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The Journal of Immunology, 2007, 179, 5693 -5700
Copyright © 2007 by The American Association of Immunologists, Inc.
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Lymphotoxin Pathway and Aire Influences on Thymic Medullary Epithelial Cells Are Unconnected1

Emily S. Venanzi, Daniel H. D. Gray, Christophe Benoist and Diane Mathis2

Department of Medicine, Section on Immunology and Immunogenetics, Joslin Diabetes Center, Brigham and Women’s Hospital; Harvard Medical School, Boston, MA 02215


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The lymphotoxin pathway is critical for the development and maintenance of peripheral lymphoid organs. Mice with deficiencies in members of this pathway lack lymph nodes and Peyer’s patches and have abnormal spleen architecture. These animals also develop autoantibodies to and lymphocytic infiltrates of multiple organs, provoking speculation that the lymphotoxin pathway may play a role in central tolerance induction. Indeed, a series of reports has claimed that lymphotoxin signals control the expression of Aire, a transcriptional regulator that is expressed in medullary epithelial cells of the thymus, mediates ectopic transcription of genes encoding a variety of peripheral tissue Ags, and promotes clonal deletion of self-reactive thymocytes. However, one report argued that lymphotoxin signals regulate the composition and organization of the thymus, particularly of the medullary epithelial compartment. Herein, we resolve this controversy in favor of the latter view. The expression and function of Aire were unaffected in medullary epithelial cells of mice lacking either lymphotoxin beta receptor or the lymphotoxin {alpha}-chain, and there was minimal overlap between the sets of genes controlled by Aire and lymphotoxin. Instead, both knockout lines showed abnormal medullary epithelial cell organization, and the line lacking the beta receptor had significantly fewer medullary epithelial cells. In short, the lymphotoxin pathway drives the developmental rather than selectional properties of thymic stromal cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lymphtoxin (LT)3 beta receptor, a TNFR superfamily member, is a key mediator of the genesis and maintenance of peripheral lymphoid tissues (1). It binds to the trimeric ligands LT{alpha}1beta2 and LIGHT and signals through TNFR-associated factors, intracellular zinc RING finger signal transducers upstream of the NF-{kappa}B family of transcription factors (2). Within the NF-{kappa}B family, the p50/RelA and p52/RelB arms are activated by LTbetaR (3), leading to the expression of many genes that regulate inflammation and lymphoid organ development and organization.

Mice deficient for the ligands and receptors of the LT pathway revealed the importance of these factors for the peripheral lymphoid system. LT{alpha} (4), LTbeta (5), and LTbetaR (6) single knockouts (KOs) all lack Peyer’s patches and most lymph nodes and have disorganized spleens. They also develop lymphocytic infiltrates of (6, 7), and produce autoantibodies against (8, 9), peripheral tissues, although the cause of this phenomenon remains unknown.

Less is known about the role of the LT pathway in the thymus. Mice deficient in factors downstream of LTbetaR, including NF-{kappa}B-inducing kinase and RelB, exhibit a disrupted thymic architecture (10, 11, 12). These results, coupled with the observation that the LT pathway is crucial for normal peripheral lymphoid organ development, suggested that this pathway might also be important in the development and organization of the thymus. In addition, the tissue infiltrates and autoantibodies in LT-deficient mice have provoked speculation that the pathway may be involved in central tolerance induction. Thymocytes express the ligands LT{alpha}1beta2 (13) and LIGHT (14), while LTbetaR is displayed by medullary epithelial cells (MECs) (15). MECs are thymic stromal cells that also express a wide array of peripheral tissue Ags (PTAs) (16), many of whose RNA transcripts are controlled by the transcriptional regulator Aire (17). Aire-deficient mice also develop lymphocytic infiltrates and autoantibodies (17, 18), exhibiting a phenotype somewhat similar to that of the LT KOs. This observation led to the suggestion that the LT and Aire influences may be somehow connected.

