Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes

Abstract

After entry into lymph nodes (LNs), B cells migrate to follicles, whereas T cells remain in the paracortex, with each lymphocyte type showing apparently random migration within these distinct areas. Other than chemokines, the factors contributing to this spatial segregation and to the observed patterns of lymphocyte movement are poorly characterized. By combining confocal, electron, and intravital microscopy, we showed that the fibroblastic reticular cell network regulated naive T cell access to the paracortex and also supported and defined the limits of T cell movement within this domain, whereas a distinct follicular dendritic cell network similarly served as the substratum for movement of follicular B cells. These results highlight the central role of stromal microanatomy in orchestrating cell migration within the LN.

Figure 1

T cells closely interact with the LN fibroblastic reticular network. (A) Confocal image of a 10 μm thick section from a LN of a Hu-CD2 GFP mouse in which all T cells express GFP (blue). The location of the FRC-secreted marker ERTR-7 (green) and the B cell marker B220 (red) were used to show the exclusion of ERTR-7 staining from the B cell follicle. Originally imaged with a 16x objective. (B) Confocal image of a section from a wild-type LN stained for desmin (red) and ERTR-7 (green), demonstrating the overlap in location of the ERTR-7⁺ FRC-derived matrix and the network formed by the desmin⁺ FRC fibers. Originally imaged with a 63x objective. See also Movie S1. (C,D) Wild-type mice were either injected i.v with 10 x 10⁶ CFSE-labeled T cells (C) or not injected (D). Twelve hours later, both groups of mice were perfused with a fixative solution. (C) A representative confocal image of a section from a recipient LN showing a transferred CFSE-labeled T cell (blue) tightly associated with FRC fibers (the arrow points to the uropod of a migrating cell where dye accumulates). Originally imaged with a 63x objective. See also Movies S2 and S3. (D) Representative SEM pictures of lymphocytes associated with FRC fibers in the T cell zone. The collagen fibrils (*) known to be encased within FRC sheaths are clearly seen, as are the microvilli extending from the T cell to the FRC fiber.

Figure 2

T cells crawl on the FRC network. (A) Protocol for generating chimeric mice with GFP⁺ FRCs. (B) 20 μm thick LN sections from chimeric (green) mice were examined using confocal microscopy after staining for desmin (blue) and ERTR-7 (red). Originally imaged with a 63x objective. See also Movie S4. (C) 20 x 10⁶ SNARF-1 labeled T cells were injected i.v into chimeric mice. Three to four hours later, popliteal LNs were imaged using intravital 2-P microscopy. Data show intravital snapshots of a single T cell (red) moving over time on FRC fibers (green) in a 12 μm thick volume. The last panel presents the superposition of all T cell positions over time, with dotted lines indicating the paths and the cell positions in the individual snapshots. See also Figure S5A and Movies S5, S6 and S8. (D) Quantitation of T cells showing turns in intravital 4D datasets with respect to their location on or off GFP-marked stromal fibers in chimeric animals.

Figure 3

T cells leave HEVs via “exit ramps”. (A,B) Confocal image of a LN section from a ubiquitin-GFP chimera (green) stained for ERTR-7 (red), PNAd (blue) (A), or desmin (blue) (B) expression, showing the thick endothelial cells of an HEV and the enveloping FRC sheath. The large lucent areas within a green endothelial cell are migrating lymphocytes. Originally imaged with a 16x and a 63x objective. (C) The popliteal LN of a chimera was surgically prepared for intravital imaging and 60 x 10⁶ SNARF-1-labeled T cells were injected i.v. The diapedesis of T cells (red) from the HEV (green vessel) to the parenchyma was imaged using 2-P microscopy. Data show intravital snapshots of T cells exiting HEV over time. See also Movie S9 (D) TEM picture of an HEV showing T cells exiting the vessel between adjacent FRCs.

Figure 4

The FRC network defines T cell migration range (territory). (A,B) 20 x 10⁶ CFSE-labeled T cells were transferred into a wild-type mouse. (A) Twelve hours later, 20 μm thick LN sections were stained for ERTR-7 (red) and B220 (blue) expression to visualize the fiber network and the B cell area, respectively, as well as the region of overlap. Picture shows a confocal image of transferred T cells (green) contacting the network at the T/B border. (B) Quantitation of T cell/ERTR-7⁺ network association in the B220⁺ enriched area. Originally imaged with a 63x objective.

Figure 5

B cells closely interact with the LN follicular dendritic cellular network. (A) Confocal image of a 10 μm thick section from a LN of a Hu-CD2 GFP mouse in which all T cells express GFP (blue). The location of the FDC network (FDC-M2⁺, green) inside the B cell area (B220⁺, red) was originally imaged with a 16x objective. (B) Confocal image of a section from a wild-type LN stained for desmin (red) and FDC-M2 (green), originally imaged with a 63x objective. (C) Wild-type mice were injected i.v with 10 x 10⁶ CFSE-labeled B cells. Twenty four hours later, mice were perfused with a fixative solution. A representative confocal image of a section from a recipient LN showing a transferred CFSE-labeled B cell (blue) tightly associated with FDC fibers was originally imaged with a 63x objective.

Figure 6

B cells crawl on the FDC network. (A) 20 x 10⁶ SNARF-1 labeled B cells were injected i.v into chimeric mice. Twenty four hours, popliteal LNs were imaged using intravital 2-P microscopy. The images show intravital snapshots of a single B cell (red) moving over time on FDC fibers (green) in a 12 μm thick volume. The last panel presents the superposition of all B cell positions over time. See also Movie S13. (B) Quantitation of B cells showing turns in intravital 4D datasets with respect to their location on or off GFP-marked stromal fibers in chimeric animals.