HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zheng, S. G. Articles by Horwitz, D. A. Search for Related Content PubMed PubMed Citation Articles by Zheng, S. G. Articles by Horwitz, D. A. Pubmed/NCBI databases Substance via MeSH Medline Plus Health Information Stem Cells

Generation Ex Vivo of TGF--Producing Regulatory T Cells from CD4⁺CD25^- Precursors¹

Division of Rheumatology and Immunology, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previously we reported that TGF- has an important role in the generation and expansion of human "professional" CD4⁺CD25⁺ regulatory T cells in the periphery that have a cytokine-independent mechanism of action. In this study we used low-dose staphylococcal enterotoxin to induce T cell-dependent Ab production. We report that TGF- induces activated CD4⁺CD25^- T cells to become Th3 suppressor cells. While stimulating CD4⁺ cells with TGF- modestly increased expression of CD25 and intracellular CTLA-4 in primary cultures, upon secondary stimulation without TGF- the total number and those expressing these markers dramatically increased. This expansion was due to both increased proliferation and protection of these cells from activation-induced apoptosis. Moreover, adding as few as 1% of these TGF--primed CD4⁺ T cells to fresh CD4⁺ cells and B cells markedly suppressed IgG production. The inhibitory effect was mediated by TGF- and was also partially contact dependent. Increased TGF- production was associated with a decreased production of IFN- and IL-10. Depletion studies revealed that the precursors of these TGF--producing CD4⁺ suppressor cells were CD25 negative. These studies provide evidence that CD4⁺CD25⁺ regulatory cells in human blood consist of at least two subsets that have TGF--dependent and independent mechanisms of action. TGF- has an essential role in the generation of both of these T suppressor cell subsets from peripheral T cells. The ability to induce CD4⁺ and CD8⁺ cells to become regulatory cells ex vivo has the potential to be useful in the treatment of autoimmune diseases and to prevent transplant rejection.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transforming growth factor- is a multifunctional cytokine with both positive and negative effects on the immune system (1). While its inhibitory effects are well known (2), this cytokine can also induce IL-2-activated human CD4 and CD8⁺ T cells to develop potent down-regulatory effects. Previously, we have reported that TGF- can induce mitogen-stimulated CD8⁺ T cells to suppress T cell-dependent Ab production (3, 4). We have also observed that TGF- can generate CD4⁺CD25⁺ cells that have very potent contact-dependent suppressive effects on alloactivated on CD8⁺ T cells (5). These suppressive effects were similar to, if not identical with, the "professional" CD4⁺CD25⁺ T cells described by others (6, 7). Here we report that TGF- costimulates CD4⁺CD25^- cells and induces them to become potent suppressors of Ab production by a TGF--dependent mechanism of action.

To study T cell/B cell interactions, we have used model systems where the confounding effects of additional APCs can be excluded. In investigating how CD8⁺ T cells become suppressor cells, we used a mitogenic combination of anti-CD2 Abs for this purpose (4). More recently, Stohl and Elliott (8) have found that the bacterial superantigen staphylococcal enterotoxin B (SEB)³ has a similar effect. This superantigen binds to T cells expressing V8 and HLA-DR on B cells and in high doses eliminates the latter cells by activation-induced apoptosis. However, in low doses SEB induces Ab production by a direct interaction of T cells and B cells without additional accessory cells (8). We have found that the magnitude of this response is controlled by TGF- produced by cells in the immediate microenvironment. Exposure of CD4⁺ cells to TGF- at the time they were activated with SEB altered the genetic program of these cells. Upon restimulation, these CD4⁺ T cells expanded, expressed high levels of CD25 and CTLA-4, and developed potent TGF--dependent suppressive activity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
The following Abs were used: anti-CD3, anti-CD8, anti-CD20, anti-CD25, anti-CTLA-4, anti-CD122, control IgG (all from BD PharMingen, San Diego, CA); anti-CD8 (OKT8), anti-CD11b (OKM1), anti-CD74 (L243) (all hybridomas from American Type Culture Collection, Manassas, VA); anti-CD16 (3G8, kindly provided by Dr. J. Unkeless, Mount Sinai Medical School, New York, NY); anti-CD103 (DAKO, Carpinteria, CA); and anti-TGF- (R&D Systems, Minneapolis, MN). SEB was purchased from Sigma-Aldrich (St. Louis, MO). TGF-1 and 1L-2 were from R&D Systems.

Lymphocyte isolation

PBMC were prepared from heparinized venous blood of healthy adult volunteers by Ficoll-Hypaque (Atlanta Biologicals, Norcross, GA) density gradient centrifugation. To prepare PBL, PBMC were added to a continuous Percoll (Pharmacia, Piscataway, NJ) density gradient and the high-density fraction was collected (9). T cells were prepared by immediate rosetting with 2-aminoethylisothiouronium bromide-treated SRBC (10). T cells were further purified from rosetting cells by staining with Abs to CD16, CD74, and CD11b and depleting reactive cells using immunomagnetic beads (Dynal Biotech, Great Neck, NY). The percentage of CD3⁺ cells in this fraction was usually >96%.

CD4⁺ cells were prepared from T cells that were stained with Abs to CD8 by negative selection using immunomagnetic beads. Purity of CD4⁺ cells was usually 95%. CD25-depleted CD4 T cells were prepared from CD4⁺ T cells by cell sorting. Before sorting, the CD4⁺CD25⁺ population was 3–5% among total CD4⁺ T cells. After sorting, the CD4⁺CD25⁺ population was <0.3%. In some experiments CD8⁺ cells were prepared by negative selection (4). To obtain B cells, nonrosetting PBMC were treated with 5 mM L-leucine methyl ester (LME) for depletion of monocytes and NK cells (11). These cells were stained with Abs to CD3, CD16, and CD11b and depleted of reactive cells by immunomagnetic beads. The resulting population was >90% CD20⁺ and <0.5% CD3⁺.

Generation and assay of regulatory CD4⁺ cells

CD4⁺ cells (2 x 10⁶) and irradiated B cells as superantigen-presenting cells (SPC) (2 x 10⁶) were cultured with SEB (0.01 ng/ml) in the presence or absence of TGF-1 (0.1–10 ng/ml) in AIM V serum-free medium (Invitrogen, Carlsbad, CA) for 5–6 days in 24-well plates (Multiwell; BD Labware, Franklin Lakes, NJ). Serum-free medium was used because TGF- binds to various serum components (12). The cells were washed and various numbers were added to fresh autologous CD4⁺ cells (5 x 10⁴/well) and B cells (5 x 10⁴/well) in 96-well flat-bottom microtiter plates (Falcon, Lincoln Park, NJ) and cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% heated-inactivated FCS (HyClone Laboratories, Logan, UT), 100 U/ml penicillin (Invitrogen), 100 g/ml streptomycin (Invitrogen), 2 mM L-glutamine (Invitrogen), 100 mM Na-pyruvate (Invitrogen), and 10 mM HEPES (Invitrogen). After culture in complete medium for 7–10 days, the supernatants were harvested and IgG content was determined by ELISA (3). The variation between triplicate wells was usually <10%. In some experiments, proliferation in secondary cultures was assessed by uptake of tritiated thymidine added for the final 18 h and assessment of cell death by annexin V staining as performed by flow cytometry according to instructions from the manufacturer (BD PharMingen).

Measurement of cytokine production

Primed CD4⁺ cells were extensively washed and restimulated with 0.01 ng/ml SEB for 24, 48, and 72 h in serum-free AIM V serum for TGF- production, and with complete medium for production of other cytokines. In some experiments, IL-2 (10 U/ml) was added to the cultures. Active TGF- was determined by mink lung epithelial cells transfected with a luciferase gene construct (13). Several concentrations of TGF- were included to generate a standard curve, and the variation between triplicate samples was always <10%. Supernatants were also tested in duplicate using ELISA kits for IL-4, IL-10, and IFN- (BioSource International, Camarillo, CA). The limits of detection of the assays performed were 7.8–500 pg/ml (for IL-4 and IL-10) and 7.8–1000 pg/ml (for IFN-).

Transwell studies

CD4⁺ T cells and CD25-depleted CD4⁺ T cells were primed with SEB with or without TGF- as described above. After 5–6 days, these cells were extensively washed, mixed with fresh CD4⁺ cells in different ratios (1:5, 1:20, and 1:100), and added to the wells of a 24-well plate containing CD4⁺ cells, B cells, and SEB. In some wells, the conditioned CD4⁺ cells and irradiated SPC were separated from responder cells by the insert of a Transwell plate (Corning Costar, Cambridge, MA). Supernatants were collected after 10 days and assayed for IgG content by an ELISA.

Immunofluorescence analysis

Cell surface Ag expression on effector CD4⁺ T cells was determined by flow cytometry. CD4⁺ T cells (10⁵) were labeled with FITC-conjugated (anti-CD4) and PE-conjugated (anti-CD25) mAbs. After 20 min at 4°C in PBS with 0.1% BSA and 0.02 mM NaN₃, the cells were washed and analyzed on a FACStar^Plus flow cytometer using CellQuest software (BD Biosciences, San Jose, CA).

For staining of intracellular CTLA-4, activated CD4⁺ T cells were harvested and washed twice with PBS. After staining for surface markers, they were fixed and permeabilized with 4% paraformaldehyde and 0.1% saponin buffer for 20 min. After two washes, the cells were incubated with normal mouse serum to inhibit nonspecific binding followed by incubation for 20 min with PE-anti-CTLA-4 or PE-conjugated control mAbs (cIgG). All staining was performed on ice and at least 10,000 viable cells were analyzed.

Statistical analysis

Significance of the results was analyzed by Student’s t test performed with GraphPad Prism software (GraphPad, San Diego, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TGF- controls SEB-induced T cell-dependent Ab production

Our initial studies suggested that the magnitude of IgG induced by low-dose SEB is controlled by TGF- produced by cells in the immediate microenvironment. In cultures containing PBMC or PBL, the dose of SEB that induced IgG production correlated inversely with the amount of TGF- detected (Fig. 1, A and B). By contrast, throughout this concentration range of SEB, IgG production remained high and levels of TGF- were minimal in cultures containing purified CD4⁺ cells and B cells. Because NK cells and monocytes are the major sources of TGF- in PBMC (4), we lysed these cells with LME and observed a marked increase in IgG production. However, adding back nanomolar concentrations of TGF- to the LME-treated PBMC decreased IgG production to background levels (Fig. 1C).

View larger version (18K): [in this window] [in a new window]   FIGURE 1. TGF- regulates T cell-dependent Ab production induced by low-dose superantigen. A, PBMC or PBL (1 x 105/well), or purified CD4 and B cells (5 x 104/well), in 96-well plates were stimulated with graded doses of SEB for 10 days and the supernatants were examined for IgG content. Unstimulated cells produced 95 ng/ml IgG. B, Supernatants from parallel cultures were harvested at 48 h and assayed for active TGF-. C, PBMC were stimulated before and after treatment with LME. A TGF- (10 or 100 pg/ml) was added as shown and stimulated with SEB as above. Each of these studies was performed at least three times with similar results.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Effect of anti-TGF- on SEB-induced IgG synthesis. A, PBMC (1 x 105/well) were stimulated with SEB (0.01 ng/ml) with or without anti-TGF- (10 µg/ml) for 10 days as in Fig. 1. B, Anti-TGF- or control IgG1 was added at the days indicated and the effect on IgG production is shown. These studies have been performed at least four times with similar results.

Priming of T cells with TGF- enables CD4⁺ cells to expand more rapidly after restimulation and protects them from activation-induced cell death

The next series of experiments revealed several costimulatory effects of TGF- on CD4⁺ cells. Others have reported that after mitogen activation of T cells in the presence of TGF- there is marked enhancement of proliferation upon restimulation (14). We have confirmed this finding with SEB and have found that, in a mixed population of human peripheral blood CD4⁺ and CD8⁺ T lymphocytes, TGF- had a selective effect on CD4⁺ cells (Fig. 3A). After restimulation of TGF--primed cells with low-dose SEB, the absolute number of CD4⁺ but not CD8⁺ cells markedly increased (Fig. 3B). This result differs from the findings of others who reported positive effects of TGF- on mouse CD8⁺ cells (15). TGF- also significantly increased the number of purified CD4⁺ cells in primary cultures (p = 0.04), but this increase was modest.

View larger version (33K): [in this window] [in a new window]   FIGURE 3. CD4+ cells primed with TGF- are selectively expanded when restimulated. T cells (2 x 106/well) and an equal number of irradiated B cells as SPC were cultured with SEB (0.01 ng/ml) with or without TGF- (0.1 or 1 ng/ml) for 5 days. The cells were washed and restimulated with SEB (0.01 ng/ml) without TGF- for 3 days. A, Cells stained by anti-CD4 Abs. B, Cells stained by anti-CD8 Abs. The bars indicate the mean and SEM of triplicate cultures. The horizontal lines indicate the starting number of CD4+ and CD8+ cells. This study is representative of nine separate experiments.

