Functions of TCR and pre-TCR subunits: lessons from gene ablation

Functions of TCR and pre-TCR subunits: lessons from gene ablation Bernard Malissen* and Marie Malissent

Recent gene-targeting experiments have highlighted the

existence of checkpoints that ensure that ap T cells do not

complete intrathymic differentiation if they have not attained

certain landmark events. These ‘proofreading’ mechanisms

operate by way of the pre-TCR and TCR complexes, which

are sequentially expressed during T-cell development.

These complexes are likely to signal via their associated

CD3 subunits. By activating intracellular effecters, the CD3

subunits probably modulate gene expression profiles and

drive the maturing aB T cells through a precise developmental

sequence.

Address Centre d’lmmunologie, INSERM-CNRS de Marseille-Luminy, Case 906 13288, Marseille, Cedex 9, France *e-mail: [email protected]

te-mail: [email protected]

Current Opinion In Immunology 1996, 8:383-393

0 Current Biology Ltd ISSN 0952-7915

Abbreviatlons

BCR

C

D

DP

ER

ES

19 IL

ITAM

J

PTK

PTP

RAG

scld

SP

TCR

TN

V

B-cell antigen receptor

constant

diversity double positive

endoptasmic reticulum

embryonic stem

immunoglobulin

interleukin

immunoreceptor tyrosine-based activation motif

joining

protein tyrosine kinase

protein tyrosine phosphatase

recombination activating gene

severe combined immune deficit

single positive

T-cell receptor

triple negative

variable

Introduction The specific recognition of antigen by T cells and its ensuing transduction into intracellular signals are accomplished by a multi-subunit transmembrane complex denoted as the TCR-CD3 complex. On the basis of the structure of the TCR antigen-binding subunits found in their TCR-CD3 complex mature T cells can be divided into two subsets. In the adult, most of them express TCR heterodimers consisting of a and B chains, whereas a minor population expresses an alternative TCR isoform consisting of y and 6 chains. The TCR a, B, y, and 6 chains each comprise an amino-terminal, clonally variable (V) region and a carboxy-terminal constant (C) region. Peptide loops homologous to immunoglobulin (Ig)

complementarity-determining regions (CDRs) are found at the membrane-distal end of the V regions, where they collectively form the binding site for antigenic peptides that are bound to products of the MHC.

Transport of the TCR heterodimers to the cell surface is dependent upon their prior assembly with a set of invariant subunits designated CD3y, CD36, CD~E, CD35, and CD3q (Fig. 1). The cytoplasmic segments of the various CD3 subunits are responsible for coupling the antigen binding TCR-aB or $5 heterodimers to intracellular signalling pathways. In contrast to the situation observed with numerous growth-factor receptors (such as those for insulin or platelet-derived growth factor), none of the CD3 subunits possesses a cytoplasmic domain endowed with recognizable enzymatic activity. However, each subunit contains one or multiple copies of a recurrent cytoplasmic sequence that fully accounts for their individual transducing capacity. These conserved sequences, referred to as the immunoreceptor tyrosine-based activation motifs (ITAMs), are also found in the transducing subunits of the antigen receptor of B lymphocytes (BCR), and the receptors for the Fc domain of IgE (FceRI) and IgG ( FcyRIIIA).

Following stimulation with antigen, protein tyrosine kinases (PTKs) belonging to the Src family (e.g. Lck, Fyn) phosphorylate conserved tyrosine residues present within the ITAMs. Once phosphorylated, the ITAMs act as high-affinity docking sites for the SH2 (src-homology 2) domains found in certain intracellular adaptor and effector molecules, among which are PTKs belonging to the Syk/ZAP-70 family (e.g. Syk, ZAP-70). The relocalization and/or clustering of Syk/ZAP-70-family PTKs into the receptor complex promote their activation and contribute to the successful progression of the activation program. Re- cruitment and activation of protein tyrosine phosphatases (PTPs) are likely to occur at later timepoints and be responsible for the termination of signal transduction. Therefore, the minimal core of the clustering-activated switch operated by the TCR-CD3 complex appears to be composed of an antigen-binding unit, a set of SHZ-docking sites (ITAMs), a few members of the Src- and Syk/ZAP-70-family of PTKs, and at least one PTI? Gene targeting experiments aimed at understanding the function of these TCR-CD3 proximal components will be the focus of this review.

Modular architecture of the TCR-CD3 complex Assembly of the TCR-CD3 complex occurs within the endoplasmic reticulum (ER) and appears to proceed in an ordered mode involving the initial asso-

304 Lymphocyte activation and effector functions

ciation of nascent TCRa and TCRB monomers with CD36c and CD3p dimers, respectively. These intermediate trimeric building blocks further combine to form CD36eTCRa-TCRBCD3w subcomplexes within which disulfide bonding of the TCR polypeptides occurs [l]. Upon association with disulfide-linked CD3-c homodimers, the complete TCR-CD3 complexes exit the ER and are efficiently transported to the cell surface.

The stoichiometry of the TCRaB, CDac, CD36e and CD35tj pairs present within a given complex is not known. Interestingly, the noncovalent nature of the interactions keeping these various polypeptide pairs together at the cell surface permits, as documented in at least two instances [2*,3*], the rapid turnover of a single pair of components independently of the rest of the complex. Moreover, the modular architecture of the TCR-CD3 complex favors the occurrence of combinatorial isoforms consisting of distinct subunits. For instance, in mouse gut intraepithelial T lymphocytes, homodimers consisting of the y chain of the FceRI can be incorporated into TCR-CD3 complexes in lieu of CD35 homodimers [4,.5]. Furthermore, in contrast to mature T cells, early thymocytes can assemble a unique TCR-CD3 isoform devoid of TCR a chain (Figure la). These results point to previously unrecognized levels of TCR-CD3 receptor diversity, and open the possibility that some of these TCR-CD3 combinatorial isoforms are responsible for coupling ligand recognition to distinct complements of intracytoplasmic effecters, providing various signal-transduction options. Finally, it is worth mentioning that the rules controlling the assembly and intracellular transport of TCR-CD3 complexes may differ according to the stage of development or between the aB- and yS T-cell lineages. For instance, in the former lineage, the evolutionary related CD3y and CD36 subunits appear unable to substitute for each other in that aB T-cell lines deprived of either CD3y or CD36 fail to express normal levels of TCR at their surface [6,7]. In contrast, the recent analysis of mice deficient in CD38 has revealed that $5 T cells lacking CD36 are still capable of expressing normal levels of functional TCR-CD3 complexes at their surface (D Kappes, personal communication). The reason for such interlineage differences has yet to be determined.

Mouse T-cell development The current approaches used for generating mice that carry intended mutations in the germline involve the introduction of null mutations into embryonic stem (ES) cells, from which homozygous mutant mice can be derived. Because the null mutations are carried in the germline, they manifest their effects throughout ontogeny and sometimes result in embryonic lethality or prevent the development of a given cell lineage. As discussed below, the constitutive inactivation of most of the genes coding for TCR-proximal components has been found to affect the processes that govern intrathymic T-cell development and has precluded the analysis of their

respective effects in mature peripheral T cells. Therefore, the experimental results discussed in this review mostly bear on the signalling functions played by the proximal components of the TCR-CD3 complex during the early phases of intrathymic T-cell development.

Intrathymic T-cell development proceeds through discrete stages, which can be defined by the expression of cell surface markers and the configuration of TCR gene loci (see Fig. 2). The most immature cells identified in the adult thymus are present in small numbers (representing 0.2% of all thymocytes) and express low levels of CD4. They carry their TCR loci in germline configuration and may develop along the aB or y8 T-cell lineages following intrathymic transfer. During the initial step of maturation, these ‘CD4low precursors’ lose their expression of CD4 to become ‘triple negative’ (TN)(CD4CD8_CD3-) cells. The differentiation of TN cells is marked by the transient expression of the IL-Z receptor a-chain (CDZ) and the loss of expression of both CD44 and CD117 (c-kit). Late TN cells can progress to the CD4+CD8+ (‘double positive’ [DP]) stage via intermediates that express either CD8 (for the majority of mouse strains) or CD4 in the absence of mature-type aB TCR complex and are therefore called ‘immature single positive’ (ISP) cells. A small percentage of the DP cells mature further into CD4+CD8- or CD4- CD8+ (‘mature SP’) cells that correspond to the end products of the intrathymic aB T-cell differentiation pathway and gradually exit from the thymus to reach peripheral lymphoid organs. It takes 11 to 15 days for the earliest CD4low precursor cells to develop into DP cells. This process is associated with two discontinuous phases of cell expansion (Fig. 2). The first starts at the CD44+CD25+ TN stage and stops during or just after the onset of downregulation of expression of CD44. Cell proliferation resumes concurrent with loss of expression of CD25 and continues up to the early DP stage at which time it declines again [8].

