Gene expression analysis of thymocyte selection in vivo

Ingo Schmitz1, Linda K. Clayton1 and Ellis L. Reinherz1

1 Laboratory of Immunobiology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA

Correspondence to: E. L. Reinherz; E-mail: ellis_reinherz{at}dfci.harvard.edu
Transmitting editor: S. Koyasu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Self versus non-self discrimination is a key feature of immunorecognition. Through TCR-activated apoptotic mechanisms, autoreactive thymocytes are purged at the CD4+CD8+ double-positive (DP) precursor stage prior to maturation to CD4+ or CD8+ single-positive (SP) thymocytes. To investigate this selection process in vivo, gene expression analysis by oligonucleotide array was performed in TCR transgenic mice. In total, 244 differentially expressed DP thymocyte genes induced or repressed by TCR triggering in vivo were identified. Genes involved in the biological processes of apoptosis, DNA recombination, antigen processing and adhesion are coordinately engaged. Moreover, analysis of gene expression in thymocyte subsets revealed that TCR ligand-induced expression profiles vary according to their developmental stage, with 48 genes showing DP preference and nine showing SP thymocyte preference. Finally, our data suggest that both the extrinsic and the intrinsic apoptosis pathways are operating in thymic selection.

Keywords: apoptosis, DNA microarray, gene profiling, T cell differentiation, thymus


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Pathogen-derived antigens are recognized by T cells through interaction of a TCR with an antigenic peptide bound to a MHC molecule (1,2). In the thymus, TCR gene segments rearrange via a combinatorial and stochastic mechanism generating billions of different {alpha}ß TCR. However, as self-reactive TCR can be generated by this process, autoreactive T cells must be deleted from the repertoire through negative selection (3). Selection acts predominantly at the CD4+CD8+ double- positive (DP) stage of T cell development where thymocytes with TCR recognizing peptide–MHC complexes (pMHC) strongly are eliminated during negative selection by apoptosis in a transcription-dependent manner (3).

There are two major apoptosis pathways, referred to as extrinsic and intrinsic. The extrinsic pathway is initiated at the cell surface by death receptors, a subgroup of the tumor necrosis factor receptor (TNFR) superfamily (4,5). Death receptor-mediated apoptosis depends on a cascade of aspartate-specific cysteinyl proteases called caspases (6). The intrinsic pathway acts on the mitochondria, and is regulated by anti-apoptotic and pro-apoptotic members of the Bcl-2 family (5,7). Anti-apoptotic family members like Bcl-2 protect the cell from mitochondrial dysfunction, while pro-apoptotic family members facilitate this process. Caspase-dependent and -independent pathways are initiated downstream of the mitochondria (8). However, within the thymus the molecular mechanisms of selection-related apoptosis remain to be fully elucidated; only a few molecules have been identified as important: (i) Nur77, an orphan steroid receptor (9), (ii) Bim, a pro-apoptotic Bcl-2 family member (10), (iii) I{kappa}BNS, a member of the I{kappa}B family (11), and (iv) caspases (12,13). The roles of Bcl-2 and the Fas (CD95/APO-1) system remain controversial (7,14).

Here, we utilize DNA oligonucleotide arrays to identify genes involved in negative selection of thymocytes. As a well-defined in vivo model of thymocyte death, we used peptide-induced negative selection in homozygous N15 TCR transgenic+/+ rag-2–/– H-2b (hereafter called N15 H-2b) mice (11,13,15). These animals possess a single TCR specificity on a well-defined genetic background and thus offer a simplified system for examining the molecular basis of selection in the absence of TCR heterogeneity or genetic background complexity. We find not only that apoptosis and transcription events are initiated during negative selection, but that other processes, including antigen processing, DNA recombination/repair and cell–extracellular matrix adhesion, are terminated. In addition, comparison of the expression profile of DP and SP subsets reveals that thymocytes respond with largely overlapping, yet distinct gene induction programs upon stimulation with the same pMHC.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Animals and peptide stimulation
The generation of N15 H-2b and N15 TCR transgenic+/+ rag-2–/– H-2d (N15 H-2d) mice has been described (15). The N15 TCR binds the vesicular stomatitis virus nucleoprotein-derived octapeptide N52–59 (VSV8) in the context of the Kb MHC class I molecule (16). Mice were maintained and bred under sterile barrier conditions at the animal facility of the Dana-Farber Cancer Institute according to institutional guidelines. The VSV8 peptide was synthesized by standard solid-phase methods. For in vivo stimulation of mice, 24 µg of peptide was dissolved in 100 µl PBS and injected i.v.

RNA preparation and Northern analysis
RNA was prepared and Northern blotting was performed as described (13,17). For detection of mRNA transcripts, cDNAs specific for the respective genes were labeled with [32P]dCTP by random primed labeling (Roche, Indianapolis, IN). For Figs 2, 3 and 4(A), DP-sorted thymocytes were employed, whereas for Fig. 4(B and C), total thymus preparations were used for Northern and Western blotting.



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Fig. 2. Gene expression profile of TCR-triggered DP thymocytes. (A) Hierarchical clustering of 244 genes differentially expressed in N15 transgenic mice treated with VSV8 peptide for the indicated times. Each row represents one gene. Green shows a lower, red a higher expression than the mean expression level over all time points for a given gene. (B) The expression profiles of representative genes from each cluster. The relative expression on the ordinate represents the model-based expression value x10–3 plotted against time.

 


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Fig. 3. Functional groups within the gene expression profile of TCR-triggered DP thymocytes. Representative genes of those 244 filtered were grouped according to their function as annotated in LocusLink (www.ncbi.nlm.nih.gov/locuslink).

 


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Fig. 4. Verification of Chip data. (A) Expression values of representative genes measured by DNA array analysis are shown as model-based expression. (B) Northern blot analysis of mRNA expression. N15 H-2b transgenic mice were treated with VSV8 for the indicated times and RNA was prepared from the whole thymus; 10 µg of total RNA (myd118, nur77, rag-1, gapdh) or 2 µg of poly(A)+ RNA (bim, c-myc, irf4) was resolved on a 1% agarose gel and transferred to a Nylon membrane. Blots were probed with DNA probes specific for the indicated genes. (C) Western blot analysis of protein expression. N15 H-2b transgenic mice were treated with VSV8 for the indicated times and cellular extracts were prepared from the thymocytes, and 20 µg of cellular extracts was resolved by SDS–PAGE and transferred to PVDF membrane. Blots were probed with specific antibodies indicated in Methods. In (B) and (C), all times points, including the 0 time point, utilized H-2b transgenic animals.

 
Microarray hybridization
Four to eight age- and sex-matched mice (3–4 weeks old) were used per experiment. After in vivo stimulation with VSV8 (24 µg/mouse; single injection), mice were sacrificed, thymic lobes dissected and single-cell suspensions prepared. Cells were stained with anti-CD4 (L3T4) and anti-CD8{alpha} (Ly-2) antibodies (PharMingen, San Diego, CA), and DP and SP thymocytes were sorted on a MoFlo (Cytomation, Fort Collins, CO). RNA was prepared from sorted thymocytes as described (13,17). Double-stranded cDNA was generated from 5 µg of total RNA with the SuperScript double-stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. T7 polymerase-driven in vitro transcription was performed on 1 µg of cDNA according to the manufacturer’s procedures with biotinylated ribonucleotides (Enzo Diagnostics, Farmingdale, NY). cRNA was purified by the RNeasy kit (Qiagen, Chatsworth, CA) and fragmented before hybridization (94°C, 35 min). Integrity of generated cRNA was confirmed on Test3 arrays (Affymetrix, Santa Clara, CA). Fragmented cRNA (15 µg) was hybridized to U74Av2 arrays overnight at 42°C. Washing and staining steps were performed according to the Affymetrix protocol EukGE-WS2.

Computational analysis
Arrays were scanned on an Agilent scanner and raw data was processed by Affymetrix GeneSuite software. Intensity value (CEL) files were imported into DNA-chip Analyzer (dChip) to calculate model-based expression values (18). Data sets were not used for analysis if array or single-probe outliers were >1.5%. In addition, the quality of each array was visually controlled. Array intensities were scaled by smoothing-spline normalization at the probe intensity level using an array with median overall intensity as the baseline array. Model-based expression values were calculated for each probe set and every array and can be found in Supplementary Table 1 (Supplementary Data available at International Immunology online). In order to analyze if the replicate experiments/arrays were more similar to each other than to any other conditions, genes were filtered in a simplistic way, excluding genes which show either little variation across the samples (0.5 < SD/mean < 10) or are not expressed in the majority of the samples (present call in arrays used >=50%). In total, 335 differentially expressed genes fulfilled these criteria and were hierarchically clustered. Supplementary Fig. 1 shows that replicate experiments indeed cluster together. To identify differentially expressed genes, the ‘compare samples’ menu of dChip was used. For analysis of DP thymocytes, the N15 H-2d samples were used as the ‘baseline’ group and compared to the ‘experiment’ groups, i.e. N15 H-2b samples after 0.5, 1 and 2 h of VSV8 stimulation. For comparison analysis genes were filtered as follows: (i) at least 3-fold difference in expression levels between the baseline (H-2d samples) and each stimulated condition with a >90% confidence level of the lower bound of fold change, (ii) difference in expression level between baseline and experiment must be at least 100, and (iii) a paired t-test was performed and the P value must be <0.05. Filtered genes had to fulfill the criteria of at least one of the experimental conditions (combine type ‘OR’ in dChip’s ‘combine comparisons’ function). Comparison of immature (DP) and mature (SP) thymocyte expression profiles was performed with the same filtering criteria. For immature thymocytes, the DP N15 H-2d samples were used as the ‘baseline’ group and 1-h VSV8-stimulated DP N15 H-2b samples as the ‘experiment’ group. For mature thymocytes, the ‘baseline’ consisted of unstimulated SP N15 H-2b samples and the ‘experiment’ consisted of 1-h VSV8-stimulated SP N15 H-2b samples. The two comparisons were combined with the Boolean operator ‘OR’ in the ‘combine comparisons’ tab of dChip. Since hierarchical clustering of arrays revealed similarity between replicate experiments, data sets from replicate arrays were merged and their mean expression values were used to calculate standardized values for hierarchical clustering, which were used for graphical presentation of the data.

Western blot analysis
Cell lysis and Western blotting were performed as described previously by Fiorini et al. (11). Antibodies were purchased from PharMingen (Nur77, clone12.14), Stressgen (Victoria, BC, Canada) (Bim, AAP-330), Santa Cruz Biotechnology (Santa Cruz, CA) (c-Myc, N-262; Irf4, M-17; TFIIH p52, C-19) and Sigma (St Louis, MO) (ß-actin, AC-74). Horseradish peroxidase-coupled goat anti-rabbit (sc-2054) and donkey anti-goat (sc-2056) secondary antibodies were from Santa Cruz Biotechnology. Horseradish peroxidase-coupled goat anti-mouse IgG1 and IgG2a were from Southern Biotechnology Associates (Birmingham, AL).

Cell-surface staining
N15 H-2b mice were stimulated in vivo by i.v. injection of VSV8 for various times. Mice were sacrificed and thymic single-cell suspensions prepared. Cells (5 x 105) were incubated with R-phycoerythrin-conjugated anti-CD4 (L3T4; PharMingen) and FITC-conjugated anti-CD8{alpha} (Ly-2; PharMingen) antibodies for 30 min at 4°C, washed with PBS, and analyzed on a FACScan cytometer (Becton Dickinson, Franklin Lakes, NJ).

Apoptosis assays
N15 H-2b mice were stimulated in vivo with VSV8 for the indicated times. Mice were sacrificed and thymic single-cell suspensions prepared. For assaying DNA fragmentation, 106 cells were washed once with PBS and resuspended in a buffer containing 0.1 % (w/v) sodium citrate, 0.1 % (v/v) Triton X-100 and 50 µg/ml propidium iodide (Sigma). After incubation in the dark at 4°C for at least 4 h, apoptotic nuclei were quantified by FACScan (Becton Dickinson). To measure {Delta}{Psi}M, 106 cells were incubated with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; 5 µg/ml) (Molecular Probes, Eugene, OR) for 15 min at 37°C in the dark, washed with PBS and analyzed on a flow cytometer (FACScan; FL-2).

Fetal thymic organ culture (FTOC)
Fetuses of N15 H-2b mice were dissected at day 15.5, with the day of the vaginal plug counted as day 1. Fetal lobes were removed and cultured in 24-well Transwell dishes (Costar, Cambridge, MA) using 0.4 ml of AIM V medium (Gibco, Rockville, MD). The samples were incubated in a humidified atmosphere with 5% CO2 for 4–5 days at 37°C. On day 5 FTOC were pre-incubated with anti-Fas ligand (FasL) blocking antibodies (MFL-3; PharMingen) or control IgG (anti-TCR{alpha}; clone H28) for 2 h. Subsequently, VSV8 peptide was added at 10 nM or 1 µM and incubated for an additional 8 h. For harvesting, lobes were ground between frosted glass slides in PBS/2% BSA/0.05% NaN3, washed and used for FACS analysis.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Kinetics of apoptosis and gene induction in thymocytes
To assess the kinetics of thymocyte deletion in vivo, N15 H-2b mice were injected with VSV8 peptide for 1, 4 and 20 h or left untreated. Thymocytes were prepared and analyzed by two-color FACS for CD4 and CD8. Deletion of DP thymocytes can be detected at 4 h post-injection and is pronounced after overnight treatment (Fig. 1A). Concurrent analyses of the mitochondrial membrane potential and DNA fragmentation, both hallmarks of apoptosis, confirm the apoptotic deletion of N15 thymocytes during these time intervals (Fig. 1B and C).



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Fig. 1. Kinetics of DP thymocyte apoptosis and deletion in vivo. (A) Thymocytes from N15 H-2b mice (age = 3 weeks) were analyzed by FACS for CD4 and CD8{alpha} expression at the indicated times after i.v. injection of VSV8 peptide. The percentages of DP thymocytes are given in each dot-plot. (B) The mitochondrial transmembrane potential ({Delta}{Psi}M) of thymocytes was determined by JC-1 staining and FACS analysis. (C) The subdiploid DNA content of thymocytes was quantified by propidium iodide staining of permeabilized cells. For (B) and (C), results are expressed as change in cell percentage at the indicated times.

 
Given the importance of understanding gene induction during the initiation phase of negative selection, we treated N15 H-2b mice with VSV8, and prepared RNA from FACS-purified DP thymocytes for microarray analysis 0.5, 1 and 2 h after VSV8 treatment. At these time points, DP thymocyte apoptosis is not yet detected (Fig. 1A–C), although nur77 and ikbns transcription is induced (11,17). RNA from FACS-sorted DP thymocytes of N15 H-2d animals was used as a control. The N15 TCR does not recognize H-2d class I molecules and therefore cannot be triggered in this background, so that neither positive nor negative selection occurs. Thus, thymocytes from N15 H-2d mice express genes representative of DP cells not stimulated by pMHC and provide a clear gene expression baseline.

cRNA was prepared and hybridized to U74A chips from Affymetrix containing >12,000 probe sets. At least two independent experiments were performed per experimental condition. Genes were filtered (see Methods) and hierarchical clustering was performed in dChip (18). In total, 244 genes fulfilled the filtering criteria (see Supplementary Table 2) building five large clusters ae (Fig. 2A). The five clusters contain genes which are either activated in a time sequential manner (clusters ad) or are rapidly repressed (cluster e). Cluster a contains genes with immediate early expression, i.e. expression is induced within 30 min of TCR triggering and declines thereafter. Cluster b contains genes whose expression peaks at 30 min to 1 h of stimulation, whereas cluster c contains genes whose expression is maximal at the 1–2 h time interval. Gene expression in cluster d shows a biphasic pattern. Examples of gene expression within each of these five clusters are shown in Fig. 2(B) and discussed further below.

