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Keywords:

  • Development;
  • Immunomodulation;
  • Regulatory T lymphocyte;
  • Thymus

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Among the several mechanisms known to be involved in the establishment and maintenance of immunological tolerance, the activity of CD4+CD25+ regulatory T lymphocytes has recently incited most interest because of its critical role in inhibition of autoimmunity and anti-tumor immunity. Surprisingly, very little is known about potential genetic modulation of intrathymic regulatory T lymphocyte development. We show that distinct proportions of CD4+CD25+FoxP3+ regulatory T cells are found in thymi of common laboratory mouse strains. We demonstrate that distinct levels of phenotypically identical regulatory T cells develop with similar kinetics in the mice studied. Our experimental data on congenic mouse strains indicate that differences are not caused by the distinct MHC haplotypes of the inbred mouse strains. Moreover, the responsible loci act in a thymocyte-intrinsic manner, confirming the latter conclusion. We have not found any correlation between thymic and peripheral levels of regulatory T cells, consistent with known homeostatic expansion and/or retraction of the peripheral regulatory T cell pool. Our data indicate that polymorphic genes modulate differentiation of regulatory T cells. Identification of responsible genes may reveal novel clinical targets and still elusive regulatory T cell-specific markers. Importantly, these genes may also modulate susceptibility to autoimmune disease.

Abbreviations:
B6:

C57BL/6

B10:

C57BL/10 

BrdU:

bromodeoxyuridine 

CD4SP:

CD4+CD8

GITR:

glucocortocoid-induced TNF-like receptor

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Immune tolerance is established by a variety of mechanisms acting in primary lymphoid organs during lymphopoiesis and in so-called “peripheral” lymphoid organs during the activation or differentiation phase of mature lymphocytes 1. Among the several mechanisms known to be involved, dominant tolerance (i.e. mediated by regulatory or suppressor lymphocytes) has incited great interest because of its potential in treatment of diseases as varied as autoimmune disorders and cancer, as well as in transplantation 24. The best studied regulatory T cell subset is of CD4+CD25+ phenotype. These cells were discovered because of their crucial role in the inhibition of multi-organ autoimmune disorder induced by thymectomy of mice at day 3 of life 5. Later, these cells were shown to inhibit inflammatory bowel disease, to fine-regulate immunity to pathogens, to inhibit anti-tumor immunity, and to protect the fetus from maternal immune aggression 69. Because of their crucial role in vivo, CD4+CD25+ regulatory T lymphocytes are very good candidates as therapeutic agents for the regulation of transplantation tolerance and inhibition of autoimmunity. It has recently been shown that these cells can inhibit graft-vs.-host disease 1014, rejection of transplanted tissue 2, 15, and autoimmune disease in experimental settings 1618.

Probably the majority of (but not all) CD4+CD25+ regulatory T lymphocytes develop in the thymus 1923. In this organ, CD25+ regulatory and CD25 effector T lymphocytes appear to have common CD4CD824 and CD4+CD8+ (our unpublished data) precursors. Similar to effector T cells, regulatory T cells are positively selected via interaction with thymic cortical epithelial cells 25. Expression of high-affinity ligands by thymic epithelial cells has been reported to favor development of regulatory T cells 19, 26, 27. A recent report suggests that this may be due to deletion of CD4+CD25 but not CD4+CD25+ precursors upon recognition of their cognate ligand expressed by thymic epithelial cells 28. Interestingly, interaction with high-affinity/avidity ligands expressed by thymic antigen-presenting cells of bone marrow origin can lead to deletion of regulatory T cell precursors 29, 30. Consistent with these observations, we and others have previously shown that the peripheral repertoire of regulatory T lymphocytes is enriched in auto-specific cells 29, 31, 32.

Surprisingly, despite the generally appreciated crucial importance of dominant tolerance, little is known about genetic control of regulatory T cell development and function. Such potential genetic variations might modulate susceptibility to a large panel of pathologies. Moreover, they would help in providing information concerning fundamental issues such as lineage choice and selection of regulatory T cell precursors in the thymus and functioning of these cells in the periphery.

Only one very rare genetic polymorphism is known to modulate differentiation of regulatory T lymphocytes. The forkhead/winged-helix transcription factor FoxP3 is preferentially (but not exclusively) expressed by regulatory T lymphocytes 3337. Transfection of effector T cells with constructs encoding this transcription factor causes these cells to exert potent suppressor-effector functions 3336. Mice carrying a natural mutation in the gene encoding FoxP3 (“scurfy”) lack regulatory T lymphocytes and die after a few weeks of life 33, 34. In humans, a natural mutation in FoxP3 causes the rare lethal autoimmune disorder IPEX 38, 39. To our knowledge this is the only genetic polymorphism known to modulate regulatory T lymphocyte development.

We here present data indicating the existence of genetic polymorphisms causing quantitative differences in regulatory T lymphocyte development in common laboratory mouse strains. We show that genes outside the MHC and acting in a thymocyte-intrinsic manner modulate intrathymic differentiation of regulatory T lymphocytes. Ultimate identification of the responsible loci should prove important for the analysis of thymic regulatory T cell lineage choice and selection, may enable identification of still elusive regulatory T cell-specific markers, and may yield more insight in mechanisms modulating susceptibility to autoimmune disease.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Distinct proportions of CD25+ regulatory T cells in thymus of different inbred mouse strains

We analyzed the proportion of CD25+ regulatory cells among CD4+CD8 (CD4SP) TCRhigh thymocytes and peripheral blood lymphocytes in the inbred mouse strains C57BL/6 (B6), C57BL/10 (B10), BALB/c, DBA/2, DBA/1, and SJL (Fig. 1A). Since regulatory CD4SP T cells express high levels of CD25 while cells expressing intermediate levels of CD25 proliferate and produce IL-2 40, 41, we only considered thymocytes of CD25high phenotype.

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Figure 1. Distinct proportions of CD25+ regulatory thymocytes in various inbred mouse strains. (A) Freshly isolated thymocytes were analyzed by four-color flow cytometry for expression of CD4, CD8, CD25 and TCR. The histograms depict TCR and CD25 levels of cells electronically gated on CD4 and CD8 as indicated. (B) The percentage of CD25+ among CD4SP TCRhi thymocytes in several inbred strains of mice (n⩾10 per strain) was calculated using gates indicated in (A). Mean values (± SD) are shown. Statistical significance between B6 and other strains is indicated (***p<0.001; NS, not significant; Student's t-test). (C) Ratios of CD25+TCRhi CD4SP (Treg) to CD4+CD8+ (DP) (upper panel) and CD25TCRhi CD4SP (Teff) to CD4+CD8+ (DP) cells (lower panel) were determined for B6, SJL, and DBA/2 mice. Depicted are mean values ± SD (n⩾4; **p<0.01, ***p<0.001; Student's t-test). (D) Thymocytes from DBA/2, SJL, and B6 mice were labeled with Ab specific for CD4, CD8, CD25, and either TCR, HSA, CD69, GITR, or FoxP3. CD4SP cells were electronically gated and analyzed for expression of indicated surface markers. (E) Percentage of FoxP3-expressing thymocytes among CD4SP thymocytes in the indicated mouse strains. Mean values (± SD) are shown (n=4; ***p<0.001, *p<0.05; Student's t-test). (F) PBMC and thymocytes from different inbred strains were stained with anti-TCR, anti-CD4, anti-CD8, and anti-CD25 Ab and analyzed by flow cytometry. The percentage of CD25+ cells among CD4SP TCRhi cells was calculated. Mean values (± SD) are shown.

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In the thymus, statistically significant different percentages of CD25high cells were observed between B6 and B10 mice on one hand, and DBA/2, BALB/c, DBA/1, and SJL strains on the other (Fig. 1B). These differences reached, in the strains analyzed, up to 1.7-fold (DBA/1 vs. B6). The quantitative differences might be caused by distinct CD25 effector (rather than CD25+ regulatory) T cell percentages. To evaluate this possibility, we analyzed the ratio of mature CD4SP TCRhighCD25high regulatory T cells to their CD4+CD8+ precursors in B6, DBA/2, and SJL mice. This ratio was significantly higher in DBA/2 and SJL mice than in B6 animals (Fig. 1C, top). On the other hand, the ratio of CD4SP CD25 to CD4+CD8+ thymocytes was similar in all three mouse-strains (Fig. 1C, bottom). These data indicate that the increased proportions of CD25+ regulatory T cells among CD4SP TCRhigh cells correspond to increased production from immature precursors.

To evaluate whether the CD4SP CD25high thymocytes found in the different mouse strains belong to the same regulatory T cell population, we assessed their surface phenotype (Fig. 1D). All CD4SP CD25high thymocytes were TCRhigh in all mouse strains studied. Interestingly, CD25high cells expressed relatively low heat-stable antigen (HSA, CD24) and CD69 levels, clearly distinguishing them from their CD25 and CD25intermediate counterparts. Moreover, in all mouse strains all CD25high cells expressed very high levels of the glucocortocoid-induced TNF-like receptor (GITR), characteristic for regulatory T cells. Most importantly, all CD4SP CD25high thymocytes expressed FoxP3. These data indicate that the CD25high cells found in the different mouse strains all belong to the same regulatory T lymphocyte lineage.

It has previously been shown that regulatory CD4+CD25+ T lymphocytes can develop in the periphery from CD25 precursors. Recent data suggest that CD25 precursors for CD25+ regulatory cells express FoxP3 42. Moreover, CD4+CD25FoxP3+ T cells inhibit T cell activation in vitro41. We therefore analyzed the percentage of FoxP3+ cells among CD4SP thymocytes by flow cytometry. As shown in Fig. 1E, substantially higher percentages of FoxP3-expressing cells were observed in DBA/2 and SJL mice than in B6 animals, confirming and extending our data on CD25high thymocytes.

We also analyzed levels of regulatory T cells in the periphery. As shown in Fig. 1F, we failed to observe a direct correlation of CD4+CD25+ percentages in thymus vs. PBMC. Similar data were obtained for secondary lymphoid organs (not shown).

Distinct proportions of thymic regulatory T cells are caused by differences in their differentiation

Distinct proportions of regulatory CD25+ cells among CD4SP thymocytes may be due to differences in their development or in thymic retention of mature thymocytes. To study the former possibility, we analyzed the kinetics of regulatory T cell development by measuring the appearance of bromodeoxyuridine (BrdU)+ cells in mice continuously fed with this nucleotide analog in their drinking water. As shown in Fig. 2, more CD4+CD25+ regulatory T lymphocytes differentiated from their dividing precursors in SJL mice than in B6 thymi. This result establishes that significant differences in thymic differentiation of these cells exist between these two mouse strains. However, it formally does not exclude the possibility that differences in thymic retention of regulatory T cells may also exist.

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Figure 2. Quantitative difference in differentiation of mature CD25+ regulatory thymocytes in B6 vs. SJL mice. BrdU was continuously administrated to mice in their drinking water. At indicated time points, thymocytes were analyzed by four-color flow cytometry using anti-CD4, anti-CD8, anti-CD25, and anti-BrdU Ab. The percentage of BrdU+CD25+ among CD4SP cells was calculated. Each point represents one mouse. Statistical significance of the difference between the two mouse strains was calculated for each day using Student's t-test (**p<0.01, ***p<0.001).

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Distinct regulatory T cell proportions are not caused by differences in MHC haplotype

We next studied whether the MHC haplotypes of the distinct mouse strains analyzed were responsible for the quantitative differences in thymic regulatory T cell generation. SJL mice have significantly higher percentages of CD25+ CD4SP thymocytes than B10 mice (Fig. 3A). Congenic B10.S mice (which carry the H-2s locus from SJL mice on a B10 genetic background) have a similar proportion of thymic regulatory T cells as B10 mice. Similarly, B10.D2 mice (carrying the DBA/2-derived H-2d locus) have CD4+CD25+ percentages similar to those in B10 mice (Fig. 3B). Therefore, the distinct MHC class I and II haplotypes of these mouse strains are not responsible for the different proportions of thymic regulatory T cells, and the genetic loci involved are not linked to the MHC.

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Figure 3. Genes modulating CD25+ regulatory T cell differentiation are located outside the MHC. (A and B) Thymocytes from indicated mouse strains were stained with anti-CD4, anti-CD8, anti-CD25 and anti-TCR Ab, and analyzed as described in the legend to Fig. 1. Mean values (± SD) are shown (n⩾5). Statistical significance was calculated using Student's t-test (**p<0.01, ***p<0.001).

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Thymocyte-intrinsic factors determine the distinct proportions of regulatory T cells

Differences in the development of regulatory T cells may be due to thymocyte-intrinsic factors or to variations in the thymic microenvironment. To distinguish between these two possibilities we generated mixed bone marrow chimeras in which thymocytes derived from the different donor mouse strains differentiate simultaneously in the same thymic microenvironment. (B6 × DBA/2)F1 (B6D2F1) hosts were lethally irradiated and reconstituted with a 1:1 mixture of B6 and DBA/2 bone marrow cells (B6 + DBA/2 <$>\rightarrow<$> B6D2F1 chimeras). Six weeks later the thymi of these chimeras were analyzed by flow cytometry.

As shown in Fig. 4A, C, among B6-derived cells from these mixed chimeras the same (lower) proportion of thymic regulatory T cells was found as in the parent strain. Among DBA/2-derived thymocytes the (higher) percentage of CD25+ cells in the CD4SP population was similar to that found in the DBA/2 parent strain. In B6 + SJL <$>\rightarrow<$> B6SJLF1 mixed bone marrow chimeras we observed a proportion of B6-derived regulatory T cells similar to that observed in the parent strain (Fig. 4B, C). Interestingly, among SJL-derived thymocytes significantly more regulatory T cells were observed than among B6-derived cells but also than in the SJL parent strain (compare Fig. 4C and 1B). The exceptionally high percentage of SJL-derived regulatory thymocytes was also observed in SJL <$>\rightarrow<$> F1 chimeras (not shown). While we currently do not have a satisfactory explanation for the high levels of SJL regulatory T cells in bone marrow chimeras, this result suggests that thymocyte-extrinsic (i.e. environmental) factors can also modulate CD4+CD25+ regulatory T cell development. Whatever the precise explanation is, these results indicate that the different levels of regulatory T cells in the distinct mouse strains studied are caused by thymocyte-intrinsic factors.

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Figure 4. Distinct proportions of CD25+ regulatory T cells are determined by a thymocyte-intrinsic mechanism. (A) Lethally irradiated B6D2F1 hosts were reconstituted with bone marrow cells from B6 (CD5.2) and DBA/2 (CD5.1) mice at a 1:1 ratio. Six weeks later, thymocytes were analyzed by four-color flow cytometry for expression of CD4, CD8, CD25, and CD5.1, using indicated electronic gates. (B) Similar experiments were performed using B6SJLF1 recipients injected with B6 (CD45.2) and SJL (CD45.1) bone marrow cells. (C) Quantitative analysis of CD25+ thymocyte development in mixed chimeras. Bar graphs depict mean values (± SD), n⩾5. Statistical significance was calculated using Student's t-test (**p<0.01, ***p<0.001).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

The data presented in this paper demonstrate that polymorphic genetic factors quantitatively control intrathymic generation of CD4+CD25+FoxP3+ regulatory T lymphocytes. In the mouse strains studied the distinct regulatory T cell levels are caused by differences in their thymic differentiation from immature precursors. Moreover, we show that thymocyte-intrinsic factors modulate regulatory T cell development. Finally, we report that the genes responsible for modulation of regulatory T cell development in the mouse strains studied are located outside the MHC locus.

Several hypotheses may explain our observation that thymocyte-instrinsic genetic factors cause quantitative differences in regulatory T cell differentiation. They may be caused by quantitative differences in commitment to the regulatory T cell lineage. The gene encoding FoxP3, located on the X chromosome (http://www.ensembl.org/Mus_musculus/geneview?gene=ENSMUSG00000039521), would therefore be among the candidate genes (see Introduction). Another candidate gene would be Notch3. Transgenic expression of a constitutively active form of Notch3 also leads to strongly increased thymic generation of regulatory T cells 43. However, Notch3 is closely linked to the MHC locus (http://www.ensembl.org/Mus_musculus/geneview?gene=ENSMUSG00000038146) and is therefore unlikely to be involved in the differences in regulatory T cell development in the inbred mouse strains reported here.

Alternatively, differences in regulatory T cell positive and/or negative selection may be responsible. While initial reports suggested that thymic CD4 vs. CD8 lineage commitment is independent of TCR specificity, more recently it has become clear that selection mechanisms are responsible. The processes of lineage commitment and selection therefore actually seem to be very closely linked 44. In a still unresolved manner, TCR-mediated signals appear to control expression of Th-POK, a zinc finger transcription factor, as well as of the chromatin remodeling protein Runx, recently identified as binary switches regulating CD4 vs. CD8 lineage commitment, respectively 4547.

