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Agonist Ligands Expressed by Thymic Epithelium Enhance Positive Selection of Regulatory T Lymphocytes from Precursors with a Normally Diverse TCR Repertoire¹

Julie Ribot^*, Paola Romagnoli^* and Joost P. M. van Meerwijk^2,*,

^* Centre de Physiopathologie Toulouse Purpan, Institut National de la Santé et de la Recherche Médicale Unité 563; Paul Sabatier University, Toulouse, France, and Institut Universitaire de France, Paris, France

	Abstract

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CD4⁺CD25⁺ regulatory T lymphocytes play a crucial role in inhibition of autoimmune pathology. In accordance with this physiological role, it is now well established that the repertoire of these lymphocytes is strongly enriched in autospecific cells. However, despite extensive investigation, the thymic mechanisms involved in development of regulatory T cells remain incompletely defined. To address the issue of selection of regulatory T cell precursors in mice with a naturally diverse TCR repertoire, we have analyzed development of superantigen-specific regulatory T cells in hemopoietic chimeras in which endogenous super-antigens are exclusively presented by thymic epithelial cells. Our results demonstrate that recognition of agonist ligands expressed by thymic epithelium does not lead to deletion but substantially enhances development of mature regulatory T cells. Interestingly, also development of a small subpopulation of CD25-expressing T cells lacking expression of the transcription factor Foxp3, thought to be autospecific, is enhanced by expression of the agonist ligand on thymic epithelium. Based on quantitative arguments, we propose that commitment to the regulatory T cell lineage is not dictated by the specificity of precursors, but that recognition of the agonist ligand expressed by thymic epithelium substantially enhances their positive selection.

	Introduction

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T lymphocyte tolerance to self-Ags is induced in the thymus during the process of negative selection (1). Despite the quantitatively impressive nature of this process (2), significant numbers of autospecific T cells migrate to the periphery (3). In the periphery, autospecific T cells are kept silent by recessive (induction of apoptosis or anergy) and dominant tolerance mechanisms (4). Dominant or "active" tolerance is assured by regulatory/suppressor T lymphocytes. The best characterized regulatory T cell (Treg)³ subset consists of CD4⁺ T cells expressing high levels of CD25, GITR, CTLA-4, and the forkhead/winged helix transcription factor Foxp3 (5, 6, 7). Although for practical purposes (i.e., isolation of Treg) the best marker is CD25, Foxp3 expression appears to correlate best with regulatory function (8). CD4⁺CD25⁺ Treg play a major role in the prevention of autoimmunity (5, 6) and inflammatory bowel disease (9), in regulating immunity to viral and parasite infections (10), in maintenance of maternal tolerance to the fetus (11), and in inhibition of antitumor immunity (12).

Interestingly, the normally diverse CD4⁺CD25⁺ regulatory thymocyte population selected on naturally expressed ligands, has higher avidity for self than CD4⁺CD25^– cells (13), resulting in a peripheral Treg repertoire highly enriched in self-reactive cells (14, 15, 16). These observations raise important questions concerning the selection of these cells in the thymus. In this organ, CD4⁺CD25⁺ Treg are positively selected by cortical epithelial cells, as are conventional CD4⁺ lymphocytes (17). Surprisingly, Treg precursors have been shown to be susceptible to thymic negative selection (14, 17, 18). However, although autospecific Treg precursors are negatively selected by APC of bone marrow origin (14), they appear relatively resistant to negative selection induced by thymic epithelium (TE) (13, 19). Therefore, TE plays an important role in development of dominant tolerance, as previously postulated based on the observation that TE can induce dominant transplantation tolerance (20).

The precise role of positive selection in development of Treg remains unclear. It has been proposed that interaction with agonist ligands directs developing thymocytes to the Treg lineage. However, studies with mice doubly transgenic for TCR and agonist ligand have led to conflicting results. In some cases, evidence of increased selection of CD4⁺CD25⁺ Treg by agonist ligand was observed (21, 22, 23, 24, 25). In other TCR/ligand doubly transgenic mice, the relatively increased percentage of CD4⁺CD25^high Treg has been attributed to deletion of CD25^– cells rather than increased development of CD4⁺CD25⁺ cells (19). The potential differences reported in the distinct doubly transgenic mice may be due to the particular avidity of the chosen TCR/ligand pair and/or to variations in the expression pattern of the ligand. Therefore, it remains unclear 1) whether TCR-mediated signals are involved in the Treg lineage choice and 2) whether differences exist between positive selection of conventional and regulatory T lymphocytes.

To gain insight into the mechanisms of thymic Treg differentiation, we analyzed the role of agonist ligands expressed by TE in the development of Treg precursors with a naturally diverse TCR repertoire. We generated bone marrow chimeras in which superantigen (sAg) presentation is limited to TE and analyzed the development of sAg-specific Treg. Our results indicate that agonist ligands considerably enhance positive selection of Treg.

	Materials and Methods

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Mice

All mice were used at 6–10 wk of age. C57BL/6 and DBA/2 mice were purchased from Janvier. B10.D2 mice were originally obtained from Harlan France and maintained in our specific pathogen-free animal facilities. C57BL/6 mice deficient in MHC expression (MHC°) because of targeted deletions in the ₂-microglobulin (26) and IA^b genes (27) were obtained from the Centre de Développement des Techniques Avancés-Centre National de la Recherche Scientifique and were maintained in our specific pathogen-free animal facility. All experiments involving animals were performed in compliance with the relevant laws and institutional guidelines (Institut National de la Santé et de la Recherche Médicale; approval no. 31-13) and have been approved by the local ethics committee (Midi-Pyrénees, France; Reference MP/01/31/10/03).

Antibodies

The following Abs were used for phenotypic analysis: FITC-labeled anti-TCR V3, 4, 5, 6, 14, 17^a, PE-labeled anti-CD25, PE-Cy7-labeled anti-CD4 (BD Pharmingen), PE-labeled anti-Foxp3, and allophycocyanin-labeled anti-CD8 and anti-CD25 Abs (eBioscience).

Flow cytometry

Thymi were homogenized, washed in medium, and resuspended in 2.4G2 (anti-FcR mAb) (28) hybridoma supernatant. After incubation of 30 min on ice, saturating concentrations of Ab were added. Twenty minutes later, cells were washed in PBS, 2.5% FCS, and 0.02% NaN₃. Labeled cells were analyzed using a FACSCalibur and CellQuest software (BD Biosciences). Dead cells were excluded using appropriate forward scatter/side scatter gates.

For TCR V analysis, thymi of three mice were pooled, depleted of CD8⁺ cells by treatment with anti-CD8 mAb 31.M (29) and complement (Saxon Europe), followed by Lympholyte-M gradient (Cederlane Laboratories). The efficiency of CD8 depletion was verified in every experiment and was routinely >99%.

Bone marrow chimeras

Irradiation bone marrow chimeras were generated by lethally irradiating (8.5 Gy gamma) C57BL/6 hosts using a ¹³⁷Cs source (7 Gy/min). Next day, irradiated mice were reconstituted by i.v. injection of 10⁷ bone marrow cells. Chimeras were kept on antibiotics containing water (0.2% Bactrim; Roche) for the complete duration of the experiment.

Statistical analysis

Statistical significance of differences between subpopulations was assessed using Student’s t test and is indicated as not significant (p 0.05); _*, p < 0.05; _**, p < 0.01; and _***, p < 0.001.

	Results

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Superantigens presented by TE enhance differentiation of CD4⁺CD8^–CD25^high Treg

To study the role of naturally expressed agonist ligands in thymic selection of Treg precursors with a normally diverse TCR repertoire, we generated irradiation chimeras in which sAg are exclusively presented by TE. As hosts, we used (lethally irradiated) DBA/2 mice which present endogenous sAg encoded by mouse mammary tumor viruses 1, 6, 7, 8, 11, and 13. These sAg are high-affinity ligands for V3, V5, and V6 but they do not interact with V4 and V14 (30). MHC-deficient (MHC°) bone marrow was used to prevent thymic deletion of Treg precursors induced by APC of hemopoietic origin (14). These MHC°DBA/2 chimeras were compared with MHC°C57BL/6 (B6) mice, in which no sAg-mediated thymic deletion is known to occur (30). Controls consisted of B6B6 and DBA/2DBA/2 chimeras.

Chimeras were analyzed by flow cytometry 6 wk after reconstitution. The percentage of thymocytes of donor origin was always superior to 99%. In the spleen, >99% of T and B lymphocytes were of donor origin. Less than 7% of host CD11c⁺CD11b^– thymic DC remained in the chimeras. Moreover, the ratio of CD8^low:CD8^high cells among remaining host DC was identical to that found in unmanipulated animals (data not shown). In the thymus of DBA/2DBA/2 chimeras, significantly higher percentages of CD25^high Treg among CD4⁺CD8^– (CD4SP) thymocytes were observed than in B6B6 chimeras (Fig. 1, A and B). This result confirms genetically determined quantitative differences in Treg development, due to thymocyte-intrinsic factors, that we have reported previously (31). Significantly reduced percentages of sAg-specific V3-, V5-, and V6-expressing CD4SP CD25^– and CD25^high cells were found in DBA/2DBA/2 as compared with B6B6 chimeras (Fig. 1, C and D). No difference in percentages of V4 and V14 T cells (which do not react with sAg presented in DBA/2 mice) was observed between the two types of chimeras. These data confirm that Treg precursors are sensitive to thymic deletion, as we and others have previously reported (14, 18).

View larger version (34K): [in this window] [in a new window]   FIGURE 1. sAg presented by TE enhance generation of CD4SP CD25high Treg. A, Analysis of CD25 expression by electronically gated CD4SP thymocytes. B, Total numbers of thymocytes (left panel), percentage of CD4SP cells among total thymocytes (middle panel), and percentage of CD25high cells among CD4SP thymocytes (right panel) in indicated chimeras. C, Flow cytometry analysis of TCR V expression by electronically gated CD4SP CD25– and CD25high thymocytes in indicated chimeras. D, Percentages of CD4SP CD25– and CD25high thymocytes expressing indicated V in the distinct chimeras. ***, p < 0.001; **, p < 0.01; ns, not significant; Student’s t test. Error bars indicate SD (B6B6 and DBA/2DBA/2: n = 3, MHC°B6 and MHC°DBA/2: n = 6).

Interestingly, a 2-fold higher percentage of CD25^– (but not CD25⁺) CD4SP V5⁺ (but not V3, V6, V4, or V14) cells was found in MHC°B6 than in B6B6 chimeras (Fig. 1D). These results show that V5-specific deletion of CD25^– but not CD25⁺ thymocytes by bone marrow-derived APC occurs in B6B6 chimeras.

In contrast to chimeras in which TE and APC express sAg, in MHC°DBA/2 mice sAg-specific V3⁺ cells were not deleted and only partial deletion of sAg-specific V5 and V6 CD4SP CD25^– cells was observed. These results are consistent with previous work documenting the limited role of thymic (medullary) epithelium in deletion of autospecific precursors (32, 33, 34). Control V4⁺ and V14⁺ thymocytes were not deleted.

As compared with MHC°B6 chimeras, in MHC°DBA/2 mice a substantial increase in the percentage of CD4SP CD25^high regulatory thymocytes expressing sAg-specific V3, V5, and V6 was observed (Fig. 1, C and D). Since in the MHC°B6 and MHC°DBA/2 chimeras the total number of thymocytes and the percentage of CD4SP cells were similar, and in MHC°DBA/2 chimeras the percentage of CD25^high Treg among CD4SP cells is increased (Fig. 1B), the increase in percentage corresponded to an increase in absolute cell numbers of sAg-specific Treg in MHC°DBA/2 chimeras. Comparable percentages of V4 and V14 CD4SP CD25^high Treg developed in both types of chimeras. These results indicate that natural agonist ligands presented by TE substantially enhance the generation of CD4SP CD25^high Treg.

sAg enhance generation of CD4⁺Foxp3⁺ Treg and a subpopulation of CD4⁺Foxp3^– thymocytes

CD25 is widely used as marker for Treg (35). However, it is also expressed on activated conventional CD4⁺ and CD8⁺ lymphocytes and does not identify all Treg. A better marker for Treg is Foxp3 (7, 8, 36), a forkhead/winged helix transcription factor. We therefore assessed whether sAg presented by TE also enhanced differentiation of CD4⁺Foxp3⁺ regulatory T cells. As compared with MHC°B6 chimeras, in MHC°DBA/2 chimeras substantially increased percentages and numbers of Foxp3⁺CD4⁺ thymocytes expressing sAg-specific V3, V5, and V6 (but not control V4 and V14) were observed (Fig. 2, A–C). In contrast, sAg-specific Foxp3^– CD4SP thymocytes were either partially deleted (V5 and 6) or not affected (V3) by sAg presented by TE (Fig. 2C).

View larger version (17K): [in this window] [in a new window]   FIGURE 2. sAg-specific CD4SP Foxp3+ T cell differentiation is enhanced by endogenous sAg presented by TE. A, Analysis of Foxp3 expression by electronically gated CD4SP thymocytes. B, Percentage of Foxp3-expressing cells among CD4SP thymocytes in indicated chimeras. C, Percentages of thymocytes expressing indicated V among CD4SP Foxp3– and Foxp3+ cells in indicated chimeras. ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, not significant; Student’s t test. Error bars indicate SD (n 4).

Foxp3⁺ CD4SP thymocytes express varying levels of CD25 (Fig. 3A) as previously reported (37). We therefore next assessed whether sAg differentially enhanced development of CD25^–, CD25^int, or CD25^high Foxp3⁺ thymocytes (Fig. 3B). sAg presented by TE substantially enhanced differentiation of all three Foxp3⁺ subpopulations (Fig. 3B, cf MHC°DBA/2 with MHC°B6 chimeras).

