- Email: [email protected]
Generation of the αβ T-cell repertoire
Generation of the CCP-T-cell’ repertoire Christophe CNRS,
The
I’INSERM
sequence
repertoire to
be
Benoist
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of events
de Chimie Biologique,
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has been known
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to a diverse
in outline
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Current
and Diane Mathis
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There have been several indications over the past year that this traditional view is far too simple. We will focus on four of these: (a) the observation that rearrangements at neither the TCR-a nor TCR-p locus are entirely random; (b) the finding th at p c h ams are displayed on the surface of immature thymocytes in the absence of c1 chains; (c) the suggestion that the CLlocus can undergo multiple rearrangements before thymocyte selection; and (d) the observation that the c@ T-cell repertoire changes markedly during ontogeny. Each of these new intricacies is an eyeopener to the fact that positive and negative selection, as we currently recognize them, are not the only forces that shape the c$l T-cell repertoire.
O$ T-cell
suggest
previously
content.
1992, 4:15&161
Non-random
The traditional description of the generation of the c$ T-cell repertoire has been a rather simple one. In outline, T-cell progenitors enter the thymus in a naive state, devoid of antigen specificity because their T-cell receptor (TCR) genes are in the unrearranged, germline conliguration. During a first ‘window’ of thymocyte differentiation, the TCR-fl locus gradually rearranges, resulting in the random joining of DJ and then VDJ segments. As part of this process, each exposed terminus undergoes endonuclease ‘nibbling’ and template-independent N nucleotide addition, creating an enormous degree of diversity at the VDJ junction. During a subsequent differentiation ‘window’, the TCR-a locus randomly rearranges and diversifies, in a similar manner to the TCR-p locus, except that no D segment is employed. TCR CLand p chains are synthesized, combine, and associate with a set of invariant CD3 chains. Once this complex multimer has been displayed on the cell surface, further TCR gene rearrangements are blocked, resulting in allelic exclusion. It is only rather late in thymocyte differentiation that specificity is imposed on the vast, essentially haphazard repertoire, through positive and negative selection events mediated by T cell-stromal cell interactions (as discussed by Pardoll and Carrera in this issue, pp 162-165).
France
form for some time. Details continue
in Immunology
Introduction
Strasbourg,
rearrangement
of TCR gene
segments It has been a common assumption that VJ rearrangements at the TCR-a and VDJ rearrangements at the TCR-P locus are randomly generated; that is, each diversity segment has an equal probability of combining with each joining segment, and every variable stretch with every diversity or joining stretch. This assumption underlies the concept of combinatorial diversity and has been incorporated into all theoretical estimates of the ap T-cell repertoire. However, it has recently been demonstrated that neither murine Vcrs and Jcls nor V(3s and Jps are joined randomly [1=,2-5,6-l. The most convincing data on the c1 locus came from a study on isolated thymocytes using polymerase chain reaction technology [ 1.1. TCR transcripts bearing Vcl segments that mapped to the 5’, central, or 3’ regions of the locus were amplified and probed with oligonucleotides corresponding to variously situated Ja segments. Particular VCLSclearly combined perferentially with particular Jcls: the V segment located nearest the 3’ end was frequently associated with the J segments nearest the 5’ end; on the other hand, the V segment located neare$ the 5’ end was joined almost exclusively to the more distal J segments (at least in adults); strikingly, two highly homologous but spatially separated members of the Vu5 family showed distinct patterns of Ja usage. These findings are highly reminiscent of results from another set of studies that used a less discriminatory Southern blotting assay [2-4]. This assay did, however, provide another piece of important information: TCR-cr rearrangements on the two homologous chromosomes often appear t9 involve J segments residing in the same region of the locus, suggesting rather strongly that the choice of a particular Ja is directed, not random. This result has recently been confirmed with T cell hybridomas [ 51. Such striking data have not so far been reported for the p locus, but evidence of directed rearrangement
Abbreviations IL-lnterleukin; Ig-immunoglobulin; PCR-poiynierase SCl[tsevere combined immunodeficiency; TCR-T-cell
156
@ Current
Biology
chain reaction; RAG-recombination activating gene; receptor; TdT-terminal deoxynucleotidyl transferase.