The genes encoding the TCR V regions are assembled via somatic site-specific DNA recombination reactions triggered by the specialized stromal microenvironment found in the thymus. These reactions, termed V(D)J rearrangements, result in the random recombination of single elements taken from two or three libraries of subexons encoding the V, D, and J gene segments, respectively. During V(D)J recombination, the accessible coding gene segment ends are generally subjected to various degrees of base deletion, addition, or both, before ligation. As a consequence, V(D)J joining reactions may result either in productive rearrangements that maintain an open-reading frame throughout the gene, or in an out-of-frame, nonfunctional gene. The products of the recombination activating genes 1 and 2 (RAG-l and RAG-Z) are the only lymphoid-specific components that are required for effecting the site-specific double strand breaks associated with V(D)J recombination [9’]. Thymocytes from RAG-deficient and severe combined

Functfons of TCR and pre-TCR subunits Maiissen and Maiissen 305

Figure 1

(a)

CD3 &

TCR

0 1996 Current Opimon in Immunology

(CD3 5)2

(W TCR TCR

CD3 &

CD3 CD3

(CD3 1)~

Putative subunit composition of the pre-TCR-CD3 (a) and TCR-CD3 (b) complexes found in immature and mature T ceils, respectively. The pre-TCR a chain (pTa) and TCR5 polypeptides are expressed within the prsTCR-CD3 complex as disuifide-linked heterodimers and possess short cytopiasmic tails (4 to 30 residues). The pTa transmembrane region contains polar residues (D,E,R,K in the single-letter amino acid code), two of which (R and K) can be rigorously aligned with the arginine and iysine found in the transmembrane segment of the TCRa and TCRG chains. (b) in the TCR-CD3 complex, the non-covalent interactions between the TCR 5 chain and CD3ys dimer are mainly controlled by the ig-like ectodomains, whereas those between the TCRa chain and CD35s dimer depend primarily on residues located within the transmembrane segment 1611. A phenyiaianine residue at position 195 of the TCR Co region (a) has been shown to be essential for the interaction of cytopiasmic CD3< homodimers with the TCRafl-CD3@ core [62]. The absence of a phenyiaianine residue in the corresponding region of pTa probably accounts for the weak association existing between the pre-TCR and CD3c [46]. The ITAMs (immunoreceptor tyrosine-based activation motifs) found in each of the CD3 subunits are shown as cylinders containing tyrosine-based docking motifs (YLYL). The cytopiasmic tails of the CDSE, CD3y and CD35 subunits each contain a single ITAM. The CD3c poiypeptide displays three concatenated copies of the ITAM. Putative N-linked carbohydrate sites are indicated by black dots. The circles show sequence segments that fold as for a C or V immunogiobuiin domain or are predicted to do so. (S-S, disuiphide bond.)

immunodeficient &id)-mice are unable to undergo V(D)J recombination and their development is blocked at the CD44-floWD25+ TN stage (Fig. 2). TCR p gene rearrangements precede rearrangements at the TCR a locus

and start at, or at the transition to, the CD44-/loWD2.5+ TN stage of development. As shown in Figure 2, TCR p rearrangements are essentially completed before the transition to the CD44-/lowCD25- TN stage. The first TCR a rearrangements, as inferred from the presence of 1.6 kb TCR a transcripts, can be detected during, or immediately after, the transition to the DP stage of development [lo].

If thymocytes fail to rearrange their TCR genes, rearrange them non-productively or express TCR a0 combinations with inappropriate specificities, they are generally arrested at discrete developmental control points (Fig. 2). Checkpoints have evolved to ensure that the transition through these control points are coupled to the attainment

of certain landmark events in T-cell development. One checkpoint controls the transition from the DP to the SP stage (a phenotypic shift often denoted as positive selection or TCR aJ3 selection, Fig. 2) and its activation is a rare event depending on both the ligand specificity of the clonally variable TCR afi dimer and the efficiency of the selection process. The other checkpoint occurs earlier in development and controls the transition from the CD44-/loWD25+ to the CD44-/loWD25- TN stage of development. A host of experiments (for examples, see [ 1 l-131) suggest that the TCR b chain plays a unique role in the activation of this early developmental checkpoint, independent of its later function as a component of the aJ3 TCR heterodimer. For instance, studies aiming at complementing RAG-deficient and scid mice with productively rearranged TCR a or TCR J3 transgenes have revealed that the expression of TCR fi chain is sufficient to relieve the developmental blockade observed in these mutant mice and to restore maturation of their thymo-


Fiaure 2

(a) - TN -

CD410 precursors

Double 44+ 44’0 44’0 ‘S

Single positive positive

25+ 25+ 25- P 4+ 3+ 4+ a-or 4- 3+

I I I I I I I I I I I I I I 2 4 6 8

zk?r I/ />IL&

t SEEDING t t

TCRP- TCRab- DEPENDENT DEPENDENT SELECTION SELECTION

TCR p rearrangement

TCR a rearrangement

RAG-l and RAG-2 transcripts

pTa transcripts

Development in mice deficient in:

RAG-l or RAG-2 RAG + CD3Qq RAG + Ick CD3-&A5 TCRP + TCRS TCRB CDs-&/q Ick CD3-6 TCRa ZAP-70 CD4 + CD8 MHC class I + class II

pTa CD45 Common cytokine receptor ychain I L-7 vav

RAG + TCRa tg RAG + TCRP tg RAG + TCRa p tg RAG + Ick Y505F tg RAG + Ha-ras’J’2 RAG + anti CD3-E (RAG + CD3@$ + anti CD3-e (RAG + Ick + anti CD3-E (RAG + hCD25/E ITAM) + anti-CD25 (RAG + hCD25/5 ITAMs) + anti-CD25 CD3-@l + anti CD3-E

Overexpression of transgene coding for:

Ick K273R (kinase-) Ick Y505F (activated form) o2, Ha-ras N17

&lEK-1 K97A (kinase-) fyn K*ssE (kinase-)

I *

Null mutations without noticeable effects: fyn, syk, CD3-q, FERIP~, TCRG, CD25 0 1996 Current Opinion m lmmunologj

Functfons of TCR and pre-TCR subunits Maiissen and Maiissen 307

cytes beyond the CD44-flowCD2.5+ TN stage [11,12]. Consistent with these observations, a null mutation of the TCR 8 locus was found to be capable of blocking thymocyte differentiation at an earlier stage than a null mutation of the TCR a locus [l&13]. Collectively, these data have led to infer that the achievement of productive TCR b rearrangements constitutes a rate-limiting factor in the assembly of a pre-TCR-CD3 complex capable of triggering the progression beyond the CD44dow CDZ+ TN stage. This early round of thymic selection is refered to as TCR b selection (see Fig. 2).

Components of the pre-TCR-CD3 complex Considering that only minute amounts of complexes containing TCR p chain can be detected at the surface of thymocytes prior to the expression of TCR a chain expression [10,11,13], most of the biochemical evidence supporting the existence of a pre-TCR-CDJ complex have been gained from the analysis of a s&derived immature T-cell line stably transfected with a productively rearranged TCR 8 chain gene [14]. In contrast to pre-T thymocytes, these transformed pre-T cells express high levels of TCR 0 polypeptides at their surface. In the absence of TCR a chain, not expressed in this scid cell line derivative, TCR fi chain was found covalently associated with a 33 kDa type I transmembrane glycoprotein containing a single extracellular Ig-like domain and a 30 residue cytoplasmic tail (Fig. la). This novel polypeptide, denoted as the pre-TCR a (pTa) chain, is encoded by a non rearranging gene that is expressed in TN cells and switched off in late DP thymocytes [15*,16,17]. The extracellular domain of pTa lacks a covalently associated Ig-like V domain. By analogy with the VpreB subunit of the pre-BCR, the pre-TCR may contain an as yet unidentified VpreT component capable of pairing with the hydrophobic surface of the VP domain [18]. Despite the

Figure 2

low levels of sequence identity existing between TCRa and pTa [16], the latter is likely to serve, in part, as a substitute for TCR a (Fig. la).

The identity of some of the CD3 chains associated with pTa-TCR p heterodimers remains controversial. For instance, although CD3-y has been identified in TCR-p-containing complexes isolated from surface-iod- inated TCR a-l- mutant thymocytes, CD38 has been more difficult to detect and is hypothesized to be only weakly, or not at all associated with the pre-TCR complex

(a) The development of a cohort of precursor ceils after

entering wild-type adult thymus (adapted from [a]). TCR t!t selection refers to the early selection event that controls the CD44AQWCD25+

to CD44-‘t”CD25- transition and, in mice deprived of $+ T cells, depends on the expression of a pre-TCR complex following in-frame

rearrangement of TCR p chain genes. Following the production of TCR a chain at the DP stage, displacement of pTa from the pre-TCR complex

and assembly of a TCR up-CD3 complex, DP ceils can undergo a second phase of selection denoted as TCR up selection. in sharp contrast

to TCR p selection, TCR afl selection is reflected in cellular differentiation rather than cellular expansion. Also shown is the pattern of expression of transcripts corresponding to the RAG-l, RAG-2 and pTa genes, the two periods at which TCRfS and TCRa genes rearrange and the position

of two major developmental checkpoints. (b) The extent of development in thymus of mice deficient and/or transgenic for a few selected

genes. For each mouse, the phases of development that proceed normally (both in terms of ceil yield and surface phenotype) are shown

as a continuous line, whereas those that are impeded are depicted by a dashed line. injection of antiCD3s antibodies or compiementation

with productively rearranged TCR fI chain (but not TCR a chain) transgenes (tg) suffice to relieve the developmental blockade observed in RAG-deficient mice However, further maturation to the SP stage was still blocked in the TCR f3 reconstituted- or anti-CD3e injected-RAG-‘- ’ mice. By contrast, the expression of matched pairs of TCR a and TCR f3 transgenes leads to further maturation into SP ceils expressing single

TCR af3 combination. Examination of the thymocytes found in mice deficient in CWrjrl 1321, bk 133.1, and pTa (381 poiypeptides indicated

that 10 to 15% of their DP ceils still express CD25. Consistent with this observation, the TN ceils found in these mice appear to ‘jump’ over the

CD44-‘twCD25- developmental stage and make a direct transition from the penultimate CD44- /lowCD25+ stage to the DP stage. As previously noted [53’], the apparent phenotypic discrepancy existing between ick-I- mice and mice overexpressing catalytically inactive form of ick (Lck

K273R) may be accounted for by the propensity of the latter to compete not only with ick, but also with alternative src-family kinases (e.g. fyn).

inactivation of the genes coding for the common cytokine receptor y chain 163,641, vav [65-67) and IL-7 [SE] drastically reduces the numbers of

thymocytes without affecting the relative representation of the CD4/CD8 subsets.

388 lymphocyte activallon and effector functions

scid mice [Z&23]. In contrast to TCRa-I- thymocytes, complexes containing TCR B expressed at the surface of these pre-T-cell lines were found associated with both CD3& and CD3y dimers. It is important to emphasize, however, that these transformed pre-T-cell lines differ markedly from bonafide pre-T thymocytes in that they express high levels of pre-TCR-CD3 complexes at their surface. The reason for such a unique property is unknown and it is presently unclear whether it may result in the expression of pre-TCR-CD3 complexes with distorted stoichiometry.

The presence of CD3c chain within the pre-TCR complex was first investigated on serological and biochemical grounds [ 19*,20,21*,22,23], and further substantiated by the analysis of mice with a targeted mutation of the CD3.c gene [24*]. This mutation, referred to as CD3&*s, was still capable of directing the synthesis of CD3& polypeptides, but in minute amounts and deprived of the extracellular domain involved in the interaction with CD36 and CD%. Unexpectedly, the CD3& mutation also reduced the rate of transcription of the neighboring CD38 and y genes. The addition of these effects prevented the formation of cD3~ and CD3& heterodimers, and resulted in the complete absence of T cells expressing detectable levels of TCR aB or yS heterodimers at their surface. Moreover, CD3&*5/*5 thymuses were found to be devoid of DP and SP cells, but retained almost normal absolute numbers of TN cells. Therefore, CD3&*5/*5 thymocytes appeared to be arrested at the very same developmental control point as thymocytes deficient in RAG (Fig. 2). To specify the degree of coincidence existing between the developmental arrest points observed in CD3&*5/*5 and RAG-l-/- thymus, their respective TN T-cell populations were monitored for the expression of CD25 and CD44. RAG-l-I- and CD3.#/*5 thymocytes were both unable to progress beyond the CD44-/lowCD25+ stage, and showed no gross distortion in the representation and Brdu-labeling profile of the CD44/CD25 subsets found prior to the developmental arrest point [Z]. Therefore, these data indicate that the CD3& mutation manifests itself first at the CD44-/loCDZS+ to CD44-/loCD25- transitional stages and suggest that the cD3~ and/or CD3& pairs are mandatory for pre-TCR assembly.

The absence of intact CD3& polypeptides has not been found to detectably affect neither DBJB nor VBDBJB rearrangements [24’]. Because CD3cAs/As thymuses constitute an enriched source of RAG+, CD44-flowCD25+ thymocytes devoid of contaminating downstream subsets, they provide the opportunity to determine whether TCR a rearrangements occur at the CD44-/lowCDZS+ stage, concurrently with TCR B gene rearrangements. PCR analyses of DNA showed that if Va-Ja rearrangements do occur in CD3+5/*5 thymocytes, it is at a frequency of at least lOO-fold lower than that observed in total wild-type thymocytes [Z]. Therefore, the vast majority of early T cells do not rearrange their TCR a loci

before the transition to the CD44-/lowCD25- stage, and this suggests that signaling through the pre-TCR contributes to the induction of a high rate of TCR a gene rearrangements. In sharp contrast, the CD3& mutation had no discernible or, more importantly, no differential effect on the occurrence and extent of TCR y and TCR 6 rearrangements [24*]. Thus, the completion of TCR y and TCR 6 rearrangements is not subjected to CD3e associated epigenetic controls similar to those operating during aB T-cell development.

CD3+ye and CD36e pairs can be expressed at low levels at the surface of TN cells before the expression of TCR B chain [19’,21’,26,27]. These dimers are associated with calnexin, a 90 kDa molecular chaperone that normally resides within the ER. Moreover, they are functionally competent in that their cross-linking with anti-CD3c monoclonal antibodies is sufficient to induce the faithful1 maturation of RAG-I- thymocytes into DP cells [ZS’]. Considering that the overexpression of various CD3a transgenes blocks thymocyte development at the CD44+CD25- TN stage, such signal-competent subcomplexes have been hypothesized to be part of a putative pro-TCR complex involved in controlling the transition beyond the CD44+CD25- stage [29*]. It is unlikely, however, that such CD3 components have a normal signaling function before the expression of TCR B, as CD3& mice produce T cells that can reach the CD44-/10w CD%+ stage and rearrange their TCR B locus [24*,253.