View larger version (25K): [in this window] [in a new window]   FIGURE 3. TE-expressed sAg differentially affect CD4+ T cell development. A, Electronically gated CD4SP thymocytes were subdivided into five different populations according to their CD25 and Foxp3 expression levels. B, Percentages of thymocytes expressing indicated V among the CD4SP Foxp3+ thymocyte subpopulations indicated in the distinct chimeras. C, Percentages of thymocytes expressing indicated V among CD25– and CD25+ CD4SP Foxp3– thymocyte subpopulations in the distinct chimeras. ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, not significant; Student’s t test. Error bars indicate SD (n 4).

sAg do not enhance positive selection of precursors for conventional T cells

The presented data indicate that generation of Treg is substantially enhanced by interaction with agonist ligand. We next investigated whether thymic-positive selection of conventional (i.e., nonregulatory) T cells can also be increased by interaction with agonist ligand. To this end, we analyzed V expression by CD69-expressing CD4⁺CD8⁺ (DP) thymocytes. Whereas it is impossible at this very early stage of development to distinguish between precursors for conventional and Treg, since the vast majority of mature thymocytes are conventional cells, analysis of the whole population in essence reflects analysis of conventional T cell precursors. Analysis of V expression by CD69⁺ DP revealed two levels of V expression: low and high (Fig. 4A). V^low thymocytes are recently activated (positively selected) cells that are precursors for mature CD4SP and CD4^–CD8⁺ (CD8SP) thymocytes (39). Using a TCR-transgenic mouse system, it has previously been shown that these cells have not yet been submitted to thymic deletion (34). V^high cells are more mature, have been submitted to thymic deletion, and are precursors for CD8SP thymocytes (34, 39, 40). We therefore analyzed the percentage of recently positively (but not yet negatively) selected V^low thymocytes in MHC°B6 and MHC°DBA/2 chimeras (Fig. 4B). No difference between the percentages of sAg-specific V5^low and V6^low and control V4^low and V14^low thymocytes in sAg-presenting vs nonpresenting chimeras was observed. We therefore conclude that positive selection of precursors for conventional T cells is not enhanced by agonist ligands.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. Positive selection of conventional T cells is not modulated by TE-expressed sAg. A, Thymocytes were analyzed by flow cytometry using Abs specific for CD4, CD8, CD69, and indicated V. FACS plots are electronically gated as indicated. B, Percentages of CD69+ DP thymocytes expressing intermediate levels of indicated V in the distinct chimeras.

	Discussion

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Specificity for sAg is determined by the V region expressed by T lymphocytes. In mice containing endogenous mouse mammary tumor viruses, sAg-specific thymocytes are deleted during T cell development in the thymus (reviewed in Ref. 30). We have previously reported that precursors for Treg are not an exception and are efficiently deleted during development (14). When sAg are only presented by TE, deletion of precursors for conventional T cells is much less efficient and tolerance is mediated by clonal anergy (41). In our MHC°DBA/2 chimeras, in which sAg are exclusively presented by TE, we indeed found that deletion of sAg-specific Foxp3^– thymocytes was much less efficient than in control DBA/2DBA/2 chimeras in which sAg can also be presented by APC. However, depending on the V region expressed, partial deletion clearly took place, confirming earlier reports showing that TE can directly induce deletion of precursors for CD4SP thymocytes (32, 33, 34).

Importantly, agonist ligand expressed by TE did not induce any deletion of Treg precursors. In contrast, it substantially enhanced development of Treg. By studying MHC class II transfer from thymic stroma to developing thymocytes, we have previously observed that precursors for CD25^–, but not CD25⁺, CD4SP thymocytes are sensitive to negative selection induced by TE (13). In TCR/ligand doubly transgenic mice in which Ag is expressed by TE, significant deletion of CD25^– but not of CD25⁺ CD4SP precursors has previously been observed (19, 21, 22, 25). However, in these studies it was unclear whether deletion was due to presentation of Ag directly by TE or indirectly by APC. In these and other transgenic mouse models, the agonist ligand induced development of increased percentages of CD25⁺ Treg among CD4SP thymocytes (19, 21, 22, 24, 25, 42). Whether or not expression of the agonist ligand led to development of more Treg, in terms of absolute cell numbers, was unclear in most (21, 22, 24, 42) but not all (25) of these reports, and this was formally dismissed in another (19). However, even if numerically more Treg developed in TCR/Ag doubly transgenic mice, this would not necessarily be due to agonist ligand-mediated recruitment into the Treg lineage or selection of these cells. In this light, we have previously reported that development of CD8SP thymocytes is limited by homeostatic mechanisms (43). When development of CD4SP thymocytes is strongly inhibited by negative selection, as in the TCR/Ag doubly transgenic mice, more Treg might develop due to homeostatic mechanisms. Such complications are related to the fact that in TCR-transgenic mice practically all precursors express the same TCR. In contrast, in the experimental model we presented here a substantially smaller fraction of precursors was involved. Indeed, the total thymocyte numbers and percentages of CD4SP were similar in the two types of irradiation chimeras we compared. As compared with chimeras in which no sAg was presented, in chimeras in which sAg were presented only by TE substantially higher numbers of sAg-specific Treg developed. Since in our experimental model it is difficult to imagine how results could be due to homeostatic mechanisms, we conclude that recognition of agonist ligand substantially enhanced development of Treg.

An unexpected observation merits particular attention. It has previously been reported that in B6 thymi V5 expression is skewed to the CD8SP population (44). This result was attributed to a less efficient positive selection of V5⁺ cells into the CD4SP than the CD8SP population. Also, peripheral V5 expression in I-E^– (C57BL/10) mice is skewed to the CD8⁺ population (45). With age the CD4:CD8 ratio of V5-expressing T cells gradually decreases, and this is known to be due to Mtv-8- and Mtv-9-mediated peripheral deletion of CD4⁺, but not CD8⁺ V5⁺ T cells (46). We observed that 2-fold more CD4SP V5⁺ thymocytes developed in absence than in presence of thymic deletion by bone marrow-derived APC. V5⁺ thymocytes are therefore partially deleted, probably by Mtv-8 and 9. Interestingly, also 2-fold more V5⁺ CD8SP thymocytes developed in MHC°B6 than in B6B6 chimeras (data not shown). Therefore, thymic skewing of V5 expression toward the CD8 lineage cannot be explained by differences in thymic deletion mediated by APC of bone marrow origin. In MHC°B6 chimeras, the percentage of V5⁺ cells among freshly positively selected CD69⁺ DP thymocytes is similar to that among CD4SP cells but significantly lower than that among CD8SP cells (data not shown). Together, these data indicate that the thymic skewing of V5 expression toward the CD8 lineage is indeed most probably explained by more efficient positive selection into the CD8 lineage. Moreover, since Mtv-8 and 9 presentation by I-A^b leads to only partial deletion of V5⁺ thymocytes, clearly low-avidity interactions are involved.

Whereas the TCR repertoires of CD25⁺ Treg and CD25^– conventional T cells show only very limited overlap (16), their TCR V repertoires are very similar (14, 18, 47). An exception to this rule is V5, which, in B6 mice is 2-fold more represented in the CD25⁺ than in the CD25^– population. In absence of negative selection by APC (in MHC°B6 chimeras), the percentage of V5⁺ cells among CD25^–, but not CD25⁺ CD4SP thymocytes was increased. As compared with conventional T cell precursors, precursors for Treg appear therefore less susceptible to APC-mediated deletion by low-avidity ligands. This conclusion is consistent with our earlier observation that the magnitude of deletion of Treg precursors by APC seemed slightly lower than that of conventional T cell precursors (14).

Interestingly, no more V5⁺ Treg developed in MHC°B6 than in B6B6 chimeras. Therefore, in contrast to high-avidity I-E^d/sAg ligands, low-avidity I-A^b/sAg ligands did not induce enhanced positive selection of Treg. This result is consistent with earlier data showing that high avidity, but not low-avidity interactions allow for Treg development in TCR/ligand doubly transgenic mice (24).

The precise mechanism involved in sAg-mediated enhanced development of Treg is uncertain. However, it appears unlikely that recognition of the agonist ligand on TE by uncommitted precursors recruits them to the Treg lineage. If this were the case, one would expect an even higher increase in development of sAg-specific Treg in MHC°DBA/2 (as compared with MHC°B6) chimeras. For example, TE expression of sAg led to deletion of 5 x 10⁵ CD25^–V5⁺ CD4SP, while only 2 x 10⁴ more CD25^highV5⁺ cells developed. Our data are more compatible with the hypothesis that recognition of the agonist ligand allows for positive selection of precursors already committed to the Treg lineage. Alternatively, recognition of the agonist ligand expressed in a thymic niche specialized in Treg development may allow for simultaneous positive selection and Treg lineage commitment. This view is consistent with the observation that thymocytes expressing Treg-derived TCR preferentially, but not exclusively, develop into Treg (16). The second scenario is also consistent with the observation that expression of Foxp3, a master switch in Treg development, requires TCR-ligand interaction (8).

To evaluate the effect of agonist ligand expression by TE on positive selection of conventional T cells, we analyzed V expression by TCRV^lowCD69⁺ DP thymocytes in MHC°B6 and MHC°DBA/2 chimeras. These cells have initiated positive selection, but have not yet been submitted to negative selection (34). Comparable percentages of sAg-specific cells were found in the two types of chimeras. We conclude therefore that positive selection of conventional T cells is not enhanced by recognition of agonist ligand. Differentiation of conventional T cells has been shown to require continued TCR-mediated signaling occurring after the initial phase of positive selection (48). Therefore, enhanced positive selection of Treg by interaction with agonist ligand may very well occur at later stages of development (e.g., in the medulla). In contrast, continued interaction of conventional T cell precursors with agonist ligands does not enhance positive selection but leads to negative selection.

Under physiological conditions, peripheral T lymphocytes of CD4⁺CD25^intFoxp3^– phenotype produce IL-2, required for in vivo maintenance of Treg (38). It has previously been proposed that this population is enriched in self-reactive T cells (8, 38). Intriguingly, we found that recognition of the agonist ligand expressed by TE enhanced development of CD4SP CD25^intFoxp3^– thymocytes. Precursors for these cells therefore appear resistant to negative selection induced by ligands expressed by TE and their positive selection is enhanced by recognition of agonist ligand. Our results strongly suggest that the autospecific CD4⁺CD25^intFoxp3^– T cell repertoire is, at least in part, generated in the thymus. These data therefore indicate that at least two apparently distinct T cell lineages, Treg and CD4⁺CD25^intFoxp3^– T cells, use identical modes of thymic selection.

In conclusion, natural agonist ligands expressed by TE do not induce deletion of Treg precursors but substantially enhance their positive selection. Importantly, medullary thymic epithelium (but not bone marrow-derived DC) expresses a large variety of "tissue-specific" Ags (49). Therefore, TE will enhance development of Treg specific for ubiquitously expressed as well as for tissue-specific Ags, but DC will induce deletion of Treg precursors specific for ubiquitously expressed Ags. Thus, a repertoire of Treg exquisitely appropriate for protection against autoimmune aggression appears to develop.

	Acknowledgments

 
We thank the staff of the IFR30 animal facility and, in particular, Maryline Calise for expert animal husbandry, and Fatima Fatima-Ezzahra L’Faqihi-Olive for help in flow cytometry. We thank Drs. Sylvie Guerder and Rob MacDonald for critical reading of this manuscript and Hans Acha-Orbea for very helpful advice and suggestions.

	Disclosures

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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 by a grant from the European Community awarded to the EuroThymaide Consortium (Contract LSHB-CT-2003-503410).

² Address correspondence and reprint requests to Dr. Joost P. M. van Meerwijk, Institut National de la Santé et de la Recherche Médicale Unité 563, BP 3028, 31024 Toulouse, Cedex 3, France. E-mail address: Joost.van-Meerwijk{at}toulouse.inserm.fr

³ Abbreviations used in this paper: Treg, regulatory T cell; CD4SP, CD4⁺CD8^– single positive; CD8SP, CD4^–CD8⁺ single positive; DP, double positive; TE, thymic epithelium; sAg, superantigen; MHC°, MHC deficient; DC, dendritic cell; int, intermediate.

Received for publication December 7, 2005. Accepted for publication May 2, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Hogquist, K. A., T. A. Baldwin, S. C. Jameson. 2005. Central tolerance: learning self-control in the thymus. Nat. Rev. Immunol. 5: 772-782. [Medline]
2. van Meerwijk, J. P. M., S. Marguerat, R. K. Lees, R. N. Germain, B. J. Fowlkes, H. R. MacDonald. 1997. Quantitative impact of thymic clonal deletion on the T cell repertoire. J. Exp. Med. 185: 377-383. [Abstract/Free Full Text]
3. Bouneaud, C., P. Kourilsky, P. Bousso. 2000. Impact of negative selection on the T cell repertoire reactive to a self-peptide: a large fraction of T cell clones escapes clonal deletion. Immunity 13: 829-840. [Medline]
4. Stockinger, B.. 1999. T lymphocyte tolerance: from thymic deletion to peripheral control mechanisms. Adv. Immunol. 71: 229-265. [Medline]
5. Sakaguchi, S.. 2004. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 22: 531-562. [Medline]
6. Piccirillo, C. A., A. M. Thornton. 2004. Cornerstone of peripheral tolerance: naturally occurring CD4⁺CD25⁺ regulatory T cells. Trends Immunol. 25: 374-380. [Medline]
7. Ramsdell, F.. 2003. Foxp3 and natural regulatory T cells: key to a cell lineage?. Immunity 19: 165-168. [Medline]
8. Fontenot, J. D., J. P. Rasmussen, L. M. Williams, J. L. Dooley, A. G. Farr, A. Y. Rudensky. 2005. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22: 329-341. [Medline]
9. Coombes, J. L., N. J. Robinson, K. J. Maloy, H. H. Uhlig, F. Powrie. 2005. Regulatory T cells and intestinal homeostasis. Immunol. Rev. 204: 184-194. [Medline]
10. Mills, K. H.. 2004. Regulatory T cells: friend or foe in immunity to infection?. Nat. Rev. Immunol. 4: 841-855. [Medline]
11. Aluvihare, V. R., M. Kallikourdis, A. G. Betz. 2004. Regulatory T cells mediate maternal tolerance to the fetus. Nat. Immunol. 5: 266-271. [Medline]
12. Terabe, M., J. A. Berzofsky. 2004. Immunoregulatory T cells in tumor immunity. Curr. Opin. Immunol. 16: 157-162. [Medline]
13. Romagnoli, P., D. Hudrisier, J. P. M. van Meerwijk. 2005. Molecular signature of recent thymic selection events on effector and regulatory CD4⁺ T lymphocytes. J. Immunol. 175: 5751-5758. [Abstract/Free Full Text]
14. Romagnoli, P., D. Hudrisier, J. P. M. van Meerwijk. 2002. Preferential recognition of self-antigens despite normal thymic deletion of CD4⁺CD25⁺ regulatory T cells. J. Immunol. 168: 1644-1648. [Abstract/Free Full Text]
15. Fisson, S., G. Darrasse-Jeze, E. Litvinova, F. Septier, D. Klatzmann, R. Liblau, B. L. Salomon. 2003. Continuous activation of autoreactive CD4⁺CD25⁺ regulatory T cells in the steady state. J. Exp. Med. 198: 737-746. [Abstract/Free Full Text]
16. Hsieh, C. S., Y. Liang, A. J. Tyznik, S. G. Self, D. Liggitt, A. Y. Rudensky. 2004. Recognition of the peripheral self by naturally arising CD25⁺CD4⁺ T cell receptors. Immunity 21: 267-277. [Medline]
17. Bensinger, S. J., A. Bandeira, M. S. Jordan, A. J. Caton, T. M. Laufer. 2001. Major histocompatibility complex class II-positive cortical epithelium mediates the selection of CD4⁺25⁺ immunoregulatory T cells. J. Exp. Med. 194: 427-438. [Abstract/Free Full Text]
18. Pacholczyk, R., P. Kraj, L. Ignatowicz. 2002. Peptide specificity of thymic selection of CD4⁺CD25⁺ T cells. J. Immunol. 168: 613-620. [Abstract/Free Full Text]
19. van Santen, H.-M., C. Benoist, D. Mathis. 2004. Number of T reg cells that differentiate does not increase upon encounter of agonist ligand on thymic epithelial cells. J. Exp. Med. 200: 1221-1230. [Abstract/Free Full Text]
20. Le Douarin, N., C. Corbel, A. Bandeira, V. Thomas-Vaslin, Y. Modigliani, A. Coutinho, J. Salaun. 1996. Evidence for a thymus-dependent form of tolerance that is not based on elimination or anergy of reactive T cells. Immunol. Rev. 149: 35-53. [Medline]
21. Kawahata, K., Y. Misaki, M. Yamauchi, S. Tsunekawa, K. Setoguchi, J.-i. Miyazaki, K. Yamamoto. 2002. Generation of CD4⁺CD25⁺ regulatory T cells from autoreactive T cells simultaneously with their negative selection in the thymus and from nonautoreactive T cells by endogenous TCR expression. J. Immunol. 168: 4399-4405. [Abstract/Free Full Text]
22. Apostolou, I., A. Sarukhan, L. Klein, H. von Boehmer. 2002. Origin of regulatory T cells with known specificity for antigen. Nat. Immunol. 3: 756-763. [Medline]
23. Jordan, M. S., M. P. Riley, H. von Boehmer, A. J. Caton. 2000. Anergy and suppression regulate CD4⁺ T cell responses to a self peptide. Eur. J. Immunol. 30: 136-144. [Medline]
24. Jordan, M. S., A. Boesteanu, A. J. Reed, A. L. Petrone, A. E. Holenbeck, M. A. Lerman, A. Naji, A. J. Caton. 2001. Thymic selection of CD4⁺CD25⁺ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2: 301-306. [Medline]
25. D’Cruz, L. M., L. Klein. 2005. Development and function of agonist-induced CD25⁺Foxp3⁺ regulatory T cells in the absence of interleukin 2 signaling. Nat. Immunol. 6: 1152-1159. [Medline]
26. Koller, B. H., P. Marrack, J. W. Kappler, O. Smithies. 1990. Normal development of mice deficient in 2M, MHC class I proteins, and CD8⁺ T cells. Science 248: 1227-1230. [Abstract/Free Full Text]
27. Chan, S. H., D. Cosgrove, C. Waltzinger, C. Benoist, D. Mathis. 1993. Another view of the selective model of thymocyte selection. Cell 73: 225-236. [Medline]
28. Unkeless, J. C.. 1979. Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J. Exp. Med. 150: 580-596. [Abstract/Free Full Text]
29. Sarmiento, M., D. P. Dialynas, D. W. Lancki, K. A. Wall, M. I. Lorber, M. R. Loken, F. W. Fitch. 1982. Cloned T lymphocytes and monoclonal antibodies as probes for cell surface molecules active in T cell-mediated cytolysis. Immunol. Rev. 68: 135-169. [Medline]
30. Luther, S. A., H. Acha-Orbea. 1997. Mouse mammary tumor virus: immunological interplays between virus and host. Adv. Immunol. 65: 139-243. [Medline]
31. Romagnoli, P., J. Tellier, J. P. M. van Meerwijk. 2005. Genetic control of thymic development of CD4⁺CD25⁺FoxP3⁺ regulatory T lymphocytes. Eur. J. Immunol. 35: 3525-3532. [Medline]
32. Ramsdell, F., T. Lantz, B. J. Fowlkes. 1989. A nondeletional mechanism of thymic self tolerance. Science 246: 1038-1041. [Abstract/Free Full Text]
33. Klein, L.. 2001. Sampling of complementing self-antigen pools by thymic stromal cells maximizes the scope of central T cell tolerance. Eur. J. Immunol. 31: 2476-2486. [Medline]
34. Gallegos, A. M., M. J. Bevan. 2004. Central tolerance to tissue-specific antigens mediated by direct and indirect antigen presentation. J. Exp. Med. 200: 1039-1049. [Abstract/Free Full Text]
35. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor -chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155: 1151-1164. [Abstract]
36. Fontenot, J. D., A. Y. Rudensky. 2005. A well adapted regulatory contrivance: regulatory T cell development and the forkhead family transcription factor Foxp3. Nat. Immunol. 6: 331-337. [Medline]
37. Fontenot, J. D., J. L. Dooley, A. G. Farr, A. Y. Rudensky. 2005. Developmental regulation of Foxp3 expression during ontogeny. J. Exp. Med. 202: 901-906. [Abstract/Free Full Text]
38. Setoguchi, R., S. Hori, T. Takahashi, S. Sakaguchi. 2005. Homeostatic maintenance of natural Foxp3⁺CD25⁺CD4⁺ regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J. Exp. Med. 201: 723-735. [Abstract/Free Full Text]
39. Lucas, B., R. N. Germain. 1996. Unexpectedly complex regulation of CD4/CD8 coreceptor expression supports a revised model for CD4⁺CD8⁺ thymocyte differentiation. Immunity 5: 461-477. [Medline]
40. van Meerwijk, J. P. M., E. M. O’Connell, R. N. Germain. 1995. Evidence for lineage commitment and initiation of positive selection by thymocytes with intermediate surface phenotypes. J. Immunol. 154: 6314-6324. [Abstract]
41. Ramsdell, F., B. J. Fowlkes. 1990. Clonal deletion versus clonal anergy: the role of the thymus in inducing self tolerance. Science 248: 1342-1348. [Abstract/Free Full Text]
42. Walker, L. S. K., A. Chodos, M. Eggena, H. Dooms, A. K. Abbas. 2003. Antigen-dependent proliferation of CD4⁺CD25⁺ regulatory T cells in vivo. J. Exp. Med. 198: 249-258. [Abstract/Free Full Text]
43. van Meerwijk, J. P., S. Marguerat, H. R. MacDonald. 1998. Homeostasis limits the development of mature CD8⁺ but not CD4⁺ thymocytes. J. Immunol. 160: 2730-2734. [Abstract/Free Full Text]
44. Bill, J., O. Kanagawa, J. Linten, Y. Utsunomiya, E. Palmer. 1990. Class I and class II MHC gene products differentially affect the fate of V5 bearing thymocytes. J. Mol. Cell. Immunol. 4: 269-279. [Medline]
45. Liao, N. S., J. Maltzman, D. H. Raulet. 1990. Expression of the V5.1 gene by murine peripheral T cells is controlled by MHC genes and skewed to the CD8⁺ subset. J. Immunol. 144: 844-848. [Abstract]
46. Blish, C. A., B. J. Gallay, G. L. Turk, K. M. Kline, W. Wheat, P. J. Fink. 1999. Chronic modulation of the TCR repertoire in the lymphoid periphery. J. Immunol. 162: 3131-3140. [Abstract/Free Full Text]
47. Kasow, K. A., X. Chen, J. Knowles, D. Wichlan, R. Handgretinger, J. M. Riberdy. 2004. Human CD4⁺CD25⁺ regulatory T cells share equally complex and comparable repertoires with CD4⁺CD25^– counterparts. J. Immunol. 172: 6123-6128. [Abstract/Free Full Text]
48. Kisielow, P., A. Miazek. 1995. Positive selection of T cells: rescue from programmed cell death and differentiation require continual engagement of the T cell receptor. J. Exp. Med. 181: 1975-1984. [Abstract/Free Full Text]
49. Derbinski, J., A. Schulte, B. Kyewski, L. Klein. 2001. Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self. Nat. Immunol. 2: 1032-1039. [Medline]

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