Ltd ISSN 0952-7915
Generation
did come from sequence analysis of a large number of TCR transcripts encoding the VP17 variable region [6*]. J segment usage by these TCRs was highly vanable, ranging from < 1% for Jp1.5 to >20% for Jp2.6. In general, there was marked preference for Jp2s rather than Jpls. This bias was not the result of positive or negative cell selection events since it was observed with Vl317a transcripts, which give rise to cell surface protein, and also with VP17b transcripts, which do not because of an interfering stop-codon. Nor was the variability peculiar to transcripts carrying vl317 since preliminary data on v~G + TCRs also showed a bias [6-l. The finding that VWJat and VpJp rearrangements are nonrandom is significant because it indicates that the T-cell repertoire that emerges in the periphery is constrained by molecular events involved in TCR gene rearrangement in addition to the various selection events mediated by cellLcell interaction, One is led to consider influences such as the relative accessibility of particular segments due to chromatin conformation, the efficacy of recombinant signals flanking individual segments and the capacity of different stretches to partake in secondary rearrangements. One must also question whether and how such influences vary in different mouse strains,
/ ‘lone’ TCR f3 chains on immature
thymocytes
_
It has been taken for granted that the TCR c1and p chains must both be expressed for either to appear on the cell surface. Results encouraging such a view were repeatedly obtained from experiments on T-cell lines that had secondarily lost the expression of one of the subunits (for example [7]) and from transfection studies where only one or the other subunit was introduced into an CL-P,recipient (for example [8]). It was a surprise, then, to discover a few years ago that transgenic mice carrying a rearranged TCR-l3 gene displayed the transgene-encoded fl chain and only that l3 on the surface of immature CD44CD8 thymocytes in the apparent absence of other TCR chains [9], During the past year, this finding has been consolidated and extended in two reports [ lO*,ll*]. Firstly, it was shown that introduction of a rearranged TCRp transgene into a severe combined immunodeliciency (SCID) genetic background provoked visible alterations in the phenotype of the thymocytes, which are usually CD4_CDWTCRin this strain due to a defect in the DNA recombination machinery [lo.]. SCID thymocytes carrying the transgene displayed p chains in the absence of TCR 01, y or 6 chains. Also, they were prompted to express CD4 and CD8, and were induced to transcribe RNA from the TCR-cl locus. Secondly, it was found that a CD4+CD8+ thymocyte line, transformed after infection with Gross virus, displayed p chains at the cell surface independently of the other TCR chains [ 11.1. These subunits carried a variable region different from that borne by the p subunits in the transgenic experiments, impart ing some generality to the phenomenon. B
of the ~1p T-cell
repertoire
Benoist
and Mathis
Having firmly established the existence of unusual cellsurface TCR p chains, both groups have proceeded with biochemical and functional analyses. It now appears that the l3 subunits are displayed as either monomers or homodimers that have an atypical liaison with CD3 subunits ([9,10~,11*,12]. These complexes are capable of signalling when stimulated by an anti-TCR monoclonal antibody; the signal can be detected by measuring calcium flux, but not proliferation or lymphokine secretion [lO*,Il*]. The critical question is, of course, what consequence does the expression of these ‘lone’ p chains have for a thymocyte in vim? Phenotypic changes observed in the transgenic mouse experiments may provide important clues. It might be that cell-surface display of p chains leads to closing down of the l3 locus to further rearrangements and opening up of the a locus to the transcription and rearrangement machinery. This would give a satisfy ing symmetry to the emerging B cell scenario, discussed at length in [lo*]. It is interesting in this regard that transgenic mice carrying a rearranged TCR-b gene with a near-complete deletion of the V region show exclusion of endogenously encoded p chains [ 131, while Ig heavy chains lacking a V region are displayed on the surface of pre-B cells and exert an important influence on the emerging repertoire [I4]. It might also be that cell-surface display of l3 chains is an important stimulus for @l T-cell differentiation, prompting expression of the CD4 and CD8 accessory molecules required by most cells to achieve terminal differentiation. SCID thymocytes do proceed to the CD4+CD8+ stage under the influence of a TCR-P transgene, but this can also occur upon expression of TCR-yG transgenes [ 151 as well as after introduction of heterologous bone marrow stem cells [ 161. Thus, one must be prepared to accept the possibility of alternative mechanisms for switching on differentiation, as well as the likelihood that the relevant signals are delivered intercellularly. Finally, it is worth considering, again on the basis of B cell precedent, that surface p chains might promote diversification of the repertoire. It has been reported that complete immunoglobulin (Ig) heavy chains displayed in the absence of light chains alter the growth requirements of pre-B cells, permitting them to proliferate in response to IL-7 in the absence of stromal cells; such proliferation is no longer possible once heavy-light chain complexes assemble at the surface [ 17-191. This led to the proposal that once a pre-B cell can synthesize a ‘good’ heavy chain, it proliferates to allow several different light chains to be combined with the same heavy chain. By analogy, a thymocyte that synthesizes a ‘good’ b subunit might proliferate because of altered lymphokine susceptibility, permitting the pairing of multiple LXchains with the same 0 chain.