View larger version (37K): [in this window] [in a new window]   FIGURE 4. Expansion of TGF--primed CD4+ cells after restimulation and protection from activation-induced apoptosis. Two million CD4+ cells and an equal number of irradiated B cells were cultured for 6 days in serum-free medium with SEB (0.01 ng/ml) and TGF-1 (10 ng/ml) (CD4TGF-) or without this cytokine (CD4con). The cells were washed, resuspended in medium containing 10% FCS, and rested overnight. Two million CD4+ cells and SPC were then restimulated with low-dose SEB plus IL-2 (10 U/ml). A, Sequential cell counts of triplicate cultures. The mean ± SEM are indicated. Significant differences between CD4con and CD4TGF- (t test) are shown (*, p < 0.05; **, p < 0.01). B, Tritiated thymidine uptake was assessed 3 days after restimulation. Six days after restimulation, viability of CD4con and CD4TGF- was assessed by flow cytometry. C and D, Cell size (forward scatter) on the abscissa and cell granularity (side scatter) on the ordinate. E, Annexin V staining. Gray line, CD4TGF-; black line, CD4con. These data are representative of four experiments.

View larger version (38K): [in this window] [in a new window]   FIGURE 5. Costimulatory effects of TGF- on CD25 and CTLA-4 expression by CD4+ cells. CD4+ cells (2 x 106) with SPC were stimulated with low-dose SEB with or without TGF- (10 ng/ml) for 6 days. CD4+ cells (2 x 106) were then restimulated with SEB as described above for an additional 3 days. Upper panels, CD4+ cells double stained for surface CD25 and intracellular CTLA-4 after primary culture. Lower panels, The effects of restimulation. The cell numbers and percentages that stained for each marker are indicated. The results are representative of six experiments.

Cytokines produced by CD4⁺ cells primed with TGF-

CD4⁺ T cells primed with TGF- had a much greater capacity to produce the active form of this cytokine. While TGF- produced by T cells is generally in the latent precursor form (16, 17), CD4⁺ cells primed in the presence of TGF- and restimulated with SEB produced greater amounts of active TGF- in comparison with controls (Fig. 6). This effect was markedly accentuated by including IL-2 in secondary cultures where a dose-dependent effect of TGF- was documented. Priming CD4⁺ cells with 10 ng/ml TGF- resulted in a much greater amount of active TGF- than cells primed with 0.1 ng/ml TGF- (Fig. 6).

View larger version (18K): [in this window] [in a new window]   FIGURE 6. CD4+ cells primed with TGF- produce the active form of this cytokine upon restimulation. CD4+ cells (2 x 106/well) were stimulated with low-dose SEB for 6 days with the concentrations of TGF- shown in nanograms per milliliter. The cells were then restimulated for 1–3 days with SEB in the presence or absence of IL-2 (10 U/ml). The supernatants were then tested for active TGF-. The results shown indicate the mean ± SEM of three independent experiments. In cultures without IL-2, the mean values of TGF- produced by priming CD4+ cells with 1 or 10 ng/ml were significantly greater than control CD4+ cells (p = 0.05–0.006) at all times studied. When CD4+ cells were restimulated with IL-2, TGF- production by CD4+ T cells primed with this cytokine were even greater, and all values were significantly higher than control CD4+ cells (p < 0.002). TGF- carry-over from primary cultures was excluded by examining the supernatants of primed CD4+ cells that were not restimulated with SEB.

View larger version (42K): [in this window] [in a new window]   FIGURE 7. IFN- and IL-10 production by CD4+ cells conditioned with TGF-. CD4+ cells (2 x 106/well) with SPC were stimulated with low-dose SEB and the doses of TGF- indicated for 6 days and restimulated with SEB. A, Time course of IFN- production and effect of anti-TGF-. B, Similar studies of IL-10 production. The mean ± SEM of three separate experiments is shown. Mean values of cytokines produced by TGF--primed CD4+ cells were significantly different from control CD4+ cells (*, p < 0.05).

The suppressive effects of CD4⁺ cells primed with TGF- and precursor phenotype of these cells

Consistent with its known immunosuppressive effects (1), treatment of CD4⁺ cells with TGF- for 48 h abolished the capacity of these cells to provide B cell help for Ab production (data not shown). However, even more interesting is the fact that the addition of TGF--primed CD4⁺ cells to fresh CD4 and B cells markedly suppressed SEB-induced IgG production. In nine separate experiments, evaluating various ratios of CD4 regulatory cells to helper cells, the addition of 5% TGF--treated CD4⁺ cells to fresh CD4⁺ cells had marked suppressive effects. In five of these experiments, the addition of only 1% of the TGF--primed CD4⁺ cells suppressed IgG production by >50%. The mean value of the suppressive activity is shown in Fig. 8A. This inhibition was completely abolished by anti-TGF- Abs (Fig. 8B). With CD4 control cells anti-TGF- had no effect on the production of IgG (Fig. 8C).

View larger version (23K): [in this window] [in a new window]   FIGURE 8. CD4+ T cells primed with TGF- develop suppressive activity mediated by this cytokine. CD4+ cells and SPC (2 x 106/well) were stimulated with low-dose SEB with 1 ng/ml TGF- (CD4reg) or without TGF- (CD4con) for 6 days. A, These cells were mixed with fresh CD4 and B cells in the percentages indicated and stimulated with SEB for an additional 10 days. B, Some cultures contained CD4reg and anti-TGF- (10 µg/ml) or a similar amount of an isotype-matched control IgG. C, Other cultures contained CD4con and anti-TGF-. The mean IgG values ± SEM from nine experiments are shown. The horizontal line indicates the mean IgG value produced by SEB-stimulated CD4 and B cells without additional CD4 primed cells. Values of p are indicated.

The phenotype and functional properties of SEB-stimulated CD4⁺CD25^- cells were indistinguishable from total CD4⁺ cells. As before, priming with TGF- resulted in a modest increase in the total cell number and those expressing CD25 and CTLA-4 in primary cultures (Fig. 9A). Upon restimulation with SEB there was a marked increase in each of these populations (Fig. 9B), and these cells developed suppressive activity that was neutralized by anti-TGF- (Fig. 9C). CD4⁺CD25^- cells primed with TGF- also produced increased levels of TGF- after restimulation with SEB (Fig. 10). The only difference noted was in the optimal dose of TGF- required for conditioning. With total CD4⁺ cells, maximal costimulatory and suppressive effects were noted with concentrations of TGF- between 0.1 and 1 ng/ml. With CD25-depleted CD4⁺ cells, maximal production of TGF- and suppressive effects required 10 ng/ml (Figs. 9C and 10).

View larger version (23K): [in this window] [in a new window]   FIGURE 9. Phenotype and function of CD4+CD25- cells conditioned with TGF-. After depletion of CD25+ cells by cell sorting, CD4+CD25- cells and SPC (2 x 106/well) were stimulated with low-dose SEB for 6 days with or without graded concentrations of TGF-. A, The total number of CD4+ cells and those expressing CD25 and CTLA-4 in primary culture. B, The phenotype of TGF--primed and control CD4+ cells after restimulation for 3 days. C, Suppressive effects of the addition of 4% of CD4+ cells primed with TGF- to fresh T cells on IgG production. The horizontal line indicates the starting cell number of CD4+ cells (A and B) or IgG production (C). These experiments have been repeated five to eight times with similar results.

