In vivo high spatiotemporal resolution visualization of circulating T lymphocytes in high endothelial venules of lymph nodes

Abstract Extravasation of leukocytes into organized lymphoid tissues and into sites of inflammation is critical to immune surveillance. Leukocyte migration to peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and Peyer's patches (PP) depends on L-selectin, which recognizes carbohydrate-bearing, sialomucin-like endothelial cell surface glycoproteins. Two of these ligands have been identified at the molecular level. One is the potentially soluble mucin, GlyCAM 1, which is almost exclusively produced by high endothelial venules (HEV) of PLN and MLN. The second HEV ligand for L-selectin is the membrane-bound sialomucin CD34. Historically, this molecule has been successfully used to purify human pluripotent bone marrow stem cells, and limited data suggest that human CD34 is present on the vascular endothelium of several organs. Here we describe a comprehensive analysis of the vascular expression of CD34 in murine tissues using a highly specific antimurine CD34 polyclonal antibody. CD34 was detected on vessels in all organs examined and was expressed during pancreatic and skin inflammatory episodes. A subset of HEV-like vessels in the inflamed pancreas of nonobese diabetic (NOD) mice are positive for both CD34 and GlyCAM 1, and bind to an L-selectin/immunoglobulin G (IgG) chimeric probe. Finally, we found that CD34 is present on vessels of deafferentiated PLN, despite the fact that these vessels are no longer able to interact with L-selectin or support lymphocyte binding in vitro or trafficking in vivo. Our data suggest that the regulation of posttranslational carbohydrate modifications of CD34 is critical in determining its capability to act as an L-selectin ligand. Based on its ubiquitous expression, we propose that an appropriately glycosylated form of vascular CD34 may act as a ligand for L-selectin-mediated leukocyte trafficking to both lymphoid and nonlymphoid sites.

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Lymphocytes perform reverse adhesive haptotaxis mediated by LFA-1 integrins

Journal of Cell Science ◽

10.1242/jcs.242883 ◽

2020 ◽

Vol 133 (16) ◽

pp. jcs242883

Author(s):

Xuan Luo ◽

Valentine Seveau de Noray ◽

Laurene Aoun ◽

Martine Biarnes-Pelicot ◽

Pierre-Olivier Strale ◽

...

Keyword(s):

T Lymphocytes ◽

Lymph Nodes ◽

Adhesion Molecules ◽

Stromal Cells ◽

Mesenchymal Cells ◽

High Endothelial Venules ◽

Integrin Ligands ◽

Inflamed Tissue ◽

Amoeboid Cells

ABSTRACTCell guidance by anchored molecules, or haptotaxis, is crucial in development, immunology and cancer. Adhesive haptotaxis, or guidance by adhesion molecules, is well established for mesenchymal cells such as fibroblasts, whereas its existence remains unreported for amoeboid cells that require less or no adhesion in order to migrate. We show that, in vitro, amoeboid human T lymphocytes develop adhesive haptotaxis mediated by densities of integrin ligands expressed by high endothelial venules. Moreover, lymphocytes orient towards increasing adhesion with VLA-4 integrins (also known as integrin α4β1), like all mesenchymal cells, but towards decreasing adhesion with LFA-1 integrins (also known as integrin αLβ4), which has not previously been observed. This counterintuitive ‘reverse haptotaxis’ cannot be explained by existing mechanisms of mesenchymal haptotaxis involving either competitive anchoring of cell edges under tension or differential integrin-activated growth of lamellipodia, because they both favor orientation towards increasing adhesion. The mechanisms and functions of amoeboid adhesive haptotaxis remain unclear; however, multidirectional integrin-mediated haptotaxis might operate around transmigration ports on endothelia, stromal cells in lymph nodes, and inflamed tissue where integrin ligands are spatially modulated.

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The Strategy of T Cell Antigen-presenting Cell Encounter in Antigen-draining Lymph Nodes Revealed by Imaging of Initial T Cell Activation

Journal of Experimental Medicine ◽

10.1084/jem.20030167 ◽

2003 ◽

Vol 198 (5) ◽

pp. 715-724 ◽

Cited By ~ 148

Author(s):

Marc Bajénoff ◽

Samuel Granjeaud ◽

Sylvie Guerder

Keyword(s):

T Cells ◽

T Cell ◽

Lymph Nodes ◽

T Cell Activation ◽

Cell Activation ◽

Strategic Positioning ◽

High Endothelial Venules ◽

Rare Cells ◽

Antigen Presenting

The development of an immune response critically relies on the encounter of rare antigen (Ag)-specific T cells with dendritic cells (DCs) presenting the relevant Ag. How two rare cells find each other in the midst of irrelevant other cells in lymph nodes (LNs) is unknown. Here we show that initial T cell activation clusters are generated near high endothelial venules (HEVs) in the outer paracortex of draining LNs by retention of Ag-specific T cells as they exit from HEVs. We further show that tissue-derived DCs preferentially home in the vicinity of HEVs, thus defining the site of cluster generation. At this location DCs efficiently scan all incoming T cells and selectively retain those specific for the major histocompatibility complex–peptide complexes the DCs present. Such strategic positioning of DCs on the entry route of T cells into the paracortex may foster T cell–DC encounter and thus optimize initial T cell activation in vivo.