Multiple
rearrangements
at the TCR-a
The regulated model of allelic exclusion that successful display of Ig heavy-light
locus
[19] postulates or TCR c&l het-
157
158
lymphocyte
development
erodimers at the cell-surface prevents further rearrangements of the light chain genes and the IX chain genes respectively. Consistent with this postulate, only about one third of B cells were found to rearrange both x loci and, with very rare exceptions, only one of these rearrangements ever gave rise to productive transcripts [20]. In addition, expression of rearranged a Igx gene in transgenic mouse B cells markedly suppressed rearrangement of the endogenous x loci [ 2 1 I. Evidence has accumulated over the past few years that the regulated model of allelic exclusion requires some modification if it is to explain allelic exclusion at the TCRa locus. Firstly, it was determined that most cloned I$ T cells have rearranged both a loci [5,22]. Secondly, there are now several examples of T cell clones or hybridomas that express productive transcripts from both IXalleles (for example [23,24]). While it became clear that some of these cells achieved allelic exclusion post-translationally [ 251, there is at least one reported case of a cell in which the two a chains paired effectively with its one p [ 261. Thirdly, it was discovered that secondary rearrangements at the a locus could occur when a transformed thymocyte line expressing ap TCRs was cultured in vitm the axpx heterodimer expressed at the surface of these thymocytes was gradually replaced by others (ay Bx, aw px, etc.) [ 271. Secondary rearrangements in thymocytes were also suggested after the isolation of DNA circles containing productively rearranged a gene segments [28]. Finally, clear evidence was provided that expression of a rearranged a gene in ap TCR transgenic mice did not effectively block rearrangements at the endogenous a loci [29,30]. In fact, a significant number of mature T cells in these mice displayed both transgene- and endogene-encoded a chains. If the signal for halting rearrangements is not expression of an af3 heterodimer on the cell surface, then what is it? An answer to this question came from two groups who realized that the recent cloning of recombination activating genes (RAG1 and RAG2) provides a new and arguably more direct means to study the control of allelic exclusion. The products of these genes are capable of inducing rearrangements in artificial substrates, and several lines of evidence [31,32] indicate that they are, in fact, critical components of the rearrangement machinery. Turku et al. [33*] have hybridized thymus sections with RAG1 and RAG2 probes and found a striking segregation of label between the cortex and medulla: almost all cortical cells, but no medullary cells, expressed RAG I and RAG2 transcripts. The significance of this result was two-fold. Since many cortical cells express TCRs at the surface, it hinted that rearrangements could still occur in cells displaying af3 heterodimers, and since positive and some negative selection appears coincident with the cortex to medulla (double-positive, CD4+CD8+, to single-positive, CD4 + or CD8+) transition, it suggested that one of these events might provide the signal that stops rearrangement. This suggestion was supported by the observation that the engagement of TCR in vitro with an anti-CD3 monoclonal antibody quickly led to down-regulation of RAG1 and RAG2 mRNA lev els. Borgulya et al. [30] demonstrated that it is indeed
positive selection that signals the termination of further rearrangements by establishing that the CD4+CD8+ TCR-/IO to CD4+CD8+TCRhi transition, known to coincide with positive selection in at least some situations, is accompanied by a signiiicant reduction in the level of RAG1 and RAG2 transcripts. Thus, there is now strong evidence that the signal halting rearrangements at the TCR-a locus is not display of an ap heterodimer on the cell surface, but instead, positive selection of those cells which have at least some affinity for self-MIX. The advantage of such a delay would again be its potential to promote diversification of the repertoire, with a thymocyte able to try different a chains combined with its particular l3.
Changes
in the ap T-cell
repertoire
during
ontogeny It is now well established that the B-cell repertoires of adult and perinatal mice are distinctly different (reviewed in [34]). A functional dichotomy was evident in the distinct spectrum of specificities exhibited by neonatal B cells: most produced low affinity, polyspecific, autoreactive antibodies. A structural dichotomy was apparent by at least two criteria: firstly, different patterns of IgH variable segment usage, and secondly, a difference in the degree of diversity contributed by N nucleotide addition (template-independent addition, probably performed by the enzyme terminal deoxynucleotidyl transferase, TdT). Similarly, it has become clear over the past few years that adult and perinatal mice express distinctly different repertoires of y6 T cells, again on the basis of both structural and functional considerations (reviewed in [35]). With mounting evidence that the as T cells from neonatal ani mals are functionally peculiar [3644], it became obvious to question whether they also express a special repertoire of receptors. The answer is a clear yes, according to the same two criteria used for B cells and y6 T cells. , First of all, it was found that TCR a chains show distinct patterns of V region usage: T cells from fetal and neonatal mice preferentially employed 3’V and 5’J segments while those from adults used a broader panoply of V and J elements, with perhaps some bias towards 5’V and 3’J segments [1*,4]. This linding was explained in terms of secondary rearrangements with the more internal V and J elements undergoing deletion upon juxtaposition of the more external V and J regions. However, this explanation begs a question: why do adults have more secondary rearrangements than neonates? It may be that RAG2 and/or RAG2 are expressed at lower levels in neonates, or alternatively, that neonatal thymocytes spend less time in the CD4+CD8+ stage before positive selection (thought to signal the end of rearrangements). Similar development fluctuations in V region usage have not been reported for the murine TCR p chain. However, this issue might be worth re-examining given the recent demonstration of two VP families in the chicken, each with a distinct developmental program of expression and distinct func-
Generation of the ct p T-cell repertoire Benoist and Mathis
tional characteristics, particularly because structural remnants of this bifurcation remain evident in the mouse VP [45,46]. That is, murine TCRs can be divided intp two structural groups and these are delineated by the same amino acids that distinguish the two chicken Vp families.
ontogeny. We must keep our eyes open to spot other unsuspected influences.
The second structural difference between the TCRs of neonatal and adult mice is the contribution that N nucleotides make to their diversity. The former have few TCR transcripts with N additions, and these are usually only one or two nucleotides long, whereas the latter have many TCR transcripts with N additions which are often as long as four to eight nucleotides [47*,48-,491. The basis for this difference has not yet been established satisfactorily. According to in situ hybridization studies, the pattern of TdT transcription is very similar to that of RAG1 and RAG2 expression, being confined to cortical cells of the thymus. However, substantial transcription is detectable only several days after birth, just preceding the appearance of sign&ant N nucleotides in the TCR transcripts of mature single-positive (CD4 + or CD8+ > thymocytes (S Gilfillan, M Bogue, C Benoist, D Mathis, unpublished data). It is not known whether the developmental switch is due to a changing cortical environment (e.g. synthesis of different cytokines) or whether the thymus is populated successively by T-cell precursors with different programs of Tflexpression. The latter possibility is made more likely by the report that fetal liver- and adult bone marrow-derived stem cells give i-ise to different populations of y6 T cells when used to reconstitute fetal thymus lobes in culture [50,51 I, and the more recent claim that this difference includes a dichotomy in N nucleotide addition [52*]. Whichever proves to be the correct mechanism, it seems that the lack of N region diversity in TCRs from neonatal mice is exaggerated by selection events in the thymus [48-l. TCR transcripts from the immature CD4_CD8+CD3_flO thymocyte subset sometimes carried a few N nucleotides; however, this source of diversity was diminished in the CD4 + CD8+ population and was essentially absent in the mature CD4- CD8+CD3+ subset most importantly, in C57 B1/6 x SJL Fl neonates, transcripts from the non-expressed VP17b allele had significantly more N nucleotides than those from the expressed Vp17a allele.
Acknowledgements
Thus, the ap T-cell repertoire changes shape during development, apparently subject to novel selection forces. The challenge now is to elucidate these forces and to discover the functional consequences of having a special neonatal repertoire.
We wish to thank M Bogue, S GilfiIlan, C Thompson Boehmer for providing unpublished information.
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Conclusions The immune system seems to exploit a large array of mechanisms to produce a diverse repertoire of T and B cells, competent to meet any form of challenge. Here we have discussed several newly apparent influences on the C$ T-cell repertoire: non random rearrangements at the TCR a and TCR b loci; cell-surface expression of ‘lone’ p chains; successive rearrangements at the TCR c1 locus; and fluctuating levels of N nucleotide addition during
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46.
47.
CHAR D, SANCHEZP, CHEN CLH, BUCH RP, C~~PER MD: A Third Sublineage of Avian T CeUs can be Identilied with a T CeU Receptor-3-specific Antibody. J Immunol 1990, 145:3547-3555. IAHTI JM, CHEN C-LH, TJOELKER LW, PICKELOM, SCHAT KA, CALNEK BW, THOMPSON CB, COPPERMD: Two Distinct a p TceU Lineages can be Distinguished by the Differential Usage of T-cell Receptor VP Gene Segments. Proc Nut1 Acad Sci (i S A 1991, 88:1095610960. FEENEY AJ: Junctional Sequences of Fetal T CelJ Receptor
48. .
B~GUE M, CANDEIASS, BENOIST C, MATHS D: A Special Repertoire of UP T Cells in Neonatal Mice. FG!4BOJ 1991,
10:3647-3654. Demonstrates that neonatal mice have a peculiar c$ T-cell repertoire, impoverished in N region diversity and shows that this paucity of N nucleotides is exaggerated by selection events in the thymus. 49.
MCCORMACKWT, TJOEIXERLW, STELW\G, POSTEMA CE, THOMPSONCB: Chicken T-cell Receptor p-chain Diver-
sity: an Evolutionarily Conserved Dp-encoded
Glycine Turn
reDertoire
Benoist
and Mathis
Within the Hypervariable CDR3 Domain. Proc Nut1 Acad Sci II S A 1991, g&769+7703. 50.
IKUTA K, KINA T, MACNEILI, UCHIDA N, PEAULTB, CHIEN YH, WEISSMANIL: A Developmental Switch in Thymic
Lymphocyte Maturation Potential Occurs at the Level of Hematopoietic Stem Cells. Cell 1990, 62:863-874. 51.
OGIMOTO M, YOSHIKAI Y, MATSU~AKIG, MATSUMOTOK, KISHIHARAK, NOMOTO K: Expression of T CeU Recep-
tor Vy5 in the Adult Thymus of Irradiated Mice after Transplantation with Fetal Liver Cells. Eur J Immunol 1990, 20:1965-1970.
p
Chains have Few N Regions. / i?q~ Med 1991, 174:115-124. demonstrates, through sequence analysis, that perinatai and adult T cells express afl TCRS with different degrees of N region diversity.
of the c1D T-ceil
IKIJTAK, WEISSM.~NIL: The Junctional Modifications of a T CeU Receptor y Chain are Determine at the Level of Thymic Precursors. J Exp Med 1991, 1741279-1282. Makes the interesting claim that T-cell precursors derived from fetal liver versus bone marrow give rise to cells displaying yS receptors with different degrees of N region diversity.
52. .
C Benoist and D Mathis, laboratoire de GPn&ique Molt-c&ire des Eucalyotes du CNRS, IJnitt- 184 de Biologie Mol&zulaire et de G&e G&@tique de I’INSERM lnstitut de Chimie Biologique, Faculte de Med&ine, Strasbourg, FrXICe.
161
The
I’INSERM
sequence
repertoire to
be
Benoist
lnstitut
of events
de Chimie Biologique,
leading
has been known
sketched
in,
to a diverse
in outline
however,
unsuspected
Current
and Diane Mathis
and
influences
Opinion
and
competent
some
of
these
on repertoire
There have been several indications over the past year that this traditional view is far too simple. We will focus on four of these: (a) the observation that rearrangements at neither the TCR-a nor TCR-p locus are entirely random; (b) the finding th at p c h ams are displayed on the surface of immature thymocytes in the absence of c1 chains; (c) the suggestion that the CLlocus can undergo multiple rearrangements before thymocyte selection; and (d) the observation that the c@ T-cell repertoire changes markedly during ontogeny. Each of these new intricacies is an eyeopener to the fact that positive and negative selection, as we currently recognize them, are not the only forces that shape the c$l T-cell repertoire.
O$ T-cell
suggest
previously
content.
1992, 4:15&161
Non-random
The traditional description of the generation of the c$ T-cell repertoire has been a rather simple one. In outline, T-cell progenitors enter the thymus in a naive state, devoid of antigen specificity because their T-cell receptor (TCR) genes are in the unrearranged, germline conliguration. During a first ‘window’ of thymocyte differentiation, the TCR-fl locus gradually rearranges, resulting in the random joining of DJ and then VDJ segments. As part of this process, each exposed terminus undergoes endonuclease ‘nibbling’ and template-independent N nucleotide addition, creating an enormous degree of diversity at the VDJ junction. During a subsequent differentiation ‘window’, the TCR-a locus randomly rearranges and diversifies, in a similar manner to the TCR-p locus, except that no D segment is employed. TCR CLand p chains are synthesized, combine, and associate with a set of invariant CD3 chains. Once this complex multimer has been displayed on the cell surface, further TCR gene rearrangements are blocked, resulting in allelic exclusion. It is only rather late in thymocyte differentiation that specificity is imposed on the vast, essentially haphazard repertoire, through positive and negative selection events mediated by T cell-stromal cell interactions (as discussed by Pardoll and Carrera in this issue, pp 162-165).
France
form for some time. Details continue
in Immunology
Introduction
Strasbourg,
rearrangement
of TCR gene
segments It has been a common assumption that VJ rearrangements at the TCR-a and VDJ rearrangements at the TCR-P locus are randomly generated; that is, each diversity segment has an equal probability of combining with each joining segment, and every variable stretch with every diversity or joining stretch. This assumption underlies the concept of combinatorial diversity and has been incorporated into all theoretical estimates of the ap T-cell repertoire. However, it has recently been demonstrated that neither murine Vcrs and Jcls nor V(3s and Jps are joined randomly [1=,2-5,6-l. The most convincing data on the c1 locus came from a study on isolated thymocytes using polymerase chain reaction technology [ 1.1. TCR transcripts bearing Vcl segments that mapped to the 5’, central, or 3’ regions of the locus were amplified and probed with oligonucleotides corresponding to variously situated Ja segments. Particular VCLSclearly combined perferentially with particular Jcls: the V segment located nearest the 3’ end was frequently associated with the J segments nearest the 5’ end; on the other hand, the V segment located neare$ the 5’ end was joined almost exclusively to the more distal J segments (at least in adults); strikingly, two highly homologous but spatially separated members of the Vu5 family showed distinct patterns of Ja usage. These findings are highly reminiscent of results from another set of studies that used a less discriminatory Southern blotting assay [2-4]. This assay did, however, provide another piece of important information: TCR-cr rearrangements on the two homologous chromosomes often appear t9 involve J segments residing in the same region of the locus, suggesting rather strongly that the choice of a particular Ja is directed, not random. This result has recently been confirmed with T cell hybridomas [ 51. Such striking data have not so far been reported for the p locus, but evidence of directed rearrangement
Abbreviations IL-lnterleukin; Ig-immunoglobulin; PCR-poiynierase SCl[tsevere combined immunodeficiency; TCR-T-cell
156
@ Current
Biology
chain reaction; RAG-recombination activating gene; receptor; TdT-terminal deoxynucleotidyl transferase.
