Thymocyte stimulation by anti-TCR- , but not by anti-TCR- , leads to induction of developmental...

Thymocyte stimulation by anti-TCR-�, but not by anti-TCR-�,leads to induction of developmental transcription program

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

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

Abstract: Anti-T cell receptor (aTCR) antibody(Ab) stimulation of T cells results in TCR down-modulation and T cell activation. Differences inthe effect of anti-�-chain and �-chain Ab have beenreported on thymocytes. Anti-�-chain Ab but notanti-�-chain reagents cause long-term TCR down-modulation. However, both types of Ab result inTCR cross-linking and activate early steps in signaltransduction. In this study, we show that TCR in-ternalization and calcium flux, hallmarks of T cellactivation, are similar with aV� and aV� treat-ment. Therefore, we have compared the gene ex-pression profiles of preselection thymocytes stimu-lated with these reagents. We find that aV� treat-ment does not cause any significant change in geneexpression compared with control culture condi-tions. In contrast, aV� stimulation results in nu-merous changes in gene expression. The alter-ations of expression of genes known to be ex-pressed in thymocytes are similar to changescaused by positive thymic selection, suggesting thatthe expression of some of the genes without knownroles in thymocyte development and of novel geneswhose expression is found to be altered may also beinvolved in this process. J. Leukoc. Biol. 77:830–841; 2005.

Key Words: microarray � positive selection � signaling � T cellactivation � TCR down-modulation

INTRODUCTION

Stimulation of T cells by different ligands can lead to vastlydifferent responses, exemplified by activation (by agonists) orantagonism of mature T cells and negative or positive selectionof thymocytes [1–3]. T cell activation by antibody (Ab)-medi-ated cross-linking of the T cell receptor (TCR) mimics mostaspects of activation by antigen (Ag), but remarkably, T cellsare activated in different ways by cross-linking via the � or the� chain of the TCR [4, 5]. Ligation of CD3 or TCR-� leads tolong-term internalization and down-modulation of the TCR,similar to agonist activation. Engagement of TCR-� cross-linksthe TCR but does not induce long-term down-modulation of theTCR. The anti (a)V� Ab used in these experiments bind to theTCR and induce cell-surface co-capping equally well as theaV� or C� reagents [5].

Positive selection in thymocytes leads to the functionalallelic exclusion of TCR-�. Cells with two different V� specieson the cell surface are relatively common in preselectionthymocytes but are rare after positive selection [6, 7]. Unlikethe � chain, where allelic exclusion operates by inhibition ofDNA rearrangement of the �-chain locus, the �-chain locusfrequently functionally rearranges the genes on both chromo-somes and expresses functional mRNA and proteins for both �chains [7]. However, mature T cells with strong expression oftwo cell-surface � chains are comparatively rare [6–9]. Thisfunctional allelic exclusion operates by a post-translationalmechanism [4, 9], involving selective retention of the positivelyselected � chain on the cell surface with down-modulation andthus, exclusion of the unselected � chain [4, 5].

In unstimulated T cells, the TCR is continuously endocy-tosed and recycled to the cell surface, resulting in a constant,steady-state level of the TCR on the cell surface. It is possiblethat when the TCR is triggered through the V� chain, it isinternalized and recycled back to the cell surface. Upon acti-vation with aV� Ab, however, the endocytosed TCR is retainedwithin the cell and degraded [10] (N. Niederberger and N. R. J.Gascoigne, in preparation). The maintenance of allelic exclu-sion is believed to be related to the endocytosis and recyclingof the TCR, as loss of c-Cbl reduces the post-translationalallelic exclusion of TCR-� chains [5]. Moreover, the negativeregulatory adaptor molecules c-Cbl and Cbl-b are important inregulating TCR down-modulation, probably as a result of theirubiquitin ligase activity targeting stimulated TCR complexesfor degradation [11].

Differences in TCR down-modulation resulting from engage-ment of either of the TCR chains must come from eventsfollowing TCR engagement. Biochemical analyses of recruit-ment and phosphorylation of molecules involved in TCR sig-naling did not find significant differences between �- or�-chain cross-linking [4]. The exception was with dynamin,involved in formation of endosomes, which was not recruited toaV�-stimulated TCR [4]. Other early events in activation fol-lowing the two types of activation, such as TCR internalizationand calcium release, might be affected. We have now tested

1 Correspondence: Department of Immunology, IMM1, The Scripps Re-search Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. E-mail:[email protected]

Received October 22, 2004; revised December 15, 2004; accepted Decem-ber 29, 2004; doi: 10.1189/jlb.1004608.

830 Journal of Leukocyte Biology Volume 77, May 2005 0741-5400/05/0077-830 © Society for Leukocyte Biology

this possibility and find no difference in the response to aV� oraV� stimulation.

We therefore decided to look for changes happening later inactivation, at the level of gene transcription. The technique ofgene expression profiling using DNA microarray hybridizationis a powerful tool to analyze signaling pathways activated as aresult of receptor ligation. In this study, we compare changes ingene transcription in preselection CD4�8� [“double positive”(DP)] thymocytes induced by Ab cross-linking through theTCR-� or -� chain. It is surprising that stimulation through theTCR-� chain induced no reproducible changes in transcriptionof mRNA among the 12,000 known genes and expressedsequence tags (ESTs) tested. In contrast, we have identified103 genes differentially expressed in response to stimulationwith aV� Ab. These include many genes known or expected tobe regulated during thymocyte-positive selection, as well asmany genes with no known role in thymocyte development.

MATERIALS AND METHODS

Mice

C57BL/6J (B6) mice were bred and maintained at The Scripps ResearchInstitute (TSRI; La Jolla, CA). Ovalbumin-specific TCR transgene (OT)-I andtransporter associated with antigen processing deficient (TAP)�/� animalswere obtained from Drs. Stephen Jameson and Kristin Hogquist (University ofMinnesota, Minneapolis). All experiments were performed in accord with theguidelines of the Animal Care and Use Committee of TSRI.

Activation of thymocytes with Ab

Tissue-culture plates were coated with phosphate-buffered saline (PBS), aloneor containing 10 �g/ml goat anti-mouse immunoglobulin G (IgG; Sigma-Aldrich, St. Louis, MO) or 10 �g/ml goat anti-rat IgG, overnight at 4°C. Plateswere washed 3� with PBS and further coated with PBS alone or containing 2�g/ml aV�5 (clone MR9-4, mouse IgG) or 2 �g/ml aV�2 (clone B20.1, ratIgG) for 3 h at 37°C. Single-cell suspensions from thymi of 4- to 8-week-oldOT-I TAP�/� mice were prepared and resuspended in RPMI-1640 mediumsupplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml pen/strep, and 50 �M �-mercaptoethanol. Thymocytes at1 � 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 otherwisenoted, the Ab used in this study were purchased from BD PharMingen (SanDiego, 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-�. The staining wasdone in the dark for 30 min at 4°C. Cells were washed, suspended in 200 �lPBS containing 1% bovine serum albumin (BSA) and 0.1% sodium azide [flowbuffer (FB)], and analyzed with a FACSCalibur (Becton Dickinson, MountainView, CA). For the microarray experiment, OT-I TAP�/� thymocytes, stimu-lated overnight with Ab, were recovered, stained with aCD4 and aCD8, andsorted for DP thymocytes. Controls were freshly isolated thymocytes (control 1)or overnight-cultured, unstimulated thymocytes (control 2). For the real-timepolymerase chain reaction (PCR) experiment, OT-I TAP�/� thymocytes werestained with aCD4 and aCD8 and sorted for DP thymocytes, whereas B6thymocytes were stained for TCR-� and CD69 and sorted for TCRintCD69�

and TCRhi thymocytes. The TCRintCD69� and TCRhi thymocyte populationswere pooled and represent thymocytes that have received a positive selectionsignal, whereas the OT-I TAP�/� DP thymocytes correspond to a population ofthymocytes that have not been selected. All stainings were done in the dark for1 h at 4°C. Cells were washed and suspended at 1 � 107/ml in filtered PBSbuffer containing 1 mM EDTA, 25 mM HEPES, and 1% FCS. Thymocyteswere 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�/�micewere prepared. Thymocytes (3�106 cells/staining) were resuspended in 100 �lPBS containing 1% BSA and 0.1% sodium azide (FB). To induce internaliza-tion, cells were incubated with biotinylated aV�2 or aV�5 for 30 min at 4°C,washed, resuspended in FB, and stained with streptavidin-Alexa Fluor 680conjugate (Molecular Probes, Junction City, OR) for 10 min on ice. Thymocyteswere finally incubated at 37°C for 10 min and fixed with 4% paraformaldehydefor 12 min at room temperature. TCR internalization was observed using aZeiss Axiovert 200 M inverted microscope. SlideBook software (IntelligentImaging Innovation, Denver, CO) was used for the capture as well as fordeconvolution.

