Epidermal T Cell Dendrites Serve as Conduits for Bidirectional Trafficking of Granular Cargo

Dendritic epidermal T cells (DETCs) represent a prototypical lineage of intraepithelial γδ T cells that participate in the maintenance of body barrier homeostasis. Unlike classical T cells, DETCs do not recirculate and they remain persistently activated through their T cell receptors (TCR) at steady state, i.e., in absence of infection or tissue wounding. The steady state TCR signals sustain the formation of immunological synapse-like phosphotyrosine-rich aggregates located on projections (PALPs) which act to anchor and polarize DETC’s long cellular projections toward the apical epidermis while the cell bodies reside in the basal layers. The PALPs are known to contain pre-synaptic accumulations of TCR-containing and lysosomal granules, but how this cargo accumulates there remains unclear. Here, we combined anti-Vγ5 TCR, cholera toxin subunit B (CTB), and LysoTracker (LT)-based intravital labeling of intracellular granules, with high resolution dynamic microscopy and fluorescence recovery after photobleaching (FRAP) to characterize the steady state composition and transport of DETC granules in steady state epidermis. Intradermal fluorescent Vγ5 antibody decorated DETCs without causing cellular depletion, dendrite mobilization or rounding up and became slowly internalized over 48 h into intracellular granules that, after 6 days, colocalized with LAMP-1 and less so with LT or early endosomal antigen-1. Intradermal CTB was likewise internalized predominantly by DETCs in epidermis, labeling a partly overlapping set of largely LAMP-1+ intracellular granules. These as well as LT-labeled granules readily moved into newly forming dendrites and accumulated at the apical endings. FRAP and spatiotemporal tracking showed that the inside tubular lengths of DETC cellular projections supported dynamic trafficking of lysosomal cargo toward and away from the PALPs, including internalized TCR and lipid raft component ganglioside GM1 (labeled with CTB). By contrast, the rate of GM1 granules transport through comparable dendrites of non-DETCs was twice slower. Our observations suggest that DETCs use chronic TCR activation to establish a polarized conduit system for long-range trans-epithelial transport aimed to accumulate mature lysosomes at the barrier-forming apical epidermis. The biological strategy behind the steady state lysosome polarization by DETCs remains to be uncovered

Longitudinal confocal microscopy imaging of solid tumor destruction following adoptive T cell transfer

A fluorescence-based, high-resolution imaging approach was used to visualize longitudinally the cellular events unfolding during T cell-mediated tumor destruction. The dynamic interplay of T cells, cancer cells, cancer antigen loss variants, and stromal cells—all color-coded in vivo—was analyzed in established, solid tumors that had developed behind windows implanted on the backs of mice. Events could be followed repeatedly within precisely the same tumor region—before, during and after adoptive T cell therapy—thereby enabling for the first time a longitudinal in vivo evaluation of protracted events, an analysis not possible with terminal imaging of surgically exposed tumors. T cell infiltration, stromal interactions, and vessel destruction, as well as the functional consequences thereof, including the elimination of cancer cells and cancer cell variants were studied. Minimal perivascular T cell infiltrates initiated vascular destruction inside the tumor mass eventually leading to macroscopic central tumor necrosis. Prolonged engagement of T cells with tumor antigen-crosspresenting stromal cells correlated with high IFNγ cytokine release and bystander elimination of antigen-negative cancer cells. The high-resolution, longitudinal, in vivo imaging approach described here will help to further a better mechanistic understanding of tumor eradication by T cells and other anti-cancer therapies

Epithelial-to-mesenchymal transition drives a pro-metastatic Golgi compaction process through scaffolding protein PAQR11

Tumor cells gain metastatic capacity through a Golgi phosphoprotein 3-dependent (GOLPH3-dependent) Golgi membrane dispersal process that drives the budding and transport of secretory vesicles. Whether Golgi dispersal underlies the prometastatic vesicular trafficking that is associated with epithelial-to-mesenchymal transition (EMT) remains unclear. Here, we have shown that, rather than causing Golgi dispersal, EMT led to the formation of compact Golgi organelles with improved ribbon linking and cisternal stacking. Ectopic expression of the EMT-activating transcription factor ZEB1 stimulated Golgi compaction and relieved microRNA-mediated repression of the Golgi scaffolding protein PAQR11. Depletion of PAQR11 dispersed Golgi organelles and impaired anterograde vesicle transport to the plasma membrane as well as retrograde vesicle tethering to the Golgi. The N-terminal scaffolding domain of PAQR11 was associated with key regulators of Golgi compaction and vesicle transport in pull-down assays and was required to reconstitute Golgi compaction in PAQR11-deficient tumor cells. Finally, high PAQR11 levels were correlated with EMT and shorter survival in human cancers, and PAQR11 was found to be essential for tumor cell migration and metastasis in EMT-driven lung adenocarcinoma models. We conclude that EMT initiates a PAQR11-mediated Golgi compaction process that drives metastasis

