The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gray, D. H. D.
Right arrow Articles by Boyd, R. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gray, D. H. D.
Right arrow Articles by Boyd, R. L.
The Journal of Immunology, 2007, 178: 4956-4965.
Copyright © 2007 by The American Association of Immunologists, Inc.

A Unique Thymic Fibroblast Population Revealed by the Monoclonal Antibody MTS-151

Daniel H. D. Gray2,*, Dedreia Tull{dagger}, Tomoo Ueno*, Natalie Seach*, Brendan J. Classon{ddagger}, Ann Chidgey*, Malcolm J. McConville{dagger} and Richard L. Boyd3,*

* Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria, Australia; {dagger} Department of Biochemistry, Melbourne University, Parkville, Melbourne, Australia; and {ddagger} Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cell differentiation in the thymus is dependent upon signals from thymic stromal cells. Most studies into the nature of these signals have focused only on the support provided by the thymic epithelium, but there is an emerging view that other stromal cells such as mesenchymal fibroblasts may also be involved. Study of the latter has been hindered by a lack of appropriate markers, particularly those allowing their isolation. In this study, we describe a new surface marker of thymic stroma, MTS-15, and demonstrate its specificity for fibroblasts and a subset of endothelial cells. Coculture experiments showed that the determinant could be transferred between cells. Extensive biochemical analysis demonstrated that the Ag bound by MTS-15 was the glycosphingolipid Forssman determinant, consistent with the distribution observed. Transcriptional analysis of purified MTS-15+ thymic fibroblasts revealed a unique expression profile for a number of chemokines and growth factors important to thymocyte and epithelial cell development. In a model of cyclophosphamide-induced thymic involution and regeneration, fibroblasts were found to expand extensively and express growth factors important to epithelial proliferation and increased T cell production just before thymic regeneration. Overall, this study identifies a useful marker of thymic fibroblasts and highlights this subpopulation as a key player in thymic function by virtue of their support of both thymocytes and epithelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The thymus is the exclusive site of differentiation for mainstream {alpha}betaT cells that are required for virtually all adaptive immune responses. The complexity of T cell differentiation is reflected in the heterogeneity of specialized thymic microenvironments, which are formed through "cross-talk" between thymocytes and thymic stromal cells (1, 2, 3). The stromal compartment includes populations of epithelium, fibroblasts, endothelium, macrophages, and dendritic cells, each of which has distinct roles in T cell development (reviewed in Ref. 4).

The diversity of the cellular and molecular components of thymic stroma has been demonstrated by panels of mAbs specific for a variety of stromal cell Ags (for example, Ref. 5). These reagents have been useful in defining the heterogeneity of thymic stromal cells, in particular the epithelium. To date, none of these Abs recognize surface determinants expressed specifically by thymic mesenchymal fibroblasts; consequently, our knowledge of this population’s function is minimal.

A role for thymic fibroblasts in the direct support of thymocyte differentiation beyond the triple negative (TN4; CD3–CD4–CD8–) CD25+CD44+ stage (TN2) has been demonstrated via their provision of extracellular matrix components (6). More recently, the mesenchyme was found to be essential for thymic epithelial cell (TEC) proliferation during embryogenesis through the production of fibroblast growth factors (FGFs) 7 and 10 (7). Whether fibroblasts have a similar role in the adult thymus through the provision of FGFs or other cytokines remains to be determined.

Although a deficiency of FGF-7 allows normal thymic development and function, FGF-7 knockout mice exhibit impaired thymic regeneration in irradiation and chemotherapy models (8). Reciprocal bone marrow chimera experiments demonstrate that expression of FGF-7 by radioresistant cells is required for normal thymic recovery, most likely a subset of stromal cells (8). The precise definition of this stromal source of FGF-7 and the roles of other growth factors in thymic regeneration are of interest given that the speed of thymic recovery following radiation or chemically induced atrophy is clinically important (9).

Within the mouse thymic stromal (MTS) panel of Abs developed in our laboratory (10), MTS-15 was distinguished by a unique, restricted expression pattern at the blood/thymus barrier. This study explores in detail the expression profile and molecular characteristics of the MTS-15 Ag on thymic fibroblasts. This marker enabled the dissection of the stromal cellular and molecular events following cyclophosphamide treatment, revealing an important role for the mesenchyme in thymic regeneration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

C57BL/6 mice were used throughout this study for organs and cells at the ages indicated. Mice were bred and maintained by the Monash University Central Animal Services (Clayton, Australia) or the Alfred Medical Research and Education Precinct Animal Centre (Melbourne, Australia) and used according to institutional guidelines. Cyclophosphamide-treated mice were injected i.p. with Cycloblastin (Pharmacia) at a dose of 100 mg/kg body weight for two consecutive days (–1 and 0) for a total dose of 200 mg/kg.

Antibodies and immunoconjugates

The ER-TR-7 and M1/87 clones (gifts from K. Shortman, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) MTS-15 and MTS-12 (anti-CD31; our unpublished observations) were grown in our laboratory and the supernatants were used for these studies. Counterstains for FACS and immunohistology were FITC-conjugated anti-CD45.2 (clone 104; BD Pharmingen), PE conjugated anti-I-A/I-E (clone M5/114.15.2; BD Pharmingen), biotinylated anti-Ly51 (clone 6C3; BD Pharmingen), allophycocyanin-conjugated anti-CD8 (clone 53-6.7; BD Pharmingen), PE-conjugated anti-CD4 (clone RM 4-5; BD Pharmingen), PE-conjugated anti-CD45RA/B220 (clone RA3-6B2, BD Pharmingen), FITC-conjugated anti-TCRbeta (clone H57-597), PE-conjugated anti-Sca-1 (clone E13-161.7; BD Pharmingen), allophycocyanin-conjugated anti-Thy 1.2 (clone 53-2.3; BD Pharmingen), and anti-keratin (wide screen) (DakoCytomation). Biotinylated and unconjugated Abs were detected with the secondary reagents CyChrome-conjugated streptavidin (BD Pharmingen), FITC-conjugated goat anti-rat Ig (Silenus Laboratories), Cy5-conjugated anti-rabbit IgG (Amersham Biosciences), and Cy3-conjugated anti-rat IgG (Amersham Biosciences). An HRP-conjugated anti-rat Ig (Caltag Laboratories) was used as a secondary Ab on immunoblots.

Immunohistology and confocal microscopy

Immunohistology was performed as described previously (11). Briefly, sections (12–14 µm) were cut on a Tissue-Tek II cryostat (Miles Scientific) at –25°C and mounted on microscope slides. These were fixed in –20°C acetone for 30 s and air dried. Sections were then incubated with 30 µl of primary mAb for 15 min in a moist box at room temperature, followed by washing three times in PBS for 5 min each. Secondary Abs (30 µl) were then applied, incubated for 15 min, and washed from the slides. For three-color immunofluorescence, 10 µl of 10%(v/v) normal rat serum was added to sections for 5 min at room temperature to block any remaining reactive sites of the secondary reagents, followed by the addition of biotinylated and direct conjugates. After incubation and washing, streptavidin conjugates were incubated and washed before mounting with fluorescent mounting medium (DakoCytomation) using coverslips.

Digital images were acquired on a Bio-Rad MRC 1024 confocal microscope mounted on a Nikon E600 upright epifluorescence microscope through x10 or x20 objective lenses with a three-line krypton/argon laser (excitation lines 488, 568, and 647 nm) using the acquisition software Bio-Rad LaserSharp version 3.2. Files were analyzed using LaserSharp processing software and scale bars were added in Adobe Photoshop version 6.0.

Flow cytometry

Lymphoid cell suspensions were prepared by gently grinding organs between two frosted glass slides with cold FACS buffer (PBS, 1% FCS, and 0.02% (w/v) NaN3). Thymic stromal cells were isolated and enriched by collagenase/DNase or dispase/DNase digestion as described previously (11). The final digests were pooled for staining and flow cytometric analysis. Lung stroma and mesenchymal cells were isolated from pools of 3–4 adult lungs. These were minced with scissors and then treated with 0.3% (w/v) collagenase and.1% (w/v) DNase (Boehringer Mannheim) in RPMI 1640 for 40 min at 37°C with agitation. After the filtration of debris and RBC lysis in 0.83% (w/v) ammonium chloride for 1 min, cells were stained as described below. Thymic and lung stromal populations were gated on the phenotypes in Table I.


View this table:
[in this window]
[in a new window]

  		Table I. Phenotypes of thymic and lung stromal cell populations

 
Cells (3.0 x 106) were washed and stained with 30 µl of primary Ab for 20 min at 4°C. Following washing in FACS buffer, secondary Abs were applied for 20 min and then washed. The remaining reactive sites on the secondary reagents were blocked with 10 µl of 10% (v/v) normal rat serum in FACS buffer for 5 min before biotinylated and direct conjugates were added, incubated, and washed, followed by streptavidin conjugates. Cells were then washed and resuspended in 200 µl of FACS buffer for acquisition. Sample data from 2 x 105 lymphocytes or 1 x 104 stromal cells were acquired on a FACScalibur (BD Biosciences) using up to four fluorescent channels. Analysis was performed using CellQuest software (BD Biosciences). A Fc receptor block (clone 2.4G2 supernatant) was used before Ab staining where appropriate.

Stromal cell sorting was performed on a FACStarPlus cell sorter (BD Biosciences) at no faster than 2.0 x 103 cells per second. Samples were collected in 30% (v/v) FCS in RPMI 1640, recovered by centrifugation and counted, and a sample was analyzed for purity. Populations were sorted to at least 95% purity gated on the same parameters used for sorting.

Reverse transcriptase PCR analysis and quantitative PCR

FACS-purified cells were washed and resuspended in TRI Reagent (Molecular Research Center). Total RNA was recovered using 1-bromo-3-chloropropane (BCP) phase separation reagent (Molecular Research Center) according to manufacturer’s instructions. RNA was reverse transcribed using Superscript II (Invitrogen Life Technologies) and oligo(dT) oligonucleotides (Invitrogen Life Technologies) were incubated at 42°C for 50 min and then inactivated at 70°C for 10 min. PCR was performed with Taq polymerase (Promega) at an annealing temperature of 56°C (28 or 32 cycles for all chemokines, stem cell factor (SCF), and IL-6; 32 or 35 cycles for IL-7 and FGFs; 35 cycles for hypoxanthine phosphoribosyltransferase) using appropriate primers.

Quantitative PCR was performed on a Corbett Rotor-Gene 3000 (Corbett Research) in 10-µl reactions using SYBR Green Supermix (Invitrogen Life Technologies) and each primer (200 nM). The primer sequences (forward then reverse) used were as follows: GAPDH, 5'-ACCATGTAGTTGAGGTCAATGAAGG-3' and 5'-GGTGAAGGTCGGTGTGAACG-3'; SCF, 5'-AGGAATGACAGCAGTAGCAG-3' and 5'-CAATTACAAGCGAAATGAGAGC-3'; IL-6, 5'-TGTATGAACAACGATGATGCACTT-3' and 5'-ACTCTGGCTTTGTCTTTCTTGTTATCT-3'; CXCL12, 5'-GCTCTGCATCAGTGACGGTACXCL12-3' and 5'-GCTCTGCATCAGTGACGGTA-3'; IL-7, 5'-GGGAGTGATTATGGGTGGTGAG-3' and 5'-TGCGGGAGGTGGGTGTAG-3'; keratinocyte growth factor, 5'-GCGCAAATGGATACTGACACG-3' and 5'-GGGCTGGAACAGTTCACACT-3'; and FGF10, 5'-GACCAAGAATGAAGACTGTCCG-3' and 5'-TACAGTCTTCAGTGAGGATACC-3'. After initial holds for 2 min at 50°C followed by 10 min at 95°C, the cDNA was amplified for 40 cycles at 95°C for 15 s and 60°C for 60 s. Target mRNA levels relative to those of GAPDH were determined using the 2–{Delta}C(T) method (12).

Cell culture

Hybridomas, cell lines, and MTE-1D/PBL or 3B6/PBL cocultures were maintained in RPMI 1640 (Invitrogen Life Technologies) supplemented with 5% (v/v) FCS and incubated at 37°C in 8% CO2 in air. PBLs were cultured alone or with either MTE-1D or 3B6 cells at a ratio of 10:1. MTE-1D cells were kindly provided by Dr. P. Naquet (Université de la Méditerranée, Marseille, France) and the 3B6 cells were a gift from Dr. D. Godfrey (Melbourne University, Melbourne, Australia).

MTS-15 Ag purification

Mouse intestinal cell membrane preparation (4 ml) was extracted using 15 ml of chloroform and methanol (1:2; v/v) with intermittent sonication for 3 h at room temperature and the extract was recovered by centrifugation. The pellet was further extracted with 5 ml of chloroform, methanol, and water (1:2:0.8; v/v) with sonication for 1.5 h at room temperature and centrifugation. Water was added to the supernatants to give a final chloroform, methanol, and water ratio of 1:2:1.4 (v/v) and then the two phases were allowed to separate overnight at 4°C. The chloroform phase was dried under N2 and resuspended in 500 µl of chloroform and methanol (1:1; v/v). An aliquot (100 µl) was applied over 17 cm of a silica gel 60 aluminum-backed high performance TLC (HPTLC) plate (Merck) and developed for 10 cm in chloroform, methanol, 1 M ammonium acetate, 13 M ammonium hydroxide, and water (180:140:9:9:23 v/v). Silica bands of 2.5 mm were scraped and glycolipids were extracted with chloroform, methanol, and water (1:2:0.8; v/v). Antigenic activity was identified by reanalyzing each fraction by HPTLC and immunoblotting with MTS-15 (see below). A duplicate HPTLC plate was stained with orcinol/sulfuric acid reagent (3 min at 80°C) for the detection of carbohydrates.

HPTLC immunoblot analysis

Chromatographed HPTLC plates were air dried and plastic coated with 0.1% (w/v) polyisobutylmethacrylate in n-hexane. Coated plates were dried and blocked in 5% (w/v) powdered skim milk in PBS and 0.02% (v/v) Tween 20 for 1 h at room temperature. Plates were probed with MTS-15 supernatant for 1 h followed by anti-rat Ig HRP-conjugated Ab (DakoCytomation) in 5% (w/v) milk in PBS and 0.02% (v/v) Tween 20 for 1 h. Blots were developed using the ECL Western detection system (Amersham Biosciences).

Gas chromatographic mass spectrometry compositional analysis

HPTLC-purified MTS-15 Ag was subjected to methanolysis in 0.5 M methanolic HCl (50 µl) for 16 h at 80°C (13). The mixture was neutralized with pyridine (10 µl), sugars re-N-acetylated by the addition of acetic anhydride (10 µl for 10 min at room temperature), and dried under vacuum. The released monosaccharides were derivatized in 15 µl of pyridine, hexamethyldisilazane, and trimethylchlorosilane (9:3:1; v/v) for 20 min at room temperature and the solvent was dried under N2. The trimethylsilyl derivatives were resuspended in 50 µl of n-hexane and detected by gas chromatography mass spectrometry in a Hewlett Packard GC system 6890 series/mass selective detector 5973 using a HP1 (Hewlett Packard) capillary column.

MALDI-TOF-MS analysis

HPTLC-purified MTS-15 Ag in 50% 1-propanol (0.3 ~ 1 µg/µl) was analyzed by MALDI-TOF/mass spectrometry on a Voyager-DE STR Perspective Biosystem (PE Biosystems) in the positive ion/reflector mode. Saturated {alpha}-cyano-4-hydrocinnamic acid in 60% 1-propanol (0.3 µl) was used as the matrix and GM1 was used as a standard.

Chemical and enzymatic treatments

Partial acid hydrolysis of the MTS-15 Ag was performed in 0.1 M trifluoroacetic acid (100 µl) for 2 h at 100°C. The sample was dried under vacuum and residual trifluoroacetic acid removed by evaporating with toluene (2 x 20 µl). Mild base hydrolysis was conducted in 0.1 M methanolic-sodium hydroxide (50 µl) for 2 h at 37°C. The samples were neutralized with 1 M acetic acid and then subjected to 1-butanol/water partitioning. The 1-butanol phase containing the glycolipids was dried under vacuum. Endoglycoceramidase II (EGCase II; 1mU/µl; Calbiochem) digestions were performed in 20 mM sodium acetate (pH 5.0) for 16 h at 30°C. Lipidic digestion products were recovered by 1-butanol and water partitioning and the 1-butanol phase was dried under vacuum. Chemical and enzymatic treatment products were resuspended in 50% 1-propanol (10 µl), resolved by HPTLC, and detected by orcinol and MTS-15 staining.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Thymic distribution of MTS-15

Immunohistological analysis of adult mouse thymus sections with MTS-15 revealed specific staining of stromal cells lining the subcapsule and large blood vessels of the corticomedullary junction (Fig. 1, A and B). At high power, MTS-15 staining appeared granular and exhibited apparent diffusion around cells adjacent to areas of high expression (Fig. 1B). Compared with markers of thymic epithelium (anti-keratin) and vasculature (anti-CD31), MTS-15 reactivity colocalized with some endothelia and pericytes, but not epithelial structures (Fig. 1B). Colocalization with the fibroblast marker ER-TR-7 (14) at the subcapsule, trabeculae, and near vasculature suggests the MTS-15 Ag is expressed by fibroblasts in these areas (Fig. 1C).


Figure 1
View larger version (68K):
[in this window]
[in a new window]

  		FIGURE 1. MTS-15 stains mouse thymic fibroblasts. A, MTS-15 staining of a young thymus was detected throughout the subcapsule and the thymic vasculature. B, Triple staining of a large blood vessel at the corticomedullary junction with MTS-15 (green), anti-CD31 (red), and anti-keratin (ker) (blue) showing colocalization of MTS-15 and CD31 (yellow). C, MTS-15 (green), ER-TR-7 (red), and anti-keratin (blue) staining revealed MTS-15 and ER-TR-7 colocalization (yellow) along the trabeculae and around some thymic blood vessels. D, All dot plots are gated on CD45– stromal cells. MHC II expression vs MTS-15 staining (upper left panel) shows a large population of MHC II–MTS-15+ cells and some MHC IIlowMTS-15+ cells. Anti-PDGFR{alpha} staining on CD45– stroma shows expression by MTS-15+ and MTS-15– populations (upper middle panel) but not CD31+ cells (upper right panel). A dot plot of MTS-15 staining vs CD31 expression on CD45– stroma with percentages of MTS-15+ CD31– and MTS-15+ CD31+ is shown (lower left panel). The histogram (lower middle panel) shows the MHC II expression of the MTS-15+ CD31+ (thick line) and MTS-15+ CD31– (shaded) thymic stromal cells. Most MTS-15+ stromal cells also express Ly51 (lower right panel). Profiles are representative of four experiments.

 
These findings were supported by flow cytometric analysis of freshly isolated thymic stromal cell preparations. MTS-15 surface staining of CD45–MHC II– stromal cells confirmed the expression of the Ag on nonepithelial cells (Fig. 1D) that could first be detected at embryonic day (E) 15 (data not shown). Because ER-TR-7 is not a cell surface marker, the {alpha}-chain of the platelet-derived growth factor receptor (PDGFR{alpha}) was used as a positive marker of thymic mesenchymal cells. Virtually all MTS-15+ cells coexpressed PDGFR{alpha} in the adult thymus but there was also a clear PDGFR{alpha}+MTS-15– population observed that were not CD31+ endothelial cells (Fig. 1D) or MHC II+ epithelial cells (data not shown). These may represent another adult mesenchymal population. Four-color labeling comparing CD31, MHC II, and MTS-15 staining on CD45– stromal cells revealed a subset of MHC IIlowMTS-15+CD31+ endothelium (Fig. 1D and Ref. 11). The remaining CD31+ cells were MTS-15low. Interestingly, ~75% of MTS-15+ stromal cells also expressed the cortical epithelium marker Ly51 (Fig. 1D); these cells may correspond to the proposed "cortical mesenchyme" population observed in a previous study (48).

An overnight culture of MTS-15+CD31–/low sorted thymic stromal cells showed the typical spindle morphology of adherent fibroblasts grown as a monolayer (data not shown). Together, these data show that MTS-15 is a cell surface marker of a subset of thymic fibroblasts that mostly line the subcapsule and perivascular spaces and of a subset of MHC IIlow thymic endothelial cells.

Tissue distribution of MTS-15

Whether MTS-15 stained similar cells in other tissues was examined by immunohistology (Table II). The spleen exhibited an MTS-15 distribution similar to that of the thymus. A strong association of MTS-15 with splenic vasculature was observed with prominent staining of the red pulp and around the central arterioles (Fig. 2A). The extension of this granular reactivity around cells adjacent to the central arterioles resembled the pattern observed around the thymic endothelium, suggesting shedding or diffusion from areas adjacent to leukocytes (Fig. 2A).


View this table:
[in this window]
[in a new window]

  		Table II. Tissue distribution of the Ag recognized by MTS-15

 

Figure 2
View larger version (73K):
[in this window]
[in a new window]

  		FIGURE 2. MTS-15 distribution on extrathymic tissues. A, Immunohistology of the spleen with MTS-15 (green) and anti-CD31 (red) revealed extensive MTS-15 reactivity throughout the red pulp regions and the surrounding central arterioles. Higher power magnification of MTS-15 (green), anti-CD31 (red), and anti-CD45 (blue) staining of a splenic central arteriole showed some colocalization with the endothelium (yellow) and adjacent leukocytes (cyan). B, Extensive MTS-15 reactivity (green) was observed in the connective tissue areas of the small intestine. Anti-CD31 (red) and anti-CD45 (blue) staining defined gastrointestinal vasculature and leukocytes. Immunohistology of small intestine with MTS-15 (green), anti-CD45 (red) and anti-keratin (ker) (blue) exhibited colocalization between MTS-15 and intestinal leukocytes (yellow). C, Scattered cells in the connective tissue of the E15 umbilical hernia expressed high levels of the MTS-15 Ag. D, MTS-15 staining did not extend into the small intestine of the E16 embryo, but scattered cells below the epidermis expressed the determinant. E, At E16, MTS-15 reactivity could be detected around the esophagus and scattered cells near the developing thymus. F, Continuous MTS-15 staining was detected along the lateral border of the left cerebral hemisphere in the E17 embryo.

 
The distribution pattern observed in lymphoid tissues contrasted with that found in the gastrointestinal tract and in female reproductive organs. Broad, intense MTS-15 staining of the connective tissue areas of the intestine showed no clear association with the vasculature (Fig. 2B). A comparison of this reactivity with CD45 and keratin demonstrated colocalization with MTS-15 on some intestinal leukocytes but not the epithelium (Fig. 2B).

Analysis of MTS-15 reactivity throughout embryogenesis demonstrated the earliest staining at E14 on scattered cells along the middle part of the central midbrain. This association with central nervous tissue continued throughout late embryogenesis (Fig. 2F) and was still evident at the blood-brain barrier in the adult (data not shown). At E15, reactivity was also detected in some cells of the umbilical hernia; however, only by E16 was connective tissue staining apparent in the esophagus (Fig. 2, C and E). At E16, intense MTS-15 staining was exhibited in scattered cells below the epithelial layers of the skin and other tissues (Fig. 2D). MTS-15 expression was first observed in the connective tissue of the small intestine at E17 and persisted thereon (Table II).

Passive acquisition of the MTS-15 Ag by leukocytes

The surface expression of the MTS-15 Ag on thymic fibroblasts coupled with the shedding apparent by immunohistology prompted flow cytometric analysis of thymocytes. Almost 20% of the thymocytes bore the Ag on their surface at low levels (Fig. 3A). The MTS-15 Ag was predominantly found on immature double negative (16%) and double positive (19%) cells, while mature CD4 and CD8 single positive thymocytes were almost negative (6 and 5% respectively) (Fig. 3B). MTS-15 staining was similar in all triple negative subsets distinguished by CD44 and CD25 staining (data not shown). The determinant was undetectable on recent thymic emigrants in the spleen assessed 24 h following intrathymic FITC injection (data not shown).


Figure 3
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 3. MTS-15 staining of leukocyte subsets and in vitro transfer onto PBL. A, Histograms showing MTS-15 staining (solid line) and isotype control staining (dotted line) on leukocytes from the tissues indicated. Proportions of cells bounded by markers set according to staining intensity are indicated. B, Histograms of MTS-15 staining of various thymocyte developmental subsets gated as shown in dot plot regions (gray line shows isotype control staining). DN, Double negative; DP, double positive; SP, single positive. C, Histograms of MTS-15 staining on major splenocyte subsets (gated according to dot plot regions) with markers set to bound MTS-15int and MTS-15high leukocytes (gray line shows isotype control staining). Representative profiles from three independent experiments are shown. D, CD45 expression was used to discriminate leukocytes (CD45+) from cell lines (CD45–) and MTS-15 expression was analyzed. The proportions of total viable cells are indicated in regions shown. Histograms of MTS-15 staining (solid line) on peripheral blood T cells (CD45+ TCR+) and B-cells (CD45+B220+) at the time points indicated show a greater proportion of MTS-15+ B cells than T cells within the markers set according to minimal background staining (<1%) of PBLs cultured alone (dotted line). Profiles are representative of three experiments.

 
Low levels of MTS-15 staining were also detected on leukocyte subsets from the spleen and lymph nodes, but not the blood (Fig. 3A). MTS-15 labeling of splenocytes distinguished three populations with high levels of staining observed on half of all nonlymphoid splenocytes (predominantly macrophages, granulocytes, and dendritic cells) (Fig. 3C). Almost 70% of splenic B-cells were MTS-15low/high, while MTS-15 staining of splenic T cells resembled that of total thymocytes (Fig. 3C). Lymph node leukocytes exhibited similar staining intensity to that of thymocytes (Fig. 3A).

This tissue-dependent, differential staining of lymphocytes with MTS-15 prompted an investigation into the possible transfer of this Ag from stromal cells to lymphocytes. To address this, an MTS-15+ TEC line (MTE-1D; Ref. 15) or an MTS-15– TEC line (3B6) was cocultured with PBLs (MTS-15–) and MTS-15 staining on the CD45+ leukocytes was analyzed by FACS (Fig. 3D). It was found that after 1 day of coculture with MTE-1D cells PBLs acquired low levels of the MTS-15 Ag on their surface, whereas PBLs cocultured with 3B6 cells remained negative (Fig. 3D and data not shown). With respect to lymphocytes, a higher proportion of B cells acquired or retained the Ag over time (from 12 to 27%), while only few T cells became MTS-15 positive (from 8 to 7%) (Fig. 3D).

This transfer of the MTS-15 Ag between cells could reflect either the interaction of a soluble Ag with a surface receptor or the acquisition of a surface Ag via membrane shedding or flipping. These possibilities were investigated by biochemical analysis of the Ag recognized by MTS-15.

Biochemical characterization of the Ag bound by MTS-15

MTS-15 immunoblots of membrane preparations from various tissues run using tricine SDS-PAGE consistently revealed activity near the dye front, suggesting that the species had an extremely low molecular mass (4–5 kDa), a high intrinsic negative charge, or was insoluble in SDS (data not shown). To address the latter issue, ultracentrifugal fractionation of antigenic activity from membrane preparations solubilized in various detergents was assayed using dot blots. These experiments indicated that the molecule detected by MTS-15 was extremely hydrophobic because nearly all of the activity was consistently detected in the insoluble pellets of all detergents tested, including SDS (data not shown). In addition, the antigenic activity was resistant to proteinase K treatment (data not shown). Together, these data indicated biochemistry consistent with a glycolipid.

To further investigate this possibility, total lipid extracts of intestinal or kidney membrane fractions were analyzed by HPTLC and probed with MTS-15. A single orcinol-reactive band was recognized by the mAb in both the crude mixture and after extensive purification by TLC (Fig. 4, A and B). Resistance of the determinant to mild base hydrolysis suggested that the Ag contained a ceramide group (i.e., a glycosphingolipid) (Fig. 4A). It was however also resistant to EGCase II, a lipase that hydrolyzes many sphingolipids except those belonging to the globo-glycosphingolipid family (Fig. 4B) (16).


Figure 4
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 4. MTS-15 recognizes a globo-glycosphingolipid. A, Orcinol staining and an MTS-15 immunoblot (IB) of HPTLC plates loaded with membrane (memb.) and chloroform/methanol (C.M.) extracts from intestine (Int.) and kidney (Kid.). Base treatment (B.T.) did not affect the mobility of the MTS-15 Ag. B, HPTLC-purified MTS-15 Ag and GM1 (control) were treated with EGCase II and the reaction products were detected by orcinol and MTS-15 staining. C, MALDI-TOF mass spectrum of purified MTS-15 glycosphingolipid (M-GSL). The molecular ions observed in the +[M-GSL] and the +[M-GSL-HexNAc]] clusters differ by 12 or 28 atomic mass units and reflect heterogeneity in the length of the lipid moiety. Inset shows MTS-15 staining vs Forssman Ag expression on CD45– thymic stromal cells.

 
Gas chromatographic mass spectrometry compositional analysis of this species revealed stoichiometric amounts of N-acetylgalactosamine (GalNAc), galactose (Gal), and glucose (Glc), also characteristic of a globo-glycosphingolipid. This was further supported by MALDI-TOF analysis of the underivatized band that revealed a cluster of putative [M+H]+ molecular ions m/z values of 1453.8, 1481.8, 1509.8, 1537.8, and 1565.8 (Fig. 4C). Together with the compositional data, this information is consistent with the MTS-15 Ag having the glycan head group HexNAc2Hex3 and a heterogeneous ceramide moiety.

A second cluster of ions was observed between m/z 1250.6 and 1334.7, corresponding to the putative MTS-15 Ag minus a HexNAc residue (Fig. 4C). Because a similar loss was observed with a standard glycosphingolipid, GM1, these data suggest that the MTS-15 Ag is capped with a GalNAc. Together with the finding that the purified glycolipid was completely resistant to {alpha}- and beta-galactosidase digestion (data not shown), the Ag detected by MTS-15 is likely to correspond to the globo-glycosphingolipid GalNAc2Gal2GlcCer. This structure forms the Forssman antigenic determinant (17). Staining of thymic stromal cells with MTS-15 and the Forssman mAb M1/87 (18) showed costaining of the same populations at similar intensities (Fig. 4C); preincubation of one before the other did not block the reactivity of either (data not shown). This suggests they detect different epitopes of the same molecule, supporting the biochemical data.

To determine whether the terminal GalNAc is a major determinant for binding of the MTS-15 Ab, the purified glycolipid was subjected to partial acid hydrolysis and reanalyzed by HPTLC. This treatment generated a number of additional glycolipid species with faster HPTLC mobility, consistent with the loss of one or more sugars (data not shown). However, none of these glycolipids was recognized by MTS-15, suggesting that the terminal GalNAc was essential for Ab recognition, a characteristic of Forssman-specific mAbs (19).

Transcription profile of MTS-15+ thymic fibroblasts

To assess whether the MTS-15 Ag has a direct role in T cell development, deoxyguanosine-treated fetal thymic organ cultures reconstituted with precursors were cultured for 6 days in the presence of saturating amounts of MTS-15 or an irrelevant Ab. Both cultures exhibited normal T cell development, indicating the Ag did not have a direct role in T cell progenitor recruitment or development (data not shown). Nevertheless, MTS-15 serves as a unique and specific marker for thymic fibroblasts. We therefore investigated further the probable functions of MTS-15+ fibroblasts within the thymic microenvironment by FACS purification of this and other stromal cell subsets for RT-PCR of various growth factors and chemokines. The major thymic stromal and lymphoid populations were analyzed separately (Fig. 5A) and then the epithelial and nonepithelial subsets (Fig. 5B) with cycle numbers titrated to highlight differences at each level (therefore, transcript levels in Fig. 5A should not be compared with those in Fig. 5B). The nonepithelial stromal component was composed of endothelial cells and MTS-15+ fibroblasts (11). These cells collectively produced significant levels of SCF (Fig. 5A), a cytokine important to the proliferation of early thymocytes (20). The low TEC transcript for this cytokine was predominantly derived from cortical TECs (cTECs) (Fig. 5B). These trends in SCF expression were confirmed by quantitative PCR, which showed that non-TEC produced 6-fold more transcript than TEC and, at the subpopulation level, fibroblasts and endothelium produced 3-fold more transcript than the cTEC (Fig. 5D). Similarly, a high signal for IL-6 was obtained in non-TECs comprising mRNA from MTS-15+ and the endothelium (Fig. 5, A, B, and D). The low levels of IL-6 transcription by whole TECs were derived mainly from the cTEC and medullary TEC low (mTEClow) subsets (Fig. 5, A, B, and D). By contrast, IL-7 was mainly expressed by cTECs and the MHC IIlow subset of mTECs while MTS-15+ fibroblasts contributed to the low non-TEC expression of this cytokine (Fig. 5, A and B).


Figure 5
View larger version (67K):
[in this window]
[in a new window]

  		FIGURE 5. Stromal cell growth factor and chemokine transcription profiles. A, Thymi from young mice were fractionated into whole CD45– thymic stromal cells (TSCs), TECs, non-TECs, whole thymocytes, or dendritic cells (DC) for RT-PCR analysis of a range of growth factors and chemokines. B, A similar analysis was performed on TECs subdivided into cTECs, MHC IIhigh (mTEChi) or MHC IIlow (mTEClo) medullary epithelial cells and non-TEC subdivided into MTS-15+ fibroblasts and CD31+ endothelium. C, Expression profiles of whole lung CD45– stroma (LSC), MTS-15+ lung mesenchyme (LMC+), MTS-15– lung mesenchyme (LMC–), and MTS-15+ thymic mesenchyme. ND, not done. D, Quantitative PCR analysis of SCF, IL-6 and CXCL12 transcripts in thymic stromal cell populations. Experiments are representative of two or three performed for each population.

 
In agreement with Ueno et al. (21), the mTEC subsets expressed high levels of CCL19 (ELC) and CCL21 (SLC) transcripts, although cTEC, MTS-15+, and CD31+ cells also contained these transcripts (Fig. 5, A and B). CXCL12 (SDF-1) mRNA was detected predominantly in nonepithelial stromal cells (Fig. 5, A and D), although as a subset cTECs expressed higher levels than fibroblasts or the endothelium (Fig. 5, B and D). Conversely, non-TEC produce almost no CCL25 (TECK) transcript, with cTEC and mTEChigh composing the main CCL25-producing cell types (Fig. 5, A and B). CXCL13 (BLC) mRNA was undetectable in the thymic populations assayed here (Fig. 5, A and B).

Of the stromal cell subsets of the thymus, MTS-15+ fibroblasts were the major producers of FGF-1, FGF-7, and FGF-10 (Fig. 5, A and B), all of which bind the FGFR2IIIb expressed by TECs (22). The FGF bands observed in the TEC subpopulations are unlikely to reflect significant FGF production by these cell types given the comparatively undetectable signals observed in whole TEC (Fig. 5A).

To gauge whether this transcription profile was unique to thymic MTS-15+ mesenchyme, it was compared with two major lung mesenchymal populations. Lung fibroblasts were purified from whole lung stromal cells (CD45–) by flow cytometry as Sca-1+CD31– (I. Bertoncello, unpublished observations) and further fractionated into MTS-15+ (lung mesenchyme cells, LMC+) and MTS-15– (LMC–). MTS-15 staining on Sca-1+ lung mesenchyme was the inverse of Thy-1 expression, thereby distinguishing two main populations of lung fibroblasts (data not shown) (23). Although the transcription profiles of these two populations were very similar, they were distinct from those of thymic MTS-15+ fibroblasts with low or negligible expression of CCL19, CCL21, IL-7, and FGF-1 (Fig. 5C). Overall, MTS-15+ thymic fibroblasts exhibited a unique transcription profile compared with thymic and lung stromal subsets, expressing a number of growth factors important to early thymocyte and epithelial cell development.

MTS-15+ fibroblasts in thymic regeneration

The expression of important thymic growth factors by MTS-15+ fibroblasts prompted an examination of their role in thymic atrophy and regeneration. We used a model of chemotherapy-induced thymic involution by cyclophosphamide treatment of young (2-mo-old) mice. In this context, the almost exclusive expression of FGFs within the CD45– compartment by MTS-15+ fibroblasts is notable given recent data showing that stromal derived FGF-7 is essential for thymic recovery following thymic damage (8).

In accordance with previous studies (24), cyclophosphamide treatment caused a drastic decrease in thymic cellularity within 3 days, predominantly due to the loss of double positive thymocytes (Fig. 6A and data not shown). A concomitant reduction in CD45– stromal cells was reflected in both TEC and MTS-15+ fibroblasts (Fig. 6A). Thymic regeneration commenced on day 5 and continued, reaching almost normal levels by day 14. Most stromal cell recovery occurred on day 7 with increases in both MTS-15+ fibroblasts and TEC (Fig. 6A). The full recovery of TEC, however, was not complete by day 14 and may require a longer period.


Figure 6
View larger version (39K):
[in this window]
[in a new window]

  		FIGURE 6. Thymic epithelium and fibroblasts contribute to regeneration following cyclophosphamide treatment. A, Thymi from 2-mo-old cyclophosphamide-treated mice were analyzed for total cell number, CD45– stromal cells, CD45–MTS-15+ fibroblasts,and TEC. Mean ± SE from five mice is shown. NY, untreated normal young controls. B, TEC and CD45–MTS-15+ fibroblasts purified from mice 3 days following cyclophosphamide (Cyclo) treatment were analyzed for transcript levels of the indicated growth factors relative to untreated controls (normalized to 1 for each). Mean ± SE representative from three experiments shown. KGF, Keratinocyte growth factor. C, Transcription of IL-6 receptor components was assessed by RT-PCR in various thymic cell types purified from untreated young mice. TSCs, thymic stromal cells; DC, dendritic cell.

 
We next quantified the transcript levels of important thymic growth factors in purified TEC and MTS-15+ fibroblasts (Fig. 6B). TEC exhibited significant increases in growth factors supporting early thymocyte development (SCF and IL-7) and intrathymic migration (CXCL12, CCL25, and CCL21) (all p < 0.01; Student’s t test). In MTS-15+ fibroblasts the highest increases were observed in the growth factors IL-6, FGF-7 (keratinocyte growth factor), and FGF-10, which support TEC proliferation (7, 25) (Fig. 6B). These increases subside over time, perhaps reflecting the restoration of thymocyte and TEC numbers upon thymic regeneration (data not shown).

The marked enhancement of IL-6 transcription during chemotherapy-induced thymic involution prompted an evaluation of responsive populations. Thymic DC, fibroblasts, cTECs, and the MHC IIlow subset of mTECs had detectable transcripts for the components of the IL-6R (Fig. 6C). Together, these data indicate that both TEC and fibroblasts recover rapidly following cyclophosphamide treatment and enhance the production of growth factors important to early thymocyte development and TEC proliferation. In addition, striking increases in IL-6 expression suggest a role for this pleiotropic cytokine in thymic regeneration following chemotherapy.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The Forssman Ag is a pentasaccharide (GalNAc{alpha}1–3GalNAcbeta1–3Gal{alpha}1–4Galbeta1–4Glu) covalently attached to ceramide (17). It is similar to many blood group Ags and others that present a major barrier to xenogeneic organ transplantation. Its function has been difficult to determine, although there is evidence for a role in cell adhesion and polarization (26). Although human cells have the means to synthesize the Forssman Ag, it is only expressed in certain mammary and colonic tumors (19, 27), presenting a potential tumor-specific determinant. In mice it is differentially expressed throughout development and in adult mesenchymal tissues (28, 29). In addition to the biochemical analysis presented here, the similarities in distribution between MTS-15 and the previously described Forssman-reactive mAbs (30) indicate that the Forssman Ag is the MTS-15 determinant.

Extensive immunohistological analysis revealed distinct staining patterns in lymphoid and nonlymphoid tissues, perhaps reflecting different functions within these sites. A feature of the staining in all organs was a granular reactivity that extended onto cells adjacent to areas of high expression. This may reflect shedding of the Forssman Ag, a process previously described following Ab cross-linking on endothelial cells (31). In this study we demonstrated that shedding occurred without cross-linking and that the Ag was transferred to peripheral blood leukocytes in a lymphocyte-specific manner in coculture assays. The variable extent of acquisition between B and T cells may reflect the different plasma membrane compositions of these cells (surface receptors, carbohydrates, and/or lipids). Low level staining observed on tissue leukocytes analyzed ex vivo is likely to reflect a transfer from MTS-15+ structures because the intensity of staining correlated with the proximity of these cells to areas of high Ag expression. The absence of MTS-15 reactivity on peripheral blood leukocytes suggests the Ag is shed or internalized when they exit organs. Further study into the nature of Forssman Ag shedding (vesicles, micelles, or monomers) should reveal more about its incorporation into other cells.

In the thymus, subsets of fibroblasts and endothelial cells express Forssman Ag. The presence of a PDGFRa+MTS-15– mesenchymal population suggests there is another significant population of fibroblasts in the adult thymus; however, these cells require further characterization. Phenotypic analyses indicated that MTS-15 represents a useful surface marker for subcapsular, perivascular, and perhaps "cortical" thymic fibroblasts, especially when combined with CD31 to allow discrimination from MTS-15+ endothelial cells. Thymic MTS-15 expression was not detectable before E15, indicating that these fibroblasts are unlikely to arise from a neural crest-derived mesenchyme because cells from this lineage are almost undetectable by E14.5 and beyond (49, 50). The precise origin of this distinct, MTS-15+ fibroblast population (and its relationship to the PDGFR{alpha}+MTS-15– population) awaits lineage tracing studies.

Given the localization of MTS-15+ fibroblasts around the proposed sites of precursor entry and T cell exit, it was of interest to find they expressed a distinctive profile of important growth factors and chemokines as compared with other thymic and lung stromal cell types. The detection of mRNA for CXCL12 in MTS-15+ fibroblasts and cTECs is consistent with the distribution found by in situ hybridization (32) and corresponds with human and mouse protein distribution (33, 34). Likewise, CCL19 and CCL21 transcription by mTECs supports previous data (34, 35), and we extend upon this to show that MTS-15+ fibroblasts also express these chemokines. This chemokine expression profile suggests that MTS-15+ subcapsular fibroblasts are important for the outward migration of TN thymocytes through the cortex toward the capsule. In addition, MTS-15+ pericytes may have a role in attracting positively selected double positive thymocytes back toward the medulla or possibly the movement of thymocyte progenitors into the thymus (36).

MTS-15+ pericytes are also situated to have a role in early thymocyte proliferation. Thymocyte precursors enter the postnatal thymus through the large blood vessels at the corticomedullary junction (37); therefore, one of the first stromal cell types they are likely to encounter are MTS-15+ fibroblasts. In the present study we found that these cells express high levels of SCF transcript, a cytokine important for the proliferation of TN1 and TN2 thymocytes (38). We also found that MTS-15+ fibroblasts express low levels of IL-7 (39); however, the higher cTEC production of this cytokine is likely to be of more importance to TN2 and TN3 proliferation, as these thymocytes traverse the cortex in contact with these cTECs (40). Likewise, the IL-7-dependent proliferation of positively selected thymocytes (41) is likely to rely on the production of this cytokine by cTECs and/or MHC IIlow mTECs.

We also found that that MTS-15+ fibroblasts are major stromal cell sources of FGF-1, FGF-7, and FGF-10, although whether they are unique in this regard awaits further characterization of the PDGFR{alpha}+MTS-15– population. These growth factors bind the receptor FGFR2IIIb expressed by TEC to induce their proliferation (7), and FGF-7 administration can enhance regeneration of the postnatal thymus following chemotherapy, irradiation, or graft-vs-host disease (42, 43, 44). The increased production of FGF-7 and FGF-10 by MTS-15+ fibroblasts following cyclophosphamide treatment found here is consistent with recent data showing that stromal-derived FGF-7 is essential for rapid thymic regeneration (8). Although single positive thymocytes also produce this cytokine (42), fibroblast-derived FGF-7 may have greater access to extracellular matrix components (such as heparan sulfate and dermatan sulfate) and, therefore, enhanced bioactivity for TEC (45). The gap of 4 days between increased FGF-7 and FGF-10 transcription and TEC expansion likely reflects the time required to produce and secrete the proteins and for sufficient TEC proliferation to exhibit increased numbers by day 7.

The large increase in IL-6 transcripts in both fibroblasts and TECs suggests this is an important stromal growth factor during thymic recovery from injury. Indeed, previous studies have indicated IL-6 is an autocrine proliferative factor for TECs (25), supported here by the overlapping expression profiles of IL-6 and IL-6R on fibroblast and TEC subsets. A role for IL-6 in thymic atrophy and regeneration has previously been implied by studies of mice rendered deficient in STAT-3 (a transducer of, among other factors, IL-6 signaling) in TEC, which showed increased susceptibility to thymic atrophy (46). However, systemic administration of high levels of IL-6 (and other gp130 family members) caused thymic atrophy in mice (47), perhaps indicating that the level and context of IL-6 production is important. Further studies are warranted to define the precise role of this cytokine in the thymic regeneration.

The limited transcription profile studied here indicated that MTS-15+ fibroblasts in the thymus appear to be similar to those of lung fibroblasts, excepting higher levels of CCL19, CCL21, IL-7, and FGF-1 in the thymus. Although there are many molecules common to the mesenchyme from different tissues, thymic MTS-15+ fibroblasts seem to have developed a unique profile to support the requirements of both epithelial cells and developing thymocytes. The definition of MTS-15 as a specific marker of thymic fibroblasts will allow more precise evaluation of the role of this "third player" (alongside TEC and thymocytes) in thymic function.


   Acknowledgments
 
We thank Drs. Philippe Naquet, Ken Shortman, and Dale Godfrey for the provision of cell lines and Abs, and Dr. Ivan Bertoncello and Professor Jim Goding for technical advice. We also gratefully acknowledge the technical assistance of Jade Barbuto, Angelina Davidson, Mark Malin, Gezza Paulovic, and Jody Zawadzki.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Australian National Health and Medical Research Council and funding from Norwood Immunology and the Australian Stem Cell Centre. Back

2 Current address: Joslin Diabetes Center, One Joslin Place, Boston, MA, 02215 Back

3 Address correspondence and reprint requests to Dr. Richard Boyd, Monash Immunology and Stem Cell Laboratories, Monash University, Wellington Road, Clayton, Victoria, Australia. E-mail address: Richard.boyd{at}med.monash.edu.au Back

4 Abbreviations used in this paper: TN, triple negative; E, embryonic day; EGCase II, endoglycoceramidase II; FGF, fibroblast growth factor; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; HPTLC, high performance TLC; MTS, mouse thymic stroma; PDGFR{alpha}, platelet derived growth factor receptor {alpha}-chain; SCF, stem cell factor; TEC, thymic epithelial cell; cTEC, cortical TEC; mTEC, medullary TEC. Back

Received for publication June 8, 2006. Accepted for publication January 19, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Boyd, R. L., C. L. Tucek, D. I. Godfrey, T. J. Wilson, N. J. Davidson, A. G. D. Bean, H. M. Ladyman, M. A. Ritter, P. Hugo. 1993. The thymic microenvironment. Immunol. Today 14: 445-459. [Medline]
  2. van Ewijk, W., E. W. Shores, A. Singer. 1994. Cross talk in the mouse thymus. Immunol. Today 15: 214-217. [Medline]
  3. Gray, D. H., T. Ueno, A. P. Chidgey, M. Malin, G. L. Goldberg, Y. Takahama, R. L. Boyd. 2005. Controlling the thymic microenvironment. Curr. Opin. Immunol. 17: 137-143. [Medline]
  4. Anderson, G., E. J. Jenkinson. 2001. Lymphostromal interactions in thymic development and function. Nat. Rev. Immunol. 1: 31-40. [Medline]
  5. Godfrey, D. I., D. J. Izon, T. J. Wilson, C. L. Tucek, R. L. Boyd. 1988. Thymic stromal elements defined by mAbs: ontogeny and modulation in vivo by immunosuppression. Adv. Exp. Med. Biol. 237: 269-275. [Medline]
  6. Anderson, G., K. L. Anderson, E. Z. Tchilian, J. J. Owen, E. J. Jenkinson. 1997. Fibroblast dependency during early thymocyte development maps to the CD25+ CD44+ stage and involves interactions with fibroblast matrix molecules. Eur. J. Immunol. 27: 1200-1206. [Medline]
  7. Jenkinson, W. E., E. Jenkinson, G. Anderson. 2003. Differential requirement for mesenchyme in the proliferation and maturation of thymic epithelial progenitors. J. Exp. Med. 198: 325-332. [Abstract/Free Full Text]
  8. Alpdogan, O., V. M. Hubbard, O. M. Smith, N. Patel, S. Lu, G. L. Goldberg, D. H. Gray, J. Feinman, A. A. Kochman, J. M. Eng, et al 2006. Keratinocyte growth factor (KGF) is required for postnatal thymic regeneration. Blood 107: 2453-2460. [Abstract/Free Full Text]
  9. van den Brink, M. R., O. Alpdogan, R. L. Boyd. 2004. Strategies to enhance T-cell reconstitution in immunocompromised patients. Nat. Rev. Immunol. 4: 856-867. [Medline]
  10. Godfrey, D. I., D. J. Izon, C. L. Tucek, T. J. Wilson, R. L. Boyd. 1990. The phenotypic heterogeneity of mouse thymic stromal cells. Immunology 70: 66-74. [Medline]
  11. Gray, D. H., A. P. Chidgey, R. L. Boyd. 2002. Analysis of thymic stromal cell populations using flow cytometry. J. Immunol. Methods 260: 15-28. [Medline]
  12. Livak, K. J., T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-{Delta}{Delta}C(T)) method. Methods 25: 402-408. [Medline]
  13. McConville, M. J., S. W. Homans, J. E. Thomas-Oates, A. Dell, A. Bacic. 1990. Structures of the glycoinositolphospholipids from Leishmania major: a family of novel galactofuranose-containing glycolipids. J. Biol. Chem. 265: 7385-7394. [Abstract/Free Full Text]
  14. Van Vliet, E., M. Melis, J. M. Foidart, W. van Ewijk. 1986. Reticular fibroblasts in peripheral lymphoid organs identified by a monoclonal antibody. J. Histochem. Cytochem. 34: 883-890. [Abstract]
  15. Naquet, P., H. Lepesant, A. Luxembourg, P. Brekelmans, C. Devaux, M. Pierres. 1989. Establishment and characterization of mouse thymic epithelial cell lines. Thymus 13: 217-226. [Medline]
  16. Ito, M., T. Yamagata. 1989. Purification and characterisation of glycosphingolipid-specific endoglycosidases (endoglycoceramidases) from a mutant strain of Rhodococcus sp.: evidence for three molecular species of endoglycoceramidase with different specificities. J. Biol. Chem. 264: 9510-9519. [Abstract/Free Full Text]
  17. Koerner, T. A., Jr, L. W. Cary, S. C. Li, Y. T. Li. 1981. Carbon-13 nuclear-magnetic-resonance spectroscopy of Forssman hapten. Biochem J. 195: 529-533. [Medline]
  18. Miller, B. A., G. Antognetti, T. A. Springer. 1985. Identification of cell surface antigens present on murine hematopoietic stem cells. J. Immunol. 134: 3286-3290. [Abstract]
  19. Sonnenberg, A., P. van Balen, T. Hengeveld, G. J. Kolvenbag, R. P. Van Hoeven, J. Hilgers. 1986. Monoclonal antibodies detecting different epitopes on the Forssman glycolipid hapten. J. Immunol. 137: 1264-1269. [Abstract]
  20. Rodewald, H. R., K. Kretzschmar, W. Swat, S. Takeda. 1995. Intrathymically expressed c-kit ligand (stem cell factor) is a major factor driving expansion of very immature thymocytes in vivo. Immunity 3: 313-319. [Medline]
  21. Ueno, T., K. Hara, M. S. Willis, M. A. Malin, U. E. Hopken, D. H. Gray, K. Matsushima, M. Lipp, T. A. Springer, R. L. Boyd, et al 2002. Role for CCR7 ligands in the emigration of newly generated T lymphocytes from the neonatal thymus. Immunity 16: 205-218. [Medline]
  22. Revest, J. M., R. K. Suniara, K. Kerr, J. J. Owen, C. Dickson. 2001. Development of the thymus requires signaling through the fibroblast growth factor receptor r2-iiib. J. Immunol. 167: 1954-1961. [Abstract/Free Full Text]
  23. Zhou, Y., J. S. Hagood, J. E. Murphy-Ullrich. 2004. Thy-1 expression regulates the ability of rat lung fibroblasts to activate transforming growth factor-beta in response to fibrogenic stimuli. Am J Pathol. 165: 659-669. [Abstract/Free Full Text]
  24. Heng, T. S., G. L. Goldberg, D. H. Gray, J. S. Sutherland, A. P. Chidgey, R. L. Boyd. 2005. Effects of castration on thymocyte development in two different models of thymic involution. J. Immunol. 175: 2982-2993. [Abstract/Free Full Text]
  25. Meilin, A., J. Shoham, L. Schreiber, Y. Sharabi. 1995. The role of thymocytes in regulating thymic epithelial cell growth and function. Scand. J. Immunol. 42: 185-190. [Medline]
  26. Zinkl, G. M., A. Zuk, P. van der Bijl, G. van Meer, K. S. Matlin. 1996. An antiglycolipid antibody inhibits Madin-Darby canine kidney cell adhesion to laminin and interferes with basolateral polarization and tight junction formation. J. Cell Biol. 133: 695-708. [Abstract/Free Full Text]
  27. Hakomori, S., S. M. Wang, W. W. Young, Jr. 1977. Isoantigenic expression of Forssman glycolipid in human gastric and colonic mucosa: its possible identity with "A-like antigen" in human cancer. Proc. Natl. Acad. Sci. USA 74: 3023-3027. [Abstract/Free Full Text]
  28. Willison, K. R., R. A. Karol, A. Suzuki, S. K. Kundu, D. M. Marcus. 1982. Neutral glycolipid antigens as developmental markers of mouse teratocarcinoma and early embryos: an immunologic and chemical analysis. J. Immunol. 129: 603-609. [Abstract]
  29. Leduc, E. H., N. Tanaka. 1956. A study of the cellular distribution of Forssman antigen in various species. J. Immunol. 77: 198-212. [Abstract/Free Full Text]
  30. Sadahira, Y., M. Mori, M. Awai, S. Watari, T. Yasuda. 1988. Forssman glycosphingolipid as an immunohistochemical marker for mouse stromal macrophages in hematopoietic foci. Blood 71: 42-48.
  31. Yuzawa, Y., J. Brett, A. Fukatsu, S. Matsuo, P. R. Caldwell, N. Niesen, F. Milgrom, G. Godman, D. Stern, G. Andres. 1995. Interaction of antibody with Forssman antigen in guinea pigs: a mechanism of adaptation to antibody- and complement-mediated injury. Am. J. Pathol. 146: 1260-1272. [Abstract]
  32. Bleul, C. C., T. Boehm. 2000. Chemokines define distinct microenvironments in the developing thymus. Eur. J. Immunol. 30: 3371-3379. [Medline]
  33. Hernandez-Lopez, C., A. Varas, R. Sacedon, E. Jimenez, J. J. Munoz, A. G. Zapata, A. Vicente. 2002. Stroma cell-derived factor-1/CXCR4 signaling is critical for early human T-cell development. Blood 99: 546-554. [Abstract/Free Full Text]
  34. Misslitz, A., O. Pabst, G. Hintzen, L. Ohl, E. Kremmer, H. T. Petrie, R. Forster. 2004. Thymic T cell development and progenitor localization depend on CCR7. J. Exp. Med. 200: 481-491. [Abstract/Free Full Text]
  35. Ueno, T., F. Saito, D. H. Gray, S. Kuse, K. Hieshima, H. Nakano, T. Kakiuchi, M. Lipp, R. L. Boyd, Y. Takahama. 2004. CCR7 signals are essential for cortex-medulla migration of developing thymocytes. J. Exp. Med. 200: 493-505. [Abstract/Free Full Text]
  36. Liu, C., T. Ueno, S. Kuse, F. Saito, T. Nitta, L. Piali, H. Nakano, T. Kakiuchi, M. Lipp, G. A. Hollander, Y. Takahama. 2005. Role of CCL21 in recruitment of T precursor cells to fetal thymus. Blood 105: 31-39. [Medline]
  37. Lind, E. F., S. E. Prockop, H. E. Porritt, H. T. Petrie. 2001. Mapping precursor movement through the postnatal thymus reveals specific microenvironments supporting defined stages of early lymphoid development. J. Exp. Med. 194: 127-134. [Abstract/Free Full Text]
  38. Shortman, K., L. Wu. 1996. Early T lymphocyte progenitors. Annu. Rev. Immunol. 14: 29-47. [Medline]
  39. Peschon, J. J., P. J. Morrissey, K. H. Grabstein, F. J. Ramsdell, E. Maraskovsky, B. C. Gliniak, L. S. Park, S. F. Ziegler, D. E. Williams, C. B. Ware, et al 1994. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J. Exp. Med. 180: 1955-1960. [Abstract/Free Full Text]
  40. Prockop, S. E., S. Palencia, C. M. Ryan, K. Gordon, D. Gray, H. T. Petrie. 2002. Stromal cells provide the matrix for migration of early lymphoid progenitors through the thymic cortex. J. Immunol. 169: 4354-4361. [Abstract/Free Full Text]
  41. Hare, K. J., E. J. Jenkinson, G. Anderson. 2000. An essential role for the IL-7 receptor during intrathymic expansion of the positively selected neonatal T cell repertoire. J. Immunol. 165: 2410-2414. [Abstract/Free Full Text]
  42. Erickson, M., S. Morkowski, S. Lehar, G. Gillard, C. Beers, J. Dooley, J. S. Rubin, A. Rudensky, A. Farr. 2002. Regulation of thymic epithelium by keratinocyte growth factor. Blood 100: 3269-3278. [Abstract/Free Full Text]
  43. Min, D., P. A. Taylor, A. Panoskaltsis-Mortari, B. Chung, D. M. Danilenko, C. Farrell, D. L. Lacey, B. R. Blazar, K. I. Weinberg. 2002. Protection from thymic epithelial cell injury by keratinocyte growth factor: a new approach to improve thymic and peripheral T-cell reconstitution after bone marrow transplantation. Blood 99: 4592-4600. [Abstract/Free Full Text]
  44. Rossi, S., B. R. Blazar, C. L. Farrell, D. M. Danilenko, D. L. Lacey, K. I. Weinberg, W. Krenger, G. A. Hollander. 2002. Keratinocyte growth factor preserves normal thymopoiesis and thymic microenvironment during experimental graft-versus-host disease. Blood 100: 682-691. [Abstract/Free Full Text]
  45. Lee, P. H., J. M. Trowbridge, K. R. Taylor, V. B. Morhenn, R. L. Gallo. 2004. Dermatan sulfate proteoglycan and glycosaminoglycan synthesis is induced in fibroblasts by transfer to a three-dimensional extracellular environment. J. Biol. Chem. 279: 48640-48646. [Abstract/Free Full Text]
  46. Sano, S., Y. Takahama, T. Sugawara, H. Kosaka, S. Itami, K. Yoshikawa, J. Miyazaki, W. van Ewijk, J. Takeda. 2001. Stat3 in thymic epithelial cells is essential for postnatal maintenance of thymic architecture and thymocyte survival. Immunity 15: 261-273. [Medline]
  47. Sempowski, G. D., L. P. Hale, J. S. Sundy, J. M. Massey, R. A. Koup, D. C. Douek, D. D. Patel, B. F. Haynes. 2000. Leukemia inhibitory factor, oncostatin M, IL-6, and stem cell factor mRNA expression in human thymus increases with age and is associated with thymic atrophy. J. Immunol. 164: 2180-2187. [Abstract/Free Full Text]
  48. Muller, S. M., G. Terszowski, C. Blum, C. Haller, V. Anquez, S. Kuschert, P. Cameliet, H. G. Augustin, H. R. Rodewald. 2005. Gene targeting of VEGF-A in thymus epithelium disrupts thymus blood vessel architecture. Proc. Nati. Acad. Sci. USA 102: 10587-10592. [Abstract/Free Full Text]
  49. Jiang, X., D. H. Rowitch, P. Soriano, A. P. McMahon, H. M. Sucov. 2000. Fate of the mammalian neural crest. Development 127: 1607-1616. [Abstract]
  50. Petrie, H. T.. 2002. Role of thymic organ structure and stromal composition in steady-state postnatal T-cell production. Immunol. Rev. 189: 8-19. [Medline]



This article has been cited by other articles:


Home page
 BloodHome page
D. A. Sultana, S. Tomita, M. Hamada, Y. Iwanaga, Y. Kitahama, N. Van Khang, S. Hirai, I. Ohigashi, S. Nitta, T. Amagai, et al.
Gene expression profile of the third pharyngeal pouch reveals role of mesenchymal MafB in embryonic thymus development
Blood, March 26, 2009; 113(13): 2976 - 2987.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y.-H. Youm, H. Yang, Y. Sun, R. G. Smith, N. R. Manley, B. Vandanmagsar, and V. D. Dixit
Deficient Ghrelin Receptor-mediated Signaling Compromises Thymic Stromal Cell Microenvironment by Accelerating Thymic Adiposity
J. Biol. Chem., March 13, 2009; 284(11): 7068 - 7077.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. L. Fletcher, N. Seach, J. J. Reiseger, T. E. Lowen, M. V. Hammett, H. S. Scott, and R. L. Boyd
Reduced Thymic Aire Expression and Abnormal NF-{kappa}B2 Signaling in a Model of Systemic Autoimmunity
J. Immunol., March 1, 2009; 182(5): 2690 - 2699.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
N. L. Alves, O. R.-L. Goff, N. D. Huntington, A. P. Sousa, V. S. G. Ribeiro, A. Bordack, F. L. Vives, L. Peduto, A. Chidgey, A. Cumano, et al.
Characterization of the thymic IL-7 niche in vivo
PNAS, February 3, 2009; 106(5): 1512 - 1517.
[Abstract] [Full Text] [PDF]

Home page
 haematolHome page
M. A. Haniffa, M. P. Collin, C. D. Buckley, and F. Dazzi
Mesenchymal stem cells: the fibroblasts' new clothes?
Haematologica, February 1, 2009; 94(2): 258 - 263.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
R. M. Kelly, S. L. Highfill, A. Panoskaltsis-Mortari, P. A. Taylor, R. L. Boyd, G. A. Hollander, and B. R. Blazar
Keratinocyte growth factor and androgen blockade work in concert to protect against conditioning regimen-induced thymic epithelial damage and enhance T-cell reconstitution after murine bone marrow transplantation
Blood, June 15, 2008; 111(12): 5734 - 5744.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. M. Muller, C. C. Stolt, G. Terszowski, C. Blum, T. Amagai, N. Kessaris, P. Iannarelli, W. D. Richardson, M. Wegner, and H.-R. Rodewald
Neural Crest Origin of Perivascular Mesenchyme in the Adult Thymus
J. Immunol., April 15, 2008; 180(8): 5344 - 5351.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. Seach, T. Ueno, A. L. Fletcher, T. Lowen, M. Mattesich, C. R. Engwerda, H. S. Scott, C. F. Ware, A. P. Chidgey, D. H. D. Gray, et al.
The Lymphotoxin Pathway Regulates Aire-Independent Expression of Ectopic Genes and Chemokines in Thymic Stromal Cells
J. Immunol., April 15, 2008; 180(8): 5384 - 5392.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
L. Kenins, J. W. Gill, R. L. Boyd, G. A. Hollander, and A. Wodnar-Filipowicz
Intrathymic expression of Flt3 ligand enhances thymic recovery after irradiation
J. Exp. Med., March 17, 2008; 205(3): 523 - 531.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gray, D. H. D.
Right arrow Articles by Boyd, R. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gray, D. H. D.
Right arrow Articles by Boyd, R. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS