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Originally published online as doi:10.1189/jlb.1004608 on January 20, 2005

Published online before print January 20, 2005
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(Journal of Leukocyte Biology. 2005;77:830-841.)
© 2005 by Society for Leukocyte Biology

Thymocyte stimulation by anti-TCR-ß, but not by anti-TCR-{alpha}, leads to induction of developmental transcription program

Nathalie Niederberger*, Lukas K. Buehler{dagger}, Jeanette Ampudia* and Nicholas R. J. Gascoigne*,1

* Department of Immunology, IMM1, The Scripps Research Institute, La Jolla, California; and
{dagger} SciScript, Inc., San Diego, California

1 Correspondence: Department of Immunology, IMM1, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. E-mail: gascoigne{at}scripps.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Anti-T cell receptor (aTCR) antibody (Ab) stimulation of T cells results in TCR down-modulation and T cell activation. Differences in the effect of anti-{alpha}-chain and ß-chain Ab have been reported on thymocytes. Anti-ß-chain Ab but not anti-{alpha}-chain reagents cause long-term TCR down-modulation. However, both types of Ab result in TCR cross-linking and activate early steps in signal transduction. In this study, we show that TCR internalization and calcium flux, hallmarks of T cell activation, are similar with aV{alpha} and aVß treatment. Therefore, we have compared the gene expression profiles of preselection thymocytes stimulated with these reagents. We find that aV{alpha} treatment does not cause any significant change in gene expression compared with control culture conditions. In contrast, aVß stimulation results in numerous changes in gene expression. The alterations of expression of genes known to be expressed in thymocytes are similar to changes caused by positive thymic selection, suggesting that the expression of some of the genes without known roles in thymocyte development and of novel genes whose expression is found to be altered may also be involved in this process.

Key Words: microarray • positive selection • signaling • T cell activation • TCR down-modulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Stimulation of T cells by different ligands can lead to vastly different responses, exemplified by activation (by agonists) or antagonism of mature T cells and negative or positive selection of thymocytes [1 2 3 ]. T cell activation by antibody (Ab)-mediated cross-linking of the T cell receptor (TCR) mimics most aspects of activation by antigen (Ag), but remarkably, T cells are activated in different ways by cross-linking via the {alpha} or the ß chain of the TCR [4 , 5 ]. Ligation of CD3 or TCR-ß leads to long-term internalization and down-modulation of the TCR, similar to agonist activation. Engagement of TCR-{alpha} cross-links the TCR but does not induce long-term down-modulation of the TCR. The anti (a)V{alpha} Ab used in these experiments bind to the TCR and induce cell-surface co-capping equally well as the aVß or Cß reagents [5 ].

Positive selection in thymocytes leads to the functional allelic exclusion of TCR-{alpha}. Cells with two different V{alpha} species on the cell surface are relatively common in preselection thymocytes but are rare after positive selection [6 , 7 ]. Unlike the ß chain, where allelic exclusion operates by inhibition of DNA rearrangement of the ß-chain locus, the {alpha}-chain locus frequently functionally rearranges the genes on both chromosomes and expresses functional mRNA and proteins for both {alpha} chains [7 ]. However, mature T cells with strong expression of two cell-surface {alpha} chains are comparatively rare [6 7 8 9 ]. This functional allelic exclusion operates by a post-translational mechanism [4 , 9 ], involving selective retention of the positively selected {alpha} chain on the cell surface with down-modulation and thus, exclusion of the unselected {alpha} chain [4 , 5 ].

In unstimulated T cells, the TCR is continuously endocytosed and recycled to the cell surface, resulting in a constant, steady-state level of the TCR on the cell surface. It is possible that when the TCR is triggered through the V{alpha} chain, it is internalized and recycled back to the cell surface. Upon activation with aVß Ab, however, the endocytosed TCR is retained within the cell and degraded [10 ] (N. Niederberger and N. R. J. Gascoigne, in preparation). The maintenance of allelic exclusion is believed to be related to the endocytosis and recycling of the TCR, as loss of c-Cbl reduces the post-translational allelic exclusion of TCR-{alpha} chains [5 ]. Moreover, the negative regulatory adaptor molecules c-Cbl and Cbl-b are important in regulating TCR down-modulation, probably as a result of their ubiquitin ligase activity targeting stimulated TCR complexes for degradation [11 ].

Differences in TCR down-modulation resulting from engagement of either of the TCR chains must come from events following TCR engagement. Biochemical analyses of recruitment and phosphorylation of molecules involved in TCR signaling did not find significant differences between {alpha}- or ß-chain cross-linking [4 ]. The exception was with dynamin, involved in formation of endosomes, which was not recruited to aV{alpha}-stimulated TCR [4 ]. Other early events in activation following the two types of activation, such as TCR internalization and calcium release, might be affected. We have now tested this possibility and find no difference in the response to aV{alpha} or aVß stimulation.

