Mye-EUNITER
 

Loré, Karin

Models available:

Studies of MRCs in the blood and skin

We have several experimental systems that my lab has developed over the years where we could analyze the function of various myeloid cells in vitro. For example we can isolate subsets of DCs, monocytes, neutrophils etc from buffy coats and apheresis products from healthy blood donors from the Dept of Transfusion Medicine, Karolinska University Hospital. High yields of specific cell subsets can be isolated using magnetic bead isolation and/or flow cytometry. We also have methods to isolate skin cells using a skin graft mesher and sequential enzymatic treatment. Upregulation of co-stimulatory markers measured by flow cytometry. Selected cytokines and chemokines in cell culture supernatants measured by ELISA and Luminex. Fluorescently-labeled antigen to visualize its internalization and intracellular compartmentalization by confocal imaging.

We have established a human skin explant model where we can administer fluorescently-conjugated antigen and identify the cells that bind and/or internalize it. We have a long-standing collaboration with the Dept of Surgery, Karolinska University Hospital Solna and the Contur clinic, Stockholm that provide us with left over skin from reconstructive surgery of healthy individuals. By staining for different markers and using confocal imaging of cryosections from ex vivo injected skin we can determine antigen localization and cell association. Also, by flow cytometry we can characterize cells that have collected antigen and migrated out of the tissue, which is naturally occurring after activation. Phenotypic differentiation (i.e. CD40, CD70, CD80) and secretion of selected cytokines (IL-10, IL-12 etc.) can also be measured.

For studies of presentation capacity, my lab has developed a novel method based on presentation of the cognate CMV pp65 antigen for which detectable memory T cell responses are common in healthy blood donors. This enables a read-out of pp65-specific responding T cells by measuring proliferation (CFSE dilution) and production of effector cytokines such as IL-2, IFN- and TNF. Whether certain myeloid subsets show significantly better antigen presenting capacity and thus represent a beneficial targets for antigens in order to augment responses can be evaluated. If activated cells can provide bystander stimulation or suppression of T cells can be evaluated by supernatant transfer experiments.

Studies of rhesus macaque MRCs

We also have access to blood and tissues samples from rhesus macaques. We have gathered a substantial volume of biopsies from different organs. Both cryoblocks and single cell suspensions are stored frozen. We have antibody panel for flow cytometry phenotyping in place. We could therefore analyze the phenotype and function of various rhesus myeloid cells in vitro.

Technology available:

  • Isolation of primary human myeloid cells from blood and skin using ficoll, magnetic beads and/or flow       cytometry sorting
  • In situ staining of biopsies (e.g. skin biopsies) and single cells   
  • Multicolor flow cytometry for phenotyping
  • Cytokine and chemokine secretion assays (flow and Elisa)
  • Antigen uptake assays
  • Antigen presentation assays (co-culture w autologous T cells)
  • Isolation of rhesus immune cells and in vitro stimulation assays

Key publications related to Mye-EUNITER:

  1. Adams WC, Gujer C, McInerney GM, Gall JDG, Karlsson Hedestam GB, Koup RA, Loré K. Adenovirus Type -35 Vectors Block Human CD4+ T Cell Activation by CD46 Ligation. Proceedings of the National Academy of Sciences USA (PNAS). 2011, 108 (18): 7499-7504. Journal impact factor: 10.52

  2. Gujer C, Sundling C, Seder RA, Karlsson Hedestam GB, Loré K. Human and Rhesus Plasmacytoid Dendritic Cell and B Cell Responses to TLR Stimulation. Immunology. 2011, 134 (3):257-69. Journal impact factor: 2.83

  3. Bond E*, Liang F*, Sandgren KJ, Smed-Sörensen A, Bergman P, Brighenti S, Adams WC, Ashenafi Betemariam S, Rangaka MX, Wilkinson RJ. Andersson J, Loré K. Plasmacytoid dendritic cells infiltrate the skin in positive tuberculin skin test indurations. *Authors contributed equally. Journal of Investigative Dematology. 2012, 132(1):114-23. Journal impact factor: 6.27

  4. Sandgren KJ, Smed-Sörensen A, Forsell MN, Soldemo M, Adams WC, Liang F, Perbeck L, Koup RA, Wyatt RT, Karlsson Hedestam GB, J, Loré K. Human Plasmacytoid Dendritic Cells Efficiently Capture the HIV-1 Envelope Glycoprotein via CD4 for Antigen Presentation. Journal of Immunology. 2013: 191(1):60-9. Journal impact factor: 7.41

Contact details:

Karolinska Institutet
Department of Medicine Solna
Clinical Immunology and Allergy Unit, L2:04
Karolinska University Hospital Solna
171 76 Stockholm
Sweden
Tel. +46852487672
Karin.Lore@ki.se