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The Journal of Immunology, 2006, 176: 2064-2068.
Copyright © 2006 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Influence of the TCR Vbeta Domain on the Selection of Semi-Invariant NKT Cells by Endogenous Ligands1

Jens Schümann2,*, Marcin P. Mycko*, Paolo Dellabona{dagger}, Giulia Casorati{dagger} and H. Robson MacDonald3,*

* Ludwig Institute for Cancer Research, Epalinges, Switzerland; and {dagger} Department of Biology and Technology, H. San Raffaele Scientific Institute, Milan, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Invariant V{alpha}14 (V{alpha}14i) NKT cells are a murine CD1d-dependent regulatory T cell subset characterized by a V{alpha}14-J{alpha}18 rearrangement and expression of mostly Vbeta8.2 and Vbeta7. Whereas the TCR Vbeta domain influences the binding avidity of the V{alpha}14i TCR for CD1d-{alpha}-galactosylceramide complexes, with Vbeta8.2 conferring higher avidity binding than Vbeta7, a possible impact of the TCR Vbeta domain on V{alpha}14i NKT cell selection by endogenous ligands has not been studied. In this study, we show that thymic selection of Vbeta7+, but not Vbeta8.2+, V{alpha}14i NKT cells is favored in situations where endogenous ligand concentration or TCR{alpha}-chain avidity are suboptimal. Furthermore, thymic Vbeta7+ V{alpha}14i NKT cells were preferentially selected in vitro in response to CD1d-dependent presentation of endogenous ligands or exogenously added self ligand isoglobotrihexosylceramide. Collectively, our data demonstrate that the TCR Vbeta domain influences the selection of V{alpha}14i NKT cells by endogenous ligands, presumably because Vbeta7 confers higher avidity binding.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Invariant V{alpha}14 (V{alpha}14i)4 NKT cells are a murine subset of CD4+ or CD4–CD8– double-negative (DN) CD1d-dependent regulatory T cells that express a semi-invariant {alpha}beta TCR repertoire and markers usually associated with NK and activated/memory T cells. Their TCR is composed of a V{alpha}14-J{alpha}18 chain, paired preferentially with a restricted beta-chain, mostly containing Vbeta8.2 or Vbeta7 (the human equivalents are V{alpha}24-J{alpha}18 and Vbeta11). V{alpha}14i NKT cells appear to efficiently regulate antitumor immunity, antimicrobial responses, and the balance between tolerance and autoimmunity. Whereas {alpha}-galactosylceramide ({alpha}GalCer), bound to CD1d, is widely used as a pan-activating agonist for V{alpha}14i NKT cells (1, 2), an endogenous ligand of V{alpha}14i NKT cells, i.e., isoglobotrihexosylceramide (iGb3), has recently been described (3).

V{alpha}14i NKT cells are a thymus-dependent population derived from CD4+CD8+ double-positive (DP) thymocytes (4, 5, 6, 7, 8). They are positively selected by CD1d-expressing DP cortical thymocytes (9, 10), which alone are sufficient for positive selection of V{alpha}14i NKT cells (11, 12). V{alpha}14i NKT cells also can be negatively selected by strong activation signals provided by thymic dendritic cells (12, 13) or DP cortical thymocytes (12). Interestingly, ectopic overexpression of CD1d resulted in a comparable reduction of frequencies of both Vbeta8.2+ and Vbeta7+ V{alpha}14i NKT cells (13), presumably representing CD1d-endogenous ligand-driven negative selection, whereas the frequency of Vbeta2+ V{alpha}14i NKT cells increased (13), possibly due to a very low avidity of CD1d-endogenous ligand binding by Vbeta2+ V{alpha}14i NKT cells.

V{alpha}14i NKT cells are most commonly identified by mouse CD1d-{alpha}GalCer tetramers (14, 15). Recently, we described novel mouse and human CD1d-{alpha}GalCer dimers that revealed an unexpected influence of the TCRbeta-chain on the avidity of CD1d-{alpha}GalCer binding. A subset of V{alpha}14i NKT cells clearly discriminated {alpha}GalCer bound to mouse or human CD1d dimers on the basis of avidity differences conferred by the Vbeta domain of the TCRbeta-chain, with Vbeta8.2 conferring higher avidity binding than Vbeta7 (16). Similarly, Stanic et al (17) described an influence of the Vbeta domain on the binding of V{alpha}14i NKT cells to CD1d complexed with other glycolipid ligands, again with Vbeta8.2 being associated with higher avidity binding than Vbeta7. However, it remains speculative whether the TCR Vbeta domain may influence the avidity of V{alpha}14i TCR binding to CD1d complexed with its endogenous ligand(s) and thereby modulate thymic selection of V{alpha}14i NKT cells. In this study, we have addressed this issue by analyzing the Vbeta repertoire of V{alpha}14i NKT cells selected in vivo under conditions where either endogenous ligand concentration or TCR{alpha}-chain avidity for endogenous ligand is suboptimal. Furthermore, we investigated in vitro whether the TCR Vbeta domain influences CD1d-dependent selection of thymic V{alpha}14i NKT cells driven by endogenous ligands or the putative V{alpha}14i NKT cell-selecting glycolipid iGb3.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Disclosures
 References
 
Mice

C57BL/6JOlaHsd mice naturally carrying the Vbetab haplotype (referred to as C57BL/6 or Vbetab/b mice throughout the text) were obtained from Harlan. Congenic B6.L-Tcrb-Va mice carrying the Vbetaa haplotype (referred to as Vbetaa/a mice) were kindly provided by A. Livingstone and have been described in more detail elsewhere (18). B6;129-CD1tm1Gru mice (referred to as CD1d–/–) (19) and B6;129-Tcra-Jtm1Tgi mice (referred to as J{alpha}18–/–) (20), kindly provided by M. J. Grusby and M. Taniguchi, respectively, were backcrossed three times to C57BL/6. Generation of B6.Cg-Tg(CD2-TRAV24-TRAJ18/Tcra-C)1Rmd mice (referred to as hV{alpha}24tg), backcrossed to B6.129P2-Tcratm1Mjo mice (referred to as C{alpha}–/–), has been described previously (21). This study has been reviewed and approved by the Service Vétérinaire Cantonal of Etat de Vaud.

Cell preparations and in vitro culture

Complete thymocytes were prepared by grinding thymi through tea strainers. For further analysis of V{alpha}14i NKT cells, thymocytes were depleted of heat-stable Ag (HSA)+ and CD8+ cells as described previously (16). In some experiments, 105 HSA/CD8-depleted thymocytes from C57BL/6 mice were cultured together with 3 x 105 total thymocytes from C{alpha}–/–CD1d+/+ or C{alpha}–/–CD1d–/– mice in the presence of iGb3 (10 µg/ml; Alexis Biochemicals) or iGb3 solvent (0.005% polysorbate 20) for 7 days. Murine IL-7 (10 ng/ml) was added to the cultures to ensure survival of V{alpha}14i NKT cells.

Flow cytometry

V{alpha}14i NKT cells were identified by mouse CD1d-{alpha}GalCer tetramers (15) or dimers (16). Cells were surface-stained with combinations of specific mAbs. For intracellular (ic) Vbeta stainings, cells were fixed with 2% paraformaldehyde after surface staining and incubated with either anti-Vbeta8.2-PE (F23-2) or anti-Vbeta7-PE (TR310) diluted in 0.5% saponin/2% FCS/PBS. Samples were passed on a FACSCanto flow cytometer (BD Biosciences).

Calculation of theoretical expected Vbeta8.2 and Vbeta7 frequencies among V{alpha}14i NKT cells in Vbetaa/b mice

Assuming equal chances for both Vbeta8.2+ and Vbeta7+ V{alpha}14i NKT cells to be selected in the thymus, the theoretical expected Vbeta8.2 and Vbeta7 frequencies among V{alpha}14i NKT cells in Vbetaa/b mice, which express only one allele for Vbeta8.2 but two alleles for Vbeta 7, are as follows: expected frequency of Vbeta8.2 in Vbetaa/b = (XVbeta8.2/2)/(100 – XVbeta8.2/2); expected frequency of Vbeta7 in Vbetaa/b = (XVbeta7)/(100 – XVbeta8.2/2). XVbeta8.2 and XVbeta7 represent the percentage of Vbeta8.2+ and Vbeta7+ cells among V{alpha}14i NKT cells in C57BL/6 (Vbetab/b) thymus.

Statistical analysis

The results were analyzed by one-way ANOVA followed by the Student-Newman-Keuls multiple comparison test. A value of p ≤ 0.05 was considered significant.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Disclosures
 References
 
Under conditions of wild-type CD1d expression, Vbeta8.2+ and Vbeta7+ V{alpha}14i NKT cells are selected to equal extents

In wild-type thymi, ~50% of V{alpha}14i NKT cells are Vbeta8.2+, whereas only ~14% are Vbeta7+ (14, 15, 16). To check for the possibility that this dominance of Vbeta8.2+ V{alpha}14i NKT cells reflects a preferred thymic selection, we compared the frequencies of Vbeta8.2+ and Vbeta7+ cells among DP thymocytes and among mature thymic V{alpha}14i NKT cells from C57BL/6 mice. Ic Vbeta staining was used to overcome the low surface expression level of TCR among DP thymocytes, whereas mature V{alpha}14i NKT cells were defined as TCRbetaint mouse CD1d-{alpha}GalCer dimer+ NK1.1+ (Fig. 1A). Both Vbeta8.2+ and Vbeta7+ V{alpha}14i NKT cells expanded to a similar extent (Fig. 1B), and the apparent dominance of Vbeta8.2+ over Vbeta7+ V{alpha}14i NKT cells simply reflects a higher frequency of Vbeta8.2+ (~9.4%) than Vbeta7+ (~2.7%) DP precursor cells. Hence, in wild-type thymi, Vbeta8.2+ and Vbeta7+ V{alpha}14i NKT cells are selected to equal extents.


Figure 1
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  		FIGURE 1. Under conditions of wild-type CD1d expression, Vbeta8.2+ and Vbeta7+ V{alpha}14i NKT cells are selected to equal extents. A, Representative staining of thymic DP precursors and mature V{alpha}14i NKT cells from C57BL/6 mice. B, The bar chart represents the mean percentages of Vbeta8.2+ and Vbeta7+ cells (± SD) among DP thymocytes from C57BL/6 (Vbetab/b J{alpha}18+/+) mice (ic Vbeta staining; left scale) and thymic mature V{alpha}14i NKT cells from C57BL/6 (Vbetab/b J{alpha}18+/+), Vbetab/b J{alpha}18+/–, or Vbetaa/b J{alpha}18+/+ mice (surface Vbeta staining; right scale), gated as shown in A (n = 3). The dotted line shows the theoretical calculated values for Vbeta8.2+ and Vbeta7+ cells among mature V{alpha}14i NKT cells in Vbetaa/b mice, as described in Materials and Methods.

 
To determine whether the Vbeta repertoire could be modified by increased competition among developing V{alpha}14i NKT cells in the presence of wild-type levels of CD1d, we also measured the frequencies of Vbeta7+ and Vbeta8.2+ V{alpha}14i NKT cells in J{alpha}18+/– mice, which can only undergo the canonical V{alpha}14-J{alpha}18 rearrangement on one allele in DP precursor cells. As shown in Fig. 1B, limitation of V{alpha}14-J{alpha}18 rearrangements in J{alpha}18+/– mice did not change the relative frequencies of mature Vbeta7+ and Vbeta8.2+ V{alpha}14i NKT cells. Furthermore, in Vbetaa/b mice, which express only one allele of the Vbeta8 gene cluster but both alleles of Vbeta7, the observed frequencies of mature Vbeta7+ and Vbeta8.2+ V{alpha}14i NKT cells precisely matched the theoretical expected values calculated assuming equal probabilities of selection (see Materials and Methods). Thus, even under competitive conditions, Vbeta7+ and Vbeta8.2+ V{alpha}14i NKT cells are selected to a similar extent in the presence of wild-type levels of CD1d, also suggesting that there is no preferential pairing of either Vbeta with V{alpha}14. Nevertheless, these data do not exclude the possibility that the Vbeta domain may influence the avidity of V{alpha}14i TCR for endogenous ligand(s), because wild-type levels of CD1d may be saturating for V{alpha}14i NKT cell selection. To further investigate this possibility, we examined the Vbeta repertoire of V{alpha}14i NKT cells under conditions of increased competition for the endogenous ligand(s).

Preferential thymic selection of V{alpha}14i NKT cells expressing Vbeta7 in CD1d+/– mice

Competition of developing V{alpha}14i NKT cells for endogenous ligand(s) was achieved by usage of CD1d+/– mice, which have been described to express half the surface CD1d levels of wild-type mice (CD1d+/+) (12, 22) and which have normal NKT cell numbers (Ref.22 and data not shown). Interestingly, whereas the frequencies of Vbeta8.2+ and Vbeta7+ cells among DP precursors were not influenced by the expression level of CD1d, the frequency of Vbeta7+ thymic mature V{alpha}14i NKT cells, but not of Vbeta8.2+ V{alpha}14i NKT cells, increased as a result of intensified competition for endogenous ligand(s), thus decreasing the Vbeta8.2-Vbeta7 ratio from ~3.5 (CD1d+/+) to ~2.2 (CD1d+/–) (Fig. 2A). Despite the shift in the Vbeta ratio, the frequencies of thymic V{alpha}14i NKT cells expressing CD4 or inhibitory NK receptors (Ly49A, Ly49C/I, and Ly49G2) were not significantly different between CD1d+/+ and CD1d+/– mice (data not shown). These results demonstrate that the TCR Vbeta domain influences thymic selection of V{alpha}14i NKT cells, presumably by contributing to the avidity of V{alpha}14i TCR binding to CD1d complexed with endogenous ligand(s). However, they do not unequivocally establish the Vbeta hierarchy of binding avidities for endogenous ligands, because the reduced Vbeta8.2-Vbeta7 ratio in CD1d+/– mice could be explained by either preferential positive selection of Vbeta7+ V{alpha}14i NKT cells or preferential negative selection of Vbeta8.2+ V{alpha}14i NKT cells at suboptimal endogenous ligand concentrations. Importantly, the frequency of Vbeta7+ V{alpha}14i NKT cells also increases in CD1d+/– mice of the Vbetaa/a genotype (Fig. 2B), where Vbeta8.2+ V{alpha}14i NKT cells are absent and thus cannot be preferentially negatively selected. We, therefore, favor the hypothesis that Vbeta7+ V{alpha}14i NKT cells have a higher avidity for CD1d complexed with its endogenous ligand(s) than V{alpha}14i NKT cells bearing other Vbetas and thus are better positively selected under more competitive conditions.


Figure 2
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  		FIGURE 2. Preferential thymic selection of V{alpha}14i NKT cells expressing Vbeta7 in CD1d+/– mice. A, Flow cytometric Vbeta8.2-Vbeta7 analysis on DP thymocytes and thymic mature V{alpha}14i NKT cells in CD1d+/+ and CD1d+/– Vbetab/b mice. The bar chart represents the mean percentages of Vbeta8.2+ (left scale) and Vbeta7+ (right scale) cells (±SD) among DP thymocytes (ic Vbeta staining) and thymic mature V{alpha}14i NKT cells (surface Vbeta staining), gated as shown in Fig. 1A (n = 3). B, Vbeta7 expression on DP thymocytes and thymic mature V{alpha}14i NKT cells in Vbeta8-deficient CD1d+/+ and CD1d+/– Vbetaa/a mice. The bar chart represents the mean percentages of Vbeta7+ cells (±SD) among DP thymocytes (ic Vbeta staining) and thymic mature V{alpha}14i NKT cells (surface Vbeta staining), gated as shown in Fig. 1A (n = 3). *, p ≤ 0.05 vs CD1d+/+.

 
Preferential thymic selection of NKT cells expressing Vbeta7 in hV{alpha}24tg mice

As a complementary approach to investigate the role of the Vbeta domain in thymic selection of V{alpha}14i NKT cells by endogenous ligands, we took advantage of mice expressing a tg human V{alpha}24i-J{alpha}18 (V{alpha}24i) chain fused with mouse C{alpha} (22). Such tg mice have been described previously to develop NK1.1+ T cells binding mouse CD1d-{alpha}GalCer tetramers (21). Selection of NK1.1+ T cells in these mice strictly depends on the presence of CD1d, because hV{alpha}24tg CD1d–/– mice were devoid of NK1.1+ T cells (data not shown). Because the human V{alpha}24i chain has presumably been evolutionarily selected to react optimally with human CD1d, it might be anticipated that the chimeric semi-invariant TCR in the tg mice would react less well with mouse CD1d than the control V{alpha}14i mouse TCR. Indeed, more detailed analysis of tg V{alpha}24i NKT cells revealed that they bound poorly mouse CD1d-{alpha}GalCer tetramers, compared with control V{alpha}14i NKT cells (Fig. 3A), strongly suggesting that the avidity of the interaction between mouse CD1d-glycolipid and the chimeric TCR in hV{alpha}24tg mice is relatively low. This interpretation was strengthened by the dramatically reduced binding of mouse CD1d-{alpha}GalCer dimers by tg V{alpha}24i NKT cells (Fig. 3A). Interestingly, the presumed low avidity V{alpha}24i NKT cell population selected in hV{alpha}24tg mice expressed a significant bias toward the usage of Vbeta7, with a Vbeta8.2-Vbeta7 ratio of 0.9, compared with 2.8 in non-tg littermates (Fig. 3B). Strikingly, this Vbeta7 bias increased dramatically when the few tetramer (or dimer) binding NKT cells present in hV{alpha}24tg mice were analyzed (Vbeta8.2-Vbeta7 ratio of 0.3 and 0.2 for tetramer+ and dimer+, respectively) (Fig. 3B). Because the Vbeta7 bias of V{alpha}24i NKT cells correlated positively with avidity for mouse CD1d complexed with {alpha}GalCer over a moderate avidity range, these data strongly suggest that the preferential thymic selection of Vbeta7+ over Vbeta8.2+ NKT cells in hV{alpha}24tg mice is due to a higher avidity of Vbeta7+ NKT cells for CD1d complexed with endogenous ligand(s) rather than less efficient negative selection of Vbeta7+ NKT cells, because negative selection should apply only to NKT cells exceeding a high avidity threshold.


Figure 3
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  		FIGURE 3. Preferential thymic selection of NKT cells expressing Vbeta7 in hV{alpha}24tg mice. A, Mouse CD1d-{alpha}GalCer tetramer and dimer binding to NKT cells from hV{alpha}24tg C{alpha}+/– mice. HSA/CD8-depleted thymocytes from hV{alpha}24tg C{alpha}+/– mice and non-tg C{alpha}+/– littermates were surface stained with tetramers or dimers and mAbs against TCRbeta and NK1.1. Numbers represent the mean percentages of positive cells (±SD) in the NKT gate (n = 3). B, Total (for ic Vbeta staining) and HSA/CD8-depleted (for surface Vbeta staining) thymocytes from hV{alpha}24tg C{alpha}+/– mice or non-tg C{alpha}+/– littermates were stained as described in Fig. 1A. The bar chart represents the mean percentages of Vbeta8.2+ and Vbeta7+ cells (±SD) among DP or DN thymocytes (ic Vbeta staining) and thymic NKT cells (surface Vbeta staining) binding either tetramers or dimers, gated as shown in A (n = 3).

 
To rule out the possibility that the very low Vbeta8.2-Vbeta7 ratio among NKT cells in hV{alpha}24tg mice has already been predetermined at the precursor stage (for example, by preferential pairing of TCR chains), we further analyzed the frequencies of ic Vbeta8.2+ and Vbeta7+ cells among thymic DN and DP cells from hV{alpha}24tg mice. Both DN and DP thymocyte subsets were analyzed because it is not clear which population is the immediate precursor of NKT cells in the hV{alpha}24tg mice, which are almost exclusively DN (21). As shown in Fig. 3B, the Vbeta8.2-Vbeta7 ratio was slightly reduced among hV{alpha}24+ DN and DP subsets from hV{alpha}24tg mice (2.1 and 2.8, respectively, vs 3.5 in DP non-tg controls). Nevertheless, these Vbeta8.2-Vbeta7 ratios are much higher than those observed in mature thymic V{alpha}24i NKT cells, indicating that preferential TCR chain pairing among NKT cell precursors plays little if any role in their thymic selection.

Preferential selection of Vbeta7+ V{alpha}14i NKT cells by iGb3 in a thymic in vitro culture

iGb3 is a CD1d-binding endogenous ligand for V{alpha}14i NKT cells that has been postulated to drive their thymic selection (3). Therefore, we directly addressed the question of whether iGb3 preferentially selects Vbeta7+ V{alpha}14i NKT cells in a 7-day in vitro culture using CD1d+/+C{alpha}–/– or CD1d–/–C{alpha}–/– thymocytes as stimulators and enriched thymic V{alpha}14i NKT cells as responders. Murine IL-7 was added to all cultures to ensure survival of V{alpha}14i NKT cells. V{alpha}14i NKT cells survived the 7-day thymic in vitro culture only in the presence of CD1d+/+C{alpha}–/– but not of CD1d–/–C{alpha}–/– thymocytes (data not shown), indicating the requirement of CD1d-dependent signals for cell survival. Exogenously added iGb3 significantly increased the frequency of Vbeta7+, but not of Vbeta8.2+, V{alpha}14i NKT cells in our in vitro system (Fig. 4). Interestingly, even in the absence of exogenously added iGb3, Vbeta7+ V{alpha}14i NKT cells were preferentially selected (Fig. 4), presumably due to interactions with constitutively expressed endogenous glycolipid Ag. These data further support our hypothesis that Vbeta7+ V{alpha}14i NKT cells have a higher avidity for CD1d complexed with endogenous ligand(s) than V{alpha}14i NKT cells bearing other Vbetas.


Figure 4
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  		FIGURE 4. Preferential selection of Vbeta7+ V{alpha}14i NKT cells by iGb3 in a thymic in vitro culture. HSA/CD8-depleted thymocytes from C57BL/6 mice were cultured together with total thymocytes from C{alpha}–/– mice in the presence of IL-7 and iGb3 or iGb3 solvent. Before culture (d0) and after 7 days (d7), cells were surface-stained with mouse CD1d-{alpha}GalCer dimers and mAbs against TCRbeta, Vbeta8.2, and Vbeta7. The bar chart represents the mean percentages of Vbeta8.2+ (left scale) and Vbeta7+ (right scale) cells (± SD) among V{alpha}14i NKT cells (TCRbetaintdimer+) (n = 3). *, p ≤ 0.05 vs d0.

 
Concluding remarks

In conclusion, our results clearly demonstrate that the TCR Vbeta domain influences thymic selection of V{alpha}14i NKT cells by CD1d complexed with endogenous ligand(s). Because selection of thymic Vbeta7+ NKT cells was favored over Vbeta8.2+ NKT cells under conditions of limiting endogenous ligand concentration and reduced TCR{alpha}-chain avidity in vivo as well as in response to iGb3 in vitro, we propose that Vbeta7+ NKT cells have a higher avidity for CD1d complexed with its endogenous ligand(s) than NKT cells bearing other Vbetas and thus are better positively selected under more competitive conditions. Moreover, because Vbeta8.2+ NKT cells bind better than Vbeta7+ NKT cells to CD1d-{alpha}GalCer complexes (16), it is tempting to speculate that distinct glycolipid ligands might preferentially activate particular subsets of V{alpha}14i NKT cells due to Vbeta-dependent differences in their binding avidities for the semi-invariant TCR.


   Acknowledgments
 
We thank M. J. Grusby (Harvard Medical School, Boston, MA) and M. Taniguchi (Chiba University, Chiba, Japan) for providing CD1d–/– and J{alpha}18–/– mice, respectively. We also are grateful to A. Khurana and M. Kronenberg (La Jolla Institute for Allergy and Immunology, San Diego, CA) for providing mouse CD1d-{alpha}GalCer tetramers, B.-Y. Wei (BD Pharmingen, San Diego, CA) for providing mouse CD1d-IgG1 dimers, and Y. Koezuka (Kirin Brewery, Gunma, Japan) for providing {alpha}GalCer.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by fellowships from the Deutsche Forschungsgemeinschaft (to J.S.) and the European Federation of Neurological Societies (to M.P.M.) and by grants from the Swiss National Science Foundation (to H.R.M.), the Human Frontiers Science Program (to H.R.M. and P.D.), and the Associazione Italiana Ricerca sul Cancro (to G.C. and P.D.). Back

2 Current address: Department of Research, University Hospital Basel, Hebelstrasse 20, 4031 Basel, Switzerland. Back

3 Address correspondence and reprint requests to Dr. H. Robson MacDonald, Ludwig Institute for Cancer Research, Chemin des Boveresses 155, 1066 Epalinges, Switzerland. E-mail address: hughrobson.macdonald{at}isrec.unil.ch Back

4 Abbreviations used in this paper: V{alpha}14i, invariant V{alpha}14; {alpha}GalCer, {alpha}-galactosylceramide; HSA, heat-stable Ag; ic, intracellular; iGb3, isoglobotrihexosylceramide; DN, double negative; DP, double positive. Back

Received for publication March 3, 2005. Accepted for publication December 20, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 

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