t-cell antigen receptor expression in the thymus

SEMINARS IN T-CELL IMMUNOBIOLOGY

T-Cell Antigen Receptor Expression in the Thymus

Oreste Acute and Luigi M. Larocca*

ABBttFVIATIONS PAGE polyacrylamid~ gel elec- PHA phy¢oheme@g|minitl

rmphoresis Tdr ~H-thymidine

I N T R O D U C T I O N

The thymus represents the major anatomical site where immunocompetent T lymphocytes are generated [1,2]. Unlike B cells developing in fetal liver and bone marrow (or the Bursa Fabricius in b~rdsL T cells do not secrete their antigen receptor and do not bind antigen alone, at least under physiological conditions. Instead, they recognize antigen in combination with membrane bound products of the major histocompatibility complex (MHC) of the immunizing individual, a phenomenon known as "MHC restriction" [3,4]. The resulting molecular interaction is still undefined in physicochemical terms. A complicated series of events takes place within the thymus where stem cells (prothymocytes) mlgradng from the liver in the fetus or bone marrow after birth populate the epithelial analgge [5]. During their thymic sejoum, they become tolerant to self-MHC [6,7], acquire the capacity to recognize the latter in combination with antigen, and express recognition accessory molecules such as the hmnan 'I'4 and T8 [8] (or the corresponding mouse homologues L3T4 and Lyt2 [1,9]) and differentiate into mature T cells able to carry out cytotoxic, helper, and suppressor functions [8-10]. This developmental process requires a transient but intense ceil division of the invading cells [11] and signals probably induced by interaction with the thymic stroma [12-14]. While only a minority of immunocompetent cells is exported to the periphery (i-2q~) [15], the vast majority of thymocytes never leaves the gland and probably dies in situ [16]. Several reviews have covered some of those aspects and illustrated the complexity of the matter [8,10,17-19]. The recent delineation of the T-cell antigen receptor and its subsequent rapid characterization, both m the functional and molecular (protein and gene) level, provides investigators with additional and perhaps more powerful tools to un- derstand the molecular basis of the "dual" antigen/MHC recognition as well as

From t/~ ga~ratery ef lmmamebieleg~. Dana.Barb¢r C¢ucer Institute and Dcpartmetst of Pathdegy. Hareard Medical Scheel. Boston. Massachusetts.

®~,;~i Larecca was on lea** of absence from t/ae Oipartimento di Anat~ia Patdogica. Unitzrsita Ce;teli~'a. Re,~e, Italy.

Adclr¢$s reprint rcqmest$ to Oreste Acute. Ph.D.. La~ratory of lmmune6idogy. Dana-Earlier Cancer Institute. 44 Binney Street. Boston. MA 02115.

Reveit,dJxne 10. 1986: accepted August 21. 1986.

Human Immunology 18, 93-109 (g987) © ELsevier Science Publishing Co., Inc., 1987 52 VaaderiM]¢ Ave., New York, NY 10017

93 0198-8859~7/$3.50

94 O. Acuto and L M. Larocca

thymic selection for the latter, to trace precisely T-cell precursor maturation and to uncover the molecular mechanisms of T-cell antigen receptor dependent activation. The scope of this paper is to summarize the recent data in this rapidly advancing area of immunology with particular emphasis on T-cell receptor development. We will first summarize the functional and molecular features of the T-cell receptor and then describe the present state of knowledge on its appearance during T-cell differentiation.

FUNCTIONAL FEATURES OF THE T.~ELL ANTIGEN RECEPTOR

The T-cell antigen receptor, hereafter referred to as Ti [20], was first identified by the use of monoclonal antibodies directed at T-cell surface epitopes unique to individual human or murine T-cell clones and tumor lines [20-24]. Anticlono- typic (anti-Ti) antibodies could inhibit both T-cell clonal proliferation to antigen and cytotoxic effector function [20,22,23,25] and increase interleukin 2 (IL-2) (previously known as T-cell growth factor) responsiveness by upregulating IL-2 receptor expression [26]. Anti-Ti antibodies immobilized by coupling to fie- pharose beads were able to specifically induce T-cell clones, an IL-2 dependent proliferation in the absence of accessory cells [27], indicating that somehow a local aggregation of Ti molecules on the cell membrane was required to induce IL-2 production. More recently, T helper cell clones stimulated with Sepharose bound anti-Ti antibodies have been found to produce a 12 kd factor (temaed IL- 4A) able, together with IL-2, to promote proliferation of resting T cells in the absence of macrophages [28].

