Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Band T lymphocytes display a wide repertoire of antigen receptors, made by random recombination of V, (D), and J segments and trimming/addition of nucleotides at the junctions between these rearranged segments (1). Besides the mainstream lymphocytes, three different types of cell subpopulations have been described that have a limited repertoire diversity: the B1 B cell subset (2, 3), some / T cell subpopulations (4), and the TCR /⁺ NK1 T cells (5). Restricted repertoires seem to define discrete lymphocyte subpopulations at the frontier between innate and adaptive immunity, as these selected repertoires may allow the presence of a high frequency of preexisting cells reactive against phylogenetically conserved antigens (6, 7). Alternatively, cells with such a restricted repertoire may play an immunoregulatory role or another physiologic function, such as the wound healing, that results from the secretion of keratinocyte growth factor (8) by / dendritic epithelial cells (DECs)¹ recognizing self-ligands (9).

B1 cells, which appear early during embryonic life, use recurrent VDJ combinations with few "N" additions (10), and seem to be selected by endogenous ligands (11). Similarly, some / T cell subsets appear early during ontogeny and use peculiar V-J combinations without N additions (12). These recurrent sequences seem to be favored by enzymatic constraints linked to the recombination process, such as homologous region-guided recombination (4). Cells bearing these invariant / TCRs seem to be also selected by endogenous ligands as, in mice deficient for these segments, they are replaced by cells expressing other VV combinations but similar clonotypic motifs (13).

NK1 T cells use an "invariant"  chain (mAV14AJ18 in mice [14], hAV24AJ18 in humans [14, 15]) with a CDR3 of constant length paired with a limited number of V segments (BV2S1, 7S1, and 8S2 in mice, BV11S1 in humans). NK1 T cells display peculiar phenotypic characteristics: they are CD3^int, CD4⁺, or CD4CD8 (DN), and have both activation (CD44^hi and 3G11^lo) and NK markers (NKRP1, Ly49) (5). In vivo development of NK1 T cells requires the MHC class Ib CD1d molecule (16), whose expression is 2-microglobulin (2m) dependent but transporter associated with antigen processing (TAP) independent (17). In both humans and rodents, mature NK1 T cells recognize some glycolipids (such as -galactosylceramide) in a CD1d-restricted fashion (18, 19) as well as some exogenous protozoan glycosylphosphatidylinositol (GPI [20]), but the exact endogenous ligand(s) presented physiologically by CD1d have not yet been identified. The most salient feature of NK1 T cells is their ability to secrete very rapidly large amounts of IL-4 in a primary response (21). They can also secrete large amounts of IFN-, especially when triggered through their NKRP1 receptor (22). However, their true in vivo functions are still enigmatic, though there is evidence that they may be involved in Th1/Th2 class polarization (23), in IL-12-induced tumor rejection (24), and in autoimmunity (25).

Mainstream / T lymphocytes express either CD8 or CD4 accessory molecules and respond to peptidic antigens presented in low amounts by either MHC class I or class II molecules. The absence of accessory molecules in the DN T cell subpopulation suggests that these cells may recognize high-density ligands (such as -galactosylceramide or glycosylphosphatidylinositol presented by CD1 for NK1 T cells) and/or display TCRs with high affinity. Indeed, most / T cells are DN or CD8, and the NK1 T cells comprise both DN (and CD8⁺ in humans [28]) and CD4⁺ cells, though the CD4 molecule in these latter does not seem to play a role in the binding affinity (14, 29). DN /⁺ T cells represent between 0.5 and 2% of PBLs in normal individuals and comprise the aforementioned NK1 T cells and other cells of unclear specificity or functions. Some of these also recognize CD1d, while others are specific for glycolipids presented by CD1a, b, or c (30).

NK1 T cells thus far represent the only known T cell subset that has conserved its TCR, its specificity, and its functional features between rodents and primates, suggesting that they may play an ancient and important physiological role (18). Here, we report on the existence of another phylogenetically conserved population of T cells in mammals, defined by the expression of a novel canonical TCR  chain comprising the hAV7S2 and AJ33 elements in humans and the homologous AV19 and AJ33 elements in mice and cattle. V7/19-J33⁺ T cells show an oligoclonal TCR  chain repertoire and are selected by a 2m-dependent but TAP-independent molecule that is neither a classical MHC class I molecule nor CD1d. The conservation between species of T cells displaying this limited repertoire and their unusual restriction pattern suggest that this population may serve a function complementary to that played by NK1 T cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Antibodies.

