Fibroblastic reticular cells guide T lymphocyte entry into and migration within the splenic T cell zone

Abstract

Although a great deal is known about T cell entry into lymph nodes, much less is understood about how T lymphocytes access the splenic white pulp (WP). We show in this study that, as recently described for lymph nodes, fibroblastic reticular cells (FRCs) form a network in the T cell zone (periarteriolar lymphoid sheath, PALS) of the WP on which T lymphocytes migrate. This network connects the PALS to the marginal zone (MZ), which is the initial site of lymphocyte entry from the blood. T cells do not enter the WP at random locations but instead traffic to that site using the FRC-rich MZ bridging channels (MZBCs). These data reveal that FRCs form a substrate for T cells in the spleen, guiding these lymphocytes from their site of entry in the MZ into the PALS, within which they continue to move on the same network.

Figure 1. MZBCs directly connect the MZ to the PALS

(A) Schematic representation of the cellular elements of a WP unit and its surrounding MZ and RP. (B) Two adjacent spleen cryostat sections from HuCD2 GFP (green = PALS) mice were stained for B220 (blue = B cell follicles), Madcam-1 (left panel; red = MZ sinus), MOMA-1 (right panel; red = MMM) and imaged using confocal microscopy. This picture is representative of 2 different experiments.

Figure 2. FRCs connect the MZ to the PALS at the MZBC

Spleen cryostat sections from HuCD2 GFP (blue) mice were stained for ERTR-7 (green = FRC-derived matrix protein), MOMA-1 (red, panels A and C) or Desmin (panel B) and imaged using confocal microscopy. In panel C, the insert shows a higher magnification of 2 MZBCs. Arrowheads point to the location of MZBCs while “B” indicates the presence of B cell follicles. This picture is representative of 3 different experiments. See also Movie S1.

Figure 3. T cells crawl on the splenic FRC network

(A) 20 μm thick spleen cryostat sections from GFP (green) chimeric mice were examined using confocal microscopy after staining for desmin (red) and ERTR-7 (blue). C.A: central arteriole. M.Z: marginal zone. (B) 5 × 10⁶ SNARF-1 labeled T cells were injected i.v into chimeric mice. One day later, spleens were sectioned using a vibratome, perfused with warm, oxygenated medium, and imaged using 2P microscopy. Data show intravital snapshots of a single T cell (red) moving over time on FRC fibers (green) in a 12 μm thick volume. (C) Quantification of T cells showing turns in 4D datasets with respect to their location on or off GFP-marked stromal fibers in chimeric animals. Data are representative of at least 3 experiments.

Figure 4. T cells access the PALS using MZBCs

(A) 10 × 10⁶ CFSE labeled polyclonal T cells (green) were transferred in recipient mice. 10, 20, 30, and 180 min later, spleen cryostat sections were stained for B220 (blue), ERTR-7 (red) and imaged using confocal microscopy in order to identify where T cells enter the WP. Arrowheads point to the location where MZBC connect the MZ. (B) Quantification of T cells entering the WP in ERTR-7⁻ and ERTR-7⁺ areas of the WP over time. Data are representative of 3 different experiments.

Figure 5. B cells access the PALS using MZBCs

10 × 10⁶ CFSE labeled polyclonal T cells (green) and 10 × 10⁶ CMTPX labeled B cells (red) were transferred i.v. into recipient mice. 3 and 8 hours later, spleens were harvested and sectioned using a cryostat. Sections were stained for ERTR-7 (white) and imaged using confocal microscopy in order to identify the MZBCs and the PALS. At t = 3 hrs, arrows indicate the B cells that are entering the WP using the MZBCs. Only T and B cells present in the WP are shown. At 8 hrs after transfer, most B cells are localized to the follicles. Data are representative of 2 different experiments.