Thymocyte Maturation: Selection for In-Frame TCR … Maturation: Selection for In-Frame TCRa-Chain...

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of July 12, 2018. This information is current as Region 3 -Chain Complementarity-Determining β Followed by Selection for Shorter TCR -Chain Rearrangement Is α In-Frame TCR Thymocyte Maturation: Selection for Maryam Yassai and Jack Gorski http://www.jimmunol.org/content/165/7/3706 doi: 10.4049/jimmunol.165.7.3706 2000; 165:3706-3712; ; J Immunol References http://www.jimmunol.org/content/165/7/3706.full#ref-list-1 , 14 of which you can access for free at: cites 33 articles This article average * 4 weeks from acceptance to publication Fast Publication! Every submission reviewed by practicing scientists No Triage! from submission to initial decision Rapid Reviews! 30 days* Submit online. ? The JI Why Subscription http://jimmunol.org/subscription is online at: The Journal of Immunology Information about subscribing to Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved. Copyright © 2000 by The American Association of 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on July 12, 2018 http://www.jimmunol.org/ Downloaded from by guest on July 12, 2018 http://www.jimmunol.org/ Downloaded from

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Page 1: Thymocyte Maturation: Selection for In-Frame TCR … Maturation: Selection for In-Frame TCRa-Chain Rearrangement Is Followed by Selection for Shorter TCR b-Chain Complementarity-Determining

of July 12, 2018.This information is current as Region 3

-Chain Complementarity-DeterminingβFollowed by Selection for Shorter TCR

-Chain Rearrangement IsαIn-Frame TCR Thymocyte Maturation: Selection for

Maryam Yassai and Jack Gorski

http://www.jimmunol.org/content/165/7/3706doi: 10.4049/jimmunol.165.7.3706

2000; 165:3706-3712; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/165/7/3706.full#ref-list-1

, 14 of which you can access for free at: cites 33 articlesThis article

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Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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Thymocyte Maturation: Selection for In-Frame TCR a-ChainRearrangement Is Followed by Selection for Shorter TCRb-Chain Complementarity-Determining Region 31

Maryam Yassai and Jack Gorski2

Thymocyte maturation consists of a number of stages, the goal of which is the production of functioning T cells that respond toforeign antigenic peptides using their clonotypic receptors. Selection of a productively rearranged TCRb-chain is the first stagein the process and occurs at the double-negative to double-positive (DP) transition. Later maturation stages are based on changesin markers such as CD5, CD69, or IL-7R. A stage in whicha-chains are selected has also been identified usingb-chain transgenicmice. Here we identify two additional selection stages in human thymocytes based on characteristics of the TCR.a selection ismeasured directly by identification of in-frame rearrangements and is associated with the appearance of CD3 on the DP thymocytesurface. The next stage has not yet been described and involves selection of thymocytes that express shorter TCRb-chaincomplementarity-determining region 3 (CDR3). This stage is associated with the acquisition of high levels of CDR3 by DP cells andthe transition to SP thymocytes. The extent of CDR3 length selection observed is a function of the TCR V and J genes. We proposethat CDR3 length selection is based on recognition of the MHC. Thus, there exist limitations on the allowable length of that portion ofthe TCR most intimately in contact with MHC and peptide. This may be a physical representation of positive selection.The Journalof Immunology,2000, 165: 3706–3712.

Production of a functional peripheral T cell repertoire re-quires a number of maturation steps to take place in thethymus. These are in part a result of the rearrangement

process, which provides receptor flexibility at a cost of generatingcells with nonproductive rearrangements. To facilitate the process,selection takes place for each TCR chain separately. In the tran-sition from double-negative (DN)3 to double-positive (DP) thymo-cytes, the quality of theb-chain rearrangement product is con-trolled by its ability to pair with the pre-Ta-chain (1). This occursin the CD442 compartment of DN mouse thymocytes (2, 3) and isreferred to asb selection (4). The subset of DN cells in which theb-chain is selected in man is not known, although recent data haveimplicated the CD41CD32CD82 immature single-positive (ISP)cells (5). Later stages, which would include pairing of the TCRb-chain with a productively rearranged TCRa-chain and recog-nition of the peptide-MHC ligand by TCRab, are less wellunderstood.