View larger version (50K): [in this window] [in a new window]   FIGURE 10. Comparison of active TGF- production by total CD4+ and CD4+CD25- cells. Total CD4+ cells or CD4+CD25- cells (2 x 106/well) and SPC were stimulated with low-dose SEB and graded concentrations of TGF- for 6 days; after extensive washing they were restimulated with SEB for 72 h. Values for active TGF- are shown. This experiment was repeated four times with similar results.

Role of cell contact in suppression of CD4⁺ cells conditioned with TGF-

Although suppression of IgG synthesis was mediated by TGF-, separation of the CD4⁺ regulatory cells from the responder cells by Transwells revealed a surprising result. With high ratios of suppressor cells to responder cells there was a partial loss of suppression with cell separation, but a complete loss was observed when the number of regulatory cells was reduced. In the experiment shown in Fig. 10, the suppressive activity by CD25-depleted CD4⁺ cells was even greater than that of total CD4⁺ cells. In other experiments the suppressive activity of each population was similar (Fig. 11).

View larger version (42K): [in this window] [in a new window]   FIGURE 11. Role of cell contact in suppression of CD4+ cells conditioned with TGF-. CD4reg or CD4con were prepared from total CD4+ cells (upper panels) or CD4+CD25- cells (lower panels) (see Materials and Methods). Various percentages of these TGF--primed CD4+ cells were added to fresh CD4 and B cells (5 x 104/well) or to Transwells that also contained SPC. The cells were stimulated with low-dose SEB for 10 days and IgG was measured. The result shown is representative of three experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This report provides further evidence that TGF- has an important role in the generation of regulatory T cells in addition to its well-known inhibitory activities on effector cell function. Here we have documented that TGF- made by cells in the immediate microenvironment controls the T cell response to the bacterial superantigen SEB. A role for TGF- in regulating the low-dose response to SEB (0.01–1 ng/ml) was initially suggested by an inverse correlation between IgG production and TGF- produced by PBL and PBMC. Whereas IgG production by purified CD4⁺ and B cells remained high in this dose range, these lymphocytes were poor producers of TGF-. Studies with a neutralizing anti-TGF- Ab provided further evidence for regulation of IgG production by TGF-.

The finding that anti-TGF- needed to be present at the start of the culture for enhancement of Ab production (Fig. 2) suggested that this cytokine was regulating early T cell activation rather than B cell differentiation. Previously, we had documented that TGF- needed to be present at the time of T cell activation for the generation of CD8⁺ regulatory T cells (4). While initial experiments revealed TGF- down-regulated helper activity provided by CD4⁺ cells, further studies indicated that TGF- induced CD4⁺ cells to inhibit IgG production by producing suppressive levels of this cytokine. An autocrine effect of TGF- has been documented by others (17).

Previously, Stohl and Elliott (8) demonstrated that the T cell response to SEB is dose dependent. Whereas high-dose SEB resulted in T cell killing of B cells, low-dose SEB stimulated T cell-dependent B cell differentiation. Similar dose-dependent regulatory effects have been described in oral tolerance. High doses of Ag administered orally result in the clonal deletion of the responding T cells, whereas low doses of Ag result in the appearance of TGF--producing regulatory T cells called Th3 cells (18).

The Th3 cells described by Weiner (32) produced large amounts of TGF- and some IL-10. Studies on the cytokines produced by our TGF--conditioned CD4⁺ cells revealed that TGF-, but not IFN-, IL-10, or IL-4, was made in large amounts. Production of IL-10 and IFN- was less than that of SEB-stimulated control CD4⁺ cells. The decreased amount of IFN- was a direct effect of TGF-, consistent with the findings of others (19). By contrast, the levels of IL-10 did not increase in the presence of anti-TGF-.

To begin to understand the mechanism of action of TGF-, we documented costimulatory effects of this cytokine on CD4⁺ cells. In preparations containing both CD4⁺ and CD8⁺ T cells, TGF- selectively costimulated CD4⁺ cells in primary cultures, and this subset expanded preferentially upon restimulation without this cytokine. The expansion of T cells primed with TGF- after restimulation has been described previously (14). In mice, one group has reported that TGF- had selective costimulatory effects on CD8⁺ T cells (15). Proliferation in response to anti-CD3 and SEB was substantially enhanced by TGF- (20). These treated CD8⁺ cells secreted IL-10 and TGF- and developed cytokine-dependent growth inhibitory activity. We also have demonstrated costimulatory effects of TGF- on purified human CD8⁺ T cells (3, 4) and have used TGF- to generate regulatory CD8⁺ T cells that have cytokine-mediated suppressive effects (21). CD4⁺ cells and CD8⁺ cells require a different length of exposure to TGF- for the induction of suppressive activity. Whereas incubation of CD8⁺ cells with TGF- for 24 h is sufficient, 5–6 days of incubation were required for CD4⁺ cells to develop suppressive effects (3, 4, 5). Thus, TGF- may have costimulatory properties upon either CD4⁺ or CD8⁺ cells, and a preferential effect on one subset may be explained by the experimental conditions used.

The next series of experiments revealed that priming CD4⁺ cells with TGF- not only enhanced the proliferation of CD4⁺ cells upon restimulation but also protected these cells from activation-induced apoptosis. Others have reported that the combination of IL-2 and TGF- has a similar protective effect on mouse CD4⁺ T cells (22). This decreased apoptosis can be explained by decreased expression of Fas ligand (23, 24) and increased expression of mitochondrial Bcl-x_L (25).

Recently, we have reported other evidence of costimulatory effects of TGF- on alloantigen-activated CD4⁺ T cells. Similar to the results described in the present study, there was increased blast transformation, increased expression of CD25 and CTLA-4, and potent, contact-dependent suppressive effects on CD8⁺ cells (5). The phenotype and functional properties of these CD4⁺ cells were similar to, if not identical with, the professional CD4⁺CD25⁺ T cells described by others where neutralizing anti-cytokine Abs had no effect on suppressive activity (reviewed in Ref. 7). Here the CD4⁺ cells produced large amounts of TGF-, and neutralizing Abs to this cytokine abrogated suppressive activity.