Human Cell Preparations.

Bovine Cell Separations.

Mice.

/

b/

/

/

b/

b/

/

Immunofluorescence Studies.

Oligonucleotides.

Nucleic Acid Preparation.

Quantitative Kinetic PCR.

Single Cell Repertoire Analysis.

Sequencing.

T-T Hybridoma Generation.

/

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

A previous analysis of TCR  chain repertoire of human DN T cells indicated that besides the invariant hAV24AJ18 TCR  chain, another TCR  chain was frequently expressed that also had a restricted AVAJ usage and recurrent junctional features (39). This  chain, which was encoded by rearranged hAV7S2AJ33 elements, had a CDR3 of constant length but some variability in the two junctional codons. A GenBank search showed that among the 20 TCR  chains sequenced in cattle, one chain (BOTCRA14) used the homologous V and J segments with a CDR3 of the same length (40). In mice, no homologous invariant  chain had been described, but the corresponding V (mAV19) and J (mAJ33) segments display high homology with their human counterparts, as there is only one amino acid difference between the murine AJ33 and its human counterpart (41, 42). Based on these observations, which suggested the existence of another conserved T cell population in mammals, we undertook an extensive analysis of the frequency and repertoire of the T cells bearing this particular combination of AVAJ elements in these three species.

Canonical hAV7S2AJ33 TCR  Chains, as well as Their Murine and Bovine Homologues, Are Abundantly Expressed in DN T Cells

To determine the frequency and coreceptor phenotype of T cells bearing hAV7S2AJ33 TCR  chains, PBLs were separated into highly purified / DN, CD4⁺, or CD8⁺ (i.e., CD8⁺ and CD8⁺) subsets, and the amount of hAV7S2AJ33-encoded transcripts in duplicate fractions was quantified by kinetic PCR. Although the amounts of C chain were similar in all samples (Fig. 1 A), there was a large shift to the left of the hAV7S2AJ33 amplification curves for the DN (8.5 cycles) and the CD8⁺ fractions (5 cycles) (Fig. 1 B) compared with the CD4⁺ fractions, indicating that DN and CD8⁺ cells contained ~150-360- and 19-32-fold more hAV7S2AJ33 transcripts, respectively, than CD4⁺ lymphocytes. hAV7S2AJ33 transcripts were enriched in DN and CD8⁺ T cell preparations from all individuals tested, and on average the enrichment levels of hAV7S2AJ33 transcripts in DN T cells were much higher than those observed for the NK1 T cell-specific invariant  chain hAV24AJ18 (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   High levels of hAV7S2/AV19-AJ33 TCR  chain are present in DN T cells from three mammal species: the indicated cell subpopulations were purified by FACS® sorting from human (A and B) or bovine (C and D) PBLs and from mouse lymph nodes (E and F). Duplicate RNA preparations were obtained and, after reverse transcription, PCR was carried out for the indicated gene segments. The amount of amplicon was quantified by ELISA at the indicated cycle. Averaged values of duplicates are shown for C-F. CD8+ fraction contains both CD8+ and CD8+ TCR /+ T cells. In cattle, no amplification could be obtained with boAV19-AJ33 primers in the CD8+ fraction, and thus the corresponding curves are not displayed.

Because a similar invariant  chain had been sequenced once in cattle (40), we examined its distribution within CD4⁺, CD8⁺, DN, or (CD4⁺ plus CD8⁺) sorted cells obtained from bovine blood. As shown in Fig. 1, C and D, the levels of boAV19AJ33 transcripts in the DN fractions were as high as those observed in humans. No boAV19AJ33 amplification could be obtained with the CD8⁺ fraction, and the comparison of CD4⁺ and CD4⁺ plus CD8⁺ amplification curves suggested that bovine CD8⁺ cells did not express high amounts of boAV19AJ33 transcripts.

In mice, no canonical TCR  chain rearrangement involving mAV19 and mAJ33, the murine elements that are homologous to hAV7S2 and hAJ33, respectively, had been described. However, quantitative PCR analysis of mAV19AJ33 transcripts within highly purified murine / DN, CD4⁺, and CD8⁺ lymphocytes indicated that, as in human and bovine blood, increased mAV19AJ33 expression was observed in the DN fraction from lymph nodes (Fig. 1, E and F). The shift of the amplification curves between DN and CD4⁺ cell preparations was lower than in humans and cattle, suggesting that mAV19AJ33-bearing cells were less abundant in mice.

To examine the length of the CDR3 regions of the hAV7S2AJ33 transcripts and their homologues in mice and cattle, we carried out polyclonal sequencing of V-C or V-J amplicons obtained from the various lymphocyte subpopulations in these species. As shown in Fig. 2 A, a readable hAV7S2AJ33 sequence was obtained from hAV7S2-C amplicons derived from DN and CD8⁺ samples but not from CD4⁺ samples. Therefore, this indicated that in the former but not the latter cells, the hAV7S2 segment was predominantly rearranged to the hAJ33 segment and comprised a CDR3 of constant length. The polyclonal hAV7S2AJ33 sequence displayed some heterogeneity at the VJ junction, in agreement with previous results from Porcelli et al. showing some variability of the two N-encoded amino acids (39).

View larger version (84K): [in this window] [in a new window]   Fig. 2.   (A) The CDR3 of hAV7S2-AJ33 TCR  chain is of constant length in DN T cells and in human CD8+ T cells. PCR amplifications from hAV7S2 to C (V-C) or to AJ33 (V-J) were carried out on cDNA from the T cell subpopulations indicated, and polyclonal sequencing was carried out using the same AV7S2/AV19 primer as used for amplification. Panel B displays polyclonal sequencing of FACS®-sorted CD8+ or CD8+ TCR /+ human PBLs.

In cattle, the picture was slightly different, as polyclonal sequencing of boAV19-C amplicons demonstrated the predominant presence of the invariant boAV19AJ33 chain in DN cells, but not in CD4⁺ or CD8⁺ cells (data not shown). At the VJ junction, the polyclonal sequence derived from DN cells displayed some junctional heterogeneity, indicating also some variability of the two amino acids encoded by N additions. In mice, polyclonal sequencing of the mAV19-C amplicons showed a fully readable sequence for the DN but not for the CD8⁺ fraction (data not shown). Moreover, the VJ sequence displayed no heterogeneity and corresponded to a "germline" junction without nucleotide trimming or N addition (see also Table II below). Altogether, these results demonstrated that in the three species, DN /⁺ T cells were enriched for cells bearing TCR  chains with highly homologous AVAJ elements (hAV7S2AJ33, mAV19AJ33, and boAV19AJ33) and constant CDR3 length as shown in the multiple alignment displayed in Fig. 3. This novel canonical TCR  chain will be referred to hereafter as the "invariant" V7/19-J33 TCR  chain.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   High homology of human, cattle, and mouse hAV7S2/AV19-AJ33  chains. Homologies were scored using the default PAM250 of ClustalW as implemented in MacVector software (Oxford Technology). Identities are bold and shaded in gray; similarities are light gray. The CDR3 region is boxed.

Direct Numbering of Cells Bearing Invariant V7/19-J33 Chains.

A large amount of invariant  chain transcripts does not formally demonstrate the existence of a large number of invariant TCR  chain-bearing cells, as this transcript could have been expressed at very high levels in a few cells. To more directly estimate the number of cells using the invariant V7-J33 chain, single /⁺ DN cells from three different subjects were sorted into PCR plates using a single cell deposition unit. FACS^®-sorted /⁺ TCR CD8⁺ or CD4⁺ cell suspensions were also serially diluted into PCR plates to get 12 replicates of the indicated cell concentrations. After cell lysis, hAV7S2AJ33 PCR amplification was carried out on genomic DNA with luminometry detection of the amplicons. As shown in Fig. 4 A, the assay was sensitive enough to detect a single cell harboring the relevant rearrangement and yielded a frequency of V7-J33⁺ DN cells of ~1/7.4. Using limiting dilution analysis (LDA), the frequency observed for CD8⁺ cells was 1/36 (confidence interval: 1/25-1/70; Fig. 4 B). Sequence analysis of amplicons from single cell preparations obtained with DN and CD8⁺ cells demonstrated the presence of invariant V7-J33 transcripts in all cases. A similar analysis on CD4⁺ cells yielded a frequency of V7-J33⁺ cells of 1/1,093 (1/751-1/2,003; Fig. 4 C). Importantly, sequencing of the positive wells at concentrations where the V7-J33 amplicons were derived from one cell showed that out of six positive wells, two corresponded to a rearrangement of the hAV7S2 to hAJ34 (which were amplified because of the short genomic distance [~700 bp] separating hAJ34 from hAJ33), three corresponded to a rearranged V7-J33 chain with a different CDR3 length, and only one corresponded to the invariant V7-J33 chain. Thus, the actual frequency of CD4⁺ cells harboring the invariant V7-J33 chain should be ~1/6,000.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Direct enumeration of AV7S2/AV19-AJ33-bearing cells by PCR-limiting dilution analysis and single cell PCR. (A) DN cells obtained from three human subjects were seeded into microtiter plates using a FACS® single cell deposition unit. After cell lysis, PCR was carried out with AV7S2 and AJ33 primers, and the amount of amplicons was quantified by ELISA. (B and C) / CD8+ or CD4+ cells were obtained by FACS® sorting, serially diluted, distributed in a microtiter plate at the indicated cell concentration, and processed as above. In C (CD4+ cells), the amplicons of the six wells indicated (which had a high probability of clonality) were sequenced. The sequences were as follows:1-GCT GTG ATG TCG AT2-GCT GTG AGA GAG ATC GGA3-GCT GTG AGA GAT4-GCT GNN ACT CGA5-GCT GTG AGA GAG GAT AAC-AJ34-intron-AJ336-GCT GTG AGG GGG GGG GAA AAC-AJ34-intron-AJ33Only one (sequence 3) corresponds to the canonical AV7S2-AJ33 rearrangement (see text for details). In B and C, frequencies were calculated by maximum likelihood analysis.

In mice, V19-J33⁺ cells were less numerous than in humans. Their frequency within / DN lymph node cells was 1/56 (Fig. 5 A), compared with ~1/7.5 in humans (Fig. 4 A).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   mAV19-AJ33+ cells are more frequent in TAP/ mice. The indicated number of DN T cells from B6 (A) or TAP/ (B) mice were seeded in PCR microplates, and the presence of the mAV19-AJ33 rearrangement was detected by PCR-ELISA.

Surface Phenotype and TCR  Repertoire of V7/19-J33-bearing Cells

Invariant V7-J33-bearing Cells Express Memory Markers and Show a Biased Usage of hBV2 and hBV13 TCR  Chains.

In the absence of anti-hAV7S2 or anti-mAV19 antibody, we used the following strategy to characterize the surface phenotype of the population expressing the invariant V7-J33 chain. / DN and CD8⁺ cells were depleted or enriched for a given marker by FACS^® sorting, and the amounts of V7-J33 transcripts in the positive and negative fractions were quantified and compared with the amounts of amplified C transcripts, in order to take into account variations in cell number. Fig. 6 displays an example of such an experiment, where we examined whether the CD8 molecules expressed by V7-J33⁺ cells were either heterodimeric () or homodimeric (), since CD8⁺ cells represent between 5 and 25% of CD8⁺ TCR /⁺ lymphocytes in human PBLs (data not shown). There were ~45-fold more V7-J33 transcripts in the CD8⁺ fraction than in the CD8⁺ fraction. This result was confirmed by polyclonal sequencing of the amplicons obtained after amplification with hAV7S2/C (V-C) or hAV7S2/hAJ33 (V-J) primers: no readable sequence was obtained from the CD8⁺ fraction, whereas the invariant  chain was present in the CD8⁺ fraction (Fig. 2 B). The absence of a readable sequence in the CD8⁺ fraction using V-J amplification indicates the near absence of the invariant  chain in this fraction. Using this strategy, it could be established that V7-J33⁺ cells had mainly a memory phenotype (i.e., CD45R0⁺CD45RA) and were CD56CD57CD28⁺CD27⁺ (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   hAV7S2-AJ33-bearing cells are CD8+ or DN. The indicated TCR /+ fractions were FACS® sorted using a four-color staining with anti-CD4-TC, anti-CD8-FITC, anti-CD8-PE, and anti-/ TCR-APC. The amounts of C (A) or hAV7S2-AJ33 (B) transcripts were quantified by kinetic PCR using the ABI Prism 7700 apparatus (Taqman). This experiment was repeated with cells from another subject with identical results. Rn, difference in fluorescence relative units.

Despite many attempts using different lymphokine mixtures, we were unable to obtain a large enough number of T cell clones expressing the invariant V7-J33 chain. Therefore, the TCR  chain repertoire of DN cells bearing invariant V7-J33 chain was directly estimated by single cell PCR analysis. After single cell deposition of DN / cells into PCR plates, cells were lysed and a first PCR reaction was carried out using hAV7S2, hAJ33, and a mixture of 29 V and 7 J. This first reaction was then amplified in a second PCR with nested AV7S2/AJ33 primers allowing us to find the wells containing V7-J33⁺ cells. Using the reaction mixture of the first PCR corresponding to the positive wells, 24 individual PCR reactions were set up using 1 (or 2) V and a mixture of 7 J primers. This allowed us to get a specific band for 0-2 V which was then sequenced using the relevant V primer. In 9 independent experiments carried out on DN cells obtained from 3 different subjects, the frequency of V7-J33⁺ wells ranged between 6 and 16/84. Among the 92 V7-J33⁺ wells studied, a  chain could be assigned and sequenced in 37 cases (40%), and we found that there was a heavy bias toward V13 and V2. Out of 13 V sequenced in donor A, 7 were BV13⁺ and 3 were BV2⁺. In donor B, 8/12 were BV13⁺ and 4/12 were BV2⁺. In donor C, 7/12 were BV13⁺ and 2/12 were BV2⁺. However, despite this biased usage of BV13 or BV2 regions, the TCR  chains derived from V7-J33⁺ cells did not show obvious restrictions in J usage or in CDR3 length (Table I). Sequence analysis of VJ junctions confirmed the presence of V7-J33 chains with the canonical CDR3 length and revealed significant variations in amino acid composition at the two N-encoded positions (Table I). Moreover, the presence of repeated TCR  and  chain junctional sequences in different cells from the same subject cloned in different PCR plates and the fact that these TCR chains were not found in other subjects indicated that this subset had undergone clonal expansion in vivo, an interpretation that is consistent with its memory phenotype (see above). Overall, V7-J33⁺ cells appear to use a semirestricted repertoire that is expanded as needed.

TCR  Chain Repertoire of V19-J33⁺ Cells.

Because the frequency of V19-J33⁺ cells is not as high in mice as in humans, the strategy followed to characterize the surface phenotype of human V7-J33⁺ cells was not successful in mice. Therefore, to characterize the TCR  chain repertoire of this subset, we took advantage of the fact that V19-J33⁺ cells are enriched in TAP^/ mice (see below). We generated T-T hybridomas from DN enriched lymph node cells from TAP^/ mice, and screened for expression of V19-J33 transcripts. In agreement with the single cell analysis (Fig. 5 B), 16/168 hybridomas studied were V19-J33⁺. As shown in Table II, in all cases, these hybridomas carried V19-J33 chains with the canonical CDR3 length and in all but two cases, the V19-J33 junction was germline encoded. The V segments used were predominantly BV8⁺ (7/16) and BV6⁺ (4/16). No restriction in the J repertoire was apparent.

Development and Selection of V7/19-J33-bearing Cells

Ontogeny of V7/19-J33-bearing Cells.

NK1 T cells are not found in neonates and accumulate after birth (5). Therefore, we examined cord blood lymphocytes for V7/ 19-J33 sequences by quantitative PCR and polyclonal sequencing of V7-C or V7-J33 amplicons on cDNA obtained from enriched DN/CD8⁺ or CD4⁺ fractions. We were unable to find significant amounts of invariant V7-J33 transcripts in cord blood in the six samples studied. However, significant amounts of this chain could be obtained in young children, albeit in lower amounts than in adults (data not shown).

Similarly, V19-J33-bearing cells were not found in spleen or thymus of mouse neonates (data not shown). They were present in lymph nodes and blood in large amounts and in smaller amounts in the spleen in the five mouse strains tested, as shown in Table III (top), which displays a summary of the studies examining the tissue distribution of V19-J33-bearing cells. Importantly, although more than half of the human invariant V7-J33⁺ cells were CD8⁺ cells, which might suggest an extrathymic development pathway (43), invariant V19-J33 rearrangements were not detected in nude mice either by quantitative PCR (Table III, top) or polyclonal sequencing (data not shown), indicating that the thymus is required for their development.