Two groups have examined the role of the LT pathway in the composition/structure of the thymic epithelium, Aire expression, and PTA gene transcription and came to conflicting conclusions. Boehm et al. (8) concluded that LTbetaR signals are required for proper MEC differentiation and organization, while the second group, led by Fu (9, 19, 20), contended that signals through LTbetaR regulate aire and PTA gene transcription and not thymus epithelium composition/structure. The latter view seems to have gained widespread acceptance (21, 22, 23, 24, 25, 26, 27, 28). In this study, we directly addressed these conflicting conclusions and further examined the role of the LT pathway in the thymus and its relationship to aire and PTA gene transcription, using a combination of histologic, flow cytometric, and gene expression-profiling approaches.


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

Aire-deficient mice were derived and genotyped as previously described (17) and backcrossed to the B6 strain for at least nine generations before intercrossing. LT{alpha}-deficient mice (7) on the B6 background were obtained from The Jackson Laboratory; LTbetaR-deficient mice on the B6 background were a gift from Dr. Y.-X. Fu (University of Chicago, Chicago, IL). For each KO line, heterozygotes were bred to each other and resulting KO and WT littermate control animals were used for the experiments in this study, unless otherwise noted. All experiments in this study were approved by the Harvard University Institutional Animal Care and Use Committee.

Thymus digestion and MEC sorting

Thymic epithelial cells were prepared according to a previous study (29) for phenotypic analysis by flow cytometry. Thymi from individual mice were serially digested, and the resulting single-cell suspensions were counted and used for staining and analysis on an LSR II flow cytometer (BD Biosciences). Thymic epithelial cells for microarray analysis were prepared according to a previous report (17). Individual thymi from young adult (3- to 5-wk-old) animals were digested, separated by density gradient centrifugation, and the thymic epithelial cell-enriched fraction was harvested and stained for sorting. MECs were sorted on a MoFlo cell sorter (DakoCytomation) according to the phenotype CD45–G8.8+Ly51intI-Abhigh.

Thymocyte analysis

Individual thymi were pushed through 40-µm mesh, liberating thymocytes, which were then washed and stained for analysis on an LSR II flow cytometer.

Abs and staining

All staining began with an incubation with the anti-FC{gamma}R Ab 2.4G2. For intracellular staining, fixation/permeabilization buffers from eBioscience were used per the manufacturer’s instructions. Abs used for MEC isolation for microarray analysis were anti-CD45-allophycocyanin (clone 30-F11), anti-Ly51-PE (clone BP-1), anti-I-Ab-FITC (clone AF6–120.1; all from BD Pharmingen, and anti-EpCam-biotin (clone G8.8a; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA). Also used was streptavidin-PE-Cy7 from BD Pharmingen. Additional Abs used for thymus cell subset analyses were anti-CD45-PerCP-Cy5.5 (clone 30-F11) and anti-CD11c-PE (clone HL3) from BD Pharmingen; anti-I-A/E-Pacific Blue (clone M5/114.15.2) from BioLegend; anti-EpCam-APC (clone G8.8a); anti-CD11b-FITC (clone M1/70) from eBioscience; anti-CD31-FITC (clone 390) and anti-rat IgG2c-PE and -FITC (clone 2C 8F1) from Southern Biotechnology Associates; MTS-15 (rat IgG2c supernatant); and anti-aire (rat IgG2c clone 5H12; gift from Dr. H. Scott, Walter and Eliza Hall Institute, Melbourne). Additional Abs used for thymocyte analyses were anti-CD4-PE-Cy7 (clone RM4-5) and anti-CD8{alpha}-allophycocyanin-Alexa750 (clone 5H10) from Caltag Laboratories; anti-V{alpha} 2-FITC (clone B20.6) and anti-Vbeta 5-PE (clone MR9.4) from BD Pharmingen; and anti-Foxp3-allophycocyanin (clone FJK-16S) from eBioscience. Additional Abs used for immunohistology were biotin-conjugated rat anti-mouse K8 (clone TROMA-1) from the Developmental Studies Hybridoma Bank; rabbit anti-mouse K5 from Covance Research Products; Cy3-conjugated donkey anti-rabbit IgG from Jackson ImmunoResearch Laboratories; and allophycocyanin-conjugated streptavidin from BD Pharmingen.