Because we had previously generated CD4⁺ suppressor cells with a cytokine-independent mechanism of action from CD25⁺ precursors, we considered that the Th3 suppressor cells described in the present study were derived from CD25^- resting CD4⁺ cells. Subsequent studies revealed that the phenotype and function of cells generated from CD4⁺CD25^- cells were indistinguishable from those derived from total CD4⁺ cells. Others have also documented suppressive properties of cells generated from the CD4⁺CD25^- fraction (26, 27). It is important to emphasize that the CD25 marker cannot distinguish "professional" regulatory cells from Th3 cells. Although the former constitutively express this marker, the latter also can display this determinant after T cell activation. Whereas "professional" CD4⁺CD25⁺ cells appear to be a unique lineage of thymic-derived T cells (6, 7), Th3 cells develop in the periphery (18).

While some workers claim that TGF- has no role in the suppressive effector activity of CD4⁺CD25⁺ cells (7), others claim that this activity is abolished by high concentrations of anti-TGF- and that latent TGF- is bound to the surface of these cells (28). It is possible that heterogeneous populations of CD4⁺CD25⁺ cells derived from different precursors explain these apparently contradictory observations.

Both the thymus-derived CD4⁺CD25⁺ cells and Th3 cells generated from CD25^- precursors in the periphery have important regulatory functions in vivo. The former block the activation of potentially aggressive, self-reactive T cells not eliminated by the thymus which are capable of causing systemic autoimmune disease (6, 7, 29, 30). CD4⁺CD25⁺ cells also regulate homeostatic T cell expansion (31). While Th3 cells can also suppress autoimmunity, they function principally as general feedback regulators of Th1 and Th2 cells (18, 32). Moreover, it is possible that these populations interact with each other in a synergistic manner. The ability to generate each of these populations ex vivo with TGF- has the potential to be useful in the treatment of autoimmune diseases and prevention of transplant rejection.

	Acknowledgments

 
We thank Harold Soucier for his skilled support in the flow cytometry analysis, and we are grateful to Dr. Gunther Dennert for his constructive comments on this manuscript. We thank Gabriela Gutierrez for her help in preparing the manuscript.

	Footnotes

 
¹ This work was supported by grants from the National Institutes of Health (AI-41768), the Nora Eccles Treadwell Foundation, and the Southern California Chapter of the Arthritis Foundation.

² Address correspondence and reprint requests to Dr. David A. Horwitz, Division of Rheumatology and Immunology, Department of Medicine, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, Hoffman Medical Research Building 711, Los Angeles, CA 90033. E-mail address: dhorwitz{at}usc.edu

³ Abbreviations used in this paper: SEB, staphylococcal enterotoxin B; SPC, superantigen-presenting cell; LME, L-leucine methyl ester.

Received for publication May 22, 2002. Accepted for publication August 7, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Letterio, J. J., A. B. Roberts. 1998. Regulation of immune responses by TGF-. Annu. Rev. Immunol. 16:137.[Medline]
2. McCartney-Francis, N. L., M. Frazier-Jessen, S. M. Wahl. 1998. TGF-: a balancing act. Int. Rev. Immunol. 16:553.[Medline]
3. Gray, J. D., M. Hirokawa, D. A. Horwitz. 1994. The role of transforming growth factor in the generation of suppression: an interaction between CD8⁺ T and NK cells. J. Exp. Med. 180:1937.[Abstract/Free Full Text]
4. Gray, J. D., M. Hirokawa, K. Ohtsuka, D. A. Horwitz. 1998. Generation of an inhibitory circuit involving CD8⁺ T cells, IL-2, and NK cell-derived TGF-: contrasting effects of anti-CD2 and anti-CD3. J. Immunol. 160:2248.[Abstract/Free Full Text]
5. Yamagiwa, S., J. D. Gray, S. Hashimoto, D. A. Horwitz. 2001. A role for TGF- in the generation and expansion of CD4⁺CD25⁺ regulatory T cells from human peripheral blood. J. Immunol. 166:7282.[Abstract/Free Full Text]
6. Sakaguchi, S., N. Sakaguchi, J. Shimizu, S. Yamazaki, T. Sakihama, M. Itoh, Y. Kuniyasu, T. Nomura, M. Toda, T. Takahashi. 2001. Immunologic tolerance maintained by CD25⁺CD4⁺ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol. Rev. 182:18.[Medline]
7. Shevach, E. M.. 2000. Regulatory T cells in autoimmunity. Annu. Rev. Immunol. 18:423.[Medline]
8. Stohl, W., J. E. Elliott. 1995. Differential human T cell-dependent B cell differentiation induced by staphylococcal superantigens (SAg): regulatory role for SAg-dependent B cell cytolysis. J. Immunol. 155:1838.[Abstract]
9. Denholm, E. M., F. M. Wolber. 1991. A simple method for the purification of human peripheral blood monocytes. J. Immunol. Methods 144:247.[Medline]
10. Abrams, S. I., Z. Brahmi. 1988. Mechanism of K562-induced human natural killer cell activation using highly enriched effector cells isolated via a new single-step sheep erythrocyte assay. Ann. Inst. Pasteur Immunol. 139:361.[Medline]
11. Thiele, D. L., P. E. Lipsky. 1985. Modulation of human natural killer cell function by L-leucine methyl ester: monocyte-dependent depletion from human peripheral blood mononuclear cells. J. Immunol. 134:786.[Abstract]
12. O’Connor-McCourt, M. D., L. M. Wakefield. 1987. Latent transforming growth factor- in serum: a specific complex with ₂-macroglobulin. J. Biol. Chem. 262:14090.[Abstract/Free Full Text]
13. Abe, M., J. G. Harpel, C. N. Metz, I. Nunes, D. Loskutoff, D. Rifkin. 1994. An assay for transforming growth factor- using cells transfected with a plasminogen activator inhibitor-1 promoter luciferase construct. Anal. Biochem. 216:276.[Medline]
14. Cerwenka, A., D. Bevec, O. Majdic, W. Knapp, W. Holter. 1994. TGF-1 is a potent inducer of human effector T cells. J. Immunol. 153:4367.[Abstract]
15. Lee, H. M., S. Rich. 1991. Costimulation of T cell proliferation by transforming growth factor-1. J. Immunol. 147:1127.[Abstract]
16. Kehrl, J. H., L. M. Wakefield, A. B. Roberts, S. Jakowlew, M. Alvarez-Mon, R. Derynck, M. B. Sporn, A. S. Fauci. 1986. Production of transforming growth factor by human T lymphocytes and its potential role in the regulation of T cell growth. J. Exp. Med. 163:1037.[Abstract/Free Full Text]
17. Seder, R. A., T. Marth, M. C. Sieve, W. Strober, J. J. Letterio, A. B. Roberts, B. Kelsall. 1998. Factors involved in the differentiation of TGF--producing cells from naive CD4⁺ T cells: IL-4 and IFN- have opposing effects, while TGF- positively regulates its own production. J. Immunol. 160:5719.[Abstract/Free Full Text]
18. Faria, A. M., H. L. Weiner. 1999. Oral tolerance: mechanisms and therapeutic applications. Adv. Immunol. 73:153.[Medline]
19. Kitani, A., I. J. Fuss, K. Nakamura, O. M. Schwartz, T. Usui, W. Strober. 2000. Treatment of experimental (trinitrobenzene sulfonic acid) colitis by intranasal administration of transforming growth factor (TGF)-1 plasmid: TGF-1-mediated suppression of T helper cell type 1 response occurs by interleukin (IL)-10 induction and IL-12 receptor 2 chain downregulation. J. Exp. Med. 192:41.[Abstract/Free Full Text]
20. Lee, H. M., S. Rich. 1993. Differential activation of CD8⁺ T cells by transforming growth factor-1. J. Immunol. 151:668.[Abstract]
21. Horwitz, D. A., J. D. Gray, K. Ohtsuka, M. Hirokawa, T. Takahashi. 1997. The immunoregulatory effects of NK cells: the role of TGF- and implications for autoimmunity. Immunol. Today 18:538.[Medline]
22. Zhang, X., L. Giangreco, H. E. Broome, C. M. Dargan, S. L. Swain. 1995. Control of CD4 effector fate: transforming growth factor 1 and interleukin 2 synergize to prevent apoptosis and promote effector expansion. J. Exp. Med. 182:699.[Abstract/Free Full Text]
23. Cerwenka, A., H. Kovar, O. Majdic., W. Holter. 1996. Fas and activation-induced apoptosis are reduced in human T cells preactivated in the presence of TGF-1. J. Immunol. 156:459.[Abstract]
24. Genestier, L., S. Kasibhatla, T. Brunner, D. R. Green. 1999. Transforming growth factor 1 inhibits Fas ligand expression and subsequent activation-induced cell death in T cells via down regulation of cMyc. J. Exp. Med. 189:231.[Abstract/Free Full Text]
25. Chen, W., W. Jin, H. Tian, P. Sicurello, M. Frank, J. M. Orenstein, S. M. Wahl. 2001. Requirement for transforming growth factor 1 in controlling T cell apoptosis. J. Exp. Med. 194:439.[Abstract/Free Full Text]
26. Stephens, L. A., D. Mason. 2000. CD25 is a marker for CD4⁺ thymocytes that prevent autoimmune diabetes in rats, but peripheral T cells with this function are found in both CD25⁺ and CD25^- subpopulations. J. Immunol. 165:3105.[Abstract/Free Full Text]
27. Annacker, O., R. Pimenta-Araujo, O. Burlen-Defranoux, T. C. Barbosa, A. Cumano, A. Bandeira. 2001. CD25⁺CD4⁺ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10. J. Immunol. 166:3008.[Abstract/Free Full Text]
28. 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 . J. Exp. Med. 194:629.[Abstract/Free Full Text]
29. Maloy, K. J., F. Powrie. 2001. Regulatory T cells in the control of immune pathology. Nat. Immunol. 2:816.[Medline]
30. Stephens, L. A., D. Mason. 2001. Characterization of thymus-derived regulatory T cells that protect against organ-specific autoimmune disease. Microbes Infect. 3:905.[Medline]
31. Gavin, M. A., S. R. Clarke, E. Negrou, A. Gallegos, A. Rudensky. 2002. Homeostasis and anergy of CD4⁺CD25⁺ suppressor T cells in vivo. Nat. Immunol. 3:33.[Medline]
32. Weiner, H. L.. 2001. Induction and mechanism of action of transforming growth factor--secreting Th3 regulatory cells. Immunol. Rev. 182:207.[Medline]

