seminars in IMMUNOLOGY, Vol 11, 1999: pp. 239]249Article No. smim.1999.0180, available onlineat http:rrwww.idealibrary.com on

Signals involved in gammarrrrrdelta T cell versusalpharrrrrbeta T cell lineage commitmentAdrian C. HaydayU, Domingo F. Barber, Nataki Douglas andEric S. HoffmanU

In seeking an explanation for the complexity of tissuedevelopment, biologists are obliged to explain lineagecommitment, the events that dictate whether or not a progen-itor cell will differentiate into one cell type or another. Suchexplanations have been sought across a broad spectrum ofbiological systems, although in no case has a full under-standing been developed. For immunologists, attention hasbeen focused on the lineage commitment of a T cell progeni-tor to becoming either a gd T cell or an ab T cell. In thisreview, we compare the signals that thymocytes may receive

( )from the pre T cell receptor preTCR with signalling fromTCRgd . These signals may determine, co-determine, facili-tate, or cement the abrgd lineage decision, in concert withsignals from additional molecules, such as Notch and cy-tokine receptors. Elucidating the pleiotropic signalling events,particularly those elicited by the preTCR, may in the nearfuture contribute to a molecular definition of lineagecommitment.

Key words: development r lineage commitment r preTCRr signalling r thymocyte

Q1999 Academic Press

Introduction to lineage commitment

ab T CELLS ARISE primarily in the thymus. Althoughmost mature ab T cells express either CD4 or CD8co-receptors, their immature thymic progenitors are

From the UDepartment of Immunobiology, Guys Kings StThomas Medical School, University of London, Guys HospitalCampus, London SE1 9RT, UK, Department of Molecular Celland Develomental Biology, Yale University, and The NationalInsitute of Allergy and Infectious Disease, National Institutes ofHealth

Q1999 Academic Press1044-5323r99r040239q11 $30.00r0

.initially double negative DN cells, expressing nei-ther CD4 nor CD8, from which arise double positive .DP cells, expressing both CD4 and CD8 simultane-

.ously reviewed in ref 1 .gd cells also develop in the thymus, but most are .DN reviewed in ref 2 . Therefore, it has been hy-

pothesised that mature gd cells split off from abprogenitors at the DN stage, prior to the acquisitionof CD4 and CD8.3 The early development of gd cellsand ab T cells from a common progenitor wouldfacilitate the common differentiated characteristics ofthe two cell types}e.g. an obligatory association ofTCR ab and TCRgd with the CD3 complex; theexpression of signalling molecules, such as lck, fyn,and ZAP70 ; and the expression of cell surfacemolecules, such as Thy1, LFA-1, CD28, and CD40L.At the same time, the two cell types have notabledifferences, particularly in the nature of their antigenrecognition, and their growth factor dependence.These differences would be consistent with the hy-pothesis that the two cell types derive from distinctlineages that diverge from a common progenitor veryearly in lymphopoiesis.

To probe further these disparate views of lineagecommitment, we and others have concentrated onmolecular events that characterise the transition ofDN thymocytes either to DP thymocytes, or to matureDN cells. When these events are better understood, itshould be possible to determine whether they pro-vide a molecular definition of lineage commitment.

ab T cell development

Operationally, DN thymocytes have been subdividedinto four subsets that develop in the following tem-

hi y.poral order: Stage I CD44 CD25 ; Stage II hi q. lo q.CD44 CD25 ; Stage III DN CD44 CD25 and

lo y. .Stage IV DN CD44 CD25 Figure 1 . Stage II DN

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Figure 1. Intrathymic T Cell Development. Cell surface markers are commonly used to define Tcell progenitors. Some of the stages of thymocyte development are shown here, starting at DN

lo q.Stage III CD44 CD25 cells. Thymocytes which undergo TCR b selection proliferate andprogress to the DP stage. By contrast, thymocytes which branch off to become gd T cells do notundergo the burst of proliferation. Indeed, TCRgd cells usually represent -2% of totalthymocytes. Still unresolved is if E cells are precommited to one of the T cell lineages orinstructed by receptor signals.

thymocytes begin to rearrange the TCR b , g , and dloci, seemingly at approximately the same time,largely completing the process in Stage III.4 ] 7

Productive TCR b gene rearrangement, indepen-dent of the TCR a chain, is critical to the movementof Stage III DN cells into Stage IV DN cells, fromwhich emerge the DP thymocytes that continue abT cell development.4 ] 6 This effect of TCR b is medi-ated by the preTCR, a complex containing TCR bwith preTa and several of the CD3 chains.8 ] 11 Theselective further development of thymocytes with aproductive TCR b gene rearrangement is known asb selection.5

Analogous studies have characterised Ig heavychain selection in the development of preB cells.12 ] 16

In the preB receptor, the IgH chain is paired with asurrogate light chain, comprising l5 and V-preBsubunits.17,18

In both B cell and T cell progenitors, the develop-mental transition provoked by the pre-antigen recep-tors is associated with rapid and significant clonalexpansion. In experiments designed to define theprecise transitional step mediated by the preTCR, weand our colleagues defined a cell cycle-arrested E

lo .cell stage within DN Stage III CD44 CD25q thatappears to be the final point reached by cells prior to

. 19b-selection Figure 1 . The immediate next stageappears to be an actively cycling Stage III cell, termedan L cell. L cells differ from E cells by a defined list

.of biochemical properties Table 1 . In particular, L

cells appear to be relieved from the cell cycle inhibi-kip1 19,20 tory effects of p27 . Coincident with this and

.possibly causally related , RAG2 protein levels aredecreased 20-fold, an event that may clearly con-tribute to allelic exclusion at the TCR b locus.19,21

These analyses of thymocytes examined directly exvivo allowed us to propose that the preTCR, like thepreBCR, is a pleiotropic signalling complex that in-duces clonal expansion, associated with additionalchanges in differentiation state. The ensuing predic-tion is that in animals in which the preTCR cannotform or function, the phenotype will include a blockin E]L lymphocyte maturation, a severe decrease inlymphocyte numbers, and a reduction in allelic ex-clusion.

Genetic dissection of preTCRsignallingsimilarities to the TCR

Data from several knockout mice fulfill this predic-tion, thereby demonstrating the importance of the

preTCR in the maturation of DN thymocytes Table. yry yry 22,232 . In Rag-1 or Rag-2 mice that cannot

undergo any TCR gene rearrangement, there is asevere inhibition of development at stage III E cellsand the thymus size is reduced G100-fold. The de-velopmental block is slightly less severe in TCR byry

mice. This difference indicates that some maturationof Stage III cells can be provoked by expression of

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Signaling in thymocyte development and lineage commitment

Table 1. Phenotypic differences between E and L cell DN CD44lowCD25q thymocytes

E cells L cells

Propidium iodide .DNA Content 2N G0rG1 98% 34%

.)2N SrG2rM 2% 66%PCR-RFLPTCR b Rearrangemennts Randomr In frame

out of frameConfocal immunofluorescenceCyclin D2 q qCyclin D3 qq qqCyclin E qqq qqqCyclin A y qqq

aCyclin B " qCDK2 q qp27 qqq yRAG2 qq "

ImmunoblotCyclin A y qqqCyclin B " qqq

.Active Rb Hyperphosphorylation y qRAG1 qq qqRAG2 qqq qry

.20-fold reductionbIP kinase assay

cdc2 1= 13=

Notes. aCytoplasmic staining onlybActivity above background

.TCRg andror TCR d chains see below . Consistentwith this, there is a very severe block in maturation inCD3eyry mice.24

Stage III cell maturation is also inhibited in micedeficient in p56 lck kinase;25 weakly inhibited inmice lacking p59 fyn kinase, and essentially com-pletely inhibited either in mice lacking both,26 or inmice overexpressing a dominant negative lck.27 Com-plementing this, the block in development imposedby the RAG1 mutation mice can be overcome intransgenic mice expressing constitutively active lck.28

The later step of DP to SP maturation is inhibited inmice deficient in p70 ZAP kinase.29 DN to DP devel-opment is negligibly inhibited in mice lacking p72syk kinase,30,31 and essentially completely inhibited inmice lacking both p70 ZAP and p72 syk.32

Taken together these studies support the view thatb-selection is mediated by a CD3-associated preTCRcomplex that initiates a lckr fyn]Zap70rsyk tyrosinekinase cascade largely if not completely identical tothat employed by mature TCR signalling. Indeed, adominant negative raf allele also inhibits the DN toDP transition,33 consistent with the progression ofpreTCR signalling through the raf-MAP kinase path-

way, again analogous to that provoked by TCR activa-tion in mature T cells. The capacity for the preTCRto signal clonal expansion of thymocytes would thenbe seen as analogous to the capacity of the TCR tosignal clonal expansion of mature T cells.

Genetic dissection of preTCRsignallingdifferences from the TCR

Notwithstanding these various results, there are signsthat there may be important differences between theway that the preTCR and the TCR function. Forexample, the detectable association of the preTCRwith CD3 components appears looser than is thecase for the TCR,11 and mutations in any of CD3g , d ,or z cause only a partial block of the DN to DPtransition.34 ] 36 Likewise, while it is probable that thecritical role of lck in thymocyte development is tosignal from the preTCR, there is currently no com-pelling biochemical paradigm for this, since in ma-ture T cells, the action of lck in TCR signalling isexplained via its binding to CD4 and CD8 co-recep-tors. Neither such co-receptor is available for binding

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Table 2. Thymocyte Development in Mutant Mice

b b!aStrain DN Cells DP Cells SP Cells Reference

cyrye 22Rag 1 Normal Absent Absent Mombaerts 1992cyrye 23Rag 2 Normal Absent Absent Shikai 1992cyrye 58 .TCR b=d Normal Absent Absent Passoni 1997c dyryf 58TCR b Normal Variable Absent Passoni 1997

yryfTCR a Normal Normal Absent Philpott 1992cyryeTCR d Normal Normal Normal Itohara 1993

yryhfPreTa Normal Reduced Reduced Fehling 1996

yry 34CD3g Normal Reduced Reduced Dave 1997yryf 35CD3d Normal Normal Reduced Haks 1998yrye 24CD3e Normal Absent Absent DeJarnette 1999

yryzrh Normal Reduced Reduced Ohno 1993yryFce RIg Normal Normal Normal Takai 1994

yryzrhrFce RIg Normal Reduced Reduced Shores 1998

yry 25lck Normal Reduced Reduced Molina 1992, Groves 1996yryfyn Normal Reduced Reduced Appleby 1992

yryx yryglck fyn Normal Absent Absent van Oers 1998g 27Dom neg lck Normal Absent Absent Levin 1993

yryg 29Zap70 Normal Normal Absent Negishi 1995, Kadlecek 1998sykyrye Normal Normal Normal Cheng 1995,30 Turner 199531

yry yrye 32Zap70 = syk Normal Absent Absent Cheng 1997

yryeiIL-7 Normal Reduced Reduced von Freeden-Jeffry 1995yryei 62IL7R Reduced Reduced Reduced Peschon 1994, He 1996

aNormal for DN cells indicates normal development up to the DN E cell stage.bReduced indicates reduced development in terms of thymocyte numbers or impaired function.c yry yry .yryWe have found that Rag1 , Rag2 and TCR b=d have normal DN thymocyte distributions, but reduced total

numbers of DN thymocytes. For example in one experiment: a typical 3 week old C57.Blr6 had 6.41 = 106 total DN; ayry 6 yry 6 .yry 6 yry 6TCR b had 1.81=10 ; a TCR d had 3.57=10 ; a TCR b=d had 1.77=10 ; and a Rag2 had 0.681=10

dTCR byry mice can have from 0.1% to 38% DP thymocytes, with an average of 14%. In real numbers, this ranges from a50-fold reduction to complete elimination of DP cells compared to normal numbers of DP in a thymus.

eThymic gd T cells do not develop normally.f Thymic gd T cells develop normally.gA few gd T cells develop from the thymus.hThe preTayry lacking both isoforms.igd T cells are more sensitive to IL-7 deficiency than ab T cells. Further, there are distinct roles for IL-7 in adult vs fetal

. yrydevelopment Crompton EJI 1998 . Finally, the earlier DN subsets may be somewhat disrupted in the IL-7R .

the preTCR in DN thymocytes. Added to these dif-ferences is a difference in assumption: while thepreTCR is proposed to signal clonal expansion ofnave thymocytes, the TCR is insufficient to achievethis in nave T cells, requiring instead additional

signals from co-stimulatory molecules reviewed in ref.37 .

The signalling potential of pTa

In considering how signalling from the preTCR maydiffer from TCR signalling, the most obvious differ -ence is the pTa chain. Ironically, its contribution to

thymocyte development has been muddied by some-what different results from two different preTa defi-cient mice. Fehling et al 38 found a DN to DP blocksimilar to that found in TCR byry mice, which con-trasted with a considerably less severe block de-scribed Xu et al39 in their strain of pTayry mice.The reasons for this seeming discrepancy are un-known. Nonetheless, a possibile explanation lies inthe discovery that there are two natural isoforms ofpTa : pTa a and pTa b.40 In pTa b, exon 2 whichencodes an Ig-like domain composing most of theextracellular portion of the molecule, is spliced out.Whereas Fehling et al mutated the transmembranedomain, precluding expression of either form of pTa ,

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Xu et al removed only exon 2, leaving open thepossibility that the mice still express pTa b.

Currently, the potential for pTa b to act as a func-tional form of pTa is under investigation. Nonethe-less, pTa b clearly encodes a detectable protein,40

and the likelihood that the protein is active is in-creased by the observations of Irving et al41 andJacobs et al42 that T cell development can be facili-tated by forms of the preTCR that lack the ectodo-mains either of pTa or of TCR b.

If the ectodomain of pTa is largely unnecessary forpreTCR function, the spotlight falls on the tran-smembrane and intracellular domains. Compared toother antigen receptor chains, pTa encodes an

.uniquely long 30 amino acids intracytoplasmic tail,traditionally the key to interaction with associatedsignalling molecules. Nonetheless, there is little evi -dence either for recognizeable signalling domains, orfor inherent enzymatic activity, and added to this, thetails of human and murine pTa are not highly con-served.43

Experimentally, the importance of the tail waslargely dismissed by the rescue of the pTayry

phenotype using a transgenic tail-less version ofpTa .44 Nevertheless, such dismissal is not obviouslynecessary. Not only was the rescue of thymocyte num-bers by the tail-less transgene incomplete, but thetail-less construct reduced thymocyte numbers innormal mice by ;50%, consistent with it acting as adominant negative construct. Additionally, tail-lesstransgenic mice displayed a novel phenotype, namelya significant fraction of DN cells with moderate levelsof detectable surface TCR b expression. While theauthors considered several explanations for this, thephenotype is consistent with the transgene acting as a

dominant negative, slowing the DN to DP transition.Is there any positive evidence for the action of the

pTa tail? The strongest homology between the hu-man and murine forms is a short proline-rich motif

.that is reiterated in the mouse gene Figure 2 . Thissequence is also conserved in the cytoplasmic tail of

.CD2 ref 45; Figure 2 , which in humans has beenstrongly implicated as a co-stimulatory molecule formature T cells. The position of the motif in the CD2tail is directly upstream of a motif recently found tobind an adaptor protein, CD2AP, that re-organizesmembrane receptor clustering.46 CD2 has beenshown to activate signalling in lck-deficient cells,47

and recently the reiterated proline-rich motif that isconserved in preTa was implicated in signalling byvirtue of its capacity to bind a protein, namedCD2BP2.48

pTa may provide signal 1 and signal 2 topromote thymocyte development

Intriguingly, CD2 signalling in a T cell line, provokeshigh levels of jnk activation, as does CD28 co-stimula-tion.49 Therefore, we proposed that the cytoplasmictail of pTa could confer on the preTCR the capacityto co-stimulate, in addition to transducing TCR-likesignals via the association of the preTCR with CD3.In this sense, the preTCR might provide to a cellsignal 1 and signal 2, that in mature cells are deliv-ered separately via the TCR and co-stimulatorymolecules, such as CD28 and CD2. Teleologically,signals 1 and 2 need only be separated in the periph-ery where the ectopic activation of T cells by selfantigen presenting cells alone needs to be prevented.

Figure 2. Proline Motifs in CD2 and preTa . There are conserved and repeated proline richmotifs in the cytoplasmic tails of CD2 and preTa from both humans and mice. In CD2 thesemotifs have been implicated in signalling. The function of the motifs in preTa is underinvesitgation.

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The precise nature of what co-stimulation providesto a cell has not been well established although twohypotheses are currently under study. One is thatco-stimulation sustains or qualitatively affects TCRstimulation by an effect on the physical nature of theTCR signalling raft at the cell surface.50 It is prema-ture to discuss this possibility regarding the preTCR,in part because a role for membrane rafts in sig-nalling as opposed to membrane protein turnover

.has not been established reviewed in ref 51 , and inpart because there is insufficient biochemical data onthe preTCR.

A second hypothesis is that co-stimulation is anti-apoptotic.52 It is commonly stated that preTCR sig-nals prevent the death by neglect of thymocytes, butthere is currently no published data attesting to this.To examine directly the capacity of the preTCR toinfluence apoptosis, we and our colleagues trans-fected the genes for TCR b , pTa a, and pTa b singlyor in various combinations into a TCRy., CD3q. cellline, 4G4. Preliminary data are that cells receivingcomplete preTCR components express higher levelsof bcl2, lower levels of fas, and higher levels ofNF B.53,78 The cells are markedly less susceptible tokinduced apoptosis than cells receiving TCR b alone,or, importantly, cells receiving TCR b plus a mutantform of pTa lacking the proline motifs.53 The valid-ity of these data was established by the re-examina-tion of thymocytes in vivo. Compared to E cells,b-selected L cells show a parallel and transient in-crease in bcl2, a decrease in fas expression, and alonger-lasting activation of NF B.53,78k

The dependence of these events on the prolinemotifs of pTa suggested that the effect might bemediated in the transfected cells by activation of jnk,analogous with the effects on T cell lines of CD3q

CD28 co-stimulation. Indeed, jnk activation was seenin cells receiving TCR bq pTa , but not those receiv -ing TCR bq pTa lacking the proline motifs. Whilethese data do not establish that jnk mediates theanti-apoptotic effects of the preTCR in vivo,54 theystrongly support the hypothesis that pTa has bonafide similarities with co-stimulatory signalling. Thusthe preTCR may reflect a single signalling complexthat provides a cell with multiple signals exceedingthose ordinarily provided by the TCR alone.

Parallel studies of defined signalling molecules willundoubtedly contribute to this picture. Thus our ownstudies have demonstrated that the c-myc gene isabsolutely required for thymocyte maturation, includ-ing development beyond Stage III, as a result ofwhich, c-mycyry thymocytes arrest following their re-

arrangement of TCRg , d and b genes N. Douglas et.al; unpublished data . Egr-1 an immediate-early gene

and transcription factor recently shown to be ex-pressed in DN thymocytes, has likewise been impli-cated in the TCR b selection step.55 Indeed, an Egr-1transgene driven by the lck proximal promoter on aRag2 deficient background provoked further matura-tion of the DN-arrested cells.56

gd cell development

The fact that the nature of gdrab lineage commit-ment is still ambiguous reflects the fact that we areuncertain of the primary developmental pathway ofgd cells. No cells have been identified that are uniqelyprecursors to gd cells, and the fact that gd cellscould be derived after transfer in vivo of Stage IV DNcells57 has fueled the notion that DN thymocyteshave developmental potential both for gd cells andab T cells.

It is clear that the development of gd cells requirestransition across a stage at which there is selection forcells with in-frame TCR d chains.58 Although the ca-pacity to detect selection on TCRg was more vari -able, there was nonetheless no strong evidence forthe action of a preTg or preTd chain, a conclusionsubsequently echoed by Raulet et al.59,60 Indeed,rather than there being a pTd chain, those authorsfound that TCRg can pair with pTa , and seeminglyprovoke a DN to DP transition. Nonetheless, the datapresented60 are compatible with the hypothesis thatthe g-pTa complex signals the DN to DP transitionvery poorly, perhaps because it is a relatively unstablecomplex. Additionally, TCRg expression is in someway suppressed in DP cells, limiting the viability of gd

cell development along that route previously termed58 .inviable route II by Passoni et al . For all these

reasons, it has come to be accepted that cells differ -entiating toward a gd cell fate receive a determin-ing, co-determining or facilitative signal fromTCRgd rather than a preTCR complex.65

TCRgd signalling

Does TCRgd signalling induce a different fate inthymocytes than preTCR signalling? In absoluteterms, the answer to this is no, since TCRgd depen-dent DP cells have been convincingly demonstratedin at least three systems.58,59,60 However, the TCRgddependent development of DP cells is extraordinarily

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Signaling in thymocyte development and lineage commitment

inefficient compared to that driven by the preTCR. .At least four reasons might explain this: i the signal

elicited by expression of TCRgd is a fate-determiningsignal that is different from that induced by the

.preTCR; ii there may be equally efficient genera-tion of DP cells, directed by expression of either

.TCRgd or the preTCR, but gd q DP cells will die .because of the extinction of TCRg expression; iii

there may be equally efficient generation of DP cells,directed by expression of another commitment factorand facilitated by either TCRgd or the preTCR, but

.again gd q DP cells will die because of the extinc- .tion of TCRg expression; and iv the differences

between signalling webs induced by preTCR andTCRgd signalling are primarily quantitative ratherthan qualitative, allowing some DP development tobe provoked by TCRgd and some DN cell matura-tion to be provoked by the preTCR.

Considering these explanations, it has become in-creasingly difficult to believe that fate is determinedentirely independently of TCR or preTCRsignalling.65 Our studies in 1995 demonstrated that gand d rearrangements in ab T cell progenitors werenot neutral events, strongly indicating that signallingfrom TCRgd influenced cell fate away from the ablineage.79 Likewise, the balance of gd cells and DPab progenitors has been significantly altered innumerous TCR transgenic mice in a way that isdifficult to reconcile with an entirely independentlineage determination mechanism. Indeed, Raulet etal found that expression merely of a TCRg transgenesignificantly disrupted commitment to the DP subset,despite the retained capacity to encode TCR b andpTa 58 The simplest explanation for these data is thatthe probability that a DN progenitor would success-fully encode TCRg was enhanced G2-fold, increas-ing by the same amount the likelihood that a cellcould express a gd heterodimer that would eitherdetermine or co-determine that cell toward matura-

.tion as a TCR q DN cell.In short, although the matter is far from unequivo-

cally resolved, there is good evidence that cells re-ceive a signal from TCRgd and that the outcome ofthat signal is most often different from that received

.via the preTCR. Because TCRgd q cells account forbetween 10- and 100-fold less thymocytes than DPcells, it has been broadly accepted that TCRgd failsto signal cell proliferation, while signalling relief fromapoptosis andror differentiation. In support of this,

lo y.Stage IV CD44 , CD25 DN cells that are activelycycling in normal mice are almost entirely stationaryin TCR byry mice.58

How could signals from TCRgd differ from thosetransduced by the preTCR? Since both the preTCRand TCRgd form complexes with CD3, the mostconspicuous difference between the complexesviewed from inside the cells would again be theextended cytoplasmic tail of pTa . Indeed, the ideathat signals from TCRgd account for only a subset ofthose transduced from the preTCR is consistent withthe argument developed above that the preTCR de-

.livers a combination of TCR-like signals signal 1 .and co-stimulatory signals signal 2 . Moreover, the

experimental finding that the preTCR can inhibitapoptosis in a pTa-dependent fashion53 may be con-sistent with the finding that gd cell development isfar more susceptible than is ab T cell developmentto deficiency in IL7, a major inhibitor of thymocyteapoptosis.62 ] 64 The hypothesis would be that in theabsence of preTCR signalling, gd cells retain a greaterdependence on IL7 for relief of apoptosis. This de-pendence may be too prolonged andror too com-plex to be relieved by provision of transgenic bcl2.

Further resolution of how thymocytes may receivedifferent signalling from TCR gd and the preTCR,respectively awaits improved biochemical analysis ofthe two signalling complexes. To date, this has beenconfounded by two factors: first, there are very few

.TCR gd q DN thymocytes, coupled with no capac-ity to identify their precise precursors; second, al-though there would be a greater number of DN andDP thymocytes expressing the preTCR, levels ofpreTCR expression are vanishingly low and have re-quired improved immunochemical techniques to de-

.tect L. Bruno et al, pers. comm. .

Lineage commitment by complete receptor orhemi-receptor signalling?

Given that thymocytes receive signals from either thepreTCR or TCRgd , and given that the nature ofthose signals will most likely be different, one canthen ask, do these molecular differences result in

disparate lineage commitment? reviewed in refs 65.and 66 We have discussed how ab T cell progeni-

tors are depleted of in-frame TCRgrd chain generearrangements, indicating that TCRgd signalling canbias cells against the ab fate.79 Likewise, there havebeen two observations that strongly favor a fate-de-termining role for the preTCR. First, pTa deficientmice harbor from 3- to 10-fold more gd cells than

. 38pTa q mice; second, it was claimed that TCR brearrangements in single gd cells sorted from

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.pTa q mice were predominantly out-of-frame,compared to an almost random pattern in pTayry

mice.67 Together these data suggested that .preTCR q cells are biased away from the gd lin-

eage.Nonetheless, there are conflicting data. Other

studies, analysing far greater sample sizes have indi-cated that TCR b rearrangements in populations ofgd cells are commonly in-frame.5,68,70 These data areconsistent with the observation that a measurable

.fraction of TCRgd q cells express intracellularTCR b protein.67 Therefore, expression of thepreTCR may not absolutely preclude gd cell develop-ment, in the same way that TCRgd signalling issometimes associated with DP cell development ratherthan DN cell maturation.58,59,61,71

To resolve this lack of absolute association, we and .independently Bruno and Owen et al have arguedthat fate determination requires a threshold level of

expression ref 65; L. Bruno and M.J. Owen, pers..comm. . Thus thymocytes expressing relatively high

levels of the preTCR will never become gd cells,whereas cells expressing lower levels are still availableto become gd cells contingent on successful rear -rangement and expression of TCRgrd chains prior

to DP differentiation L. Bruno and M.J. Owen, pers..comm. . This argument can draw on striking prece-

dent. The fate outcome for DP cells depends verymuch on the avidity of the interaction of TCR abwith peptide-containing major histocompatibility

.complexes MHC : low avidity provokes maturation;high avidity provokes a different signalling outcomethat induces apoptosis. Although in this system avidityis regulated by the nature and density of the ligand,one can speculate that at the late DN stage, the fateof thymocytes will vary according to their levels ofexpression of TCRgd or the preTCR, respectively.

If the primary difference in signalling betweenTCRgd and the preTCR is the presence in the latterof pTa , then one can predict that ectopic expressionon DN cells of a complete TCR ab would also commitcells to DN maturation rather than DP progression.Indeed this can be the case: numerous TCR abtransgenic mice in which the TCR a chain is ex-pressed ectopically early, display small thymi with

.heightened numbers of DN TCR q cells, and re-duced numbers of DP cells.72,73 Hence, we have pro-posed that operationally, signals received by thymo-cytes will vary according to whether they are induced

.by a complete TCR gd or ab or an hemi-TCR . 65preTa .b .

Cell-autonomous or induced lineagecommitment?

It has been shown that the preTCR must escape fromthe endoplasmic reticulum in order that it effectivelypromote thymocyte maturation.74 By any orthodoxviewpoint, this escape to the cell surface would allowthe preTCR to interact with a ligand, most likely on astromal cell, facilitating preTCR signalling. However,there has been no success in identifying one or morepreTCR ligands, just as there has been no success inidentifying a preBCR ligand, despite the fact that thisissue has been pursued for approximately a decade.

Therefore, it has been proposed that the preTCRdoes not engage a ligand, but that it signals simply byclustering on the thymocyte surface. This is consis-tent with functional activity from a form of the

.preTCR lacking the ectodomains ref 41; see above .Although this model can be glibly invoked, there hasuntil recently been no obvious precedent for it in cellbiology. Rather, all the experimental models for sig-nalling cascades involving tyrosine phosphorylation,GTP-dependent ras activation, and subsequent ser-ine]threonine kinase activity of the MAP-kinase familymembers, have been built on receptor cross-linkinginduced by ligand binding. Nonetheless, Goedell et alrecently identified a cell protein that inhibits the

.spontaneous capacity of tumor necrosis factor TNFreceptor complexes to aggregate and to signal in aligand-independent fashion.75 Therefore, the preTCRmay be a receptor that signals in a ligand-indepen-dent fashion. Interestingly, the same may be true indevelopment for TCRgd and for TCR ab. Althoughligands for TCRgd are insufficiently well charac-terised to examine this question directly, there is noevidence that MHC interactions dictate or even in-fluence the DN cell maturation provoked by trans-genic TCR ab expression. The major significance ofthis issue is its reflection of whether or not DN cellmaturation events are cell autonomous or induced bystromal cells. Unquestionably, thymocyte maturationis stromal cell-dependent, but during the DN period,this may primarily reflect the provision of growthfactors, notably IL7, prior to b-selection.

Other views of lineage commitment are very dif-ferent, attaching immense importance to stromalcell]thymocyte interactions. Predominant among the

molecules invoked to facilitate this are Notch ex-. pressed on thymocytes and Notch ligands expressed

.on stromal cells . Robey and her colleagues found

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Signaling in thymocyte development and lineage commitment

that transgenic TCRgd cells on a Notch transgenicbackground more often differentiated as DP cells,toward an ab T cell fate, than did so in the unma-nipulated TCRgd transgenic mice.76 Mechanistically,Notch could be increasing the level of some signallingmolecule which influences this decision. Likewise,there is an increased representation of DPs inTCR byry mice that express an activated Notch trans-gene. Again, an increase in the concentration of thesignalling molecule could accomplish this. A corrol-lary experiment showed that proportionately moregd cells developed from bone marrow heterozygousfor Notch, than developed from Notchqrq bone mar-row. Nevertheless, the effects of Notch do not repre-sent an alternative, TCR-independent pathway of lin-eage commitment, since the authors showed that

.TCR b y thymocytes that developed as DPs wereselected for in-frame d genes. Hence completeandror hemi-TCR signals seem to be required atminimum for facilitating commitment.

Defining the precise interactions of Notch withcomplete- and hemi-TCR signalling in T cell develop-ment will not likely to be easy. From studies in myriadbiological systems, it would appear that Notch is highlypleiotropic, with among other effects, a capacity toinhibit apoptosis. Therefore, the overexpression ofNotch may simply enhance the survival of cells thatwould ordinarily have died.77 Likewise Notch and rasare so commonly involved in fate decisions that it isunclear, by the very fact of their widespread involve-ment, that these molecules can confer real specificity.Possibly they act as facilitative factors that modulatethe capacity of a cell to respond to specific determin-ing factors.

Such issues await resolution. We reiterate that animproved definition of thymocyte signalling pathwaysand webs that are activated by engagement of thepreTCR, TCRgd , and Notch should clarify whetherNotch is upstream of, downstream of, or parallel topreTCR signalling, and whether any one set of signalsare more obviously those that commit cells to oneidentifiable fate, while precluding others. For a signalto determine fate in this way, it will presumably leadto irreversible changes in chromatin conformationthat terminally affect gene expression. Since webelieve that preTCR signalling and TCRgd signallingcan achieve this, as measured by allelic exclusion ofgene rearrangement,69 those molecules and the sig-nals they provoke remain strong candidates for eitherdeterminants or co-determinants of lineage commit-ment.

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249

Introduction to lineage commitmentT cell developmentFigure 1.

Genetic dissection of preTCR signalling similarities to the TCRTable 1.

Genetic dissection of preTCR signalling differences from the TCRTable 2.

The signalling potential of pTapTa may provide signal 1 and signal 2 to promote thymocyte developmentFigure 2.

cell developmentTCRgd signallingLineage commitment by complete receptor or hemi- receptor signalling?Cell-autonomous or induced lineage commitment?References