Immunohistology

Air-dried cryosections of thymus (8 µm) were stained with titrated concentrations of primary Abs for 30 min, then subjected to three 5-min washes in PBS. Secondary Abs were incubated for 30 min followed by further washes and mounting with coverslips in aqueous mounting medium (Biomeda). Images were acquired on an Axiovert 200M confocal microscope (Zeiss) using a xenon arc lamp in a Lambda DG-4 wavelength switcher (Sutter Instrument) and processed with Slidebook imaging software (Intelligent Imaging).

RNA isolation

Total RNA was prepared from the sorted MECs using the TRIzol reagent (Invitrogen Life Technologies). The standard manufacturer’s protocol for isolation of total RNA from cultured cells was followed. RNA was resuspended in sterile, RNase-free water. It was quantitated using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies).

RNA amplification and microarray hybridization

Total MEC RNA was amplified using a T7 polymerase-based method. First- and second-strand cDNA synthesis, in vitro transcription of complementary RNA, and a second round of first- and second-strand cDNA synthesis were performed using a MessageAmp aRNA Kit (Ambion) as directed. The resulting cDNA was used in a second round of in vitro transcription, performed using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics) as directed, incorporating biotinylated ribonucleotides into the aRNA. The labeled aRNA was purified using the RNeasy Mini Kit (Qiagen). A 5x fragmentation buffer (200 mM Tris-acetate (pH 8.1), 500 mM KOAc, 150 mM MgOAc) was added to 15 µg of the biotinylated aRNA, and this mixture was heated at 94°C for 35 min. Fragmented samples were then hybridized to Affymetrix Mouse Genome 430 2.0 chips. The arrays were hybridized for 16 h at 45°C with constant rotation at 60 rpm. Next, they were washed and conjugated with streptavidin-PE in the Fluidics Station 400 (Affymetrix). Finally, each chip’s fluorescence was read on a GeneArray Scanner (Affymetrix), an argon laser detector. The GAPDH and actin 3':5' ratios of the amplified aRNA probes were normally between 2.5 and 10.

Microarray data analysis

The microarray data in this report are available at www.ncbi.nlm.nih.gov/geo, accession number GSE8563. The raw probe-level chip data (CEL files) were normalized by the robust multiarray average algorithm (30) using the ExpressionFileCreator module of the GenePattern 2.0 software package (31). Further analysis was performed using the MultiPlot module in GenePattern (R. Melamed and G. Hyatt, unpublished data). Random data sets were generated on a Gaussian distribution created for each gene. This distribution was centered on the gene-wise mean across all samples, with a SD that was the mean of the per-class (class = set of replicates) SDs for that gene. The false discovery rate is the frequency at which a given fold change (FC) value is higher in random data than in experimental data, for FC increments of 0.1. Values of p were calculated using Welch’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Reduced MEC numbers and aberrant organization in LTbetaR-deficient thymi

To address the question of whether the LT pathway affects the cellular composition of the thymus, we used flow cytometry to perform a thorough accounting of the numbers of stromal cells in individual wild-type (WT), LTbetaR–/– and LT{alpha}–/– thymi. As shown in Fig. 1A, we could identify and count cortical epithelial cells (CECs), MHC class II-high and -low MECs, Aire-expressing MHC class II-high MECs, dendritic cells (DCs), macrophages, fibroblasts and endothelial cells. The numbers of MECs, of all subsets, were significantly lower in LTbetaR–/– than in WT thymi (Table I). This difference was most significant when comparing the numbers of Aire-expressing class II-high MECs (Fig. 1B). However, MEC numbers were not similarly affected in LT{alpha}–/– thymi, which contained near WT levels of all subsets. Most other cell populations we evaluated were of comparable abundance in all mice examined (Table I), demonstrating an effect of LTbetaR specifically on MECs.


Figure 1
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  		FIGURE 1. MEC numbers and distribution are affected by deficiencies in LT pathway components. A, Gating scheme for analysis of thymic populations as initially described in Ref. 29 . Among CD45– cells, CECs (G8.8+Ly51high) and MECs (G8.8+Ly51int) (top, second panel), endothelial cells (CD31+MTS-15–), and fibroblasts (MTS-15+) (second row, first panel) were identified. MECs were further subdivided into MHC class II-high and -low populations (top, third panel), and the aire expression of MHC class II-high MECs was examined (top, right panel). Among CD45+ cells, DCs (CD11c+MHC class II+) (second row, middle panel) and macrophages (CD11b+CD11c–) (second row, right panel) were identified. B, Amount of MHC class IIhighaire+ MECs per LTbetaR–/– and LT{alpha}–/– thymus relative to WT littermate controls. Each bar represents the average ± SD of the cell amounts in each of three individual thymi. *, p < 0.00026 by Student’s t test. C, Cryosections of WT, LTbetaR–/–, or LT{alpha}–/– thymi were stained with UEA-1 (medullary subset, green), anti-K5 (medulla, red), and anti-K8 (cortex, blue) and images were acquired at low magnification. D, Anti-Aire (green) staining of WT, LTbetaR–/–, and LT{alpha}–/– thymi at low magnification. Cortical and medullary regions were delineated by K5 staining (data not shown). E, Anti-Aire (green), anti-K5 (red), and anti-K8 (blue) staining of WT, LTbetaR–/–, and LT{alpha}–/– thymi at high magnification. Images are representative of two independent experiments. *, MEC-free areas containing lymphocytes. C, Cortex; M, medulla. WT immunofluorescence controls in C–E were littermates of LTbetaR–/– animals.

 

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  		Table I. Cell numbers of thymic subsets in WT- and LT-deficient animalsa

 
To determine whether thymus architecture was affected by deficiencies in the LT pathway, we stained sections from WT, LTbetaR–/– and LT{alpha}–/– thymi with reagents recognizing MEC markers and analyzed them by confocal microscopy. The distribution of keratin (K) 5 and K8 allowed distinction of K8+ CEC and K5+ MEC as previously reported (32). However, in contrast to Klug et al. (32), but consistent with a recent study by Gillard et al. (33), we observed coexpression of K8 and K5 by thymic epithelial cells in the medulla of WT mice, in addition to those at the corticomedullary junction (Fig. 1, C and E). The medullary regions of LTbetaR–/– thymi were smaller and more sparsely populated by K5+ MECs than were those of WT thymi (Fig. 1C, cf center and left panels). Ulex europeaus agglutinin 1 (UEA-1)-positive MECs were detected on LTbetaR–/– thymus sections at a lower frequency than on WT sections and were localized in small clusters (Fig. 1C), consistent with previous observations (8). Frequent MEC-free regions (e.g., those asterisked in the center panel, Fig. 1E) containing lymphocytes revealed by 4',6-diamidino-2-phenylindole staining (data not shown) were observed on LTbetaR–/– sections. LT{alpha}–/– thymi (Fig. 1C, right panel) tended to have small medullary islets, but were not disrupted to the extent of their LTbetaR–/– counterparts. Specific anti-Aire staining showed a clustering pattern of Aire+ MECs in LTbetaR–/– thymi reminiscent of that of UEA-1+ MECs, in contrast to the even distribution throughout medullary areas in WT or LT{alpha}–/– mice (Fig. 1D). The characteristic punctate, nuclear localization of Aire was apparent in MECs of LTbetaR–/– and LT{alpha}–/– mice at fluorescent intensities similar to those of WT littermates (Fig. 1E), indicating normal cellular localization and expression levels.

Normal Aire protein levels in MECs of mice lacking LT pathway members

The amount of Aire in MECs of WT, LTbetaR–/– and LT{alpha}–/– thymi was compared by intracellular staining. Although MEC numbers were reduced in LTbetaR–/– thymi, the amount of Aire expressed by the remaining MECs was at the WT level, as it was in LT{alpha}–/– MECs (Fig. 2A). Calculating the mean fluorescence intensities (MFIs) of the Aire-positive, class II-high MECs confirmed this observation (Fig. 2B).


Figure 2
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  		FIGURE 2. Aire protein levels are unaffected in MECs of mice with LT pathway deficiencies. A, Assessment of Aire protein levels by intracellular staining with rat anti-Aire mAb. Cells plotted are CD45–G8.8+Ly51intMHC class IIhigh. Aire–/– thymi were stained as negative controls. Plots are representative of three animals of each genotype. KOs and corresponding WT controls were littermates. B, MFIs of the CD45–G8.8+Ly51intMHC class IIhighAire+ cell populations were determined for each individual, and LTbetaR–/– and LT{alpha}–/– MFIs were compared with WT littermate controls. Bars represent the average ± SD of the relative MFIs for each of three individual thymi. Brightness and proportion of Aire+ MECs differ slightly from experiment to experiment due to normal variation in the extent of thymic digestion and intracellular Aire staining.

 
LT pathway effects on MEC gene expression profiles

To further examine the effect of a deficiency in LT pathway members on MECs, we performed gene expression-profiling using Affymetrix Mouse Genome 430 2.0 microarrays, which represent ~34,000 genes, essentially the entire mouse genome. Using flow cytometry, we isolated MECs of the phenotype CD45–G8.8+Ly51intAbhigh from single-cell suspensions of thymi from individual LTbetaR–/– and LT{alpha}–/– animals, as well as from WT littermate controls. RNA prepared from these MEC isolates was amplified and biotinylated, then used to probe the Affymetrix chips. MECs from aire–/– and WT littermate controls were subjected to microarray analysis in the same manner, to permit direct comparison of the effects of Aire vs LT-pathway deficiencies. Three WT/KO pairs for each genotype were analyzed, all mice being males of 3–4.5 wk of age on the B6 genetic background.

Fig. 3A displays expression values for all genes on the 430 2.0 microarrays, plotting average levels of expression of a given gene in MECs from KO vs WT littermates. Aire expression was not substantially affected in the LT KOs, as indicated by its proximity to the x = y diagonal in the top two plots of Fig. 3A, confirming the flow cytometric data of Fig. 2. Many more genes had a different expression level in WT vs Aire-KO MECs than in WT vs LTbetaR- or LT{alpha}-KO MECs. Although the numbers of genes that were changed by at least 2-fold in the LTbetaR or LT{alpha} comparisons were quite low in relation to those in the Aire comparison (Fig. 3A), they were still not as low as were found with random data (data not shown; at most, five genes with a FC > 2 and four genes with a FC < 0.5).


Figure 3
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  		FIGURE 3. Gene expression profiling of MECs from mice deficient in LT pathway components vs Aire. A, Two-dimensional scatter plots of gene expression values for all genes on the mouse 430 2.0 microarray, comparing expression in KOs and WT littermate controls. Each expression value is the mean of three independent replicate values. Gray diamonds indicate the location of Aire on the top two plots. Gray lines mark arbitrary FC cutoffs of 2 and 0.5, with gray numbers showing how many genes have FC values surpassing the cutoffs. B, KO vs WT expression value ratio comparisons (derived from the average expression values in A) for all genes on the mouse 430 2.0 microarray. The diagonal line on each plot denotes x = y. Note the different x-axis scale on the bottom plot.

 
We wondered whether the LT-controlled genes were a subset of those affected by Aire or, rather, represented a distinct group. The FC vs FC plots in Fig. 3B demonstrate that different sets of genes were affected by the LT pathway and by Aire, as revealed by the lack of genes on the x = y diagonal in the upper two plots. However, comparison of the effects of LTbetaR and LT{alpha} generated a very different profile, with many genes along the x = y diagonal (Fig. 3B, lower panel). In fact, a linear regression on these data produced a line with a slope of 1.002, pointing to an overall trend of LTbetaR and LT{alpha} regulating very similar genes. To further compare Aire’s effects on gene expression with those of LT pathway members, we calculated the mean LTbetaR WT/KO and LT{alpha} WT/KO FC values for the 1,383 genes having an Aire WT/KO FC of more than two and obtained values of 1.012 for LTbetaR and 1.020 for LT{alpha}. For the 408 genes with an Aire WT/KO FC < 0.5, the mean FC values were 1.038 for LTbetaR and 1.001 for LT{alpha}. Therefore, the group of genes most affected by Aire was, overall, minimally affected by either LTbetaR or LT{alpha}.

