Cortical sinus probing, S1P1-dependent entry and flow-based capture of egressing T cells

Abstract

The cellular dynamics of the egress of lymphocytes from lymph nodes are poorly defined. Here we visualized the branched organization of lymph node cortical sinuses and found that after entry, some T cells were retained, whereas others returned to the parenchyma. T cells deficient in sphingosine 1-phosphate receptor type 1 probed the sinus surface but failed to enter the sinuses. In some sinuses, T cells became rounded and moved unidirectionally. T cells traveled from cortical sinuses into macrophage-rich sinus areas. Many T cells flowed from medullary sinuses into the subcapsular space. We propose a multistep model of lymph node egress in which cortical sinus probing is followed by entry dependent on sphingosine 1-phosphate receptor type 1, capture of cells in a sinus region with flow, and transport to medullary sinuses and the efferent lymph.

Figure 1. Morphology and distribution of LYVE-1⁺ cortical sinuses

(a) Projection of confocal images spanning a 6 μm thick section of inguinal lymph node with transferred GFP⁺ T cells (green) stained with LYVE-1 specific antibody (red). Scale bar, 200 μm. (b) Confocal microscopy of a lymph node section stained with LYVE-1 specific antibody (red) and with MOMA-1 specific antibody (green) to detect sinus-lining macrophages. A macrophage free cortical sinus (arrow) connects to LYVE-1⁺ sinuses near the medulla that are filled with MOMA-1⁺ macrophages, some of which are also LYVE-1⁺. Scale bar, 100 μm. (c) Schematic of connections between subcapsular, cortical and medullary LYVE-1⁺ sinuses in an inguinal lymph node. The major structures and regions are labeled. Gray circle shading indicates macrophage-rich areas. Positions of the objectives indicate cortical and medullary areas of inguinal lymph node subjected to two-photon intravital microscopy. (d) Projection view of a LYVE-1⁺ cortical sinus structure in an explanted inguinal lymph node observed using two-photon microscopy. Shown as a projection view of a 214 μm z-stack. LYVE-1⁺ sinuses are labeled with LYVE-1 specific antibody (green) and T cells with CMTMR (red). Scale bar, 30 μm. See also Supplementary Movie 1. Data in a, b and d are representative of inguinal lymph nodes from at least 3 mice.

Figure 2. T cell entry into LYVE-1⁺ cortical sinuses is dependent on S1P₁

(a-b) Two-photon intravital imaging of inguinal lymph node at its cortical side. Edg1^+/+ (GFP⁺, green) and Edg1^−/− (labeled with CMTMR, red) T cells were transferred 12 hours prior to imaging. LYVE-1⁺ sinuses are also labeled red. 21 μm z projection. Elapsed time is shown as mm:ss. (a) Shows a video frame containing a LYVE-1⁺ cortical sinus. White line follows contours of the LYVE-1⁺ sinus wall. Dotted yellow square indicates the area presented in the time lapse images in b. Arrow points to the Edg1^−/− T cell followed in the top panels of b. Scale bar, 30 μm. (b) Corresponding time-lapse images of Edg1^−/− (red, top panel) and Edg1^+/+ (green, bottom panel) T cells in relation to a sub-region of the LYVE-1⁺ cortical sinus. The path of an Edg1^−/− cell is tracked (arrow heads) from 1:20 to 5:20 min and then the path of an Edg1^+/+ cell is tracked from 5:20 to 7:00 min. See also Supplementary Movie 2. (c) Frequency of entry to LYVE-1⁺ sinuses for Edg1^+/+ and Edg1^−/− T cells. Three-dimensional reconstruction of fluorescently labeled LYVE-1⁺ structures was performed as described in Methods. T cell positions relative to the outside, inside and border of the cortical sinuses were assessed in every z plane of the image. Entry frequency is the ratio of entry events for cells starting at the outer sinus border versus the total events leaving the outer border. In a small number of cases clear discrimination of whether a cell was at the border of the sinus or transmigrated inside was not possible. Therefore, calculation of the entry frequency was performed both excluding these cases (top panel) or using a frequency-overestimating assumption that all of the indistinguishable cells did enter the structure (lower panel). Gray bars, Edg1^+/+ and white bars, Edg1^−/− T cells. Calculations were performed for 2 intravital movies (IVM) (2 mice) with observation from the cortical side (C) and 2 movies of explanted lymph nodes (2 mice) with one imaged from the cortical and one from the medullary side (M). For each experiment n indicates tracked number of events when T cells leave outer border of sinus either into parenchyma or inside the sinus. (d) Distribution of time spent at the outer border of cortical LYVE-1⁺ sinuses for Edg1^+/+ T cells (top panel) and Edg1^−/− T cells (lower panel) combined from two intravital experiments (2 mice). Light gray bar shows data for cells that contacted the sinus border and left within the tracking time; dark gray bar shows data for cells that arrived at the border before the tracking began or were still at the border at the end of the tracking. (e) Velocities of Edg1^+/+ and Edg1^−/− T cells outside LYVE-1⁺ sinuses (light gray) (median velocities 16.1 and 14.0 μm/min) or at their borders (dark gray) (median velocities 16.2 and 14.5 μm/min). Data are from two intravital experiments (2 mice). (f) Relative propensity of Edg1^+/+ and Edg1^−/− T cells to move towards LYVE-1⁺ sinuses. The ratio of Edg1^+/+ and Edg1^−/− T cell transition frequency into the area within 0 −10, 10 − 20 and 20 − 30 μm away from the sinus, calculated for 2 intravital (black and gray circles) and 1 explant (white circles) movie (3 mice) was determined as detailed in Methods.

