The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Masuyama, J.-i.
Right arrow Articles by Minota, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Masuyama, J.-i.
Right arrow Articles by Minota, S.
The Journal of Immunology, 2002, 169: 3710-3716.
Copyright © 2002 by The American Association of Immunologists

A Novel Costimulation Pathway Via the 4C8 Antigen for the Induction of CD4+ Regulatory T Cells1

Jun-ichi Masuyama2,*, Shuji Kaga{dagger}, Shogo Kano* and Seiji Minota*

* Division of Rheumatology and Clinical Immunology, Department of Medicine, Jichi Medical School, Tochigi, Japan; and {dagger} First Department of Medicine, Showa University, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD4+CD25+ regulatory T (Treg) cells naturally occur in mice and humans, and similar Treg cells can be induced in vivo and in vitro. However, the molecular mechanisms that mediate the generation of these Treg cell populations remain unknown. We previously described anti-4C8 mAbs that inhibit the postadhesive transendothelial migration of T cells through human endothelial cell monolayers. We demonstrate in this work that Treg cells are induced by costimulation of CD4+ T cells with anti-CD3 plus anti-4C8. The costimulation induced full activation of CD4+ T cells with high levels of IL-2 production and cellular expansion that were comparable to those obtained on costimulation by CD28. However, upon restimulation, 4C8-costimulated cells produced high levels of IL-10 but no IL-2 or IL-4, and maintained high expression levels of CD25 and intracellular CD152, as compared to CD28-costimulated cells. The former cells showed hyporesponsiveness to anti-CD3 stimulation and suppressed the activation of bystander T cells depending on cell contact but not IL-10 or TGF-{beta}. The suppressor cells developed from CD4+CD25-CD45RO+ cells. The results suggest that 4C8 costimulation induces the generation of Treg cells that share phenotypic and functional features with CD4+CD25+ T cells, and that CD25- memory T cells may differentiate into certain Treg cell subsets in the periphery.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ability to discriminate between self and nonself is a fundamental feature of the immune system. Immune tolerance plays a crucial role in the discrimination, and its dysfunction may induce a breakdown of immune homeostasis and the development of autoimmune disease. Central tolerance is a well-established mechanism that leads to clonal deletion of self-reactive T cells by negative selection in the thymus (1, 2). However, some self-reactive lymphocytes that escape selective deletion migrate from the thymus to the periphery. Although these cells have the potential to respond to extrathymic self-antigens, their harmful response is prevented by mechanisms of peripheral tolerance, including clonal anergy (3), ignorance (4), and clonal deletion (5). Moreover, a number of recent studies have provided firm evidence for an additional active mechanism of peripheral tolerance that could regulate autoimmunity and alloreactions in transplantation (6, 7, 8, 9). A distinct subset of CD4+ T cells that is defined by coexpression of CD25 or CD45RBlow has been elucidated to prevent the development of autoimmune disease in mice and rats (10, 11, 12, 13). It was demonstrated that the transfer of disease-inducing CD4+CD25- T cells from normal mice into syngeneic nude mice results in the development of organ-specific autoimmune disease, whereas cotransfer of the CD4+CD25+ population prevented the induction of disease (12, 13). Subsequent studies confirmed the existence of these regulatory T (Treg)3 cells in human peripheral blood with the same phenotypic and functional characteristics as those in mice (14, 15, 16, 17). Although CD4+CD25+ T cells are in an anergic state associated with the failure of IL-2 secretion, they potently suppress the proliferation of bystander CD25- T cells on TCR stimulation with soluble anti-CD3 in the presence of APC. The suppressive effect is exerted by inhibiting the production of IL-2 by the responder cells, but is overcome by the addition of exogenous IL-2 and anti-CD28 (18). Suppression requires TCR stimulation of the suppressor cells and is mediated by cell-cell contact, but not by inhibitory cytokines such as IL-10 and TGF-{beta}. However, once activated, CD4+CD25+ cells show suppressor function in an Ag-nonspecific manner (19). Further characterization revealed that the CD25+ population constitutively expresses high levels of intracellular CTLA-4 (CD152) (20, 21). In addition to naturally occurring CD4+CD25+ cells that arise first in the thymus (22, 23), different suppressor types of CD4+ T cells can be generated by repetitive Ag stimulation in vivo and in vitro, including Th3 cells derived from oral tolerant animals (24), T regulatory 1 (Tr1) cells induced by repetitive stimulation with APC in the presence of IL-10 (25), and Tr1-like cells developed by cocultures with allogeneic immature dendritic cells (iDC) (26). These cells secrete large amounts of IL-10 and/or TGF-{beta} and inhibit Ag-specific immune responses in a cytokine- or cell contact-dependent manner.

Concerning the molecular mechanisms behind the generation of regulatory cells, it has been shown that the interaction between CD28 and B7 molecules is involved in the induction of CD4+CD25+ T cells (27, 28). More recently, Tr1 cells have been shown to develop after costimulation of TCR with CD2 (29). However, the precise molecular pathway of the generation remains obscure. In addition, whether de novo differentiation of Treg cells occurs in the periphery remains to be determined, because mice thymectomized as adults can develop T cells with the ability to suppress the induction of autoimmune disease (30).

Previously, we reported anti-4C8 mAbs that inhibit the transmigration of T cells through, but not their tight adhesion to, human endothelial cell monolayers (31). This molecule is expressed on CD3+ T cells, NK cells, monocytes, and eosinophils, but not neutrophils or endothelial cells. In this study, we demonstrate that costimulation with anti-CD3 plus anti-4C8 induced full activation of CD4+ T cells with high levels of IL-2 production and cellular expansion. Most importantly, we found that the phenotype and immune functions of 4C8-costimulated cells were entirely different from those of CD28-costimulated cells, which resembled in many ways CD4+CD25+ cells or iDC-induced Tr1-like T cells. Furthermore, the results revealed that the suppressor cells induced by 4C8 costimulation were generated from CD4+CD25-CD45RO+ cells. These findings support the hypothesis that memory CD4+CD25- T cells may differentiate into CD4+CD25+-like Treg cells through a development pathway with an active stimulation process in the periphery.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
mAbs and reagents

Purified anti-CD3 mAb (OKT3) was a gift from Dr. S. Kashiwagi (Japan Immunoresearch Laboratories, Gunma, Japan). Anti-CD28 (clone CD28.2) and FITC-conjugated and purified anti-CD25 mAbs were purchased from BD PharMingen (San Diego, CA). Quantum Red-conjugated anti-CD4 and PE-conjugated anti-CD152 (CTLA-4) mAbs were purchased from Sigma-Aldrich (St. Louis, MO). Anti-4C8 mAb was purified from hybridoma supernatants by using a protein A-Sepharose column as described elsewhere (31). Anti-IL-10 and anti-TGF-{beta}1, 2, and 3 (1D11) mAbs were obtained from Genzyme (Cambridge, MA) and R&D Systems (Minneapolis, MN), respectively. Anti-CD45RA (2H4) and anti-CD45RO (UCHL-1) were purchased from Beckman Coulter (Miami, FL). IL-2 was obtained from PeproTech (London, U.K.).

Isolation of T cell subsets

PBMC were separated from heparinized venous blood of healthy adult human donors by centrifugation over Ficoll-Hypaque (Amersham Biotech, Buckinghamshire, U.K.). CD4+ T cells were negatively selected from PBMC using magnetic colloid beads according to the manufacturer’s instructions (StemCell Technologies, Vancouver, Canada). Using a MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany), the isolated CD4+ T cells were further divided into CD45RA+ and CD45RO+ cells by negative selection with anti-CD45RO and anti-CD45RA, respectively, and magnetic beads coated with sheep anti-mouse IgG. Cell purification was in the range of 91–96% for each fraction as determined by flow cytometry (FACScan; BD Biosciences, Mountain View, CA). In some experiments, CD25+ cells were removed from CD4+ T cells by the MACS magnetic immunodepletion using anti-CD25 (BD PharMingen). The CD4+CD25- population contained no detectable (<1%) CD25+ cells. PBMC irradiated at 50 Gy were used as APC.

T cell proliferation assays

The proliferation of CD4+ T cells was assessed in 96-well flat-bottom microtiter plates (Falcon; BD Biosciences), with each well containing 2 x 105 cells in a final volume of 200 µl of RPMI 1640 with 10% FCS (Hyclone Laboratories, Logan, UT), penicillin (100 U/ml), streptomycin (100 µg/ml), and 2 mM glutamine. Immobilization of the plates with mAbs was achieved by 24-h incubation at 4°C of PBS (100 µl/well) containing anti-CD3 (0.1 µg/ml). Plate binding of anti-4C8 (10 µg/ml) was similarly carried out after anti-CD3 immobilization. Anti-CD28 mAb (5 µg/ml) was added at the initiation of cultures. Cells were cultured for 72 h in the plates at 37°C in a 5% CO2 humidified incubator and pulsed with 0.5 µCi of [3H]thymidine (New England Nuclear, Boston, MA) for the last 12 h. The time of thymidine pulse and the concentration of mAbs used were determined based on the results of preliminary studies for a time course and dose-dependent response of costimulation. Incorporated radioactivity was quantified by scintillation counting. The experiments were performed in triplicate. Viable cells in a pool of T cells from three wells were counted by the trypan blue dye exclusion test. To assess the regulatory properties of costimulated T cells, first, purified CD4+ T cells were costimulated for 3 days with immobilized anti-CD3 (0.1 µg/ml) plus either soluble anti-CD28 (5 µg/ml) or immobilized anti-4C8 (10 µg/ml). These cells were harvested, washed, and further rested for 4–6 days. For assessment of the anergic state of cells, the costimulated cells (1 x 105/well) were stimulated for 3 days with soluble anti-CD3 (25 ng/ml) in the presence of irradiated (50 Gy) PBMC (4 x 105/well) with or without IL-2 (100 U/ml) in a final volume of 200 µl of complete medium in 96-well round-bottom plates (Flow Laboratories, McLean, VA). For experiments on suppression, freshly isolated CD4+ cells (1 x 105/well) as responders were cocultured for 3 days with irradiated PBMC (4 x 105/well) and irradiated costimulated cells (1 x 105/well). Freshly isolated CD4+ cells (1 x 105/well) were also irradiated and used as control for costimulated cells. Proliferation was measured as above. The combination of cells was syngeneic in all experiments.

Cytokine assays

Cytokine production was assessed at different time points by the analysis of supernatants from cultures using human IL-2, IL-4, IL-10, and IFN-{gamma} ELISA kits (BioSource International, Camarillo, CA).

Flow cytometry

For staining of cell surface Ags, 2–3 x 105 cells were incubated for 20 min with FITC-conjugated anti-CD25, PE-conjugated anti-CTLA-4, and Quantum Red-conjugated anti-CD4 mAbs. After two washes with 1 ml of PBS containing 0.1% BSA and 0.05% sodium azide, cells were resuspended and fixed in 0.5 ml of PBS with 1% paraformaldehyde. For intracellular staining of CTLA-4, cells were first incubated for 20 min with Quantum Red-conjugated anti-CD4 mAb. After two washes, they were fixed and permeabilized with PermeaFix (Ortho Diagnostics, Raritan, NJ) and stained with PE-conjugated anti-CTLA-4 according to the manufacturer’s instructions. Stained cells were examined on a FACScan (BD Biosciences) and 5000 events were routinely collected and analyzed using CellQuest software (BD Biosciences).