Development of V7-J33⁺ Cells Is Not Affected in MHC Class II- and TAP-deficient Patients.

The restriction element of V7-J33⁺ cells could be either a class I or class II MHC molecule, as they do not express CD4 or CD8 accessory molecules. To address this issue, we quantified the expression of the V7-J33 invariant chain in PBLs obtained from MHC-deficient patients. MHC class II- deficient patients harbor low but significant numbers of CD4⁺ / lymphocytes whose V repertoire is grossly normal (44). CD4⁺, CD8⁺, and DN enriched fractions were prepared and analyzed by quantitative PCR. Although the amount of C was much lower in the DN than in the CD4⁺ samples (Fig. 7 A), the amounts of V7-J33 transcripts were comparable in both samples (Fig. 7 B), therefore indicating an enrichment of V7-J33 transcripts within DN cells and, presumably, selection of invariant V7-J33-bearing cells in this patient. Accordingly, the predominance of the invariant V7-J33 sequence within DN cells was confirmed by polyclonal sequencing (data not shown). Similar results were obtained in the two other patients studied. Taken together, these results strongly suggest that the restriction element of the invariant V7-J33⁺ cells is not an MHC class II molecule.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   AV7S2-AJ33-bearing cells are present in MHC class II- and TAP-deficient patients. The indicated cell subpopulations were obtained from an MHC class II- (A and B) or a TAP-deficient (C and D) patient. The amounts of C or hAV7S2-AJ33 transcripts were quantified by kinetic PCR-ELISA. Two other MHC class II- deficient patients and another TAP-deficient patient were studied with similar results.

There is no MHC class I-deficient patient described to date, but we could obtain PBLs from two TAP-deficient siblings (45). In these patients, /⁺ TCR CD8⁺ cells are reduced in numbers at birth but may expand with age. /⁺ TCR CD4⁺ and CD8⁺ cells were separated in one of these patients, and the amount of C and V7-J33 transcripts was quantified in the two fractions. Despite lower amounts of C in the CD8⁺ fraction, the amount of V7-J33 transcripts was much higher in the CD8⁺ fraction (Fig. 7, C and D). Enrichment for V7-J33 transcripts was also demonstrated in a PHA-stimulated CD8⁺ line derived from the other TAP-deficient patient, when compared with either unsorted PBL-derived cell line from the same patient or to CD8⁺ cells from healthy donors (data not shown). Actually, the larger amounts of V7-J33 transcripts in the CD8⁺ / T cells of TAP-deficient patients is in agreement with the higher proportion of CD8⁺ cells in these patients (data not shown). This indicates that T cells bearing this invariant  chain are probably not selected by a TAP-dependent MHC class I molecule.

Thus, the selecting molecule for the invariant V7-J33⁺ cells appeared to be neither an MHC class II nor a classical MHC class I molecule. However, because the defect in MHC class II expression may not be complete and the level of MHC class I expression in TAP-deficient patients is still 1% of normal, no definitive conclusion could be drawn from these studies. Therefore, we turned to the mouse, where carefully controlled studies using well-characterized MHC-deficient mice can be performed.

The Selecting Ligand for V19-J33⁺ Cells Is 2m-dependent, TAP-independent, and Distinct from CD1.