Also the distinct proportions of regulatory cells among mature CD4+ thymocytes may be a consequence of differences in thymic selection and/or lineage commitment. Since thymocyte-intrinsic factors determine quantitative variations in regulatory T cell development, adhesion or signaling molecules may be involved. These molecules would probably also play important roles in function of peripheral regulatory T cells. Thus, one of the many candidate regions is the diabetes susceptibility locus Idd5. Within this locus three genes are located that encode proteins expressed by regulatory T cells: CD28, CTL-associated antigen (CTLA)-4, and inducible costimulator protein (ICOS). CD28 is known to play a crucial role in regulatory T cell development and homeostasis 4850. Very closely linked is the gene encoding CTLA-4, which is critically involved in regulatory T cell function 51. Moreover, Ctla-4 is a diabetes susceptibility gene in humans 52, and has been reported to modulate thymic negative selection of effector T cells in mice 53. A third gene within the Idd5 locus, ICOS, is also known to play an important role in regulatory T cell function 18, 54. However, none of these genes has thus far been shown to modulate regulatory T cell development.

Identification of the responsible gene(s) may also reveal entirely novel factors critically involved in regulatory T cell development and function, and thus enable better understanding of these processes. Such factors may also constitute unique markers for regulatory T cells, which have thus far proven elusive, and become clinical targets. Whatever the precise explanation for genetic modulation of regulatory T cell development is, it may have important consequences for regulatory T cell repertoire and/or function and thus modulate susceptibility, e.g. to autoimmune diseases. It would therefore be important to assess regulatory T cell differentiation in autoimmune-prone animals, and to identify responsible genetic loci.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Mice

B6, SJL, DBA/2, DBA/1, BALB/c, (B6 × DBA/2)F1 (B6D2F1), and (B6 × SJL)F1 (B6SJLF1) females of 5–7 wk of age were purchased from Janvier (Le Genest St Isle, France). B10 mice were purchased from Charles River (Les Oncins, France). B10.D2, B10.S, and B10.D1 (B10.Q) mice were bred in our facilities. All experiments involving animals were performed in compliance with the relevant laws and institutional guidelines (INSERM; approval No. 31-13, ethical review No. MP/02/32/10/03).

Antibodies

The following Ab and secondary reagents were used for phenotypic analysis: PE-Cy7- or allophycocyanin-labeled anti-CD4 (GK1.5), FITC- or allophycocyanin-labeled anti-CD8 (53.6.7), allophycocyanin- or PE-labeled anti-CD25 (PC61), FITC-labeled anti-HSA (M1/69), FITC-labeled anti-CD69 (H1.2F3), FITC-labeled anti-TCRβ, FITC-labeled anti-CD45.1, PE-labeled anti-FoxP3, FITC-labeled anti-CD5.1, PE-Cy5.5-labeled streptavidine (eBioscience, San Diego, CA). Biotin-labeled anti-GITR was purchased from R&D (Lille, France).

Bone marrow chimeras

Bone marrow from femurs and tibias was collected in Dulbecco's modified Eagle's medium supplemented with 10% FCS. Thy1+ cells were eliminated using AT83 hybridoma supernatant and rabbit complement (Saxon Europe, Suffolk, UK). Cells from each donor were injected intravenously into lethally γ-irradiated hosts (8.5 Gy; 137Cs source, 6.3 Gy/min) that were kept on antibiotic-containing water (0.2% of Bactrim; Roche) for the complete duration of the experiment (6 wk).

Flow cytometry

Thymocytes or PBMC were incubated 30 min on ice in 2.4G2 (anti-FcγR mAb) hybridoma supernatant. Cells were then incubated 20 min with saturating concentrations of Ab. Intracellular FoxP3 staining on CD8-depleted thymocytes (using anti-CD8 mAb 31 M and complement) was performed according to the instructions of the manufacturer. Labeled cells were analyzed using a FACSCalibur cytometer and CellQuest software (BD Biosciences, San Diego, CA).

BrdU incorporation studies

Mice were continuously exposed to the thymidine analogue BrdU (0.8 mg/mL) in their drinking water. Extracellular staining of thymocytes with mAb against CD4, CD8, and CD25 was performed as described above. Cells were fixed, permeabilized, and stained with FITC-labeled anti-BrdU using the BrdU Flow Kit (BD Pharmingen, Heidelberg, Germany).

Statistical analysis

Statistical significance of the data was analyzed using Student's t-test.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

We thank the staff of the IFR30 animal facility, and in particular Maryline Calise, for expert animal husbandry, and Drs. Gilbert Fournié and Jean-Charles Guéry for critical reading of the manuscript.