We therefore decided to look for changes happening later in activation, at the level of gene transcription. The technique of gene expression profiling using DNA microarray hybridization is a powerful tool to analyze signaling pathways activated as a result of receptor ligation. In this study, we compare changes in gene transcription in preselection CD4+8+ ["double positive" (DP)] thymocytes induced by Ab cross-linking through the TCR-{alpha} or -ß chain. It is surprising that stimulation through the TCR-{alpha} chain induced no reproducible changes in transcription of mRNA among the 12,000 known genes and expressed sequence tags (ESTs) tested. In contrast, we have identified 103 genes differentially expressed in response to stimulation with aVß Ab. These include many genes known or expected to be regulated during thymocyte-positive selection, as well as many genes with no known role in thymocyte development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
C57BL/6J (B6) mice were bred and maintained at The Scripps Research Institute (TSRI; La Jolla, CA). Ovalbumin-specific TCR transgene (OT)-I and transporter associated with antigen processing deficient (TAP)–/– animals were obtained from Drs. Stephen Jameson and Kristin Hogquist (University of Minnesota, Minneapolis). All experiments were performed in accord with the guidelines of the Animal Care and Use Committee of TSRI.

Activation of thymocytes with Ab
Tissue-culture plates were coated with phosphate-buffered saline (PBS), alone or containing 10 µg/ml goat anti-mouse immunoglobulin G (IgG; Sigma-Aldrich, St. Louis, MO) or 10 µg/ml goat anti-rat IgG, overnight at 4°C. Plates were washed 3x with PBS and further coated with PBS alone or containing 2 µg/ml aVß5 (clone MR9-4, mouse IgG) or 2 µg/ml aV{alpha}2 (clone B20.1, rat IgG) for 3 h at 37°C. Single-cell suspensions from thymi of 4- to 8-week-old OT-I TAP–/– mice were prepared and resuspended in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml pen/strep, and 50 µM ß-mercaptoethanol. Thymocytes at 1 x 107/well in a final volume of 1 ml were incubated at 37°C/5% CO2 overnight in 24-well tissue-culture plates precoated with Ab. Unless otherwise noted, the Ab used in this study were purchased from BD PharMingen (San Diego, CA).

Flow cytometry and cell sorting
For flow cytometry, OT-I TAP–/– thymocytes, stimulated overnight ± Ab, were recovered and stained with aCD4, aCD8, and aTCR-ß. The staining was done in the dark for 30 min at 4°C. Cells were washed, suspended in 200 µl PBS containing 1% bovine serum albumin (BSA) and 0.1% sodium azide [flow buffer (FB)], and analyzed with a FACSCalibur (Becton Dickinson, Mountain View, CA). For the microarray experiment, OT-I TAP–/– thymocytes, stimulated overnight with Ab, were recovered, stained with aCD4 and aCD8, and sorted for DP thymocytes. Controls were freshly isolated thymocytes (control 1) or overnight-cultured, unstimulated thymocytes (control 2). For the real-time polymerase chain reaction (PCR) experiment, OT-I TAP–/– thymocytes were stained with aCD4 and aCD8 and sorted for DP thymocytes, whereas B6 thymocytes were stained for TCR-ß and CD69 and sorted for TCRintCD69+ and TCRhi thymocytes. The TCRintCD69+ and TCRhi thymocyte populations were pooled and represent thymocytes that have received a positive selection signal, whereas the OT-I TAP–/– DP thymocytes correspond to a population of thymocytes that have not been selected. All stainings were done in the dark for 1 h at 4°C. Cells were washed and suspended at 1 x 107/ml in filtered PBS buffer containing 1 mM EDTA, 25 mM HEPES, and 1% FCS. Thymocytes were sorted with a FACSVantage. All the Ab used for staining were conjugated directly to phycoerythrin (PE), fluorescein isothiocyanate, allophycocyanin, and peridinin chlorophyll protein.

Internalization of the TCR
Single-cell suspensions of thymocytes from 4- to 8-week-old OT-I TAP–/– mice were prepared. Thymocytes (3x106 cells/staining) were resuspended in 100 µl PBS containing 1% BSA and 0.1% sodium azide (FB). To induce internalization, cells were incubated with biotinylated aV{alpha}2 or aVß5 for 30 min at 4°C, washed, resuspended in FB, and stained with streptavidin-Alexa Fluor 680 conjugate (Molecular Probes, Junction City, OR) for 10 min on ice. Thymocytes were finally incubated at 37°C for 10 min and fixed with 4% paraformaldehyde for 12 min at room temperature. TCR internalization was observed using a Zeiss Axiovert 200 M inverted microscope. SlideBook software (Intelligent Imaging Innovation, Denver, CO) was used for the capture as well as for deconvolution.

Calcium flux
OT-I TAP–/– thymocytes were washed and resuspended at 1 x 106/ml in indo-1 loading buffer described previously [12 ]. Indo-1 AM (Molecular Probes), at 5 mg/ml in dimethyl sulfoxide, was added to the cells at a final concentration of 7 µM. Thymocytes were incubated for 30 min at 37°C, washed with RPMI-1640 medium, and supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml pen/strep, and 50 µM ß-mercaptoethanol. Cells were incubated in RPMI media for an additional hour at 37°C, washed, and resuspended at 2 x 106/500 µl RPMI. Thymocytes were kept on ice in the dark until flow cytometry analysis. Before analysis, cells were incubated at 37°C for 5 min. The change in fluorescence intensity of Indo-1 at 405/20 and 530/30 for bound and free probe, respectively, following ultraviolet excitation, was measured with a FACS Digital LSRII instrument (BD Biosciences, Mountain View, CA) using FlowJo software (Tree Star, Inc., Ashland, OR). After 30 s of analysis, primary Ab, aV{alpha}2 (10 µg/ml) or aVß5 (10 µg/ml), were added, and 1 min later, streptavidin (20 µg/ml) was supplemented. As negative controls, primary Ab aV{alpha}2 or aVß5 (10 µg/ml) or streptavidin (20 µg/ml) only were added to the cells. Ionomycin at 2 µg/ml was used as a positive control.