Transcription factor genes
Figure 3 presents some well-defined functional groups found within these 244 differentially expressed genes. Thus, in agreement with a facet of TCR-induced apoptosis being transcription-dependent (19), multiple transcriptional regulators are induced. For example, ets2 in cluster e is down-regulated upon TCR stimulation while its corresponding repressor, Ets2 repressor factor (erf), is up-regulated at 30 min and hence belongs to cluster a. Recently, ets2 has been linked to thymocyte maturation, proliferation and survival (20). Thus, down-regulation of this survival factor and concomitant up-regulation of its repressor may favor apoptotic deletion of negatively selected thymocytes. Other transcription factors with an immediate early expression profile (cluster a) include fosB, and the early growth response (egr) genes egr1, egr2 and nab2. Nab2 is a regulator of Egr proteins (21) and shows the same expression profile as the two egr genes, suggesting that these three genes are transcriptionally co-regulated. The inhibitor of DNA binding 3 gene (idb3) is another interesting example of immediate early expression since Id3-deficient mice have defects in thymic selection (22). In contrast, idb2 is an example of a gene with later induction kinetics (cluster c) as it does not show high expression until 2 h after TCR stimulation. Additionally, the duration of gene induction varies between transcription factors. For example, nur77 and the NF{kappa}B family members nf{kappa}b1, nf{kappa}b2 and relB are induced early, and their expression remains high. On the other hand, expression of irf4 increases progressively, while hivep1, a zinc finger transcription factor, is transiently induced at the 1-h time point.

Apoptosis-inducing genes
The apoptosis-related group contains bim (cluster d) and nur77 (cluster b). This validates the DNA array analysis herein because these two genes have been reported to be important for negative selection in vivo (9,10). Differential expression of representative genes, including bim and nur77, was verified at the mRNA level by Northern blot and at the protein level by Western blot analysis (Fig. 4). Other identified apoptotic genes are the FasL (fasL/CD95L), the transcription factor and cell cycle regulator c-myc, myd118, and pdcd1, the gene encoding the inhibitory co-stimulatory receptor PD-1. Interestingly, PD-1-deficient mice show a defect in peripheral tolerance (23). Although negative selection seemed normal in mice with a T cell-specific inducible c-Myc transgene, activation of c-Myc induced apoptosis of thymocytes in vitro (24). myd118, also known as gadd45ß, is the most highly induced gene in our analysis (72-fold induction; see Supplementary Table 2) and is involved in growth arrest, differentiation and apoptosis (25). One possible mechanism of Myd118-mediated apoptosis is the activation of p38 MAPK via MEKK4 (26) since p38 MAPK is implicated in negative selection (27). Note that some disparities between model-based expression (Fig. 4A) and Northern analysis (Fig. 4B), e.g. as seen with Myd118, may be a consequence of usage of sorted thymocytes in the former and total thymus in the latter.

The p85{alpha} subunit of phosphatidylinositol 3-kinase, a kinase with anti-apoptotic functions, is down-regulated in VSV8-stimulated DP thymocytes. Nevertheless, acute down-regulation of p85{alpha} may facilitate progression of apoptosis in negatively selected thymocytes. In line with this, loss of PTEN expression, a negative regulator of the phosphatidylinositol 3-kinase pathway, in T cells results in defective negative selection (28). In p85{alpha}–/– mice B cell, but not T cell, development is impaired (29,30), which may be explained by other isoforms of the regulatory subunit of phosphatidylinositol 3-kinase compensating for the loss of p85{alpha} in T cells.

Surprisingly, we observe the seemingly paradoxical up-regulation of the anti-apoptotic genes bcl-2, bcl2a1b and bcl2a1d. All three anti-apoptotic Bcl-2 family members are preferentially induced in certain subsets of thymocytes and their possible role in regulation of thymocyte apoptosis is discussed below.

Cell-surface receptor and ligand genes
Another large group of genes comprise cytokines and cell-surface molecules. Cell–cell communication seems to be significantly enhanced, in line with the observation that, upon induction of negative selection, thymic stromal cell activation is initiated via thymocyte-derived cytokines/chemokines (17). Among the induced genes are the chemokines cxcl10 (ip-10) and ccl4 (mip1ß) as well as the chemokine receptor ccr7. Chemokines and their receptors maybe important in homing of thymocytes to certain locations of the thymic microenvironment. They might also mediate integrin activation and thereby adhesion (31). However, specific functions of a given chemokine or its receptor in T cell development remain elusive.