Self-RNA–antimicrobial peptide complexes activate human dendritic cells through TLR7 and TLR8

Dendritic cell (DC) responses to extracellular self-DNA and self-RNA are prevented by the endosomal seclusion of nucleic acid–recognizing Toll-like receptors (TLRs). In psoriasis, however, plasmacytoid DCs (pDCs) sense self-DNA that is transported to endosomal TLR9 upon forming a complex with the antimicrobial peptide LL37. Whether LL37 also interacts with extracellular self-RNA and how this may contribute to DC activation in psoriasis is not known. Here, we report that LL37 can bind self-RNA released by dying cells, protect it from extracellular degradation, and transport it into endosomal compartments of DCs. In pDC, self-RNA–LL37 complexes activate TLR7 and, like self-DNA–LL37 complexes, trigger the secretion of IFN-α without inducing maturation or the production of IL-6 and TNF-α. In contrast to self-DNA–LL37 complexes, self-RNA–LL37 complexes also trigger the activation of classical myeloid DCs (mDCs). This occurs through TLR8 and leads to the production of TNF-α and IL-6, and the differentiation of mDCs into mature DCs. We also found that self-RNA–LL37 complexes are present in psoriatic skin lesions and are associated with mature mDCs in vivo. Our results demonstrate that the cationic antimicrobial peptide LL37 converts self-RNA into a trigger of TLR7 and TLR8 in human DCs, and provide new insights into the mechanism that drives the auto-inflammatory responses in psoriasis

Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma

Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde–derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde–derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma

Visualization of Protein Interactions in Living Cells

Ligand binding to cell membrane receptors sets off a series of protein interactions that convey the nuances of ligand identity to the cell interior. The information may be encoded in conformational changes, the interaction kinetics and, in the case of multichain immunoreceptors, by chain rearrangements. The signals may be modulated by dynamic compartmentalization of the cell membrane, cellular architecture, motility, and activation—all of which are difficult to reconstitute for studies of receptor signaling in vitro. In this paper, we will discuss how protein interactions in general and receptor signaling in particular can be studied in living cells by different fluorescence imaging techniques. Particularly versatile are methods that exploit Förster resonance energy transfer (FRET), which is exquisitely sensitive to the nanometer-range proximity and orientation between fluorophores. Fluorescence correlation microscopy (FCM) can provide complementary information about the stoichiometry and diffusion kinetics of large complexes, while bimolecular fluorescence complementation (BiFC) and other complementation techniques can capture transient interactions. A continuing challenge is extracting from the imaging data the quantitative information that is necessary to verify different models of signal transduction

Photobleaching-Corrected FRET Efficiency Imaging of Live Cells

Fluorescent resonance energy transfer (FRET) imaging techniques can be used to visualize protein-protein interactions in real-time with subcellular resolution. Imaging of sensitized fluorescence of the acceptor, elicited during excitation of the donor, is becoming the most popular method for live FRET (3-cube imaging) because it is fast, nondestructive, and applicable to existing widefield or confocal microscopes. Most sensitized emission-based FRET indices respond nonlinearly to changes in the degree of molecular interaction and depend on the optical parameters of the imaging system. This makes it difficult to evaluate and compare FRET imaging data between laboratories. Furthermore, photobleaching poses a problem for FRET imaging in timelapse experiments and three-dimensional reconstructions. We present a 3-cube FRET imaging method, E-FRET, which overcomes both of these obstacles. E-FRET bridges the gap between the donor recovery after acceptor photobleaching technique (which allows absolute measurements of FRET efficiency, E, but is not suitable for living cells), and the sensitized-emission FRET indices (which reflect FRET in living cells but lack the quantitation and clarity of E). With E-FRET, we visualize FRET in terms of true FRET efficiency images (E), which correlate linearly with the degree of donor interaction. We have defined procedures to incorporate photobleaching correction into E-FRET imaging. We demonstrate the benefits of E-FRET with photobleaching correction for timelapse and three-dimensional imaging of protein-protein interactions in the immunological synapse in living T-cells

video_4_Epidermal T Cell Dendrites Serve as Conduits for Bidirectional Trafficking of Granular Cargo.mp4

