An enlarged subpopulation of T lymphocytes bearing two distinct 
TCR in an HIV-positive patient
Jean-Luc Taupin,
Franck Halary1,
Julie Déchanet,
Marie-Alix Peyrat1,
Jean-Marie Ragnaud2,
Marc Bonneville1 and
Jean-Franciois Moreau
CNRS UMR 5540, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France
1 INSERM U463, Institut de Biologie, 9 Quai Moncousu, 44035 Nantes Cedex, France
2 Service de Médecine Interne et Maladies Infectieuses, Hôpital Pellegrin, Place Amélie Raba-Léon, 33076 Bordeaux Cedex, France
Correspondence to:
J.-L. Taupin
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Abstract
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Although T cell clone monospecificity is ensured by several allelic exclusion processes operating at either the genotypic or phenotypic levels, clones expressing two distinct
ß or 
TCR have been described in several instances. Thus far, the origin of dual TCR-expressing cells and the homeostatic mechanisms controlling the size of this subset in the periphery remain poorly understood. In the course of a phenotypic analysis of 
T cells in HIV-infected patients, we detected the presence of a T cell subset stained by both V
2- and V
3-specific mAb, which represented a large fraction (up to 16.5%) of 
peripheral blood lymphocytes (PBL) in one HIV patient. The presence of two distinct functional
chains on these cells was confirmed by phenotypic and molecular analysis of TCR transcripts expressed by V
2+V
3+ T cell clones derived from this patient. For 18 months, the absolute number of these cells varied similarly to the other PBL subsets, before becoming undetectable in blood samples. Moreover, most of these cells expressed CD8 receptors, which are classically found on activated, but not resting, 
T cells. Taken together, these data suggest that dual TCR-expressing T cells are subjected to peripheral expansions and contractions presumably following antigen recognition, which would argue against a systematic counter-selection of these cells during peripheral antigen-driven responses.
Keywords: dual TCR, flow cytometry, 
T lymphocytes
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Introduction
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Most peripheral blood T lymphocytes express TCR encoded by TCR
and ß rearranged genes. A minor peripheral blood lymphocyte (PBL) subset displays a second type of TCR composed of
and
subunits (
TCR). The latter, unlike the former, are generally devoid of CD4 and CD8 co-receptors, and are thus referred to as `double negative' (DN). In normal individuals, the DN T cell subset accounts for <5% of total PBL, among which 90% use the V
2 (DV102S1) and V
9 (GV2S1) region genes to form their TCR. The remaining 
PBL mainly express TCR
chains comprising V
1 (DV101S1) or V
3 (DV103S1) regions. The reverse situation is observed in other tissues such as thymus, spleen and epithelia, where non-DV102S1 
T cells predominate.
Recent studies have shown that a high proportion (up to one-third) of circulating
ß T cells co-expresses two distinct TCR
chains (1). A similar finding was reported for 
T cells, where co-expression of two distinct TCR
chains are frequently found in association with the same
chains on PBL-derived T cell clones (2). By contrast, T cells co-expressing two different ß or
chains are seldom (35). As a whole, and in apparent contradiction with early dogmas, a significant fraction of T lymphocytes can display two different TCR and, therefore, may have two distinct antigen specificities. However, the ability of these cells to respond to their antigen(s) remains a matter of debate. In particular because T cell activation requires engagement of a relatively large fraction of TCR and because dual TCR-bearing cells express only intermediate levels of each TCR (presumably due to limiting amounts of available CD3 components), it is anticipated that dual TCR expressors will be diluted out by single expressors in the course of an antigen-driven response.
T cells bearing 
TCR have been found expanded in the course of various infectious diseases but so far the functions of these cells remain largely unknown. Several lines of evidence suggest a particular role of 
T cells in immunity against tumors and intracellular pathogens, among which are mycobacteria and unicellular parasites such as plasmodium, toxoplasma and leshmania (6). In HIV-infected patients, expansions of 
T cells are often observed (711). In particular in patients with CD4 T cell counts <100/mm3, an increase in DN 
T cells was recently reported as associated with the onset of opportunistic infections by Mycobacterium avium complex (12). To better characterize these cells, we undertook a phenotypic study of the variable regions of the TCR
chains displayed by DN T cells in 200 of these patients. In the course of this analysis, we found one patient with a large fraction of his circulating 
T cells bearing two distinct TCR
chains. This cell subset was further characterized and its size variations followed during an 18 month period of time.
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Methods
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Patient's clinical status
Patient R was tested seropositive for HIV in June 1987. No clinical or biological follow-up was performed until July 1993, when the patient consulted for persistent lymphadenopathies. At that time, CD4+ lymphocytes were determined at 316/mm3. In January 1995, CD4+ lymphocytes were at 65/mm3, adenopathies persisted and a monotherapy with AZT was started. In March 1995, the patient had lost 5% of his body weight since his last visit and CD4+ cells had reached 110/mm3. In June 1995, the patient consulted for persistent diarrhea, CD4+ cells were decreased 2-fold and AZT was replaced with ddC. Symptoms were first blunted, but peripheral neuropathies occured. On July 27 (day 1 in our study), they led to the initiation of a bitherapy associating ddI and AZT. On day 180 (1/23/1996), severe diarrhea required hospitalization and cryptosporidiosis was diagnosed. HIV therapy was maintained and diarrhea diminished upon anti-parasitic treatment. On day 272 (4/23/1996), CD4+ cells were at 5/mm3, and oesophagus candidosis was diagnosed and treated. An anti-HIV tritherapy was installed, associating ritonavir, ddC and AZT. Patient clinical status rapidly improved, as well as CD4+ cells number which rose to 57/mm3 on day 310 (5/31/1996). HIV viral load was quantitated at 105,000 copies/ml on 6/10/1996. However, on day 368 (7/28/1996), diarrhea had reappeared and CD4+ cells had dropped down to 28/mm3. On October 7 (day 439), diarrhea persisted despite treatment and CD4+ cells had decreased to 10/mm3, while HIV viral load remained elevated, at 108,000 copies/ml. Tritherapy was modified by replacing ddC by 3TC and this association was not changed from that date on.
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Flow cytometry (FCM) analysis
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FCM analysis of PBL was performed on peripheral blood samples collected on anticoagulant EDTA. For the determination of T lymphocyte subpopulations (CD4 and CD8 single-positive and DN T cells), 50 µl of blood was incubated for 15 min at 4°C with 5 µl of Tetrachrome (Coulter, Hialeah, FL), which is a combination of CD45FITC, CD4phycoerythrin (PE), CD8ECD and CD3PECy5. Red blood cells were lysed and white blood cells were fixed using the Immunoprep solution (Coulter), according to the manufacturer's recommendations. Lymphocytes were then analyzed by double-gating on the basis of the forward and side scatter parameters as well as the positivity for CD45 staining, using a Coulter Epics XL four-color FCM, calibrated using Flowcheck fluorospheres.
For the analysis of the DN subpopulation, 100 µl of whole blood was incubated for 15 min at 4°C with a mixture of mAb against CD8
chain (clone B9.11), and against the constant region (anti-TCRDC, clone IMMU510) and the DV102S1 (V
2, clone IMMU389) variable portion of the TCR
chain. These antibodies were from Immunotech (Marseille, France) and were coupled respectively to PECy5, PE and FITC. Red blood cells were then lysed and white blood cells fixed using the FACS Lysing Solution (Becton Dickinson, Mountain View, CA) according to the manufacturer's recommendations. Lymphocytes were then analyzed on the basis of the forward and side scatter parameters, using a FACScan flow cytometer equipped with CellQuest 1.1.2 software and calibrated with the CaliBRITE III beads (Becton Dickinson) and FACScomp software.
TCR 
variable regions were determined by indirect staining using a set of mAb against the different TCRGV regions GV1S2/S3/S4 (V
2/3/4, clone 23D12), GV1S4 (V
4, clone 94), GV1S8 (V
8, clone R-4.5.1) and GV2S1 (V
9, clone IMMU360), and against the TCRDV regions DV101S1 (V
1, clone R-9.12.6.2), DV102S1 (V
2, clone IMMU389 and clone TiV
2) and DV103S1 (V
3, clone P-8.6b and clone P11.5b), all described in (13,14). We also used a mAb that recognizes all TCRD chains except those carrying the DV102S1 region (clone 178). Whole blood (100 µl) was incubated with each of these mAb for 30 min at 4°C or a combination of IgG1 and IgG2a isotype-matched control antibodies. Cells were then washed twice with PBS and stained with PE-labeled goat anti-mouse (Immunotech) for another 30 min. Cells were washed again and incubated with the FITC-labeled anti-DV102S1 antibody. Samples were then treated as described above for the TCRDC staining.
The different subpopulations were expressed as percentages of total lymphocytes. Absolute numbers were computed from these percentages and the absolute count of lymphocytes as determined by an automatic hemocytometer (H2 Technicon, Bayer, Germany).
Generation of T cell lines and clones
Lymphocytes were isolated from peripheral blood by gradient sedimentation on Ficoll (Eurobio, Les Ulis, France) and sorted using immunomagnetic beads as described (13). In brief, cells were incubated with TCRDV102S1-specific mAb for 45 min, washed once and rotated for 4 h at 4°C with magnetic beads coated with sheep anti-mouse IgG (Dynal, Oslo, Norway). After eight washes, bead-adherent cells were expanded in RPMI 1640, 10% pooled human serum, 1 mM L-glutamine, rIL-2 (150 IU), leukoagglutinin (0.5 µg/ml) (Sigma, St Louis, MO), and 5x104 irradiated (30 Gy) allogeneic PBL and 5x103 irradiated B lymphoblastoid cells. These culture conditions allow expansion of 70100% of T cells and do not introduce any bias in the TCR repertoire of
ß and 
T cells (our unpublished observations and 14). As described (13), T cell cloning was performed by seeding 30 cells/100 wells in 96-well microtiter plates (round bottom) in 200 µl of RPMI 1640, supplemented with 8% human serum, 1 mM L-glutamine, rIL-2 (150 IU), leukoagglutinin (0.5 µg/ml), irradiated PBL and B lymphoblastoid cells/well. After 2 weeks, T cell colonies were re-stimulated in 96-well plates under the above conditions and eventually transferred to culture flasks for further expansion.
mAb-redirected killing assay
This assay was performed as described in (15). Briefly, FcR-bearing P815 cells were labeled with 51Cr (5 µCi/106 cells), for 1 h at 37°C. Then 3000 cells/well were seeded in round-bottom 96-well plates in RPMI with 10% FCS, in the presence of serial dilutions of either anti-TCRDV102S1 mAb IMMU389, anti-TCRDV103S1 mAb P8.6.b1 or an isotype-matched control antibody, at concentrations ranging from 1 µg/ml to 0.1 ng/ml. Each condition was performed in triplicates. Dual 
TCR-expressing effector clones Rv6 or Rv9 were added in each well at a effector:target ration of 5:1. After 4 h at 37°C, 51Cr release was measured and specific lysis was calculated.
Molecular analysis of TCR transcripts
Amplification and sequencing of TCRG and TCRD transcripts was performed as previously described (2). T cell clone RNA was reversed transcribed and then amplified using the following primers: TCRGC 5'-TAG AGC TCT ATG TTC CAG CCT TCT GGA G; TCRDC 5'-GCG ACA TTT GTT CCA TTT TTC; TCRGV1 5'-CTA CAT CCA CTG GTA CCT ACA; TCRDV102S1 5'-TTG CAA AGA ACC TGG CTG; and TCRDV103S1 5'-TCA CTT GGT GAT CTC TCC. After purification on low-melting point agarose, amplified DNA was sequenced according to the USB Sequenase kit procedure for a double-strand template, using previously described primers (2). For designation of TCR
(TCRG) and
(TCRD) genes, we have followed the recommendations of the WHOIUIS Nomenclature Sub-Committee on TCR Designation (15).
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Results
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Evidence for circulating T cells bearing two different 
TCR
In a recent study, we described an increase in CD4/CD8 DN T cells in peripheral blood of HIV-infected patients with numbers of CD4+ T cells <100/mm3 and suffering from opportunistic infections. In an attempt to characterize the TCR structural features of these expanded T cell populations, we recently studied in more detail the PBL repertoire of HIV patients using pan-
(anti-TCRDC) and DV102S1-specific mAb. In the course of this analysis, we found one patient (patient R) displaying highly unusual staining patterns with the above antibodies. This patient had very low numbers of circulating CD4+ T lymphocytes and DN T cells at his entry in this study in July 1995, but no clinical signs characteristic of advanced AIDS. Two-color FCM analysis using a combination of FITC-labeled CD4 and CD8 mAb, and either of PE-labeled pan-
or pan-ß mAb indicated that the majority of DN T cells (95%, corresponding to 6.57% of total lymphocytes) expressed a 
TCR (data not shown), among which 25% (1.64% of total lymphocytes) bore a TCRDV102S1 region (Fig. 1
). However, the staining profiles of the patient's PBL using the anti-DV102S1 mAb clearly differed from that of most other patients or from healthy individuals (Fig. 1
). Indeed while in the latter, the DV102S1+ subpopulation was homogeneously stained by mAb 389 (Fig. 1A
and data not shown), the DV102S1+ PBL from patient R showed a bimodal staining profile, with 38 and 62% of DV102S1+ cells (0.63 and 1.02% of total lymphocytes) being dimly and brightly stained by mAb 389 respectively (Fig. 1B
). These two subsets were similarly stained by the pan-
mAb when compared between each other [mean fluorescence intensity (MFI) = 274 and 382 respectively] and with a control patient (MFI = 387), thereby showing that comparable densities of TCRD chains were expressed on the cell surface. Furthermore, the 389bright subset in patient R displayed MFI similar to the control patient (595 versus 627). Therefore, it seemed that 389dim cells (MFI = 109) either displayed an abnormally low number of V
2 regions or that their TCR DV102S1+ chains adopted an unusual conformation that was barely recognized by mAb 389.
TCRGV and TCRDV region FCM analysis of patient R PBL
TCRDV region expression was studied by two-color FCM using mAb directed against the three major TCRDV regions, DV101S1, DV102S1 and DV103S1, which altogether cover on average 98% of peripheral blood 
T cells. As expected, the DV102S1 and DV102S1bright subsets were stained in a mutually exclusive fashion by the three TCRDV-specific mAb: DV102S1 cells were recognized by either DV101S1 or DV103S1-specific mAb, whereas DV102S1bright cells were stained by none of these mAb (Fig. 2B and C
). In contrast, all DV102S1dim were DV103S1+, thus suggesting that they carried two distinct TCR
chains on their surface (Fig. 2B
and Table 1
). Analysis of TCRGV region expression (Fig. 2DG
) on these cells by two-color FCM using either TCRGV1S2/S3/S4-, GV1S4-, GV1S8- and GV2S1-specific mAb in conjunction with TCRDV102S1 mAb indicated that all DV102S1bright cells were GV2S1+ (Fig. 2D
), whereas all DV102S1dim cells expressed either a GV1S2 or GV1S3 region (Fig. 2E and F
, and data not shown), but no GV1S8 region (Fig. 2G
).
Molecular evidence that DV102S1dim cells carry two distinct TCRD chains
To formally prove that DV102S1dim DV103S1+ cells expressed distinct TCRD chains, we performed an extensive phenotypic and molecular analysis of TCR expressed by several DV102S1+ T cell clones derived from patient R PBL. To this end, we first isolated DV102S1+ cells by immunomagnetic sorting using mAb 389 and cloned them after a short-term in vitro expansion. In agreement with FCM data obtained with fresh PBL, T cell clones expressing either bright (Rv1, Rv10 and Rv12) or dim (Rv6 and Rv9) levels of DV102S1 chains could be isolated (Table 1
). Moreover, unlike DV102S1bright clones, DV102S1dim ones were also stained by the DV103S1-specific mAb (Table 1
), and two distinct anti-DV102S1 and two distinct anti-DV103S1 mAb displayed identical results when tested on clone Rv6 (Table 1
). Phenotypic analysis of TCRGV region expression confirmed that DV102S1bright clones were GV2S1+, whereas DV102S1dim clones were stained by anti-GV1S2/S3/S4 mAb but not by GV1S4 mAb, thus indicating the presence of either GV1S2 or GV1S3 chains on the latter (data not shown). The junctional regions of rearranged TCRG and TCRD genes derived from clones Rv6 and Rv9 were then sequenced after reverse transcription and amplification of TCR transcripts using pairs of TCRV/TCRC primers. Both T cell clones carried a single productive rearrangement which comprised the GV1S3 region gene and two distinct in frame TCRD sequences comprising either the DV102S1 or the DV103S1 region gene (Table 2
). Furthermore, clones Rv6 and Rv9 displayed identical junctional sequences (Table 2
), thus strongly suggesting that the peripheral DV102S1dim subset was oligo- or monoclonal.
Clones bearing two distinct 
TCR remain functional
To determine whether the two distinct 
TCR expressed by clones Rv6 and Rv9 were functional, their cytotoxicity was tested against FcR-bearing P815 target cells in a redirected killing assay using either anti-TCRDV102S1 mAb IMMU389 or anti-TCRDV103S1 mAb P8.6.b1. Figure 3
shows that clone Rv6 was capable of killing P815 target cells in the presence of each of the two stimulating anti-TCR mAb, in a dose-dependent fashion. Similar cytotoxic activity was found for clone Rv9. This strongly suggested that the two distinct TCR expressed on these cells were capable of transducing activation signal following binding to their specific antigen(s).
Activation status and time-course evolution of DV102S1dim cells
While CD8 is a differentiation marker that is closely linked to particular MHC-restriction patterns of TCR
ß T cells, several studies suggest that its expression on 
T cells correlates with their activation status. Significantly, a relatively high proportion of DV102S1bright (32%) and DV102S1dim (62%) T cells expressed CD8, meaning that part of dual TCR
expressing cells were activated in the periphery (Fig. 4
). We also looked for the expression on the DV102S1dim subset, of other activation markers such as HLA-DR or CD25. Sixty percent of these cells were stained with HLA-DR on day 272, whereas no expression of CD25 was detectable. We did not show these results since this staining was performed on only one blood sample, in contrast with the CD8 expression which was found positive all along this study. As a whole, these data suggest that these cells display an activated phenotype.
To evaluate the stability of this circulating subset, 10 determinations of the T cell subpopulations were performed throughout an 18 month long period of time. Figure 5
displays the kinetics of the following parameters: CD4+, TCRD+ and the two DV102S1+ T cell subpopulations. In Fig. 5(A)
results are expressed in percentages of total lymphocytes and Fig. 5(B)
results are expressed as absolute numbers per mm3, along with the total number of blood lymphocytes. A progressive decrease in CD4+ T cells required an initiation of a tritherapy at day 285, which induced a significant increase in CD4+ cells within the first 4 weeks, but which did not last further. Absolute numbers of DV102S1bright and DV102S1dim T cells were also decreasing regularly before the start of the tritherapy. They subsequently increased in similar proportions but with a delay of 7 weeks, which corresponded to the interval between the sixth and seventh blood samplings. Similarly to CD4+ cells, both DV102S1 subpopulations subsequently decreased, although with a steeper slope for DV102S1dim cells, which finally became undetectable 4 months later (not shown).
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Discussion
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In a previous study, we reported on an increase in DN T cells bearing a 
TCR, in the peripheral blood of HIV-infected patients with <100 CD4+ T cells/mm3 and suffering from opportunistic infections (12). While studying the proportion of TCRDV102S1+ cells among the TCR 
T lymphocytes, we noticed the existence of a subset of 
T cells with highly unusual staining patterns among PBL from one patient. Indeed out of 200 patients routinely followed for TCRDC/TCRDV102S1 expression, whose lymphocytes systematically showed a bimodal staining pattern (i.e. negative or bright) using a TCRDV102S1-specific mAb, patient R was unique in that he carried a subset of 
T cells brightly stained by pan-
mAb but dimly stained by TCRDV102S1-specific mAb. Subsequent FCM analyses using a larger set of TCR V region-specific mAb strongly suggested that DV102S1dim cells expressed another TCRD chain which comprised a DV103S1 region. This assumption was thereafter formally proven by molecular analysis of TCR transcripts expressed by DV102S1dim T cell clones.
Dual expression of distinct TCR chains on T cells has already been reported. In this respect T cell clones bearing distinct TCR chains belonging to all possible isotypes (i.e. encoded by TCRA, TCRB, TCRG or TCRD genes) have been described in several instances. However, these studies have also shown that the fraction of dual TCR-expressing T cells was highly variable and mainly depended on the isotype of the TCR chains considered. For instance, T cell clones bearing distinct TCRA or TCRG chains usually represent a very significant fraction (up to one-third) of
ß or 
T cells respectively (1,2). In contrast, the average frequency of
ß and 
T cells bearing respectively distinct TCRB and TCRD chains was shown to be at most 1% (35). Such differences are likely to be explained by distinct mechanisms controlling rearrangements of given TCR isotypes. Analyses of mice carrying functional TCRB transgenes indicate that expression of a functional transgenic TCRB chain leads to strong inhibition of rearrangements involving endogenous TCRB loci, thus suggesting occurrence of a stringent allelic exclusion process acting at the genotypic level. In stark contrast, endogenous TCRA chains are frequently found in mice carrying functional transgenic TCRA chains. Therefore while the low fraction of dual TCRB-expressing T cells would be the likely consequence of an escape mechanism, the frequent occurrence of T cell clones bearing distinct TCRA chains would presumably reflect the absence of a tight TCRA genotypic exclusion. Whether similar mechanisms could explain the observed differences between the frequency of cells carrying either distinct TCRG or TCRD chains is yet unclear. It is likely that the high frequency of dual TCRG-expressing clones is accounted for by absence of a tight genotypic exclusion of TCRG gene rearrangements, as suggested by available data (2) (F. Davodeau et al., unpublished data). However, explanations for the paucity of dual TCRD-expressing clones, which was suggested in a previous study (5), are still lacking. In particular, the occurrence of a tight interallelic control of TCRD gene rearrangements has not been proven yet and is not supported by developmental studies, which have so far failed to demonstrate any dissociation between the onset/regulation of TCRG and TCRD gene rearrangements. At first sight, the present demonstration that T cells carrying two distinct TCRD chains represent a very significant fraction (up to one-third) of 
PBL in one patient fits well with the above-mentioned studies, suggesting lack of stringent mechanisms controlling TCRD allelic exclusion. However, since such a high frequency of dual TCRD expressors was observed in one patient only out of 200 studied, it would seem that in a more general fashion, dual TCRD expression is a rather infrequent event.
Despite reaching 1% of total lymphocytes of patient R, double TCRD-bearing T cells seemed oligo- or monoclonal, as suggested by the following observations. (i) All DV102+DV103+ T cells carried TCRG chains comprising a homogeneous set of GV regions (i.e. GV103S1), as suggested by FCM. (ii) A molecular analysis of junctional regions of GV1S3, DV102S1 and DV103S1 transcripts derived from two randomly chosen clones yielded identical sequences. (iii) Both clones also expressed identical levels of TCRDV102S1 and TCRDV103S1 receptors, which were comparable to those observed on fresh DV102S1dim cells. In this regard, in all previous studies describing co-expression of two different TCR on the same cells carrying either two TCRG (2,17), two TCRA (1) or two TCRB (3) chains, the relative expression of each TCR chain was very stable for any given clone, but varied considerably from one clone to another. Therefore in the present study, if the TCRDV102S1/TCRDV103S1 had been polyclonal, one would have expected several discrete populations, which was clearly not the case.
The fate of the TCRDV102S1/TCRDV103S1 population has been studied over an 18 month long period. The cells persisted all along this time, albeit undergoing several contractions and expansions. Later on, they progressively declined down to frequencies below FCM detection thresholds. Importantly these fluctuations also concerned in a similar fashion other 
T lymphocytes subpopulations as well as CD4+ T lymphocytes. Therefore, the double TCR 
T cells did not undergo uncontrolled proliferation which could have reflected a leukemic or pre-leukemic behavior. Conversely, they probably underwent an antigen-driven expansion, which could also explain their reduced clonality and the presence on part of these cells of CD8
homodimers, a well-known activation marker on 
and NK cells (1820). This marker was expressed on a significant percentage of these cells all along the study, with up to 62% of CD8+ cells. Unfortunately the presence of other activation markers could not be studied and thus no definitive conclusions concerning their actual activation status can be drawn yet.
Since this peculiar population of cells in this patient seems to be antigen controlled, this raises the question of the effective functionality of such double TCR+ 
T cells and more generally of their physiological role. Studies performed on TCR
ß T cells showed that they were indeed less effective (21,22). Addressing such an issue in the present case would require identification of the specific antigen. However, our inability to associate frequency alterations of this subset with any particular clinical event (e.g. opportunistic infections) make this task impossible at this time.
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Acknowledgments
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We are indebted to J.-C. Carron, M. Garcie and N. Berrié for excellent technical assistance. This work was supported by grants from the Ligue contre le Cancer de Gironde, the Ligue Nationale contre le Cancer and Sidaction 95.
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Abbreviations
|
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DN | double negative |
FCM | flow cytometry |
MFI | mean fluorescence intensity |
PE | phycoerythrin |
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Notes
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Transmitting editor: A. Fischer
Received 2 April 1998,
accepted 15 December 1998.
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References
|
---|
-
Padovan, E., Casorati, G., Dellabona, P., Meyer, S., Brockhaus, M. and Lanzavecchia, A. 1993. Expression of two T cell receptor
chains: dual receptor T cells. Science 262:422.[ISI][Medline]
-
Davodeau, F., Peyrat, M. A., Houde, I., Hallet, M. M., De Libero, G., Vié, H. and Bonneville, M.1993. Surface expression of two distinct functional antigen receptors on human

T cells. Science 260:1800.[ISI][Medline]
-
Davodeau, F., Peyrat, M. A., Romagné, F., Necker, A., Hallet, M. M., Vié, H. and Bonneville, M. 1995. Dual TCR-ß chain expression on human T lymphocytes. J. Exp. Med. 181:1391.[Abstract]
-
Padovan, E., Giachino, C., Cella, M., Valiutti, S., Acuto, O. and Lanzavecchia, A. 1995. Normal T lymphocytes can express two different ß chains: implications for the mechanism of allelic exclusion. J. Exp. Med. 181:1591.
-
Peyrat, M. A., Davodeau, F., Houde, I., Romagné, F., Necker, A., Leget, C., Cervoni, J. P., Cerf-Bensussan, N., Vié, H., Bonneville, M. and Hallet, M. M. 1995. Repertoire analysis of human peripheral blood lymphocytes using a human V
3 region-specific monoclonal antibody. Characterization of dual T cell receptor (TCR)
-chain expressors and
ß T cells expressing V
3J
C
-encoded TCR chains. J. Immunol. 155:3060.[Abstract]
-
Haas, W., Pereira, P. and Tonegawa, S. 1993. Gamma/delta cells. Annu. Rev. Immunol. 11:637.[ISI][Medline]
-
Autran, B., Triebel, F., Katlama, C., Rozenbaum, W., Hercend, T. and Debré, P. 1989. T cell receptor gamma delta lymphocytes subsets during HIV-1 infection. Clin. Exp. Immunol. 75:206.[ISI][Medline]
-
Margolick, J. B., Scott, E. R., Okada, N. and Saah, A. J. 1991. Flow cytometric analysis of gamma delta T cells and natural killer cells in HIV-1 infection. Clin. Immunopathol. 58:126.[ISI]
-
De Maria, A., Ferrazin, A., Ferrini, S., Ciccone, E., Terragne, A. and Moretta, L. 1992. Selective increase of a subset of T cell receptor gamma delta T lymphocytes in the peripheral blood of patients with human immunodeficiency virus type 1 infection. J. Infect. Dis. 1665:917.
-
Hinz, T., Wesch, D., Friese, K., Reckziegel, A., Aden, B. and Kabelitz, D. 1994. T cell receptor

repertoire in HIV-1 infected individuals. Eur. J. Immunol. 24:3044.[ISI][Medline]
-
Boullier, S., Cochet, M., Poccia, F. and Gougeon, M. L. 1995. CDR3-independent

V
1+ T cell expansion in the peripheral blood of HIV-infected persons. J. Immunol. 154:1418.[Abstract/Free Full Text]
-
Moreau, J. F., Taupin, J. L., Dupon, M., Carron, J. C., Ragnaud, J. M., Marimoutou, C., Bernard, N., Constans, J., Texier-Maugein, J., Barbeau, P., Journot, V., Dabis, F., Bonneville, M. and Pellegrin, J. L. 1996. Increases in CD3+CD4CD8 T lymphocytes in AIDS patients with disseminated Mycobacterium avium-intracellular complex infection. J. Infect. Dis. 174:969.[ISI][Medline]
-
Kabelitz, D., Ackermann, T., Hinz, T., Davodeau, F., Band, H., Bonneville, M., Janssen, O., Arden, B. and Schondelmaier, S. 1994. New monoclonal antibody (23D12) recognizing three different V
elements of the human 
T cell receptor. 23D12+ cells comprise a major subpopulation of 
T cells in postnatal thymus. J. Immunol. 152:3128.[Abstract/Free Full Text]
-
Romagné, F., Peyrat, M. A., Leget, C., Davodeau, F., Houde, I., Necker, A., Hallet, M. M., Vié, H. and Bonneville, M. 1996. Structural analysis of

TCR using a novel set of TCR
and
chain-specific monoclonal antibodies generated against soluble 
TCR. Evidence for a specific conformation adopted by the J
2 region and for a V
1 polymorphism. J. Immunol. Methods 189:25.[ISI][Medline]
-
WHOIUIS Nomenclature Sub-Committee on TCR Designation. 1995. Nomenclature for T cell receptor (TCR) gene segments of the immune system. Immunogenetics 42:451.[Medline]
-
Azuma, M., Cayabyab, M., Phillips, J. H. and Lanier, L. L. 1993. Requirements for CD28-dependent T cell-mediated cytotoxicity. J. Immunol. 150:2091.[Abstract/Free Full Text]
-
Hinz, T., Marx, S., Nerl, C. and Kabelitz, D. 1996. Clonal expansion of

T cells expressing two distinct T-cell receptors. Br. J. Haematol. 94:62.[ISI][Medline]
-
Lefrancois, L. and Goodman, T. 1989. In vivo modulation of cytolytic activity and Thy-1 expression in TCR-

+ intraepithelial lymphocytes. Science 243:1716.[ISI][Medline]
-
Jarry, A., Cerf-Bensussan, N., Brousse, N., Selz, F. and Guy-Grand, D. 1990. Subsets of CD3+ (T cell receptor
ß or 
) and CD3 lymphocytes isolated from normal human gut epithelium display phenotypical features different from their counterparts in peripheral blood. Eur. J. Immunol. 20:1097.[ISI][Medline]
-
Deusch, K., Luling, F., Reich, K., Classen, M., Wagner, H. and Pfeffer, K. 1991. A major fraction of human intraepithelial lymphocytes simultaneously expresses the gamma/delta T cell receptor, the CD8 accessory molecule and preferentially uses the V
1 gene segment. Eur. J. Immunol. 21:1053.[ISI][Medline]
-
Blichtfeldt, E., Munthe, L. A., Rotnes, J. S. and Bogen, B. 1996. Dual T cell receptor T cells have a decreased sensitivity to physiological ligands due to reduced density of each T cell receptor. Eur. J. Immunol. 26:2876.[ISI][Medline]
-
Valitutti, S. and Lanzavecchia, A. 1997. Serial triggering of TCRs: a basis for the sensitivity and specificity of antigen recognition. Immunol. Today 18:299.[ISI][Medline]