DNA repair and recombination genes
The most down-regulated in the peptide-stimulated DP thymocytes is the recombinase activating gene (rag)-1, which is essential for antigen receptor rearrangement. As rag-2, terminal deoxynucleotide transferase (tdt) and the thyroid autoantigen 70 (ku70/xrcc6) are down-regulated as well, the entire recombination machinery appears to be shut down, in agreement with published results (32). The finding that rag-2 is down-regulated in the rag-2–/– background may seem surprising. However, only a segment of the rag-2 gene was deleted in this knockout mouse (33), leaving the promotor and the chip array target sequence intact, and thus detectable in this assay. Ku70 protein, a component of the DNA-dependent protein kinase, is also involved in DNA repair. Likewise, the gene for the p52 subunit of TFIIH (gtf2h4), a component of the general transcription complex of RNA polymerase II, which is implicated in nucleotide excision repair, is down-regulated. In contrast, the exo- and endonuclease apex (ape/ref-1) implicated in base excision repair is up-regulated. In addition to its role in DNA repair, however, APEX protein stimulates DNA-binding activity of transcription factors such as Jun, Fos and NF{kappa}B (34). In this regard, as stated above, fos and nf{kappa}b genes are induced during negative selection.

Antigen presentation genes
Strikingly, a number of genes involved in antigen presentation are down-regulated and fall within cluster e. This functional group includes the MHC class I h-2d gene and MHC-related molecules cd1d1, mr1, h2-t10 and h2-t3. The latter two genes encode thymus leukemia (TL) antigens which bind CD8{alpha} homodimers (35). The non-classical MHC class I molecule MR1 has recently been shown to be involved in the selection of mucosal-associated invariant T cells (36). Also part of this group are ctse and ctsl, the cathepsins E and L genes, which are involved in antigen processing (37,38). CD1d1 and cathepsin L are important for the development of NKT cells (39,40). Finally, the ifngr gene is also down-regulated. Signaling by the IFN-{gamma} receptor induces many genes within the MHC locus and its down-regulation may thus enhance the decrease in MHC class I molecules noted above (41).

Cell adhesion-mediating genes
Upon induction of negative selection, cellular adhesion is regulated in a complex manner. Discoidin domain receptor 1 (ddr1), a collagen-binding receptor gene, and fibulin 2, a gene encoding an extracellular matrix protein with epidermal growth factor-like domains, are down-regulated, suggesting a reduced capacity of thymocytes to attach to the extracellular matrix. In contrast, cell–cell adhesion is enhanced. The integrin ligand intercellular adhesion molecule (icam)-1 and bystin-like (bysl), the mouse homologue of a human cytoplasmic component of a cell adhesion complex, are up-regulated. Furthermore, the proline–serine–threonine phosphatase-interacting protein 1 (pstpip1), also known as cd2bp1, is down-regulated, leading to enhanced CD2-mediated adhesion (42). Thus, apoptotic thymocytes apparently loosen their binding to the extracellular matrix while tightening their interactions with neighboring cells. The shift in adhesion may allow more efficient engulfment by phagocytic cells within the thymus. Cytoskeletal genes, which may be involved in adhesion processes, are regulated in a similar complex manner as adhesion-mediating genes (Fig. 3).

Gene expression profiles of DP and SP thymocytes
Mainly DP thymocytes are subjected to thymic selection processes although superantigen-mediated deletion affects also CD4+ SP thymocytes (3). CD8+ SP thymocytes have already undergone positive selection, thereby differentiating to a stage refractory to VSV8-triggered deletion in N15 H-2b mice (13,15). Thus, gene-induction common to DP and SP thymocytes can be principally viewed as related to T cell lineage activation, whereas genes preferentially expressed in SP thymocytes relate to more mature function. In contrast, genes that are exclusively induced in DP thymocytes are likely to be pivotal in selection. In order to determine which genes encode components of the DP thymocyte selection process rather than elements of a more general T-lineage-activation pathway, we compared the expression profile of CD4+CD8+ DP thymocytes with that of CD8+ SP thymocytes.

cRNA was prepared from FACS-sorted SP thymocytes from untreated or 1-h VSV8 stimulated N15 H-2b mice (Fig. 5A) and hybridized to U74Av2 oligonucleotide arrays. This SP data set was compared to the data derived from DP thymocytes of unstimulated N15 H-2d and 1-h VSV8 stimulated N15 H-2b mice. Genes were filtered by the aforementioned criteria and expression profiles of the two thymocyte populations compared. In total, 118 genes fulfilled the filtering criteria in either of the two cell populations and a clustering of these genes is shown in Fig. 5(B). A complete list of these genes can be found in Supplementary Table 3. The overlapping and specific sets of genes are illustrated in a Venn diagram in Fig. 5(C). Although the same TCR is triggered by a single pMHC ligand, certain genes are expressed selectively in DP or SP thymocytes (see clusters 5 and 2, respectively). Forty-eight genes are primarily induced (cluster 5) or repressed (cluster 1) in DP thymocytes, while nine genes are predominantly induced in SP thymocytes (cluster 2).



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Fig. 5. Comparison of DP and SP thymocyte expression profiles. (A) DP and SP thymocytes from unstimulated or 1-h VSV8-injected mice were sorted and the post-sort purity of a representative sample is shown. (B) Hierarchical clustering of 118 genes comparing the stimulation-dependent expression profiles of DP and SP thymocytes. (C) Overlapping and specific sets of pMHC-stimulated genes in DP and SP thymocytes. Numbers in the overlapping region of the Venn diagram represent common genes; numbers in the non-overlapping regions indicate genes preferentially expressed in either DP or SP thymocytes. (D) Expression data for each of four example genes from clusters 2, 4 and 5. The maximal expression value of each gene was set to 100%.