Calcium flux

OT-I TAP�/� thymocytes were washed and resuspended at 1 � 106/ml inindo-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 concentrationof 7 �M. Thymocytes were incubated for 30 min at 37°C, washed withRPMI-1640 medium, and supplemented with 10% heat-inactivated FCS, 2 mML-glutamine, 100 U/ml pen/strep, and 50 �M �-mercaptoethanol. Cells wereincubated in RPMI media for an additional hour at 37°C, washed, andresuspended at 2 � 106/500 �l RPMI. Thymocytes were kept on ice in thedark until flow cytometry analysis. Before analysis, cells were incubated at37°C for 5 min. The change in fluorescence intensity of Indo-1 at 405/20 and530/30 for bound and free probe, respectively, following ultraviolet excitation,was measured with a FACS Digital LSRII instrument (BD Biosciences, Moun-tain View, CA) using FlowJo software (Tree Star, Inc., Ashland, OR). After 30 sof analysis, primary Ab, aV�2 (10 �g/ml) or aV�5 (10 �g/ml), were added, and1 min later, streptavidin (20 �g/ml) was supplemented. As negative controls,primary Ab aV�2 or aV�5 (10 �g/ml) or streptavidin (20 �g/ml) only wereadded to the cells. Ionomycin at 2 �g/ml was used as a positive control.

RNA preparation and microarray procedures

Total RNA from sorted OT-I TAP�/� DP thymocytes was isolated using theRNeasy mini-kit method (Qiagen, Valencia, CA). The RNA was then processedaccording to Affymetrix protocols. Briefly, the total RNA (5.0–20 �g) was usedas template to generate double-stranded cDNA using the GeneChip T7-Oligo(dT) promoter primer kit (Affymetrix, Santa Clara, CA) for primingfirst-strand cDNA and the Superscript II kit (Invitrogen Life Technologies,Carlsbad, CA). Biotin-labeled cRNA was synthesized in an in vitro transcrip-tion reaction by using the ENZO BioArray™ High Yield™ RNA transcript-labeling kit (Affymetrix). The cRNA was cleaned and fragmented. Finally, thefragmented, biotinylated cRNAs were hybridized to Affymetrix MG-U74Av2arrays for 16 h. Arrays were washed and stained with streptavidin-PE andfinally read using the GeneArray scanner. The gene expression values werequantified and analyzed using the Affymetrix Microarray Suite (MAS) software,Version 5.0. A global scaling method, where the average intensity (signalvalue) of every array is scaled to a common value of 250 (target intensity), wasapplied, making possible the comparison between arrays.

Microarray data analysis

Dendrograms to compare the relationship between arrays were done using theGeneCluster and TreeView programs [13]. This method makes it possible todistinguish whether biological replicates of each experiment are more similarto each other than to any other conditions. It compares the signal strength (aquantitative measure of the relative abundance of a transcript) of 7151 genesout of 12,488 sequences, represented on the MG-U74Av2 array after genesfound absent in all conditions are removed from the data. Data signalsrepresent average difference signals from Affymetrix data files. Hierarchicalclustering of arrays, but not genes, was performed using an “average linkageclustering” mode. Scatter plots were produced to compare pair-wise conditions:aV�2 versus overnight control, aV�5 versus overnight control, and aV�5versus aV�2. The scatter plot compares global data on hybridized chips andincludes 7151 genes, after genes found absent in all conditions were removedfrom the data. The log2 signals of the average difference call values for each

Niederberger et al. Transcription program initiated by aV� but not aV� 831

of the genes were used, such that, for example, a 2� increase or decrease inexpression is 1 in the log2 scale, and a 4� increase or decrease in expressionis 2. In the scatter plots, genes whose expression is increased show a higherlog2 value in the y-condition compared with the x-condition and are foundabove the diagonal, whereas genes whose expression is decreased show a lowerlog2 value in the y-condition compared with the x-condition and are foundbelow the diagonal. A log2 representation allows a better signal discriminationfor these data than a linear scale. A mean-difference plot was produced tocompare aV�5 versus overnight control. It compares the log2 ratio versusaverage log intensity (“MA plot”) for the 7151 genes left after absent geneswere eliminated. The log2 ratio is the ratio of an experimental signal fromaV�5-treated thymocytes over a control signal, as computed by Affymetrix filecomparison software (MAS 5.0). The log2 intensity value is calculated as (logaV� signal�log control signal)/2. The log2 intensity scale spreads the genesalong the horizontal, allowing for easier viewing of the genes. In the MA scatterplot, genes whose expression is increased in aV�5-stimulated thymocytescompared with the overnight control are located above the horizontal, corre-sponding to a log2 ratio value of zero, and genes whose expression is decreasedin aV�5-stimulated thymocytes compared with the overnight control are lo-cated below the same horizontal line. The hierarchical clustering of selectedgenes was done using GeneCluster [13]. Array data from two independenthybridizations were combined (biological replicates). To select genes withsignificant variation of expression across the samples, several criteria wereapplied. Genes considered absent in all conditions were excluded. Among thegenes kept, those with a no-change (NC; P�0.05) call across the samples wereremoved. Of the 2689 remaining genes, only those with a “change P value”0.01 were selected. The Affymetrix statistical algorithms implemented in theanalysis software (MAS 5.0) determed the change P values, based on Wilcox-on’s signed rank test. These define the confidence of the call (increase,decrease, or NC compared with the control). The 547 genes that conformed tothese selection criteria were hierarchically clustered. Average difference sig-nals were adjusted using log transformation and median centering (medianvalue0) prior to gene clustering (average linkage clustering mode). Clusterswere visualized using TreeView [13].

Quantitative real-time PCR

A control RNA was required to construct standard curves. Total RNA fromOT-I thymi between 4 and 8 weeks old was isolated using the RNeasy mini-kitmethod (Qiagen). The RNA was treated with DNase I for 15 min at roomtemperature (DNase I amplification grade, Invitrogen Life Technologies). Thereaction was stopped by the addition of 25 mM EDTA, and the DNase wasinactivated at 65°C for 10 min. To confirm the results of the gene-chip analysis,the RNA from aV�- or V�-treated and untreated, overnight control thymocyteswas the same samples as used for hybridization to Affymetrix MG-U74Av2arrays. To measure protein kinase C (PKC)�, PKC�, nuclear factor of activatedT cells (NFAT)c, CD5, Egr-1, Bcl-XL, CD4, and Ets-2 transcript levels andcompare them between OT-I TAP�/� DP thymocytes, which did not receive aselection signal, and B6 thymocytes, which have undergone positive selection(TCRintCD69� plus TCRhi populations), RNA from the different populations ofthymocytes was isolated as described above for the standard RNA. All theRNA samples were used as template to synthesize cDNA using the Super-script™ first-strand synthesis system for reverse transcriptase (RT)-PCR (In-vitrogen Life Technologies), following the manufacturer’s recommendations.Oligonucleotide primers were designed using Primer Express 1.5 software (PEApplied Biosystems, Foster City, CA). Primer sequences are presented inSupplementary Table S1. Quantitative real-time PCR reactions were run onthe ABI Prism 7700� sequence detection system using SYBR Green PCRmaster mix from PE Applied Biosystems. The PCR mix was optimized forSYBR Green reactions and contained SYBR Green 1 dye, AmpliTaqGold DNApolymerase, and deoxy-unspecified nucleoside 5 -triphosphates with deoxy-uridine 5 -triphosphate. Each reaction was performed in a 25-�l final volumeand contained the SYBR Green PCR master mix, cDNA, and 150 nM eachprimer. For each gene, two independent experiments were done, and for bothexperiments, all the reactions were performed in duplicate. A standard curveusing a twofold serial dilution of the standard cDNA was generated for thetarget genes as well as for the housekeeping gene �-actin. cDNAs (20 ng/reaction) from unstimulated, overnight control, aV�- or aV�-stimulated sam-ples, OT-I TAP�/� DP thymocytes, and B6 thymocytes (TCRintCD69� plusTCRhi pooled populations) were used as template for real-time PCR. Thestandard curves were used to determine the relative concentrations of amplified

transcripts. The transcript levels of each sample were first normalized using thevalues of �-actin. The normalized sample values were finally divided by thenormalized control (unstimulated, overnight control) values to obtain the foldchange of mRNA expression between unstimulated, overnight control andaV�- or aV�-stimulated samples or by the OT-I TAP�/� DP values to obtainthe fold change of mRNA expression between OT-I TAP�/� DP thymocytesand B6 (TCRintCD69� � TCRhi) thymocytes.

RESULTS

Early events in activation following triggering ofTCR with Ab against the � or � chain are similar

Previous studies showed that Ab-mediated cross-linking ofTCR by Ab recognizing the � chain leads to down-modulationof the TCR, and ligation of the � chain does not, suggestingthat the two distinct chains of the TCR can induce differentsignals following their engagement with Ab [4, 5] (Fig. 1A).Early events in activation tested previously were similar withthe two types of stimulation, including the recruitment ofsignaling molecules and the patterns of phosphorylation [4].Different early events in activation might be affected, andtherefore, we performed two additional experiments. First, wetested whether aV�2 and aV�5 Ab are equally effective atinducing internalization of the TCR. Therefore, OT-I TAP�/�

thymocytes were incubated with biotinylated aV�2 or aV�5Ab. Alexa Fluor 680-labeled streptavidin was added, and thethymocytes were incubated at 37°C for 10 min. The reactionwas stopped with 4% paraformaldehyde, and internalizationwas visualized by microscopy. Both Ab were able to induceinternalization of the TCR (Fig. 1B). Second, we investigatedintracellular calcium mobilization in response to OT-I TAP�/�

TCR cross-linking. Thymocytes were preloaded with the cal-cium dye Indo-1, and flow cytometry was used to monitorintracellular calcium concentration following activation of theTCR with biotinylated aV�2 or aV�5 Ab, followed by cross-linking with streptavidin. As seen in Figure 1C, the kineticsand amplitudes of the calcium response were similar with thetwo types of stimulation. Both Ab were effective at inducing aninitial transient peak of intracellular calcium concentrationwithin a few minutes of TCR cross-linking. The peak graduallydecreased to reach the baseline level.

Comparison of gene expression changes inCD4�CD8� thymocytes following engagement ofTCR with aV� or aV� Ab: aV� does not inducechanges in gene expression

To identify genes differentially expressed following engage-ment of either of the TCR chains, a DNA microarray approachwas taken. Thymocytes from the OT-I TCR transgenic mouse(V�2V�5) [14] bred onto the TAP-1�/� background wereisolated. These thymocytes are arrested at the CD4�CD8� DPstage of development as a result of lack of major histocompat-ibility complex (MHC) class I-peptide complexes to stimulatepositive or negative selection [15]. Isolated thymocytes werecultured overnight at 37°C on plates coated with aV�2 or aV�5Ab or on noncoated plates. Freshly isolated thymocytes wereused as an additional control. DP thymocytes were identifiedusing aCD4 and aCD8 Ab, purified by single-cell sorting, and

832 Journal of Leukocyte Biology Volume 77, May 2005 http://www.jleukbio.org

used to isolate total RNA. Biotin-labeled cRNA was preparedand hybridized to DNA arrays representative of 12,000 murinegenes and ESTs (MG-U74Av2 arrays, Affymetrix).

To determine whether expression profiling could identifydifferences between aV�- and aV�-induced TCR signaling,two datasets with four samples each were analyzed usingCluster and Treeview programs [13]. The samples were fromaV�- or aV�-treated thymocytes, nonstimulated cells incu-bated at 37°C overnight (control 2), or freshly isolated DPthymocytes (control 1). Gene Expression Omnibus database(www.ncbi.nlm.nih.gov/geo/) accession numbers are as follows:First dataset: overnight control, GSM 24449; aV�2, GSM24450; aV�5, GSM 24451; freshly isolated thymocytes, GSM24455. Second dataset: overnight control, GSM 24452; aV�2,

GSM 24453; aV�5, GSM 24454; freshly isolated thymocytes,GSM 24456. The mRNA expression data showed a distinctpattern. The biological replicates of aV�5 incubation weremore similar to each other than to any other condition, whereasthe aV�2 results clustered with their respective overnightcontrol treatments rather than with each other (Fig. 2A). Thisindicates first, that aV�5 incubation induced distinct changesin the overall gene expression patterns as compared withcontrol and aV�2 treatment and second, that aV�2 incubationis indistinguishable from overnight incubation without Ab. It issurprising that variations in gene expression between biologi-cal repeats of aV�-treated thymocytes were bigger than thosebetween the aV�2 treatments and their unstimulated, over-night controls. This analysis was an indication that no signif-

Fig. 1. Changes induced by incubation of OT-I TAP�/� thymocytes overnight or by treatment with aTCR V� or V�. (A) TCR down-modulation induced by aV�(light, solid line) but not by aV� (dotted line) treatment overnight. The aV� treatment shows no difference from unstimulated cells cultured for the same period(bold, solid line). (B) aV�2 (left panel) and aV�5 (right panel) Ab are equally effective at inducing TCR internalization. OT-I TAP�/� thymocytes were incubatedwith biotinylated aV�2 or aV�5 Ab followed by TCR cross-linking with streptavidin Alexa Fluor 680 conjugate for 10 min at 37°C. Cells were fixed and analyzedby fluorescence microscopy. One representative experiment out of at least three is shown. (C) The calcium response induced following TCR engagement throughV� or V� chain is similar. OT-I TAP�/� thymocytes were loaded with the dye Indo-1 and then stimulated by the addition of 10 �g/ml biotinylated aV�2 or aV�5Ab at 30 s and 20 �g/ml streptavidin at 90 s. Ionomycin (2 �g/ml) was added at 180 s and serves as a positive control. Biotinylated aV�2 (10 �g/ml) Ab or aV�5Ab 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 overa period of time with a 4-min minimum. The kinetics data were analyzed with FlowJo software.


icant variation in gene expression was induced by engagementof the TCR with aV� Ab. This does not mean that the aV�biological replicates are dissimilar but rather that there aresome differences between the individual mice used in theseparate experiments and/or day-to-day variation between theexperiments, leading to clustering of the control and aV�treatments for each of the separate experiments. Therefore,aV� and overnight control are essentially two identical nega-tive controls of the same animal: Only aV� treatment producesspecific changes that override the baseline differences betweenthe individual mice used. The freshly isolated thymocytes weresomewhat different from the overnight-cultured controls, re-flecting changes as a result of culture conditions, which areknown to affect the thymocytes [16]. The scatter plots pre-sented in Figure 2B further confirm this result. Genes whoseexpression is increased show a higher log2 value in the y-condition compared with the x-condition and are found abovethe diagonal, whereas genes whose expression is decreasedshow a lower log2 value in the y-condition compared with thex-condition and are found below the diagonal. In the left panel,only a few minor changes were observed following activation ofTCR with aV� Ab compared with the unstimulated, overnightcontrol. We saw that most of the genes with an intermediate orhigh expression level were localized on or very close to thediagonal. Two genes changed between control and aV� treat-ments. One of these is a pseudogene (nucleotide sequencedatabase ID L04848), which was increased ninefold in oneexperiment and decreased twofold in the other. The other geneis triosephosphate isomerase, which was increased 2.8-fold inone experiment and decreased 2.6-fold in the other. As thesewere not reproducible between experiments, they were consid-ered as being not significant and are not further described. Weobserved many changes upon aV� activation compared withovernight control (Fig. 2B, middle panel) and aV� conditions(Fig. 2B, right panel).