Separate cocultures

Using a Cell Culture Insert system (Cell Culture Insert, 0.4 µm, high-density pore, 24-well format; BD Labware, France), responder CD4+ T cells (5 x 105) plus irradiated PBMC (2 x 106) were placed in the lower chamber and irradiated 4C8-costimulated cells (5 x 105) plus irradiated PBMC (2 x 106) were placed in the upper chamber in a 24-well plate for use with Cell Culture Inserts. Cells in both chambers were stimulated with anti-CD3 (25 ng/ml). As a control, responder T cells and irradiated 4C8-costimulated cells alone or in combination were also stimulated with anti-CD3 in the presence of irradiated PBMC. After 3 days of coculture, 1 x 105 responder T cells were transferred to round-bottom 96-well plates and pulsed with [3H]thymidine for 12 h as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of 4C8 costimulation on T cell proliferation

First, anti-CD28 and anti-4C8 mAbs were simultaneously compared for their costimulatory effects on proliferative responses of CD4+, CD4+CD45RA+, and CD4+CD45RO+ T cells in the presence of immobilized anti-CD3. While anti-CD3 alone led to a minimal increase in the T cell response, CD28 and 4C8 costimulation comparably induced [3H]thymidine incorporation into CD4+ T cells (Fig. 1GoA). Costimulation with anti-CD3 and different doses of anti-4C8 showed a dose-dependent response, but anti-4C8 alone had no effects (data not shown). In the responses of CD45RA+ and CD45RO+ T cells, anti-CD28 induced predominant costimulation of CD45RO+ cells, whereas anti-4C8 strongly stimulated both naive and memory T cells. The response of CD45RA+ cells to CD28 costimulation reached levels comparable to those observed with CD45RO+ cells at day 4 (data not shown). It has been reported that costimulation by T cell molecules other than CD28 leads to a transient increase of DNA synthesis but fails to expand the number of T cells (32). Thus, we examined whether the number of viable cells recovered from cultures is actually increased during the period from days 2 to 5 after CD28 and 4C8 costimulation. As shown in Fig. 1GoB, CD28 costimulation induced a gradual proliferation of CD4+ T cells initiated at day 3, whereas 4C8 costimulation led to a faster increase in the number of viable cells than CD28 costimulation. In contrast, stimulation with anti-CD3 or anti-4C8 alone decreased the number of cells. Collectively, these findings indicate that the 4C8-induced costimulatory effects are equivalent to those of CD28 costimulation with regard to full activation of CD4+ T cells with cellular expansion.



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 1. Proliferative responses of CD4+ T cells to CD28 and 4C8 costimulation. A, Whole CD4+, CD4+CD45RA+, and CD4+CD45RO+ T cells (2 x 105 cells/well), which were purified from PBMC of a donor, were cultured in 96-well plates precoated with anti-CD3 mAbs (0.1 µg/ml) in the presence of soluble anti-CD28 (5 µg/ml) or immobilized anti-4C8 (10 µg/ml) mAbs. The proliferation of cells was assessed by the addition of [3H]thymidine after 3 days of culture for the final 12 h. The results are presented as the mean ± SD of triplicate determinants. [3H]Thymidine uptake with anti-4C8 alone in the absence of anti-CD3 was consistently <500 cpm. One representative experiment of six is shown. B, Viable cells were counted at various time points by trypan blue dye exclusion under the same culture conditions as in A. Results were similar in four independent experiments.

 
Cytokine profile of 4C8-costimulated CD4+ T cells

Costimulation by non-CD28 molecules such as CD9 or CD11a constitutively expressed on resting T cells fails to sustain T cell proliferation because of a poor ability to stimulate IL-2 production (32). The weak effect on IL-2 production results in apoptosis of activated T cells followed by a decrease in the number of viable cells. In contrast, CD28 signaling induces high levels of IL-2 and consequently promotes T cell proliferation and survival while preventing apoptosis. Thus, the level of IL-2 production is essential for cellular expansion of T cells. To investigate the cytokine production profiles of CD4+ T cells activated by costimulation, we measured IL-2, as well as IL-4, IL-10, and IFN-{gamma}, in supernatants released from cultures costimulated by CD28 and 4C8 (Table IGo). Furthermore, to determine the changes in the cytokine production pattern after costimulation, we performed a second restimulation of costimulated cells with anti-CD3 plus anti-CD28. In the first stimulation culture, CD28 and 4C8 costimulation induced secretion of IL-2 at similar levels, a finding consistent with the cellular expansion observed on costimulation by 4C8. IL-4 was detected to a lesser extent than IL-2 in both costimulated cultures. Ample amounts of IL-10 were produced by CD28- and 4C8-costimulated cells at comparable levels. In the second stimulation culture, the overall levels of cytokines produced were reduced as compared to the first stimulation. However, high levels of IL-10 with no IL-2 or IL-4 were consistently found in cultures after 4C8 costimulation, whereas both IL-10 and IL-4 were produced at low levels by CD28-costimulated cells. This cytokine pattern was similar to that of Tr1 cells, which predominantly produce IL-10 but not IL-2 or IL-4, suggesting that 4C8 costimulation promotes the differentiation of CD4+ T cells into Tr1-like cells but not Th2 cells.


View this table:
[in this window]
[in a new window]
  		Table I. Cytokine profile of CD4+ T cells induced by costimulationa

 
4C8-costimulated cells highly express CD25 and intracellular CD152

High levels of CD25 are necessary for the continuous proliferation of activated T cells. We compared the induction of CD25+ T cells between CD28 and 4C8 costimulation at day 3 of culture and subsequently at days 6 and 9 during resting cultures. As shown in Fig. 2GoA, a high level of CD25 expression was maintained during the resting period after 4C8 costimulation compared to CD28 costimulation. In addition, it has been shown that CD4+CD25+ T cells have high levels of intracellular CD152 despite an insignificant expression on the cell surface (16). While CD28 costimulation led to a transient and marginal increase in cell surface expression of CD152, 4C8 costimulation greatly upregulated the expression until day 6, followed by a rapid return to the basal level at day 9 (Fig. 2GoB). However, when surface CD152 was untraceable, the majority of 4C8-costimulated cells expressed intracellular CD152, compared to only a small population of CD28-costimulated cells (Fig. 3Go).



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 2. CD25 and CD152 induced by CD28 and 4C8 costimulation. CD4+ T cells were stimulated with anti-CD3 plus anti-CD28 ({triangleup}) or anti-4C8 ({circ}). After 3 days of culture, the cells were washed and rested for 6 days. Cell surface expression of CD25 (A) and CD152 (B) on CD4+ T cells was assessed by flow cytometeric analysis at the indicated time points. One representative experiment of six is shown.

 


View larger version (84K):
[in this window]
[in a new window]
  		FIGURE 3. 4C8-costimulated cells express low levels of cell surface but high levels of intracellular CD152. On day 5 of a resting period after 3 days of costimulation, CD4+ T cells were examined for cell surface and intracellular CD152 as described in Materials and Methods. Similar results were obtained in four independent experiments.

 
Hyporesponsiveness of 4C8-costimulated T cells to anti-CD3 stimulation

Generally, Treg cells are unresponsive or hyporesponsive to polyclonal or Ag-specific stimulation (7). We determined the responsiveness of 4C8-costimulated cells to anti-CD3 stimulation in the presence of irradiated PBMC. Compared to control CD4+ T cells and CD28-costimulated cells, 4C8-costimulated cells showed hypoproliferative responses to anti-CD3 stimulation, but the weak proliferation was markedly augmented by the addition of IL-2 and anti-CD28 (Fig. 4Go). This result also indicates that the hyporesponsiveness is not due to activation-induced cell death.



View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 4. Hyporesponsiveness of 4C8-costimulated T cells to anti-CD3 stimulation and reversal of hyporesponsiveness by the addition of IL-2. CD4+ T cells (1 x 106/well) were stimulated in 48-well plates precoated with anti-CD3 (0.1 µg/ml) in the presence of soluble anti-CD28 (5 µg/ml) or immobilized anti-4C8 (10 µg/ml). After 3 days of culture, the activated cells were washed and further rested for 4 days without stimulation or exogenous IL-2. Subsequently, 1 x 105 costimulated cells, as well as freshly isolated CD4+ T cells as control, were incubated for 3 days with anti-CD3 (25 ng/ml) in the presence of 4 x 105 irradiated PBMC. Proliferation of cells was assessed as described in Fig. 1GoA. The results are representative of eight experiments.

 
Suppressive effects of 4C8-costimulated T cells on polyclonal activation of bystander T cells

CD4+ Treg cells possess the ability to suppress the proliferative responses of bystander T cells. We investigated the suppressive activity of 4C8-costimulated T cells using a coculture system in which freshly isolated CD4+ T cells (responders) are stimulated with soluble anti-CD3 and irradiated PBMC in the absence or presence of costimulated T cells (suppressor) that had been irradiated to abrogate their [3H]thymidine uptake. Irradiated CD4+ T cells also were used as a control for costimulated cells. Compared to control and CD28-costimulated cells, polyclonal activation of responder cells was suppressed up to 70% by 4C8-costimulated cells when added to cocultures at a 1:1 (responder:suppressor) ratio (Fig. 5GoA). However, the suppression may be due to the effect of CD4+CD25+ cells contained in the CD4+ population, because the regulatory cells could be activated and expanded by 4C8 costimulation. We purified CD4+ cells and CD4+CD25- cells from the same donor and cultured them with 4C8 costimulation. There was no difference in suppression between CD4+ and CD4+CD25- cells, suggesting that CD25- cells acquired suppressive activity by 4C8 costimulation (Fig. 5GoB). A strong and sustained expression of CD25 also was induced in 4C8-costimulated CD4+CD25- cells (data not shown). Moreover, we confirmed that CD45RO+ cells, but not CD45RA+ cells, differentiated into suppressor cells on costimulation by anti-4C8 (Fig. 6Go). CD28 costimulation did not induce such development in either population. The addition of exogenous IL-2 (100 U/ml) to cocultures with CD45RO+ cells partially restored the suppression.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 5. 4C8-costimulated cells induce suppression of polyclonal activation of bystander CD4+ T cells. Whole CD4+ T cells (A) and CD4+CD25- cells (B) were incubated for 3 days under costimulation culture conditions as indicated in Fig. 1GoA. After a 4-day resting period, 1 x 105 freshly isolated responder CD4+ cells were stimulated with anti-CD3 (25 ng/ml) in the presence of 4 x 105 PBMC and 1 x 105 costimulated cells, both of which were irradiated at 50 Gy, in 96-well round-bottom plates. Freshly isolated CD4+ cells (1 x 105/well) were also irradiated and used as control for costimulated cells. After 3 days, the proliferation of responder T cells was measured. The combination of cells was syngeneic in all experiments. The data are representative of >10 separate experiments in A and six separate experiments in B.

 


View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 6. CD4+CD45RO+ memory T cells, but not CD4+CD45RA+ naive T cells, display suppressor activity after 4C8 costimulation. CD4+CD45RO+ and CD4+CD45RA+ T cells were negatively isolated by magnetic separation. 4C8-costimulated cells were prepared and assessed for suppressor function as described in Fig. 5Go. Recombinant human IL-2 (100 U/ml) was added at the initiation of the suppression assay.

 
Dose-dependent reduction of IL-2 in coculture supernatants with 4C8-costimulated T cells

Previous reports have suggested that the suppressive effects of Treg cells are exerted by inhibiting IL-2 production by bystander T cells (18). To determine this, we measured the IL-2 concentration in coculture supernatants 24 h after costimulated T cells were added to cocultures at responder:suppressor ratios from 1:0 to 1:2 (Fig. 7Go). The addition of CD28-costimulated cells induced a slight increase in IL-2 production by responder CD4+ T cells at the higher ratios, 1:1 and 1:2, but had no effect at any ratio in assays with control CD4+ T cells. In sharp contrast, reduction of IL-2 was observed in cocultures with 4C8-costimulated cells in a dose-dependent fashion, in which IL-2 was undetectable in the supernatants at a ratio of 1:2.



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 7. Reduction of IL-2 in coculture supernatants with 4C8-costimulated T cells. CD28- and 4C8-costimulated CD4+ T cells were prepared and then suppression experiments were performed as described in Fig. 5Go. Different doses of costimulated cells or control CD4+ cells were titrated into wells containing 1 x 105 responder CD4+ cells. Culture supernatants were taken 24 h later for analysis of IL-2 production by ELISA. The results were presented as the mean of duplicate determinants. Similar results were obtained in four independent experiments.

 
Suppression by 4C8-costimulated cells is mediated in a cell contact-dependent, but an IL-10- and TGF-{beta}-dependent, manner

CD4+CD25+ T cells require cell contact for suppression, while Tr1 cells exert their suppressive effects in an IL-10-dependent manner (25, 33). Finally, we determined whether suppression by 4C8-costimulated cells is mediated by cell contact or inhibitory cytokines, IL-10 and TGF-{beta}. The proliferative responsiveness of responder CD4+ T cells was inhibited up to 70% by cocultures with irradiated 4C8-costimulated cells at a ratio of responders:suppressors of 1:1 (Fig. 8GoA). However, when the two populations were stimulated separately in chambers in the same well using a Cell Culture Insert system, significant suppression was not observed (Fig. 8GoA). The results suggest that the suppressive function of 4C8-costimulated cells requires physical contact with the responder population. Consistent with the results, the addition of neutralizing mAbs against IL-10 and TGF-{beta} alone or in combination had no effect on the suppression at a ratio of 1:2 (Fig. 8GoB).



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 8. Suppression by 4C8-costimulated cells requires cell-cell contact but is independent of IL-10 and TGF-{beta}. A, Freshly isolated responder CD4+ T cells (5 x 105) and/or irradiated 4C8-costimulated cells (5 x 105) were cultured in the presence of irradiated PBMC (2 x 106) in a 24-well plate. In addition, responder CD4+ T cells plus irradiated PBMC and irradiated 4C8-costimulated cells plus irradiated PBMC were placed in the lower and upper chambers of a Cell Culture Insert system, respectively (bottom bar). Cells were stimulated with anti-CD3 (25 ng/ml). After 3 days of coculture, 1 x 105 responder T cells were transferred to 96-well plates and pulsed with [3H]thymidine. B, Neutralizing Abs to IL-10 (5 µg/ml) and TGF-{beta} (5 µg/ml) alone or in combination were added to cultures for suppression at a ratio of responders:suppressors of 1:2.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although many molecules other than CD28 have been reported as having costimulatory function to enhance T cell proliferation (34), their costimulatory effect does not appear to induce prolonged proliferation with cellular expansion, mainly because of insufficient induction of IL-2 and Bcl-xL (32, 35, 36). Regarding cytokine production, CD28 costimulation promotes production of both Th1-type (IL-2 and IFN-{gamma}) and Th2-type (IL-4 and IL-10) cytokines (37), whereas signals via certain non-CD28 costimulatory molecules, such as CD2 and CD11a, predominantly lead to IL-10 production with no IL-2 or IL-4 (38, 39, 40). In the present study, the results of T cell proliferation and cytokine profile in the first costimulation cultures suggest that the 4C8 Ag functions as a costimulatory molecule comparable to CD28 but not non-CD28 molecules. Nevertheless, this Ag is clearly different from CD28 in some costimulatory functions. For instance, 4C8 costimulation induces very high expression of CD25 and CD152 on CD4+ T cells, indicating that it delivers more potent signals than CD28 costimulation, although this finding may be merely due to potent activation by costimulation with the 4C8 Ag, which is expressed on all T cells at high density compared to CD28 (31). Upon restimulation, the cytokine pattern of 4C8-costimulated cells became similar to that of IL-10-producing Tr1 cells (25), i.e., production of large amounts of IL-10 but no IL-2 or IL-4. The difference between CD28 and 4C8 costimulation is probably qualitative rather than quantitative. In fact, this is supported by the finding that 4C8-costimulated cells differentiated into CD4+ T cells with suppressive activity.

Suppressive properties are a critical feature of Treg cells. It has been reported that different types of Treg cells can be developed by in vitro and in vivo experimental procedures. Th3-type Treg cells induced by oral tolerance exert suppressive effects on other T cells, primarily through TGF-{beta} secretion (24, 41). Tr1 cells derived from naive CD4+ T cells by repetitive stimulation in the presence of IL-10 secrete large amounts of IL-10 which result in inhibition of naive T cell activation without cell contact (25). IL-10-producing Tr1-like T cells that were induced after several stimulations with allogeneic iDC require direct cell contact to exert their suppressive effect, but not soluble factors such as IL-10 and TGF-{beta} (26). In contrast, it is now accepted that a small subset of CD4+ T cells that coexpress CD25 naturally occur as professional suppressor cells to control peripheral tolerance or the development of autoimmune disease in mice and in humans (12, 13, 14, 15, 16, 42). Treg cells induced by 4C8 costimulation (termed 4C8 Treg cells) displayed not only such suppressor activity but also important characteristics reported on Treg cells: they showed high-level expression of intracellular CD152 and they were obviously hyporesponsive to anti-CD3 stimulation, although they were not as anergic as CD4+CD25+ T cells and Tr1 cells (18, 25). We also found that 4C8 Treg cells developed from CD4+CD25- T cells, suggesting that the suppression by 4C8 Treg cells is not due to CD4+CD25+ cells contained in the initial CD4+ population, and that a subpopulation of Treg cells may be originated from CD25- T cells in vivo.

Several mechanisms by which 4C8 Treg cells display suppressor function could be considered, including inhibition of IL-2 production by responder T cells, secretion of inhibitory cytokines, and direct suppression through cell-cell contact. Dose-dependent reduction of IL-2 in the supernatants from cocultures with 4C8 Treg cells suggests that IL-2 may play an important role to control the functions of the Treg cells. This notion is supported by the finding that the addition of exogenous IL-2 profoundly affected their hyporesponsiveness to anti-CD3 and suppressive activity (Figs. 4Go and 6Go). Inhibitory cytokines such as IL-10 and TGF-{beta} are thought to exert suppressive effects of Th3 and Tr1 regulatory cells. A more recent report has shown that cell surface-bound TGF-{beta} expressed on stimulated CD4+CD25+ T cells mediates their suppressive activity in mice (43). In our coculture system, irradiated 4C8 Treg cells and PBMC can still secrete a significant level of IL-10 (~300 pg/ml) in response to anti-CD3 (data not shown). However, a predominant suppressive role of IL-10 as well as TGF-{beta} is very unlikely from the results of Ab-blocking studies. The studies of separate cocultures strongly suggest that suppressive functions of 4C8 Treg cells are primarily mediated through a cell contact-dependent mechanism. Suppression by CD4+CD25+ T cells requires TCR activation in addition to cell-cell contact, indicating that a cell surface molecule(s) induced by activation mediates the suppression. Similarly, 4C8 costimulation may promote expression of suppression-mediating molecules on the cell surface of CD4+ T cells.

Although little information is available about the molecular basis for the generation of Treg cells, recent reports have suggested that costimulatory molecules play an important role in Treg cell differentiation. It has been shown that CD28 costimulation is required for the induction of CD4+CD25+ cells in nonobese diabetic mice because B7-deficient mice develop severe diabetes due to a profound decrease in regulatory cells (28). Blockade of B7-mediating signaling also prevents tolerance induction (27, 44). Even if CD4+CD25+ cells do originate from 4C8 Treg cells in vivo, the present findings do not necessarily conflict with the notion that the generation of Treg cells is dependent upon the B7/CD28 interaction. It should be noted that 4C8 Treg cells were derived from CD45RO+ memory cells, but not CD45RA+ naive cells, which had been developed by the conventional costimulation pathway via CD28. There is increasing evidence that, rather than CD28, non-CD28 costimulatory molecules may contribute to the differentiation of Treg cells. Ag presentation by murine APC overexpressing Serrate l induced Ag-specific Treg cells in vivo that can transfer tolerance to naive mice (45). It is possible that Serrate1/ligand Notch signaling functions as a costimulatory pathway to induce Treg cells. As described above, it has been proven that, while TCR stimulation alone causes anergy, costimulation by CD2, but not CD11a, results in the production of Tr1 cells (29). These findings suggest that Treg cells might be induced by an active process through multiple costimulation pathways.

Where could 4C8 Treg cell induction occur? In this regard, we emphasize that 4C8 Treg cells were derived from CD45RO+ memory T cells. The memory and naive T cell subsets exhibit distinct patterns of recirculation; the former have the ability to migrate through vascular endothelium into peripheral tissues, whereas the latter directly enter lymphoid organs from the circulation (46). Considering the periphery-prone migratory capacity of CD45RO+ T cells, 4C8 Treg cells may be generated in nonlymphoid peripheral tissues as well as in lymphoid tissues. This hypothesis is consistent with the suggestion that Ag presentation by parenchymal cells to CD45RO+ T cells plays an important role in the induction of peripheral tolerance (47, 48). When CD4+CD45RA+ and CD45RO+ T cells were cocultured with allogeneic, HLA-DR-expressing epithelial cells, a suppressive ability was found only in CD45RO+ T cells, whereas allospecific hyporesponsiveness was observed in both populations. Indeed, in renal transplant recipients, donor-specific CD4+ T cell hyporesponsiveness occurs predominantly in CD4+CD45RO+ T cells with a capacity for moving through the graft (49).

In summary, this study suggests that nonregulatory CD4+CD45RO+ T cells in the periphery may have the capacity to differentiate into Treg cells depending on an active process via 4C8 costimulation. Our data also support the possibility that there are multiple non-CD28 costimulation pathways to induce Treg cells in the periphery.


   Acknowledgments
 
We thank Mamiko Senba for her expert technical assistance.


   Footnotes
 
1 This work was partially supported by a grant from the Japan Rheumatism Foundation. Back

2 Address correspondence and reprint requests to Dr. Jun-ichi Masuyama, Division of Rheumatology and Clinical Immunology, Department of Medicine, Jichi Medical School, Minami-Kawachi, Tochigi, 329-0498, Japan. E-mail address: jmas{at}jichi.ac.jp Back

3 Abbreviations used in this paper: Treg, regulatory T; iDC, immature dendritic cell; Tr1, T regulatory 1; 4C8 Treg cell, Treg cell induced by 4C8 costimulation. Back

Received for publication February 20, 2002. Accepted for publication July 31, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Nossal, G. J.. 1994. Negative selection of lymphocytes. Cell 76:229.[Medline]
  2. Rocha, B., H. von Boehmer. 1991. Peripheral selection of the T cell repertoire. Science 251:1225.[Abstract/Free Full Text]
  3. Schwartz, R. H.. 1990. A cell culture model for T lymphocyte clonal anergy. Science 248:1349.[Abstract/Free Full Text]
  4. Miller, J. F., W. R. Heath. 1993. Self-ignorance in the peripheral T-cell pool. Immunol. Rev. 133:131.[Medline]
  5. Webb, S., C. Morris, J. Sprent. 1990. Extrathymic tolerance of mature T cells: clonal elimination as a consequence of immunity. Cell 63:1249.[Medline]
  6. Roncarolo, M. G., M. K. Levings. 2000. The role of different subsets of T regulatory cells in controlling autoimmunity. Curr. Opin. Immunol. 12:676.[Medline]
  7. Shevach, E. M.. 2000. Regulatory T cells in autoimmunity. Annu. Rev. Immunol. 18:423.[Medline]
  8. Sakaguchi, S.. 2001. Policing the regulators. Nat. Immunol. 2:283.[Medline]
  9. Waldmann, H., S. Cobbold. 2001. Regulating the immune response to transplants: a role for CD4+ regulatory cells?. Immunity 14:399.[Medline]
  10. Fowell, D., D. Mason. 1993. Evidence that the T cell repertoire of normal rats contains cells with the potential to cause diabetes: characterization of the CD4+ T cell subset that inhibits this autoimmune potential. J. Exp. Med. 177:627.[Abstract/Free Full Text]
  11. Powrie, F., M. W. Leach, S. Mauze, L. B. Caddle, R. L. Coffman. 1993. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in CB-17 scid mice. Int. Immunol. 5:1461.[Abstract/Free Full Text]
  12. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor {alpha}-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151.[Abstract]
  13. Asano, M., M. Toda, N. Sakaguchi, S. Sakaguchi. 1996. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 184:387.[Abstract/Free Full Text]
  14. Dieckmann, D., H. Plottner, S. Berchtold, T. Berger, G. Schuler. 2001. Ex vivo isolation and characterization of CD4+CD25+ T cells with regulatory properties from human blood. J. Exp. Med. 193:1303.[Abstract/Free Full Text]
  15. Stephens, L. A., C. Mottet, D. Mason, F. Powrie. 2001. Human CD4+CD25+ thymocytes and peripheral T cells have immune suppressive activity in vitro. Eur. J. Immunol. 31:1247.[Medline]
  16. Jonuleit, H., E. Schmitt, M. Stassen, A. Tuettenberg, J. Knop, A. H. Enk. 2001. Identification and functional characterization of human CD4+CD25+ T cells with regulatory properties isolated from peripheral blood. J. Exp. Med. 193:1285.[Abstract/Free Full Text]
  17. Levings, M. K., R. Sangregorio, M. G. Roncarolo. 2001. Human CD25+CD4+ T regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function. J. Exp. Med. 193:1295.[Abstract/Free Full Text]
  18. Thornton, A. M., E. M. Shevach. 1998. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188:287.[Abstract/Free Full Text]
  19. Thornton, A. M., E. M. Shevach. 2000. Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J. Immunol. 164:183.[Abstract/Free Full Text]
  20. Takahashi, T., T. Tagami, S. Yamazaki, T. Uede, J. Shimizu, N. Sakaguchi, T. W. Mak, S. Sakaguchi. 2000. Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 192:303.[Abstract/Free Full Text]
  21. Read, S., V. Malmstrom, F. Powrie. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192:295.[Abstract/Free Full Text]
  22. Itoh, M., T. Takahashi, N. Sakaguchi, Y. Kuniyasu, J. Shimizu, F. Otsuka, S. Sakaguchi. 1999. 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. 162:5317.[Abstract/Free Full Text]
  23. 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.[Medline]
  24. Chen, Y., V. K. Kuchroo, J. Inobe, D. A. Hafler, H. L. Weiner. 1994. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265:1237.[Abstract/Free Full Text]
  25. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. de Vries, M. G. Roncarolo. 1997. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389:737.[Medline]
  26. Jonuleit, H., E. Schmitt, G. Schuler, J. Knop, A. H. Enk. 2000. Induction of interleukin 10-producing, nonproliferating CD4+ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J. Exp. Med. 192:1213.[Abstract/Free Full Text]
  27. Liu, L., V. K. Kuchroo, H. L. Weiner. 1999. B7.2 (CD86) but not B7.1 (CD80) costimulation is required for the induction of low dose oral tolerance. J. Immunol. 163:2284.[Abstract/Free Full Text]
  28. Salomon, B., D. J. Lenschow, L. Rhee, N. Ashourian, B. Singh, A. Sharpe, J. A. Bluestone. 2000. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12:431.[Medline]
  29. Wakkach, A., F. Cottrez, H. Groux. 2001. Differentiation of regulatory T cells 1 is induced by CD2 costimulation. J. Immunol. 167:3107.[Abstract/Free Full Text]
  30. Taguchi, O., K. Kontani, H. Ikeda, T. Kezuka, M. Takeuchi, T. Takahashi. 1994. Tissue-specific suppressor T cells involved in self-tolerance are activated extrathymically by self-antigens. Immunology 82:365.[Medline]
  31. Masuyama, J., T. Yoshio, K. Suzuki, S. Kitagawa, M. Iwamoto, T. Kamimura, D. Hirata, A. Takeda, S. Kano, S. Minota. 1999. Characterization of the 4C8 antigen involved in transendothelial migration of CD26hi T cells after tight adhesion to human umbilical vein endothelial cell monolayers. J. Exp. Med. 189:979.[Abstract/Free Full Text]
  32. Yashiro, Y., X. G. Tai, K. Toyo-oka, C. S. Park, R. Abe, T. Hamaoka, M. Kobayashi, S. Neben, H. Fujiwara. 1998. A fundamental difference in the capacity to induce proliferation of naive T cells between CD28 and other co-stimulatory molecules. Eur. J. Immunol. 28:926.[Medline]
  33. Roncarolo, M. G., R. Bacchetta, C. Bordignon, S. Narula, M. K. Levings. 2001. Type 1 T regulatory cells. Immunol. Rev. 182:68.[Medline]
  34. Watts, T. H., M. A. DeBenedette. 1999. T cell co-stimulatory molecules other than CD28. Curr. Opin. Immunol. 11:286.[Medline]
  35. Boise, L. H., M. Gonzalez-Garcia, C. E. Postema, L. Ding, T. Lindsten, L. A. Turka, X. Mao, G. Nunez, C. B. Thompson. 1993. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74:597.[Medline]
  36. Boise, L. H., A. J. Minn, P. J. Noel, C. H. June, M. A. Accavitti, T. Lindsten, C. B. Thompson. 1995. CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-xL. Immunity 3:87.[Medline]
  37. Romagnani, S.. 1996. Th1 and Th2 in human diseases. Clin. Immunol. Immunopathol. 80:225.[Medline]
  38. Bleijs, D. A., R. de Waal-Malefyt, C. G. Figdor, Y. van Kooyk. 1999. Co-stimulation of T cells results in distinct IL-10 and TNF-{alpha} cytokine profiles dependent on binding to ICAM-1, ICAM-2 or ICAM-3. Eur. J. Immunol. 29:2248.[Medline]
  39. Bullens, D. M., K. Rafiq, L. Charitidou, X. Peng, A. Kasran, P. A. Warmerdam, S. S. Van Gool, J. L. Ceuppens. 2001. Effects of co-stimulation by CD58 on human T cell cytokine production: a selective cytokine pattern with induction of high IL-10 production. Int. Immunol. 13:181.[Abstract/Free Full Text]
  40. Dong, H., G. Zhu, K. Tamada, L. Chen. 1999. B7–H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat. Med. 5:1365.[Medline]
  41. Fukaura, H., S. C. Kent, M. J. Pietrusewicz, S. J. Khoury, H. L. Weiner, D. A. Hafler. 1996. Induction of circulating myelin basic protein and proteolipid protein-specific transforming growth factor-{beta}1-secreting Th3 T cells by oral administration of myelin in multiple sclerosis patients. J. Clin. Invest. 98:70.[Medline]
  42. Shevach, E. M., A. Thornton, E. Suri-Payer. 1998. T lymphocyte-mediated control of autoimmunity. Novartis Found. Symp. 215:200.[Medline]
  43. Nakamura, K., A. Kitani, W. Strober. 2001. Cell contact-dependent immunosuppression by CD4+CD25+ regulatory T cells is mediated by cell surface-bound transforming growth factor {beta}. J. Exp. Med. 194:629.[Abstract/Free Full Text]
  44. Tsitoura, D. C., R. H. DeKruyff, J. R. Lamb, D. T. Umetsu. 1999. Intranasal exposure to protein antigen induces immunological tolerance mediated by functionally disabled CD4+ T cells. J. Immunol. 163:2592.[Abstract/Free Full Text]
  45. Hoyne, G. F., I. Le Roux, M. Corsin-Jimenez, K. Tan, J. Dunne, L. M. Forsyth, M. J. Dallman, M. J. Owen, D. Ish-Horowicz, J. R. Lamb. 2000. Serrate1-induced notch signalling regulates the decision between immunity and tolerance made by peripheral CD4+ T cells. Int. Immunol. 12:177.[Abstract/Free Full Text]
  46. Mackay, C. R.. 1993. Homing of naive, memory and effector lymphocytes. Curr. Opin. Immunol. 5:423.[Medline]
  47. Marelli-Berg, F. M., R. I. Lechler. 1999. Antigen presentation by parenchymal cells: a route to peripheral tolerance?. Immunol. Rev. 172:297.[Medline]
  48. Marelli-Berg, F. M., A. Weetman, L. Frasca, S. J. Deacock, N. Imami, G. Lombardi, R. I. Lechler. 1997. Antigen presentation by epithelial cells induces anergic immunoregulatory CD45RO+ T cells and deletion of CD45RA+ T cells. J. Immunol. 159:5853.[Abstract]
  49. Baker, R. J., M. P. Hernandez-Fuentes, P. A. Brookes, A. N. Chaudhry, R. I. Lechler. 2001. The role of the allograft in the induction of donor-specific T cell hyporesponsiveness. Transplantation 72:480.[Medline]



This article has been cited by other articles:


Home page
 ReproductionHome page
Y. Lin, H. Wang, W. Wang, S. Zeng, Y. Zhong, and D.-J. Li
Prevention of embryo loss in non-obese diabetic mice using adoptive ITGA2+ISG20+ natural killer-cell transfer
Reproduction, June 1, 2009; 137(6): 943 - 955.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. Feunou, S. Vanwetswinkel, F. Gaudray, M. Goldman, P. Matthys, and M. Y. Braun
Foxp3+CD25+ T Regulatory Cells Stimulate IFN-{gamma}-Independent CD152-Mediated Activation of Tryptophan Catabolism That Provides Dendritic Cells with Immune Regulatory Activity in Mice Unresponsive to Staphylococcal Enterotoxin B
J. Immunol., July 15, 2007; 179(2): 910 - 917.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Karim, C. I. Kingsley, A. R. Bushell, B. S. Sawitzki, and K. J. Wood
Alloantigen-Induced CD25+CD4+ Regulatory T Cells Can Develop In Vivo from CD25-CD4+ Precursors in a Thymus-Independent Process
J. Immunol., January 15, 2004; 172(2): 923 - 928.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
S. Vigouroux, E. Yvon, H.-J. Wagner, E. Biagi, G. Dotti, U. Sili, C. Lira, C. M. Rooney, and M. K. Brenner
Induction of Antigen-Specific Regulatory T Cells following Overexpression of a Notch Ligand by Human B Lymphocytes
J. Virol., October 15, 2003; 77(20): 10872 - 10880.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Masuyama, J.-i.
Right arrow Articles by Minota, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Masuyama, J.-i.
Right arrow Articles by Minota, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS