    French, Paul M. W.
    Davis, Daniel M.
    Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria2006In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 177, no 12, p. 8476-8483Article in journal (Refereed)
    Abstract [en]

    We report that two classes of membrane nanotubes between human monocyte-derived macrophages can be distinguished by their cytoskeletal structure and their functional properties. Thin membrane nanotubes contained only F-actin, whereas thicker nanotubes, i.e., those > similar to 0.7 mu m in diameter, contained both F-actin and microtubules. Bacteria could be trapped and surf along thin, but not thick, membrane nanotubes toward connected macrophage cell bodies. Once at the cell body, bacteria could then be phagocytosed. The movement of bacteria is aided by a constitutive flow of the nanotube surface because streptavidin-coated beads were similarly able to traffic along nanotubes between surface-biotinylated macrophages. Mitochondria. and intracellular vesicles, including late endosomes and lysosomes, could be detected within thick, but not thin, membrane nanotubes. Analysis from kymographs demonstrated that vesicles moved in a stepwise, bidirectional manner at similar to 1 mu m/s, consistent with their traffic being mediated by the microtubules found only in thick nanotubes. Vesicular traffic in thick nanotubes and surfing of beads along thin nanotubes were both stopped upon the addition of azide, demonstrating that both processes require ATP. However, microtubule destabilizing agents colchicine or nocodazole abrogated vesicular transport but not the flow of the nanotube surface, confirming that distinct cytoskeletal structures of nanotubes give rise to different functional properties. Thus, membrane nanotubes between macrophages are more complex than unvarying ubiquitous membrane tethers and facilitate several means for distal interactions between immune cells.

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