Stimulation by aV� induces a transcriptionalprogram in DP thymocytes

Stimulation of immature DP thymocytes through the TCR caninduce differentiation (positive selection) or apoptosis (nega-tive selection) at least partly in relation to the ligand-bindingstrength [1, 14, 17, 18]. Earlier studies had shown that stim-ulation of thymocytes with Ab to TCR-� induces gene expres-sion patterns expected for cells undergoing positive selectionin vivo [19, 20]. DNA array technology allows a more system-atic and broader approach to identify changes in the steady-

state levels of mRNA species that are affected by stimulationthrough the TCR. The OT-I TAP-1�/� TCR transgenic thymo-cytes provide a relatively homogeneous source of DP thymo-cytes, which are blocked at the stage of positive selection butcan be activated to progress by TCR stimulation [15]. Ageneral view of transcripts regulated by engagement of TCR-�is presented in Figure 3. In this scatter plot, genes whoseexpression is increased in aV�5 compared with the overnightcontrol are located above the horizontal line corresponding to alog2 ratio value of zero, and those genes whose expression isdecreased are located below this line. Among the transcriptsregulated by engagement of TCR-�, several have been de-scribed previously as being regulated after TCR engagement ofDP thymocytes. Bcl2, CD5, and Egr1 transcripts were in-creased by aV� stimulation, whereas RAG1, CD8, and CD4transcripts were decreased, as expected from previous work[19–21]. This reinforces the idea that aV� stimulation of DPthymocytes mimics aspects of thymocyte maturation.

The expression-profiling technique provides us with theability to identify patterns of expression shared between genesusing statistical techniques. To select genes showing signifi-cant variation across the samples out of the 12,000 genes andESTs tested using the U74Av2 array, several criteria wereapplied. First, genes that were absent in all conditions wereexcluded. Second, those that showed NC (P�0.05) across allsamples 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 andwere hierarchically clustered. To discard false-positive signals,

Fig. 2. (A) A dendrogram comparingthe relationship between arrays [13].(B) The transcription data for the threedifferent treatments, showing little dif-ference among the unstimulated, cul-tured control and aV�-stimulated cellsbut many differences between aV�compared with aV� or overnight, un-stimulated cells.

log2

rat

io

log2 intensity

Bcl-2 Egr-1

CD5

CD8

Rag1CD4

Fig. 3. General view of transcripts regulated in aV�-stimulated DPCD4�CD8� thymocytes. This scatter-plot analysis compares the log ratioversus average log intensity (MA plot) for 7151 genes. The arrows point to a fewwell-known genes regulated during thymocyte maturation and showing differ-ential expression upon V� treatment.


array data from two independent hybridizations were com-bined. The pattern of gene induction is shown in Figure 4. Thegreen color represents genes that were repressed, and the redcolor identifies genes that were induced in one of the comparedconditions. The brighter the color, the bigger the change inexpression. White indicates NC in gene expression. The clus-tering results in two main subclusters. Cluster 1 shows genesthat were repressed in aV�5-stimulated thymocytes comparedwith the overnight, unstimulated thymocyte control (green), andCluster 2 identifies genes that were induced upon aV� stimu-lation (red). Only those genes showing at least a twofold changein aV� versus the overnight-cultured control were selected. Intotal, 103 genes were differentially regulated as a result of aV�stimulation. Out of these genes, 63 were up-regulated, and 40were down-regulated. They are presented in Table 1. Tovalidate our array data, the expression of eight genes in non-

stimulated or aV�- or aV�-stimulated OT-I TAP�/� thymo-cytes was estimated using quantitative real-time PCR, whichconfirmed the results of the gene-chip analysis (Fig. 5A,described in detail below).

Changes in the expression pattern of genes byaV� stimulation

Among the 103 genes differentially regulated upon aV� stim-ulation, a large diversity of functional gene classes is repre-sented (Table 1), including cell-surface proteins and receptors,signaling molecules, mediators of TCR gene rearrangement,transcription factors, regulators of apoptosis, as well as anumber of ESTs representing unknown genes. The up-regula-tion or down-regulation of various cell-surface proteins orreceptors is induced by TCR stimulation, and therefore, it wasnot surprising that we found several cell-surface proteinsshown previously to be stimulated by positive selection in theup-regulated group, including CD5, CD2, CD6, CD53, andItm2a (Table 1, Fig. 5A) [19, 20, 22–31]. CD69 was notrepresented on the U74Av2 array and so could not be checkedagainst its known up-regulation by positive selection. CD4,CD8, Ly6d, and TL (thymus leukemia antigen, a nonclassicalMHC class I protein) expression were decreased, which alsocorrelates with the delivery of a positive selection signal [15,19, 20, 27–32]. To avoid repetition, the main description of thegenes found to be regulated by aV� treatment is presented inDiscussion.

Changes in the expression pattern of genesinduced by aV� are similar to changes occurringduring thymocyte selection in vivo

As many genes regulated upon aV� stimulation have alreadybeen shown to be regulated during thymocyte development, wedecided to use quantitative RT-PCR to test whether the ex-pression of the eight genes we used to validate our array datawould behave in a similar way during thymocyte developmentin vivo. Therefore, the transcript levels of PKC�, PKC�,NFATc, CD5, Egr-1, Bcl-XL, CD4, and Ets-2 genes from OT-ITAP�/� DP thymocytes, which are arrested at the DP stage ofdevelopment, and from B6 TCRintCD69� plus TCRhi thymo-cytes, which have undergone positive selection, were com-pared. As presented in Figure 5B, all the genes tested showeda similar pattern of expression following stimulation of the TCRwith aV� or when undergoing normal thymocyte selection invivo. The only exception was CD4, and its expression wasslightly induced instead of reduced in the B6 TCRintCD69�

plus TCRhi thymocytes; this difference is explained by the factthat part of this B6 thymocyte population contains some single-positive thymocytes that have already up-regulated the core-ceptor CD4.

DISCUSSION

Early events in activation following cross-linkingof the TCR with Ab against the � or � chain areidentical

Cross-linking of TCR with Ab typically results in TCR down-modulation. We and others have previously shown that cross-

Fig. 4. View of the hierarchical clustering of selected genes performed usingGeneCluster and TreeView programs [13]. Data from two biological replicatesof aV� and overnight, unstimulated thymocytes were combined and compared.Among the 12,000 genes and ESTs present on the array, only 547 genes hada P value 0.01 and were selected. These were hierarchically clustered usingaverage difference signals between aV� and overnight, nonstimulated samples.The clustering results in two subclusters: Cluster 1 shows aV� down-regulatedgenes (green color) and Cluster 2, aV� up-regulated genes (red color). If athreshold change of 2� is applied to this list, only 103 genes remain.


TABLE 1. Genes and ESTs Showing Differential Expression after Stimulation of OT-I TAP–/– Thymocytes with aV�

Functional gene classesAccession

# Gene nameFold changesin expression Description

Signal transductionUp-regulated genes L14543 Sh3bp2 (3BP2) 5.65 Src homology (SH)3 domain-binding protein 2

D90242 Prkch (PKC-�) 4.6 PK-C�U09268 Dusp2 (PAC1) 2.93 Dual-specificity phosphatase 2. Dephosphorylates

mitogen-activated protein (MAP) kinasesAI847399 Rgs10 2.64 Regulator of G protein signaling 10AW120722 Mapkapk2 (MK2) 2.46 MAP kinase-activated protein kinase 2

Down-regulated genes AV374868 Socs3 3.73 Suppressor of cytokine signaling 3Guanosine 5�-triphosphate (GTP)-

binding proteinsUp-regulated genes L38444 Tgtp 9.18 T cell-specific GTPase

AW047476 Gbp3 3.86 GTP-binding protein 3AJ007970 Gbp2 3.36 GTP-binding protein 2AJ223782 Sept7 (Cdc10) 2.46 Septin 7 (GTP-binding protein or GTPase)

Cell-surface protein and receptorsUp-regulated genes X67914 Pdcd1 (PD-1) 39.39 Programmed death-1. Member of CD28 family

L38971 Itm2a (E25) 10.19 Integral membrane protein 2A (up-regulatedduring pos. sel.)

AB023132 Icos 5.46 Inducible T cell costimulatorX00246 H2-D1 5.27 Histocompatibility 2, D region locus 1X06143 CD2 4.75 CD2 antigenM15177 CD5 4.6 CD5 antigen (up-regulated in pos. and neg. sel.)X97227 CD53 4.28 CD53 antigen (up-regulated in pos. and neg. sel./

tetraspanin)AF029215 Mox2 (CD200/

OX2)3.25 Costimulatory molecule. Ig superfamily

U39827 Gpr65 (Gpcr25/TDAG8)

3.25 G protein-coupled receptor 65

U12434 CD6 3 CD6 antigenAI839286 Lrp10 2.38 Low-density lipoprotein receptor-related protein

10Down-regulated genes D86082 H2-T18 (TL) 4.14 Nonclassical MHC class I molecule: thymus

leukemia antigenAB000713 Cldn4 (CPE-R) 4 Claudin 4M17080 CD4 3.14 CD4 antigenX63782 Ly6d (Thb) 2.93 Lymphocyte antigen 6 complex, locus D (down-

reg. by pos. sel.)AF010452 Mr1 2.83 Nonclassical MHC class I-related geneAV316162 CD8b 2.83 CD8 antigen, � chainU34881 CD8a 2.46 CD8 antigen, � chain

Gene expressionUp-regulated genes AF087434 Nfatc1 (NFAT2/

NFATc)8.87 NFAT, cytoplasmic, calcineurin dep.

M24377 Egr2 (Krox-20) 6 Early growth response 2X16995 Nr4a1 (Nur77) 5.65 Nuclear receptor subfamily 4, group A, member

1U47543 Nab2 4.92 Ngfi-A-binding protein 2. Corepressor of Egr-1,2

activityZ54283 Pou2af1 (BOB.1/

OBF.1)4.75 POU domain, class 2, associating factor 1. Binds

October,2 promoter bind. prot.U20735 JunB 4.28 Jun-B oncogene, part of activated protein (AP)-1

transcription complexM28845 Egr1 (Krox-24/

NGFI-A)4.14 Early growth response 1

M21065 Irf1 3.86 Interferon (IFN) regulatory factor 1D16503 Lef-1s 3.36 Lymphoid enhancer-binding factor 1 (alternative

splice form)V00727 Fos (c-fos) 3.25 Fos, part of AP-1 transcription complexM64068 Bmi1 2.83 Zn-finger protein. PcG (polycomb-group) geneAF006492 Zfpm1 (friend of

GATA1/FOG)2.64 Zinc finger protein, multitype 1. Synergistic with

GATA-1


TABLE 1. (Continued)

Functional gene classes Accession # Gene nameFold changesin expression Description

Down-regulated genes AF038029 Cbfa2t3h (ETO2) 7.21 Core-binding factor, runt domain, � subunit 2,translocated to 3 homolog (human)

U09504 Nr1d2 (Rev-erb) 4.6 Nuclear receptor subfamily 1, group D, member2

J04103 Ets2 4.6 Transcription factor Ets2AW047343 Dbp 4.44 D-site albumin promoter-binding proteinAI850881 Gtf2h4

(p52/TFIIH)4.28 General transcription factor II H (TFIIH),

polypeptide 4AI842472 Zdhhc14 3.36 Zinc finger, Asp-His-His-Cys (DHHC) domain

containing 14AW123880 Xbp1 (TREB5) 2.38 X-box-binding protein 1

DNA-bindingproteins/recombination/replication/repair

Up-regulated genes D90374 Apex1 2.83 Apurinic/apyrimidinic endonuclease 1Down-regulated genes M64796 Rag2 16.56 Recombination-activating gene 2

M29475 Rag1 5.64 Recombination-activating gene 1X04123,

AV312871TdT 4.75 Terminal deoxynucleotidyltransferase

X05862 Hist1h2ai 3.6 Histone 1, H2aiAJ003133 Orc1l 3.48 Origin recognition complex, subunit 1-like

(Saccharomyces cerevisiae)L22977 Xlr3b 3.48 X-linked lymphocyte-regulated 3bM38700 G22p1 (Ku70) 2.93 DNA repair protein induced in TCR/Ig gene

rearrangementApoptosisUp-regulated genes U23778 Bcl2a1b 4.75 B-cell leukemia/lymphoma 2-related protein A1b

L31532 Bcl2 3.86 B-cell leukemia/lymphoma 2, antiapoptotic geneU23781 Bcl2a1d 2.83 B cell leukemia/lymphoma 2-related protein A1d

Down-regulated genes L35049 Bcl21 (Bcl-XL) 2.55 Bcl2-like antiapoptotic. Down-regulated by pos.and neg. sel. in DP

Calcium and cell structureUp-regulated genes AW124113 Basp1 5.09 Calmodulin-binding protein

AA711704 Aim1 4.44 Nonlens ��-crystallin protein. Binds Ca2�

AI835858 Tpm4 4.14 Tropomyosin 4. Binds actin-filamentsZ19543 Cnn2 3.73 Calponin 2. Binds actin, tropomyosin, and

calmodulinD37837 Lcp1 (Plastin 2,

L)3.6 Binds Ca2�, calmodulin, actin. Stabilizes actin

filamentsAI852553 Tmsb10 3.6 Thymosin, � 10. Induces F-actin

depolymerizationDown-regulated genes M72414 Mtap4 (MAP4) 2.73 Microtubule-associated protein 4

X15373,M21530

Itpr1 (IP3R) 2.64 Inositol 1,4,5-triphosphate receptor 1.Intracellular Ca2�-release channel

X65979 Mylc2pl (PLRLC) 2.38 Myosin light chain 2, precursor lymphocyte-specific

Miscellaneous and cell metabolismUp-regulated genes U89491 Ephx1 18.38 Epoxide hydrolase 1, microsomal

AF022992 Per1 5.27 Period homolog 1 (Drosophila)X75129 Xdh 4.14 Xanthine dehydrogenaseM59821 Ier2 (Pip92) 3.36 Immediate early response 2. Growth factor-

inducible proteinAF064635 Hsd17b12 3.36 Hydroxysteroid (17-�) dehydrogenase 12AW122030 Psat1 2.93 Phosphoserine aminotransferase 1AW123026 Gnpnat1 2.83 Glucosamine-phosphate N-acetyltransferase 1U18975 Galgt1

(GalNAcT)2.64 Uridine 5 -diphosphate (UDP)-N-acetyl-�-D-

galactosamine. Adds core 1 O-linked glycan toprotein

AI842065 Kctd12 2.55 Potassium channel, tetramerization domain-containing 12

L40406 Hsp105 2.46 Heat shock protein 105. Chaperone proteinAI835257 Ndfip1 2.46 Nedd4 family-interacting protein 1. Nedd4 is an

E3 ubiquitin ligaseAJ002387 Hspa5 (Bip) 2.3 Heat shock 70 kD protein 5 or Ig-H chain-

binding protein. Chaperone prot.AI853226 Etnk1 2.3 Ethanolamine kinase 1


linking with aTCR V� reagents gives a different outcome, withno long-term down-modulation, despite a similar ability of theAb to bind, cross-link, and induce clustering of TCR, asanti-�-chain reagents [4, 5]. aV� and aV� stimulation inducessimilar patterns of tyrosine-phosphorylated proteins and ofrecruitment of proteins to the signalsome, the exception beingdynamin, which is not recruited with aV� [4]. As TCR trig-gering induces a lot of different signals within the cell, we werecurious to see the outcome of earlier events in activationfollowing the two types of stimulation. From our results, it isclear that aV�2 and aV�5 Ab were able to induce internal-ization of the TCR as well as a calcium flux response, clearlysuggesting that all early events in activation following cross-linking of the TCR with Ab against the � or � chain areidentical. Even so, aV� and aV� Ab have a different long-termeffect on TCR expression.

aTCR V� stimulation does not induce changes ingene expression

This similarity led us to compare gene expression programsinduced in thymocytes by cross-linking of TCR through the �chain or the � chain using DNA arrays. We were amazed tofind that there were no significant and reproducible changes ingene expression induced by aV�-mediated cross-linking ofTCR. In contrast, many changes were induced by aV�.

This means that despite the similarities of early eventsafter TCR cross-linking by the Ab, the �-chain (or CD3)cross-linking activates a program of gene expression,whereas �-chain cross-linking is unnoticed by the cell inthe long-term. The fact that �-chain-cross-linked TCR re-appears on the T cell surface, whereas �-chain-stimulatedTCR does not suggests that the former remains in the normalpool of TCR, which undergoes constitutive endocytosis andrecycling [10], whereas the latter is diverted to lysosomes.We have recent evidence indicating that this differentialsorting is indeed the case (N. Niederberger and N. R. J.Gascoigne, in preparation), but it remains unclear how suchapparently similar stimuli could give such different out-comes. The answer likely lies in how the Ab cross-link theTCR proteins in relation to the natural cross-linking of TCRby Ag recognition and to the architecture of the TCR com-plex and its coreceptors. Unfortunately, this is largely therealm of speculation, as so little is known of these details.The orientation of the TCR in its binding to MHC peptideand the interaction sites of CD4 and CD8 with MHC indicatethat the coreceptor is more closely associated with the�-chain side of the TCR than the �-chain side [3, 33].Therefore, the different response to aV� reagents could bea result of the Ab separating the TCR from the coreceptorand therefore, from its attached kinase (Lck).

