Superresolution Microscopy Reveals Nanometer-Scale Reorganization of Inhibitory Natural Killer Cell Receptors upon Activation of NKG2D

Pageon, Sophie V.; Cordoba, Shaun; Owen, Dylan M.; Rothery, Stephen; Oszmiana, Anna; Davis, Daniel M.

doi:10.1126/scisignal.2003947

Cited by 75 publications

(98 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Therefore, we hypothesize NK signal integration occurs by the relative sizedependent colocalization of receptors at the NK IS, and this is the explanation for the functional differences observed for elongated ligands. Considering the differences observed at a nanometer-scale, our results further support recent evidence that immune cell receptors show a nanometer-scale organization, with functional associated implications [19,22,50,[52][53][54][55]. Interestingly, there is mounting evidence that the molecular mechanisms behind immune inhibition are in part spatially restricted and common to various immune cells such as T, B and NK cells [56][57][58][59].…”

Section: Discussionsupporting

confidence: 86%

“…Thus, previous studies have used NK cell-like lines and artificial target cell surfaces, e.g. lipid bilayers or cross-linking antibody surfaces to study the NK IS [22,32,45,50]. However, neither the use of cell lines or lipid bilayers or antibody-coated surfaces are ideal to reproduce the exact molecular conditions observed in a bona fide NK IS cell-cell interface.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Nanoscale colocalization of NK cell activating and inhibitory receptors controls signal integration

Tomaz

Pereira

Guerra

et al. 2018

Preprint

View full text Add to dashboard Cite

NK cell responses depend on the balance of signals from inhibitory and activating receptors. However, how the integration of antagonistic signals occurs upon NK cell-target cell interaction is not fully understood. Here, we provide evidence that NK cell inhibition via the inhibitory receptor Ly49A is dependent on its relative colocalization at nanometer-scale with the activating receptor NKG2D upon immune synapse (IS) formation. NKG2D and Ly49A signal integration and colocalization was studied using NKG2D-GFP and Ly49A-RFP-expressing primary NK cells, forming ISs with NIH3T3 target cells, with or without expression of single chain trimer (SCT) H2-D d and an extended form of SCT H2-D d -CD4 MHC-I molecules. Nanoscale colocalization was assessed by Förster resonance energy transfer (FRET) between NKG2D-GFP and Ly49A-RFP and measured for each synapse. In the presence of their respective cognate ligands, NKG2D and Ly49A colocalize at a nanometer scale leading to NK cell inhibition. However, increasing the size of the Ly49A ligand reduced the nanoscale colocalization with NKG2D consequently impairing Ly49A-mediated inhibition. Thus, our data shows NK cell signal integration is critically dependent on the dimensions of NK cell ligand-receptor pairs by affecting their relative nanometer-scale colocalization at the IS. Together, our results suggest the balance of NK cell signals, and NK cell responses, are determined by the relative nanoscale colocalization of activating and inhibitory receptors in the immune synapse. NK cell signal integration | FRET nanoscale localization | NKG2D | Ly49ACorrespondence:david.tomaz@kcl.ac.uk, r.henriques@ucl.ac.uk, k.gould@imperial.ac.uk Introduction NK cell activation is dependent on a different array of germline-encoded receptors capable of triggering effector responses against infection [1][2][3] and cellular transformation [4,5], and yet maintaining tolerance towards healthy cells and tissues [6,7]. NK cell functionality is widely characterized by a balance between activating and inhibitory receptors [8,9]. However, upon immune synapse formation, how NK cells integrate signals from functionally antagonistic receptors, is not yet fully understood [10]. One major hypothesis to explain immune signal integration is based on the kinetic segregation (K-S) model [11]. This model states that the balance of antagonistic signals is mediated by the equilibrium of phosphorylation (kinases) and dephosphorylation (phosphatases), which depends on the relative colocalization of receptors and their associated signaling molecules upon synapse formation. This hypothesis has been validated using T cells, in the context of TCR triggering [12,13] and, more recently, NK cells [14,15]. The K-S model suggests immune inhibition, upon immune synapse formation, is maintained by the dephosphorylation of tyrosines in stimulatory receptors (e.g ITAMs-associated receptors such as TCR-CD3 or NKG2D-DAP12), due to a nanoscale colocalization with receptors bearing phosphatase activity (e.g. CD45, CD148) or with t...

show abstract

Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Nanoscale colocalization of NK cell activating and inhibitory receptors controls signal integration