  • 1

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  • 2

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  • 3

    WILEY-VCH

  • 4

    WILEY-VCH

  • 1
    Stockinger, B., T lymphocyte tolerance: From thymic deletion to peripheral control mechanisms. Adv. Immunol. 1999. 71: 229265.
  • 2
    Waldmann, H., Graca, L., Cobbold, S., Adams, E., Tone, M. and Tone, Y., Regulatory T cells and organ transplantation. Semin. Immunol. 2004. 16: 119126.
  • 3
    Sakaguchi, S., Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 2004. 22: 531562.
  • 4
    Piccirillo, C. A. and Shevach, E. M., Naturally-occurring CD4+CD25+ immunoregulatory T cells: Central players in the arena of peripheral tolerance. Semin. Immunol. 2004. 16: 8188.
  • 5
    Asano, M., Toda, M., Sakaguchi, N. and Sakaguchi, S., Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 1996. 184: 387396.
  • 6
    Singh, B., Read, S., Asseman, C., Malmstrom, V., Mottet, C., Stephens, L. A., Stepankova, R. et al., Control of intestinal inflammation by regulatory T cells. Immunol. Rev. 2001. 182: 190200.
  • 7
    Mills, K. H., Regulatory T cells: Friend or foe in immunity to infection? Nat. Rev. Immunol. 2004. 4: 841855.
  • 8
    Sakaguchi, S., Sakaguchi, N., Shimizu, J., Yamazaki, S., Sakihama, T., Itoh, M., Kuniyasu, Y. et al., Immunological tolerance maintained by CD4+CD25+ regulatory T cells: Their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol. Rev. 2001. 182: 1832.
  • 9
    Aluvihare, V. R., Kallikourdis, M. and Betz, A. G., Regulatory T cells mediate maternal tolerance to the fetus. Nat. Immunol. 2004. 5: 266271.
  • 10
    Trenado, A., Charlotte, F., Fisson, S., Yagello, M., Klatzmann, D., Salomon, B. L. and Cohen, J. L., Recipient-type specific CD4+CD25+ regulatory T cells favor immune reconstitution and control graft-versus-host disease while maintaining graft-versus-leukemia. J. Clin. Invest. 2003. 112: 16881696.
  • 11
    Edinger, M., Hoffmann, P., Ermann, J., Drago, K., Fathman, C. G., Strober, S. and Negrin, R. S., CD4(+)CD25(+) regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation. Nat. Med. 2003. 9: 11441150.
  • 12
    Taylor, P. A., Lees, C. J. and Blazar, B. R., The infusion of ex vivo activated and expanded CD4(+)CD25(+) immune regulatory cells inhibits graft-versus-host disease lethality. Blood 2002. 99: 34933499.
  • 13
    Hoffmann, P., Ermann, J., Edinger, M., Fathman, C. G. and Strober, S., Donor-type CD4(+)CD25(+) regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation. J. Exp. Med. 2002. 196: 389399.
  • 14
    Cohen, J. L., Trenado, A., Vasey, D., Klatzmann, D. and Salomon, B. L., CD4(+)CD25(+) immunoregulatory T cells: New therapeutics for graft-versus-host disease. J. Exp. Med. 2002. 196: 401406.
  • 15
    Joffre, O., Gorsse, N., Romagnoli, P., Hudrisier, D. and van Meerwijk, J. P., Induction of antigen-specific tolerance to bone marrow allografts with CD4+CD25+ T lymphocytes. Blood 2004. 103: 42164221.
  • 16
    Tang, Q., Henriksen, K. J., Bi, M., Finger, E. B., Szot, G., Ye, J., Masteller, E. L. et al., In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J. Exp. Med. 2004. 199: 14551465.
  • 17
    Tarbell, K. V., Yamazaki, S., Olson, K., Toy, P. and Steinman, R. M., CD25+ CD4+ T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes. J. Exp. Med. 2004. 199: 14671477.
  • 18
    Herman, A. E., Freeman, G. J., Mathis, D. and Benoist, C., CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion. J. Exp. Med. 2004. 199: 14791489.
  • 19
    Apostolou, I., Sarukhan, A., Klein, L. and von Boehmer, H., Origin of regulatory T cells with known specificity for antigen. Nat. Immunol. 2002. 3: 756763.
  • 20