The FC vs FC plots comparing the effects of LTbetaR and LT{alpha} (Fig. 3B) revealed several genes whose expression was uniquely regulated by either LT{alpha} or LTbetaR, but not by both. There were 27 probe sets with a LT{alpha} WT/KO FC of >2 or <0.5, but a LTbetaR WT/KO FC of between 1.25 and 0.8. These 27 probe sets were specific for 24 unique genes and we considered the expression of these loci to be dependent on LT{alpha} but not on LTbetaR. Of these genes, 13 mapped to chromosome 17, in or near the MHC. This phenomenon may have been an artifact generated by using the LT{alpha} KO, considering that the LT{alpha} gene itself is located in the MHC. Because this KO was produced by gene targeting in embryonic stem cells of the 129 strain (4), followed by backcrossing to mice of the B6 strain, some genes from the 129 MHC region were probably carried along with the KO allele. Although both the B6 and 129 strains bear an H-2b MHC haplotype, these regions are not identical. Any genes in the region whose transcription is regulated differentially between the two strains would appear to be expressed differentially between LT{alpha} KOs and WT mice. In addition, the neomycin-resistance cassette present in this LT{alpha} KO affects gene transcription (34), further suggesting that some of the gene expression differences observed for the LT{alpha} KOs may be experimental artifacts. Among the 12 genes with a LTbetaR WT/KO FC of >2 or <0.5 but a LT{alpha} WT/KO FC of between 1.25 and 0.8, 4 were found on chromosome 6, near the LTbetaR gene.

In total, there were only 80 genes whose LT{alpha} WT/KO FC or LTbetaR WT/KO FC was >2 or <0.5. Clearly, then, LT signals do not promote large-scale up-regulation of PTA expression, as does Aire (17, 22). For example, the expression level of the PTA insulin 2 did not differ by >1.2-fold in either LT WT/KO comparison nor did insulin 1 expression. These findings do not support the recent suggestion of the Fu group (19) that LTbetaR is involved in an Aire-independent PTA expression pathway in MECs. Their conclusion was based on the observation that expression of one Ag, type II collagen (CII), was regulated by the LT pathway, but not by Aire. The microarray data presented here indicated that the level of CII transcription was essentially unaffected in the LT KOs. The mean relative expression level of CII in the LT{alpha} KO MECs was 81.24 vs 90.08 in the WT littermates. The expression in LTbetaR KO MECs was 97.98 vs 92.22 in the WT littermates. The Fu group’s finding (19) may have resulted from taking K14 gene expression as a control for the RT-PCR analyses. In fact, our microarray data revealed an increase (~1.3-fold) in K14 expression in the LTbetaR KOs, which could well explain an augmentation in CII relative to K14 expression.

Lists of genes whose expression was affected by LTbetaR or LT{alpha} can be found in the supplemental material.4 Among the loci regulated by LT{alpha}, LTbetaR, or both, were those encoding proteins involved in the control of cell structure, such as epiplakin 1 and cortactin-binding protein 2. Other genes encoded proteins mediating cell-cell contact, including cadherin 2 and protocadherins 17 and 20. That the LT pathway regulates these types of genes is consistent with our immunofluorescence data and with the conclusion of Boehm et al. (8) that it is involved in controlling MEC organization within the medulla.

The LT pathway has little influence on thymocyte clonal deletion

Although LT did not affect aire or PTA gene expression, we wondered whether it might somehow control another important function of MECs, clonal deletion of self-reactive thymocytes. To evaluate the effect of LT deficiency on clonal deletion, we bred LTbetaR-deficient mice to mice transgenic for the rat insulin promoter (RIP)-mOVA Ag and the OT-II TCR. OT-II recognizes the 323–339 peptide from chicken OVA in the context of the MHC class II molecule Ab (35). Thus, most single-positive thymocytes (Fig. 4A, left panels) and mature T cells (data not shown) in OT-II-transgenic mice are CD4+ and express a high level of the OT-II clonotype V{alpha}2/Vbeta 5. The RIP drives a high level of membrane-bound OVA expression in the pancreas and lower-level expression in the thymus and kidney (36). In double-transgenic mice, most OT-II thymocytes, in particular those displaying high levels of clonotype, were clonally deleted (Fig. 4A, center panels) since they encounter their cognate Ag OVA during differentiation in the thymus. Clonal deletion in LTbetaR-deficient mice was also very effective (Fig. 4A, right panels). There was a slight, but consistent, increase in the fraction of OVA-specific OT-II thymocytes, but this was minimally reflected in the absolute numbers of CD4 single-positive and V{alpha}2/Vbeta 5 clonotype-high and -low thymocytes (Fig. 4B). The number of Foxp3+CD4+ regulatory T cells was also unaffected by the absence of LTbetaR (Fig. 4B).