Figure 3. Wild-type and S1P₁-deficient T cells extend processes into LYVE-1⁺ cortical sinuses during entry decision-making

a-d, Time-series images obtained by two-photon microscopy of explanted inguinal lymph node. Edg1^+/+ T cells double-labeled with CFSE and CMTMR (a, b) and Edg1^−/− T cells labeled with CMTMR (c, d) are shown approaching LYVE-1⁺ cortical sinuses, labeled with LYVE-1 specific antibody (green). White arrows indicate the location of cell processes. z-projections are from two z planes 3 μm apart (a, c and d), or a single z plane (b). Elapsed time is shown as mm:ss. Data in a-d are from two independent experiments (2 mice). Scale bar, 10 μm. See also Supplementary movie 3.

Figure 4. Directional flow and T cell retention inside LYVE-1⁺ cortical sinuses

(a) Three-dimensional reconstruction of LYVE-1⁺ structures imaged by two-photon intravital microsopy of inguinal LN. The structure was subdivided into macrophage-rich sinus area (red) and cortical sinuses (yellow) as described in the Methods. Colored tracks represent trajectories of Edg1^+/+ T cells inside various interconnected regions of LYVE-1⁺ positive sinuses. See also Supplementary movie 5. (b) Superimposed tracks of cells in regions I, II (first 2 panels) and IV, V (last 2 panels) of a, in the xy plane, setting the starting coordinates to the origin. Scale bar, 30 μm. (c) Axis ratio of cells outside LYVE-1⁺ cortical sinuses (white triangle), in the regions of the sinuses with random cell movement (gray diamond) and in the regions with directional cell movement (gray circle). Data are combined from two intravital experiments (2 mice) and medians are shown by horizontal lines. (d) Two-photon microscopy image of T cell morphology in the parenchyma and inside a LYVE-1⁺ cortical sinus with directional cell movement. The image is a 6 μm z projection and the cells and structures were labeled as indicated. (e) Velocities of Edg1^+/+ T cells from experiment illustrated in a. Velocities are shown for cells outside LYVE-1⁺ cortical sinuses (white triangles), and in sinus regions without flow (diamonds) and with flow (circles). Color-coding corresponds to the cortical sinus regions in a. (f) Velocities of Edg1^+/+ T cells from three intravital experiments (3 mice). Median velocities were calculated for each period of time that cells spent outside sinuses (white triangles) or inside sinuses without flow (diamonds) and with flow (circles). (g) Distribution of time spent by Edg1^+/+ T cells inside cortical LYVE-1⁺ sinuses in regions without flow (top panel) and with flow (lower panel). Data are pooled from three intravital experiments (3 mice). Light gray bars indicate tracks that entered and left the sinus within the tracking time. Dark gray bars indicate cells that transmigrated inside before the tracking began or were still inside at the end of the tracking. (h) Fraction of cells inside LYVE-1⁺ cortical sinuses that returned to the parenchyma within 30 min of tracking in regions without flow and with flow. Data are from three intravital experiments (3 mice). Bars indicate means. (i) Trajectories of two T cells tracked in green and blue migrating within a LYVE-1⁺ cortical sinus from a region without flow into an adjacent region where they are “captured” by flow at the position indicated by the red arrow. Image shows a fragment of region II from a. (j and k) Changes in the apparent velocities and axis ratio of the T cells shown in i. Red arrow indicates the location where the green and blue tracks cross into the region of flow. Velocity data are shown for the entire track duration whereas cell shape index data are shown for part of each track as indicated, where time zero indicates the start of imaging. Dashed lines indicate the median for cells in the absence of flow (long dashes) and in the presence of flow (short dashes), from c and f. Data in a, b, e are from one experiment that is representative of three (3 mice) and i-k are from one experiment that is representative of two (2 mice).

Figure 5. Comparison of Edg1^+/+ and Edg1^−/− T cell behavior in the medullary region and Edg1^+/+ T cell exit into the subcapsular space

(a) Three-dimensional view of medullary LYVE-1⁺ structures (green) and trajectories of Edg1^+/+ (white tracks) and Edg1^−/− (red tracks) T cells. See also Supplementary movie 7. (b) Median cell velocities of Edg1^+/+ and Edg1^−/− T cells in the medullary region. Horizontal lines indicate medians. (c) Edg1^+/+ and Edg1^−/− T cell velocities with respect to the LYVE-1⁺ medullary structures. Cell velocities were evaluated in the areas outside (light gray), on the border (dark gray) and inside (black) the LYVE-1⁺ structures. Horizontal lines indicate medians. n indicates number of events. Data in a are representative of three independent experiments and the tracking analysis in b and c was performed in one experiment. (d) Medullary side of the lymph node where T cells flow into the hilar region. Three-dimensional view of the LYVE-1⁺ structures (green) and Edg1^+/+ T cells (yellow). Dotted line outlines contour of the subcapsular space. The tracks are of cells that flow through the medullary sinus into the subcapsular region. White arrow indicates direction of movement. Data are representative of two intravital imaging experiments. See also Supplementary movie 8.