TABLE 1. (Continued)

Functional gene classesAccession

# Gene nameFold changesin expression Description

Down-regulated genes D12713 Sec23a 9.85 SEC23A (S. cerevisiae). Ortholog. May beinvolved in endoplasmic reticulum (ER)vesicle budding

AA763874 Arl6ip2 3.73 Adenosine 5 -diphosphate (ADP) ribosylation-like factor 6-interacting protein 2

M95545 Pp11r (Tcl-30) 3.6 Placental protein 11-relatedAF033565 Clk3 (cdc2/

CD28-likeprot. kinase 3)

3.14 Cell division control (CDC)-like kinase 3.Implicated in pre-mRNA splicing

M25944 Car2 3.14 Carbonic anhydrase 2AF052453 Papss2 3 3 -Phosphoadenosine 5 -phosphosulfate synthase

2AI606257 Cdca7 2.73 Cell division cycle-associated 7AA822174 Dhrs8

(Hsd17b11)2.64 Dehydrogenase/reductase [short-chain

dehydrogenase/reductase (SDR) family]member 8

AW120868 Tiparp 2.38 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP-ribose) polymerase

UnknownUp-regulated AW124401 2210402C18 9.51 RIKEN cDNA

AW121801 5033405D03 4 RIKEN cDNAAW122893 1810015C04 3.73 RIKEN cDNAAW046694 4631408O11 3.25 RIKEN cDNAAW047643 EST 2.93AW125453 EST 2.83AI851387 5830436L09 2.22 RIKEN cDNAAW214164 3110002K08 2.14 RIKEN cDNA

Down-regulated genes AV367683 EST 9.19 Weakly similar to tuftelin 1AW061306 9130427A09 4 RIKEN cDNAAI851014 BC028975 3.6AW120676 1110007A06 2.55 RIKEN cDNAAI839232 EST 2.46


Genes regulated by aV� stimulation

For the most part, the pattern of genes up- or down-regulatedby aV� stimulation corresponds to those that are also regulatedby natural positive selection stimuli, as shown in other studiesusing microarrays [28–31, 34]. Our results suggest the possi-bility that positive selection signals may be mediated throughthe TCR-� chain. The two coreceptors CD4 and CD8 arerepressed upon aV� stimulation, consistent with DNA microar-ray data showing down-regulation of these genes in response toTCR activation and positive selection in vivo [29, 31]. Thesame is true for genes encoding proteins involved in TCRvariable(diversity)joining-region rearrangement such as recom-bination-activating gene 1 (RAG1), RAG2, Ku70, and terminaldeoxynucleotidyl transferase [29, 31, 35, 36]. In addition, ahistone and Orc1l, involved in DNA replication, were down-regulated, and an endocuclease (Apex1 or Ape1) involved innucleotide incision repair of oxidatively damaged DNA wasup-regulated.

Various cell-surface molecules, which apparently fine-tuneselection signaling threshold, such as CD2, CD5, CD6, andCD53, were up-regulated. Whereas CD2, CD5, and CD53 haveearlier been reported to be regulated during thymic selectionby 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 expressionparallels the expression of CD69 and increases when thymo-cytes are positively selected [39]. Other cell-surface receptors,whose expression was affected, included up-regulation of theinducible costimulatory receptors, inducible costimulator andprogram death-1, both members of the CD28/cytotoxic T lym-phocyte-associated antigen 4 family. The G protein-coupledreceptor Gpcr25 was also up-regulated. Although little isknown about Gpcr25, it could be involved in the differentiationof T cells [40].

Expression of proteins involved in cell signaling was alsoaffected. The expression levels of two IFN-induced GTPases,Gbp-2 and Gbp-3 [41], were up-regulated, as was IFN-regula-tory factor (Irf)1, which has been shown to be involved in CD8T cell development [42]. Others, such as T cell-specific GT-Pase (Tgtp) [43], SH3 domain-binding protein [44], the MAPkinase phosphatase Dusp2/pituitary adenylate cyclase-activat-ing polypeptide receptor 1 [45], and MAP kinase-activatedprotein kinase (MAPKAP) 2 [46], have been shown to beinvolved in T cell signaling, but their precise function inthymocyte development is still unknown. PKC� (Prkch) ex-pression was increased, but PKC� expression was unchanged(Table 1, Fig. 5A). PKC� is of great importance in T cellsignaling, where it is strongly recruited to the central supramo-lecular activation cluster of the synapse [47]. The fact thatPKC� is up-regulated during thymocyte maturation suggests apotential involvement of this protein in thymocyte development(N. Niederberger, in preparation) [47, 48].

Signaling pathways culminate in the activation or repressionof genes through the activity of transcription factors. Nineteentranscription factors or proteins regulating transcription wereexpressed differentially (Table 1, Fig. 5A), many of which havepreviously been shown to be involved in thymocyte develop-ment, 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 Ets2positive selection [21, 49]. Ets2, like several other transcrip-tion factors identified in these experiments (Pou2af1, Lef-1 s,Bmi1, and Zfpm1), has not been shown to be involved inthymocyte maturation so far [50].

The regulation of antiapoptotic molecules such as Bcl2 andBcl-XL was also consistent with positive selecting signals, andBcl2 was up-regulated and Bcl-XL down-regulated [29, 31,51–53]. Both molecules are important regulators of pro-grammed cell death, able to protect cells against apoptosis.They might therefore play a role in thymocyte survival duringthe thymic selection process. Parenthetically, PKC� is alsoreported to be antiapoptotic [54].

Several genes showing differential expression have also beenshown to be modulated during negative selection or afteraCD3/CD28 stimulation [28, 29, 31, 34]. It has been shownthat aTCR-� stimulation alone can induce developmentalchanges typical of maturation and survival that happen duringpositive selection, whereas costimulation with CD28 is re-quired to induce negative selection in vitro [20, 55, 56]. Someof our thymocytes might undergo apoptosis, as they were incu-bated overnight for 16 h in culture. This could explain theexpression of Nur77, a gene exclusively regulated during neg-ative selection. A dominant-negative mutant of Nur77 affects

Fig. 5. Quantitative real time RT-PCR. (A) Compares RNA samples fromaV� (open bars) or aV�-treated (solid bars) thymocytes. Results are shown asfold change compared with unstimulated cells cultured overnight. (B) Theaverage fold change in transcript levels of eight genes from B6 thymocytes(pooled TCRintCD69� plus TCRhi), which have undergone positive selectionrelative to the corresponding control OT-I TAP�/� thymocytes that are arrestedat the DP stage of development, Error bars indicate the SE.


negative selection but not positive selection [57]. However,other genes typically regulated during negative selection, suchas Bim and E2F1, were not detected in our assay. Other genesidentified in our screen are also differentially expressed innegative selection. Egr1, Ets2, Egr2, and Nab2 interact witheach other. Nab2 is a negative regulator of Egr1 and Egr2. Allof these are early-response genes and could therefore beneeded in early stages of positive and negative selection [28].Alternatively, fine-tuning in expression of these genes mightcontribute to the final decision. CD53, like CD69, could serveas marker of activated thymocytes following engagement of theTCR and therefore, be up-regulated in positive and negativeselection [25, 38]. Calponin and plastin bind actin and there-fore, might play a role in the actin polymerization eventsoccurring after TCR engagement [58]. The MAPKAP2 genewas shown to be differentially regulated in two other studies ofthymic selection [29, 34].

Perhaps most interesting are the genes that have not beendescribed previously as being regulated during thymocyte dif-ferentiation. Of potential interest are PKC� (Prkch), GTPasesSeptin7 (cdc10), and the T cell-specific GTPase (Tgtp), as wellas a G protein-coupled receptor and Rgs10, a regulator of Gprotein signaling. Among the transcription factors, Bmi1 is apolycomb-group Zn-finger protein, which has not been de-scribed in thymocytes. A component of the general TFIIH isdown-regulated. TFIIH interacts with the facilitates chromatintranscription (FACT) complex in ensuring the fidelity of tran-scription initiation and elongation [59]. We previously showedthat FACT140 (Supt16h, the mouse homologue of yeast Spt16/Cdc68) is highly up-regulated in prepositive-selection thymo-cytes and would be expected to be down-regulated by thisstimulus [60]. Unfortunately, FACT140 was not represented onthis gene chip.

If aV� stimulation mimics at least partly positive selectionin vivo, as suggested by the pattern of genes up- or down-regulated by aV� stimulation and further confirmed with ourRT-PCR, then the genes that we have identified that do nothave known roles in thymocyte development may in fact beimportant in positive selection.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health GrantR01 GM48002 to N. R. J. G. We are grateful to Kris Hogquistand Steve Jameson (University of Minnesota, Minneapolis) formice and reagents. This is manuscript Number 16543-IMMfrom The Scripps Research Institute.

REFERENCES

1. Gascoigne, N. R. J., Zal, T., Alam, S. M. (2001) T-cell receptor bindingkinetics in T-cell development and activation. Exp. Rev. Mol. Med. 12February, 1–17 (http://www-ermm.cbcu.cam.ac.uk/01002502h.htm).

2. Starr, T. K., Jameson, S. C., Hogquist, K. A. (2003) Positive and negativeselection of T cells. Annu. Rev. Immunol. 21, 139–176.

3. Werlen, G., Hausmann, B., Naeher, D., Palmer, E. (2003) Signaling lifeand death in the thymus: timing is everything. Science 299, 1859–1863.

4. Boyd, R., Kozieradzki, I., Chidgey, A., Mittrucker, H-W., Bouchard, D.,Timms, E., Kishihara, K., Ong, C. J., Chui, D., Marth, J. D., Mak, T. W.,

Penninger, J. M. (1998) Receptor-specific allelic exclusion of TCR V�-chains during development. J. Immunol. 161, 1718–1727.

5. Niederberger, N., Holmberg, K., Alam, S. M., Sakati, W., Naramura, M.,Gu, H., Gascoigne, N. R. J. (2003) Allelic exclusion of the TCR �-chainis an active process requiring TCR-mediated signaling and c-Cbl. J. Im-munol. 170, 4557–4563.

6. Alam, S. M., Crispe, I. N., Gascoigne, N. R. J. (1995) Allelic exclusion ofmouse T cell receptor � chains occurs at the time of thymocyte TCRup-regulation. Immunity 3, 449–458.

7. Gascoigne, N. R. J., Alam, S. M. (1999) Allelic exclusion of the TCR�-chain: developmental regulation of a post-translational event. Semin.Immunol. 11, 337–347.

8. Heath, W. R., Carbone, F. R., Bertolino, P., Kelly, J., Cose, S., Miller,J. F. A. P. (1995) Expression of two T cell receptor � chains on the surfaceof normal murine T cells. Eur. J. Immunol. 25, 1617–1623.

9. Alam, S. M., Gascoigne, N. R. J. (1998) Post-translational regulation ofTCR V� allelic exclusion during T cell differentiation. J. Immunol. 160,3883–3890.

10. Liu, H., Rhodes, M., Wiest, D. L., Vignali, D. A. A. (2000) On thedynamics of TCR:CD3 complex cell surface expression and downmodu-lation. Immunity 13, 665–675.

11. Jang, I. K., Gu, H. (2003) Negative regulation of TCR signaling and T-cellactivation by selective protein degradation. Curr. Opin. Immunol. 15,315–320.

12. Taddie, J. A., Hurley, T. R., Hardwick, B. S., Sefton, B. M. (1994)Activation of B- and T-cells by the cytoplasmic domains of the B-cellantigen receptor proteins Ig-� and Ig-�. J. Biol. Chem. 269, 13529–13535.

13. Eisen, M. B., Spellman, P. T., Brown, P. O., Botstein, D. (1998) Clusteranalysis and display of genome-wide expression patterns. Proc. Natl.Acad. Sci. USA 95, 14863–14868.

14. Hogquist, K. A., Jameson, S. C., Heath, W. R., Howard, J. L., Bevan, M. J.,Carbone, F. R. (1994) T cell receptor antagonist peptides induce positiveselection. Cell 76, 17–27.

15. Hogquist, K. A., Tomlinson, A. J., Kieper, W. C., McGargill, M. A., Hart,M. C., Naylor, S., Jameson, S. C. (1997) Identification of a naturallyoccurring ligand for positive selection. Immunity 6, 389–399.

16. Roehm, N., Herron, L., Cambier, J., DiGuisto, D., Kappler, J., Marrack, P.(1984) The major histocompatibility complex-restricted antigen receptoron T cells: distribution on thymus and peripheral T cells. Cell 38,577–584.

17. Alam, S. M., Travers, P. J., Wung, J. L., Nasholds, W., Redpath, S.,Jameson, S. C., Gascoigne, N. R. J. (1996) T cell receptor affinity andthymocyte positive selection. Nature 381, 616–620.

18. Holmberg, K., Mariathasan, S., Ohteki, T., Ohashi, P. S., Gascoigne,N. R. J. (2003) TCR binding kinetics measured with MHC class Itetramers reveal a positive selecting peptide with relatively high affinityfor TCR. J. Immunol. 171, 2427–2434.

19. Kearse, K. P., Takahama, Y., Punt, J. A., Sharrow, S. O., Singer, A. (1995)Early molecular events induced by T cell receptor (TCR) signaling inimmature CD4�CD8� thymocytes: increased synthesis of TCR-� proteinis an early response to TCR signaling that compensates for TCR-�instability, improves TCR assembly, and parallels other indicators ofpositive selection. J. Exp. Med. 181, 193–202.

20. Groves, T., Parsons, M., Miyamoto, N. G., Guidos, C. J. (1997) TCRengagement of CD4�CD8� thymocytes in vitro induces early aspects ofpositive selection, but not apoptosis. J. Immunol. 158, 65–75.

21. Shao, H., Kono, D. H., Chen, L. Y., Rubin, E. M., Kaye, J. (1997)Induction of the early growth response (Egr) family of transcription factorsduring thymic selection. J. Exp. Med. 185, 731–744.

22. Azzam, H. S., DeJarnette, J. B., Huang, K., Emmons, R., Park, C. S.,Sommers, C. L., El-Khoury, D., Shores, E. W., Love, P. E. (2001) Finetuning of TCR signaling by CD5. J. Immunol. 166, 5464–5472.

23. Teh, S. J., Killeen, N., Tarakhovsky, A., Littman, D. R., Teh, H. S. (1997)CD2 regulates the positive selection and function of antigen-specificCD4-CD8� T cells. Blood 89, 1308–1318.

24. Singer, N. G., Fox, D. A., Haqqi, T. M., Beretta, L., Endres, J. S.,Prohaska, S., Parnes, J. R., Bromberg, J., Sramkoski, R. M. (2002) CD6:expression during development, apoptosis and selection of human andmouse thymocytes. Int. Immunol. 14, 585–597.

25. Tomlinson, M. G., Hanke, T., Hughes, D. A., Barclay, A. N., Scholl, E.,Hunig, T., Wright, M. D. (1995) Characterization of mouse CD53: epitopemapping, cellular distribution and induction by T cell receptor engage-ment during repertoire selection. Eur. J. Immunol. 25, 2201–2205.

26. Kirchner, J., Bevan, M. J. (1999) ITM2A is induced during thymocyteselection and T cell activation and causes downregulation of CD8 whenoverexpressed in CD4(�)CD8(�) double positive thymocytes. J. Exp.Med. 190, 217–228.


27. Tiong Ong, S., Ly, C., Nguyen, M., Kay Brightman, B., Fan, H. (2003)Expression profiling of a transformed thymocyte cell line undergoingmaturation in vitro identifies multiple genes involved in positive selection.Cell. Immunol. 221, 64–79.

28. Schmitz, I., Clayton, L. K., Reinherz, E. L. (2003) Gene expressionanalysis of thymocyte selection in vivo. Int. Immunol. 15, 1237–1248.

29. Huang, Y. H., Li, D., Winoto, A., Robey, E. A. (2004) Distinct transcrip-tional programs in thymocytes responding to T cell receptor, Notch, andpositive selection signals. Proc. Natl. Acad. Sci. USA 101, 4936–4941.

30. McCarty, N., Shinohara, M. L., Lu, L., Cantor, H. (2004) Detailed analysisof gene expression during development of T cell lineages in the thymus.Proc. Natl. Acad. Sci. USA 101, 9339–9344.

31. Mick, V. E., Starr, T. K., McCaughtry, T. M., McNeil, L. K., Hogquist,K. A. (2004) The regulated expression of a diverse set of genes duringthymocyte positive selection in vivo. J. Immunol. 173, 5434–5444.

32. Reese, J. T., Mehta, H., Chappell, C. H., Bamezai, A. (2001) Downregu-lated expression of Ly-6-ThB on developing T cells marks CD4�CD8�subset undergoing selection in the thymus. Dev. Immunol. 8, 107–121.

33. Sim, B-C., Lo, D., Gascoigne, N. R. J. (1998) Preferential expression ofTCR V� regions in CD4/CD8 subsets: class discrimination or co-receptorrecognition? Immunol. Today 19, 276–282.

34. DeRyckere, D., Mann, D. L., DeGregori, J. (2003) Characterization oftranscriptional regulation during negative selection in vivo. J. Immunol.171, 802–811.

35. Kouskoff, V., Vonesch, J-L., Benoist, C., Mathis, D. (1995) The influenceof positive selection on RAG expression in thymocytes. Eur. J. Immunol.25, 54–58.

36. Bogue, M., Gilfillan, S., Benoist, C., Mathis, D. (1992) Regulation ofN-region diversity in antigen receptors through thymocyte differentiationand thymus ontogeny. Proc. Natl. Acad. Sci. USA 89, 11011–11015.

37. Sasada, T., Reinherz, E. L. (2001) A critical role for CD2 in both thymicselection events and mature T cell function. J. Immunol. 166, 2394–2403.

38. Puls, K. L., Hogquist, K. A., Reilly, N., Wright, M. D. (2002) CD53, athymocyte selection marker whose induction requires a lower affinityTCR-MHC interaction than CD69, but is up-regulated with slower kinet-ics. Int. Immunol. 14, 249–258.

39. Singer, N. G., Fox, D. A., Haqqi, T. M., Beretta, L., Endres, J. S.,Prohaska, S., Parnes, J. R., Bromberg, J., Sramkoski, R. M. (2002) CD6:expression during development, apoptosis and selection of human andmouse thymocytes. Int. Immunol. 14, 585–597.

40. Choi, J. W., Lee, S. Y., Choi, Y. (1996) Identification of a putative Gprotein-coupled receptor induced during activation-induced apoptosis of Tcells. Cell. Immunol. 168, 78–84.

41. Boehm, U., Guethlein, L., Klamp, T., Ozbek, K., Schaub, A., Futterer, A.,Pfeffer, K., Howard, J. C. (1998) Two families of GTPases dominate thecomplex cellular response to IFN-�. J. Immunol. 161, 6715–6723.

42. Penninger, J. M., Sirard, C., Mittrucker, H. W., Chidgey, A., Kozieradzki,I., Nghiem, M., Hakem, A., Kimura, T., Timms, E., Boyd, R., Taniguchi,T., Matsuyama, T., Mak, T. W. (1997) The interferon regulatory transcrip-tion factor IRF-1 controls positive and negative selection of CD8� thy-mocytes. Immunity 7, 243–254.

43. Carlow, D. A., Marth, J., Clark-Lewis, I., Teh, H. S. (1995) Isolation of agene encoding a developmentally regulated T cell-specific protein with aguanine nucleotide triphosphate-binding motif. J. Immunol. 154, 1724–1734.

44. Maeno, K., Sada, K., Kyo, S., Miah, S. M., Kawauchi-Kamata, K., Qu, X.,Shi, Y., Yamamura, H. (2003) Adaptor protein 3BP2 is a potential ligand

of Src homology 2 and 3 domains of Lyn protein-tyrosine kinase. J. Biol.Chem. 278, 24912–24920.

45. Grumont, R. J., Rasko, J. E., Strasser, A., Gerondakis, S. (1996) Activationof the mitogen-activated protein kinase pathway induces transcription ofthe PAC-1 phosphatase gene. Mol. Cell. Biol. 16, 2913–2921.

46. Salmon, R. A., Foltz, I. N., Young, P. R., Schrader, J. W. (1997) The p38mitogen-activated protein kinase is activated by ligation of the T or Blymphocyte antigen receptors, Fas or CD40, but suppression of kinaseactivity does not inhibit apoptosis induced by antigen receptors. J. Im-munol. 159, 5309–5317.

47. Monks, C. R. F., Freiberg, B. A., Kupfer, H., Sciaky, N., Kupfer, A. (1998)Three-dimensional segregation of supramolecular activation clusters in Tcells. Nature 395, 82–86.

48. Arendt, C. W., Albrecht, B., Soos, T. J., Littman, D. R. (2002) Proteinkinase C-�: signaling from the center of the T-cell synapse. Curr. Opin.Immunol. 14, 323–330.

49. Robinson, L., Panayiotakis, A., Papas, T. S., Kola, I., Seth, A. (1997) ETStarget genes: identification of egr1 as a target by RNA differential displayand whole genome PCR techniques. Proc. Natl. Acad. Sci. USA 94,7170–7175.

50. Alberola-Ila, J., Hernandez-Hoyos, G. (2003) The Ras/MAPK cascade andthe control of positive selection. Immunol. Rev. 191, 79–96.

51. Linette, G. P., Grusby, M. J., Hedrick, S. M., Hansen, T. H., Glimcher,L. H., Korsmeyer, S. J. (1994) Bcl-2 is upregulated at the CD4� CD8�stage during positive selection and promotes thymocyte differentiation atseveral control points. Immunity 1, 197–205.

52. Grillot, D. A., Merino, R., Nunez, G. (1995) Bcl-XL displays restricteddistribution during T cell development and inhibits multiple forms ofapoptosis but not clonal deletion in transgenic mice. J. Exp. Med. 182,1973–1983.

53. Chao, D. T., Korsmeyer, S. J. (1997) BCL-XL-regulated apoptosis in T celldevelopment. Int. Immunol. 9, 1375–1384.

54. Matsumura, M., Tanaka, N., Kuroki, T., Ichihashi, M., Ohba, M. (2003)The � isoform of protein kinase C inhibits UV-induced activation ofcaspase-3 in normal human keratinocytes. Biochem. Biophys. Res. Com-mun. 303, 350–356.

55. Campbell, J. J., Hashimoto, Y. (1993) Recombinase activating gene ex-pression in thymic subpopulations: a transitional cell type has lost RAG-2but not RAG-1. J. Immunol. 150, 1307–1313.

56. Punt, J. A., Osborne, B. A., Takahama, Y., Sharrow, S. O., Singer, A.(1994) Negative selection of CD4�CD8� thymocytes by T cell receptor-induced apoptosis requires a costimulatory signal that can be provided byCD28. J. Exp. Med. 179, 709–713.

57. Calnan, B. J., Szychowski, S., Chan, F. K., Cado, D., Winoto, A. (1995) Arole for the orphan steroid receptor Nur77 in apoptosis accompanyingantigen-induced negative selection. Immunity 3, 273–282.

58. Barda-Saad, M., Braiman, A., Titerence, R., Bunnell, S. C., Barr, V. A.,Samelson, L. E. (2005) Dynamic molecular interactions linking the T cellantigen receptor to the actin cytoskeleton. Nat. Immunol. 6, 80–89.

59. Mason, P. B., Struhl, K. (2003) The FACT complex travels with elongatingRNA polymerase II and is important for the fidelity of transcriptionalinitiation in vivo. Mol. Cell. Biol. 23, 8323–8333.

60. McGuire, M. V., Suthipinijtham, P., Gascoigne, N. R. J. (2001) The mouseSupt16h/Fact140 gene, encoding part of the FACT chromatin transcriptioncomplex, maps close to Tcra and is highly expressed in thymus. Mamm.Genome 12, 664–667.


Thymocyte stimulation by anti-TCR- , but not by anti-TCR- , leads to induction of developmental...

Documents

Transcript of Thymocyte stimulation by anti-TCR- , but not by anti-TCR- , leads to induction of developmental...