Human Tissue Samples

Tonsils (n = 5) were removed from patients undergoing routine tonsillectomy, and mesenteric LNs (n = 1) and livers were removed from patients during liver transplantation (normal liver, n = 1; PBC liver, n = 4; two contained well-defined TLT structures at several levels). Sjögren's syndrome salivary gland tissue (n = 10; two contained well-defined TLT structures at several levels) was obtained for biopsy. All human tissue samples were frozen in liquid nitrogen. Ethical approval for the use of tissue samples taken during this study was obtained from the Birmingham and local Research Ethics Committee (REC 2002/088, LREC 5735, and 06/Q2706/66).

Immunofluorescence Microscopy on Human Tissue Samples

Acetone-fixed 5-μm sections of snap-frozen tissues were prepared and stained, as previously described,43 with the antibodies listed in Table 1. Slides were mounted in 2.4% w/v 1,4-diazabicyclo(2,2,2)octane (Aldrich, Gillingham, England) in 10% v/v glycerol (Fisons Scientific, Loughborough, UK) in PBS (pH, 8.6) and analyzed by confocal microscopy (LSM 510; Carl Zeiss Ltd, Welwyn Garden City, England).

Immunofluorescence Microscopy on Murine Tissues

To visualize functional conduits, Texas Red–labeled 10-kDa dextran (Invitrogen AG, Basel, Switzerland) was injected i.v. and mice were sacrificed 5 minutes later. The pancreas was removed, fixed in four percent paraformaldehyde at 4°C, and saturated in 30% sucrose for 3 hours at 4°C before embedding in Optimal Cutting Compound (OCT, Tissue-Tek; Sakura), followed by freezing in an ethanol dry ice bath. For all other experiments, dissected tissues were embedded in OCT without prior fixation. Immunofluorescence stainings were performed on cryostat sections (8 μm) with the antibodies listed in Table 1, as previously described.10 For podoplanin and CCL21, primary antibodies were revealed using biotinylated secondary antibodies and streptavidin–horseradish peroxidase, followed by tyramide signal amplification (kit 22; Invitrogen), according to the manufacturer's instructions; however, a borate buffer (0.1 mol/L in PBS, pH 8.5) was used for tyramide dilution. Images were acquired on a microscope (Axioplan) with AxioCam MRm (Zeiss) or on a different microscope (DM IRE2) with laser-scanning confocal head TCS SP2 AOBS (acoustic optical beam splitter; Leica, Nunningen, Switzerland).

Vibratome Sections and Immunofluorescence Staining

Isolated peripheral LNs and pancreas were fixed overnight at 4°C in freshly prepared one percent paraformaldehyde (in PBS), washed, and embedded in four percent low-gelling agarose (Sigma) in PBS; 100- to 200-μm sections were cut using a vibratome (Microm HM 650V). Sections were blocked with one percent bovine serum albumin for 1 hour and stained for at least 3 hours or overnight with the antibodies listed in Table 1. Subsequently, sections were washed extensively in PBS and embedded using Mowiol 4-88 (Merck, Darmstadt, Germany). Images were taken with an upright microscope (Axio Imager with ApoTome; Zeiss). The 3D image reconstructions were made with Axio Vision software version 4.8.1 (Zeiss).

Isolation of LTi Cells

Peripheral LNs, spleen, or pancreas was dissected from euthanized mice. The capsule of the LNs was opened with 26-gauge syringe needles; the spleen and pancreas were cut into small pieces. The LN, spleen, and pancreas pieces were then digested with gentle stirring for 20 minutes at 37°C in 2 mL of RPMI 1640 medium containing 1 mg/mL collagenase IV (Worthington, Lakewood, NJ), 40 μg/mL DNase I (Roche, Basel, Switzerland), and two percent (v/v) FBS. Collagenase D, 1 mg/mL (Roche), was added; and digestion continued for another 20 minutes. Every 10 minutes, the suspension was gently pipetted to break up remaining aggregates until no visible fragments remained. During the last pipetting, EDTA was added to a final concentration of 5 mmol/L to further reduce cell aggregates. Cells were then passed through a 40-μm mesh, washed twice, and resuspended in RPMI 1640 medium containing two percent (v/v) FBS and 5 μg/mL DNase I at a maximum concentration of 2 × 10⁷ cells/mL. The cell suspension was then underlayed with 2.5 mL of mixed Lympholyte M (Cedarlane, Burlington, VT) at 25°C and centrifuged at 750 × g for 20 minutes. The cells at the gradient interphase were collected and used for flow cytometry.