RNA preparation and microarray procedures
Total RNA from sorted OT-I TAP–/– DP thymocytes was isolated using the RNeasy mini-kit method (Qiagen, Valencia, CA). The RNA was then processed according to Affymetrix protocols. Briefly, the total RNA (5.0–20 µg) was used as template to generate double-stranded cDNA using the GeneChip T7-Oligo(dT) promoter primer kit (Affymetrix, Santa Clara, CA) for priming first-strand cDNA and the Superscript II kit (Invitrogen Life Technologies, Carlsbad, CA). Biotin-labeled cRNA was synthesized in an in vitro transcription reaction by using the ENZO BioArrayTM High YieldTM RNA transcript-labeling kit (Affymetrix). The cRNA was cleaned and fragmented. Finally, the fragmented, biotinylated cRNAs were hybridized to Affymetrix MG-U74Av2 arrays for 16 h. Arrays were washed and stained with streptavidin-PE and finally read using the GeneArray scanner. The gene expression values were quantified and analyzed using the Affymetrix Microarray Suite (MAS) software, Version 5.0. A global scaling method, where the average intensity (signal value) of every array is scaled to a common value of 250 (target intensity), was applied, making possible the comparison between arrays.

Microarray data analysis
Dendrograms to compare the relationship between arrays were done using the GeneCluster and TreeView programs [13 ]. This method makes it possible to distinguish whether biological replicates of each experiment are more similar to each other than to any other conditions. It compares the signal strength (a quantitative measure of the relative abundance of a transcript) of 7151 genes out of 12,488 sequences, represented on the MG-U74Av2 array after genes found absent in all conditions are removed from the data. Data signals represent average difference signals from Affymetrix data files. Hierarchical clustering of arrays, but not genes, was performed using an "average linkage clustering" mode. Scatter plots were produced to compare pair-wise conditions: aV{alpha}2 versus overnight control, aVß5 versus overnight control, and aVß5 versus aV{alpha}2. The scatter plot compares global data on hybridized chips and includes 7151 genes, after genes found absent in all conditions were removed from the data. The log2 signals of the average difference call values for each of the genes were used, such that, for example, a 2x increase or decrease in expression is 1 in the log2 scale, and a 4x increase or decrease in expression is 2. In the scatter plots, genes whose expression is increased show a higher log2 value in the y-condition compared with the x-condition and are found above the diagonal, whereas genes whose expression is decreased show a lower log2 value in the y-condition compared with the x-condition and are found below the diagonal. A log2 representation allows a better signal discrimination for these data than a linear scale. A mean-difference plot was produced to compare aVß5 versus overnight control. It compares the log2 ratio versus average log intensity ("MA plot") for the 7151 genes left after absent genes were eliminated. The log2 ratio is the ratio of an experimental signal from aVß5-treated thymocytes over a control signal, as computed by Affymetrix file comparison software (MAS 5.0). The log2 intensity value is calculated as (log aVß signal+log control signal)/2. The log2 intensity scale spreads the genes along the horizontal, allowing for easier viewing of the genes. In the MA scatter plot, genes whose expression is increased in aVß5-stimulated thymocytes compared with the overnight control are located above the horizontal, corresponding to a log2 ratio value of zero, and genes whose expression is decreased in aVß5-stimulated thymocytes compared with the overnight control are located below the same horizontal line. The hierarchical clustering of selected genes was done using GeneCluster [13 ]. Array data from two independent hybridizations were combined (biological replicates). To select genes with significant variation of expression across the samples, several criteria were applied. Genes considered absent in all conditions were excluded. Among the genes kept, those with a no-change (NC; P>0.05) call across the samples were removed. Of the 2689 remaining genes, only those with a "change P value" <0.01 were selected. The Affymetrix statistical algorithms implemented in the analysis software (MAS 5.0) determed the change P values, based on Wilcoxon’s signed rank test. These define the confidence of the call (increase, decrease, or NC compared with the control). The 547 genes that conformed to these selection criteria were hierarchically clustered. Average difference signals were adjusted using log transformation and median centering (median value=0) prior to gene clustering (average linkage clustering mode). Clusters were visualized using TreeView [13 ].