The amount of V19-J33 transcripts was examined in /⁺ DN lymph node cells from several MHC-deficient strains. Because the frequency of V19-J33⁺ cells and the percentage of DN cells are lower in mice, the differences between positive and negative samples are smaller in mice than in humans. However, despite higher levels of C in the 2m^/ samples, there were fewer V19-J33 transcripts than in control or I-A^b/ mice (Fig. 8, A and B). Polyclonal sequencing of V19-J33 amplicons from 2m^/ samples showed the complete absence of invariant V19-J33 transcripts. This was consistent with an MHC class I or 2m-dependent class I-like selection of the invariant V19-J33⁺ cells. However, the relevant molecule is not a classical MHC class I molecule because the amount of invariant V19-J33 transcripts was higher in the DN cells from K^b/D^b/ mice than in controls, though the C levels were lower. These results suggest that the MHC class I selecting molecule is not a classical one and that, in the absence of classical class I molecules, the invariant chain-bearing DN cells are less diluted by mainstream cells. In Fig. 8, C and D, V19-J33 transcripts were quantified in CD8-, CD1-, and TAP-deficient mice, B6 and 2m^/ mice being used as positive and negative controls, respectively. There was no decrease in the amounts of V19-J33 transcripts in CD1-deficient mice, indicating that V19-J33⁺ cells were not selected by CD1 or a CD1-bound ligand. The curves for TAP^/ samples were actually shifted to the left, indicating an increased frequency of V19-J33⁺ cells in TAP^/ mice, in agreement with the result shown in Fig. 5 B where the frequency of V19-J33⁺ cells in DN TAP^/ mice was 1/8.5 (i.e., a sixfold increase compared with B6; Fig. 5 A). The V19-J33 curve corresponding to the CD8-deficient mice is similar to that of B6, indicating the presence of V19-J33 invariant chains in CD8^/ mice. This was confirmed by polyclonal sequencing (data not shown). Altogether, these results, summarized in Table III (bottom), indicate that V19-J33⁺ cells do not require CD8 for their selection and that the selecting molecule is a 2m-dependent, TAP-independent molecule distinct from CD1.

View larger version (23K): [in this window] [in a new window]   Fig. 8.   AV19-AJ33+ cells are selected by a 2m-dependent, TAP-independent nonclassical MHC-like molecule distinct from CD1. DN lymph node cells were obtained from the indicated mice, and the amounts of C (A and C) and mAV19-AJ33 (B and D) transcripts were quantified by kinetic PCR-ELISA. All results were confirmed by polyclonal sequencing.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Invariant V7.2-J33 TCR  chain and its murine and bovine homologues were found in three different mammalian species and, thus, define a new phylogenetically conserved T cell population using a canonical TCR  repertoire. In humans, DN and CD8⁺ cells bearing invariant V7-J33 chain accumulate after birth and become quite abundant, as they represent ~0.1-0.2% of all human PBLs. Therefore, it is legitimate to wonder why such cells have not been previously reported. This may be due to the lack of a stringent V repertoire restriction, or to the difficulties in growing clones expressing this invariant chain (our unpublished observations), together with methodological limitations linked to TCR  chain repertoire analysis (e.g., lack of allelic exclusion [46]). However, this invariant  chain had already been described in DN cells by Porcelli et al. (39) and, among the 317 random CDR3 sequences reported by Moss and Bell (47), 4 (1.2%) had a sequence corresponding to the V7-J33 invariant  chain. In the same study, this canonical sequence was not found in cord blood samples or in CD4⁺ cells, but represented 3/40 (7.5%) CDR3 sequences derived from CD8⁺ cells. This age and cell distribution is in agreement with our own measurements (data not shown). In cattle, this CDR3 was found in 1/20 TCR  chains randomly sequenced (40). In mice, random cloning of TCR  chains revealed one example of this invariant V19-J33  chain (48). It should be stressed that as shown in Fig. 3, the AJ33 segments are quasi-identical in the three species. The mouse and human J loci are entirely homologous, with the same genomic organization and an average similarity of 71% including the J coding sequences (41, 42, 49). The AJ33 is the most similar J segment between mouse and human sequences with only one amino acid difference; the two other most similar human/ mouse pairs (AJ23 and AJ24) have four and three amino acid differences, respectively (41). This quasi identity between mouse and human AJ33 segments suggests a strong selective pressure for these segments.

In humans and cattle, the TCR  chain displayed some junctional diversity, whereas in mice a genomic sequence without trimming or N additions was found in most cases (Table II). However, a murine sequence made by trimming and reconstitution of the canonical sequence through N additions was found in two hybridomas (11F1 and 4H1 in Table II), indicating that the TCR in mice is selected at the protein level. In humans, the invariant TCR  chain was associated with a limited number of V segments (mostly hBV2 and hBV13), and the same TCR with the same nucleotide sequence was present in different cells from two subjects, suggesting oligoclonal expansions. Despite this combinatorial restriction, TCR  chain diversity of invariant V