Flow Cytometry

Stainings were performed with 1 × 10⁵ to 1.5 × 10⁶ cells in 96-well V-bottom plates (Falcon; Milian AG, Geneva, Switzerland). Cells were blocked with one percent normal mouse serum and stained with the antibodies listed in Table 1 in 25 μL of PBS containing two percent FBS, 2 mmol/L EDTA, and 0.1% NaN₃; the cells were washed twice with 200 μL of this buffer after each step. Unconjugated primary antibodies were detected with the secondary reagents listed in Table 1. Data were acquired on a flow cytometer (FACSCanto; BD Biosciences, Basel, Switzerland) and analyzed with Flowjo software version 9.1 (TreeStar, Ashland, OR). To detect surface expression of LTβR, cells were pretreated with FcR blocking antibody (2.4.G2; BD Biosciences) and 0.5% normal mouse and rat serum. The LTβR-Fc (provided by J. Browning) and the FN14-Fc (provided by P. Schneider) (both human IgG1) were added and detected using a biotinylated goat anti-human IgG (Jackson ImmunoResearch, Newmarket, UK) pretreated for 30 minutes with four percent normal mouse and rat serum. Finally, streptavidin-phycoerythrin (eBioscience, San Diego, CA) was added, along with other surface markers.

In Situ Hybridization

Mice were anesthetized and perfused with PBS, followed by four percent paraformaldehyde in PBS. The pancreas was then removed and further fixed in four percent paraformaldehyde overnight and saturated in 30% sucrose for 3 hours at 4°C before embedding in OCT, followed by freezing in an ethanol–dry ice bath. Frozen sections (8 μm) were then treated as previously described9 and hybridized overnight at 60°C with a CCL21-antisense digoxigenin-labeled riboprobe in hybridization solution. After washing at high stringency, sections were incubated with sheep anti-digoxigenin (Roche), followed by alkaline phosphatase–coupled donkey anti-sheep antibody (Jackson ImmunoResearch Laboratories) and developed with Nitroblue-tetrazoliumchloride (NBT, Bio-Rad) and 5-Bromo-4-chloro-3-indolyl phospate (BCIP, Sigma).

Podoplanin⁺ Stromal Cells in Human Tissues Share Many Properties with Murine TRCs

To test whether these TRC-like cells observed in human tissues share other properties with TRCs characterized in murine LNs and spleen, we further defined these cells histologically. In T zones of LNs and tonsils, podoplanin⁺ cells formed a dense reticular network that associated with ECM molecules (ie, collagen-1, laminin, and fibronectin) (Figure 2, data not shown) in structures that resemble, in their composition and morphological features, the conduits found in murine SLOs.13 Podoplanin⁺ cells colocalized with CCL21 protein and were in close contact with CD11c-positive dendritic cells (Figure 2). Similarly, at sites of chronic inflammation, such as PBC liver and Sjögren's syndrome salivary gland, but not in healthy liver, ECM fibers were observed in association with podoplanin⁺ cells in T cell–rich infiltrates (Figure 2). To a variable extent, TRC-like cells displayed some detectable CCL21 protein and were associated with DCs. Together, these data indicate a high similarity of the stromal compartments of the T zone between murine SLOs and human lymphoid tissues.

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Figure 2

The podoplanin⁺ CD21⁻ cell network in human tissues is associated with ECM fibers, the chemokine CCL21 and CD11c⁺ dendritic cells. Immunofluorescence analysis of human SLOs, such as LNs and tonsils; and TLTs, such as inflamed PBC liver and inflamed salivary gland [Sjögren's syndrome (SS)]. Healthy liver served as a control. Podoplanin (green), collagen-1 (red), laminin (blue), CCL21 (red), and CD11c (red) in CD3⁺ T-cell areas are shown. Scale bars = 100 μm.