This article has been cited by other articles:

				Y. Han, Q. Guo, M. Zhang, Z. Chen, and X. Cao CD69+CD4+CD25- T Cells, a New Subset of Regulatory T Cells, Suppress T Cell Proliferation through Membrane-Bound TGF-{beta}1 J. Immunol., January 1, 2009; 182(1): 111 - 120. [Abstract] [Full Text] [PDF]

				W. Chen, X. Liang, A. J. Peterson, D. H. Munn, and B. R. Blazar The Indoleamine 2,3-Dioxygenase Pathway Is Essential for Human Plasmacytoid Dendritic Cell-Induced Adaptive T Regulatory Cell Generation J. Immunol., October 15, 2008; 181(8): 5396 - 5404. [Abstract] [Full Text] [PDF]

				D. R. Tonkin, J. He, G. Barbour, and K. Haskins Regulatory T Cells Prevent Transfer of Type 1 Diabetes in NOD Mice Only When Their Antigen Is Present In Vivo J. Immunol., October 1, 2008; 181(7): 4516 - 4522. [Abstract] [Full Text] [PDF]

				Z. Zhao, S. Yu, D. C. Fitzgerald, M. Elbehi, B. Ciric, A. M. Rostami, and G.-X. Zhang IL-12R{beta}2 Promotes the Development of CD4+CD25+ Regulatory T Cells J. Immunol., September 15, 2008; 181(6): 3870 - 3876. [Abstract] [Full Text] [PDF]

				N. B. Marshall, W. R. Vorachek, L. B. Steppan, D. V. Mourich, and N. I. Kerkvliet Functional Characterization and Gene Expression Analysis of CD4+CD25+ Regulatory T Cells Generated in Mice Treated with 2,3,7,8-Tetrachlorodibenzo-p-Dioxin J. Immunol., August 15, 2008; 181(4): 2382 - 2391. [Abstract] [Full Text] [PDF]

				S. G. Zheng, J. Wang, and D. A. Horwitz Cutting Edge: Foxp3+CD4+CD25+ Regulatory T Cells Induced by IL-2 and TGF-{beta} Are Resistant to Th17 Conversion by IL-6 J. Immunol., June 1, 2008; 180(11): 7112 - 7116. [Abstract] [Full Text] [PDF]

				M. Elrefaei, C. A. R. Baker, N. G. Jones, D. R. Bangsberg, and H. Cao Presence of Suppressor HIV-Specific CD8+ T Cells Is Associated with Increased PD-1 Expression on Effector CD8+ T Cells J. Immunol., June 1, 2008; 180(11): 7757 - 7763. [Abstract] [Full Text] [PDF]

				N. Perez, S. Karumuthil-Melethil, R. Li, B. S. Prabhakar, M. J. Holterman, and C. Vasu Preferential Costimulation by CD80 Results in IL-10-Dependent TGF-{beta}1+-Adaptive Regulatory T Cell Generation J. Immunol., May 15, 2008; 180(10): 6566 - 6576. [Abstract] [Full Text] [PDF]

				Y. A. Taher, B. C. A. M. van Esch, G. A. Hofman, P. A. J. Henricks, and A. J. M. van Oosterhout 1{alpha},25-Dihydroxyvitamin D3 Potentiates the Beneficial Effects of Allergen Immunotherapy in a Mouse Model of Allergic Asthma: Role for IL-10 and TGF-{beta} J. Immunol., April 15, 2008; 180(8): 5211 - 5221. [Abstract] [Full Text] [PDF]

				M. Razmara, B. Hilliard, A. K. Ziarani, Y. H. Chen, and M. L. Tykocinski CTLA-4{middle dot}Ig converts naive CD4+CD25- T cells into CD4+CD25+ regulatory T cells Int. Immunol., April 1, 2008; 20(4): 471 - 483. [Abstract] [Full Text] [PDF]

				T. L. Sumpter, K. K. Payne, and D. S. Wilkes Regulation of the NFAT pathway discriminates CD4+CD25+ regulatory T cells from CD4+CD25- helper T cells J. Leukoc. Biol., March 1, 2008; 83(3): 708 - 717. [Abstract] [Full Text] [PDF]

				R. K. Selvaraj and T. L. Geiger Mitigation of Experimental Allergic Encephalomyelitis by TGF-{beta} Induced Foxp3+ Regulatory T Lymphocytes through the Induction of Anergy and Infectious Tolerance J. Immunol., March 1, 2008; 180(5): 2830 - 2838. [Abstract] [Full Text] [PDF]

				W. Niedbala, B. Cai, H. Liu, N. Pitman, L. Chang, and F. Y. Liew Nitric oxide induces CD4+CD25+ Foxp3 regulatory T cells from CD4+CD25 T cells via p53, IL-2, and OX40 PNAS, September 25, 2007; 104(39): 15478 - 15483. [Abstract] [Full Text] [PDF]

				J. A. Kapp, K. Honjo, L. M. Kapp, K. Goldsmith, and R. P. Bucy Antigen, in the Presence of TGF-beta, Induces Up-Regulation of FoxP3gfp+ in CD4+ TCR Transgenic T Cells That Mediate Linked Suppression of CD8+ T Cell Responses J. Immunol., August 15, 2007; 179(4): 2105 - 2114. [Abstract] [Full Text] [PDF]

				R. K. Selvaraj and T. L. Geiger A Kinetic and Dynamic Analysis of Foxp3 Induced in T Cells by TGF-beta J. Immunol., June 15, 2007; 178(12): 7667 - 7677. [Abstract] [Full Text] [PDF]

				K. N. Couper, D. G. Blount, J. B. de Souza, I. Suffia, Y. Belkaid, and E. M. Riley Incomplete Depletion and Rapid Regeneration of Foxp3+ Regulatory T Cells Following Anti-CD25 Treatment in Malaria-Infected Mice J. Immunol., April 1, 2007; 178(7): 4136 - 4146. [Abstract] [Full Text] [PDF]

				J. Stein-Streilein and A. W. Taylor An eye's view of T regulatory cells J. Leukoc. Biol., March 1, 2007; 81(3): 593 - 598. [Abstract] [Full Text] [PDF]

				V. C. Liu, L. Y. Wong, T. Jang, A. H. Shah, I. Park, X. Yang, Q. Zhang, S. Lonning, B. A. Teicher, and C. Lee Tumor Evasion of the Immune System by Converting CD4+CD25- T Cells into CD4+CD25+ T Regulatory Cells: Role of Tumor-Derived TGF-beta J. Immunol., March 1, 2007; 178(5): 2883 - 2892. [Abstract] [Full Text] [PDF]

				M. Elrefaei, F. L. Ventura, C. A. R. Baker, R. Clark, D. R. Bangsberg, and H. Cao HIV-Specific IL-10-Positive CD8+ T Cells Suppress Cytolysis and IL-2 Production by CD8+ T Cells J. Immunol., March 1, 2007; 178(5): 3265 - 3271. [Abstract] [Full Text] [PDF]

				S. G. Zheng, J. Wang, P. Wang, J. D. Gray, and D. A. Horwitz IL-2 Is Essential for TGF-beta to Convert Naive CD4+CD25- Cells to CD25+Foxp3+ Regulatory T Cells and for Expansion of These Cells J. Immunol., February 15, 2007; 178(4): 2018 - 2027. [Abstract] [Full Text] [PDF]

				S. Nadkarni, C. Mauri, and M. R. Ehrenstein Anti-TNF-{alpha} therapy induces a distinct regulatory T cell population in patients with rheumatoid arthritis via TGF-{beta} J. Exp. Med., January 22, 2007; 204(1): 33 - 39. [Abstract] [Full Text] [PDF]

				K. S. Seo, S. U. Lee, Y. H. Park, W. C. Davis, L. K. Fox, and G. A. Bohach Long-Term Staphylococcal Enterotoxin C1 Exposure Induces Soluble Factor-Mediated Immunosuppression by Bovine CD4+ and CD8+ T Cells Infect. Immun., January 1, 2007; 75(1): 260 - 269. [Abstract] [Full Text] [PDF]

				M.-L. Cheng, H.-W. Chen, J.-P. Tsai, Y.-P. Lee, Y.-C. Shih, C.-M. Chang, and C.-C. Ting Clonal restriction of the expansion of antigen-specific CD8+ memory T cells by transforming growth factor-{beta} J. Leukoc. Biol., May 1, 2006; 79(5): 1033 - 1042. [Abstract] [Full Text] [PDF]

				S. E. Weber, J. Harbertson, E. Godebu, G. A. Mros, R. C. Padrick, B. D. Carson, S. F. Ziegler, and L. M. Bradley Adaptive islet-specific regulatory CD4 T cells control autoimmune diabetes and mediate the disappearance of pathogenic Th1 cells in vivo. J. Immunol., April 15, 2006; 176(8): 4730 - 4739. [Abstract] [Full Text] [PDF]

				S. G. Zheng, J. H. Wang, W. Stohl, K. S. Kim, J. D. Gray, and D. A. Horwitz TGF-beta Requires CTLA-4 Early after T Cell Activation to Induce FoxP3 and Generate Adaptive CD4+CD25+ Regulatory Cells J. Immunol., March 15, 2006; 176(6): 3321 - 3329. [Abstract] [Full Text] [PDF]

				S. G. Zheng, L. Meng, J. H. Wang, M. Watanabe, M. L. Barr, D. V. Cramer, J. D. Gray, and D. A. Horwitz Transfer of regulatory T cells generated ex vivo modifies graft rejection through induction of tolerogenic CD4+CD25+ cells in the recipient Int. Immunol., February 1, 2006; 18(2): 279 - 289. [Abstract] [Full Text] [PDF]