Figure 4
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  		FIGURE 4. Clonal deletion of self-reactive thymocytes was only slightly altered in LTbetaR-deficient animals. A, Flow cytometric plots showing CD4 and CD8 expression by total thymic lymphocytes (top row) and V{alpha} 2/Vbeta 5 OT-II clonotype expression by CD4 single-positive thymocytes (bottom row). Representative of three independent experiments. B, Total number of cells of each indicated subset per thymus in mice of each genotype. Each bar represents the average ± SD of the cell numbers in each of three individual thymi. All mice were between 4 and 6 wk of age and were age and gender matched.

 
Previously, Aire was found to be necessary for normal clonal deletion in OT-II/RIP-mOVA double-transgenic animals as well as in several other TCR neo-self-Ag double-transgenic systems (37, 38), in some cases independently of Aire’s regulation of PTA expression (37, 39, 40). If LTbetaR does influence clonal deletion, it does so only very weakly, demonstrating that, in addition to having no influence on Aire’s regulation of PTA expression, the LT pathway also does not significantly influence its regulation of clonal deletion.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have analyzed the effects of deficiencies in LT pathway components on MEC number, organization, and function to resolve conflicting prior conclusions on these issues.

Our examinations of MEC numbers by flow cytometry and MEC distribution by immunofluorescence revealed a significant decrease in MEC numbers in LTbetaR KOs and abnormal medullary architecture in both LTbetaR and LT{alpha} KOs. These observations were consistent with those of Boehm et al. (8), who compared, by both immunohistochemistry and flow cytometry, the number and distribution of MECs in thymi of LTbetaR KO mice and WT littermates, demonstrating that there were significantly fewer MECs, exhibiting a disrupted localization pattern, in the former. Our finding that MEC numbers were reduced in LTbetaR KO, but not in LT{alpha} KO, thymi points to a role for another ligand, or ligands, of LTbetaR in controlling the MEC number. This is also the case for the findings of Boehm et al. (8). In addition to examining LTbetaR KO animals, they also studied LTbeta KO and LTbeta/LIGHT double-KO mice, finding that UEA-1-positive MECs from those KOs were present at normal numbers, but displayed the same abnormal organization as those in the LTbetaR KOs. Based on their observations, it seems that LIGHT, the only other known LTbetaR ligand, may not have much of an effect on MEC distribution and that another, as-yet-unidentified ligand may be involved. Analysis of LIGHT single-knockout thymi would strengthen this conclusion. A search for other thymic ligands for LTbetaR, perhaps using LTbetaR-FC fusion proteins (41), may also prove fruitful.

In agreement with our findings, Rossi et al. (42) recently found, using immunofluorescence, normal numbers of Aire-expressing MECs in thymi of LT{alpha}–/– animals. The Fu group (9) came to a somewhat different conclusion on this point. They did find normal numbers of MECs in the LT{alpha}–/– thymi, as Rossi et al. (42) and we did; however, relying on visual comparisons of immunofluorescent labeling of WT and LT{alpha}-KO thymus sections, they concluded that the distribution of UEA-1+ MECs in the latter was normal, which differed from our results. They did not show immunofluorescence data for LTbetaR KOs, nor did they include LTbeta or LIGHT KOs in their analyses. They also did not perform flow cytometry to assess MEC numbers.

