Sofia Johansson Project
Receptor dynamics on Natural Killer cells at the molecular scale
We are interested in the molecular dynamics of immune cells. We study how immune cell receptors are localized and move at the cell surface. Our aim is to characterize how such molecular dynamics relate to immune cell function. We use a number of novel fluorescence-based techniques to reach this aim.
Most of our studies take their start in that some cytotoxic immune cells are more efficient killers than other cells. Often it is not known why this is; the functional activity has not been related to any specific phenotype. Our basic idea is that the molecular dynamics can be used as phenotypical markers, just as for instance expression of a certain protein can. The quantitative information we gain about molecular dynamics can also be used for instance for mathematical modelling, to better predict how immune cells will react in different situations.
Fluorescence Correlation Spectroscopy (FCS)
Video: Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy
FCS measures the diffusion and concentration of fluorescently labelled molecules with down to single molecule sensitivity. In the ideal case, it is a truly quantitative technique that measures the exact number of molecules within the focus, and their diffusion rate. FCS was partly invented at KI and KTH. We have taken part in the development to enable FCS measurements also in cell membranes. By a cross-correlation FCS (FCCS) one can also measure the fraction of two proteins which are interacting within the cell cytoplasm or cell membrane.
Super resolution microscopy
Stimulated Emission by Depletion (STED) enables a resolution up to 30 nm (at present), compared to 250 nm with confocal resolution at best . This allows for the visual exploration of protein localization, for instance in lipid rafts or other nano-domains. 2-color STED also gives a more accurate account of protein co-localization, since the scale is close to the single-molecule level, whereas confocal microscopy fails to detect distances relevant for protein interaction.
TIRF-iMSD is a recently developed method that distinguish between free diffusion and different types of hindered diffusion in cell membranes. Diffusion can be hindered either by the cytoskeleton, or by confinement of molecules in nanodomains.
In two of our projects we study how cytokine stimulation and NK cell education are related to the molecular dynamics at the NK cell surface.
What are NK cells?
NK cells are lymphocytes which belong to the innate immune system. They can both kill abnormal cells directly, and release cytokines to alert other immune cells. NK cells rely on the balance of input from a set of different activating and inhibitory receptors to quickly recognize virus infected or tumorigenic cells. The activating ligands can be both endogenic, e.g. upregulated on the body’s own cells upon stress, or be of pathogenic origin. NK cells are currently used in cell-based therapies against several cancer types, mostly leukemia, due to their efficient cytotoxic capacity.
NK cells and cytokines
Several cytokines stimulate NK cell killing. Interleukin-2 (IL-2) is the prototypical cytokine that is most commonly used for clinical NK cell activation. IL-2 induces the expression of numerous proteins, including several adhesion molecules, and enhances the IFN-γ production and cytotoxicity against target cells in vitro and in vivo. Type I interferons, like interferon alpha and beta (IFN-α+β), are also strong inducers of NK cell cytotoxicity.
NK cell education
A particular capability of NK cells is to recognize the lack of “self” Major Histocompatibility Complex (MHC) class I molecules on cells. Cells who lack expression of self MHC class I will be killed by NK cells in a “missing-self” response. This depends on the expression of a set of inhibitory MHC-specific receptors on NK cells. The receptors are structurally unrelated but exhibit the same function and expression pattern in humans and in mice. In humans they are called Killer Immunoglobulin-Like receptors (KIR), and in mice Ly49 receptors. Different KIR/Ly49 receptors are specific for different MHC class I alleles. Since the MHC class I alleles differ hugely between different individuals within the human population, a “missing-self” response can for instance be triggered by donor NK cells against leukemic cells in a patient who are not, or only partially, MHC class I allele-matched.
Some NK cells express self-MHC specific receptors, and some do not. The maturing NK cells will thus need to learn whether they have receptors for MHC class I alleles which are “self”. This happens through a largely uncharacterized process called “education”. NK cells expressing receptors specific for self-MHC class I alleles become educated, whereas NK cells lacking such receptors remain uneducated, and do not take part in missing-self killing. The mechanism behind NK cell education is currently largely unknown. There are no proteins or protein combinations that are expressed uniquely by educated NK cells, that could serve as a phenotypical marker of educated NK cells. We have previously shown that the effectiveness of a particular NK cell is tuned according to how much inhibition it has received during maturation. This refers to both the number of self-specific inhibitory receptors the NK cell express, and the strength of the inhibitory interaction, in a quantitative fashion. Therefore, NK cells can be more or less educated, and thus kill with different efficiency on a gradual scale.
Ass. Prof. Petter Höglund, Karolinska Institutet (KI), Department of Medicine, Huddinge
Prof. Jerker Widengren, Experimental Biomolecular Physics, Applied Physics, Royal Institute of Technology.
Prof. Ramit Mehr, The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
IL-15, TIM-3 and NK cells subsets predict responsiveness to anti-CTLA-4 treatment in melanoma patients
Rossana Tallerico, Costanza M. Cristiani, Elina Staaf, Cinzia Garofalo, Rosa Sottile, Mariaelena Capone et al
OncoImmunology vol 6, 2017 issue 2