Thymic selection has been divided into two conceptual frame-works, referred to as positive selection and negative selection(6, 7). Negative selection is easily understood as elimination of Tcells whose receptor/coreceptor affinity for self-peptide-MHC istoo high (8). Positive selection can be defined quite broadly, rang-ing from the rescue of thymocytes from programmed cell death to

the specific stimulation of a thymocyte by a peptide mimic of thefuture Ag. The most accepted definition of positive selection im-plicates only those events in which the thymocyte is interactingwith self-MHC-peptide (see Ref. 9 for review). Historically, thisform of selection has been closely linked to lineage selection,which was used as the readout. There have been recent studiesindicating a division between positive selection of thymocytes andlineage selection (10, 11). There has been a large effort in deter-mining the roles of peptides in the selection process (12–14).

Selection has also been assayed independent of the lineagemarkers using TCRb-chain Tg mice. It has been reported that insome cases theb-chain pairs preferentially witha-chains similarto those with which it was paired in the hybridoma of origin. Byassaying the stage at which thisa-chain selection is observed, thisform of positive selection has been mapped to the CD691 subsetof DP cells (15).

We have been investigating the rearrangement status of the TCRa- andb-chain loci during human thymocyte maturation. Evidencefor b- anda-selection was obtained. As part of these studies wehave identified an additional stage in the maturation process thatinvolves the accumulation of SP cells that contain TCRb-chainswith shorter lengths of complementarity-determining region 3(CDR3). These results are discussed in terms of our current un-derstanding of thymic selection.

Materials and MethodsCells

Thymi were obtained as surgical tissue discards from The Children’s Hos-pital of Wisconsin. PBMC were obtained as discards after removal of in-dwelling catheters. All materials were obtained under an institution reviewboard-approved protocol.

Fluorescent staining and sorting

Thymi were disaggregated by passing them through a wire mesh. Cellswere suspended in RPMI medium (Life Technologies, Gaithersburg, MD),0.1% sodium azide, and 2% FCS. To determine whether the thymi werenormal, 0.53 106 cells were stained using mouse mAbs to human cell

The Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwau-kee WI 53201

Received for publication March 13, 2000. Accepted for publication July 17, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Blood Center Research Foundation.2 Address correspondence and reprint requests to Dr. Jack Gorski, The Blood Re-search Institute, The Blood Center of Southeastern Wisconsin, POB 2178, Milwau-kee, WI 53201-2178. E-mail address: [email protected] Abbreviations used in this paper: DN, double negative; DP, double positive; ISP,immature single positive; CDR, complementarity-determining region; RF, relativefrequency; FAM, 59-carboxyfluorescein.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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surface markers; CD3-FITC conjugate, TCRab-FITC conjugate, CD4-Tri-color conjugate, and CD8-R-PE conjugate (Caltag, San Francisco, CA).The stained cells were analyzed using FACScan (Becton Dickinson, SanJose, CA). Thymi that had normal CD3, CD4, and CD8 profiles were thenstained for sorting. Three color sorts were performed using FACStar (Bec-ton Dickinson), and different populations were collected. Primary gatingwas on the CD3 marker, which resolved the thymocytes into three popu-lations, referred to as CD3neg, CD3low, and CD3high (Fig. 1A). These threepopulations were then further divided on the basis of CD4 and CD8 ex-pression (Fig. 1A). Cells were collected into 0.5 ml of FCS so that the finalconcentration in the tube was 10% (5-ml final volume).

Preparation of nucleic acids from sorted cells

For DNA, sorted cells were spun down and resuspended in nucleic lysisbuffer, pH 8.2 (10 mM Tris, 0.4 M NaCl, and 2 mM EDTA), in the pres-ence of SDS and proteinase K, then the cells were incubated overnight at45 C to ensure the complete lysis. After the incubation, proteins wereprecipitated by adding 5.3 M NaCl, and DNA was isolated from the su-pernatant by ethanol precipitation (16). RNA was made using TRIzol re-agent (Life Technologies, Gaithersburg, MD).

Rearrangement analysis

Rearrangement analysis was performed by PCR amplification of the CDR3using V and J region-specific primers. A description of the methods hasbeen published (17–19). The J region primer was labeled with 59-carboxy-fluorescein (FAM), the PCR products were analyzed on denaturing poly-acrylamide gels, and the fluorescent PCR products were quantitated usinga FluorImager (Molecular Dynamics, Sunnyvale, CA). Data were collectedas a 16-bit Tiff file. Band intensities could be further analyzed using Im-ageQuant and spreadsheet software. For calculation of CDR3 lengthchanges, band intensities originally measured as relative fluorescence unitsby the FluorImager were converted to the relative frequency (RF) of eachband over the total band intensity. The relative band intensities correct forminor fluctuations in the data. The use of RF to calculate shortening isshown in Fig. 5, and a general description is given in Ref. 19.

Rearrangement analysis was performed on DNA samples that hadbeen titrated to insure equal efficiency of amplification of theb-chainDNA constant region. An example of such a titration is shown in Fig.

1B. Volumes of the DNA preparations were chosen so that the signalswould be identical. The titration procedure was described in greaterdetail previously (19).

RT-PCR

Levels of pre-Ta and a-chain mRNA were measured by RT-PCR usingprimers specific for each cDNA. One microgram of total RNA was con-verted to cDNA using Moloney murine leukemia virus reverse transcrip-tase. The cDNA from a different population of thymocytes was titrated todetermine the amount needed to obtain an equivalent actinb-chain mRNAsignal. Serial dilutions of cDNA were amplified for 24 cycles using twoprimers, one in exon 2 and the other in the exon 3 of the actinb-chainlocus. Based on theb-actin titration, levels of pre-Ta anda-chain mRNAwere measured using primers specific for each cDNA. Three concentra-tions of cDNA were used for the PCR to insure a linear response of flu-orescent signal to input. The sequences of the primers used are as follows:b-actin direct, 59-CGTGTGGCTCCCGAGGAGCACC-39;b-actin anti-Fam labeled, 59-CCCTGTACGCCTCTGGCCGTACCAC-39; pre-Ta di-rect, 59-GGCACACCCTTTCCTTCTCTG-39; pre-Ta anti-Fam labeled,59-GCTTCTACAGCCAGGACCTGC-39; Ca direct, 59-GATATCCAGAACCCTGACCC-39; and Ca anti-Fam labeled, 59-ATGACGCTGCGGCTGTGGTCCAG-39.

ResultsRecombination analysis of thymocyte Ag receptors

We have used the variations in intensity of CDR3 length of theTCR b-chain to assay changes in the T cell repertoire. It was ofsome interest to extend these studies to thymocytes, as mature SPthymocytes represent the immediate precursor of naive circulatingT cells. The recombination assay consists of generating a PCRproduct that amplifies the CDR3 using V family-specific and Jregion-specific primers. The length of the CDR3 thus amplified isresolved on denaturing acrylamide gels. We have published ananalysis of the thymic rearrangement profiles of normal V genescompared with pseudogenes (18) and have also used this methodfor analysis of the relationship ofgd thymocytes toab thymocytes(17). The technique and approach are similar to those described byHayday and colleagues (4, 20).

Analysis of TCR Vb-chain genes during thymocyte maturation

The thymic maturation series in man, as assayed by the surface ex-pression of CD3, CD4, and CD8, consists of the following stages,CD3negCD4negCD8neg cells (triple negative) followed by two DPcompartments, CD3negCD41CD81 and CDlowCD41CD81. This di-vision of DP thymocytes into almost equivalent numbers of CD3neg

and CD3low is specific to man and is not found in nontransgeniclaboratory mice. In man, triple-negative cells proceed to the DPCD3neg stage through an ISP CD32CD41CD82 compartment (21).The most mature cells are SP, showing the following markers:CD3highCD41CD82 and CD3highCD42CD81 (reviewed in Ref. 22).The flow cytometric profile of a typical human thymus is shown inFig. 1A. The three levels of CD3 expression are shown in the firstpanel, and the CD4 and CD8 profiles of the three CD3 gates areshown in the following panels.

An example of a typical recombination analysis is shown in Fig.2. The amount of DNA used for each amplification has been nor-malized by titration using a common set of primers amplifyingexon 1 of theb constant region gene. The banding pattern showsa 3-bp spacing indicative of in-frame selection. The intensity ofrearranged genes is the same throughout the maturation series,indicating no furtherb-chain rearrangements. We have analyzedsix human thymi, representing all age groups in which a reasonableamount of thymic tissue is still found, and have observed the samepatterns of BV rearrangement in these five subsets.

FIGURE 1. Titration of DNA from human thymocyte subsets.A, Hu-man thymocyte subsets fractionated on the basis of three surface markers.The first panel shows the CD3neg (R1), CD3low (R2), and CD3high (R3)populations. The next three panels show the CD4 and CD8 profiles of eachof the three CD3 populations. Arrows identify regions used for each thymicsubpopulation.B, Titration of DNA isolated from five different subsets ofthe same thymus. PCR is performed at a number of different DNA con-centrations to insure that the amplified product is a function of input DNA.The subsets are identified in the inset.

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Analysis of TCR Va-chain genes during thymocyte maturation

Recent experiments usingb-chain TCR transgenic mice have iden-tified a stage occurring in DP cells in which thea-chain is selected(10). These took advantage of a propensity of theb-chain to pairwith a-chains similar to that with which it was paired in the hy-bridoma of origin (15, 23). This identifies a stage that can be re-ferred to asa selection. A candidate for such a stage in man is theCD3low DP subset. Expression of detectable CD3 on the surfaceimplies that both TCR chains are expressed on the surface, i.e., thatpairing has taken place.

We tested this supposition by using rearrangement analysis todetermine whether the TCRa-chain was selected in the CD3low

DP cells. Fig. 3Ashows analysis of the same five thymocyte sub-sets as were analyzed for BV rearrangement in Fig. 2. The data arefor the AV8 family. It is obvious that there are very few rearrangeda-chain genes in the CD4 ISP and CD3neg DP compartments.There is a large jump in the overall intensity of rearranged AVgenes in the CD3low DP population, which would indicate thatrearrangement is taking place at this stage. The increases in inten-

sity in the SP populations indicate further rearrangement. How-ever, the rearrangement profiles show a single base pair spacingthroughout the different compartments. This is probably due to theamplification of rearranged genes that are on excised circles gen-erated as part of the continuing rearrangement process (24, 25).The accumulation of circles with rearrangeda-chain genes, mostof which are out-of-frame, thwarts the ability of the analysis todefine at which point thymocytes with in-frame rearrangements areselected.

To overcome the difficulty posed by recombination circles weused available AV (26) and AJ (27) gene maps to confine therecombination analysis to those genes that cannot be further ex-cised because of their distal (AV) or proximal (AJ) positions.Analysis was performed using the AV1S1 locus, which is the firstAV locus and thus must be retained on the chromosome. Thisanalysis shows that the selection for in-frame AV genes is firstobserved in the CD3low DP compartment (Fig. 3B), whereas theCD3neg DP compartment has accumulated thymocytes with out-of-frame rearrangements for the same AV gene. The analysis wasalso performed with AJ4 primers. AJ4 is the first AJ locus that isused to any great extent in AVJ joining and thus must be main-tained on the chromosome. In the analysis with AJ4 and AV12S2primers (Fig. 3C), the same in-framea selection was observedbetween the two DP stages. The results in Fig. 3,B andC, are fromtwo different thymi, and similar results were obtained with threeother thymi.

Further data supporting the observation thea selection takesplace between the CD3neg and CD3low DP compartments comefrom analysis of pre-Ta and TCRa-chain mRNA levels. Beforepairing with thea-chain, the TCRb-chain is paired with the pre-Ta. It would be expected that at the point at whicha selection hasoccurred, the levels of pre-Ta mRNA would decrease, whereas thelevels of TCRa-chain mRNA would increase. We performed suchan analysis on the RNA from three thymocyte subsets, and theresults are shown in Fig. 4. Quantitative RT-PCR was performed

FIGURE 2. Analysis of BV2-BJ2.7 rearrangements in thymocyte subsets.Subsets are identified above each lane and have been defined in the text.

FIGURE 3. Recombination analysis of AV TCR in thymocyte subsets.A, Analysis of AV8-AJ49 recombinations in thymocyte subsets. The DNAused in these analysis is the same as that used for the BV2-BJ2.7 analysisin Fig. 2. B, Analysis of AV1S2-AJ4 rearrangements in the two DP thy-mocyte subsets from thymus T112. The subsets are identified above eachlane. The lane labeled RNA shows PCR products from peripheral T cellcDNA and identifies the in-frame CDR3 sizes.C, Analysis of AV12S1-AJ4 rearrangements in the two DP subsets from thymus T111.

FIGURE 4. RT-PCR analysis of pre-Ta and VA mRNA levels in thy-mocyte subsets.A, Pre-Ta cDNA. B, VA constant cDNA. The subsetsanalyzed are identified in the inset. cDNA concentrations for the threecompartments were normalized on the basis of a titration withb-actinmRNA primers. PCRs for pre-Ta and AV were performed using the nor-malized amount of cDNA as well as half and twice the amount, respec-tively, to insure a linear response relative to the input.

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at a number of dilutions of the cDNA for either pre-Ta (Fig. 4A)or TCR a-chain (Fig. 4B). Before the analysis, the cDNA wastitrated to determine the amount needed to obtain an equivalentb-chain mRNA signal. The data are presented as a dilution seriesto ensure that the response of the PCR is a linear function of theinput cDNA. It can be seen that there is a very significant decreasein pre-Ta mRNA between the two DP stages. Likewise, the mostsignificant increase in TCRa-chain mRNA was observed betweenthe two DP stages. This fits witha selection taking place at theCD3neg to CD3low DP boundary.

Maturation of SP thymocytes includes selection of cells withshorter CDR3

One of the more interesting data to come from the rearrangementanalyses is the observation that the average length of the CDR3shortens between the DP CD3low and SP stages. This can be ob-served to some extent in the data presented in Fig. 2, although it isnot overtly apparent. For BV2-J2.7 rearrangements (Fig. 2), visualinspection of the data is difficult, so we will use these data tointroduce a more quantitative approach for analysis of the bandfluorescence intensities. The steps involved in this are shown inFig. 5. The FluorImager data are converted to relative fluorescenceunits using ImageQuant software (Fig. 5A). This is shown for theCD3low DP and the CD4 SP lanes. The intensity of each band isconverted into the RF of the band with respect to the total bandintensity (Fig. 5B). The difference of the RF of equivalent bands inany two samples compared yields theDRF. If two band distribu-tions are similar, theDRF will hover around zero. If there is short-ening, then theDRF will be positive for the higher m.w. bands(right) and negative for the lower mw bands (left). The data willshow a well-defined shift from negative to positive values. Thisform of analysis shows that for BV2-BJ2.7 rearrangements there isindeed shortening between the CD3low DP and CD4 SP popula-tions (Fig. 5C).

Shortening is observed between the CD3low DP and SP stages

To determine whether the selection of thymocytes with shorterCDR3 is a continuing process or whether there is a particular stageat which this occurs, we analyzed the CDR3 length distributions inthe two DP compartments and the SP compartments of the thymus.An example of such an analysis is shown for the BV7 and BV5.1families (Fig. 6). The shortening for these families between theCD3low DP and CD4 SP is readily discernible by visual inspectionof the gel data (Fig. 6A). TheDRF analysis clearly shows that thereis shortening that occurs when the CD3low DP is compared withthe CD4 SP (Fig. 6B). The same is not seen when the CD3negDPis compared with the CD3low DP (Fig. 6C). Thus, a characteristicof SP thymocytes is that they tend to have shorter TCR BV CDR3.

To show the general nature of these observations, thymocytepopulations prepared from two different thymi are shown in Fig. 6.As a control for theDRF analysis, the two CD3negDP populationsfrom two thymi were compared. Formally these profiles should besimilar and represent the CDR3 size distribution of the initial re-arrangement process if no selection has taken place. Comparisonof the CD3neg DP from the two thymi gave very similar profiles(Fig. 6D), as evidenced by the lowDRF. In all, we have clearlyobserved the phenomenon in five different thymi and for 10 dif-ferent BV genes.

Thus far, the data have shown the selection for CD4 SP thymo-cytes. The same can be observed for CD8 SP thymocytes. A rep-resentative gel analysis of a recombination analysis (Fig. 7A)shows that the average CDR3 length of CD4 SP and CD8 SP arevery similar. TheDRF data for a number of V gene rearrangementsfrom two different thymi (Fig. 7,B and C) show that this is a

general phenomenon. Thus, there does not appear to be a differ-ence between the two major SP thymocyte lineages with respect tothis phenotype.

The possible role of BJ genes was also explored. Thus far, allthe data we have presented used BJ2.7. This J gene was chosenbecause it is the most frequently used J and thus provides increasedsignal strength. The shortening observed is not just a function ofthis J gene, as analysis of rearrangements using the BJ2.1 showedthe same phenomenon (not shown). TheDRF data show evidenceof shortening, although the overall pattern of shortening differedby the J gene used.

The average CDR3 length of CD3high DP thymocytes is shorterthan that of the other DP subsets

While a majority of DP thymocytes show either no or low expres-sion of CD3 on the surface, there is a small, but distinct, population

FIGURE 5. Measurement of CDR3 shortening. Data from the CD3low

DP and CD4 SP lanes of Fig. 2 are analyzed.A, The gel data are convertedto fluorescence intensity, expressed as relative fluorescence units. The orig-inal gel lanes are shown above and below the graph. Bands correspondingto shorter CDR3 are on the left.B, Conversion of raw peak height data intothe RF for each band.C, Generating theDRF by subtracting the RF of theCD4 SP bands from that of the CD3low DP bands. A positiveDRF indicatesthat the DP band is more intense. A negativeDRF indicates that the SPband is more intense.

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of DP thymocytes that express higher levels of CDR3. It is pos-tulated that CD3high DP cells may the precursors of CD3high SPcells (28, 29). The generation of SP thymocytes from transferredCD3low DP thymocytes has been reported (30), but a direct pre-cursor relationship of CD4high DP and SP thymocytes has not yetbeen shown. The rearrangement status of the CD3high DP popula-tion was investigated to determine whether the selection was al-ready taking place at this stage. The CD3high thymocytes (Fig. 8A)were fractionated by their CD4 and CD8 expression (Fig. 8B), andthe DP and the CD4 SP cells were collected. CD3neg DP andCD3low DP were collected from the respective populations as de-scribed previously (see Fig. 1). The four thymocyte populationswere analyzed, and an example is shown in Fig. 8C. The resultsshow that the selection for shorter CDR3 begins to be observed inthe CD3high DP population. The difference between the CD3low

DP and CD3high DP is not obvious by visual inspection (Fig. 8C),but DRF analysis shows that there is a selection step between thetwo stages (Fig. 8E) that continues between the CD3high DP andCD4 SP stages (Fig. 8F). As shown before, there was no evidencefor selection between the CD3negDP and CD3low stages (Fig. 8D).These data are compatible with the three stages, CD3low DP,CD3high DP, and CD4 SP, constituting a sequential maturationpathway characterized by increasing selection for short CDR3.

The relationship of shortening to thymus transit

There is a simple explanation for the accumulation of thymocyteswith shorter CDR3 in the SP subset. This is that the SP thymocyteswith longer CDR3 rapidly exit the thymus, and thymocytes withshorter CDR3 are retained. While it is has been impossible for usto obtain both thymus tissue discards and peripheral blood cellsfrom the same individual, the general nature of this phenomenonshould insure that comparison of thymus and peripheral T cellsbetween different individuals is sufficient. We compared two pairsof age-matched samples, one from thymus and one from PBMC.Rearrangement analysis of total thymocytes (predominantly DPcells) and peripheral T cells showed easily visualized evidence ofCDR3 shortening between the two compartments (Fig. 9). Themean length in the periphery was similar to that observed in SPthymocytes. Thus, it is thymocytes with short CDR3 that are ex-ported and accumulate in the periphery.

DiscussionIn addition to the relatively well understood stage ofb-selection,our data have identified two additional stages of thymocyte selec-tion. The second stage identified involvesa-chain gene rearrange-ment and pairing of thea-chain with theb-chain. This takes placewithin the DP thymocyte population and results in DP cells that areCD3low, as they now express TCRab on the surface. Commensu-rate with this division of DP thymocytes is the decrease in levelsof pre-Ta mRNA and the increase in AV mRNA in the CD3low DP

FIGURE 6. Measurement of CDR3 shortening for two different BVfamilies in two different thymi.A, Rearrangement analysis gel data. TheBV family and thymus analyzed are identified above each gel. Subsets areidentified above each lane.B, DRF calculation for CD3low DP and CD4 SPsubsets.C, Measurement ofDRF between CD3neg DP and CD3low DPsubsets.D, Measurement ofDRF between the CD3neg DP subsets of thy-mus T114 and T111.

FIGURE 7. Analysis of CD4 SP and CD8 SP subsets.A, Gel data forthe BV5.1 rearrangement for thymus 114.DRF data for results from T108(B) and T114 (C) are shown. The V families analyzed are identified in theinset. All rearrangements are for BJ2.7.

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population. A similar observation was reported previously, al-though the DP populations were not resolved (31). Direct evidencefor a selection is obtained from rearrangement analyses of AV-AJrearrangements that must be maintained on the chromosome. Forsuch rearrangements, the first thymocyte population in which in-frame rearrangements are observed is the CD3low DP population.

The third stage of maturation is characterized by accumulationof SP thymocytes that have shorter CDR3. This is an unexpectedcharacteristic of thymocyte maturation. The shorter CDR3 ob-served in the CD4 SP and CD8 SP subsets is not a result of rapidexit of thymocytes with longer CDR3, as peripheral T cells alsohave short CDR3. The short length of peripheral TCR BV CDR3had been noted previously (32), but it was not clear whether thiswas a function of the rearrangement mechanism itself or of a short-ening postrearrangement. The selection for thymocytes withshorter CDR3 can be observed at the CD3high DP stage, stronglysupporting the precursor relationship between CD3low and CD3high

DP cells (10, 15, 28, 29).The shortening phenomenon is a general one, having been ob-

served for a number of individuals. However, it is not clearwhether it will be observed for the same V-J combination in allindividuals. Within any thymus, the phenomenon is characterizedby a dependence on the BV-BJ combination used for the analysis.For example, it is less evident for the BV2 family used to obtainthe data in Fig. 2, whereas it is much more evident in the data forthe other families shown. The same sensitivity to the recombinedJ gene has also been observed. While our studies have not beenexhaustive, the data have always shown some evidence of short-ening, no matter which V or J gene was studied. In contrast, com-parison of the CD3neg DP subset from different thymi does notshow any evidence for shortening. This would be expected if thisless mature subset had not undergone any selection and the com-

ponents of the rearrangement machinery responsible for determin-ing CDR3 length were not polymorphic.

We propose that the observed accumulation of thymocytes withshorter CDR3 in the transitions between the two TCR-expressingDP populations, CD3low and CD3high, as well as that leading to SPcells is a direct result of selection on the TCR ligand, i.e., peptide-MHC molecules. This is the most reasonable interpretation of theobserved dependence of the selection on the V-J combination be-ing analyzed. Direct evidence for the role of peptide-MHC in theshortening process will have to come from work in the mouse,where inbred strains and mutants are available. We have observedselection of thymocytes with shorter CDR3 in CD4 SP thymocytesin the mouse. Results using inbred mouse strains show that thereis an effect on the extent of shortening observed for a particular V-Jcombination if MHC disparate or recombinant strains are exam-ined (our manuscript in preparation). The extent of shortening ismuch higher in 129 and B10 (H2b) than in B10.PL and PL (H2u)mice, indicating that the MHC plays a role in the process. Thesemouse data also speak to the generality of the CDR3 lengthselection.

TCR and Ig employ the same machinery for generating recom-binational diversity, whereas the recognition events for these twoclasses of immune receptors are different. Therefore, it is not sur-prising that the recognition of peptide-MHC may require differ-ences in the length of the contact specificity portion of the mole-cule. Shorter CDR3 could more easily form the flatter recognitionsurface characteristic of those observed in TCR-MHC crystalstructures (33–35).

In addition to the role of the interaction of TCR with MHC:peptide, another molecule that may dictate a need for shorterCDR3 is the coreceptor, CD4 or CD8. The coreceptors are presentat the time of selection and may also impose structural limitationson the preferred length of the CDR3.

Because the stage at which TCRa-chains are selected is differ-ent from that at which the shortening takes place, we do not thinkthat the shortening is related to pairing of the two chains. Our dataimply thata selection is a distinct phenomenon from the selectionfor thymocytes with shorterb-chain CDR3. If CDR3 shorteningrepresents the first selection on peptide-MHC molecules, then theobservation thata selection precedes CDR3 shortening would im-ply that a selection could be solely based on pairing and not onpeptide-MHC recognition.

While the data presented here provide a novel measure of thy-mocyte maturation, there remain a number of interesting issuesthat will require further investigation. For example, it would be ofgreat interest to determine how the short CDR3 phenotype fits withcurrent models of positive selection at the DP to SP boundary (10,

FIGURE 8. Analysis of the CD3high DP subset. The FACS profile forCD3 (A) and CD4 vs CD8 (B) are shown to define the sorted population.C, An example of the gel data.D–F, TheDRF of the analyses for fourdifferent BV families with BJ2.7. TheDRF are calculated for CD3neg DP-CD3low DP (D), CD3low DP-CD3high DP (E), CD3high DP-CD4 SP (F). BVfamilies are identified in the inset (D).

FIGURE 9. Rearrangement analysis comparing thymocytes and periph-eral T cells. An example of the gel data (BV7) is shown at the left. Thetissue source, thymus (T) or PBMC (P), is identified above the lane. TheDRF for three different BV families are shown in the panel on the right.The data for the BV5.3 family are from one thymus-PBMC pair. The datafor the BV6.1 and BV7 families are from another thymus-PBMC pair.

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15, 29). If there is coreceptor involvement, the possible role ofCDR3 length selection in lineage commitment could be explored.In the context of our current understanding of thymic maturation,two opposing explanations for the selection process could be en-visaged, falling under the rubric of either positive or negative se-lection. If only short CDR3 are compatible with the interaction ofthe TCR with peptide-MHC (coreceptor) complexes needed tomaintain viability, this could be considered positive selection, withelimination of unselected thymocytes by the “neglect” mechanism.While perhaps less likely, it is possible that a longer CDR3 dem-onstrates a high affinity interaction with the peptide-MHC liganddue to the generation of deeper, more complex, Ig-like contacts.This would result in elimination of these thymocytes by negativeselection mechanisms. It will be interesting to determine whichmechanism is at work. The data presented here provide a physicalbasis for considering the issues involved in thymocyte selectionand open up further avenues for studying the process.

AcknowledgmentsWe thank Drs. Burt Litwin and James Tweddell, Children’s Hospital ofWisconsin, for the thymic tissue. The many discussions with our col-leagues and especially members of the Gorski laboratory is gratefullyacknowledged.

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