 
Cluster 2 contains nine different genes including the transcription factor nurr1, the cell cycle regulator cyclin D2 (ccnd2), the cell-surface receptors cd83, tnfr2 and cd137 (4-1bb), the chemokine ccl4 (mip1ß), the anti-apoptotic genes bcl2a1b and bcl2a1d, and the cytotoxic and regulatory T cell molecule gene crtam. Seven of these genes are also induced in DP thymocytes, but to a lesser extent. Two genes, i.e. cd83 and crtam, members of the Ig superfamily, show virtually no induction in DP thymocytes stimulated for 1 h. CD83 is a sialic acid-binding Ig-like lectin mediating interaction of activated T cells with dendritic cells (43,44). crtam was found to be expressed in MHC class I-restricted cells of the T and NKT lineage (45). CD137, a member of the TNFR superfamily, is a T cell co-stimulatory molecule and mediates survival of CD8+ T cells (46). Thus, cluster 2 contains genes linked to mature T cell functions, including survival (bcl2a1b, bcl2a1d, cd137), proliferation (nurr1, ccnd2) and co-stimulation (cd137, cd83).

Cluster 5 contains 12 different genes, which are preferentially induced in DP thymocytes, with expression levels at least 2-fold higher in DP than in SP thymocytes. This group contains both signaling molecules and transcription regulators. Signaling molecules include suppressor of cytokine signaling-1 (socs-1), TNFR-associated factor 3 (traf3), SH3 domain binding protein 2 (3bp2), prostaglandin E2 receptor EP4 (ptger4) and the small GTPase rad. The Rad protein belongs to the RGK (Rad/Gem/Kir) subfamily within the Ras superfamily and has been shown to antagonize Rho signaling by binding to the downstream effector Rho kinase (ROK/ROCK) (47). Thus, in addition to the GTPases Rho and Rac-1 that are implicated in survival and selection of thymocytes (48), rad appears to be similarly involved. TRAF proteins mediate signal transduction of the TNFR superfamily. TRAF3 in particular binds to CD40 and LTßR, and has been implicated in LTß-mediated cell death (49). Interestingly, LTß is induced upon VSV8 injection (see Fig. 3) suggesting an LTß–TRAF3-mediated apoptotic pathway in thymic selection. Transcriptional regulators in cluster 5 are the NF{kappa}B inhibitor i{kappa}b{alpha}, egr1, the Egr1-inhibitor nab2 and DNA methyltransferase 3A (dnmt3a), which mediates transcriptional silencing by methylation of DNA (50). The majority of cluster 5 genes are induced rapidly after VSV8 injection, implying regulation at the level of early signaling initiated by the TCR.

Apoptosis pathways in thymic selection
The Bcl-2 family is a major regulator of the intrinsic apoptosis pathway (7) and certain members of this family are differentially regulated in our study. For example, up-regulation of bim was found in both DP and SP thymocytes, with a higher induction in DP thymocytes (Fig. 5D). The BH3-only protein Bim seems to play a major role in negative selection, suggesting that this process is driven by the intrinsic apoptosis pathway (10). Consequently, the induction of bim in DP and SP thymocytes raises the question as to why SP thymocytes are refractory to deletion. One explanation might be the high induction of the anti-apoptotic genes bcl2a1b and bcl2a1d, which are predominantly induced in SP thymocytes (Fig. 5D). In line with this, thymocytes from mice transgenic for A1, an anti-apoptotic member of the Bcl-2 family, are resistant to a variety of apoptotic stimuli, but remain Fas sensitive (51). Interestingly, these transgenic mice show an increased thymic cellularity. In contrast, bcl-2 is induced by VSV8 in DP thymocytes (Fig. 5D) and previously has been associated with positive selection (7). However, bcl-2 expression herein clearly fails to prevent negative selection, suggesting an inability to counteract the pro-apoptotic activity of bim. In line with our finding, bcl-2 is induced upon TCR stimulation in the absence of selection processes in vitro (52) and maybe rather an activation marker. In fact, bcl-xL rather than bcl-2 is important for survival of DP thymocytes (7). Of note, bcl-xL levels are not altered upon TCR triggering with VSV8 (data not shown).

Although analysis of thymic development in mice with mutations in the Fas system did not reveal an absolute requirement in selection, other studies suggest a role for the Fas system at least at high antigen concentrations (14). Here we find fasL expression to be preferentially induced in DP thymocytes, suggesting a functional role for this apoptosis pathway in thymic selection (Fig. 5D). This induction in DP thymocytes, particularly in light of earlier controversy regarding the role of FasL in negative selection, prompted us to examine whether FasL is involved in deletion of N15 TCR transgenic thymocytes upon VSV8 stimulation. To this end, we performed antibody blocking experiments in FTOC. Thymic lobes were treated with 10 nM VSV8 for 8 h in the absence or presence of a monoclonal anti-FasL antibody or a control hamster IgG. Addition of the anti-FasL antibody blocks VSV8-mediated deletion of DP thymocytes, while the control antibody is without effect, so that DP thymocytes are deleted (Supplementary Fig. 2). However, stimulation with higher VSV8 concentrations (Supplementary Fig. 2) or longer treatment (data not shown) resulted in deletion of DP thymocytes, indicating that under these conditions apoptosis can proceed in a Fas-independent manner.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
To identify genes involved in thymic selection in a global manner we performed oligonucleotide microarray analysis of peptide-induced negative selection. We took advantage of the well-defined N15 TCR transgenic model in which selection can be studied both in organ culture and in vivo (11,13,15). In this model, deletion of DP thymocytes can be rapidly induced by a single injection of the negatively selecting peptide. Exposure of DP thymocytes to a negatively selecting pMHC ligand influences several biological processes in parallel (Fig. 6). These include transcription, apoptosis, adhesion, cell–cell communication and DNA recombination/repair. To our knowledge, the present study shows for the first time that these different processes are coordinately regulated during negative selection. Such an orchestration of events is likely to be important to ensure that autoreactive thymocytes commit apoptotic suicide and subsequently are engulfed by phagocytes without induction of inflammation. While an alteration of RNA levels does not mandate concurrent changes in the proteome, the observed changes in expression of coordinate groups of genes implies a general function in negative selection.



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Fig. 6. A model for negative selection. The interactions of a thymocyte with a thymic epithelial cell, a macrophage and the extracellular matrix are shown. Upon stimulation of the TCR by specific ‘high-affinity’ pMHC ligands, several genes involved in antigen (Ag) presentation and DNA recombination are down-regulated. Apoptotic molecules like Bim and FasL are up-regulated. Bim induces mitochondrial dysfunction. FasL is secreted and binds in an autocrine fashion to its receptor Fas. Adhesion to the extracellular matrix is weakened by down-regulation of DDR1, while cell–cell adhesion is enhanced by up-regulation of ICAM1 and down-regulation of CD2BP1, a negative regulator of CD2-mediated adhesion.

 
We have chosen to use the N15 H-2d thymocytes as the baseline group to avoid any ongoing negative selection that would be present in the N15 H-2b thymocytes. This raises the caveat that there may be some positive selection differences as well when these two samples are compared. However, the low level of constitutive positive selection observed in the N15 system (15,53) is negligible compared to the acute negative selection response induced within these short time points by cognate antigen exposure.

Previous in vivo analysis of negative selection by peptide injection has been hampered by cytokine- and glucocorticoid-mediated thymocyte apoptosis resulting from more protracted and repetitive activation of peripheral T cells (54). While we cannot completely rule out effects of peripheral T cells in our in vivo model, we believe that these are negligible for the following reasons. (i) Cytokine- and glucocorticoid-mediated thymocyte apoptosis by peripheral T cells has been described upon repetitive activation, i.e. three consecutive injections of peptide (54); in contrast, we induce negative selection by a single injection of VSV8. (ii) We use a 4 times lower concentration of peptide than Martin and Bevan. (iii) Cytokine- and glucocorticoid-mediated thymocyte apoptosis by peripheral T cells occurs within days (55); we analyzed gene induction in a time frame of a few hours. (iv) We observe deletion of DP thymocytes in N15 H-2b mice in FTOC, i.e. in the absence of peripheral T cells.

The finding that antigen processing/presentation is rapidly shut down in DP thymocytes is surprising as this function is believed to be mediated by epithelial and dendritic cells within the thymus. What might be the reason for this event? Preventing stromal cells from presenting peptide or lipid antigens from dying thymocytes may be important in maintaining the diverse array of pMHC complexes comprising self-substituents required for the thymic education process. Dendritic cells, in particular, can cross-present pMHC I and II ligands from dying cells (56,57). Furthermore, as TL molecules are linked to TCR-independent CD8 function including cellular activation (35), their continued expression is unnecessary. A reduction in cd8{alpha} gene expression is consistent with this view (see Fig. 3). An alternative, but not mutually exclusive, explanation might be that thymocytes act as antigen-presenting cells. In line with this possibility, presentation of lipid antigens by thymocytes is important for the development of NKT cells (40). Interestingly, thymocyte-derived cathepsin L and CD1d are crucial for this process (39), and their genes are down-regulated upon induction of negative selection (see Fig. 3).

We find expression of rag-1/-2, egr1 and bcl-2 genes preferentially regulated in DP thymocytes stimulated with a negatively selecting peptide. Such modulation has also been associated with positive selection (7,32,58). Likewise, differential expression of tdt, cd53 and itm2A has been associated with thymocyte maturation (5961). Although endogenous positive selection events occur in N15 H-2b mice in contrast to H-2d mice, it seems unlikely that positive selection is enhanced by the negatively selecting VSV8 peptide. Rather, the induction of these genes in the N15 model strongly suggests that these genes are markers for TCR stimulation in general similar to CD69. In the case of egr1, there might be an alternative explanation for involvement in both selection processes. egr1 transgenic mice show enhanced negative selection (62), but thymic deletion is unimpaired in the absence of egr1 expression (58). This might be explained by functional redundancy of Egr proteins and, interestingly, we detected induction of egr2 mRNA in thymocytes. Alternatively, the function of egr1 might be modulated by cofactors like nab2 which is induced in DP, but not SP, thymocytes.

Among the few genes known to be involved in negative selection of thymocytes are the orphan steroid receptor nur77 and the pro-apoptotic Bcl-2 family member bim (9,10). Both genes are identified by our microarray analysis, validating the use of this approach. Negative selection is impaired in transgenic mice expressing a dominant-negative mutant form of nur77 (9). While Bim induces mitochondrial dysfunction (5), the molecular mechanism of nur77-mediated apoptosis is unclear. Although fasL and cd30 have been proposed as target genes, nur77-mediated apoptosis is not impaired by the absence of these genes (9). A recent report suggests a transcription-independent apoptotic function of nur77 directly targeting the mitochondria (63). Yet, nur77-mediated apoptosis in T cells is not impaired by Bcl-2 overexpression (9) and transcriptional activity of Nur77 correlates with apoptosis in thymocytes (64) making this possibility in thymocytes less likely.

We also identified fasL as an apoptotic gene induced during thymic selection. Whereas analysis of thymic development in mice with mutations in the Fas system did not reveal an absolute requirement in selection, other studies suggest a role for the Fas system at least at high antigen concentrations (14). Here, fasL expression was found to be preferentially induced in DP thymocytes, supporting a functional role for this apoptosis pathway in thymic selection. This possibility was confirmed by functional analysis in FTOC since deletion of DP thymocytes was impaired in the presence of a blocking anti-FasL mAb.

The FasL drives death receptor-mediated apoptosis through Fas (CD95/Apo-1) (4). Fas-mediated apoptosis in thymocytes is independent of the intrinsic pathway as apaf-1–/– and caspase-9–/– thymocytes are Fas-sensitive (6567). Hence, Bcl-2 or A1 overexpression does not prevent Fas-mediated apoptosis in this cell type (51,68). It follows that cells highly expressing bcl-2 after TCR stimulation may undergo apoptosis. These cells are presumably deleted via an autocrine mechanism involving the FasL as is known to exist in single-cell in vitro cultures (69). If this notion is correct, then the Fas system might act as a safeguard mechanism in order to delete autoreactive thymocytes resistant to Bim-mediated deletion.

The fact that the two most prominent pro-apoptotic genes identified in this study, bim and fasL, are inducers of the intrinsic and extrinsic pathway respectively is worthy of note. In addition, Myd118-induced activation of p38 MAPK and LTß–TRAF3-mediated cytotoxicity may contribute to negative selection. Thus, induction of independent apoptotic pathways may be important to ensure deletion of autoreactive thymocytes and to prevent autoimmunity. If one apoptotic pathway is perturbed, deletion might proceed via one of the other pathways. These results may also explain conflicting findings obtained in different transgenic models which concentrated on one single molecule (or pathway) in negative selection like studies on Apaf-1, Bcl-2, Fas, FasL and FADD (7,14,7073). Moreover, different negative selecting signals might predominantly act via the extrinsic or intrinsic pathway. In line with this, DR3–/– mice show impaired negative selection in the H-Y TCR model, but not in response to endogenous superantigens (74). In contrast to these initiator molecules, blocking of caspases impairs thymocyte deletion in several models of peptide-induced negative selection (12,13,75), indicating that the pathways described herein converge at the level of executioner molecules.

Microarrays have been previously used for gene expression analysis of lymphoid tumors, peripheral lymphocytes and dendritic cells (7679). Here we used microarrays to analyze thymic selection in vivo. About 250 genes are differentially regulated in DP thymocytes upon triggering with a negatively selecting peptide. This is an order of magnitude less than in the gene expression program observed during activation of the innate immune system dendritic cells by danger signals, suggesting a more limited, but highly specified, set of genes under ligand-stimulated modulation (76,77). Further studies are necessary to elucidate the functional role of these genes in thymic selection and to identify the signaling pathways responsible for their induction.


    Note added in proof
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
While this study was under review, a related study was published by DeRyckere et al. (J. Immunol. 171:802, 2003). Their list of most up-regulated genes is similar, confirming the results herein. Differences among the two studies may be related to the use of an MHC class II-restricted TCR transgene by DeRyckere et al. compared to the MHC class II-restricted TCR transgene used in the current analysis.


    Acknowledgements
 
We thank Drs Fred Alt, Ron DePinho, William Lafuse and Dan Liebermann for generously providing plasmids, and the Dana-Farber Flow Cytometry facility for cell sorting. We are grateful to Dr Sabina Chiaretti for help with Chip technology and discussions, to Stephanie Osborn for help with FTOC, and to Dr Hsiu-Ching Chang for the H28 antibody. We also thank Drs Jerome Ritz and Christoph Benoist for critically reading the manuscript, Michael Boutros and Marek Svoboda for discussions, as well as Robert Gentleman, Xiachun Li and Ronghui Xu for help with the statistics. This work is supported by NIH grants AI50900 and AI45022 to E. L. R., and an Emmy-Noether fellowship of the Deutsche Forschungsgemeinschaft to I. S. (SCHM1586/1-1).


    Abbreviations
 
DP—double positive

FasL—Fas ligand

FTOC—fetal thymic organ culture

N15 H-2b—N15 TCR transgenic+/+ rag-2–/– H-2b

N15 H-2d—N15 TCR transgenic+/+ rag2–/– H-2d

pMHC—peptide–MHC complex

SP—single positive

TL—thymus leukemia

TNFR—tumor necrosis factor receptor

VSV8—vesicular stomatitis virus nucleoprotein-derived octapeptide N52–59


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 

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