Boehm et al. (8) and the Fu group (9) also examined the expression of Aire and PTAs in mice deficient in LT pathway components. Boehm et al. (8) cytofluorimetrically isolated MECs from WT and LTbetaR KO thymi and performed semiquantitative RT-PCR for transcripts encoding Aire, insulin, and several other PTAs. They observed no differences in gene expression levels in MECs of WT and KO mice. The Fu group assayed transcription of aire and insulin by quantitative RT-PCR of cDNA from whole, unfractionated thymi from LTbetaR- and LT{alpha}-deficient mice. They observed ~3- to 6-fold lower levels of Aire and insulin mRNA in both KOs. Because they performed their analyses on whole thymi, rather than on sorted MECs, it is possible, in the case of the LTbetaR KOs, that they were measuring deficiencies in total MEC number rather than Aire levels expressed by MECs. Furthermore, Aire+ MECs make up only ~0.01–0.03% of the total cells in WT thymi; therefore, these assays may have been close to the limit of detection for Aire and insulin, leading to imprecise results. This may explain why, in the case of the LT{alpha}-deficient thymi, in which MEC numbers were normal, that the Fu group (9) still observed lower Aire and PTA expression levels. In addition, from immunofluorescent staining of LT{alpha}-deficient thymus sections with polyclonal anti-Aire Ab, the Fu group concluded that Aire protein levels were much reduced; however, no quantification was performed nor was an Aire-deficient thymus control included.

The view of the Fu group (9, 19, 20), that the LT pathway controls Aire and PTA expression, seems to have taken hold in the literature (21, 22, 23, 24, 25, 26, 27, 28). However, our results on Aire expression by flow cytometry, immunofluorescence and gene expression-profiling of isolated MECs were consistent with those of Boehm et al. (8). We observed Aire protein at normal levels in its expected punctate nuclear distribution (43, 44) in LTbetaR–/– and LT{alpha}–/– thymus sections, rather than in the atypical perivascular localization shown by Chin et al. (9). Like Boehm et al. (8), we found no difference in Aire expression, at either the protein or transcript level, in LTbetaR- or LT{alpha}-deficient MECs. Additionally, our microarray data revealed that only a few PTA transcripts were controlled by the LT pathway and that there was little overlap in the genes regulated by Aire and the LT pathway.

Control of Aire and subsequent PTA expression is, then, independent of the LT pathway. However, recent work by Rossi et al. (42) has proposed that another TNF/TNFR family pathway may be responsible for these phenomena. They found that the RANK ligand, produced by CD4+3– lymphoid tissue inducer cells, was required for induction of Aire and PTA expression by MECs that express RANK (42).

In conclusion, the LT pathway remains an important player in the domain of thymus development/organization and appears to exit as a critical protagonist in the realm of Aire-mediated thymocyte tolerance.


   Acknowledgments
 
We thank Rachel Melamed for assistance with microarray data analysis, Dr. Hamish Scott for his gift of anti-aire Ab, and Dr. Yang-Xin Fu for his gift of LTbetaR knockout mice.


   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 National Institutes of Health Grant RO1 DK60027 and Young Chair funds (to D.M. and C.B.) and by Joslin’s National Institutes of Diabetes and Digestive and Kidney Diseases-funded Diabetes and Endocrinology Research Center core facilities. E.S.V. received support from National Institutes of Health Training Grant T32 DK07260 and D.H.D.G. received support from an Australian National Health and Medical Research Council C. J. Martin Overseas Biomedical Fellowship. Back

2 Address correspondence and reprint requests to Dr. Christophe Benoist and Dr. Diane Mathis, Section on Immunology and Immunogenetics, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. E-mail address: cbdm{at}joslin.harvard.edu Back

3 Abbreviations used in this paper: LT, lymphotoxin; KO, knockout; PTA, peripheral tissue Ag; MEC, medullary epithelial cell; WT, wild type; CEC, cortical epithelial cell; K, keratin; UEA-1, Ulex europeaus agglutinin 1; FC, fold change; CII, type II collagen; RIP, rat insulin promoter; MFI, mean fluorescence intensity; DC, dendritic cell. Back

4 The online version of this article contains supplemental material. Back

Received for publication May 14, 2007. Accepted for publication September 5, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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