Induction of antigen-specific immunologic tolerance by in vivo and in vitro antigen-specific expansion of naturally arising Foxp3+CD25+CD4+ regulatory T cells

Eiji Nishimura1,2, Toshiko Sakihama1,5, Ruka Setoguchi1, Koichi Tanaka2 and Shimon Sakaguchi1,3,4

1 Department of Experimental Pathology, Institute for Frontier Medical Sciences and 2 Department of Transplantation and Immunology, Faculty of Medicine, Kyoto University, Kyoto 606-8507, Japan
3 Laboratory of Immunopathology, Research Center for Allergy and Immunology, Institute for Physical and Chemical Research, Yokohama 230-0045, Japan
4 Core Research for Evolutional Science and Technology (CREST) Program, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan
5 Present address: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, the University of Tokyo, Tokyo 153-8904, Japan

Correspondence to: S. Sakaguchi; E-mail: shimon{at}frontier.kyoto-u.ac.jp


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Naturally arising CD25+CD4+ regulatory T (TR) cells can be exploited to establish immunologic tolerance to non-self antigens. In vivo exposure of CD25+CD4+ T cells from normal naive mice to alloantigen in a T cell-deficient environment elicited spontaneous expansion of alloantigen-specific CD25+CD4+ TR cells, which suppressed allograft rejection mediated by subsequently transferred naive T cells, leading to long-term graft tolerance. The expanded TR cells, which became CD25low in the absence of other T cells, stably sustained suppressive activity, maintained expression levels of other TR cell-associated molecules, including Foxp3, CTLA-4 and GITR, and could adoptively transfer tolerance to normal mice. Furthermore, specific removal of the TR cells derived from originally transferred CD25+CD4+ TR cells evoked graft rejection in the long-term tolerant mice, indicating that any TR cells deriving from CD25CD4+ naive T cells minimally contribute to graft tolerance and that natural TR cells are unable to infectiously confer significant suppressive activity to other T cells. Similar antigen-specific expansion of TR cells can also be achieved in vitro by stimulating naturally present CD25+CD4+ T cells with alloantigen in the presence of IL-2. The expanded CD25+CD4+ T cells potently suppressed even secondary MLR in vitro and, by in vivo transfer, established antigen-specific long-term graft tolerance. Thus, in vivo or in vitro, direct or indirect ways of antigen-specific expansion of naturally arising Foxp3+CD25+CD4+ TR cells can establish antigen-specific dominant tolerance to non-self antigens, and would also be instrumental in re-establishing self-tolerance in autoimmune disease and antigen-specific negative control of pathological immune responses.

Keywords: CTLA-4, GITR, transplantation tolerance


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
It is to be hoped that immunologic tolerance to certain non-self antigens, such as alloantigens in organ transplants, can be established as stably as natural tolerance to self-constituents. Accumulating evidence indicates that naturally arising CD25+CD4+ regulatory T cells (TR cells) play a crucial role in the maintenance of immunologic self-tolerance and negative control of immune responses (13). Elimination of CD25+CD4+ TR cells, for example, leads to spontaneous development of various autoimmune diseases in normal animals and provokes effective tumor immunity in otherwise non-responding animals (48). The elimination also enhances immune responses to non-self antigens including allogeneic transplantation antigens (4). In this report we have addressed the issue of how natural CD25+CD4+ TR cells can be expanded in vivo and in vitro in an antigen-specific manner and what conditions are required to establish long-term tolerance to non-self-antigens with such antigen-specific TR cells.

Natural CD25+CD4+ TR cells are at least in part produced by the normal thymus as a functionally mature T-cell subpopulation (9). They are functionally unique in that they proliferate poorly in response to antigenic stimulation in vitro unless IL-2 is provided and in that, upon in vitro stimulation with specific antigens, they potently suppress the activation/proliferation of other T cells in an antigen-nonspecific manner, seemingly through cell to cell interactions on antigen-presenting cells (APC) (912). Phenotypically, they constitutively express CTLA-4, certain members of Toll-like receptors, CD103 ({alpha}E integrin) and GITR (glucocorticoid-induced TNF receptor family-related gene) at high levels (1319). They specifically express the transcription factor Foxp3, which appears to act as a master control gene for their development and function (2022). These findings collectively indicate that naturally arising CD25+CD4+ TR cells are a developmentally, phenotypically and functionally distinct subpopulation of T cells (1). Furthermore, recent studies have shown by utilizing CD25+CD4+ T cells from TCR transgenic mice that antigen-specific TR cells can expand in vivo upon antigen stimulation along with potent adjuvant or mature DCs (2325). Natural CD25+CD4+ TR cells also show homeostatic proliferation in a T cell-deficient environment (26,27), and a fraction of them are proliferating in normal animals presumably by recognizing self-antigens (28,29). It remains to be determined, however, how this in vivo proliferative capacity of natural TR cells can be exploited for inducing antigen-specific tolerance to non-self-antigens and negative control of immune responses. It is also unclear whether natural TR cells should play key roles in long-term immunologic tolerance induced to non-self antigens, such as allografts, by short-term administration of monoclonal antibodies or drugs that control the activation/proliferation of T cells, or whether antigen-specific TR cells generated by these treatments should originate from natural TR cells or naive T cells (3035). Furthermore, it should be determined whether natural TR cells can confer a suppressive activity to naive T cells in an ‘infectious’ manner in immunologic tolerance (3638).

In this report, we demonstrate that alloantigen-specific TR cells present in the naturally arising CD25+CD4+ TR cell population can be expanded in vivo by sensitization to alloantigens without any adjuvant, in contrast to their in vitro hypoproliferation upon allogeneic stimulation. Notably, permanent graft tolerance can be achieved if this antigen-specific expansion of natural TR cells is allowed to the extent that they are sufficient in number and suppressive activity to control the expansion/activation of allo-reactive effector T cells. Alloantigen-specific CD25+CD4+ TR cells can also be expanded in vitro in the presence of IL-2 and used for inducing similar antigen-specific graft tolerance in vivo. We further assess the stability of the phenotype and function of these antigen-expanded TR cells in vivo, in particular whether they can confer regulatory activity to non-TR cells or whether other T cells can give rise to TR cells. This simple way of antigen-specific TR cell expansion, either by directly stimulating them in vitro with specific antigens or indirectly preparing an in vivo immunological condition that allows their spontaneous antigen-specific expansion, can also be used for re-establishing self-tolerance in autoimmune disease.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Mice
Seven-week-old female BALB/c, DBA/2, C57BL/6 (B6), C3H and BALB/c athymic nude mice were purchased from Clea Japan (Tokyo, Japan). BALB/c-Thy 1.1 congenic mice were established in our laboratory (5). All these mice were maintained in our animal facility and cared for in accordance with the institutional guidelines for animal welfare.

Antibodies and reagents
FITC-, PE-, CyChrome-labeled, or biotinylated mAbs to CD25 (7D4), CD4 (RM4-5 and H129.19), CD4 (H129.19), Vß6 (RR4-7) and Vß10 (B21.5) were purchased from PharMingen (San Diego, CA). FITC-labeled or biotinylated anti-Thy 1.1 mAbs were from Serotec (Oxford, UK). PE- or CyChrome-streptavidin, as secondary reagents, were also from PharMingen. Anti-GITR mAb, DTA-1 (16) was made from ascites in SCID mice and purified by 40% ammonium sulfate precipitation twice. Affinity-purified goat anti-rat IgG (specific for whole IgG molecule) and purified normal hamster IgG were purchased from ICN Pharmaceuticals (Aurora, OH). Normal rat IgG was purchased from Sigma (St Louis, MO). Murine recombinant IL-2 (rIL-2) (3.89 x 106 U/mg) was a gift from Shionogi Co. (Osaka, Japan).

Preparation of T cell subpopulations
To enrich CD4+ T cells, spleen and lymph node cells were treated with anti-CD8 (3.155) and anti-CD24 (J11d) mAbs and incubated on plastic dishes pre-coated with goat anti-rat IgG at 37°C for 30 min. Non-adherent cells (>85% of which were CD4+) were stained with biotin-anti-CD25 mAb, then with PE–streptavidin and FITC-anti-CD4 mAb; and CD25+ or CD25CD4+ T cells were purified by Epics–ALTRA cell sorter (Beckman Coulter, Miami, FL). Purity of the CD25+ or CD25-CD4+ population was >98% and ~99%, respectively. Cells were also stained with biotin-anti-CD25 mAb, then with PE–streptavidin, CyChrome-anti-CD4 and FITC-anti-Thy 1.1 mAb; and CD25hi, CD25int or CD25 CD4+ Thy 1.1+ T cells were purified by cell sorter. In some experiments, CD25+ cells were enriched by the MACS system (Magnetic Cell Sorting; Miltenyi Biotech). Briefly, enriched CD4+ T cells were stained with biotin-anti-CD25 mAb, PE–streptavidin and then incubated with anti-PE microbeads according to the manufacturer's instruction. Cells were positively selected on an LS separation column. CD25+CD4+ population was further prepared from the positive fraction by using an LS column again. The negative fraction was incubated with anti-CD4 microbeads and the CD25CD4+ population positively selected on an LS column. Purity of CD25+ or CD25CD4+ population was >93% and ~99%, respectively. To prepare unfractionated whole T cells, spleen and lymph node cells were depleted of B cells and adherent cells by treatment with J11d mAb and panning on anti-rat IgG-coated plastic dishes. Non-adherent cells were further treated with rabbit complement (C) (Cederlane Laboratories, Ontario, Canada). CD8+ T cells were enriched by incubation with J11d and anti-CD4 (GK1.5) mAbs, panning and C treatment as described above. In some experiments, lymphocytes were depleted of CD25+ cells by treatment with anti-CD25 (7D4) mAb and C, as previously described (4,5).

Skin grafting and cell transfer
Full thickness B6, C3H, DBA/2 or BALB/c tail skin (0.5 cm2) was grafted onto the backs of BALB/c nude mice. The grafted sites were wrapped for 7 days with gauze and bandages. After a further 7 days, the mice were reconstituted with 2 x 105 unfractionated T cells from naive BALB/c mice. Some mice received 2–6 x 105 BALB/c CD25+CD4+ T cells at the same time or 1 week before reconstitution with normal, untreated T cells from naive BALB/c mice. Grafts were inspected four times a week and considered rejected when no viable donor skin was present. Statistical analysis of graft survival was made by the log-rank method.

Mixed lymphocyte reaction (MLR)
BALB/c whole, CD4+, CD8+ or CD25CD4+ T cells (5 x 104/well) together with various numbers of CD25+CD4+ T cells (0–5 x 104/well) were cultured with RBC-lysed and X-irradiated (20 Gy) B6 or C3H splenocytes (1 x 105/well) as stimulators for 6 days in 96-well round-bottom plates (Corning Costar, Cambridge, MA) in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (PAA Laboratories, Newport Beach, CA), penicillin (100 U/ml), streptomycin (100 µg/ml) and 50 µM 2-ME. Cultures were pulsed with [3H]thymidine ([3H]TdR) (37 kBq/well) (Du Pont/NEN) for the last 16 h. To assess the secondary MLR, BALB/c CD25CD4+ T cells (5 x 104/well) were pre-cultured with irradiated B6 splenocytes for 7 days, washed, and then cultured with X-irradiated fresh B6 splenocytes for 5 days in the presence or absence of CD25+CD4+ T cells. To assess anti-Mls-1 responses, BALB/c T cells were cultured with mitomycin C (MMC)-treated DBA/2 splenocytes for 5 days.

In vitro stimulation of CD25+CD4+ T cells
BALB/c CD25+CD4+ T cells (2.5 x 104/well) were co-cultured with X-irradiated B6 or C3H splenocytes, or with MMC-treated DBA/2 splenocytes (1 x 105/well) for 7 days in the presence of 100 U/ml rIL-2. Cells were washed and added to the MLR or inoculated to the skin-grafted nude mice. For analysis of Vß6 or Vß10-expressing cells, freshly prepared BALB/c CD4+ T cells or CD25+CD4+ T cells were co-cultured with DBA/2 splenocytes plus IL-2 (100 U/ml) for 7 days, stained with biotin-anti-CD25 mAb and FITC-anti-Vß6 or Vß10 mAb, then with PE–streptavidin. The proportion of Vß6- or Vß10-expressing cells was analyzed by flow cytometry (Epics-XL, Beckman Coulter).

RT–PCR
Total cellular RNA was extracted from 1–5 x 105 sorted cells supplemented with 10 µg glycogen using Isogen reagent (Nippon Gene, Tokyo, Japan). The total amount of RNA was reverse transcribed using Superscript II reverse-transcriptase and oligo(dT)12–18 primer (Invitrogen Japan, Tokyo, Japan) in a final volume of 20 µl. Foxp3 mRNA levels were quantified by real-time PCR as described previously (20). Normalized value for Foxp3 mRNA expression in each sample was calculated as the relative quantity of Foxp3 divided by the relative quantity of HPRT (x100). All samples were run in triplicate.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
In vivo expansion of antigen-specific CD25+CD4+ TR cells upon sensitization to allografts
To determine whether natural CD25+CD4+ TR cells can expand upon allogeneic antigen stimulation without any adjuvant, we took advantage of the anti-Mls-1a (Mtv-7) response of BALB/c (Mls-1b) mice; i.e. expansion of BALB/c Vß6+ T cells is stimulated specifically by exposure to the Mls-1 antigen of DBA/2 mice (39). BALB/c-Thy1.1+CD25+CD4+ T cells were transferred to nude mice with DBA/2 or BALB/c skin grafts, and the percentage and the number of Thy1.1+CD4+ splenic T cells expressing Mls-1-reactive Vß6 or non-reactive Vß10 was assessed every week after transfer (Fig. 1A and B). Analysis 1 week after transfer showed that both the percentage and number of Vß6+ cells increased (~5-fold and ~20-fold, respectively) in the nude mice with DBA/2 grafts but not in those with BALB/c grafts, contrasting with no increase of the percentage or number of Vß10+ cells in both groups (Vß6+ or Vß10+ cells constituted ~10% and ~6%, respectively, of CD25+CD4+ T cells in normal naive BALB/c mice; see legend to Fig. 1 for detailed percentage and number). The time course study revealed that maximal Vß6+ cell expansion occurred 1 week after transfer and showed significant differences from the expansion in nude mice with syngeneic BALB/c grafts (P < 0.05) (Fig. 1B). Although it was difficult to obtain a sufficient number of Vß6+ T cells from these nude mice to assess their in vitro Mls-1-specific suppressive activity (see below), they exerted potent suppression upon anti-CD3 stimulation (data not shown).



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Fig. 1. In vivo alloantigen-specific expansion of CD25+CD4+ TR cells. (A) 2 x 105 BALB/c-Thy1.1 CD25+CD4+ T cells were transferred to BALB/c nude mice with DBA/2 or BALB/c skin grafts. Percentage (upper panel) and number (lower panel) of Vß6+ or Vß10+ cells among Thy1.1+CD4+ splenic T cells were analyzed 1 week after transfer. Before transfer, Vß6+ or Vß10+ cells constituted 10.2 ± 1.2% and 5.6 ± 0.5%, respectively, of CD25+CD4+ T cells (n = 4), indicating that inoculated 2 x 105 BALB/c-Thy1.1 CD25+CD4+ T cells contained 2.0 ± 0.2 x 104 Vß6+ cells and 1.1 ± 0.1 x 104 Vß10+ cells. The means with SDs of four independent experiments are shown. (B) Time course of the expansion of Vß6 or Vß10 cells in the spleen (n = 4 at each time point). The values on day 0 are the percentages and numbers of Vß6+ or Vß10+ splenic T cells before transfer (see above). Note different scales for Vß6 or Vß10 in (A) and (B). (C) 1 x 106 BALB/c-Thy1.1 CD25+CD4+ were transferred to BALB/c nude mice with B6 or C3H skin grafts; 1 week later graded numbers of Thy-1.1+ cells recovered from the nude mice were co-cultured at indicated ratios with CD25CD4+ T cells freshly prepared from normal BALB/c mice, and stimulated with B6 or C3H stimulator cells. A representative result of two independent experiments is shown.

 
To determine then whether CD25+CD4+ TR cells could generate an antigen-specific suppressive activity upon exposure to alloantigen, we transferred CD25+CD4+ T cells, prepared from BALB/c-Thy-1a congenic mice, to BALB/c nude mice with B6 or C3H skin grafts, and assessed 1 week later whether Thy-1.1+ cells recovered from the mice suppressed the in vitro MLR responses of BALB/c naive T cells to B6 or C3H stimulator cells (Fig. 1C). Although the total numbers of Thy1.1+ T cells recovered in each group of mice were comparable (data not shown; see also Fig. 6B), the Thy-1.1+ cells from either group of mice exhibited ~4-fold more potent MLR suppression when the stimulator cells were from the same strain as the graft donor, compared with the stimulator cells from the other strain.



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Fig. 6. Antigen-specific suppression of graft rejection by ex vivo antigen-stimulated CD25+CD4+ TR cells. (A) BALB/c nude mice engrafted with B6 skin grafts received 2 x 105 B6 or C3H-prestimulated BALB/c CD25+CD4+ T cells, 1 x 106 B6-prestimulated BALB/c CD25+CD4+ T cells, or PBS alone as control. Seven days later, all mice received 2 x 105 naive BALB/c whole T cells. The result is the total of five independent experiments. (B) BALB/c nude mice engrafted with C3H skin grafts received 2 x 105 B6 or C3H-prestimulated BALB/c CD25+CD4+ T cells, or PBS alone as control. Seven days later, all mice received 2 x 105 naive BALB/c whole T cells. The data shown are the total of six independent experiments. (C) BALB/c nude mice engrafted with B6 skin grafts received 2 x 105 BALB/c-Thy1.1 CD25+CD4+ T cells freshly prepared or prestimulated with B6 splenocytes and 100 U/ml rIL-2 for 1 week. Seven days later, spleen and lymph node cells were harvested and the number of Thy1.1+CD4+ cells was calculated from the total number of harvested cells and the percentage of Thy1.1+CD4+ cells among them. The result shown is the total of three independent experiments.

 
These results taken together indicate that antigen-specific TR cells in the CD25+CD4+ T cell population can substantially expand in vivo within a week upon sensitization to allo-antigens and that such stimulated TR cells show antigen-specific suppression.

Induction of allograft tolerance by naturally occurring CD25+CD4+ TR cells
We subsequently examined whether CD25+CD4+ TR cells can suppress in vivo allogeneic immune responses more effectively after expansion. We transferred 2 x 105 naive T cells to B6 skin-grafted BALB/c nude mice with an equal number of CD25+CD4+ T cells either simultaneously or 7 days after the transfer of the CD25+CD4+ population (Fig. 2A). Although the co-transfer of CD25+CD4+ T cells and naive T cells significantly prolonged graft survival compared with transfer of naive T cells alone (median survival time (MST): 42 days vs 31 days, n = 12 and 29, respectively; P = 0.002), inoculation of CD25+CD4+ T cells 7 days prior to naive T-cell transfer prolonged graft survival further (MST: 64 days, n = 13, P = 0.0002) with 23% of the mice showing long-term (>100 days) graft acceptance. Furthermore, when a 3-fold excess (6 x 105) of CD25+CD4+ T cells to naive T cells was transferred 7 days apart, 73% of the recipients showed long-term acceptance of the grafts (MST: >100 days, n = 15, P < 0.00001).



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Fig. 2. Induction of allograft tolerance by natural CD25+CD4+ TR cells. (A) Indicated number of BALB/c nude mice with B6 skin grafts were reconstituted with normal naive BALB/c T cells (2 x 105) only or together with BALB/c CD25+CD4+ T cells (2 x 105) on day 0; some mice were transferred with CD25+CD4+ T cells (2 x 105 or 6 x 105) 7 days prior to the transfer of naive T cells (2 x 105) on day 0. Control mice received naive T cells only. The result shown is the total of 21 independent experiments. (B) BALB/c nude mice retaining B6 skin grafts for >100 days were engrafted on each flank with B6 or C3H skin fragments. Survival of original B6 grafts after secondary grafting is also shown. The result of a total of five independent experiments is shown. (C) Adoptive transfer of graft tolerance by T cells. Spleen and lymph node cells from tolerant nude mice shown in Fig. 2(A) were treated with anti-CD25 mAb and C or C only, as shown by staining with anti-CD4 and anti-CD25 after treatment (upper panel); and 2~4 x 107 treated cells were i.v. transferred to BALB/c nude mice with B6 skin grafts. After 100 days, both groups of mice i.v. received 2 x 105 naive BALB/c T cells. Another group of nude mice (open circles) received lymphocytes from the nude mice that had previously rejected B6 skin grafts. The result of total five independent experiments is shown.

 
Grafting of B6 or C3H skin grafts on each flank of these tolerant mice to determine the antigen-specificity of tolerance resulted in rapid rejection of C3H grafts in all the mice and, interestingly, gradual rejection of the fresh B6 grafts in 60% of recipients while all the mice retained the original B6 grafts (Fig. 2B).

We then attempted to determine whether the graft tolerance can be adoptively transferred to other mice (Fig. 2C). When spleen and lymph node cells were transferred from the mice with long-term surviving (>100 days) B6 grafts, shown in Fig. 2(A), to naive nude mice with B6 skin grafts, the recipients showed long-term acceptance of the skin grafts (MST: >100 days), contrasting with rapid graft rejection by the transfer of lymphocytes from the donors that had rejected a B6 skin graft (MST: 18 days). Interestingly, not only untreated lymphocytes but also CD25+ cell-depleted lymphocytes prepared from the tolerant mice by in vitro anti-CD25 mAb and C-treatment were able to transfer the tolerance (Fig. 2C; and see below). Furthermore, inoculation of naive BALB/c T cells (2 x 105 cells) sufficient in number to reject B6 grafts in naive nude mice failed to elicit graft rejection in the majority of these nude mice transferred with either whole or CD25 cells.

Thus, CD25+CD4+ T cells in normal naive mice can induce transplantation tolerance by suppressing graft-rejecting T cells, especially when the former is allowed to expand for a limited period in the absence of the latter; and this dominant tolerance has antigen-specificity and can be adoptively transferred to other mice by T cells.

Phenotypic and functional stability of CD25+CD4+ TR cells
The result in Fig. 2 that CD25 T cells were able to adoptively transfer allograft tolerance indicate that some, at least, of naturally arising CD25+CD4+ TR cells may lose their CD25 expression while retaining suppressive function; alternatively, CD25CD4+ T cells may differentiate to functional TR cells. To assess these possibilities, we first examined the stability of the expression of TR cell-associated molecules, the suppressive activity in vitro, and the level of Foxp3 gene expression, when CD25+CD4+ T cells from naive mice were transferred to nude mice (Fig. 3).



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Fig. 3. Phenotypic and functional stability of CD25+CD4+ TR cells upon in vivo transfer. (A) 2 x 105 naive BALB/c-Thy1.1 CD25+ or CD25CD4+ T cells were transferred to BALB/c nude mice with B6 skin grafts. Seven days later, CD25, GITR, CD103 and CTLA-4 expression of Thy1.1+CD4+ cells was analyzed (thick line). The expression level of each molecule on CD25+ or CD25CD4+ T cells from normal naive BALB/c mice is shown by thin line, and control staining as shaded area. (B) BALB/c nude mice with B6 skin grafts were transferred with 2 x 106 naive BALB/c-Thy1.1 CD25+ or CD25CD4+ T cells; 10 days later, spleen and lymph node cells were stained with anti-Thy-1.1, anti-CD4 and anti-CD25, and CD4/CD25 staining of Thy1.1+ cells is shown (upper panel). Thy1.1+CD4+ spleen and lymph node cells were sorted into the indicated compartments by cell sorter and cultured with freshly prepared BALB/c CD25CD4+ T cells at various ratios and irradiated BALB/c splenocytes in the presence of anti-CD3 mAb. a, b, a', b' and c' correspond to those in the subpopulations in the upper panel. (C) Foxp3 mRNA expression of each population was examined by quantitative RT–PCR. A representative result of three independent experiments is shown in (A–C).

 
When CD25+ or CD25CD4+ T cells prepared from BALB/c-Thy1.1 mice were transferred to BALB/c nude mice with B6 skin grafts, the levels of CD25 expression on the inoculated CD25+CD4+ T cells were much lower 7 days after transfer than pre-transfer while the inoculated CD25CD4+ T cells up-regulated CD25 expression to a similar level to that of the CD25+CD4+-derived T cells (Fig. 3A and B). The inoculated CD25CD4+ T cells also gave rise to a small population, constituting ~5% of the CD25CD4+-derived cells, that expressed high levels of CD25 and moderately down-regulated CD4 expression (Fig. 3B, upper panel). Although the expression of GITR and CTLA-4 was up-regulated in both populations after transfer, the expression patterns, which are normally higher on CD25+CD4+ T cells than CD25CD4+ T cells (1317), were well preserved. Expression of CD103, which is expressed by a fraction of CD25+CD4+ T cells in normal naive mice (18), was also retained after transfer.

To assess the in vitro suppressive activity of CD25+ and CD25CD4+-derived T cells in nude mice, the CD25-positive or CD25-negative fraction was co-cultured with freshly prepared BALB/c CD25CD4+ T cells and stimulated with anti-CD3 (Fig. 3B, lower panel). The CD25+CD4+-derived cells, either CD25-positive or -negative, showed suppressive activity equivalent to that of freshly prepared BALB/c CD25+CD4+ T cells; and they themselves scarcely proliferated upon TCR stimulation (Fig. 3B). Furthermore, they sustained the levels of Foxp3 mRNA expression equivalent to 50~110% of the natural CD25+CD4+ T-cell population present in normal mice (Fig. 3C). On the other hand, the CD25CD4+-derived T cells, either CD25-positive or -negative, were non-suppressive, proliferated upon anti-CD3 stimulation, and scarcely expressed Foxp3 (Fig. 3B and C). Interestingly, however, CD25high CD4low cells, a fraction that had developed from CD25-CD4+ T cells and constituted ~5% of the CD4+ T cells (fraction c' in Fig. 3B and C), was hypoproliferative upon TCR stimulation, exhibited a suppressive activity equivalent to natural CD25+CD4+ TR cells, and expressed Foxp3 at a high level. This TR population appeared to have differentiated/expanded from CD45RBlow GITRhigh cells in the CD25CD4+ population since transfer of CD25CD4+ T cells after depletion of CD45RBlow cells or GITRhigh cells to nude mice resulted in the development of lower numbers (i.e. 1/4~1/5) of such CD25highCD4low TR cells than the transfer of whole CD25CD4+ T cells (Supplementary fig. 1, available at International Immunology Online).

These results collectively indicate that CD25+CD4+ TR cells may lose their CD25 expression presumably in the absence of other T cells while still retaining their suppressive activity, and that a small subpopulation of CD25CD4+ T cells can differentiate to CD25+ TR cells which are functionally similar in vitro to natural CD25+CD4+ TR cells although the majority of CD25CD4+ T cells are kept non-regulatory.

Potent and persistent suppressive activity of antigen-stimulated CD25+CD4+ TR cells
To determine then which TR cells, either derived from CD25+ or CD25CD4+ T cells, are responsible for maintaining allograft tolerance (Figs 2 and 3), we prepared tolerant mice by transferring BALB/c-Thy-1.1+CD25+CD4+ T cells to B6-skin-grafted BALB/c nude mice 7 days prior to inoculation of normal BALB/c (Thy-1.2+) CD25+ cell-depleted T cells at the ratio of 10:1. Eighty percent of the mice stably retained the skin grafts 100 days after the transfer of CD25 T cells. These mice also retained both Thy-1.2+ cells and Thy-1.1+ cells; two-thirds of the Thy-1.1+ T cells expressed CD25 at levels as high as naturally present CD25+CD4+ TR cells. Since nude mice develop some endogenous CD4+ cells (4,40), the Thy-1.2+ fraction contained a larger number of cells than Thy1.1+ cells (3.4% vs 1.1%), irrespective of the transfer of a larger number of the latter cells (Fig. 4A). Transfer of lymphocytes from these tolerant mice to newly prepared B6-skin-grafted BALB/c nude recipients resulted in no graft rejection in the majority, whereas transfer of lymphocytes after elimination of Thy1.1+ cells by anti-Thy1.1 mAb and C-treatment led to rapid graft rejection in all the recipients (Fig. 4B). The result indicates that TR cells derived from natural CD25+CD4+ T cells are mainly responsible for the maintenance of dominant transplantation tolerance in the donor mice and that other TR cells, including those derived from CD25CD4+ T cells (Fig. 3), may not be sufficiently potent per se to be able to suppress rejection. Furthermore, judging from the result that one-third of CD25+CD4+ TR cells had lost CD25 expression in tolerant mice (Fig. 4A), successful transfer of tolerance by CD25 cells in Fig. 2(C) can be attributed to the original CD25+CD4+ TR cells that had lost CD25 expression, rather than TR cells derived from CD25CD4+ T cells.



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Fig. 4. Persistence of in vivo suppressive activity of CD25+CD4+ TR cells. BALB/c nude mice with B6 skin grafts were made tolerant by transfer of 1 x 106 BALB/c-Thy1.1 CD25+CD4+ T cells 7 days prior to inoculation of 1 x 105 BALB/c (Thy 1.2) CD25+-depleted T cells. (A) After 100 days, the expression level of CD25 on Thy-1.1+ or Thy1.1CD4+ T cells from graft-tolerant nude mice was analyzed. Control staining is shown by shadow. (B) Spleen and lymph node cells from graft-tolerant nude mice were treated with anti-Thy1.1 and C or C only; 2 x 107 treated cells were transferred to naive nude mice with B6 skin grafts and graft survival was monitored. The data presented are the total of two independent experiments.

 
In vitro expansion of alloantigen-specific CD25+CD4+ TR cells
Naturally present CD25+CD4+ TR cells exhibit poor proliferative responses in primary allogeneic MLR in vitro, and suppress the allo-reactive proliferation of both CD4+ T cells and CD8+ T cells; and once TR cells are activated by alloantigen stimulation, they suppress allo-responses of third party T cells as well (Supplementary fig. 1). Thus, the mode of in vitro CD25+CD4+ TR cell-mediated suppression is similar between T cell responses to histocompatibility antigens and soluble protein antigens (10,12).

Such alloreactive CD25+CD4+ TR cells expanded in vitro upon allo-antigen stimulation in the presence of IL-2 (Fig. 5A). BALB/c CD25+CD4+ T cells stimulated with X-irradiated B6 splenocytes along with a high dose IL-2 (e.g. 100 U/ml) expanded ~2-fold in 1 week, and ~4-fold in 4 weeks. These expanded cells sustained Foxp3 expression (Fig. 5B). When these cells were washed and restimulated with B6 cells, they scarcely proliferated but potently suppressed the primary MLR of freshly prepared BALB/c CD25CD4+ T cells (Fig. 5C). Compared with freshly prepared CD25+CD4+ T cells, much smaller numbers of these prestimulated T cells sufficed to suppress the MLR completely. Furthermore, 1- or 4-week-prestimulated TR cells could potently suppress a secondary MLR, whereas the same number of freshly prepared CD25+CD4+ T cells could not (Fig. 5C). The suppressive activity of the 4-week-stimulated CD25+CD4+ T cells was much higher than the activity of the 1-week-stimulated ones.



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Fig. 5. In vitro expansion of allo-antigen-specific CD25+CD4+ TR cells and resulting enhancement of their antigen-specific suppression. (A) BALB/c CD25CD4+ T cells, CD25+CD4+ T cells, or the two populations mixed at the ratio of 1:1 were stimulated with B6 splenocytes in the presence of various concentrations of IL-2. (B) Foxp3 expression of BALB/c CD25+CD4+ T cells freshly prepared, prestimulated with B6 splenocytes plus IL-2 (100 U/ml) for 1 or 4 weeks were examined by quantitative RT–PCR. (C) BALB/c CD25+CD4+ T cells freshly prepared, or prestimulated with B6 splenocytes plus IL-2 for 1 or 4 weeks were mixed with BALB/c CD25CD4+ T cells either freshly prepared (left) or prestimulated with B6 splenocytes for 7 days (right). The cell mixtures were stimulated with X-irradiated B6 splenocytes. (D) BALB/c CD25+CD4+ T cells either freshly prepared or prestimulated with B6 or C3H spleen cells plus IL-2 for 7 days were mixed with freshly prepared BALB/c CD25CD4+ T cells at various ratios and stimulated with either X-irradiated B6 (left) or C3H (right) splenocytes. (E) Percentage of Vß6+ or Vß10+ cells in the BALB/c CD25+CD4+ population unstimulated or stimulated with MMC-treated DBA/2 splenocytes plus IL-2 for 7 days. (F) BALB/c CD25+CD4+ T cells freshly prepared or prestimulated with DBA/2 splenocytes plus IL-2 for 7 days were mixed with freshly prepared BALB/c CD25CD4+ T cells at various ratios and stimulated with MMC-treated DBA/2 splenocytes for 5 days. A representative result of more than three independent experiments is shown in (A–F).

 
Furthermore, titration of the number of TR cells required for a particular degree of suppression revealed that B6-prestimulated BALB/c CD25+CD4+ T cells were more (nearly 8-fold) potent than C3H-prestimulated BALB/c CD25+CD4+ T cells in suppressing the response of BALB/c CD25CD4+ T cells to B6-stimulation; the C3H-prestimulated CD25+CD4+ T cells showed a comparable suppressive activity to that of freshly prepared BALB/c CD25+CD4+ T cells (Fig. 5D). Likewise, C3H-prestimulated BALB/c CD25+CD4+ T cells more potently suppressed the response of BALB/c CD25CD4+ T cells to C3H stimulation than B6-prestimulated or freshly prepared BALB/c CD25+CD4+ T cells.

To determine then whether this in vitro antigen-specific enhancement of suppression was due to expansion of alloantigen-specific TR cells, we assessed the degree of expansion of Vß6+ T cells in BALB/c CD25+CD4+ T cells stimulated with DBA/2 splenocytes and IL-2 for 7 days (Fig. 5E). Vß6+ cells among CD25+CD4+ T cells increased 5-fold in percentage (11.3% and 54.6% on days 0 and 7, respectively) and 10-fold in actual cell number, whereas Vß10+ cells showed no increase in percentage (6.0% and 2.7% on days 0 and 7, respectively) or cell number. Moreover, compared with non-stimulated BALB/c CD25+CD4+ T cells, 16- to 32-fold fewer of the DBA/2-prestimulated BALB/c CD25+CD4+ T cells sufficed to completely suppress anti-Mls-1 response of BALB/c CD25CD4+ T cells (Fig. 5F). This marked increase in the magnitude of suppression, compared with increase in the percentage of Vß6+ cells, indicates that the suppressive activity of individual antigen-specific CD25+CD4+ TR cells also increased.

Thus, in vitro stimulation of CD25+CD4+ T cells with allogeneic cells and IL-2 can enhance their suppressive activity in an alloantigen-specific fashion. Judging from the increase in suppressive activity and the cell number, this enhancement can be mainly attributed to the expansion of antigen-specific TR cells and to a lesser degree to an increase in the suppressive activity of individual TR cells.

Antigen-specific suppression of graft rejection by ex vivo stimulated CD25+CD4+ TR cells
To examine the ability of the ex vivo prestimulated CD25+CD4+ TR cells to suppress graft rejection in vivo, BALB/c CD25+CD4+ T cells in vitro stimulated with B6 APCs with 100 U/ml IL-2 for 1 week were transferred to BALB/c nude mice with B6 skin grafts (Fig. 6A). The transfer significantly prolonged graft survival in a cell dose-dependent fashion (MST = 67 days at 1:1 ratio of prestimulated CD25+CD4+ T cells vs freshly prepared whole T cells, n = 10, and MST >100 days at 5:1 ratio, n = 9; P = 0.00004 and 0.00003 vs control, respectively). This prolongation appeared to be antigen-specific since B6-prestimulated CD25+CD4+ T cells significantly prolonged the survival of B6 grafts at 1:1 ratio of TR cells and naive T cells; the survival was significantly longer compared with the transfer of C3H-prestimulated CD25+CD4+ T cells (MST = 62 vs 40 days, P =0.03) (Fig. 6A). Similarly, C3H-prestimulated CD25+CD4+ T cells significantly prolonged the survival of C3H grafts than B6-prestimulated CD25+CD4+ T cells (MST = 36 vs 23 days, P = 0.017) (Fig. 6B).

It was noted in the above experiments that the kinetics of graft survival was almost equivalent between ex vivo pre-stimulated CD25+CD4+ T cells and the same number of freshly prepared cells (Fig. 2A vs Fig. 6A), irrespective of marked expansion of alloantigen-specific TR cells in the former. To analyze this discrepancy, we assessed the survival and expansion of inoculated CD25+CD4+ T cells (Fig. 6C). Transfer of B6-prestimulated or freshly prepared BALB/c-Thy1.1 CD25+CD4+ T cells to BALB/c nude mice with B6 skin grafts revealed that, in contrast to the 2-fold expansion of freshly prepared CD25+CD4+ T cells within 1 week after transfer, the in vitro pre-stimulated CD25+CD4+ T cells were reduced to half the number of the inoculated cells.

Taken together, the in vitro pre-stimulated CD25+CD4+ T cells can effectively prolong the graft survival in an antigen-specific manner, although a significant fraction of them may die within 1 week after transfer or, as another possibility, they might migrate into non-lymphoid tissues.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
A key finding in this report is that in vivo exposure of naturally arising Foxp3+CD25+CD4+ TR cells to alloantigen in the absence of other T cells elicited spontaneous expansion of alloantigen-specific TR cells, which suppressed allograft rejection mediated by subsequently transferred naive T cells and established long-term graft tolerance. In addition, in vitro allogeneic stimulation of natural TR cells in the presence of IL-2 also elicited their antigen-specific proliferation and apparently enhanced suppressive activity in individual antigen-specific TR cells. This in vivo or in vitro, direct or indirect induction of antigen-specific expansion of natural Foxp3+CD25+CD4+ TR cells would be instrumental for establishing transplantation tolerance and re-establishing self-tolerance in autoimmune disease.

We took advantage of Mls-specific expansion of T cells that express a particular Vß TCR subfamily to monitor in vivo and in vitro TR cell expansions and differentiate their antigen-specific expansion from possible antigen-nonspecific homeostatic proliferation in the T cell-deficient environment of nude mice. This approach clearly showed alloantigen-specific expansion of natural TR cells. It can be argued that in contrast with this Mls-disparate stimulation through the direct pathway of antigen sensitization, in vivo T-cell activation by MHC-disparate allo-stimulation may be mainly through the indirect pathway (41). It is highly likely, however, that allogeneic MHC stimulation can expand TR cells through the indirect pathway as well, since in vivo stimulation of TCR-transgenic mice-derived CD25+CD4+ TR cells with a specific antigen and CFA or with antigen-loaded mature DCs can evoke their proliferation (2325). Although the peak of TR cell expansion with Mls stimulation was ~1 week in nude mice, the precise kinetics of TR cell expansion upon allo-MHC stimulation must be determined to confer more distinct antigen specificity to TR cells and establish more stable antigen-specific tolerance.

Given that natural CD25+CD4+ TR cells are pre-committed to be suppressive in function before antigen exposure, this in vivo and in vitro antigen-specific TR cell expansion has the following implications. First, the expansion of antigen-specific TR cells forms the cellular basis of antigen-specificity in TR cell-mediated dominant tolerance, even when the expanded TR cells may exert antigen-nonspecific or bystander suppression of other T cells to a certain extent (Supplementary fig. 2). Second, if alloantigen-specific CD25+CD4+ TR cells are allowed to expand in the absence of allo-reactive effector T cells (as the transfer of CD25+CD4+ T cells alone to T cell-deficient nude mice), they can expand in a short time to the extent that they are sufficient in number and suppressive activity to stably control allo-reactive effector T cells and thereby to establish dominant transplantation tolerance. Third, a dynamic balance is attained and maintained between such allo-specific CD25+CD4+ TR cells and allo-reactive effector T cells in graft-tolerant animals, with the former continuously and actively suppressing the latter. Supporting this notion, removal of TR cells from long-term tolerant mice evoked graft rejection (Fig. 4B), and inoculation of normal naive T cells failed to elicit rejection in tolerant animals (Fig. 2C). Furthermore, the expanded natural TR cells are apparently unable to confer significant suppressive activity, if at all, to naive T cells in an ‘infectious’ manner during their co-existence in the tolerant host, since specific removal of the expanded TR cells derived from CD25+CD4+ TR cells sufficed to evoke graft rejection in tolerant animals (Fig. 4B). The result, however, does not exclude the possibility that, while the expanded CD25+CD4+ TR cells restrain the activation of allo-reactive T cells, CD25CD4+ T cells may give rise to certain TR cells (Fig. 3; and see discussion below).

It was noted in our experiments that the long-term tolerant animals gradually rejected secondary grafts while stably retaining the original grafts (Fig. 2B). This illustrates several key features of dominant graft tolerance. First, allo-reactive effector T cells potentially capable of rejecting graft have persisted in tolerant animals under the dominant control by TR cells. Second, activation of graft-rejecting effector T cells by secondary grafts is insufficient to trigger rejection of the original graft. Third, the maintenance of graft tolerance may partly depend on the local activity of TR cells (42) or stable settlement of TR cells in their ‘niche’ (43). Thus, at the original graft site, a stable local balance appears to have been established between TR cells and effector T cells that have been recruited to the site, whereas the two populations may be in a more delicate and vulnerable balance at the site of secondary graft. Monitoring the recruitment and persistence of TR cells, e.g. as Foxp3+ cells (see discussion below), and effector T cells at the graft site may help to assess the local balance between the two populations and thereby the local state of graft tolerance.

The antigen-expanded CD25+CD4+ TR cells are functionally and phenotypically stable, more potent in suppression than TR cells derived from CD25CD4+ T cells, and able to adoptively transfer graft tolerance to naive mice. For example, expression of Foxp3, which appears to be a master control gene for the development and function of natural CD25+CD4+TR cells, is stably maintained and shows a good correlation with the level of in vitro suppressive activity irrespective of the phenotypes of cell surface molecules or the origin of TR cells (see below). The expression levels of CTLA-4, GITR and CD103 were also kept higher in CD25+CD4+ TR cells or a fraction of them, compared with T cells derived from CD25CD4+ T cells. These findings indicate that CTLA-4, GITR, CD103 and Foxp3 in particular, are good markers for monitoring natural TR cells. In contrast to these molecules, natural TR cells may decrease the level of CD25 expression under certain conditions, such as their transfer to nude mice (Fig. 3A) or SCID mice (26,27). Noteworthy, however, is that CD25 expression by CD25+CD4+ TR cells was moderately maintained when they were co-transferred with CD25CD4+ T cells to nude mice (Fig. 4A). It is likely that IL-2 is required for the maintenance of CD25 expression on natural TR cells and that, in the nude mice, IL-2 from the co-transferred normal T cells was responsible for the maintenance (4446). IL-2 is also necessary for in vivo survival of natural CD25+CD4+ TR cells (47). Once natural TR cells establish transplantation tolerance, in vivo administration of IL-2 may contribute to the maintenance of their CD25 expression, survival and function, thereby to stable graft tolerance.

CD25CD4+ T cells, especially CD45RBlowCD25CD4+ T cells, which express Foxp3 at a low level (20), gave rise to a small number of CD25high Foxp3moderately high TR cells with in vitro suppressive activity (Fig. 3B and C). This TR subpopulation was, however, much less potent in suppressing graft rejection than CD25+CD4+-derived TR cells (Fig. 4B). Similar findings were also made in other experimental systems; e.g. the CD45RBlowCD25CD4+ T cell population contains TR cells capable of preventing autoimmune disease or colitis, or transferring transplantation tolerance induced by anti-CD4 mAb treatment. In these experiments, however, the regulatory activity of this fraction was much lower than CD25+CD4+ TR cells, and insufficient to transfer graft tolerance (26,32,34,4850). Moreover, such CD25CD4+ TR cells became CD25+ upon antigenic stimulation in vitro and in vivo (Fig. 3B; M. Ono and S. Sakaguchi, unpublished data). The origin of these CD25CD4+ TR cells remains to be determined; e.g. they may be derived from natural CD25+CD4+ TR cells that have lost CD25 expression or from pre-committed TR cells naturally present in the CD25CD4+ T cell population, especially in the CD45RBlowGITRhighCTLA-4+ fraction [(51); Supplementary fig. 1; M. Ono, J. Shimizu, T. Yamaguchi, Z. Fehervari, Y. Miyachi and S. Sakaguchi, manuscript in preparation); alternatively, naive CD25CD4+ non-TR cells may differentiate to CD25± TR cells under certain conditions of antigen-presentation and/or cytokine milieu (37,38,5254).

Alloantigen-specific CD25+CD4+ T cells can be expanded in vitro by allogeneic stimulation and high dose IL-2. They showed potent alloantigen-specific suppressive activity in vitro, suppressed even a secondary MLR, and could prevent graft rejection in vivo. However, they appear to have a short life span in vivo upon transfer or change their homing patterns, making them less effective in vivo compared with their potent in vitro suppressive activity, as also suggested by others in treatment of GVHD (55,56). The possible poor survival of in vitro activated CD25+CD4+ TR cells could be attributed to their apoptosis upon sudden withdrawal of high dose IL-2 at the time of cell transfer (57). Use of other cytokines for TR cell expansion, delivery of apoptosis-inhibitory signals to expanding TR cells, or use of mature allogeneic DCs for stimulation without exogenous IL-2 may overcome the problem. The present results nevertheless indicate that, compared with a large number of TR cells required for inducing tolerance to full MHC-mismatched skin grafts as shown here, a much lower number of in vitro expanded TR cells may suffice to establish tolerance to other organs with minor histoincompatibility even if many of the transferred TR cells may die after transfer.

Taken together, the results in this report suggest two possible ways of exploiting naturally occurring CD25+CD4+ TR cells for induction of graft-specific tolerance, without hampering immune responses to other antigens. One is to control non-TR cells for preparing an immunological condition facilitating antigen-specific spontaneous expansion of natural TR cells; e.g. to reduce the number or block the activation of non-TR cells as specifically as possible, and meanwhile to sensitize the remaining CD25+CD4+ TR cells to alloantigens, allowing alloantigen-specific TR cells to expand to the extent that they can dominantly suppress allo-reactive T cells recovering from the reduction or blockade. Certain monoclonal antibodies or drugs whose administration for a limited period can induce long-term TR cell-dependent graft tolerance may have this effect as a common mechanism of tolerance induction (1,3035,58). Another way of tolerance induction with natural TR cells is to isolate CD25+CD4+ TR cells from the recipient of the organ transplant, stimulate them ex vivo with donor stimulator cells, and transfer back the expanded antigen-specific TR cells to the recipient before organ transplantation, as is possible in living donor transplantation (59). In this setting, suppression of the recipient's allo-reactive T cells by the inoculated TR cells may allow any of the recipient's residual natural TR cells, which may be CD25+ or CD25, to expand spontaneously in a graft-specific fashion, potentiating graft tolerance synergistically with the inoculated TR cells. This possible mechanism of ‘infectious tolerance’ is currently under investigation. These ways of inducing immunologic tolerance to organ transplants could, in principle, be applied for re-establishing self-tolerance in autoimmune disease and controlling aberrant or excessive immune responses to non-self-antigens.


    Supplementary data
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Supplementary data are available at International Immunology Online.


    Acknowledgements
 
We thank Dr K. J. Wood for helpful discussion, and Drs T. Takahashi, T. Nomura, S. Hori and Z. Fehervari for critical reading of the manuscript. A part of this work was presented at the FASEB summer research conference on transplantation immunology in Vermont in June 2002, and the annual meetings of Japanese Society of Immunologists, December 2002 and December 2003. This work was supported by grants-in-aids from the Ministry of Education, Science, Sports and Culture, the Ministry of Human Welfare, and Japan Science and Technology Agency.


    Abbreviations
 
APC   antigen-presenting cell
CTLA-4   cytotoxic T lymphocyte-associated antigen-4
GITR   glucocorticoid-induced TNF receptor superfamily-related gene
MLR   mixed lymphocyte reaction
MMC   mitomycin C
MST   median survival time

    Notes
 
Transmitting editor: K. Okumura

Received 3 April 2004, accepted 2 June 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 

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