Ontogeny of stromal organizer cells during lymph node development

Abstract

The development of secondary lymphoid organs, such as lymph nodes (LNs), in the embryo results from the reciprocal action between lymphoid tissue inducer (LTi) cells and stromal cells. However, the initial events inducing LN anlagen formation before the LTi stromal cells cross-talk interactions take place are not fully elucidated. In this study, we show that the inguinal LN anlagen in mouse embryos developed from mesenchymal cells surrounding the lymph sacs, spherical structures of endothelial cells that bud from veins. Using inguinal and mesenteric LNs (mLNs), we provide evidence supporting a two-step maturation model for stromal cells: first, ICAM-1(-)VCAM-1(-) mesenchymal precursor cells become ICAM-1(int)VCAM-1(int) cells, in a process independent of LTi cells and lymphotoxin beta receptor (LTbetaR) signaling. The second step involves the maturation of ICAM-1(int)VCAM-1(int) cells to ICAM-1(high)VCAM-1(high) mucosal addressin cell adhesion molecule-1(+) organizer cells and depends on both LTi cells and LTbetaR. Addition of alphaLTbetaR agonist to LN organ cultures was sufficient to induce ICAM-1(int)VCAM-1(int) cells to mature. In LtbetaR(-/-) embryos, both inguinal and mLN stromal cells showed a block at the ICAM-1(int)VCAM-1(int) stage, and, contrary to inguinal LNs, mLNs persist longer and contained LTi cells, which correlated with the sustained gene expression of Il-7, Cxcl13, and, to a lesser degree, Ccl21. Taken together, these results highlight the importance of the signals and cellular interactions that induce the maturation of stromal cells and ultimately lead to the formation of lymphoid tissues.

FIGURE 1

The LN anlagen developed at the site of contact between mesenchymal and endothelial cell populations. A, Structure of an E13 iLN primordium. At this stage, the iLN primordium consists of a bud of endothelial cells stained with gp38/podoplanin (green) surrounded by layers of mesenchymal cells stained with ER-TR7 (red) (×40/1.4 numerical aperture [NA] water lens). B, Immunofluorescence staining of E15 iLN sections (×40/1.4NAwater lens) showing the central endothelium stained with gp38/ podoplanin, ICAM-1, CCL21 (green), collagen type I (red), laminin α5 (green), and perlecan (red) and the surrounding mesenchyme stained with PDGFRα, fibronectin, ER-TR7 (red), and CD44 (green). The left panels of all sections show the overlay with DAPI (white) staining the cell nuclei.

FIGURE 2

Mesenchymal cells invade the endothelial bud around E17 in iLNs. Immunofluorescence stainings of E17 iLN sections (×25/1.4 NA water lens) showing the endothelial markers perlecan in green (A), collagen type I in red, (B, F, H, and J), and laminin α5 in red (D). The following molecules expressed by mesenchymal cells have been stained in green: PDGFRα (G), fibronectin (C and E), and CD44 (I). Note that fibronectin also stained some cells of the vasculature and CD44 also stained hematopoietic cells. A white line has been drawn encircling the endothelial cell compartment. The bottom row shows the DAPI (white) staining the cell nuclei. Scale bar, 50 µm (A).

FIGURE 3

Ontogeny of stromal organizer cells. Emergence of the I^highV^high stromal organizer cells in LNs. FACS analysis of single-cell suspensions of iLN at E15 and E17 showing the recruitment of CD45⁺ hematopoietic cells and the concomitant phenotypic changes in the CD45⁻ stromal cells. Percentages shown in histogram correspond to CD45⁻ stromal cells and CD45⁺ hematopoietic cells. A, In iLNs, four different stromal cell populations were distinguished according to their level of expression of ICAM-1 and VCAM-1, and the levels of PDGFRα, gp38/podoplanin, MAdCAM-1, and VEGFR3 are shown for each stromal population: I⁻V⁻ (black) expressed PDGFRα, I^intV^int (blue) expressed gp38/podoplanin and PDGFRα, I⁺V⁻ (gray) expressed gp38/podoplanin, MAdCAM-1, and VEGFR-3 (E15 and E17), and I^highV^high (red) expressed PDGFRα, gp38/podoplanin, and MAdCAM-1 (E17). The ICAM-1/VCAM-1 expression profile of the CD45⁻ cells from iLNs changed between E15 and E17 independently of whether cells were harvested and analyzed on the same day or in different days. The gates in A have been set up to fit according to the profile of the different ICAM and VCAM expression levels in the cell populations rather than using the same for both E15 and E17. B, In mLNs, the same four stromal cell populations were identified. The I⁺V⁻ (gray) and I^highV^high (red) expressed MAdCAM-1 (E15 and E17). Percentages shown in scatter plots correspond to CD45⁻ stromal cells according to their expression of ICAM-1 and VCAM-1. Results are representative of at least three independent experiments.

FIGURE 4

Structures of E18 mLN and iLN. Immunofluorescence staining of E18 mLN sections (×25/1.4 NA and ×40/1.4 NA water lens) and E18 iLN sections (×40/1.4 NA water lens) showing the Lyve-1⁺ capsule (green), the CD4⁺ LTi cells (blue), and RANKL (top row), MAdCAM-1 (middle row), or IL-7Rα (bottom row) (red). Results are representative of at least three independent experiments.

FIGURE 5

Gradient of expression of stromal organizer markers during maturation of the mesenchyme. Cell sorting and gene expression analysis of E18 mLN I⁻V⁻ (black), I^lowV^low (blue), and I^highV^high (red) stromal cell populations. Real-time RT-PCR analysis of LtβR, RelB, Ccl21, Ccl19, Cxcl13, Il-7, RankL, Mmp9, and Tnfr1 genes. Ratio of gene of interest to β-actin is shown. Results are representative of at least three independent experiments.

FIGURE 6

LtβR signaling induce maturation of stromal cells from I^intV^int to I^highV^high. A, FACS analysis of single-cell suspensions from WT, LtβR^−/−, and Rorγ^−/− E15 and E17 iLNs and mLNs showing the recruitment of CD45⁺ cells and the concomitant phenotypic changes in the CD45⁻ stromal cells. Percentages shown in histograms correspond to CD45⁻ stromal cells and CD45⁺ hematopoietic cells. The different ICAM-1 and VCAM-1 CD45⁻ stromal cell populations described in Fig. 3 are shown with corresponding percentages. Note the normal development of the LtβR^−/− and Rorγ^−/− E15 iLN and the absence of the I^highV^high stromal cell populations in the LtβR^−/− and Rorγ^−/− E15 mLNs and E17 iLNs and mLNs. Results are representative of at least three independent experiments. B, LTβR stimulation induces the maturation of stromal cells from I^intV^int to I^highV^high in E14 mLN and E15 iLN organ cultures. FACS analysis of single-cell suspensions of E14 mLNs (left panels) and E15 iLNs (right panels) in organ cultures for 3 d showing the phenotypic changes in the CD45⁻ stromal cells induced by agonistic αLTβR Ab (bottom panels). Percentages shown in histograms correspond to CD45⁻ stromal cells. Three different stromal cells populations were identified: I⁻V⁻ (black), I^intV^int (blue), and I^highV^high (red). Expression levels of MAdCAM-1 for each cell population are shown in histograms. Results are representative of three independent experiments.

FIGURE 7

LTβR is not fully required for the recruitment and retention of LTi cells to mesenteric LNs. A, Cell sorting and gene expression analysis of the WT and LtβR^−/− E15 mLN I⁻V⁻ (black), I^intV^int (blue), and I⁺V⁻ (gray) stromal cell populations. Real-time RT-PCR analysis of the Ccl21, Il-7, and Cxcl13 genes. Ratio of gene of interest to β-actin is shown. Results are representative of two independent experiments. B, LTβR is required for the recruitment of LTi cells to iLNs but not mLNs. FACS analysis of single-cell suspensions from WT and LtβR^−/− E15 and E17 iLNs and mLNs showing the percentage of CD45⁺ cells and CD4⁺IL-7Rα⁺ LTi cells. Percentages shown in histograms correspond to CD45⁺ hematopoietic cells. CD45⁺ hematopoietic cell dot plot analysis of CD4 and IL-7Rα showed the percentages of LTi cells. Note the presence of LTi cells in the LtβR^−/− E17 mLN and their absence in iLNs. C, Immunofluorescence staining of WT and LtβR^−/− E16 mLN sections (×40/1.4 NA water lens) showing the Lyve-1⁺ capsule (green), CD4⁺ LTi cells (blue), and in red, IL-7Rα (left panels), MAdCAM-1 (middle panels), or RANKL (right panels). Note in the LtβR^−/− mLNs the presence of the capsule expressing Lyve-1 and MAdCAM-1, the presence of LTi cells coexpressing IL-7Rα and CD4, and the absence of RANKL expression. Results are representative of two independent experiments.

FIGURE 8

Model of LN stromal cell maturation. Based on our immunfluorescence and FACS analysis data (summarized in the top row in capital letters) and qPCR data (summarized in the bottom row in italics), we propose a model for the maturation of stromal cells during LN development. The I⁻V⁻ mesenchymal precursor cells (dark gray), upon stimulation by an unknown signal, will become I^intV^int primed stromal cells (blue) that upon upregulating the expression of RelB will be able to respond to LTβR signals upon engagement by the lymphotoxin α1β2 ligand expressed by LTi cells. Signals through the LTβR and other receptors induce the maturation of the I^intV^int cells (red) to become I^highV^high stromal organizer cells (see Discussion).