Quantitative real-time PCR
A control RNA was required to construct standard curves. Total RNA from OT-I thymi between 4 and 8 weeks old was isolated using the RNeasy mini-kit method (Qiagen). The RNA was treated with DNase I for 15 min at room temperature (DNase I amplification grade, Invitrogen Life Technologies). The reaction was stopped by the addition of 25 mM EDTA, and the DNase was inactivated at 65°C for 10 min. To confirm the results of the gene-chip analysis, the RNA from aV{alpha}- or Vß-treated and untreated, overnight control thymocytes was the same samples as used for hybridization to Affymetrix MG-U74Av2 arrays. To measure protein kinase C (PKC){theta}, PKC{eta}, nuclear factor of activated T cells (NFAT)c, CD5, Egr-1, Bcl-XL, CD4, and Ets-2 transcript levels and compare them between OT-I TAP–/– DP thymocytes, which did not receive a selection signal, and B6 thymocytes, which have undergone positive selection (TCRintCD69+ plus TCRhi populations), RNA from the different populations of thymocytes was isolated as described above for the standard RNA. All the RNA samples were used as template to synthesize cDNA using the SuperscriptTM first-strand synthesis system for reverse transcriptase (RT)-PCR (Invitrogen Life Technologies), following the manufacturer’s recommendations. Oligonucleotide primers were designed using Primer Express 1.5 software (PE Applied Biosystems, Foster City, CA). Primer sequences are presented in Supplementary Table S1. Quantitative real-time PCR reactions were run on the ABI Prism 7700® sequence detection system using SYBR Green PCR master mix from PE Applied Biosystems. The PCR mix was optimized for SYBR Green reactions and contained SYBR Green 1 dye, AmpliTaqGold DNA polymerase, and deoxy-unspecified nucleoside 5'-triphosphates with deoxy-uridine 5'-triphosphate. Each reaction was performed in a 25-µl final volume and contained the SYBR Green PCR master mix, cDNA, and 150 nM each primer. For each gene, two independent experiments were done, and for both experiments, all the reactions were performed in duplicate. A standard curve using a twofold serial dilution of the standard cDNA was generated for the target genes as well as for the housekeeping gene ß-actin. cDNAs (20 ng/reaction) from unstimulated, overnight control, aVß- or aV{alpha}-stimulated samples, OT-I TAP–/– DP thymocytes, and B6 thymocytes (TCRintCD69+ plus TCRhi pooled populations) were used as template for real-time PCR. The standard curves were used to determine the relative concentrations of amplified transcripts. The transcript levels of each sample were first normalized using the values of ß-actin. The normalized sample values were finally divided by the normalized control (unstimulated, overnight control) values to obtain the fold change of mRNA expression between unstimulated, overnight control and aVß- or aV{alpha}-stimulated samples or by the OT-I TAP–/– DP values to obtain the fold change of mRNA expression between OT-I TAP–/– DP thymocytes and B6 (TCRintCD69+ + TCRhi) thymocytes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Early events in activation following triggering of TCR with Ab against the {alpha} or ß chain are similar
Previous studies showed that Ab-mediated cross-linking of TCR by Ab recognizing the ß chain leads to down-modulation of the TCR, and ligation of the {alpha} chain does not, suggesting that the two distinct chains of the TCR can induce different signals following their engagement with Ab [4 , 5 ] (Fig. 1A ). Early events in activation tested previously were similar with the two types of stimulation, including the recruitment of signaling molecules and the patterns of phosphorylation [4 ]. Different early events in activation might be affected, and therefore, we performed two additional experiments. First, we tested whether aV{alpha}2 and aVß5 Ab are equally effective at inducing internalization of the TCR. Therefore, OT-I TAP–/– thymocytes were incubated with biotinylated aV{alpha}2 or aVß5 Ab. Alexa Fluor 680-labeled streptavidin was added, and the thymocytes were incubated at 37°C for 10 min. The reaction was stopped with 4% paraformaldehyde, and internalization was visualized by microscopy. Both Ab were able to induce internalization of the TCR (Fig. 1B) . Second, we investigated intracellular calcium mobilization in response to OT-I TAP–/– TCR cross-linking. Thymocytes were preloaded with the calcium dye Indo-1, and flow cytometry was used to monitor intracellular calcium concentration following activation of the TCR with biotinylated aV{alpha}2 or aVß5 Ab, followed by cross-linking with streptavidin. As seen in Figure 1C , the kinetics and amplitudes of the calcium response were similar with the two types of stimulation. Both Ab were effective at inducing an initial transient peak of intracellular calcium concentration within a few minutes of TCR cross-linking. The peak gradually decreased to reach the baseline level.



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  		Figure 1. Changes induced by incubation of OT-I TAP–/– thymocytes overnight or by treatment with aTCR V{alpha} or Vß. (A) TCR down-modulation induced by aVß (light, solid line) but not by aV{alpha} (dotted line) treatment overnight. The aV{alpha} treatment shows no difference from unstimulated cells cultured for the same period (bold, solid line). (B) aV{alpha}2 (left panel) and aVß5 (right panel) Ab are equally effective at inducing TCR internalization. OT-I TAP–/– thymocytes were incubated with biotinylated aV{alpha}2 or aVß5 Ab followed by TCR cross-linking with streptavidin Alexa Fluor 680 conjugate for 10 min at 37°C. Cells were fixed and analyzed by fluorescence microscopy. One representative experiment out of at least three is shown. (C) The calcium response induced following TCR engagement through V{alpha} or Vß chain is similar. OT-I TAP–/– thymocytes were loaded with the dye Indo-1 and then stimulated by the addition of 10 µg/ml biotinylated aV{alpha}2 or aVß5 Ab at 30 s and 20 µg/ml streptavidin at 90 s. Ionomycin (2 µg/ml) was added at 180 s and serves as a positive control. Biotinylated aV{alpha}2 (10 µg/ml) Ab or aVß5 Ab or streptavidin (data not shown) were added at 30 s and serve as a negative control. The fluorescence between bound and unbound Indo-1 was analyzed over a period of time with a 4-min minimum. The kinetics data were analyzed with FlowJo software.