Tomaz

Pereira

Guerra

et al. 2018

Preprint

View full text Add to dashboard Cite

show abstract

“…However, weak interactions leading to nanodomain formation may allow for diffusion of individual TCR as has been observed for Ras and GPI anchored proteins in nanodomains 62, 63 . In NK cells, there have to our knowledge been no super-resolution investigations of CD16 distribution, although micrometer-scale clusters have been observed 17 , however other NK cell receptor nanoclusters have been sized at 110 nm (KIR2DL1 measured by ground state depletion (GSD) and PALM 20 ).…”

Section: Discussionmentioning

confidence: 99%

“…More recently, advanced microscopy approaches including total internal reflection fluorescence microscopy (TIRF) 12 , and super-resolution techniques such as PALM 13 have revealed submicron clusters of cell surface receptors including the T cell antigen receptor (TCR) 12, 13 , Lat 13, 14 , ZAP-70 7, 15, 16 and SLP-76 7, 16 . Similarly, NK cell immunological synapses have revealed clusters of both activating (CD16 17, 18 and NKG2D 19 ) and inhibitory (KIR2DL1 20 ) receptors.…”

mentioning

confidence: 99%

Nanoscale Ligand Spacing Influences Receptor Triggering in T Cells and NK Cells

Delcassian¹,

Depoil²,

Rudnicka³

et al. 2013

Nano Lett.

Self Cite

116

112

View full text Add to dashboard Cite

Bioactive nanoscale arrays were constructed to ligate activating cell surface receptors on T cells (the CD3 component of the TCR complex) and NK cells (CD16). These arrays are formed from biofunctionalized gold nanospheres with controlled interparticle spacing in the range 25 – 104 nm. Responses to these nanoarrays were assessed using the extent of membrane-localized phosphotyrosine in T cells stimulated with CD3-binding nanoarrays, and the size of cell contact area for NK cells stimulated with CD16-binding nanoarrays. In both cases, the strength of response decreased with increasing spacing, falling to background levels by 69 nm in the T cell/anti-CD3 system and 104 nm for the NK cell/anti-CD16 system. These results demonstrate that immune receptor triggering can be influenced by the nanoscale spatial organization of receptor/ligand interactions.

show abstract

“…These protein islands coalesce into micrometer-scale clusters, or "microclusters," after T-cell activation (13). Although many other membrane proteins form similar nanometer-sized compartments, including lipid-anchored proteins (15)(16)(17), integrins (18,19), immune receptors (13,14,20,21), growth factor receptors (22) and neurotransmitter receptors (23,24), these have not been observed in tissue. In particular, nanometer-scale TCR organization and rearrangement after antigen recognition have not been observed in the physiological environment of T cells.…”

mentioning

confidence: 99%

Superresolution imaging reveals nanometer- and micrometer-scale spatial distributions of T-cell receptors in lymph nodes

Cang

Lillemeier

2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

T cells become activated when T-cell receptors (TCRs) recognize agonist peptides bound to major histocompatibility complex molecules on antigen-presenting cells. T-cell activation critically relies on the spatiotemporal arrangements of TCRs on the plasma membrane. However, the molecular organizations of TCRs on lymph node-resident T cells have not yet been determined, owing to the diffraction limit of light. Here we visualized nanometer- and micrometer-scale TCR distributions in lymph nodes by light sheet direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM). This dSTORM and SIM approach provides the first evidence, to our knowledge, of multiscale reorganization of TCRs during in vivo immune responses. We observed nanometer-scale plasma membrane domains, known as protein islands, on naïve T cells. These protein islands were enriched within micrometer-sized surface areas that we call territories. In vivo T-cell activation caused the TCR territories to contract, leading to the coalescence of protein islands and formation of stable TCR microclusters.

show abstract

Superresolution Microscopy Reveals Nanometer-Scale Reorganization of Inhibitory Natural Killer Cell Receptors upon Activation of NKG2D

Cited by 75 publications

References 60 publications

Nanoscale colocalization of NK cell activating and inhibitory receptors controls signal integration

Nanoscale colocalization of NK cell activating and inhibitory receptors controls signal integration

Nanoscale Ligand Spacing Influences Receptor Triggering in T Cells and NK Cells

Superresolution imaging reveals nanometer- and micrometer-scale spatial distributions of T-cell receptors in lymph nodes

Contact Info

Product

Resources

About