Ltd ISSN 0952-7915
Generation
did come from sequence analysis of a large number of TCR transcripts encoding the VP17 variable region [6*]. J segment usage by these TCRs was highly vanable, ranging from < 1% for Jp1.5 to >20% for Jp2.6. In general, there was marked preference for Jp2s rather than Jpls. This bias was not the result of positive or negative cell selection events since it was observed with Vl317a transcripts, which give rise to cell surface protein, and also with VP17b transcripts, which do not because of an interfering stop-codon. Nor was the variability peculiar to transcripts carrying vl317 since preliminary data on v~G + TCRs also showed a bias [6-l. The finding that VWJat and VpJp rearrangements are nonrandom is significant because it indicates that the T-cell repertoire that emerges in the periphery is constrained by molecular events involved in TCR gene rearrangement in addition to the various selection events mediated by cellLcell interaction, One is led to consider influences such as the relative accessibility of particular segments due to chromatin conformation, the efficacy of recombinant signals flanking individual segments and the capacity of different stretches to partake in secondary rearrangements. One must also question whether and how such influences vary in different mouse strains,
/ ‘lone’ TCR f3 chains on immature
thymocytes
_
It has been taken for granted that the TCR c1and p chains must both be expressed for either to appear on the cell surface. Results encouraging such a view were repeatedly obtained from experiments on T-cell lines that had secondarily lost the expression of one of the subunits (for example [7]) and from transfection studies where only one or the other subunit was introduced into an CL-P,recipient (for example [8]). It was a surprise, then, to discover a few years ago that transgenic mice carrying a rearranged TCR-l3 gene displayed the transgene-encoded fl chain and only that l3 on the surface of immature CD44CD8 thymocytes in the apparent absence of other TCR chains [9], During the past year, this finding has been consolidated and extended in two reports [ lO*,ll*]. Firstly, it was shown that introduction of a rearranged TCRp transgene into a severe combined immunodeliciency (SCID) genetic background provoked visible alterations in the phenotype of the thymocytes, which are usually CD4_CDWTCRin this strain due to a defect in the DNA recombination machinery [lo.]. SCID thymocytes carrying the transgene displayed p chains in the absence of TCR 01, y or 6 chains. Also, they were prompted to express CD4 and CD8, and were induced to transcribe RNA from the TCR-cl locus. Secondly, it was found that a CD4+CD8+ thymocyte line, transformed after infection with Gross virus, displayed p chains at the cell surface independently of the other TCR chains [ 11.1. These subunits carried a variable region different from that borne by the p subunits in the transgenic experiments, impart ing some generality to the phenomenon. B
of the ~1p T-cell
repertoire
Benoist
and Mathis
Having firmly established the existence of unusual cellsurface TCR p chains, both groups have proceeded with biochemical and functional analyses. It now appears that the l3 subunits are displayed as either monomers or homodimers that have an atypical liaison with CD3 subunits ([9,10~,11*,12]. These complexes are capable of signalling when stimulated by an anti-TCR monoclonal antibody; the signal can be detected by measuring calcium flux, but not proliferation or lymphokine secretion [lO*,Il*]. The critical question is, of course, what consequence does the expression of these ‘lone’ p chains have for a thymocyte in vim? Phenotypic changes observed in the transgenic mouse experiments may provide important clues. It might be that cell-surface display of p chains leads to closing down of the l3 locus to further rearrangements and opening up of the a locus to the transcription and rearrangement machinery. This would give a satisfy ing symmetry to the emerging B cell scenario, discussed at length in [lo*]. It is interesting in this regard that transgenic mice carrying a rearranged TCR-b gene with a near-complete deletion of the V region show exclusion of endogenously encoded p chains [ 131, while Ig heavy chains lacking a V region are displayed on the surface of pre-B cells and exert an important influence on the emerging repertoire [I4]. It might also be that cell-surface display of l3 chains is an important stimulus for @l T-cell differentiation, prompting expression of the CD4 and CD8 accessory molecules required by most cells to achieve terminal differentiation. SCID thymocytes do proceed to the CD4+CD8+ stage under the influence of a TCR-P transgene, but this can also occur upon expression of TCR-yG transgenes [ 151 as well as after introduction of heterologous bone marrow stem cells [ 161. Thus, one must be prepared to accept the possibility of alternative mechanisms for switching on differentiation, as well as the likelihood that the relevant signals are delivered intercellularly. Finally, it is worth considering, again on the basis of B cell precedent, that surface p chains might promote diversification of the repertoire. It has been reported that complete immunoglobulin (Ig) heavy chains displayed in the absence of light chains alter the growth requirements of pre-B cells, permitting them to proliferate in response to IL-7 in the absence of stromal cells; such proliferation is no longer possible once heavy-light chain complexes assemble at the surface [ 17-191. This led to the proposal that once a pre-B cell can synthesize a ‘good’ heavy chain, it proliferates to allow several different light chains to be combined with the same heavy chain. By analogy, a thymocyte that synthesizes a ‘good’ b subunit might proliferate because of altered lymphokine susceptibility, permitting the pairing of multiple LXchains with the same 0 chain.
Multiple
rearrangements
at the TCR-a
The regulated model of allelic exclusion that successful display of Ig heavy-light
locus
[19] postulates or TCR c&l het-
157
158
lymphocyte
development
erodimers at the cell-surface prevents further rearrangements of the light chain genes and the IX chain genes respectively. Consistent with this postulate, only about one third of B cells were found to rearrange both x loci and, with very rare exceptions, only one of these rearrangements ever gave rise to productive transcripts [20]. In addition, expression of rearranged a Igx gene in transgenic mouse B cells markedly suppressed rearrangement of the endogenous x loci [ 2 1 I. Evidence has accumulated over the past few years that the regulated model of allelic exclusion requires some modification if it is to explain allelic exclusion at the TCRa locus. Firstly, it was determined that most cloned I$ T cells have rearranged both a loci [5,22]. Secondly, there are now several examples of T cell clones or hybridomas that express productive transcripts from both IXalleles (for example [23,24]). While it became clear that some of these cells achieved allelic exclusion post-translationally [ 251, there is at least one reported case of a cell in which the two a chains paired effectively with its one p [ 261. Thirdly, it was discovered that secondary rearrangements at the a locus could occur when a transformed thymocyte line expressing ap TCRs was cultured in vitm the axpx heterodimer expressed at the surface of these thymocytes was gradually replaced by others (ay Bx, aw px, etc.) [ 271. Secondary rearrangements in thymocytes were also suggested after the isolation of DNA circles containing productively rearranged a gene segments [28]. Finally, clear evidence was provided that expression of a rearranged a gene in ap TCR transgenic mice did not effectively block rearrangements at the endogenous a loci [29,30]. In fact, a significant number of mature T cells in these mice displayed both transgene- and endogene-encoded a chains. If the signal for halting rearrangements is not expression of an af3 heterodimer on the cell surface, then what is it? An answer to this question came from two groups who realized that the recent cloning of recombination activating genes (RAG1 and RAG2) provides a new and arguably more direct means to study the control of allelic exclusion. The products of these genes are capable of inducing rearrangements in artificial substrates, and several lines of evidence [31,32] indicate that they are, in fact, critical components of the rearrangement machinery. Turku et al. [33*] have hybridized thymus sections with RAG1 and RAG2 probes and found a striking segregation of label between the cortex and medulla: almost all cortical cells, but no medullary cells, expressed RAG I and RAG2 transcripts. The significance of this result was two-fold. Since many cortical cells express TCRs at the surface, it hinted that rearrangements could still occur in cells displaying af3 heterodimers, and since positive and some negative selection appears coincident with the cortex to medulla (double-positive, CD4+CD8+, to single-positive, CD4 + or CD8+) transition, it suggested that one of these events might provide the signal that stops rearrangement. This suggestion was supported by the observation that the engagement of TCR in vitro with an anti-CD3 monoclonal antibody quickly led to down-regulation of RAG1 and RAG2 mRNA lev els. Borgulya et al. [30] demonstrated that it is indeed
positive selection that signals the termination of further rearrangements by establishing that the CD4+CD8+ TCR-/IO to CD4+CD8+TCRhi transition, known to coincide with positive selection in at least some situations, is accompanied by a signiiicant reduction in the level of RAG1 and RAG2 transcripts. Thus, there is now strong evidence that the signal halting rearrangements at the TCR-a locus is not display of an ap heterodimer on the cell surface, but instead, positive selection of those cells which have at least some affinity for self-MIX. The advantage of such a delay would again be its potential to promote diversification of the repertoire, with a thymocyte able to try different a chains combined with its particular l3.
Changes
in the ap T-cell
repertoire
during
ontogeny It is now well established that the B-cell repertoires of adult and perinatal mice are distinctly different (reviewed in [34]). A functional dichotomy was evident in the distinct spectrum of specificities exhibited by neonatal B cells: most produced low affinity, polyspecific, autoreactive antibodies. A structural dichotomy was apparent by at least two criteria: firstly, different patterns of IgH variable segment usage, and secondly, a difference in the degree of diversity contributed by N nucleotide addition (template-independent addition, probably performed by the enzyme terminal deoxynucleotidyl transferase, TdT). Similarly, it has become clear over the past few years that adult and perinatal mice express distinctly different repertoires of y6 T cells, again on the basis of both structural and functional considerations (reviewed in [35]). With mounting evidence that the as T cells from neonatal ani mals are functionally peculiar [3644], it became obvious to question whether they also express a special repertoire of receptors. The answer is a clear yes, according to the same two criteria used for B cells and y6 T cells. , First of all, it was found that TCR a chains show distinct patterns of V region usage: T cells from fetal and neonatal mice preferentially employed 3’V and 5’J segments while those from adults used a broader panoply of V and J elements, with perhaps some bias towards 5’V and 3’J segments [1*,4]. This linding was explained in terms of secondary rearrangements with the more internal V and J elements undergoing deletion upon juxtaposition of the more external V and J regions. However, this explanation begs a question: why do adults have more secondary rearrangements than neonates? It may be that RAG2 and/or RAG2 are expressed at lower levels in neonates, or alternatively, that neonatal thymocytes spend less time in the CD4+CD8+ stage before positive selection (thought to signal the end of rearrangements). Similar development fluctuations in V region usage have not been reported for the murine TCR p chain. However, this issue might be worth re-examining given the recent demonstration of two VP families in the chicken, each with a distinct developmental program of expression and distinct func-
Generation of the ct p T-cell repertoire Benoist and Mathis
tional characteristics, particularly because structural remnants of this bifurcation remain evident in the mouse VP [45,46]. That is, murine TCRs can be divided intp two structural groups and these are delineated by the same amino acids that distinguish the two chicken Vp families.
ontogeny. We must keep our eyes open to spot other unsuspected influences.
The second structural difference between the TCRs of neonatal and adult mice is the contribution that N nucleotides make to their diversity. The former have few TCR transcripts with N additions, and these are usually only one or two nucleotides long, whereas the latter have many TCR transcripts with N additions which are often as long as four to eight nucleotides [47*,48-,491. The basis for this difference has not yet been established satisfactorily. According to in situ hybridization studies, the pattern of TdT transcription is very similar to that of RAG1 and RAG2 expression, being confined to cortical cells of the thymus. However, substantial transcription is detectable only several days after birth, just preceding the appearance of sign&ant N nucleotides in the TCR transcripts of mature single-positive (CD4 + or CD8+ > thymocytes (S Gilfillan, M Bogue, C Benoist, D Mathis, unpublished data). It is not known whether the developmental switch is due to a changing cortical environment (e.g. synthesis of different cytokines) or whether the thymus is populated successively by T-cell precursors with different programs of Tflexpression. The latter possibility is made more likely by the report that fetal liver- and adult bone marrow-derived stem cells give i-ise to different populations of y6 T cells when used to reconstitute fetal thymus lobes in culture [50,51 I, and the more recent claim that this difference includes a dichotomy in N nucleotide addition [52*]. Whichever proves to be the correct mechanism, it seems that the lack of N region diversity in TCRs from neonatal mice is exaggerated by selection events in the thymus [48-l. TCR transcripts from the immature CD4_CD8+CD3_flO thymocyte subset sometimes carried a few N nucleotides; however, this source of diversity was diminished in the CD4 + CD8+ population and was essentially absent in the mature CD4- CD8+CD3+ subset most importantly, in C57 B1/6 x SJL Fl neonates, transcripts from the non-expressed VP17b allele had significantly more N nucleotides than those from the expressed Vp17a allele.
Acknowledgements
Thus, the ap T-cell repertoire changes shape during development, apparently subject to novel selection forces. The challenge now is to elucidate these forces and to discover the functional consequences of having a special neonatal repertoire.
We wish to thank M Bogue, S GilfiIlan, C Thompson Boehmer for providing unpublished information.
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p
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of the c1D T-ceil
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52. .
C Benoist and D Mathis, laboratoire de GPn&ique Molt-c&ire des Eucalyotes du CNRS, IJnitt- 184 de Biologie Mol&zulaire et de G&e G&@tique de I’INSERM lnstitut de Chimie Biologique, Faculte de Med&ine, Strasbourg, FrXICe.
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