Similar functional effects were observed when T-cell clones or peripheral T cells were reacted with monoclonal antibody directed at the nonpolymorphic T3 surface marker expressed on all mature human T cells [29,30]. The latter was demonstrated to be intimately associated to Ti both by biochemical and cellular studies [25,31,32] and the complex is called T3-Ti. Anti-T3 antibodies bound to macrophages through their Fc receptors [33] or immobilized onto Sepharose beads in the presence of macrophages [33] were able to induce an IL-2 dependent proliferation of resting T cells. Induction of proliferation in resting T cells seems to require, therefore, at least two conditions: (1) cross-linking of T3-Ti as for T-cell clones [27]; and (2) the presence of a second signal provided by macrophages, probably IL-1 [33,34]. Other factors such as B-cell growth factor (BCGF) and B-cell differentiation factor (BCDF) were produced when T-cell clones or peripheral T cells were stimulated with Sepharose bound anti-T3 antibodies [35]. Thus, it appears that T3-Ti cross-linking reproduces the physiologic effects induced by antigen when presented on the cell surface of accessory cells with the appropriate MHC products.

Interaction of ligands with their receptors in many cellular systems results in rapid generation of breakdown products of phosphatidylinositol 4,5-bis phos- phate (PIP2) (see [36] for review). Inositol triphosphate (IP~) and diacylglycerol (DG) which are generated in the breakdown process seem to be intermediates in the activation of cell growth and function [36]. IP.~ can release Ca 2 + from intracellular stores and DG is thought to activate protein kinase C [36,37]. The latter event is an important step to induce a kinase C'dependent intracellular phosphorylation of specific protein substrates [38] and probably activates a cas- cade of reactions [36]. Kinase C can also be activated by phorbol esters such as phorbol myristate acetate (PMA) [38]. Similar to lectins or antigens/MHC, phorbol esters together with Ca ~ ~ ionophores can mimic the early events in T-cell activation leading to lymphokine production [39]. Anti-T3-Ti antibodies induced a rapid appearance of lPs and increased intracellular Ca 2 ÷ released by intracellular stores in Jurkat, a T cell derived tumor cell line that could produce IL-2 in

T-Cell Antigen Receptor Expression 95

response to a combination of PHA and PMA [40]. Nevertheless, anti-T3-Ti antibodies induced IL-2 in Jurkat only after PMA addition [40] indicating that additional signals were required for complete activation.

In T-cell clones, immobilized anti-T3-Ti antibodies were able to increase [Ca 2 + ]i and to induce IL-2 dependent proliferation g4 ~ I- Proliferation was dependent on extracellular Ca 2 + since it could be iohib~¢cd by addition of EGTA to the medium [4 I]. Taken together, these results indicate that in T cells, release of Ca 2 + from internal stores and Ca 2 + influx are both required for activation and IL-2 production. Recently, [Ca 2 + I] increase has been demonstrated in T-cell clones after stimulation with antigen and macroph~kges of the appropriate MHC genotype [42].

The above data and other evidence (discussed in a recent review, see [43]) clearly indicate that the T3-Ti complex is linked to transmembrane signalling that utilizes IP, and DG as "second messengers" leading to activation of protein kinase C [36]. The picture arising from experimental evidence seems, therefore, to suggest the following scheme. When Ti on a mature T cell binds antigen and MHC with sufficient avidity, Ti "cross-linking" is induced. Signals are then trans- mitted to the T3 complex and delivered to the cytoplasm and to the nucleus where many genes start to be transcribed providing T cells with proliferating potential ~,.ad the capacity to produce a number of differentiation and growth factors.

Ti PROTEIN AND GENE STRUCTURES

Anti-Ti antibodies were shown to be directed at disulfide linked membrane bound heterodimers of 80-90 kd, composed ot two giycosyhted subunits: an acidic alpha chain of 43-53 kd and a basic chain of 43 kd [21,22,24A4]. In the human, three additional nonpolymorphic subunits termed gamma of 25-27 kd, delta of 21-23 kd, and epsilon of 20 kd are noncovalently associated with Ti [31,32t and constitute the T3 complex. Recently, a T3-1ike molecule has been documented in the mouse [45] although no specific antibody to it has yet been obtained. Similar to immunoglobulin molecules, both alpha and beta subunits were demonstrated to contain constant and variable domains by pep[]de map analysis [46]. Amino acid sequencing [47-49] and molecular cloning [50-54] have led to the identification and characterization of the T-cell receptor alpha and beta cDNAs and the genes encoding them. These studies have revealed that variable (V), joining O), and constant (C) segments of similar size and sequence to the corresponding immunoglobulin elements are present in both the alpha [52-54] and beta [51,55] subunit. For the latter, a diversity (D) element has been found [55] but not for alpha so far [52-54]. Several structural features of the Ti heterodimer (depicted in F/gure 1) can be inferred from the translated sequences of cloned cDNAs [50-54]. Both alpha and beta comprise an N-terminal variable (V) domain (VDJ or VJ) including approximately I00 amino acid (AA) residues sta- bilized by an intrachain disulfide bridge forming a loop of similar size as the |g V donmin. The constant (C) domain of approximately 150-170 AA contains: (1) an intrach~dn stabilizing disulfide bridge; (2) a connecting pep[]de probably of different length in the two chains; (3) a transmembrane hydrophobic region (20-30 AA); and (4) an hydrophylic C-terminal intracytoplasmic tail (5 AA residues for both human alpha and beta). Within the connecting peptides are the cysteine residues likely to form the interchain disulfide bridge. Also indicated in the figure are the three subun/ts (gamma, delta, epsilon) of the 2"3 complex. Only the gene encoding the delta subunit has been cloned [56] whose deduced AA sequence predicts the ex/stence of a 43 AA long intracytophsmic domain, an extracellular domain of 79 AA, and a transmembrane segment of 26 AA. The


Antigen ÷ MHC V

FIGURE ! Schemat.lc view of the T3-Ti molecular complex. V: variable domain; C: constant domain. (P) indicates a possible phosphorylation site of the gamma subunit. Although not indicated, the delta subunit may be intracellularly phosphorylated but to a lesser extent compared to gamma [58]. S: cysteine residues. The location of the delta and epsilon subunits is only speculative.

gamma subunit appears to be closely linked to the Ti beta subunit t57]. Phos- phorylation of the gamma and to a lesser extent, the delta, subu~.ies has been detected after treatment of anti-T3 activated T cells with phorbol esters [58].

Restriction enzyme analysis ofgenomic DNA using alpha and betagene probes has shown that the latter are rearranged in T but not in B cells [51,59], suggesting that like immunoglobulins, V, D, J, and C elements are dispersed within the genome and are brought together by DNA rearrangements giving rise to a tran- scriptional active unit. Analysis of DNA isolated from antigen specific T-cell clones or lines has confirmed that this is indeed the case for the beta gene [55,58]. cDNAs isolated from thymus libraries have revealed the presence of aberrant rearrangements that could not lead to a translatable product [51 ]. The alpha gene has an unusu.! organization compared to other members of the Ig gene super- family in that J segments lie on a considerably long segment of DNA (>60 kb) [60,61]. Due to the long distance between the C and J segments, rearrangements have been demonstrated using only V gene but not constant gene probes [52-541. Nevertheless, the genomic organization of the alpha locus [60,61] implies the occurrence of DNA rearrangements as in the case of the beta gene. Two constant region genes have been found for the beta [62L designated C-betal and C-beta2, which show >95% homology to each other [63]. The products of C-betal or C-beta2 do not represent functional isoty~s [64i.

Each C-beta is preceded by seven J segments, six of which are functional [62j and at least one D el.. aent [62]. Only one alpha constant gene has been found [52-54] preceded by the corresponding J segments, probably in the order of 40-50 [65]. Northern analysis has revealed that two transcripts of 1.3 and 1.0 kb homologous to beta cDNA could be detected in almost every T-cell line analyzed [66,67]. The 1.3 kb mRNA represent a full transcript comprising the leader sequence, V, D, J, and C segments, whereas in the 1.0 kb mRNA, no V gene segment is present [66,67]. Similarly, alpha RNAs of 1.6 a~ld 1.4 kb have been found with the latter lacking the V geae segment [67]. Truncated Ig heavy chain gene transcripts, derived from DJ ioining, have been described in immature


pre-B cells [68] indicating that D to J joining is the first event in Ig gene rearrangements.

V-alpha genes seem to be organized as regards to homologies in larger families than V-beta genes [69-72] and the latter are often found to be composed of single members [69-7 I]. On a statistical basis, the V-bern repertoire (24 or more genes with 95% confidence) appears to be more limited than the V-alpha (40 or more) [69,70], although the determination of the upper limits probably has to await the sequencing of many more V genes. As for immunoglobulins, diversity seems therefore to be generated by {a) the presence of a multiplicity of germline V gene segments; (b) combinatorial diversity generated by association of different gene segments during gene rearrangements (V, D, and J for beta chain and V, J and possibly D for alpha chain); (c) junctional diversity, created by variations iu the ioining sites of V gene segments; and (d) association mediated diversity, arising from association of differe1~t alpha and beta chains.

Studies by Hood and collaborators have concluded that only three identifiable hypervariable regions could be found in both alpha and bern vaciable genes [65,71]. Data comparing the predictable secondary structure and AA residues conservation of the variable regions also supports the striking similarity between |g and Ti molecules [65].

Despite the overall similarity between Ig and Ti, somatic hypermuration mechanism does not seem to contribute to the generation of diversity [59,65]. A third type of T-cell specific rearranging gene, named gamma (not to be confused with T3 gamma subunit), has been identified which bates homology to the alpha and beta genes [73]. The gamma gene appears to give rise to limited variability [74] but its protein product has not yet been identified. It is nevertheless interesting that gamma is the first gene to be rearranged during ontogeny (see below) and it seems to be preferentially expressed in cytotoxic T-cell clones specific for class I MHC [74].

Ti EXPRESSION IN THE T H Y M U S With the rapid ~cquisition of knowledge on Ti structure and gene organization, several studies have tried to assess first whether Ti is acquired within the thymus and if so, whether the corresponding genes are rearranged and their products expressed during T-ceil development in an orderly manner as in the case of the lg gene f~mily [68]. The intent of these initial studies has been to attempt to provide a rational basis for impoix,m¢ questions such as where T-cell tolerance is acquired and when thymocytes become susceptible to repertoire selection, to explain rhymocyte death within the thymus and to unambiguously define developmental stages of the various thymus subpopulations previously identified by antibodies directed at T-cell surface markets. Alpha and beta gene rearrangement and expression of their products in adult and/or feral thymocytes have been investigated both in human and mouse by using eDNA and antibody probes. We will summarize these data with particular emphasis on the human system and refer to studies in the mouse (such as fe~al thymus) which have not yet been performed in the human.

Human Thymic Subpopulacions The thymus is invaded by lymphocytes during the eighth and ninth week of gestation in the human [75] and day 11 or 12 in the mouse [5]. In the remaining time before birth, they exponentially increase in number and gradually become identifiable by surface marker expression as mature T cells. Monoclonal anti-

98 O. Acum and L M. Larocca

bodies directed at the human T-cell surface antigens T l l , T6, T8, T4, and T3 allow to identify three major thymic populations [8,76]. An immature population (T11 + T 6 - T 3 - , mostly T4 - T 8 - [see below]) composed mainly of large blast- like cells represents 3% {77] of total adult thymocytes and expresses the sheep erythrocyte receptor T11, a marker found throughout the T-cell lineage [78]. Only in this popuhdon is it possible m demonstrate a sizable proportion (20-400~) of IL-2 receptor (TAC antigen) positive cells [77] similar to immature Lyt2 -L3T4 - mouse thymocytes [79~ These cells can respond to high doses ofIL-2 (LM Larocca et al., submitted) and vigorously proliferate to PHA and iL-2 [77]. A second population (T11 + T6 + T8 + Tzi + T3 + / - ), comprising >70o~ of roml thymocytes, expresses the T6 marker found only within the thymus and the 1"4 and T8 antigens (coexpsessed) [8]. T6 + thymocytes are located almost exclusively in the cortical compartment as demonstrated by in situ staining [80]. They are composed mainly of resting cells [77] which, like cortical mouse cells [ 16,17,20), are believed to be destined to die within the thymus, although a proportion of cortical thymocytes may represent a transition stage towards complete maturation [76]. T6 + cells are poorly responsive in vitro to mit~genic st;muli when compared to the other thymic populations [77] in spite of the existence of large blast- like T6 + cells which show the highest levels of DNA synthesis [77]. Consistent with the evidence that T3 + thymocytes are found scattered in the cortex [80], T6 + thymocytes express T3 in various proportions. This point is illustrated in Figure 2 where T6 + thymocytes have been analyzed by direct immunofiuores- cence with an anti-T3 antibody. Of the total population, 15% displayed a bright fluorescence whereas 37% identifiable as a discrete peak showed approximately eight- to tenfold lower T3 fluorescence. Whether the latter population represents a transition stage towards higher T3 expression or cells that have negatively modulated T3-Ti perhaps as a result of interaction with their ligand, as suggested by previous studies in mature T cells [25], is not keown. Finally, the more mature intrathymic pool (T11 + T 6 - T 3 +T4 + orT8 + ) consists of thymocytes that lack the cortical T6 marker and represents approximately 20% of total thymocytes

FIGURE 2 Distribution of T3 antigen in isolated T6+ thymocytes. T6+ thy,~ocytes were separated by indire~, '~,~mune rosette method as described by using an anti-T6 monodonal antibody [84]. After several washings, ceils were stained with an anti-T3 monoclonal antibody (Leu4) directly labeled with fluorescein ~ analyzed on an Epics V cell sorter (thick line). As a negative control (near thin line), an irrelevant antibody dil~cdy labeled with fluorescein was used. Purified T6 + thymocytes stained with ~d-T6 antibody were >95% T6+ (not shown). Numbers in abscissa are given in arbitrary units. The percentage of cells expressing different levels of posidvity was determined using a com- puter data processing system by integration of the area right at the point of intersection of the two adjacent peaks.

I Neg 1

o

lO loo Fluorescence intensity

I - - 37%---k- 14%--:

T-Cell Antigen Receptor Express/on 99

[8]. The density of T3 on these cells is similar to peripheral T cells [8] and to the cortical population expressing high levels of the molecule [80]. Within this subset, 2"4 and T8 segregate into discrete cell populations that define the maiocity of helper/inducer and cytotoxic/suppressor peripheral T-cell compartments respectively [8,81]. T3 + T 6 - thymocytes appear to be comqaed to the medullary compartment [80], although the results presented in Figure 2 do not exclude t~e possibility that a small proportion of T3 + T 6 - thymocy~s may be located in the cortex.

Recent studies have added some medifica.'ions to this initial scheme. For example, recent evidence indicates that within T 3 - T 6 - cells, a p o p ~ d o n of approximately 300~ expresses T4 and/or "1"8 ([77], and L.M. Iarocca et al., submitted), suggesfmg that the appem~nce of the htter cmi precede T6 expression as previously reported in ~ntogenetic studies [82]. Furthermo~, a T3 + T S - T 4 - population of approximately 0 .5-1% of total thymocytes has re- ceudy been described ([83] and Larocca, unpublished). This cell population could be m~dnt~ned in culture with recombinant IL-2 and disphyed cytotoxic activity [83].

T 3 - T i Mo|ectflm" Complex Expre~ion in Thymu,

Previous studies have demonstrated a physical and functional association of the mature T-cell m~ker T3 to Ti in individual T-cell clones {25,31,32]. Consistent with the coexpression of T3 and Ti in peripheral T cells, Ti heterodimer, were found only among T3 + thymocytes analyged by two.dimcn,donal PAGE [44] and by immonoprecipitation using both and-Ti alpha and bern constant region antirel~ [84]. There results were also confirmed by the obrervadon that Ti alphe and beta surface heterodimeea were found only in T3 + thymus-derived tumor cell iines but not in T 3 - cell lines [44]. Given the evidence that 0 .5-1% of total thymocytes express T3 in the absence of 2"4 and T8 antigens [83], it is perhaps not surprising that small amounts of 1"/heterodimel~ have been found in mouse L y t 2 - L 3 T 4 - immature thymocytes r85L Whether T 3 + T 4 - T 8 - thymof~ytes derive from T3 + T8 ~- T4 + cells that have lost T4 and T8 or whether they represent a lineage independent of T4/T8 expression is not cie~. Although the above data indicted a strong linkage between the expre~ion of T3 and Ti in thymocytes, they did not rule out the possibility that the T3 complex could be expressed in the ab~nce of Ti. A direct d e m o n s ~ i o n of ibis point would require the ure of antibodies reacting with the constant portion of Ti to be used for repc~ting the Ti + population. Several attempts to p~gtuee such reagents have failed since anti-Ti constant region obtained in several hboratories do im- munoprecipimte Ti molecules from thymocyees but do not react with the intact surface Ti molecule [86,87]. Althov4gh not directly demonstrated, such failure has been attributed to steric hindrance of epitopes due to interactions of the molecules forming the T3 complex. Despite these difficulties, indepe,,Men, lines of evidence indicate that T3 and Ti must be coexpresred on the cell surface of individual T cells. For example, all of the mutants produced from Jurkat tumor ceil line *hat had been immunoselected for the loss ofT3 or Ti on the cell surface fai|ed ~ express Ti or T3, respectively {88]. All the T 3 - / T i - mutants had T3 proteins in the cympl~m and Northern bloc ~nalysis reveMed in some that only the 1.0 kb beta mgNA (immature) w ~ present. Furthermore, in one of there mutants lacking the mature 1.3 kb mRNA T3-Ti surface expression could be induced by ~l~ansfecting the beta gene {89].

Collectively, the above data strongly suppo,~x the idea of the T3 protein being the obligatory pmners for Ti expression and vice versa. Nevertheless, they do

100 O. Acum and L M. Larocca

not exclude that some components of the T3 molecular complex can be expressed in the thymus in the absence of the others. In this regard, recent experiments of antibody staining in thymic tissue sections [90] indicated that the delta subunit was present in the medulla at higher levels than in the cortex, most likely cor- relating with Ti expression [80]. Anti-epsilon subunit antibodies, on the contrary, stained thymocytes in both compartments but with equal intensity. It is nevertheless not clear whether epsilon was present on the cell surface of the positive cells [90].

A monoclonal antibody (KJ16-133) directed towards a V beta gene product expressed in 20% of peripheral T cells in the mouse has been used to directly assess the presence of Ti in different thymic populations by FAC$ analysis and tissue staining {91,92]. The results of those studies have basically confirmed findings in the human with anti-T3 antibodies ([78,80], and above results). Med- ullary cells were found to stain more intensely than cortkal cells, few of which were strongly positive, Thymocytes negative with KJ 16-133 (approximately 50o/6) we~c ~nfined to the cortical compartment.

Gene Rearrangements and mRNA of Alpha and Beta

In order to investigate whether rearrangements occur in tempor, d sequence for the lg gene family and to determine the developmental stage of the various thymic populations, thymocytes have been analyzed for the presence of alpha and beta rearrangements and mRNA expression. Human thymocytes were separated into T3 + and T3 - and the latter population further sepmmed into T6 + and T 6 - [84]. By using a cDNA encoding the constant portion of the beta gene isolated from the tumor cell line REX [44], both betal [84] and beta2 (O. Acuto, unpublished observarion) were found in germline configuration in the imnmture T3 - T 6 - thymic population. The T11 marker was expressed in > 9 0 ~ of the isolated population and thymic macroph~es were virtually absent. Subsequent analysis of mRNA isolated from the stone population showed low but detectable amounts of both 1.3 and 1.0 kb in roughly equal amounts [93]. By using an alpha eDNA probe isolated from REX [93], virtually no mess,ge for the latter was found in these cells, strongly suggesting that no re~rangement of the alpha locus had taken place. This latter point could not be directly demonstrated at the population level for the reasons explained above. In T 3 - T 6 + thymocytes, beta was undetectable in germline configuration [84L Consistently, the level of the 1.3 kb mRNA was tenfold higher than in T 3 - T 6 - cells [93]. in T 3 - T 6 + cells, the 1.3 kb species appeared to be approximately tenfold higher than the 1.0 kb form. The higher representativity of the 1.0 kb beta mRNAs in the T 3 - T 6 - cells when compared to T 3 - T 6 + cells implied that a substantial fractio~t of Ti beta transcripts at the earlier s t~e were derived from recharged DJ segments lacking the V segments. This observation suggests that as for lg {68], D to J joining takes place before V to J joining. Alpha mRNAs could he first detected in T 3 - T 6 + thymocytes although in minute amounts {92], but it did not lead apparently to the expression of the detectable amounts of T3-Ti complex [84]. Whether this meat,s that some components of the T3-Ti complex are still not fully expressed at thet stage is not known.

T3 + cells contained beta mRNA surprisingly in smaller amounts than in T 3 - T 6 + cells (2.5 to 5 times less). The level of alpha mRNAs was, on the contrary, increased three to fourfold in T3 + thymocytes, the only population in which Ti molecules were detected on the cell surface. From the above evid.-nce, it appears that maximal expressions of alpha and beta transcripts in the v~rious differentiation stages are distinct. Ti alpha transcription initiates in cortical cells and increases with maturation. In contrast, regulation of Ti beta tran,cripes


seems more complex, initially induced in the most immature thym/c population, maxh-,mi in T6 + T 3 - , and reduced in T3 + mature cells. Several mechva~isms such as RNA processing, transport, and degr0Aation could explain this observation. Nevertheless, these results would argue for possible positive and negative regulatory elements playing a role in Ti beta mmscripdon. On the other hand, it is possible that Ti hem transcripts (and/or their products) may control the activation of the Ti alpha gene. This mechanism has been proposed for the com~rol of Ig light chain gene expression by the heavy chain gene products to explain the appearance of kappa light chains only when productive rearrangements of heavy chain takes place [94]. The obvious concll sion from ~he ~bove studies is that the Ti beta gene is the first to undergo rearrangement ~ d to be expressed followed by the Ti alpha gene during T-cell differentiation. The sequential expression of beta and Mpha, respectively, has been confirmed by pm~lel studies using several T3 + an 4 T3 - thymus derived tumor cell !ines representing the ma l~an t counterpart of thymocytes derived from different s ~ s of intr~iivtaic devel- optaent [84,93]. All the T3 - m t a o r lines examined (five) contained bern mRNA and the corresponding polypeptide [94]. In no c,~e was alpha found to be expressed alone. Since in all these lines the beta gene was found to be r e ~ , it is reasonable to assume that the lack of alpha expr~sion was due to its gene still being in germline configuration. Such T 3 - lines may therefore represent the pre-T ceil equivalent of pre-B cells.

Analysis of alpha, beta, arid gamma rearrangements m~d/or expression in mouse fetal thymus (day 14-17) indicates that gamma transcripts ap~r first, followed by beta, and finally alpha [65,85,95,96]. The levels of gamma mRNA decrease at the time when alpha appears [95]. No hem mRNA has been found in fetal liver, although some gamma rearrangements were apparently detected in this organ [65]. Interestingly, incomplete mRNAs (D-J-C) always preceded the expression of V gene segments con~ning mRNAs [65L Similarly, immature L y t 2 - L 3 T 4 - adult thymocytes largely resembling day 14-15 feral thymocytes expressed little or no bern mRNA and no alpha when compared to total thymocytes [97,98]. Thus, it appears that the ~ e or very similar maturation pro- cesses that take place during fer~l development are operating in the adult thymus in double negative ceils (Lyt2 - L3T4 - or most o f T 6 - T3 - ceils in the human).

CONCLUSIONS A N D PERSPECTIVES With r e s e t to the expression of the T-cell antigen receptor in the thymus, the data discussed above allow the following conclusions: (D ~he recognition unit of T ceils (Ti) is acquired within the thymus where the genes coding for it are expresssed in an orderly manner; (2) the strong similari~ between |g and T-ceil receptor developmemal gene expression suggests that the two systems may utilize distinct but similar control mechanisms; (3) given the mechani~n by which T cells generate dive~ity, it is conceivable that at least pm~ of the ceil death obse~ed in the thymus is due to the necessary waste generated by unproductive rear- rangemems [51]; and (4) since no machanism for somatic mutations appears to take place for the Tocell receptor, the acqmsltion of a functional repertoire for antigen and MHC recognition cannot be accounted for by this mechanism as prevlo~l~iy hypothesized [99].

Nevertheless, several points still remain unclear at present. For example, in the 8~xsence of complete data on the appearance of the gmmma, delra, a~d epsilon (and maybe other) T3 subunits, it is not dear whether one of them or none represents the rate limiting step for T3-Ti surface expression. Without knowledge of the interaction between Ti and antigen]MHC in physicochemical terms, it is difficult to speculate on the molecular mechanisms underlying chymic selec-

102

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E~

T-Cell Antigen Recep¢or E~ression 103

tion. The role of gamma (rearranging) expression as a possible signal of com- mitment to T-cell lineage or to one T-cell snbpop~adon (cytotoxi¢?) [74t or its contribution to T-cell specificity [951 is not supported by direct evidence. Finally, the precise precuranr-prodnct relationship of the various thymic pop~3~ations is at present rather speculative and the new findings add perhaps more com#kadons to the models already existing.

in this regard, we have attempted to illustrate in Figure 3 an exemp~fied scheme of T-cell development taking into account the dam discussed above as well as data available from the vast literature on the ~gmnent. B~cic~Lly, two models have been proposed to explain at large T-cell n ~ t ~ d o n within the thymus [8,10]. In both, mature medullary cells me derived from coixical pre- cumors (located mosdy in the outer cortex). These populations are defined T l l + T 3 - T S - T 4 - T 6 - in the human and L?~2-L3T4-duJILyl in the mouse [18L The nmin discordance between these models is whether mature thymc~ytes derive direcdy from those p rec~ocs or from do~ble positive ( T 6 + T 4 + T S + or Ly¢2+L3T4 +) cells. In the first model, donble positive cell, represent an obligetory degiend in line with the low responsiveness to ~ n k s¢~ulL I~ also predkts that immature cells sbo~d be present in the m e d ~ co~p~men t . Much evidence has been proposed to support this view (dhcmsed in [10]). in suppor~ of a direct derivation of m e d ~ cells from do~bie positive cells is the evidence tlmt a sm,dl proportion of cortical peanut ,~ggtndain (PNA)+ T3 + thymocyre* respond to mitogenic as well as antigenic s~hn~ {76~]. F ~ h e ~ o r e , the changes in beta e x p r e ~ n from low levels in immature cells to high level, in T6 + ce~s where alpha mRNA begins to be detected [93] and t ~ ~ncre~ng expression of Ti in the latter popnlation would s~ppoix the second hypothesis. Finally, the presence of T3=Ti in a considerable propoi~ion of cells that express a co~ic~l pheno~ype (T3 + T6 + T8 + T4 + ) m ~ e s a d~rect derivaikm of meduiim~ f~,~m a do~ble negative cells more complicated to expMn, ~tbocgh ~ of these co~icM cells may die ~s a re,~lt, for exan~ple, of a ne~tive selection of ceils with h ~ dfinity for selfoMHC or with aberrant antigen/MHC binding c h ~ t e r i , d c s . in the absence of a clear answer, we have indicated both p~thw~s (perhaps no~ m ~ t ~ l y excl~aive?) in onr scheme.

The present and f~ture ch~cteri~adon of the m o l ~ l ~ and fnnction~ features of the T<ell ant i~n ~eceptor wiU ~dlow ~o address more precise questions about T ceUs and their p~:~r~rs . The knowledge of the biochemic~ mecimaii~s u~erlying T3-Ti depe~en~ ~tivadon (e.g., the role of Ca2 + and khmse C) n~y ,dtow ~s to better define the f~nc~ionM features of ~:e v~ious ~hymic pop- ~ d o n , . ~n this respect, the ~ecent delineation of ~he T~ 1 dependent T<ell acdwtion and its role in thymocy~e ~dwf ion ~ ~ 00,101 ]open, s new pe~pecdves. New antibody probes directed ~ both Ti giph~ and b e ~ u ~ ; ~ for sen~tive in ~ tis,ue s~ning techniques, ma~ direcd~ indicate if and where th~moc~tes in~e~t with strom~ cells (e0g., e p i ~ I M , m~roph~ge, dendritic cells) thocght ~o de t e~ ine T<ell thymic ,election [ 12,13#2]. Fin~dly, the po~ibility m cult~e in vitro immature th~mocytes ([102] and L Lgrocca et ~d., s~bmltted) and perhaps their p ~ u r ~ r , , ~ u l d allow ~he ,¢~dy of ,dmuli nccess~y to induce ~hymocy~e differentiation and help to clarify the precursor-produc~ relationship between d~erent ~hymic cell populations.

ACKNOWleDGMENTS We wish to th~k i~. Ellis Reinhe~ for reviewing the manuscript and fox" suppo~ of our work. We gl~ wish ~o ~¢knowledge Dr,. Andre, A~¢over, Robeix Siih:iano, ~ d Neii Pdchard,on for ~dlng the ~uv'~ip¢ ~md providing su~stioas.


Dr. Larocca was a recipient of a fellowship from the A~oc~ione IudLm~ gicerca sul Cancro (AIRC).

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97. Sl~mel,o~ LI~, Li~M~e~ T, Fow|kes BJ, v~n den Ei,e~ P, Terho~¢ C, EM~s MM, Gen-n~n RN, ~ h w ~ RH: ~ k ~ of ~nes of ~ e T cell ,mtigen r c c e ~ r complex in p~ecm~r ~hymocy~e,. Ngt~re 315:76% 1985.

98. T r o w M ~ IS, Le~eF J, T ~ e ~ J, Hym~r~ R: Thymocy~e subpop~mion enriched for p ~ n i ~ o e , wieh ~ u n r e ~ T cell ~e~c~r be¢~ chain gene. N ~ , ~ ~15:666, 198%

99, Jetne HK: The ~o~6c geneeg~h~n of ~mn~u~ recognition, l~ur J i r e~no l I:I, 1971,

I00. g e i n ~ ~2.: A mol~l~r b ~ for ~h~c ~ct{oo: reg~h~io~ of Ti I i~d~'d

T o ~ y 6:75, 19~%

lOl, Akover A, Wei,, MJ, {:)~leyJF, geinher~ EL: TI l # y o g i n is f ~ e ~ d o ~ tinked eo ~ c~kium ¢h~r~l in precug, or ~ d m ~ r e T ~ n ¢ ~ cells. ~ ~ ! A¢~t Sci USA $3:2614, 1986 . . . .

102. P ~ : k ~ R, yon ~ h m e r H: Requiremems for growth o~ ~ r e ~hy~noey~e, ~ from f e ~ ~ d ~ult mice in vitro. E~rJ lmmunol 16:12, 1986.

103. C~kt DP, l ~ k JM, Pet~ey CL, Cho M, C o l i ~ J, W ~ y JN, Teri~e,¢ C: I ~ of cDNA c kmes encoding the 20K mm-#ycos~a~ed poiypei~kie ch in of the h ~ T ceil re~:e~ot~T3 complex. I~la~ure 321:43L I 9~ .

104. B~nner MB, bicLemtJ, D~yrms DP, StromingerJL, 8m[thjA, O~en FL. Seklnmn JG, lp S, Rosend F, Kr~gel MS: Idendf/cadon of a pueative secom! T cell lx~cepme. Nacum 322:145, 1986.

110 O. Acuto and L M . Larocca

105. Bank I, DePinho RA, Brenner MB, Cassimeris J, Air FW, Chess L: A functional T3 molecule associated with a novel heterodimer on the surface of immature human thymocytes. Nature 322:179, 1986.

Note added in proof: Recendy the gene coding for the T3 el~ilon subunit has been cloned [ 1031. Moreover, the elusive product of the gamma rem~n#ng gene has been identified and found to be expressed in association with T3 in a small subset of peripheral T cells [104] and thymocytes [105].

t-cell antigen receptor expression in the thymus

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