 
Comparison of gene expression changes in CD4+CD8+ thymocytes following engagement of TCR with aV{alpha} or aVß Ab: aV{alpha} does not induce changes in gene expression
To identify genes differentially expressed following engagement of either of the TCR chains, a DNA microarray approach was taken. Thymocytes from the OT-I TCR transgenic mouse (V{alpha}2Vß5) [14 ] bred onto the TAP-1–/– background were isolated. These thymocytes are arrested at the CD4+CD8+ DP stage of development as a result of lack of major histocompatibility complex (MHC) class I-peptide complexes to stimulate positive or negative selection [15 ]. Isolated thymocytes were cultured overnight at 37°C on plates coated with aV{alpha}2 or aVß5 Ab or on noncoated plates. Freshly isolated thymocytes were used as an additional control. DP thymocytes were identified using aCD4 and aCD8 Ab, purified by single-cell sorting, and used to isolate total RNA. Biotin-labeled cRNA was prepared and hybridized to DNA arrays representative of 12,000 murine genes and ESTs (MG-U74Av2 arrays, Affymetrix).

To determine whether expression profiling could identify differences between aV{alpha}- and aVß-induced TCR signaling, two datasets with four samples each were analyzed using Cluster and Treeview programs [13 ]. The samples were from aV{alpha}- or aVß-treated thymocytes, nonstimulated cells incubated at 37°C overnight (control 2), or freshly isolated DP thymocytes (control 1). Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo/) accession numbers are as follows: First dataset: overnight control, GSM 24449; aV{alpha}2, GSM 24450; aVß5, GSM 24451; freshly isolated thymocytes, GSM 24455. Second dataset: overnight control, GSM 24452; aV{alpha}2, GSM 24453; aVß5, GSM 24454; freshly isolated thymocytes, GSM 24456. The mRNA expression data showed a distinct pattern. The biological replicates of aVß5 incubation were more similar to each other than to any other condition, whereas the aV{alpha}2 results clustered with their respective overnight control treatments rather than with each other (Fig. 2A ). This indicates first, that aVß5 incubation induced distinct changes in the overall gene expression patterns as compared with control and aV{alpha}2 treatment and second, that aV{alpha}2 incubation is indistinguishable from overnight incubation without Ab. It is surprising that variations in gene expression between biological repeats of aV{alpha}-treated thymocytes were bigger than those between the aV{alpha}2 treatments and their unstimulated, overnight controls. This analysis was an indication that no significant variation in gene expression was induced by engagement of the TCR with aV{alpha} Ab. This does not mean that the aV{alpha} biological replicates are dissimilar but rather that there are some differences between the individual mice used in the separate experiments and/or day-to-day variation between the experiments, leading to clustering of the control and aV{alpha} treatments for each of the separate experiments. Therefore, aV{alpha} and overnight control are essentially two identical negative controls of the same animal: Only aVß treatment produces specific changes that override the baseline differences between the individual mice used. The freshly isolated thymocytes were somewhat different from the overnight-cultured controls, reflecting changes as a result of culture conditions, which are known to affect the thymocytes [16 ]. The scatter plots presented in Figure 2B further confirm this result. Genes whose expression is increased show a higher log2 value in the y-condition compared with the x-condition and are found above the diagonal, whereas genes whose expression is decreased show a lower log2 value in the y-condition compared with the x-condition and are found below the diagonal. In the left panel, only a few minor changes were observed following activation of TCR with aV{alpha} Ab compared with the unstimulated, overnight control. We saw that most of the genes with an intermediate or high expression level were localized on or very close to the diagonal. Two genes changed between control and aV{alpha} treatments. One of these is a pseudogene (nucleotide sequence database ID L04848), which was increased ninefold in one experiment and decreased twofold in the other. The other gene is triosephosphate isomerase, which was increased 2.8-fold in one experiment and decreased 2.6-fold in the other. As these were not reproducible between experiments, they were considered as being not significant and are not further described. We observed many changes upon aVß activation compared with overnight control (Fig. 2B , middle panel) and aV{alpha} conditions (Fig. 2B , right panel).



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  		Figure 2. (A) A dendrogram comparing the relationship between arrays [13 ]. (B) The transcription data for the three different treatments, showing little difference among the unstimulated, cultured control and aV{alpha}-stimulated cells but many differences between aVß compared with aV{alpha} or overnight, unstimulated cells.