Biochemical studies have suggested that the pre-TCR complex maintains a weak association with CD3< [ZO,ZZ]. These initial clues have been recently conforted by the analysis of the signaling function of the pre-TCR expressed at the surface of transformed pre-T cell lines [30’], and the development of several independent lines of mice lacking both the CD3< and eta polypeptides [4,5,31]. CD3-5&/- mutant mice have small thymuses (2.4~ 107 cells on average) containing cells that show a profound reduction in the surface levels of TCR complexes and are arrested at the DP stage of development. Within a given CD3</n-I- inbred line, the number of DP-cells was found to vary considerably from animal to animal. For instance, CD3-5/n-/- thymuses can contain from two- to 30-fold less DP cells than those from age-matched wild-type littermates. Substantial interindividual variations in the number of DP cells were also noticed in TCR B-l- thymuses [ll]. The source of such variations is presently unclear. The CD3</n-/- mutation had no effect on the rearrangements of the TCRB, y, and 6 genes [4,5]. Analysis of the TCR a gene rearrangements found in CD3</n-/- thymocytes revealed that their frequency was about 30-fold lower than in wild-type thymocytes [25]. Consistent with this estimate, CD3&l-l- thymocytes showed low levels of TCR a transcripts, providing a potential for the synthesis of only limited amounts of TCR a polypeptides [25,31]. In the absence of CD3t;

Functions of TCR and pm-TCR subunits Malissen and Malissen 389

and CD3q polypeptides, it is likely that the few resulting subcomplexes (TCRao CD3ye CD36.a) are sequestered within the DP cells and thus prevented from inducing further maturation to the SP stage. The reduced numbers of DP cells found in CD35&/- mice, their limited content of rearranged TCR a gene segments [ZS], their residual expression of CD25 [32], as well as their resistance to dexamethasone-induced apoptosis [33*], concur to indicate that the CD35/71 mutation does affect both quantitatively and qualitatively the generation of DP cells. Therefore, disruption of the CD3& gene appears to influence a8 T-cell development at two distinct timepoints: first, by limiting first the generation and/or survival of DP cells; and second, by blocking the transition to the SP stage of the few DP cells capable of synthesizing appropriate TCR ab combinations. As shown in Figure 2, by contrast to the CD3& or CD36 mutations, the CD3qn mutation does not properly fit into a twocheckpoint model of a0 T-cell development in that it corresponds primarily to a leaky mutation of the checkpoint operating at the CD44-~l~WD25+-CD44-~l~~CDZ.S- transition.

The differential contribution of the modules containing CD3e and CD3< [34] to the pre-TCR complex may result from their unique signaling properties or distinct scaffolding role. According to the latter hypothesis, the lack of CD3e may prevent the assembly of pTaTCRp and CD355 dimers into functional pre-TCR subcomplexes, whereas, in the absence of CD31;, pre-TCR subcomplexes made of pTaTCRb, CD3yr~ and/or CD36e dimers may still assemble and activate, albeit inefficiently, the CD~~-/~~~CDZ~+~CD~~-/~OWCDZS- transition. Alterna- tively, subcomplexes made of pTaTCRp and CD3<< may readily assemble in the absence of CD3a, but the CD3t; ITAMs may be devoid of any signaling activity at this stage of development. To determine whether the ITAMs found in the cytoplasmic segments of CD3& and CD35 have distinct signaling capacities once expressed in CD44-/lowCDZS+ TN cells, chimeric molecules including the extracellular and transmembrane regions of the human IL-Z receptor a chain (hCDZ5) and the cytoplasmic tail of either CD3& or CD3< have been expressed separately at the surface of RAG-deficient thymocytes [35*]. Upon cross-linking with anti-hCD25 antibodies, both chimeras were found equally capable of inducing the progression to the DP stage (Fig. 2). Therefore, these data indicate that the CD3e ITAM does not mediate a specific signaling function during TCRB selection and suggest that the mandatory contribution of the module containing CD3e to the operation of the CD~~-/~~WCDZS+~CD~~-~~“CDZS- transition checkpoint is rather related to its unique nucleating role during the assembly of pre-TCR complexes, According to this view, CD3< homodimers would merely constitute a dispensable amplification module for the pre-TCR complex, increasing its assembly rate and stability [36], as well as contributing, by way of additional

and redundant ITAMs [33*,3.5*], to the strengthening of the signals induced by the pre-TCR complex.

Role of the pre-TCR complex The pre-TCR/CD3 complex appears to constitute a molecular sensor operating at the CD44-/loWDZS+ TN stage and coupling further maturation to the prior achievement of productive TCR fi gene rearrangements. It is not yet clear whether the pivotal role played by the pre-TCR during TCRfi selection requires pre-TCR expression at the cell surface or relies on its ability to sense some intracellular features associated with the presence of TCR g polypeptides. Along that line, it is interesting to note that RAG-l-I- thymocytes expressing a rearranged TCRfl transgene with a near complete deletion of the Vj3 region can reach the DP stage [19’]. The cascade of events triggered by the pre-TCR is likely to permit the selective survival, expansion and differentiation of TCRfl positive CD44-fl”WDZ5+ cells. Mice deficient in TCR p polypeptide should resemble ~~~~~~~~~~~~ 5 mice and be ranked as ‘non-leaky’ pre-TCR checkpoint mutants. However, the presence of small numbers of both CD44-/lW2DZ- TN and DP cells in TCRB-I- mutant mice [11,37*] apparently contradicts the above views as it shows that the TCR g chain is sufficient, but not necessary, for the progression of development beyond the CD44-floWD25+ stage [38]. Similarly, disruption of the gene coding for the pTa subunit should have prevented the assembly of pre-TCR complex and halted the progression beyond the CD44-/loWDZS+ TN stage. Nevertheless, despite the expected absence of CD44-/loWCD25- cells, pTa-I- mice were found to contain small numbers of DP and SP cells that expressed normal levels of TCR ap dimers on the cell surface [39’]. These data suggest that the pre-TCR complex may constitute a leaky molecular sensor, capable of being occasionally fired in the absence of pTaTCRg dimers. However, the finding that thymocytes present in RAG-/; TCRfi-I-xTCRW as well as CD3&*5/*5 mutant mice do not progress beyond the CD44-/lowCDZ.S+ TN stage [24*,37*] permits the latter possibility to be dismissed and further suggests the existence of maturation pathway(s) independent of pre-TCR that are capable of inefficiently promoting the transition through the CD44+WD25+ to CD44-/l~WD25- control point. It is worth emphasizing that TCRB- and pTa- deficient mice differ from RAG-/; TCRf?j-xTCRG-/- and CD3&*5/*5 mutant mice in that they contain y6T cells. The latter may provide, directly or indirectly (i.e. via interaction with the thymic stroma), intercellular factors that promote the survival of a few CD44-/lowCD25+ cells and permit their differentiation to the DP stage in the absence of clonal expansion. Consistent with this latter possibility, thymocytes with defective V(D)J rearrangement from scid mice are blocked at the CD44-floWDZS+ stage, but can be induced to become DP by ‘tram signals emanating from adoptively


transfered TCRafi+ [40] or TCRyG+ [41] thymocytes. In this context, it is interesting to note that the induction of DNA damage can also trigger the generation of small numbers of DP cells in RAG-/- thymus [42,43].

The findings described above suggest that differentiation to the DP stage can be uncoupled from the presence of pre-TCR and point to an alternative model of pre-TCR function in which its primary role may be enabling, rather than inductive. According to this model, and as previously hypothesized for hematopoietic growth factor receptors [44] and the pre-BCR [45], the function of the pre-TCR may not involve the induction of differentiation toward the ap T-cell lineage, but rather be limited to trigger signals that promote the survival and/or proliferation of the TCRB+, CD44-/lowCDZS+ cells and, indirectly, permit the subsequent unfolding of the ap T-cell differentiation program.

Downstream effecton of the pm-TCR complex The cytoplasmic tail of pTa may have confered some unique signal transduction properties to the pre-TCR complex. There are no obvious differences, however, in the proximal signaling events elicited by cross-linking pre-TCR or mature-type TCR complexes as they result in tyrosine phosphorylation of CD3& and CD35 subunits, recruitment and phosphorylation of ZAP-70 and syk PTKs, and rapid rise in intracellular calcium levels [20,22,30’]. In that context, it should be emphasized that the 127 amino acids that are predicted to comprise the human pTa cytoplasmic segment do not show any identity with the 30 amino acid residues comprising the mouse pTa cytoplasmic segment [46]. Collectively, these results argue against an essential signaling function of pTa and suggest that the associated CD3 ITAMs probably account for most of the transducing capacity of the pre-TCR complex.

Mutation of the genes coding for the PTKs acting downstream of the pre-TCR and TCR complexes resulted in rather distinct phenotypes. For instance, the disruption of thekn 1471 and syk [48*,49*] genes had no discernible effect on ap T-cell development, and this lack of phenotype was attributed to compensation by other members of the src- and syk/ZAP-70-family kinases, respectively. In contrast, mutation of Ick [SO] and ZAP-70 [51*] severely arrested T cell development at two distinct stages (Fig. 2). ZAP-70-l- thymuses contained normal numbers of DP cells with surface levels of TCR-CD3 complexes indistinguishable from wild-type DP thymocytes. The loss of ZAP-70, however, prevented these receptor complexes from inducing maturation further to the SP stage. By contrast, peripheral CD4+ T cells expressing signaling-deficient TCR-CD3 complexes have been found in a patient deficient in ZAP [X?]. This interspecies difference probably results from the fact that, in the human, syk may compensate for ZAP-70 during TCR afJ selection of CD4+ lineage cells. The presence

of normal numbers of DP thymocytes in ZAP-70-- mice and humans indicates that ZAP-70 is dispensable for TCR p selection and suggest that the pre-TCR may not be coupled to ZAP-70 or, more likely, that syk may compensate for ZAP-70 at this very stage of afi T-cell development [30’].

Mice carrying a mutation in the /CR gene display a pronounced thymic atrophy associated with a dramatic reduction in the number of DP cells and an almost complete absence of SP cells [SO]. However, the few double cells present in Ick-l- thymus differ from wild-type double positive cells as they express residual levels of CD25 [33’], and intermediate to high levels of ap TCR complexes at their surface [SO]. In the absence of Ick, however, these TCR complexes or their CD4/CD8 co-receptors, are probably unable to trigger signals required for TCR ab selection. Similarly, the loss of /CR probably prevents the pre-TCR complex from inducing an efficient progression to the DP stage.

Three additional observations support the pivotal role played by kR during TCR f3 selection. First, overexpression of a constitutively active form of Ick (termed Ick YSOSF in Fig. 2) was capable of restoring the progression of RAG-/- thymocytes to the DP stage and increasing the number of DP cells to within the range (or slightly higher) of that of wild type [53*]. Interestingly, the signaling properties of Ick YSOSF were independent of its ability to interact with the CD4 and CD8 co-receptors [53*]. Second, thymocytes overexpressing a transgene coding for a dominant negative (catalytically inactive) form of Ick (termed Ick K273R in Figure 2) are arrested at the very same early stage as RAG-/- thymocytes ([54]; see also Fig. 2). In contrast to RAG-deficient mice, however, and as expected from the fact that Ick acts downstream of TCRfl, the effect of the kk K273R transgene cannot be reversed by the coexpression of a productively rearranged TCR b transgene ([54]; see also [55]). Third, injection of anti-CD3e antibodies into Ick-I-XRAG-l-l- double deficient mice results in the production of DP cells, however with a CD25+ phenotype and in numbers that do not exceed 15% of that obtained in RAG-l-/- mice injected with anti-CD3c antibodies [33*]. Considering that the Ick and CD3</n mutations do prevent the development of mature yS T cells [4,5,56], it seems valid to compare their direct effects on ap T-cell development. Both correspond primarily to leaky mutations of the pre-TCR checkpoint and secondarily prevent the emerging DP cells from undergoing TCR ap selection. Such similarity suggests that CD3gq and Ick constitute distinct elements of the pre-TCR signaling cassette. In that context, the DP cells present in mice deficient in CD3gn and lck are probably attributable to inefficient signaling via CD3+ or fyn, respectively.

The generation of DP thymocytes was apparently unaf- fected in transgenic mice overexpressing either a dominant

Functions of TCR and pre-TCR subunlts Malissen and Malissen 391

negative p2 lras protein (termed Ha-ras N17 in Fig. 2, [57*]), or a catalytically inactive form of a MAP kinase kinase (referred to as MEK-1 A97 in Fig. 2, [W]). This suggests that either the pre-TCR complex is not coupled to the ras-+raf+MEK+ERK signaling cassette, or that the signals initiated during TCR f&selection transit via ras and MEK-1 but need higher levels of these dominant negative forms to be impaired. Consistent with the latter, expression of an activated form of ras (termed Ha-rasv12 in Figure 2) into RAG-l+ thymocytes was capable of restoring their ability to mature into DP thymocytes and expand their number to wild-type levels [59*].

Conclusion The results reviewed here show that progression beyond the CD44-flowCD25+ TN stage of intrathymic T-cell development depends on signals triggered by the pre- TCR-CD3 complex. These signals are probably relayed by a wealth of components including, ras, Ick and CD45 [60*]. Nevertheless much remains to be learned about the sequence of events which facilitate the survival, expansion and differentiation of CD44_floWD25+ triple negative

thymocytes.

Acknowledgements Some of the dcscribcd

Cancer. WC thank Pierre Golstcin and Yujiro Tanaka for commcnfs on the manuscript,

Dietmar Nicholas Holmes, Lcvclt, Wojcicch Swat and Anne Wilson for unpublished Corinnc Beziers-La-Fossc graphic art, and No~llc Guglictca

reading Papers of particular interest, published review, have been highlighted as:

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Functions of TCR and pre-TCR subunits: lessons from gene ablation