<p>Dendritic epidermal T cells (DETCs) represent a prototypical lineage of intraepithelial γδ T cells that participate in the maintenance of body barrier homeostasis. Unlike classical T cells, DETCs do not recirculate and they remain persistently activated through their T cell receptors (TCR) at steady state, i.e., in absence of infection or tissue wounding. The steady state TCR signals sustain the formation of immunological synapse-like phosphotyrosine-rich aggregates located on projections (PALPs) which act to anchor and polarize DETC’s long cellular projections toward the apical epidermis while the cell bodies reside in the basal layers. The PALPs are known to contain pre-synaptic accumulations of TCR-containing and lysosomal granules, but how this cargo accumulates there remains unclear. Here, we combined anti-Vγ5 TCR, cholera toxin subunit B (CTB), and LysoTracker (LT)-based intravital labeling of intracellular granules, with high resolution dynamic microscopy and fluorescence recovery after photobleaching (FRAP) to characterize the steady state composition and transport of DETC granules in steady state epidermis. Intradermal fluorescent Vγ5 antibody decorated DETCs without causing cellular depletion, dendrite mobilization or rounding up and became slowly internalized over 48 h into intracellular granules that, after 6 days, colocalized with LAMP-1 and less so with LT or early endosomal antigen-1. Intradermal CTB was likewise internalized predominantly by DETCs in epidermis, labeling a partly overlapping set of largely LAMP-1⁺ intracellular granules. These as well as LT-labeled granules readily moved into newly forming dendrites and accumulated at the apical endings. FRAP and spatiotemporal tracking showed that the inside tubular lengths of DETC cellular projections supported dynamic trafficking of lysosomal cargo toward and away from the PALPs, including internalized TCR and lipid raft component ganglioside GM1 (labeled with CTB). By contrast, the rate of GM1 granules transport through comparable dendrites of non-DETCs was twice slower. Our observations suggest that DETCs use chronic TCR activation to establish a polarized conduit system for long-range trans-epithelial transport aimed to accumulate mature lysosomes at the barrier-forming apical epidermis. The biological strategy behind the steady state lysosome polarization by DETCs remains to be uncovered.</p

video_9_Epidermal T Cell Dendrites Serve as Conduits for Bidirectional Trafficking of Granular Cargo.avi

<p>Dendritic epidermal T cells (DETCs) represent a prototypical lineage of intraepithelial γδ T cells that participate in the maintenance of body barrier homeostasis. Unlike classical T cells, DETCs do not recirculate and they remain persistently activated through their T cell receptors (TCR) at steady state, i.e., in absence of infection or tissue wounding. The steady state TCR signals sustain the formation of immunological synapse-like phosphotyrosine-rich aggregates located on projections (PALPs) which act to anchor and polarize DETC’s long cellular projections toward the apical epidermis while the cell bodies reside in the basal layers. The PALPs are known to contain pre-synaptic accumulations of TCR-containing and lysosomal granules, but how this cargo accumulates there remains unclear. Here, we combined anti-Vγ5 TCR, cholera toxin subunit B (CTB), and LysoTracker (LT)-based intravital labeling of intracellular granules, with high resolution dynamic microscopy and fluorescence recovery after photobleaching (FRAP) to characterize the steady state composition and transport of DETC granules in steady state epidermis. Intradermal fluorescent Vγ5 antibody decorated DETCs without causing cellular depletion, dendrite mobilization or rounding up and became slowly internalized over 48 h into intracellular granules that, after 6 days, colocalized with LAMP-1 and less so with LT or early endosomal antigen-1. Intradermal CTB was likewise internalized predominantly by DETCs in epidermis, labeling a partly overlapping set of largely LAMP-1⁺ intracellular granules. These as well as LT-labeled granules readily moved into newly forming dendrites and accumulated at the apical endings. FRAP and spatiotemporal tracking showed that the inside tubular lengths of DETC cellular projections supported dynamic trafficking of lysosomal cargo toward and away from the PALPs, including internalized TCR and lipid raft component ganglioside GM1 (labeled with CTB). By contrast, the rate of GM1 granules transport through comparable dendrites of non-DETCs was twice slower. Our observations suggest that DETCs use chronic TCR activation to establish a polarized conduit system for long-range trans-epithelial transport aimed to accumulate mature lysosomes at the barrier-forming apical epidermis. The biological strategy behind the steady state lysosome polarization by DETCs remains to be uncovered.</p