				S. Sugita, T. F. Ng, P. J. Lucas, R. E. Gress, and J. W. Streilein B7+ Iris Pigment Epithelium Induce CD8+ T Regulatory Cells; Both Suppress CTLA-4+ T Cells J. Immunol., January 1, 2006; 176(1): 118 - 127. [Abstract] [Full Text] [PDF]

				J. Shimizu, R. Iida, Y. Sato, E. Moriizumi, A. Nishikawa, and Y. Ishida Cross-Linking of CD45 on Suppressive/Regulatory T Cells Leads to the Abrogation of Their Suppressive Activity In Vitro J. Immunol., April 1, 2005; 174(7): 4090 - 4097. [Abstract] [Full Text] [PDF]

				H.-K. Kang, M. A. Michaels, B. R. Berner, and S. K. Datta Very Low-Dose Tolerance with Nucleosomal Peptides Controls Lupus and Induces Potent Regulatory T Cell Subsets J. Immunol., March 15, 2005; 174(6): 3247 - 3255. [Abstract] [Full Text] [PDF]

				M. R. Walker, B. D. Carson, G. T. Nepom, S. F. Ziegler, and J. H. Buckner De novo generation of antigen-specific CD4+CD25+ regulatory T cells from human CD4+CD25- cells PNAS, March 15, 2005; 102(11): 4103 - 4108. [Abstract] [Full Text] [PDF]

				Y. Cong, A. Konrad, N. Iqbal, R. D. Hatton, C. T. Weaver, and C. O. Elson Generation of Antigen-Specific, Foxp3-Expressing CD4+ Regulatory T Cells by Inhibition of APC Proteosome Function J. Immunol., March 1, 2005; 174(5): 2787 - 2795. [Abstract] [Full Text] [PDF]

				P. E. Rao, A. L. Petrone, and P. D. Ponath Differentiation and Expansion of T Cells with Regulatory Function from Human Peripheral Lymphocytes by Stimulation in the Presence of TGF-{beta} J. Immunol., February 1, 2005; 174(3): 1446 - 1455. [Abstract] [Full Text] [PDF]

				B. Gunnlaugsdottir, S. M. Maggadottir, and B. R. Ludviksson Anti-CD28-induced co-stimulation and TCR avidity regulates the differential effect of TGF-{beta}1 on CD4+ and CD8+ naive human T-cells Int. Immunol., January 1, 2005; 17(1): 35 - 44. [Abstract] [Full Text] [PDF]

				B. E. Anderson, J. M. McNiff, C. Matte, I. Athanasiadis, W. D. Shlomchik, and M. J. Shlomchik Recipient CD4+ T cells that survive irradiation regulate chronic graft-versus-host disease Blood, September 1, 2004; 104(5): 1565 - 1573. [Abstract] [Full Text] [PDF]

				C. Vasu, B. S. Prabhakar, and M. J. Holterman Targeted CTLA-4 Engagement Induces CD4+CD25+CTLA-4high T Regulatory Cells with Target (Allo)antigen Specificity J. Immunol., August 15, 2004; 173(4): 2866 - 2876. [Abstract] [Full Text] [PDF]

				H.-B. Park, D.-J. Paik, E. Jang, S. Hong, and J. Youn Acquisition of anergic and suppressive activities in transforming growth factor-{beta}-costimulated CD4+CD25- T cells Int. Immunol., August 1, 2004; 16(8): 1203 - 1213. [Abstract] [Full Text] [PDF]

				S. M. Wahl, J. Swisher, N. McCartney-Francis, and W. Chen TGF-{beta}: the perpetrator of immune suppression by regulatory T cells and suicidal T cells J. Leukoc. Biol., July 1, 2004; 76(1): 15 - 24. [Abstract] [Full Text] [PDF]

				T. L. Sumpter and D. S. Wilkes Role of autoimmunity in organ allograft rejection: a focus on immunity to type V collagen in the pathogenesis of lung transplant rejection Am J Physiol Lung Cell Mol Physiol, June 1, 2004; 286(6): L1129 - L1139. [Abstract] [Full Text] [PDF]

				S. G. Zheng, J. H. Wang, J. D. Gray, H. Soucier, and D. A. Horwitz Natural and Induced CD4+CD25+ Cells Educate CD4+CD25- Cells to Develop Suppressive Activity: The Role of IL-2, TGF-{beta}, and IL-10 J. Immunol., May 1, 2004; 172(9): 5213 - 5221. [Abstract] [Full Text] [PDF]

				S. G. Zheng, J. H. Wang, M. N. Koss, F. Quismorio Jr., J. D. Gray, and D. A. Horwitz CD4+ and CD8+ Regulatory T Cells Generated Ex Vivo with IL-2 and TGF-{beta} Suppress a Stimulatory Graft-versus-Host Disease with a Lupus-Like Syndrome J. Immunol., February 1, 2004; 172(3): 1531 - 1539. [Abstract] [Full Text] [PDF]

				L. Cosmi, F. Liotta, E. Lazzeri, M. Francalanci, R. Angeli, B. Mazzinghi, V. Santarlasci, R. Manetti, V. Vanini, P. Romagnani, et al. Human CD8+CD25+ thymocytes share phenotypic and functional features with CD4+CD25+ regulatory thymocytes Blood, December 1, 2003; 102(12): 4107 - 4114. [Abstract] [Full Text] [PDF]

				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]

				D. A. Horwitz, S. G. Zheng, and J. D. Gray The role of the combination of IL-2 and TGF-{beta} or IL-10 in the generation and function of CD4+ CD25+ and CD8+regulatory T cell subsets J. Leukoc. Biol., October 1, 2003; 74(4): 471 - 478. [Abstract] [Full Text] [PDF]

				T. Mizobuchi, K. Yasufuku, Y. Zheng, M. A. Haque, K. M. Heidler, K. Woods, G. N. Smith Jr., O. W. Cummings, T. Fujisawa, J. S. Blum, et al. Differential Expression of Smad7 Transcripts Identifies the CD4+CD45RChigh Regulatory T Cells That Mediate Type V Collagen-Induced Tolerance to Lung Allografts J. Immunol., August 1, 2003; 171(3): 1140 - 1147. [Abstract] [Full Text] [PDF]

				Z.-m. Chen, M. J. O'Shaughnessy, I. Gramaglia, A. Panoskaltsis-Mortari, W. J. Murphy, S. Narula, M. G. Roncarolo, and B. R. Blazar IL-10 and TGF-{beta} induce alloreactive CD4+CD25- T cells to acquire regulatory cell function Blood, June 15, 2003; 101(12): 5076 - 5083. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zheng, S. G. Articles by Horwitz, D. A. Search for Related Content PubMed PubMed Citation Articles by Zheng, S. G. Articles by Horwitz, D. A. Pubmed/NCBI databases Substance via MeSH Medline Plus Health Information Stem Cells