    Chen, W., Jin, W., Hardegen, N., Lei, K.-J., Li, L., Marinos, N., McGrady, G. and Wahl, S. M., Conversion of peripheral CD4+CD25 naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor FoxP3. J. Exp. Med. 2003. 198: 18751886.
  • 21
    Walker, M. R., Kasprowicz, D. J., Gersuk, V. H., Benard, A., Van Landeghen, M., Buckner, J. H. and Ziegler, S. F., Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25 T cells. J. Clin. Invest. 2003. 112: 14371443.
  • 22
    Liang, S., Alard, P., Zhao, Y., Parnell, S., Clark, S. L. and Kosiewicz, M. M., Conversion of CD4+ CD25 cells into CD4+ CD25+ regulatory T cells in vivo requires B7 costimulation, but not the thymus. J. Exp. Med. 2005. 201: 127137.
  • 23
    Apostolou, I. and von Boehmer, H., In vivo instruction of suppressor commitment in naive T cells. J. Exp. Med. 2004. 199: 14011408.
  • 24
    Itoh, M., Takahashi, T., Sakaguchi, N., Kuniyasu, Y., Shimizu, J., Otsuka, F. and Sakaguchi, S., Thymus and autoimmunity: Production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J. Immunol. 1999. 162: 53175326.
  • 25
    Bensinger, S. J., Bandeira, A., Jordan, M. S., Caton, A. J. and Laufer, T. M., Major histocompatibility complex class II-positive cortical epithelium mediates the selection of CD4+CD25+ immunoregulatory T cells. J. Exp. Med. 2001. 194: 427438.
  • 26
    Jordan, M. S., Boesteanu, A., Reed, A. J., Petrone, A. L., Holenbeck, A. E., Lerman, M. A., Naji, A. and Caton, A. J., Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2001. 2: 301306.
  • 27
    Kawahata, K., Misaki, Y., Yamauchi, M., Tsunekawa, S., Setoguchi, K., Miyazaki, J.-I. and Yamamoto, K., 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. 2002. 168: 43994405.
  • 28
    van Santen, H.-M., Benoist, C. and Mathis, D., Number of T reg cells that differentiate does not increase upon encounter of agonist ligand on thymic epithelial cells. J. Exp. Med. 2004. 200: 12211230.
  • 29
    Romagnoli, P., Hudrisier, D. and van Meerwijk, J. P. M., Preferential recognition of self-antigens despite normal thymic deletion of CD4+CD25+ regulatory T cells. J. Immunol. 2002. 168: 16441648.
  • 30
    Pacholczyk, R., Kraj, P. and Ignatowicz, L., Peptide specificity of thymic selection of CD4+CD25+ T cells. J. Immunol. 2002. 168: 613620.
  • 31
    Fisson, S., Darrasse-Jeze, G., Litvinova, E., Septier, F., Klatzmann, D., Liblau, R. and Salomon, B. L., Continuous activation of autoreactive CD4+ CD25+ regulatory T cells in the steady state. J. Exp. Med. 2003. 198: 737746.
  • 32
    Hsieh, C. S., Liang, Y., Tyznik, A. J., Self, S. G., Liggitt, D. and Rudensky, A. Y., Recognition of the peripheral self by naturally arising CD25+ CD4+ T cell receptors. Immunity 2004. 21: 267277.
  • 33
    Khattri, R., Cox, T., Yasayko, S. A. and Ramsdell, F., An essential role for scurfin in CD4(+)CD25(+) T regulatory cells. Nat. Immunol. 2003. 3: 337342.
  • 34
    Fontenot, J. D., Gavin, M. A. and Rudensky, A. Y., FoxP3 programs the development and function of CD4(+)CD25(+) regulatory T cells. Nat. Immunol. 2003. 3: 330336.
  • 35
    Hori, S., Nomura, T. and Sakaguchi, S., Control of regulatory T cell development by the transcription factor FoxP3. Science 2003. 299: 10571061.
  • 36
    Yagi, H., Nomura, T., Nakamura, K., Yamazaki, S., Kitawaki, T., Hori, S., Maeda, M. et al., Crucial role of FoxP3 in the development and function of human CD25+CD4+ regulatory T cells. Int. Immunol. 2004. 16: 16431656.
  • 37
    Roncador, G., Brown, P. J., Maestre, L., Hue, S., Martinez-Torrecuadrada, J. L., Ling, K. L. et al., Analysis of FoxP3 protein expression in human CD4(+)CD25(+) regulatory T cells at the single-cell level. Eur. J. Immunol. 2005. 35: 16811691.
  • 38
    Wildin, R. S., Ramsdell, F., Peake, J., Faravelli, F., Casanova, J. L., Buist, N., Levy-Lahad, E. et al., X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat. Genet. 2001. 27: 1820.
  • 39
    Bennett, C. L., Christie, J., Ramsdell, F., Brunkow, M. E., Ferguson, P. J., Whitesell, L., Kelly, T. E. et al., The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FoxP3. Nat. Genet. 2001. 27: 2021.
  • 40
    Setoguchi, R., Hori, S., Takahashi, T. and Sakaguchi, S., 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. 2005. 201: 723735.
  • 41
    Fontenot, J. D., Rasmussen, J. P., Williams, L. M., Dooley, J. L., Farr, A. G. and Rudensky, A. Y., Regulatory T cell lineage specification by the forkhead transcription factor FoxP3. Immunity 2005. 22: 329341.
  • 42
    Zelenay, S., Lopes-Carvalho, T., Caramalho, I., Moraes-Fontes, M. F., Rebelo, M. and Demengeot, J., FoxP3+ CD25 CD4 T cells constitute a reservoir of committed regulatory cells that regain CD25 expression upon homeostatic expansion. Proc. Natl. Acad. Sci. USA 2005. 102: 40914096.
  • 43
    Anastasi, E., Campese, A. F., Bellavia, D., Bulotta, A., Balestri, A., Pascucci, M., Checquolo, S. et al., Expression of activated Notch3 in transgenic mice enhances generation of T regulatory cells and protects against experimental autoimmune diabetes. J. Immunol. 2003. 171: 45044511.
  • 44
    Singer, A. and Bosselut, R., CD4/CD8 coreceptors in thymocyte development, selection, and lineage commitment: Analysis of the CD4/CD8 lineage decision. Adv. Immunol. 2004. 83: 91131.
  • 45
    He, X., Dave, V. P., Zhang, Y., Hua, X., Nicolas, E., Xu, W., Roe, B. A. and Kappes, D. J., The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment. Nature 2005. 433: 826833.
  • 46
    Sato, T., Ohno, S., Hayashi, T., Sato, C., Kohu, K., Satake, M. and Habu, S., Dual functions of Runx proteins for reactivating CD8 and silencing CD4 at the commitment process into CD8 thymocytes. Immunity 2005. 22: 317328.
  • 47
    Sun, G., Liu, X., Mercado, P., Jenkinson, S. R., Kypriotou, M., Feigenbaum, L., Galera, P. and Bosselut, R., The zinc finger protein cKrox directs CD4 lineage differentiation during intrathymic T cell positive selection. Nat. Immunol. 2005. 6: 373381.
  • 48
    Salomon, B., Lenschow, D. J., Rhee, L., Ashourian, N., Singh, B., Sharpe, A. and Bluestone, J. A., B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 2000. 12: 431440.
  • 49
    Tang, Q., Henriksen, K. J., Boden, E. K., Tooley, A. J., Ye, J., Subudhi, S. K., Zheng, X. X. et al., Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+ regulatory T cells. J. Immunol. 2003. 171: 33483352.
  • 50
    Tai, X., Cowan, M., Feigenbaum, L. and Singer, A., CD28 costimulation of developing thymocytes induces FoxP3 expression and regulatory T cell differentiation independently of interleukin 2. Nat. Immunol. 2005. 6: 152162.
  • 51
    Takahashi, T., Tagami, T., Yamazaki, S., Uede, T., Shimizu, J., Sakaguchi, N., Mak, T. W. and Sakaguchi, S., Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 2000. 192: 303310.
  • 52
    Ueda, H., Howson, J. M., Esposito, L., Heward, J., Snook, H., Chamberlain, G., Rainbow, D. B. et al., Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 2003. 423: 506511.
  • 53
    Buhlmann, J. E., Elkin, S. K. and Sharpe, A. H., A role for the B7-1/B7-2:CD28/CTLA-4 pathway during negative selection. J. Immunol. 2003. 170: 54215428.
  • 54
    Greve, B., Vijayakrishnan, L., Kubal, A., Sobel, R. A., Peterson, L. B., Wicker, L. S. and Kuchroo, V. K., The diabetes susceptibility locus Idd5.1 on mouse chromosome 1 regulates ICOS expression and modulates murine experimental autoimmune encephalomyelitis. J. Immunol. 2004. 173: 157163.