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In vivo volumetric imaging of calcium and glutamate activity at synapses with high spatiotemporal resolution

Nature Communications ◽

10.1038/s41467-021-26965-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Wei Chen ◽

Ryan G. Natan ◽

Yuhan Yang ◽

Shih-Wei Chou ◽

Qinrong Zhang ◽

...

Keyword(s):

Adaptive Optics ◽

Temporal Resolution ◽

Simultaneous Recording ◽

High Temporal Resolution ◽

Spatiotemporal Resolution ◽

Volumetric Imaging ◽

High Spatiotemporal Resolution ◽

Mouse Cortex ◽

Two Photon Fluorescence

AbstractStudying neuronal activity at synapses requires high spatiotemporal resolution. For high spatial resolution in vivo imaging at depth, adaptive optics (AO) is required to correct sample-induced aberrations. To improve temporal resolution, Bessel focus has been combined with two-photon fluorescence microscopy (2PFM) for fast volumetric imaging at subcellular lateral resolution. To achieve both high-spatial and high-temporal resolution at depth, we develop an efficient AO method that corrects the distorted wavefront of Bessel focus at the objective focal plane and recovers diffraction-limited imaging performance. Applying AO Bessel focus scanning 2PFM to volumetric imaging of zebrafish larval and mouse brains down to 500 µm depth, we demonstrate substantial improvements in the sensitivity and resolution of structural and functional measurements of synapses in vivo. This enables volumetric measurements of synaptic calcium and glutamate activity at high accuracy, including the simultaneous recording of glutamate activity of apical and basal dendritic spines in the mouse cortex.

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T Lymphocyte Migration to Lymph Nodes Is Maintained during Homeostatic Proliferation

Microscopy and Microanalysis ◽

10.1017/s1431927608080215 ◽

2008 ◽

Vol 14 (3) ◽

pp. 211-224 ◽

Cited By ~ 10

Author(s):

Masanari Kodera ◽

Jamison J. Grailer ◽

Andrew P-A. Karalewitz ◽

Hariharan Subramanian ◽

Douglas A. Steeber

Keyword(s):

T Cells ◽

T Lymphocytes ◽

Lymph Nodes ◽

Homeostatic Proliferation ◽

Cell Numbers ◽

Naïve T Cells ◽

Memory Type ◽

Migration Assays

AbstractThe immune system maintains appropriate cell numbers through regulation of cell proliferation and death. Normal tissue distribution of lymphocytes is maintained through expression of specific adhesion molecules and chemokine receptors such as L-selectin and CCR7, respectively. Lymphocyte insufficiency or lymphopenia induces homeostatic proliferation of existing lymphocytes to increase cell numbers. Interestingly, homeostatic proliferation of T lymphocytes induces a phenotypic change from naïve- to memory-type cell. Naïve T cells recirculate between blood and lymphoid tissues whereas memory T cells migrate to nonlymphoid sites such as skin and gut. To assess effects of homeostatic proliferation on migratory ability of T cells, a murine model of lymphopenia-induced homeostatic proliferation was used. Carboxyfluorescein diacetate, succinimidyl ester-labeled wild-type splenocytes were adoptively transferred into recombination activation gene-1-deficient mice and analyzed by flow cytometry, in vitro chemotactic and in vivo migration assays, and immunofluorescence microscopy. Homeostatically proliferated T cells acquired a mixed memory-type CD44high L-selectinhigh CCR7low phenotype. Consistent with this, chemotaxis to secondary lymphoid tissue chemokine in vitro was reduced by 22%–34%. By contrast, no differences were found for migration or entry into lymph nodes during in vivo migration assays. Therefore, T lymphocytes that have undergone homeostatic proliferation recirculate using mechanisms similar to naïve T cells.

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Adhesive mechanisms governing interferon-producing cell recruitment into lymph nodes

Journal of Experimental Medicine ◽

10.1084/jem.20051035 ◽

2005 ◽

Vol 202 (5) ◽

pp. 687-696 ◽

Cited By ~ 86

Author(s):

Thomas G. Diacovo ◽

Amanda L. Blasius ◽

Tak W. Mak ◽

Marina Cella ◽

Marco Colonna

Keyword(s):

Lymph Nodes ◽

Nk Cell ◽

High Endothelial Venules ◽

Cell Recruitment ◽

Cell Responses ◽

Peripheral Lymph ◽

Chemokine Receptor Ccr5 ◽

Gain Access ◽

Receptor Ccr5

Natural interferon-producing cells (IPCs) are found in peripheral lymph nodes (PLNs), where they support NK cell, T cell, and B cell responses to pathogens. However, their route of entry and the adhesive mechanisms used to gain access to PLNs remain poorly defined. We report that IPCs can enter PLNs via a hematogenous route, which involves a multistep adhesive process, and that transmigration is enhanced by inflammation. Results indicate that L-selectin on IPCs is required for efficient attachment and rolling on high endothelial venules in vivo in both nonstimulated and inflamed PLNs. IPCs, however, also possess functional ligands for E-selectin that contribute to this process only in the latter case. In conjunction with selectin-mediated adhesion, both β1- and β2-integrins participate in IPC attachment to the inflamed vessel wall, whereas chemotaxis relies in part on the chemokine receptor CCR5. Identification of the adhesive machinery required for IPC trafficking into PLNs may provide opportunities to regulate immune responses reliant on the activity of these cells.

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