 
Stimulation by aVß induces a transcriptional program in DP thymocytes
Stimulation of immature DP thymocytes through the TCR can induce differentiation (positive selection) or apoptosis (negative selection) at least partly in relation to the ligand-binding strength [1 , 14 , 17 , 18 ]. Earlier studies had shown that stimulation of thymocytes with Ab to TCR-ß induces gene expression patterns expected for cells undergoing positive selection in vivo [19 , 20 ]. DNA array technology allows a more systematic and broader approach to identify changes in the steady-state levels of mRNA species that are affected by stimulation through the TCR. The OT-I TAP-1–/– TCR transgenic thymocytes provide a relatively homogeneous source of DP thymocytes, which are blocked at the stage of positive selection but can be activated to progress by TCR stimulation [15 ]. A general view of transcripts regulated by engagement of TCR-ß is presented in Figure 3 . In this scatter plot, genes whose expression is increased in aVß5 compared with the overnight control are located above the horizontal line corresponding to a log2 ratio value of zero, and those genes whose expression is decreased are located below this line. Among the transcripts regulated by engagement of TCR-ß, several have been described previously as being regulated after TCR engagement of DP thymocytes. Bcl2, CD5, and Egr1 transcripts were increased by aVß stimulation, whereas RAG1, CD8, and CD4 transcripts were decreased, as expected from previous work [19 20 21 ]. This reinforces the idea that aVß stimulation of DP thymocytes mimics aspects of thymocyte maturation.



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  		Figure 3. General view of transcripts regulated in aVß-stimulated DP CD4+CD8+ thymocytes. This scatter-plot analysis compares the log ratio versus average log intensity (MA plot) for 7151 genes. The arrows point to a few well-known genes regulated during thymocyte maturation and showing differential expression upon Vß treatment.

 
The expression-profiling technique provides us with the ability to identify patterns of expression shared between genes using statistical techniques. To select genes showing significant variation across the samples out of the 12,000 genes and ESTs tested using the U74Av2 array, several criteria were applied. First, genes that were absent in all conditions were excluded. Second, those that showed NC (P>0.05) across all samples were removed. Third, out of the 2689 remaining genes, only those with a more stringent P value (<0.01) were selected. A total of 547 genes conformed to these selection criteria and were hierarchically clustered. To discard false-positive signals, array data from two independent hybridizations were combined. The pattern of gene induction is shown in Figure 4 . The green color represents genes that were repressed, and the red color identifies genes that were induced in one of the compared conditions. The brighter the color, the bigger the change in expression. White indicates NC in gene expression. The clustering results in two main subclusters. Cluster 1 shows genes that were repressed in aVß5-stimulated thymocytes compared with the overnight, unstimulated thymocyte control (green), and Cluster 2 identifies genes that were induced upon aVß stimulation (red). Only those genes showing at least a twofold change in aVß versus the overnight-cultured control were selected. In total, 103 genes were differentially regulated as a result of aVß stimulation. Out of these genes, 63 were up-regulated, and 40 were down-regulated. They are presented in Table 1 . To validate our array data, the expression of eight genes in nonstimulated or aVß- or aV{alpha}-stimulated OT-I TAP–/– thymocytes was estimated using quantitative real-time PCR, which confirmed the results of the gene-chip analysis (Fig. 5A , described in detail below).



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  		Figure 4. View of the hierarchical clustering of selected genes performed using GeneCluster and TreeView programs [13 ]. Data from two biological replicates of aVß and overnight, unstimulated thymocytes were combined and compared. Among the 12,000 genes and ESTs present on the array, only 547 genes had a P value <0.01 and were selected. These were hierarchically clustered using average difference signals between aVß and overnight, nonstimulated samples. The clustering results in two subclusters: Cluster 1 shows aVß down-regulated genes (green color) and Cluster 2, aVß up-regulated genes (red color). If a threshold change of 2x is applied to this list, only 103 genes remain.

 

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  		Table 1. Genes and ESTs Showing Differential Expression after Stimulation of OT-I TAP–/– Thymocytes with aVß

 


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  		Figure 5. Quantitative real time RT-PCR. (A) Compares RNA samples from aV{alpha} (open bars) or aVß-treated (solid bars) thymocytes. Results are shown as fold change compared with unstimulated cells cultured overnight. (B) The average fold change in transcript levels of eight genes from B6 thymocytes (pooled TCRintCD69+ plus TCRhi), which have undergone positive selection relative to the corresponding control OT-I TAP–/– thymocytes that are arrested at the DP stage of development, Error bars indicate the SE.

 
Changes in the expression pattern of genes by aVß stimulation
Among the 103 genes differentially regulated upon aVß stimulation, a large diversity of functional gene classes is represented (Table 1) , including cell-surface proteins and receptors, signaling molecules, mediators of TCR gene rearrangement, transcription factors, regulators of apoptosis, as well as a number of ESTs representing unknown genes. The up-regulation or down-regulation of various cell-surface proteins or receptors is induced by TCR stimulation, and therefore, it was not surprising that we found several cell-surface proteins shown previously to be stimulated by positive selection in the up-regulated group, including CD5, CD2, CD6, CD53, and Itm2a (Table 1 , Fig. 5A ) [19 , 20 , 22 23 24 25 26 27 28 29 30 31 ]. CD69 was not represented on the U74Av2 array and so could not be checked against its known up-regulation by positive selection. CD4, CD8, Ly6d, and TL (thymus leukemia antigen, a nonclassical MHC class I protein) expression were decreased, which also correlates with the delivery of a positive selection signal [15 , 19 , 20 , 27 28 29 30 31 32 ]. To avoid repetition, the main description of the genes found to be regulated by aVß treatment is presented in Discussion.

