T Cell Development in TCRb Enhancer-Deleted Mice:Implications for ab T Cell Lineage Commitment andDifferentiation 1
Isabelle Leduc, William M. Hempel,2 Noelle Mathieu, Christophe Verthuy, Gaelle Bouvier,Francoise Watrin,3 and Pierre Ferrier4
T cell differentiation in the mouse thymus is an intricate, highly coordinated process that requires the assembly of TCR complexesfrom individual components, including those produced by the precisely timed V(D)J recombination of TCR genes. Mice carryinga homozygous deletion of the TCRb transcriptional enhancer (Eb) demonstrate an inhibition of V(D)J recombination at thetargeted TCRb locus and a block inab T cell differentiation. In this study, we have characterized the T cell developmental defectsresulting from the Eb2/2 mutation, in light of previously reported results of the analyses of TCRb-deficient (TCRb2/2) mice.Similar to the latter mice, production of TCRb-chains is abolished in the Eb2/2 animals, and under these conditions differen-tiation into cell-surface TCR2, CD41CD81 double positive (DP) thymocytes depends essentially on the cell-autonomous expres-sion of TCRd-chains and, most likely, TCRg-chains. However, contrary to previous reports using TCRb2/2 mice, a minorpopulation of TCR gd1 DP thymocytes was found within the Eb2/2 thymi, which differ in terms of T cell-specific gene expressionand V(D)J recombinase activity, from the majority of TCR2, ab lineage-committed DP thymocytes. We discuss these data withrespect to the functional role of Eb in driving ab T cell differentiation and the mechanism ofab T lineage commitment. TheJournal of Immunology,2000, 165: 1364–1373.
T he T lymphocytes belong to distinct lineages that expresseitherab or gd TCRs on their cell surface and, withinabT cells, either the CD4 or CD8 coreceptors. T cell devel-
opment in the thymus proceeds from a common precursor (1) andrequires the ordered rearrangements of TCR genes (i.e., throughV(D)J recombination) as well as a series of selection eventsthrough which newly assembled TCR complexes signal for cellsurvival, proliferation, and differentiation (2). Due to the inherentimprecision of the DNA joining step during V(D)J recombination(3), only rearrangements that generate an open reading frameacross the recombined TCR gene(s) (i.e., productive rearrange-ments) allow for expression of the corresponding polypeptide(s)and ensuing selection. It is not yet clear whether TCR complexes,once assembled, instruct the developing thymocytes to commit toand differentiate along one or the other lineages (“instructive”model of lineage commitment) or, alternatively, whether lineage
commitment occurs stochastically before receptor assembly (“sto-chastic selection” model) (4–6).
During the past few years, a comprehensive view ofab T celldevelopment in the mouse thymus has started to emerge. TCRbgene recombination, starting with Db-to-Jb rearrangement, is firstdetected within a subpopulation of CD42CD82 double negative(DN)5 cells expressing the CD25 and CD44 markers (DNCD441CD251); Vb-to-DJb assembly is completed within thesubsequent DN CD442CD251 cell stage (7–9). Productive rear-rangements allow for the expression of a TCRb-chain which, whenassociated with the pTa-chain and signal-transducing CD3 pro-teins, forms the pre-TCR (10). Pre-TCR-expressing cells possess aselective advantage to differentiate along theab developmentalpathway, resulting in cell populations in which in-frame TCRbrearrangements are thus overepresented (11, 12). Passage throughthis check-point, referred to asb selection, is coupled to the down-modulation of CD25, massive cell proliferation, the arrest ofTCRb gene rearrangement to mediate allelic exclusion, and theonset of TCRa gene rearrangement. Cells emerging from theseprocesses are CD41CD81 double positive (DP) thymocytes ex-pressing low levels of theab TCR-CD3 complexes. At this stage,through receptor/coreceptor interaction with MHC products, asmall proportion of DP cells are positively selected (13). Positiveselection results in the arrest of V(D)J recombination, an increasein ab TCR-CD3 expression, and the modulation of coreceptorexpression (14) to yield CD41 or CD81 single positive (SP) cells,which eventually migrate to the periphery.
In contrast toab T cells,gd thymic cell development is far lessunderstood, partly due to their small number (as compared withab1 thymocytes) and lack of phenotypic markers (other than the
Centre d’Immunologie de Marseille-Luminy, Institut National de la Sante´ et de laRecherche Medicale-Centre National de la Recherche Scientifique, Marseille, France
Received for publication June 17, 1999. Accepted for publication May 22, 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 institutional grants from Institut National de la Santeet de la Recherche Medicale and Centre National de la Recherche Scientifique and byspecific grants from the Association pour la Recherche sur le Cancer, the Commissionof the European Communities, the Fondation Princesse Grace de Monaco (to P.F.),and the Ligue Nationale Contre le Cancer (to I.L. and P.F.).2 Current address: Institut de Recherche Jouveinal/Parke-Davis, 3–9 rue de la Loge,94265 Fresnes Cedex, France.3 Current address: Institut National de la Sante et de la Recherche Medicale U491, 27Boulevard Jean Moulin, 13385 Marseille Cedex 5, France.4 Address correspondence and reprint requests to Dr. Pierre Ferrier, Centred’Immunologie de Marseille-Luminy, Institut National de la Sante et de la RechercheMedicale, Centre National de la Recherche Scientifique, Case 906, 13288 MarseilleCedex 9, France. E-mail address: [email protected]
5 Abbreviations used in this paper: DN, double negative; DP, double positive; SP,single positive; Eb, TCRb gene enhancer; LR-PCR, long-range PCR; LM-PCR, li-gation-mediated PCR; SE, signal end; WT, wild type; HSA, heat-stable antigen; DSB,double strand break; RAG, recombinase-activating gene.
gd TCR). Recent studies indicate that a significant proportion ofTCRd and TCRg rearrangements are completed earlier than thosefor TCRb, at the DN CD441CD251 stage (8, 9). Also, analysis ofmice deficient for TCRb-chain expression (TCRb2/2 mice thatproducegd1 T cells only (15)) has identified thymic cell popula-tions in which in-frame TCRd rearrangements are enriched, sug-gesting that development in thegd pathway similarly goes througha selection event(s) (16). However, the precise stage at which thisprocess would take place in normal mice and the composition ofthe receptor complexes involved are still unclear.
With respect to the aforementioned models of lineage commit-ment, the question remains as to what are the forces that drive thechoice between the TCRab andgd differentiation pathways. Per-haps productive TCRb or gd rearrangements divert differentiationaway from thegd or ab lineage, respectively (17–19). Silencing ofthe TCRg gene and/or deletion of the TCRd gene (i.e., by Va-to-Ja recombination) would be factors which, subsequently, con-tribute to lock the developing cells into theab cell lineage (4, 20).Lack of pTa expression and/or inhibition of Vb-to-DJb joining byexpressedgd TCRs would play a similar role in thegd develop-mental pathway (5). However, these views have been challengedby the findings that the differentiation of small populations ofaband gd lineage-committed T cells from several transgenic andknock-out mouse models may be independent of the nature of theexpressed receptors (18, 21, 22).
Recently, we have described knock-out mice that show reducedlevels of TCRb gene recombination and a block inab T celldifferentiation after deletion of the only defined transcriptionalTCRb gene enhancer (Eb) at the TCRb locus (23). In the presentstudy, we characterize more precisely the TCRb gene expressionand T cell developmental defects in the Eb2/2 thymus. Our resultsemphasize the role of Eb in ab T cell development and provideadditional implications with respect to the processes ofab vs gdlineage commitment and differentiation.
Materials and MethodsMice
Wild-type (WT) C57BL/6J mice, single knockout TCRb-deficient(TCRb2/2), and TCRd-deficient (TCRd2/2) mice (15, 24), heterozygous(Eb1/2) and homozygous (Eb2/2) Eb-deleted (23) mice, and doubleknock-out TCRd-deficient (TCRd2/2) Eb2/2 mice (25) were used in thisstudy. TCRb2/2 and TCRd2/2 mice, both on a C57BL/6J genetic back-ground, were obtained from The Jackson Laboratory (San Diego, CA). Allmice were housed in a specific pathogen-free animal facility in accordancewith institutional guidelines. Mice were sacrificed for analysis between 4and 6 wk of age. Initial analyses were performed using Eb2/2 mice bredon a mixed (S1293 BALB/c) genetic background; subsequent studiesusing Eb2/2 animals bred on a C57BL/6J background gave essentially thesame results.
Antibodies
Biotinylated, FITC-, PE-, and allophycocyanin-conjugated mAbs againstCD8 (53-6.7), CD4 (H129.19), CD44 (Pgp-1), CD25 (7D4), TCRb (H57-597), TCRgd (GL3), Vb3 TCR (KJ25), CD3-e (2C11), and CD24/heat-stable Ag (HSA; M1/69) were purchased form PharMingen (San Diego,CA). Biotinylated mAbs against the B220 (RA3-6B2; B cell-specific),Mac-1 (M1/170; macrophage-specific), and Gr-1 (RA6-8c5; granulocyte-specific) markers were from Caltag (Tebu, Le Perray en Yvelines, France).Biotinylated mAbs were revealed using streptavidin tricolor (Caltag).
Flow cytometry and cell cycle analysis
Lymphocyte preparation and cell-staining with saturating levels of mAbswere conducted according to published protocols (for example, see Ref.26). For cell-surface analyses, 5–503 105 gated events were acquiredusing a FACScan flow cytometer (Becton Dickinson, Mountain View, CA)and were analyzed with the Lysis II software. Cell sorting was performedas described previously (26, 27) using a FACStarPlus (Becton Dickinson).For cell sorting of Eb2/2 DP gd1 andgd2 thymocytes, the sorting win-dows were defined in such a way that 1) only cells expressing high levels
of both CD4 and CD8 were purified, and 2) within the selectedCD41CD81 cells, thegd1 andgd2 sorting gates do not overlap.
For propidium iodide staining of DNA, 53 105 sorted cells werewashed in PBS-0.2% BSA and then fixed in PBS-70% ethanol for 30 minat 4°C. Cells were washed with PBS-0.1% glucose, treated with RNase A(Boehringer Mannheim, Meylan, France; 1mg/ml) for 15 min at roomtemperature, and stained with propidium iodide (Sigma-Aldrich, Saint-Quentin Fallavier, France; 25mg/ml in PBS-0.1% glucose) for 20 min atroom temperature and then for 10 min at 4°C. Analysis was performed ona FACScan (Becton Dickinson) with appropriate doublet discrimination.
Nucleic acid extraction, reverse transcription, and long-rangePCRs (LR-PCRs)
Total RNA and genomic DNA were simultaneously extracted from purifiedcell populations (;53 105 sorted cells) using TRIzol (Life Technologies,Cergy Pontoise, France) as recommended by the manufacturer. RNA sam-ples were treated with RNase-free DNase I (Pharmacia, Orsay, France) andwere converted to cDNA by reverse transcription, using the Ready-to-GoT-primed first strand kit (Pharmacia).
Analysis of T cell-specific transcripts and gene rearrangements by RT-PCR and LR-PCR assays, using cDNA or genomic DNA templates andlocus-specific primers, were performed essentially as described previously(25, 27). RT-PCR was performed for 22 cycles of 30 s at 94°C, 30 s at58°C, and 1 min at 72°C; LR-PCR was performed for 25 cycles of 30 s at94°C, 1 min at 58°C, and 1 min at 72°C. Individual RT-PCR used thefollowing pairs of forward and reverse primers: Vd4-Cd, 59-CCGCTTCTCTGTGAACTTCC-39 and 59-GCTGCTAGGAAAACTCTCCT-39;Vg4-Cg1, 59-TGTCCTTGCAACCCCTACCC-39and 59-ATTGCCACAGACAGATGTTGT-39; Va8-Ca, 59-ACCCAGACAGAAGGCCTGGTCACT-39 and 59-GAGGGAGCTGAGTGGGTG-39; pTa, 59-CTGCAACTGGGTCATGCTTC-39 and 59-GTCCAAATTCTGTGGGTGGG-39;RAG-2, 59-CACATCCACAAGCAGGAAGTACAC-39and 59-GGTTCAGGGACATCTCCTACTAAG-39; CD4, 59-GAGAAGACGCTGGTGCTGGG-39 and 59-CCCACAACTCCACCTCCTC-39; CD8, 59-CAAGCATCTACTGGCTGCGGG-39and 59-GTGGGGGAACGGGCATTGCTT-39;andb-actin, 59-GTGGGCCGCTCTAGGCACCAA-39and 59-CTCTTTGATGTCACGCACGATTTC-39. Individual LR-PCR used the following pairsof forward and reverse primers: Vd4- or Vd5-Jd1, 59-CCGCTTCTCTGTGAACTTCC-39 or 59-CAGATCCTTCCAGTTCATCC-39and 59-CAGTCACTTGGGTTCCTTGTCC-39; Vg1-Jg4, 59-CCGGCAAAAAGCAAAAAAGTT-39 and 59-ACTACGAGCTTTGTCCCTTTG-39; Vg2-Jg2, 59-TACCGGCAAAAAACAAATC-39 and 59-CAGAGGGAATTACTATGAGC-39;Vg4-Jg1, 59-TGTCCTTGCAACCCCTACCC-39 and 59-CAGAGGGAATTACTATGAGC-39; and Cb2, 59-TGTGGCAGGCTCTAATTAAAT-39 and59-GCTATAATTGCTCTCCTTGATGGCCTG-39. After amplification, PCRproducts were electrophoresed on 1% agarose/0.5% NuSieve gels, transferredto Nylon membranes (Gene Screen Plus; NEN Life Science Products, LeBlancMesnil, France), and hybridized with [g-32P]-labeled locus-specific oligonu-cleotide probes internal to the corresponding primers. Images were generatedby use of a phosphoimager (BAS 1000; Fuji, Raytest France S.A.R.L., France)and quantified using MacBAS software.
Ligation-mediated PCR (LM-PCR)
Detection of in vivo-generated signal ends (SEs) by LM-PCR, usinggenomic DNA ligated to the unidirectional BW linker (28) as template, aswell as verification for the presence of the DNA in the BW linker-ligatedsamples by PCR using primers specific for CD14 (59-GCTCAAACTTTCAGAATCTACCGAC-39 and 59-AGTCAGTTCGTGGAGGCCGGAAATC-39), were conducted as described previously (25, 29). Depending onthe TCR gene segment analyzed, the following primers were used in twosuccessive rounds of amplification: Ja50 (round 1, 59-CCACGTCCAGATGCCAACTTGAAA-39; round 2, 59-GAGAGGAGTGCTGAAAACAGCCTT-39); Jd1 (round 1, 59-TGTTGTTCCCACATGCTGCTCAAAC-39;round 2, 59-AACCTCCTGTAAGCTAACCCATCCT-39); and Jg1 (round1, 59-CCAACTGAACTCCTTCTATTTTCTGTTGGTG-39; round 2, 59-AACTCCAGGGAGAACAGTGTATGAG-39). PCR products were ana-lyzed as described in the previous section.
PCR-RFLP
Diverse TCRd and TCRg gene rearrangements were studied by PCR-RFLP, according to published protocols (11, 30). Briefly, genomic DNAfrom sorted thymocytes was PCR-amplified using appropriate pairs ofTCRd and TCRg primers as follows: Vd4- or Vd5-Jd1, 59-CCATCGATGGCCGCTTCTCTGTGAACTTCC-39or 59-CCATCGATGGCAGATCCTTCCAGTTCATCC-39 and 59-CAGTCACTTGGGTTCCTTGTCC-39;
Vg2-Jg2, 59-CCATCGATGGTACCGGCAAAAAACAAATC-39 and 59-TGAATTCCTTCTGCAAATACCTTG-39; and Vg4-Jg1, 59-CCATCGATGGTGTCCTTGCAACCCCTACCC-39and 59-TGAATTCCTTCTGCAAATACCTTG-39. PCR products were gel purified, digested with theClaI restriction enzyme (forward primers contain aClaI site (ATCGAT) ontheir 59side) to generate fragments of predicted size that spanned the poly-morphic junctions, and then labeled using T4 polynucleotide kinase and[g-32P]ATP (Amersham, Les Ulis, France). After ethanol precipitation, thelabeled products were resolved on 5% denaturing polyacrylamide gels,parallel to calibrated sequencing ladders from DNA fragments of knownsize. Gels were used to generate images and quantification data, as de-scribed above. Lengths of predicted in-frame joints were calculated frompublished sequences.
Calculation of PCR-RFLP values for TCRd and TCRg selection
Assuming that rearrangements occur with an equal frequency in all threereading frames, within a population of rearranging TCRd cells, 1/3 willcarry an in-frame (d1) allele after the initial attempt and 2/3 will carry anout-of-frame (d2) allele. Given the absence of allelic exclusion at theTCRd locus (31), rearrangement could proceed to the second allele in theformer subpopulation, yielding cells that have either an additionald1 allele(d1/d1; 1/33 1/3) or ad2 allele (d1/d2; 2/33 1/3). Among the remaining2/3 cells, rearrangement on the second allele yields cells with either ad1
allele (d2/d1; 1/3 3 2/3) or an additionald2 allele (d2/d2; 2/3 3 2/3). Intotal, all d alleles in the cell population undergo rearrangement, and 1/31(1/3 3 1/3) 1 (1/3 3 2/3) ared1. Cells carrying ad1 rearrangement(s)(corresponding to a total of 2[1/31 (1/33 2/3)] d rearranged alleles) enterthe selected pool, whereas thed2/d2 cells are eliminated. Therefore, whenscanning TCRd selected cells by PCR-RFLP, the theoretical ratio (N) be-tween the number ofd1 alleles (numerator) vs that of total rearranged (d1
andd2) alleles (denominator) is as follows.
N 51/31 ~1/33 1/3! 1 ~1/33 2/3!
2@1/31 ~1/33 2/3!#5
1/3~1 1 1/31 2/3!
2 3 1/3~1 1 2/3!5 3/55 0.6
Allelic exclusion may not apply to TCRg gene recombination either (32).Moreover, each of the two TCRg1, g2, andg4 alleles (33) may attemptrearrangement in any one cell. Therefore, cells carrying ag2 joint on bothalleles at a giveng locus (for example, theg1 locus) may theoretically berescued into the selected pool after ag1 rearrangement at any one of thefour remaining alleles (i.e., the twog2 andg4 alleles). Because PCR-RFLPanalyzes only one locus at a time, theN ratio when scanning TCRg1selected cells, for example, is decreased according to the following revisedformula, in which the number ofg1 alleles (the numerator) remains un-changed, whereas that of theg2 alleles (the denominator) is augmented in
proportion to the number ofg12/g12 cells rescued in the sampled popu-lation after an in-frameg2 or g4 rearrangement.
Note that, in this situation in which up to six TCRg alleles could be rear-ranged, the predicted value of in-frame junctions in the case of TCRgselection is close to that associated with random recombination (0.33).
ResultsLack of TCRb chain expression in Eb2/2 T cells
The Eb2/2 mouse presents a severe defect in TCRb gene recom-bination and a block inab T cell development (23). ImpairedabT cell development is best characterized by reduced cellularity andaltered cellular profiles in thymuses from the Eb2/2 animals com-pared with those from WT or heterozygous (Eb1/2) littermates(Tables I and II, Fig. 1, and data not shown). On average, totalthymocytes in the Eb2/2 mutants are decreased by.15-fold (Ta-ble I), even though we noted important variations (from 3.53 106
to 423 106) among Eb2/2 individuals. When stained for CD4 andCD8 surface expression, Eb2/2 thymocytes contain an abnormallyhigh proportion of DN cells and, conversely, a reduced proportionof DP cells; genuine CD4high and CD8high SP cells are missing(Fig. 1A, upper middleand upper left panels). Reported to theabsolute cell numbers, Eb2/2 DN thymocytes are present in nor-mal or slightly diminished numbers, whereas both Eb2/2 DP andSP cells are severely reduced, accounting for the collapse in thy-mus cellularity (Table I).
The cellular profiles described above, notably the severe reduc-tion but not disappearance of DP cells, are similar to those reportedfor TCRb-chain-deficient (TCRb2/2) mice which, after gene tar-geting, carry a large (.15-kb) deletion within the TCRb locus(15). In TCRb2/2 mice, it has been shown that the production ofDP thymocytes is highly, if not entirely, dependent on TCRd-chainexpression, because these cells were almost completely absentwhen the mutation was introduced onto a TCRd-deficient(TCRd2/2) background (15, 16, 34). To test whether a similareffect occurs in the Eb2/2 mice, these were crossed with TCRd2/2
Table I. Absolute numbers and percentage of thymocytes in Eb1/2, Eb2/2, and TCRd2/2 Eb2/2 mice: anti-CD4/-CD8 staining of total thymocytesa
a Average cell numbers and percentage for the individual cell subsets were calculated from the indicated number (n) of mice for each line. Numbers between parenthesesindicate the minimal and maximal percentages found in the given subset. In the Eb2/2 line, absolute numbers of CD41/CD81 DP thymocytes were highly variable; also, CD41
and CD81 cell stainings within the Eb2/2 SP thymocyte subsets were of low to intermediate intensities.
Table II. Absolute numbers and percentage of thymocytes in Eb1/2, Eb2/2, and TCRd2/2 Eb2/2 mice: anti-CD44/-CD25 and anti-gd stainings ofCD42/CD82 DN thymocytesa
mice (24), and thymuses from double mutant (TCRd2/2 Eb2/2)animals were analyzed as above (Table I and Fig. 1A, upper rightpanel). Strikingly, thymus cellularity was further reduced in theTCRd2/2 Eb2/2 mice, showing little interindividual variation.Moreover, TCRd2/2 Eb2/2 thymuses essentially lacked DP andSP cells, which is indicative of a drastic block in early T celldifferentiation. This was confirmed by analysis of the CD44/CD25profiles from DN thymocytes (Table II and Fig. 1A, lower panels).Thus, although CD441CD251 and CD442CD251 DN cells areproportionally increased in the TCRd2/2 Eb2/2 thymus, thesecells fail to efficiently populate the CD442CD252 compartment. Asimilar, but less marked, block of T cell differentiation was ob-served for Eb2/2 DN cells. We conclude that, as in the TCRb2/2
mice, deletion of Eb leads to a block in early T cell developmentthat can be bypassed, albeit inefficiently, by expression ofTCRd-chains.
The above results strongly argue that the Eb mutation is notpermissive for the residual expression of TCRb-chains (otherwiseDP and SP cells would presumably be found in the TCRd2/2
Eb2/2 thymus). To confirm this, thymus and lymph node cells
from an Eb2/2 mice were analyzed using the H57-597 mAb,which reacts against pan-TCRb-chains; Eb1/2 and TCRb2/2
cells were used as positive and negative controls, respectively(Fig. 1B). As expected, surface H57-597 staining was observedfor cells from the Eb1/2 mouse (both H57-597low and H57-597high
cells were found in the Eb1/2 thymus, whereas only H57-597high
cells were found in the lymph nodes) but not forthose from theTCRb2/2 mouse. Importantly, thymus and lymph node cells fromthe Eb2/2 mouse were also H57-597-negative. These results werereproduced upon testing several individual animals (I.L., unpub-lished data), establishing the Eb2/2 mouse as an additional modelof TCRb-chain (hence pre-TCR) deficiency. It is noteworthy thatgd1 cells are found in increased numbers and at a higher percent-age among the Eb2/2 DN thymocytes (Table II), implying that theEb deletion does not blockgd T cell development.
Cellular characteristics of the Eb2/2 DP thymocytes
Examples of pre-TCR-independent DP cell development in severalmodels of natural or engineered genetic mouse mutants have fu-eled speculation about the underlying developmental mechanismsand their relevance to the physiology of thymic cell differentiation.Issues of particular concern are 1) the actual lineage (ab or gd) ofthe resulting TCRb2 DP thymocytes, 2) the extent to which theDN to DP cell transition depends upon acis-autonomous or, al-ternatively,trans-induction mechanism, and 3) the nature of thespecific receptors that drive this stage of thymocyte differentiation(4–6). Because we wished to understand how our Eb2/2 mice fitinto the present picture, we undertook a series of cytofluorometricanalyses focusing on the DP cells present in the mutant thymus;thymocytes from heterozygous Eb1/2 mice were also analyzed.The results are reported in Fig. 2 and Table III. Tricolor stainingusing anti-CD4, -CD8, and -gd mAbs indicated the presence of aminor population of anti-gd reactive cells (hereafter referred to asgd1) within the DP compartment from the Eb2/2 thymuses (Fig.2). On average, thegd1 cells account for 16.6% of Eb2/2 DPthymocytes, which is lower than the proportion ofgd1 cells in theEb2/2 DN compartment (mean value of 24%) but significantlyhigher than that of Eb2/2 DP thymocytes which were nonspecifi-cally stained by an anti-Vb3 TCR mAb (mean value of 5.2%;Tables I–III, Fig. 2, and data not shown). Within the Eb2/2 thy-mus,gd1 high expressors were mainly found in the DN cell com-partment, whereas the Eb2/2 DP gd1 thymocytes consistedmostly of low to intermediate with a few highgd1 expressors (asjudged by cytofluorograph profiles and comparison of the meanvalues of rate fluorescence intensity). Under these experimentalconditions, only a few percent of specific, anti-gd-reacting cellswas seen among the DP thymocytes from either heterozygousEb1/2 or WT mice (0.5%–,2%) as well as the DP thymocytesfrom TCRb2/2 mice (,4%) (Fig. 2 and data not shown).
Four-color cytofluorometric analyses using additional mAbsagainst the T cell-specific surface markers CD24/HSA, CD3-e, orCD25 allowed us to further compare thegd1 and gd-unreactive(hereafter referred to asgd2) subsets within the Eb2/2 DP com-partment (Table III). Both HSA and CD25 staining are indicatorsof thymocyte immaturity, the latter often being associated withreduced cell proliferation at the DN-to-DP transition (35). CD3-estaining usually correlates with high levels of TCR expression.High percentages of HSA-expressing cells were found in all DPsubsets, whether Eb1/2 or Eb2/2, with equally prominent valuesin both the Eb2/2 DP gd1 andgd2 subsets (mean rates$94%)and a slightly lower value in the Eb1/2 DP subset (mean rate586.3%). On the other hand, mean percentages of cells expressinghigh levels of CD3-e were lower in the Eb2/2 DP subpopulations
FIGURE 1. Flow cytofluorometric analysis of T lineage cells from4-wk-old Eb1/2, Eb2/2, TCRd2/2 Eb2/2, and TCRb2/2 mice. A, Totalthymocytes were analyzed by flow cytometry for the expression of CD4 vsCD8 (upper panels), and DN thymocytes were analyzed for the expressionof CD44 vs CD25 (lower panels). For the CD44/CD25 studies, DN Tlineage cells were analyzed after staining of thymocytes with anti-CD44-PE, anti-CD25-FITC, and anti-CD3, -CD4, -CD8, -B220, -Mac-1, and-Gr-1 (all biotinylated and revealed with streptavidin tricolor) mAbs andgating on the CD3-, CD42, CD82, B220-, -Mac-1, and -Gr-1 negativewindow. The absolute number of thymocytes found in each type of mouseis shown on the top; quadrant percentages are indicated in the lower rightquadrant (CD4/CD8 analysis) or the upper right quadrant (CD44/CD25analysis).B, Thymocytes (left panel) and lymph node cells (right panel)were analyzed for the cell surface expression of TCRb-chains using theCb-specific H57.597 mAb. Note that H57.597 staining was slightly lowerin the case of the Eb2/2 mouse compared with that of the TCRb2/2
compared with Eb1/2 DP thymocytes, thegd2 subset being pre-dominantly affected (0.5 vs 20.3%, a 40-fold difference). Notably,a similar comparison for CD25 staining demonstrated moderatelyhigher levels of expression within the Eb2/2 DP gd1 and gd2
subsets (15.7 and 6 vs,2%, respectively).
Finally, after cell sorting and propidium iodide staining, we an-alyzed the Eb2/2 DP gd1 andgd2 subsets with respect to theirlevel of .2N DNA content (reflecting the extent of cell cycling;Table III). Compared with the Eb1/2 total DP controls, the Eb2/2
DP cells displayed lower percentages of cells with greater than adiploid DNA content. The lowest value (3.7%) was found for thegd1 population, which is consistent with this subset containingmostly nondividing cells. However, thegd2 population exhibiteda higher level of dividing cells (9.5%).
In summary, our cytofluorometric analyses demonstrate thatEb2/2 DP thymocytes consist primarily of moderately dividingcells that are TCR2 CD3-e2/low, although a sizable population ofslowly dividing TCRgd1 cells is also present. Both populationsinclude a significant percentage of cells that exhibit a relativelyimmature phenotype, as defined from anti-HSA and -CD25stainings.
Lymphoid gene expression in Eb2/2 DP gd1 and gd2
thymocytes
The presence ofgd1 cells within the DP compartment ofTCRb2/2 mice has been questioned (15, 22). Indeed, these studieslead to the conclusion that TCRb2/2 DP thymocytes correspond to“ab -like” T cells, based on the expression of TCRa transcripts(15) and the presence of V(D)J recombinase-mediated DNA dou-ble strand breaks (DSBs) flanking the upstream Ja gene segments(22). To verify that the Eb2/2 DP gd1 andgd2 thymocytes rep-resent truly distinct entities and further clarify their lineage, weconducted analyses of selected gene activities within the two sub-sets. First, we used RT-PCR to analyze expression of several Tcell-specific genes in total RNA from sorted Eb2/2 thymocytes,including rearranged TCRd, g, anda genes and the pTa gene. Ascontrols, RNA from purified Eb1/2 thymocytes and cultured Bcells were included in the analyses. Representative data are shown,which emphasize the distinction between the Eb2/2 DP gd1 andgd2 cell subsets (Fig. 3A, lanes 4and5). Thus, as predicted, highlevels of Vd4-Cd and Vg4-Cg1 transcripts were detected withinthe Eb2/2 DP gd1 subset, as opposed to the Eb2/2 DP gd2
subset, where these transcripts were barely detectable. In the lattersubset, Vd4-Cd transcripts slightly predominated over Vg4-Cg1transcripts. However, conversely, mature-sized Va8-Ca tran-scripts and pTa transcripts of both the pTaa and pTab isotypes(35) were found in the Eb2/2 DP gd2 subset but not in the Eb2/2
DP gd1 subset. As expected for the Eb1/2 control, TCRd andTCRg as well as pTa transcripts were detected within the DNpopulation whereas, conversely, mature TCRa transcripts werefound to predominate in the DP cells (lanes 1and2; note that thepattern of pTa gene expression in the Eb1/2 thymus, showing
Table III. Flow cytometric analysis of T cell-specific cell surface markers and extent of cell cycling withinthe DP thymocytes from the Eb1/2 and Eb2/2 mice
a Average percentages6 SEM of cells in the DP total,gd1, andgd2 subsets expressing the TCRgd or the CD24/HSA,CD3-«, or CD25 cell surface markers, as indicated. Only cells expressing high level of surface CD3-«were gated. Results arefrom two separate experiments in which thymocytes from two Eb1/2 mice and three Eb2/2 mice were analyzed (n5 4 and6, respectively). For calculation of the average value of anti-gd staining within the Eb2/2 DP compartment, the percentage ofnonspecific Vb3 staining was subtracted from that ofgd staining for each analyzed sample.
b Percentages of cells in the DP subsets exhibiting a greater than 2NDNA content (hence in the S/G2/M phases of the cellcycle); thymocytes from one Eb1/2 mouse and one Eb2/2 mouse were analyzed.
FIGURE 2. A minor population ofgd1 thymocytes is present within theEb2/2 DP cell compartment. Total thymocytes from the indicated micewere triple-stained with anti-CD4, -CD8 and -gd, or -Vb3 TCR mAbs and,using flow cytofluorometry, CD42CD82/DN and CD41CD81/DP thymo-cytes were analyzed forgd cell surface expression (leftandmiddle panels,respectively), and CD41CD81/DP thymocytes were analyzed for Vb3 cellsurface expression (right panels; the anti-Vb3 TCR mAb, which has beenproduced in the same species and possesses the same biochemical prop-erties as the anti-gd mAb, is used here as control for nonspecific stainingof the Eb2/2 DP thymocytes). Eb2/2 #1 and #2 correspond to two mutantindividuals. The percentages ofgd1 or Vb31 staining are indicated. Asubpopulation of Vb31 cells was detected within the SP compartmentsfrom both Eb1/2 and TCRd2/2 thymi (e.g., accounting for 4.8 and 3.6%of their CD42CD81/SP thymocytes, respectively; data not shown).
high and low levels in DN and DP thymocytes, respectively, re-produces that described for WT thymus (36)). Predictably, B cellslacked TCRd, g, a, and pTa transcripts (lane 6). Consistent withthe above results, Vd5-Cd, Vg7-Cg1, Vg2-Cg2, and Vg1-Cg4transcripts were more prevalent within the Eb2/2 DP gd1 subset,whereas Va2-Ca and Va5-Ca transcripts strongly predominatedwithin the Eb2/2 DP gd2 subset (I.L., unpublished data). cDNAproducts derived from Eb2/2 DP gd2 cells and amplified usingVa and Ca primers did not hybridize to a Jd1-specific probe (notshown), suggesting that they correspond to VJa-Ca messengersrather than to alternatively spliced VDJd-Ca hybrids (37), an assump-tion also consistent with the finding of Va-Ja rearrangements ingenomic DNA from Eb2/2 DP gd2 cells (I.L., unpublished data).
Additional RT-PCR analyses demonstrated the production ofhigh levels of CD4 and CD8 transcripts in both the Eb2/2 DPgd1
andgd2 subsets but, as expected, not in purified Eb2/2 DN gd1
cells (Fig. 3B,lanes 5–7), a finding that strengthens our cytofluo-rometric results and shows that the Eb2/2 DP gd1 cells are notmerely contaminants from the DN compartment. Finally, recom-binase-activating gene 1 (RAG1) and RAG2 transcripts werefound in cells from thegd2 subset only (Fig. 3Aand data notshown; the RAG1 and RAG2 transcripts code for the core com-ponents of the V(D)J recombinase (3)). The latter findings stronglysuggested the possibility of ongoing V(D)J recombination in theEb2/2 DP gd2 but not thegd1 subset. This was confirmed byusing LM-PCR to detect the blunt-ended intermediates (SEs) pro-duced by RAG-mediated DNA DSB cleavage (29). DSBs weredetected at the TCR-Ja50, -Jd1, and -Jg1 segments in genomicDNA from the DPgd2 but not the DPgd1 subset (Fig. 4). DSBprofiles within the Eb2/2 DP gd2 subset, including prominentTCR-Ja50 SEs and less intense Jd1 and Jg1 SEs, appear related tothose in Eb1/2 DP cells and distinct from those in both the Eb1/2
and Eb2/2 DN subsets in which Jd and Jg cleaved products pre-dominate (comparelanes 1-3and5). Altogether, these results con-firm, at the molecular level, the existence of two distinct subpopu-lations of Eb2/2 DP gd1 and TCR2 thymocytes. TCRd and gtranscription and the absence of ongoing V(D)J recombinationwithin the former subset are consistent with itsgd1 cellular phe-notype. Conversely, the preferential expression of pTa andVJa-Ca products together with the presence of Ja SE products ofV(D)J recombination within the Eb2/2 DP gd2 subset indicatethat it consists mostly of “ab -like” differentiating T cells. How-ever, the production of pTa transcripts within these cells at levelsclose to those in DN thymocytes underscores the relatively imma-ture phenotype of this population with respect to that of conven-tional DP thymocytes.
FIGURE 4. V(D)J recombination SE intermediates at the TCRad andggene loci in Eb2/2 DPgd2 thymocytes. Genomic DNA was prepared fromsorted Eb1/2 and Eb2/2 thymocytes and analyzed by LM-PCR. Briefly,DNA from the indicated cells was ligated to the BW linker (28) and usedas a template in PCR reactions with primers to detect broken SEs associ-ated with Ja50-, Jd1-, and Jg1-flanking recombination signal sequences(Ja50 SE, Jd1 SE, or Jg1 SE, respectively). Negative controls were per-formed using linker-ligated genomic DNA from a WT kidney (Kd.WT).The bottom panelshows ethidium bromide-stained amplification productsfrom DNA-PCR reactions using CD14-specific primers to control for theamount of sample DNA loaded in the individual reactions.
FIGURE 3. T cell-specific gene expression in Eb2/2 DP gd1 andgd2
thymocytes.A, Thymocytes from 4-wk-old Eb1/2 or Eb2/2 mice weresorted by flow cytofluorometry into the indicated DN, DP, and DPgd1 orgd2 subpopulations, respectively. Total RNA was isolated from the sortedcells and from the 7A.4 B cell line used here as a negative control (B cells)and was analyzed by RT-PCR using standard protocols. The amplified genetranscripts are indicated to the right of the panels.B, Same asA, except thatRNA from WT thymus and kidney, as well as that from sorted Eb2/2 DNgd1 thymocytes, was also used.Lanes 1-3show a titration of the indicatedtranscripts in the WT background. Input material was kept constant (50 ngof cDNA) by using decreasing amounts of thymus and increasing amountsof kidney samples, respectively.Lane 1, undiluted thymus;lanes 2and3,1:3 and 1:9 dilutions, respectively. In bothA andB, thebottom panelshowsthe amplified products fromb-actin to control for the amount and qualityof RNA.
TCRd and g gene rearrangements in Eb2/2 DP gd1 and gd2
thymocytes
What is the driving force behind pre-TCR-independent thymocytedevelopment in the Eb2/2 thymus and is there a developmentalrelationship between the two subpopulations of DPgd1 and “ab-like” cells? Although distinct according to cytofluorometric andgene expression criteria, both depend on TCRd-chain expressionfor development (see above). In pre-TCR-deficient animals, devel-opment of DN precursors into DP cells was proposed to dependeither on the self-expression of TCRd-chains, possibly in associ-ation with TCRg-chains (16, 18, 22) or, alternatively, on interac-tions with pre-existinggd1 cells present in the mutant thymus(38). In the first situation, it is predicted that the resulting DP cellscarry in-frame TCRd and TCRg rearrangements despite the factthat they may not express these products as a possible consequenceof their commitment to theab lineage. Such cells would thus differfrom DP cells of normal mice in which in-frame TCRd and TCRgjoints appear to be counterselected (17, 30). Conversely, in thesecond situation, rearrangements at the TCRd and TCRg lociwould at least be expected to not be enriched for in-frame junc-tions. To define which mechanism could be responsible for thegeneration of the DPgd2 thymocytes in the Eb2/2 mice and an-alyze their developmental relationship withgd1 cells, we per-formed two sets of experiments. First, we used semiquantitativeLR-PCR assays on genomic DNA prepared from sorted thymo-cytes to analyze the relative levels of TCRd andg gene rearrange-ments within the Eb2 DPgd2 vsgd1 subpopulations. Second, we
used PCR-RFLP to qualitatively analyze the same rearrangements.The latter technique permits the determination of the ratio of in-frame vs out-of-frame rearrangements at any V(D)J recombinedlocus in a given cell population, hence the probability that thepopulation has been selected (or counterselected) based on a par-ticular TCR chain expression profile (11, 30). Considering thatPCR-RFLP samples only the fraction of a cell population in whicha given rearrangement has occurred, conclusions with respect toTCR chain selection, based on results obtained using this tech-nique, are dictated by the levels of rearranged loci present withinthis population. Results from these analyses are shown in Figs. 5and 6, respectively. Thus, Vd4-to-Jd1 and Vd5-to-Jd1 rearrange-ments were detected at roughly equivalent levels in Eb2/2 DPgd1 and DPgd2 thymocytes, which are close to those found inDN gd1 thymocytes from the same mice (Fig. 5,top panelandlegend for details on quantification, by phosphorimager scanning,of 32P emission from the amplified products). Detection of TCRdjoints in “ab -like” DP cells is in agreement with published resultsdemonstrating the persistence of these products as extrachromo-somal circles in cells that have performed Va-to-Ja rearrangement(17). Similarly, levels of Vg1-to-Jg4, Vg2-to-Jg2, and Vg4-to-Jg1rearrangements were also found to be equivalent within the Eb2/2
gd1 (either DN or DP) and DPgd2 subsets (Fig. 5,middle pan-els). Parallel PCR-RFLP assays emphasized the rearrangementprofile similarities between the two Eb2/2 DP gd1 andgd2 sub-sets (Fig. 6). Overrepresentation of in-frame Vd4-to-Jd1 and Vd5-to-Jd1 junctions was obvious for both Eb2/2 DP gd1 and gd2
FIGURE 5. Levels of Vd-to-Jd and Vg-to-Jg joints in Eb2/2 DP gd1
andgd2 thymocytes. LR-PCR assays were used to detect Vd4-to-Jd1 re-arrangements (top panel) and Vg1-to-Jg4 and Vg2-to-Jg1 rearrangements(middle panels) using genomic DNA from sorted DPgd1 and gd2 thy-mocytes. Briefly, DNA was amplified using V-specific forward and J-spe-cific reverse primers. At the TCRd and TCRg loci, the unrearranged V andJ segments are too far apart to be amplified within a common germlinefragment. To control for the amount of DNA in the individual reaction, thenonrearranging TCRb2 constant region gene (Cb2) was amplified usingforward and reverse primers located 59of and inside exon 1, respectively.The source of DNA is indicated above each lane; PCR products corre-sponding to specific fragments are indicated to the right.Lanes 1–3showa titration of sorted DNgd1 DNA from an Eb2/2 mouse. Input materialwas kept constant (100 ng of genomic DNA) by using decreasing amountsof DN gd1 DNA and increasing amounts of Eb2/2 kidney DNA. Lane 1,undiluted thymus;lanes 2and3, 1-to-5 and 1-to-10 dilutions, respectively.Densitometric analysis using a phosphorimager indicated that levels ofgene rearrangements in Eb2/2 DP gd1 and gd2 thymocytes relative tothose in DNgd1 thymocytes were, respectively, 71.6 and 78.4% (Vd4-to-Jd1); 85.8 and 104% (Vg1-to-Jg4); and 77 and 89.7% (Vg2-to-Jg2), aftercorrecting for differences in the amount of loaded DNA as determined byscanning of Cb2 PCR products.
FIGURE 6. TCRd and TCRg rearrangement status in Eb2/2 DP gd1
and DPgd2 thymocytes. PCR-RFLP analyses for Vd4-to-Jd1, Vd5-to-Jd1,Vg2-to-Jg2, and Vg4-to-Jg1 rearrangements are shown. DNA from sortedthymocytes was PCR amplified, radiolabeled, and electrophoresed througha sequencing gel. The origin of DNA is indicated above each lane (gd1 orgd2, DP gd1 or gd2 thymocytes, respectively). The position of in-framerearrangements, determined from parallel DNA sequencing ladders (notshow), is indicated by bars on the right of each panel. The frequency ofsuch joints as a percent of the total signal, determined by densitometricanalysis using a phosphorimager, is shown below each lane.
cells, showing percentages above 63% in all cases, the lowest val-ues being consistently observed in the latter subset (Fig. 6,upperpanels). Analysis of Vg2-to-Jg2, Vg4-to-Jg1, and Vg1-to-Jg4junctions gave lower rates of in-frame joints, ranging from 36 to59% (Fig. 6, lower panels, and data not shown). Overall, thesevalues are in general agreement with those predicted in the case ofTCRd and TCRg selection, respectively (seeMaterials and Meth-odsfor calculation), although this may be difficult to conclude forthe junctions and cell subsets showing the lowest percentages (e.g.,the Vg2-to-Jg2 rearrangements within thegd2 subset) becausesuch values are close to that associated with random recombina-tion. However, after cloning and sequencing of a total of 48 Vg2-to-Jg2 junctions from Eb2/2 DP gd2 thymocytes, we found that43.7% were in-frame (data not shown), a result which tends tofurther support a role for TCRg selection in generating the sam-pled population. Also consistent with this argument is the higherpercentage of in-frame junctions observed for the Vg4-to-Jg1joints (Fig. 6,lower right panel). This is most likely because of thefact that the Vg4 gene carries an in-frame STOP codon at its 39extremity (39) that precludesg-chain synthesis unless eliminatedby the recombination reaction (i.e., being that STOP codons arenot recognized by PCR-RFLP, an excess of in-frame joints involv-ing this particular gene segment relative to other types of Vg-Jgjunctions is precisely expected in the case of TCRg selection).
In conclusion, our analyses showing high levels of predomi-nantly in-frame TCRd joints in the Eb2/2 DP gd2 thymocytes areconsistent with a role for TCRd selection in determining autono-mous development of this cell subset. Similarly, LR-PCR, PCR-RFLP, and sequencing data also support TCRg selection of most,if not all, cells within this subpopulation.
DiscussionWe have analyzed the effects of TCRb enhancer deletion on thy-mic cell differentiation, with the goal of evaluating the resulting Tcell developmental defects compared with those described in theexisting pre-TCR-deficient mouse models. We have shown that,similar to TCRb2/2 mice (15), there is a specific lack of TCRb-chain expression in the Eb2/2 animals. In addition, we haveshown that the DP cells that develop in the Eb2/2 thymus arecomprised of distinct TCR2 and TCRgd1 subsets, in dissimilarproportions. The former, predominant subset includes relativelyimmature T cells committed to theab lineage (as evidenced no-tably by ongoing TCR-Ja recombination), although they carrymostly productive TCRd gene rearrangements and, most likely,productive TCRg gene rearrangements as well. Eb2/2 gd1 DPthymocytes, on the other hand, exhibit no V(D)J recombinationactivity and, as discussed further below, may represent either im-mediate precursors to theab-committed DP thymocytes or, alter-natively, a minor branch of terminally rearrangedgd T cells. Theseresults illustrate the critical function of Eb in regulatingab T celldifferentiation and shed further light on pre-TCR-independent pro-cess(es) ofab-T cell development.
Essential role of Eb during ab T cell differentiation
The defect in TCRb-chain expression in Eb-deleted T cells suffersfrom no leakiness; otherwise, DP thymocytes would have devel-oped in the TCRd2/2 Eb2/2 mice. This is a remarkable phenotypeconsidering the limited extent (560 bp in length) of the Eb deletion(23) compared with the large size (.500 kb in the mouse) andcomplex structure of the TCRb locus (40). As demonstrated by usand by others (23, 25, 41), deletion of Eb severely affects V(D)Jrecombination ofcis-linked gene segments, although the precisemechanism(s) by which this effect is mediated is still under inves-
tigation. Strikingly, knock-out deletion of enhancer elements fromother TCR and Ig genes generally results in a consistent but lesssevere defect (i.e., V(D)J recombination is decreased at the tar-geted locus, but production of the corresponding polypeptide andmature lymphocytes in the relevant lineage is not completely abol-ished (42, 43)). This particularity could be related to the fact that,whereas the TCRb locus has only one known enhancer, other Igand TCR loci carry at least two such elements. However, it isequally possible that, depending on the locus considered, othercis-regulatory elements (e.g., different from transcriptional en-hancers (44)) could provide on their own and/or complement, atleast so some extent, the recombination enhancing function. Intro-ducingcis-linked mutations in Ag receptor gene loci (45) will helpto clarify these issues.
Origin, mode of differentiation, and fate of the Eb2/2 DPthymocytes
Analysis of Eb2/2 mice adds to the general picture that, paradox-ically, the lack of a pre-TCR does not impair the development ofa few DP cells (2, 5). Our cellular and molecular studies identifydistinctgd1 andgd2 subsets within the Eb2/2 DP compartment.The DPgd1 subset represents a minority of cells in the mutantthymus. It exhibits the highest rate of CD3-e staining and lowestrate of cell divisions. Molecular analyses confirm thegd1 pheno-type and detected no sign of V(D)J recombination in this popula-tion. It has been reported thatgd1 cells are not found within theDP compartment from TCRb2/2 thymi (22), although the pres-ence of a small population of TCR-d-positive cells has occasion-ally been described (15, 16). However, these cells have not beencharacterized in detail, although one report does show that theyexhibit a high rate of proliferation (16), opposite to the slow di-viding rate that we found for the Eb2/2 DP gd1 thymocytes,suggesting that these two types ofd1 DP cells may be different.Conversely, in the same study of TCRb2/2 mice, populations ofslowly dividing CD442CD251 and CD442CD252 thymocytesthat develop along thegd pathway have been identified (16). TheEb2/2 DP gd1 thymocytes may be derived from similar cells andtherefore may represent a minor branch of cells in thegd lineagethat for unknown reasons express the CD4 and CD8 coreceptors.The possible basis for the discrepancy between the Eb2/2 andTCRb2/2 mouse strains in terms ofgd gene expression within theDP cell compartment is currently unclear. It is unlikely to be theresult of a difference in the genetic background, because all strainswere bred on the C57BL6/J background for several (n . 9) gen-erations. An intriguing possibility would be that it results, by anunknown mechanism(s), from their significant differences in theengineered genomic alterations at the TCRb locus (e.g., differ-ences in the extent of the targeted deletion; presence or not of theselectableNeogene (15, 23). Finally, it may be noteworthy that theEb2/2 DP gd1 subset appears roughly equivalent, in terms ofabsolute cell numbers, to a minute population of CD41CD81 DPd1 thymocytes identified in the normal thymus (16), suggestingthat both may belong to the same pathway and that the Eb2/2 DPgd1 subset is not peculiar to this mutant strain. Indeed, a signif-icant proportion of late embryonic thymicgd1 T cells are DP (46),and the promotion of CD4/CD8 surface expression by thegd TCRalone has been reported (47).
The remaining TCR2 thymocytes constitute a majority of theDP cell subset in the Eb2/2 thymus. Although comprised ofmostly CD3-e2 cells and consisting of an elevated proportion ofCD251 cells, this subset obviously includesab lineage-committedthymocytes. Despite the fact that our analyses were performed oncell populations rather than on individual cells, the high level ofin-frame TCRd rearrangements found in thegd2 cells indicates
that most cells within this subset have gone through a process ofdselection, in agreement with genetic evidence from the analysis ofTCRd2/2 Eb2/2 double knock-out mice. Most probably, selectionfor g-chain expression also occurred for a majority of cells in thissubset, as supported by LR-PCR and PCR-RFLP profiles and se-quencing analysis. A similar population of “ab -like” TCR-nega-tive cells, of which development would be promoted by thegdTCR, has been postulated to arise within the TCRb2/2 thymus(16, 18, 22). It has been argued that such a population follows adevelopmental pathway distinct from that ofgd1 cells. Our cel-lular and molecular analyses, notably those showing distinct di-viding rates (Table III), T cell-specific gene expression profiles,and V(D)J recombination activity between the Eb2/2 DP gd1 andgd2 subsets (Figs. 3 and 4), support this view (but also see below).As emphasized by Livak et al. (22), the possibility that cells car-rying the same type of receptor adopt distinct developmentalgd orab lineages strongly supports a “stochastic selection” mechanismof early T cell commitment and differentiation, later amplified bylateral cell-cell interactions that may influence the final outcome(48). However, based on the overall low rates of productive Vg-to-Jg joints found in TCRb2/2 DP thymocytes, Hayday and col-leagues (16) have discussed another possibility that developmentof somed-selected cells in this population may be TCRg-indepen-dent, leading then to a more complex picture in which “stochasticselection” and “instructive” modes ofab/gd lineage commitmentmay coexist. Proposed basis for this type of selection includedreceptors made of the association of the TCRd-chain with anotherpolypeptide such as, for example, pTa (for discussion, see Ref.16). In this regard, we found little support for such a hypothesisbecause introducing the Eb2/2 mutation onto a pTa- or TCRa-deficient background (hence testing for two factors that are ex-pressed in the Eb2/2 DP gd2 subset; Fig. 3) had no obvious effecton DP cell development in the double mutants (I.L., unpublisheddata). Along the same lines, a significant role for TCRg-pTa com-plexes in mediating the DN-to-DP cell transition (34) was alsoruled out because DP cells never exceeded 0.5% of total TCRd2/2
Eb2/2 thymocytes (Table I and Fig. 1A). The possibility remainsthat production of DSBs at the TCRb locus in Eb2/2 andTCRb2/2 animals (Ref. 25; W.M.H. and P.F., unpublished data)may trigger an intracellular signaling pathway(s) that facilitatesDP development, by analogy with the induction of the DN-to-DPtransition in RAG-deficient mice by sublethal gamma-irradiation(a treatment known to induce DNA DSBs) (49, 50). However, it isdifficult to explain how such a process could preferentially affectTCR d-selected cells. Finally, it has to be stressed that the utili-zation of agd TCR (or a putatived-based TCR) to commit and/ordifferentiate along theab pathway may be a feature that is readilyobservable in genetically engineered TCRb-deficient mice but thatis marginal in the normal situation(confined, for example, todevelopment of a few cells that have failed to productivelyrearrange the TCRb locus). However, in normal mice the pre-TCR would readily bypass these relatively inefficient processes toplay an instructive role that actively boostsab development, asrecently proposed (19).
Whereas the Eb2/2 “ab -like” thymocytes must be eliminatedat the DP stage because of the absence of a TCR and lack ofpositive selection, the behavior of DP cells within thegd1 subsetis more uncertain. Elevated levels of Annexin V staining found inthis population (as in the “ab -like” DP subset) imply that a largeproportion is committed to die (I.L., unpublished data). However,some cells may represent the precursors either of the CD41/CD81
SP gd1 cells present in the peripheral lymphoid tissues in theEb2/2 mouse (I.L. and C.V., unpublished data) or, alternatively,of the Eb2/2 DP gd2 cells discussed above (a developmental
progression that would imply extensive changes in gene expres-sion programs, e.g., the reactivation ofrag gene expression andV(D)J recombination activity, of pTa gene expression, increasedcell proliferation, etc.). These possibilities, which could possiblycoexist, are currently under investigation.
AcknowledgmentsWe thank Drs. M. Bonneville, S. Candeias, and P. Naquet for criticalreading of this manuscript, C. Beziers La Fosse for preparing the artwork,N. Brun-Roubereau and M. Barrad for helping in the cytofluorometric anal-yses, and M. Pontier and G. Warcollier for maintaining the mouse colonies.
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