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.
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We thank A. Bullen and M. Krummel for help with the two-photon microscope; R. Proia (US National Cancer Institute) for S1P1-deficient mice; L. Shiow for help with movie preparation; and J. An for colony management. Supported by the Cancer Research Institute (I.L.G.), the Irvington Institute for Immunological Research (S.R.S.), the National Health and Medical Research Council and American Australian Association (T.G.P.), the Boyer Graduate Program in the Biochemical Sciences (T.H.M.P.), the Howard Hughes Medical Institute (J.G.C.) and the National Institutes of Health.
LYVE-1+ cortical sinus structure. The first part of the movie shows a three-dimensional (3-D) reconstruction of a 214 μm z-stack through a LYVE-1+ structure imaged by two photon microscopy from the cortical side of an explanted inguinal lymph node. The second part of the movie shows a z stack of this same region that starts close to the capsule and extends deeper into the lymph node. LYVE-1+ sinuses were labeled with anti-LYVE-1 (green) and T cells with CMTMR (red). LYVE-1+ structures filled with LYVE-1+ signal are capsule-proximal regions packed with LYVE-1+ macrophages (MΦ). Deeper into the lymph node sinuses have well-defined borders and no meshwork of LYVE-1+ signal inside.
Cortical sinus entry of T cells is dependent on S1P1 (30 minutes). Time-lapse image sequence of 18 μz-projection shows a fragment of a LYVE-1+ cortical sinus labeled to detect LYVE-1 (red) and Edg1+/+ (GFP, green) and Edg1−/− (CMTMR, red) T cells. In the first image sequence, the tracks of several T cells that contacted the sinus border are highlighted by dots. The dots change from white to yellow if cell entry occurs. For clarity only some of the T cells that made contact are displayed with such tracks. The second image sequence is a replay of the first, now using white and yellow arrows to register correspondingly Edg1+/+ and Edg1−/− T cells in contact with the outer border of the LYVE-1+ sinus. The arrows appear only if the next step of the T cell is transmigration or departure from the sinus border within the time and z stack of the movie. White circles indicate the time and location where a T cell transmigrated into the sinus. The asterix highlights two cells that entered at a similar location. The score indicates the number of T cell contacts with the sinus outer border versus the number of entries into the sinus. Elapsed time is shown as mm:ss.
LYVE-1+ cortical sinus probing by Edg1+/+ and Edg1−/− T cells during entry decision-making (30 minutes). Two-photon microscopy of explanted inguinal lymph node. Four time-lapse image sequences of 2, 1, 2 or 2 z-planes, correspondingly, that contain fragments of LYVE-1+ cortical sinuses (green) and T cells approaching them from the parenchyma (Edg1+/+, double labeled with CFSE and CMTMR, yellow, or Edg1−/−, CMTMR, red). White arrows point to the imaged cells and their processes. Elapsed time is shown as mm:ss.
Drainage of PE into the medullary sinuses (30 minutes). Two-photon intravital microscopy of the medullary region of the inguinal lymph node ∼5 min after R-PE injection into the tail of the mouse. The two time-lapse image sequences represent superficial (closer to the capsule) and somewhat deeper 15 μm z-projections. The movie shows gradual drainage of R-PE (red) into the medullary sinuses of the lymph node. Medullary LYVE-1+ structures are labeled by anti-LYVE-1 (green). Edg1+/+ T cells (GFP, yellow) and Edg1−/− T cells (red) were both labeled with CMTMR. Elapsed time is shown as mm:ss.
Flow of cells inside LYVE-1+ cortical sinuses. Two-photon intravital microscopy of inguinal lymph node. Cortical LYVE-1+ structure was volume-rendered at the mid-point of the imaging period. The structure was subdivided into MΦ–rich sinus area (red) and cortical sinuses (yellow) as described in Methods. Tracks in various colors represent trajectories of Edg1+/+ T cells inside interconnected regions of LYVE-1+ sinuses. The first part of the movie shows a rotation of the 3-D image with rendered structures and cell trajectories. The second segment is a 30 min movie that shows movement of Edg1+/+ T cells (green, center of their coordinates are indicated with gray spheres) in the regions of LYVE-1+ sinuses with flow. Note that there was a small shift in the lymph node over the course of the experiment and this causes the single volume-rendered structure that is displayed to not fully superimpose with track locations at all time points. Elapsed time is shown as mm:ss.
Flow of cells in medullary region (30 minutes). Two-photon intravital microscopy of inguinal lymph node from the medullary side. LYVE-1+ medullary structures were labeled with anti-LYVE-1 (green). Edg1+/+ T cells (GFP, yellow) and Edg1−/− T cells (red) were both labeled with CMTMR. Wild-type B cells (CFP, blue) are also present. The two time-lapse image sequences represent a superficial (closer to the capsule) 15 μm z-projection and a somewhat deeper 18 μm z-projection. The left segment of the deeper view shows cells moving from a sinus that is surrounded by T cells and containing little internal LYVE-1 (macrophage) signal, into a sinus region with few surrounding T cells and containing internal LYVE-1 signal. This view is suggested to correspond to a cortical to medullary sinus connection. Elapsed time is shown as mm:ss.
T cell motility in the medullary region (30 minutes). Two-photon intravital microscopy of inguinal lymph node from the medullary side. Labeled to detect LYVE-1+ medullary structures (green), Edg1+/+ T cells (GFP, yellow), Edg1−/− T cells (red) and B cells (CFP, blue). Time-lapse image sequences represent 18 μm z-projection. Elapsed time is shown as mm:ss.
Cell exit from medullary sinuses into subcapsular space (30 minutes). Two-photon intravital microscopy of Edg1+/+ (yellow) T cells in the medullary region of the lymph node. The time-lapse image sequence represents a superficial 18 μm z-projection and shows cells that are exiting through the medullary sinus into the space beneath the capsule. Medullary LYVE-1+ structures are labeled by anti-LYVE-1 (green). Arrows in both movies indicate the same cells. Elapsed time is shown as mm:ss. See also Figure 5D.
Flow of cells beneath the capsule at the medullary side (20 minutes). Two-photon intravital microscopy of the medullary region of the lymph node. Labeled to detect medullary LYVE-1+ structures (green), Edg1+/+ T cells (GFP, yellow), Edg1−/− T cells (red), and B cells (CFP, blue). The two time-lapse image sequences represent a superficial (closer to the capsule) 15 μm z-projection, and a deeper 15 μm z-projection. Elapsed time is shown as mm:ss.