Changes in the expression pattern of genes induced by aVß are similar to changes occurring during thymocyte selection in vivo
As many genes regulated upon aVß stimulation have already been shown to be regulated during thymocyte development, we decided to use quantitative RT-PCR to test whether the expression of the eight genes we used to validate our array data would behave in a similar way during thymocyte development in vivo. Therefore, the transcript levels of PKC{theta}, PKC{eta}, NFATc, CD5, Egr-1, Bcl-XL, CD4, and Ets-2 genes from OT-I TAP–/– DP thymocytes, which are arrested at the DP stage of development, and from B6 TCRintCD69+ plus TCRhi thymocytes, which have undergone positive selection, were compared. As presented in Figure 5B , all the genes tested showed a similar pattern of expression following stimulation of the TCR with aVß or when undergoing normal thymocyte selection in vivo. The only exception was CD4, and its expression was slightly induced instead of reduced in the B6 TCRintCD69+ plus TCRhi thymocytes; this difference is explained by the fact that part of this B6 thymocyte population contains some single-positive thymocytes that have already up-regulated the coreceptor CD4.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Early events in activation following cross-linking of the TCR with Ab against the {alpha} or ß chain are identical
Cross-linking of TCR with Ab typically results in TCR down-modulation. We and others have previously shown that cross-linking with aTCR V{alpha} reagents gives a different outcome, with no long-term down-modulation, despite a similar ability of the Ab to bind, cross-link, and induce clustering of TCR, as anti-ß-chain reagents [4 , 5 ]. aV{alpha} and aVß stimulation induces similar patterns of tyrosine-phosphorylated proteins and of recruitment of proteins to the signalsome, the exception being dynamin, which is not recruited with aV{alpha} [4 ]. As TCR triggering induces a lot of different signals within the cell, we were curious to see the outcome of earlier events in activation following the two types of stimulation. From our results, it is clear that aV{alpha}2 and aVß5 Ab were able to induce internalization of the TCR as well as a calcium flux response, clearly suggesting that all early events in activation following cross-linking of the TCR with Ab against the {alpha} or ß chain are identical. Even so, aV{alpha} and aVß Ab have a different long-term effect on TCR expression.

aTCR V{alpha} stimulation does not induce changes in gene expression
This similarity led us to compare gene expression programs induced in thymocytes by cross-linking of TCR through the {alpha} chain or the ß chain using DNA arrays. We were amazed to find that there were no significant and reproducible changes in gene expression induced by aV{alpha}-mediated cross-linking of TCR. In contrast, many changes were induced by aVß.

This means that despite the similarities of early events after TCR cross-linking by the Ab, the ß-chain (or CD3) cross-linking activates a program of gene expression, whereas {alpha}-chain cross-linking is unnoticed by the cell in the long-term. The fact that {alpha}-chain-cross-linked TCR reappears on the T cell surface, whereas ß-chain-stimulated TCR does not suggests that the former remains in the normal pool of TCR, which undergoes constitutive endocytosis and recycling [10 ], whereas the latter is diverted to lysosomes. We have recent evidence indicating that this differential sorting is indeed the case (N. Niederberger and N. R. J. Gascoigne, in preparation), but it remains unclear how such apparently similar stimuli could give such different outcomes. The answer likely lies in how the Ab cross-link the TCR proteins in relation to the natural cross-linking of TCR by Ag recognition and to the architecture of the TCR complex and its coreceptors. Unfortunately, this is largely the realm of speculation, as so little is known of these details. The orientation of the TCR in its binding to MHC peptide and the interaction sites of CD4 and CD8 with MHC indicate that the coreceptor is more closely associated with the {alpha}-chain side of the TCR than the ß-chain side [3 , 33 ]. Therefore, the different response to aV{alpha} reagents could be a result of the Ab separating the TCR from the coreceptor and therefore, from its attached kinase (Lck).

Genes regulated by aVß stimulation
For the most part, the pattern of genes up- or down-regulated by aVß stimulation corresponds to those that are also regulated by natural positive selection stimuli, as shown in other studies using microarrays [28 29 30 31 , 34 ]. Our results suggest the possibility that positive selection signals may be mediated through the TCR-ß chain. The two coreceptors CD4 and CD8 are repressed upon aVß stimulation, consistent with DNA microarray data showing down-regulation of these genes in response to TCR activation and positive selection in vivo [29 , 31 ]. The same is true for genes encoding proteins involved in TCR variable(diversity)joining-region rearrangement such as recombination-activating gene 1 (RAG1), RAG2, Ku70, and terminal deoxynucleotidyl transferase [29 , 31 , 35 , 36 ]. In addition, a histone and Orc1l, involved in DNA replication, were down-regulated, and an endocuclease (Apex1 or Ape1) involved in nucleotide incision repair of oxidatively damaged DNA was up-regulated.

Various cell-surface molecules, which apparently fine-tune selection signaling threshold, such as CD2, CD5, CD6, and CD53, were up-regulated. Whereas CD2, CD5, and CD53 have earlier been reported to be regulated during thymic selection by flow cytometric analysis or differential expression studies [22 , 29 , 31 , 37 , 38 ], CD6 expression has only recently been shown to play a role in positive selection. CD6 expression parallels the expression of CD69 and increases when thymocytes are positively selected [39 ]. Other cell-surface receptors, whose expression was affected, included up-regulation of the inducible costimulatory receptors, inducible costimulator and program death-1, both members of the CD28/cytotoxic T lymphocyte-associated antigen 4 family. The G protein-coupled receptor Gpcr25 was also up-regulated. Although little is known about Gpcr25, it could be involved in the differentiation of T cells [40 ].