Podoplanin⁺ Stromal Cells Are Present in Murine TLTs

To study the development and properties of TLT-associated TRCs in greater detail, we tested several mouse models displaying lymphocytic tissue infiltration for the presence of these stromal cells: NOD mice that are a model of autoimmune diabetes mellitus and develop mostly smaller pancreatic lymphoid infiltrates44 and RIP-LTα transgenic and RIP-CXCL13 mice that form large infiltrates of mostly naïve T and B cells in kidney and pancreas, respectively, without causing overt disease.21, 29, 30 Similar to murine LNs, reticular podoplanin⁺ cell networks were present in the T- but not the B-cell–rich areas in TLTs of all three mouse models. These fibroblastic cells showed a surface phenotype distinct from podoplanin⁻ CD31^high blood vessels and podoplanin^+/− lyve1⁺ lymphatic vessels associated with the infiltrate, in addition to their difference in cell morphological features and organization (Figure 3, A and B; see Supplemental Figure S4 at http://ajp.amjpathol.org). Although large and intermediate infiltrates with well-developed T-cell areas were always associated with a reticular podoplanin⁺ cell network, smaller infiltrates without a T zone were often lacking it (not shown). A higher magnification showed that the podoplanin⁺ stromal cells also expressed desmin and surrounded conduit-like structures recognized by the ER-TR7 antibody, similar to LNs (Figure 3C). Contrary to a previous report,21 we occasionally found CD35⁺ FDCs inside the B-cell follicles of large RIP-CXCL13 infiltrates. However, the podoplanin⁺ cell network in the T zone was always CD35⁻ (Figure 3D), suggesting the presence of two distinct stromal cell networks. Therefore, TLTs in all three tissue infiltration models displayed reticular podoplanin⁺ cell networks with a close resemblance to the TRC network of the LN T zone.

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Figure 3

The TLTs developing in murine models of chronic inflammation form a network of podoplanin⁺ stromal cells in the T zone, similar to LNs. Immunofluorescence analysis of frozen sections of pancreas from NOD and RIP-CXCL13 tg⁺ mice and kidney from RIP-LTα tg⁺ mice and peripheral LNs. A: B220⁺ B cells (green) and CD3⁺ T cells (red), the TRC marker podoplanin (podo, red) combined with the endothelial marker CD31 (green) in serial sections. Tg⁻ pancreata did not show reticular podoplanin staining typical for TRCs (data not shown). Scale bar = 100 μm. i indicates islet. B: Infiltrate labeled with podoplanin (red), the lymphatic vessel marker Lyve-1 (green), and DAPI⁺ nuclei (blue). Arrows indicate lymphatic vessels; and arrowheads, the more abundant fibroblastic reticular cells. Scale bar = 100 μm. C: Higher-magnification images of the T-cell–rich zone of the infiltrate labeled with podoplanin (red) and desmin and the conduit marker ER-TR7 (green). Scale bar = 20 μm. D: The podoplanin (red) and CD35 (green) staining showing two distinct stromal cell networks in infiltrates of RIP-CXCL13⁺ pancreas. Scale bar = 100 μm. Data are representative of at least three independent experiments.

T-Zone Reticular Networks Share Phenotypic and Functional Features with LN TRCs

To further compare the phenotype of podoplanin⁺ CD31⁻ stromal cells found in TLTs with their counterparts in LNs,10, 12 a panel of markers was tested on pancreatic infiltrates of RIP-CXCL13–transgenic mice, which, among the three mouse models, displayed the highest frequency of large infiltrates and lymphoid tissue structures. Confocal microscopic images showed that many TRC markers, including BP-3, vascular cell adhesion molecule-1 (VCAM-1), platelet-derived growth factor receptor (PDGFR)α/β, and LTβR, colocalized with podoplanin staining (Figure 4A). Comparable to LNs,10 most CCL21 protein was localized in conduit-like structures that were enwrapped by podoplanin⁺ stromal cells (Figure 4B). By using in situ hybridization analysis, we detected Ccl21 mRNA expression on HEVs and in a reticular pattern in the T-cell–rich zone of pancreatic infiltrates, indicating that the CCL21 protein is locally produced (Figure 4C). To determine whether TLTs also form a conduit system, as found in SLOs,13, 14 ectopic pancreatic infiltrates of RIP-CXCL13 transgenic (tg) mice were labeled with antibodies to podoplanin and various ECM molecules. A 3D reconstruction of confocal microscopic images, obtained from thick vibratome sections, confirmed that TLTs develop 3D networks of podoplanin⁺ cells wrapping around laminin⁺ matrix structures, similar to LNs (Figure 4D). These structures consisted of a cellular layer expressing podoplanin, a basement membrane containing fibronectin and laminin, and a core containing collagen-1 (Figure 4E). Pancreatic infiltrates in prediabetic NOD mice displayed matrix structures with the same composition (Figure 4E). The functionality of the conduits present in RIP-CXCL13 tg⁺ pancreata was demonstrated by the rapid transport of i.v. injected fluorescent tracer (dextran–Texas Red) that highlights the ER-TR7⁺ reticular network within minutes after injection (Figure 4F). Similar to LNs, major histocompatibility complex class II⁺ DCs were associated on the outside of the podoplanin⁺ cells, possibly retrieving antigen from the conduit lumen13 (Figure 4G). In summary, functional conduits develop in T-cell–rich areas of TLTs and are tightly associated with TRCs, the likely producers of these ECM molecules. Because of the high phenotypic and functional resemblance of TRC-like cells in TLTs with their counterpart in SLOs, we will refer to them as TRCs as well.

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Figure 4

Phenotype of podoplanin⁺ stromal cells and matrix structures in T-cell–rich zones of pancreatic infiltrates in RIP-CXCL13 tg⁺ mice. A: Confocal immunofluorescence analysis of sections stained with markers for podoplanin (red) and BP-3, VCAM-1, PDGFRα, PDGFRβ, and LTβR (all green). Scale bars = 10 μm. B: Immunofluorescence analysis of pancreatic sections of RIP-CXCL13 tg⁺ mice labeled with antibodies against CCL21 (green) and podoplanin (red). Scale bar = 2 μm. Data are representative of three independent experiments. C:In situ hybridization analysis for Ccl21 transcripts in pancreatic infiltrates of RIP-CXCL13 tg⁺ mice. Tg⁻ pancreas showed only Ccl21 transcripts on lymphatic vessels distant from the islets (not shown). Scale bar = 100 μm. B indicates B zone; T, T zone; i, islet. Confocal immunofluorescence images from vibratome (D and G) or 8-μm-thick sections (E and F). D: The 3D reconstruction of images from T-cell–rich zones of pancreatic infiltrates of RIP-CXCL13 tg⁺ mice and of LNs labeled with podoplanin (red) and laminin (green). Scale bars = 5 μm. E: T-cell–rich zone in sections from RIP-CXCL13 tg⁺ and NOD pancreas labeled with antibodies against the TRC marker podoplanin (red) and the conduit markers fibronectin (green), laminin (red), and collagen-1 (green). Scale bars = 2 μm. F: Pancreas sections of RIP-CXCL13 tg⁺ mice that were injected i.v. with the tracer dextran–Texas Red 10 minutes before isolating and fixing the tissue. Sections were costained with antibodies against ER-TR7 (green) and podoplanin (blue). Scale bar = 2 μm. G: T-cell–rich zone of infiltrates in RIP-CXCL13 tg⁺ pancreas labeled with podoplanin (red), laminin (green), and MHC II (blue). Scale bar = 5 μm. Data are representative of at least two independent experiments.

Maintenance of Ectopic TRC Networks and Conduits Is Largely LTαβ Dependent

The formation and maintenance of several lymphoid structures in CXCL13-transgenic pancreata is known to be LTα and LTβR dependent, including HEVs, BP-3⁺, and CCL21⁺ stromal cells.21 To determine whether LTβR signaling was also involved in the maintenance of podoplanin⁺ TRC networks and conduits in RIP-CXCL13 tg⁺ mice, they were treated for 22 days with LTβR-Fc fusion protein, leading to the disappearance of most large infiltrates (see Supplemental Figure S5A at http://ajp.amjpathol.org).21 The remaining infiltrates showed small T-cell–rich zones that correlated with a marked reduction in the extent of the reticular TRC network (podoplanin⁺ CD31⁻Lyve1⁻) and of CD31⁺ blood vessels and Lyve1⁺ lymphatic vessels (Figure 5, A and B). Surprisingly, staining with antibodies against podoplanin, fibronectin, laminin, and collagen-1 revealed that the overall structure of the small remaining TRC network and conduits was not altered in LTβR-Fc–treated mice (Figure 5C, see Supplemental Figure S5B at http://ajp.amjpathol.org). In some infiltrates, the expression of the chemokine CCL21 was still detectable after LTβR-Fc treatment but always in a much reduced area (Figure 5D), confirming an earlier report21 and correlating with the smaller podoplanin⁺ TRC network. Therefore, the maintenance of functional and well-developed T zones in RIP-CXCL13 tg⁺ mice, including TRC networks and their associated conduits, is partially dependent on continuing LTβR signaling.

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Figure 5

Maintenance of the ectopic TRC network and the conduit system in RIP-CXCL13 tg⁺ pancreas is partially LTβR dependent. Immunofluorescence analysis of 8-μm-thick pancreas sections from adult RIP-CXCL13 tg⁺ mice (aged 4 months) treated twice per week for 22 days with 100 μg of soluble LTβR-Fc or control LFA-3-Fc fusion proteins. Data are representative of three mice per group. A: Serial frozen sections were stained for CD3⁺ T cells (red) and B220⁺ B cells (green) and for podoplanin (red) and CD31 (green). Scale bar = 100 μm. B: Staining of an infiltrated islet for podoplanin (red), lyve-1 (green), and DAPI⁺ nuclei (blue), with the clustered DAPI⁺ cells representing mainly infiltrating T and B cells. Lymphatic vessels appear in yellow (podoplanin⁺lyve1⁺); and reticular fibroblasts in red (podoplanin⁺lyve1⁻). Right: A higher magnification of the infiltrate is shown with the vascular marker CD31 instead of DAPI (blue). Most podoplanin⁺ cells (arrowheads) show reticular morphological features and absence of vascular markers (Lyve1⁻CD31⁻). C: Confocal images showing podoplanin⁺ cells (red) and fibronectin⁺ conduits (green) in the T-cell–rich region. Scale bar = 10 μm. D: Immunofluorescence analysis of CCL21 (green) expression in the podoplanin⁺ region. Scale bar = 100 μm.

LTi Cells Are Present in Adult Secondary and TLTs

To test if LTi cells could play a role as the LTαβ source in the formation of TLTs and LTβR⁺ TRC networks in particular, we crossed RIP-CXCL13 tg⁺ mice to mice deficient in the transcription factor RORγ, which lack LTi cells and, consequently, all LNs and PPs.41 We confirmed by flow cytometry that few CD4⁺ CD3⁻ CD45⁺ LTi cells expressing IL-7Rα and LTβR ligands can be found in the LNs and spleen of adult wild-type mice (Figure 6A), as previously reported.35, 36, 45 Consistent with the RORγ requirement of LTi cell development, cells with this phenotype were completely absent from spleens of adult RORγ^−/− mice (Figure 6A), confirming an earlier report35 on RORγ^−/− bone marrow chimeras. The CD4⁺ CD3⁻ cells expressing IL-7Rα and LTβR ligands were also detected in the pancreas of adult RIP-CXCL13–transgenic mice, but not in nontransgenic littermates or transgenic mice on an RORγ-deficient background (Figure 6B, data not shown). In the three mouse strains, we did not obtain strong evidence for CD4⁻ LTi cells because most CD4⁻ CD3⁻ cells were negative for IL-7Rα and LTβR ligands.46 Strikingly, in RIP-CXCL13–transgenic pancreas, the LTi frequency was in a similar range as in the SLOs of adult mice (Figure 6, A and B). Histological analysis showed that most IL-7Rα⁺ CD3⁻ cells localized to perifollicular regions in the pancreatic infiltrates, similar to adult LNs and spleen (Figure 6C).45 The phenotype and localization of these cells in adult tissues suggest they are LTi cells. This finding raises the possibility of cross talk between these LTi cells and LTβR⁺ TRCs found in the same zone.

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Figure 6

The RORγ-dependent LTi cells are present in adult wild-type spleen and pancreatic infiltrates of adult RIP-CXCL13 tg⁺ mice and are critical for the development of larger pancreatic infiltrates with TRC networks and conduit systems. A: Flow cytometric analysis of single-cell suspensions prepared from adult peripheral LNs and the spleen of RORγ^+/+ and RORγ^−/− mice. Dot blots are pregated for CD45⁺ cells. Gates indicate CD4⁺ CD3⁺ (T cells; red) and CD4⁺ CD3⁻ CD11c⁻ CD19⁻ (LTi like; blue) populations (n = 3). Histograms show IL-7Rα expression on these two populations and LTβR ligand expression on the CD4⁺ CD3⁻ LTi cell population, detected with LTβR-Fc compared with the control FN14-Fc fusion protein. B: Hematopoietic cells were isolated from the pancreas of adult RIP-CXCL13 tg⁺ mice on an RORγ^+/+ or RORγ^−/− background. Dot blots are pregated for CD45⁺ cells and show CD4⁺ CD3⁻ CD11c⁻ CD19⁻ (LTi-like; blue) and CD4⁻ CD3⁻ CD11c⁻ CD19⁻ (red) populations (n = 3). Histograms show IL-7Rα and LTβR ligand expression on these two populations. C: Serial frozen sections of peripheral LNs, spleen, and pancreas of RIP-CXCL13 tg⁺ mice were stained with antibodies to IL-7Rα (red) and CD3/CD11c (green). Arrowheads indicate IL-7Rα⁺ CD3⁻ CD11c⁻ LTi-like cells in perifollicular regions. Scale bar = 100 μm. A through C: Data are representative of at least three independent experiments. B, B cell-rich zone; T, T cell rich zone. D: Pancreata from RIP-CXCL13 tg⁺ mice crossed onto the RORγ^−/− and TCRβδ^−/− background were sectioned (10 μm), and every 15^th section was stained with hematoxylin and analyzed for the frequency and size of islet-associated mononuclear infiltrates. Results were derived from 100 to 200 islets in each of the three transgenic mice per group (littermates aged 7 to 12 weeks).21 The frequencies of small (5 to 30 hematopoietic cells/infiltrate in a section), medium (31 to 300 cells), and large (greater than 300 cells) infiltrates and total infiltrates are shown. Arrow indicates the striking reduction of large infiltrates in tg⁺ mice on a RORγ^−/− background. E: Sections of RIP-CXCL13 tg⁺ pancreata on an RORγ^+/−, RORγ^−/−, or TCRβδ^−/− background were labeled for CD3⁺ T cells (red) and B220⁺ B cells (green). Examples of the largest infiltrates in each mouse are shown. Scale bar = 100 μm. The white squares indicate the area chosen in the consecutive sections to show staining for podoplanin⁺ cells (red) and laminin⁺ (red) and collagen-1⁺ (green) conduits.

We thank Karin Hirsch for expert technical help; Mathias Heikenwälder for critical reading; Jeff Browning and Pascal Schneider for Fc-fusion proteins; Max D. Cooper, Willem van Ewijk, Beat Imhof, and Shin-ichi Nishikawa for antibodies or hybridomas; and Hans Acha-Orbea, Mathias Heikenwälder, and Dan Littman for mice.