Expression of proteins involved in cell signaling was also affected. The expression levels of two IFN-induced GTPases, Gbp-2 and Gbp-3 [41 ], were up-regulated, as was IFN-regulatory factor (Irf)1, which has been shown to be involved in CD8 T cell development [42 ]. Others, such as T cell-specific GTPase (Tgtp) [43 ], SH3 domain-binding protein [44 ], the MAP kinase phosphatase Dusp2/pituitary adenylate cyclase-activating polypeptide receptor 1 [45 ], and MAP kinase-activated protein kinase (MAPKAP) 2 [46 ], have been shown to be involved in T cell signaling, but their precise function in thymocyte development is still unknown. PKC{eta} (Prkch) expression was increased, but PKC{theta} expression was unchanged (Table 1 , Fig. 5A ). PKC{theta} is of great importance in T cell signaling, where it is strongly recruited to the central supramolecular activation cluster of the synapse [47 ]. The fact that PKC{eta} is up-regulated during thymocyte maturation suggests a potential involvement of this protein in thymocyte development (N. Niederberger, in preparation) [47 , 48 ].

Signaling pathways culminate in the activation or repression of genes through the activity of transcription factors. Nineteen transcription factors or proteins regulating transcription were expressed differentially (Table 1 , Fig. 5A ), many of which have previously been shown to be involved in thymocyte development, including Ets2, Egr1, Egr2, JunB, NFATc1, and Lef-1. For example, Egr1 is up-regulated and is a target of Ets2, which is down-regulated, suggesting the involvement of Ets2 positive selection [21 , 49 ]. Ets2, like several other transcription factors identified in these experiments (Pou2af1, Lef-1 s, Bmi1, and Zfpm1), has not been shown to be involved in thymocyte maturation so far [50 ].

The regulation of antiapoptotic molecules such as Bcl2 and Bcl-XL was also consistent with positive selecting signals, and Bcl2 was up-regulated and Bcl-XL down-regulated [29 , 31 , 51 52 53 ]. Both molecules are important regulators of programmed cell death, able to protect cells against apoptosis. They might therefore play a role in thymocyte survival during the thymic selection process. Parenthetically, PKC{eta} is also reported to be antiapoptotic [54 ].

Several genes showing differential expression have also been shown to be modulated during negative selection or after aCD3/CD28 stimulation [28 , 29 , 31 , 34 ]. It has been shown that aTCR-ß stimulation alone can induce developmental changes typical of maturation and survival that happen during positive selection, whereas costimulation with CD28 is required to induce negative selection in vitro [20 , 55 , 56 ]. Some of our thymocytes might undergo apoptosis, as they were incubated overnight for 16 h in culture. This could explain the expression of Nur77, a gene exclusively regulated during negative selection. A dominant-negative mutant of Nur77 affects negative selection but not positive selection [57 ]. However, other genes typically regulated during negative selection, such as Bim and E2F1, were not detected in our assay. Other genes identified in our screen are also differentially expressed in negative selection. Egr1, Ets2, Egr2, and Nab2 interact with each other. Nab2 is a negative regulator of Egr1 and Egr2. All of these are early-response genes and could therefore be needed in early stages of positive and negative selection [28 ]. Alternatively, fine-tuning in expression of these genes might contribute to the final decision. CD53, like CD69, could serve as marker of activated thymocytes following engagement of the TCR and therefore, be up-regulated in positive and negative selection [25 , 38 ]. Calponin and plastin bind actin and therefore, might play a role in the actin polymerization events occurring after TCR engagement [58 ]. The MAPKAP2 gene was shown to be differentially regulated in two other studies of thymic selection [29 , 34 ].

Perhaps most interesting are the genes that have not been described previously as being regulated during thymocyte differentiation. Of potential interest are PKC{eta} (Prkch), GTPases Septin7 (cdc10), and the T cell-specific GTPase (Tgtp), as well as a G protein-coupled receptor and Rgs10, a regulator of G protein signaling. Among the transcription factors, Bmi1 is a polycomb-group Zn-finger protein, which has not been described in thymocytes. A component of the general TFIIH is down-regulated. TFIIH interacts with the facilitates chromatin transcription (FACT) complex in ensuring the fidelity of transcription initiation and elongation [59 ]. We previously showed that FACT140 (Supt16h, the mouse homologue of yeast Spt16/Cdc68) is highly up-regulated in prepositive-selection thymocytes and would be expected to be down-regulated by this stimulus [60 ]. Unfortunately, FACT140 was not represented on this gene chip.

If aVß stimulation mimics at least partly positive selection in vivo, as suggested by the pattern of genes up- or down-regulated by aVß stimulation and further confirmed with our RT-PCR, then the genes that we have identified that do not have known roles in thymocyte development may in fact be important in positive selection.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grant R01 GM48002 to N. R. J. G. We are grateful to Kris Hogquist and Steve Jameson (University of Minnesota, Minneapolis) for mice and reagents. This is manuscript Number 16543-IMM from The Scripps Research Institute.

Received October 22, 2004; revised December 15, 2004; accepted December 29, 2004.


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 DISCUSSION
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