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The Journal of Immunology, 2007, 179, 2105 -2114
Copyright © 2007 by The American Association of Immunologists, Inc.
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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 Responses1

Judith A. Kapp2,*,{dagger}, Kazuhito Honjo{dagger}, Linda M. Kapp*, Kelly Goldsmith{dagger} and R. Pat Bucy{dagger}

* Department of Ophthalmology and {dagger} Department of Pathology, University of Alabama AL 35233-7331


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD4+CD25+ regulatory T cells (Tregs) inhibit immune responses to a variety of Ags, but their specificity and mechanism of suppression are controversial. This controversy is largely because many studies focused on natural Tregs with undefined specificities and suppression has frequently been measured on polyclonal T cell responses. To address the issue of specificity further, we have bred Kd-specific, CD4+ TCR (TCR75) transgenic mice to Foxp3gfp knockin reporter mice to permit sorting of Tregs with a known specificity. Foxp3gfp.TCR75 mice did not express significant numbers of natural FoxP3+ Tregs expressing the TCR75 transgenes, but FoxP3 expression was induced by stimulating with Kd plus TGF-beta. The resulting GFP+ TCR75 cells were anergic, whereas the GFP– TCR75 cells proliferated upon restimulation with Kd peptide. Yet both exhibited severely reduced expression of intracellular IFN-{gamma} and TNF-{alpha} upon restimulation. GFP+, but not GFP–, TCR75 T cells suppressed responses by naive TCR75 T cells and by nontransgenic spleen cells stimulated with anti-CD3. GFP+ TCR75 cells also inhibited polyclonal C57BL/6 anti-Kd CTL responses if the APC expressed Kd and both MHC class I and class II, and responses by OT1 T cells to B6.Kd.OVA but not B6.Kd plus OVA expressing APC, demonstrating linked-suppression of CD8 responses. Thus, Tregs exhibit a greater degree of specificity in vitro than previously appreciated. The observation that Tregs and responder T cells must recognize the same APC provides a mechanistic explanation for the observation that Tregs must be in direct contact with effector T cells to suppress their responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The concept that T cells can mediate immunologic tolerance through active suppression was first demonstrated over 30 years ago by the seminal experiments of Gershon and Kondo (1), who showed that lymphocytes from tolerant mice adoptively transferred Ag-specific tolerance to naive mice. Interest in this pathway waxed and waned in the intervening years, but it has again become widely accepted on the basis of the observations that a distinct subset of naturally occurring CD4+ T cells (2), referred to as regulatory T cells (Tregs),3 has the capacity to prevent autoimmune diseases mediated by endogenous, self-reactive T cells (3, 4). The expression of CD25 by T cells from naive mice allowed them to be physically separated from other CD4+ T cells and shown to have immunosuppressive activity (2). Molecular analysis of Tregs identified the unique expression of a transcription factor from the forkhead/winged helix family (FoxP3) (5, 6), which is now considered to be a master switch driving differentiation of naive T cells into the Treg lineage (6, 7, 8, 9, 10, 11) and maintaining the regulatory phenotype (12). Development of knockin reporter strains of mice whose CD4+ T cells express fluorescent markers upon activation of the FoxP3 gene allowed the FoxP3+ subset of CD4+ T cells to be purified and shown to mediate regulatory activity (13, 14).

Although there is a burgeoning literature on the characteristics of Tregs, several dichotomies have yet to be resolved. For example, Tregs proliferate to peptides presented in the context of MHC class II in vivo (15, 16), but they appear to be anergic in vitro (17). Natural Tregs must interact with Ag in the periphery to inhibit autoimmune responses specifically (18), yet their suppressive activity has been reported to be nonspecific in vitro (17, 19). The observations that Tregs can be induced from CD4+CD25– peripheral cells by stimulation with exogenous Ags presented via a tolerogenic route (20, 21) or by activation in the presence of TGF-beta (22, 23) and that these induced Tregs also express FoxP3 (23, 24) suggest that most, if not all, CD4+ may be capable of becoming Tregs under the appropriate conditions. If the latter hypothesis is correct, then the apparent differences between natural and induced Tregs may reflect differences in chronic vs acute activation rather than any inherent differences between these cells.

In vitro, Tregs require direct contact with the population to be suppressed as shown by the failure of Tregs to inhibit responder T cells if they are separated by a semipermeable membrane (17, 25, 26). These observations suggest that suppression is not mediated by secreted cytokines because soluble cytokines would cross the membrane. However, the pathways by which Tregs inhibit immune responses are controversial and may depend upon the nature of the responses being regulated. For example, membrane-bound TGF-beta appears to play a role in some Treg systems (27, 28, 29, 30) but not others (31, 32). Despite the variable role TGF-beta seems to play in the effector activity of various Tregs, TGF-beta plays an essential role in preventing autoimmune responses as shown by the massive, multifocal infiltration of lymphocytes and myeloid cells into most organs and the ultimate death of TGF-beta knockout mice (33, 34). TGF-beta is also required for the up-regulation of FoxP3 and the maintenance of the inhibitory function of Tregs (12).

To study the function of CD4+ Tregs, we have taken a reductionist approach using transgenic (Tg) mice to isolate the key elements of this complex system. To rule out the confounding effects of multiple autoantigens, we generated a monoclonal population of CD4+ Tregs using T cells from TCR (TCR75) Tg mice. TCR75 T cells react with a peptide derived from the H-2Kd MHC class I molecule presented by the I-Ab class II molecule (Kd54–68/I-Ab) on the C57BL/6 (B6) background (35). Recombinase sufficient TCR75 mice do not contain a significant population of natural Tregs expressing the TCR transgenes but, FoxP3 was found to be up-regulated in a subset of TCR75 T cells by activation in the presence of TGF-beta. To assess the functional activity of purified FoxP3+ T cells, the TCR75 mice were bred to Foxp3gfp knockin mice (13). The results reported in this study demonstrate that TGF-beta-activated GFP+ but not GFP– T cells from Foxp3gfp.TCR75 mice caused linked suppression of CD8 T cell responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Experimental animals

B6 (H-2b) mice were bred in our colony from stock obtained from The Jackson Laboratory. The C57BL/6-Tg(TcraTcrb)TCR75Rpb (TCR75) Tg mice, which express {alpha}beta TCR V{alpha}1.1, Vbeta8.3 specific for I-Ab/H-2Kd54–68 epitope, were previously characterized (35). TCR75 Tg mice were bred to FoxP3gfp knockin mice provided by Dr. A. Rudensky (University of Washington, Seattle, WA) and obtained from Dr. C. Weaver (University of Alabama, Birmingham, AL). Heterozygous, F1 hybrids that expressed both the GFP and TCR75 transgenes (referred to as FoxP3gfp.TCR75 mice) were used in these experiments. The FoxP3gfp gene is integrated into the X chromosome and thus only about one-half of FoxP3+ cells in heterozygous female F1 mice also express GFP (data not shown) due to random X chromosome inactivation (5). Consequently, male FoxP3gfp.TCR75 mice were used for the experiments in this study. FoxP3gfp.TCR75 mice were also mated to B6.Rag–/– mice and the F1 mice backcrossed to B6.Rag–/– mice to produce Rag–/–.FoxP3gfp.TCR75 for some experiments. C57BL/6-Tg(TcraTcrb)1100Mjb (36) Tg mice, referred to as OT1, which express V{alpha}2, Vbeta5 and recognize OVA257–264 in the context of H-2Kb were a gift of Dr. M. Bevan (University of Washington, Seattle, WA). Rag–/–.OT1 Tg mice were made by crossing and backcrossing them to B6.Rag1–/– mice.

C57BL/6-Tg(Kd)Rpb (B6.Kd) mice, express the full genomic sequence of H-2Kd, which has a tissue distribution indistinguishable from the expression of endogenous H-2Kd in B10.D2 mice (37). B6.Kd mice were also crossed to MHC class II-deficient mice (I-Abeta–/– mice) (38) and to mutant mice with a defective beta2-microglobulin (beta2M) gene (B6.beta2M–/– mice) (39), obtained from The Jackson Laboratory and intercrossed to yield the homozygous knockouts, I-Abeta–/–.Kd and beta2M–/–.Kd. Mice expressing a membrane form of the chicken OVA gene under the chicken actin promoter, C57BL/6-Tg(ACTB-OVA)916Jen (40), referred to as B6.OVA, were provided by M. Jenkins (University of Minnesota, Minneapolis, MN). Spleen cells from I-Abeta–/–.Kd, beta2M–/–.Kd, B6.Kd, B6.OVA, and F1(B6.Kd x B6.OVA), referred to as B6.Kd.OVA, mice were used as a source of APC in these studies.

We have also bred several of these Tg strains to express homozygous polymorphisms for either the CD451/1 (Ly-5) or CD901/1 (Thy-1) alleles on the B6 (CD452/2 and CD902/2) background to allow simultaneous tracking of different populations in cultures containing cell mixtures. All procedures on animals were conducted with approval by the University of Alabama Animal Care and Use Committee.

Induction and assay of Ag-specific, CD4+ TCR Tg Tregs

We have adapted the assay system that we previously used to study polyclonal alloreactive T suppressor cells (41, 42) to the analysis of TCR Tg T cells in vitro. This system is a "two-step" culture in which the first culture is used to activate naive T cells into a regulatory phenotype and the second step culture measures their inhibitory activity on the induction of primary responses by CD8+ B6 or OT1 TCR Tg T cells. To activate Treg activity, spleen cells from FoxP3gfp.TCR75 mice (1.0 x 106) were incubated with (1.0 x 106) irradiated (2500 rad) spleen cells from B6 mice, as a source of APC, plus 0.3 µM Kd54–68 peptide, 100 U/ml IL-2 and 3 ng/ml recombinant human TGF-beta (R&D Systems) in 48-well plates unless otherwise stated. Sufficient anti-TGF-beta1, -beta2, -beta3 (1D11; R&D Systems) to block the function of 3 ng/ml TGF-beta was added at culture initiation in some experiments. The cultured cells were harvested 6 days later, and analyzed for suppressive activity, as described below.

In some cultures, cell division was determined by incubating cells purified splenic T cells with 1 µM CFSE (Molecular Probes) using the method described by Lyons and Parish (43). The geometric mean fluorescence intensity of CFSE staining was measuring by Cell Quest software (BD Biosources), and corrected for linearity by calibration with Sphero Rainbow calibration particles (Spherotech) to determine the intensity in mean equivalent soluble fluorescence units. The mean cycle number of the population was then determined by the equation (logCFSEc – logCFSEex)/log2, where CFSEc is the CFSE intensity of control cells and CFSEex is the CFSE intensity of the experimental cells. Thus, the loss of CFSE that is not associated with cellular division is taken into account at each time point.

In other experiments, proliferative responses were measured 96-well round-bottom plates containing 5 x 104 lymphoid cells and 15 x 104 irradiated (2500 rad) spleen cells each from the indicated strains of mice as stimulator cells. Various numbers of FoxP3gfp.TCR75 cells, activated with Kd54–68 with or without exogenous TGF-beta, were added to these cultures, which were incubated for 3 days and pulsed overnight with 1 µCi [3H]thymidine (Amersham Biosciences) harvested, and counted in a beta-counter.

To analyze the suppressive effect of Treg on CTL responses, splenic T cells from B6 or OT1 mice were incubated with spleen cells, treated with 0.5 mg/ml collagenase VIII (Sigma-Aldrich) in 2 ml of culture medium for 45 min in CO2 incubator and irradiated (2000 rad), as a source of APC plus various numbers of Tregs. After 4–5 days, the cultures were harvested and assayed for lytic activity using the H-2b tumor cell line (EL4, an MHC class II-negative T cell lymphoma) (44), E.G7-OVA (EL4 cell line that expresses the chicken OVA gene (44), and the H-2d cell line (P815) as targets for the CTL assays. CTL activity was measured using a modification of published flow cytometry methods using CFSE-labeled targets (45, 46) to directly count the number of viable target cells, as previously described (47). The ratio of viable Ag+/Ag– cells in each experimental tube divided by the same ratio in the control tubes gives a measure of Ag-specific cytotoxicity by the formula: percentage of specific cytotoxicity = 100 x (1 – (Ag+/Ag–)experimental/(Ag+/Ag–)control).

Representative data are shown for all experiments, which were repeated at least twice with similar results.

Immunofluorescent staining

Single-cell suspensions were stained for cells surface Ags by incubating with PE-, FITC-, or biotin-labeled mAbs specific for CD90.2 (30-H12), CD90.1 (OX-7), CD45.1 (A20), CD11c (HL3), CD25 (7D4) (BD Biosciences) or glucocorticoid-induced TNFR (GITR, DTA-1; eBioscience). Intracellular stains used IFN-{gamma} (clone XMG1.3), TNF-{alpha} (MP6-XT22), IL-10 (clone JES5-16E3), and IL-17 (TC11-18H10) (BD Biosciences), and FoxP3 (PE FJK-16s; eBioscience). TCR75 T cells express Vbeta8.3, which was detected with PE-labeled Ab from clone B21.14 (BD Biosciences), whereas OT1 cells express Vbeta5, which was detected with PE-labeled Ab from clone MR9-4 (BD Biosciences). After cell surface staining, cells were fixed, permeabilized, and stained for intracellular expression of FoxP3 (PE FJK-16s), using the staining set reagents, all of which were obtained from eBioscience according to the manufacturer’s directions. Biotinylated Abs were detected with streptavidin-Red 670 obtained from Invitrogen Life Sciences or streptavidin-CyChrome (BD Biosciences).

Intracellular cytokine (ICC) expression was determined by secondary activation of cultured T cells with Ag for 2 h, after which brefeldin A (GolgiPlug; BD Biosciences) was added and the cultures incubated an additional 2 h. The T cells were washed and stained for cell surface Ags as described and fixed, permeabilized using Cytofix/Cytoperm Plus kit, and then stained with PE-labeled Abs specific for IFN-{gamma}, TNF-{alpha}, IL-10, and IL-17.

Fluorescent 10-µm diameter beads (Molecular Probes) were added to each staining tube as a volume calibrator to determine the cell yield in various cultures by flow cytometry. Analysis of the flow cytometry data was performed using either a FACScan or FACSCalibur instrument (BD Biosciences). In some experiments, 7-aminoactinomycin D was added to allow gating on viable cells. The data were analyzed using either CellQuest Pro (BD Biosciences) or FlowJo software (Tree Star).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Induction of Ag-specific CD4+ TCR75 Tregs

Preliminary data indicated that very few but somewhat variable numbers of naive Vbeta8.3+ T cells from TCR75 Tg mice expressed intracellular FoxP3 but incubation with Kd plus TGF-beta-induced FoxP3 expression (data not shown). Unfortunately, Abs to CD25 cannot be used to purify the induced FoxP3+ TCR75 cells from these cultures because activated FoxP3+ and FoxP3– cells both express CD25. Because Abs to intracellular proteins, such as FoxP3, cannot be used to purify viable cells for functional studies, the TCR75 mice were bred to Foxp3gfp knockin mice (13) so that FoxP3+ cells could be sorted by flow cytometry based on GFP expression.

During the production of the double Tg B6 mice expressing both TCR75 and FoxP3gfp, we found that the offspring could be rapidly phenotyped by flow cytometric analysis of PBLs (Fig. 1A). As expected, CD4+ T cells from nontransgenic B6 mice expressed ~5% Vbeta8.3+ cells and no background GFP+ cells. B6 mice expressing FoxP3gfp, but not the TCR75 transgenes, also expressed ~5% Vbeta8.3+ cells and ~1% of the CD4+ T cells expressed GFP, which constituted ~3% of both Vbeta8.3+ and Vbeta8.3– CD4+ subsets. The ~95% of CD4+ T cells in the TCR75 Tg mice expressed Vbeta8.3 and no detectable GFP+ cells, whereas the double Tg mice expressed a total of ~1.3% GFP+ cells, of which only 0.3% expressed the TCR Vbeta8.3 Tg gene. Whether these few GFP+ Vbeta8.3+ cells also expressed the TCR75 V{alpha}1.1 transgene cannot be determined for lack of an appropriate Ab for V{alpha}1.1. However, it is clear that the proportion of GFP+ cells were highly enriched (1%) GFP+ among the (6.8%) Vbeta8.3– cells and diminished (0.3%) GFP+ in the (91.9%) Vbeta8.3+ T cells in the double Tg mice. Thus, few if any TCR75+ T cells express FoxP3 (and presumably natural Treg activity) in naive mice.


Figure 1
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  		FIGURE 1. Expression of FoxP3gfp by Tg T cells. Peripheral blood was collected from nontransgenic mice or mice expressing TCR75 and/or FoxP3gfp transgenes, and the erythrocytes were lysed and stained with Abs specific for CD4 and Vbeta8.3. A, After gating on CD4+ cells, the samples were analyzed for expression of Vbeta8.3 and GFP. The numbers in each quadrant represent the percentage of CD4+ cells expressing the markers shown on the axes. Spleen cells from mice expressing TCR75 and FoxP3gfp transgenes (FoxP3gfp.TCR75) were cultured with irradiated B6 filler cells, 0.3 µM Kd54–68 peptide, 100 U/ml IL-2, or 3 ng/ml TGF-beta, as described in Materials and Methods. Cells were harvested after 6 days and stained with Abs to CD4 and Vbeta8.3 or FoxP3. After gating on CD4+ cells, the samples were analyzed for expression of Vbeta8.3 and GFP (top) or FoxP3 and GFP (bottom) (B) and CD4, CD25, or GITR vs GFP (C).

 
After stimulation of spleen cells from the double Tg mice with the Kd peptide with or without TGF-beta, >99% of the recovered CD4+ T cells expressed the TCR Vbeta8.3 (Fig. 1B, top), suggesting that the few Vbeta8.3– cells observed in naive mice (Fig. 1A) died for lack of stimulation by Kd peptide. Few, if any, of the Vbeta8.3+ cells expressed FoxP3 or GFP in cultures activated by Kd in the absence of TGF-beta (Fig. 1B, top left) or if incubated with TGF-beta but no Kd peptide (data not shown). However, activation with Kd in the presence of TGF-beta up-regulated GFP expression in ~23% of the TCR75 (Vbeta8.3+) T cells (Fig. 1B, top right). Few if any GFP+ Vbeta8.3– cells were detected, which rules out the possibility that significant down-modulation of the TCR occurred during the induction culture. To verify that GFP expression equated with FoxP3 expression, the CD4+ cells were examined simultaneously for expression of GFP and intracellular FoxP3. After activation with Kd and TGF-beta, the data (Fig. 1B, bottom right) demonstrate that virtually all of the GFP+ cells expressed FoxP3 and vice versa. Moreover, dual staining gave a narrow diagonal pattern, which indicates that the levels of GFP and FoxP3 were coordinately regulated in these cultured cells.

After activation with Kd peptide, virtually all the TCR75 cells expressed CD25, which was very heterogeneous ranging from low to high levels, and low levels of GITR but no GFP (Fig. 1C, left). After activation with Kd peptide plus TGF-beta, GFP+ and GFP– TCR75 T cells expressed similar patterns that were positive for CD25 and GITR albeit slightly dimmer than TCR75 activated by Kd alone. These results illustrate that neither Abs specific for CD25 nor GITR can be used to separate induced FoxP3+ from FoxP3– TCR75, whereas the FoxP3+ are clearly separable from FoxP3– TCR75 by expression of GFP.

To determine whether the GFP+ TCR75+ T cells arose in these cultures by differentiation of GFP– precursors or by expansion of the very small population of GFP+ T cells detected in naive mice (Fig. 2, top left), GFP+ T cells were depleted from naive FoxP3gfp.TCR75 spleen cells before culture initiation (Fig. 2, bottom left). After stimulation with Kd peptide and TGF-beta, naive GFP– cells developed similar or slightly higher levels of GFP+ Vbeta8.3+ cells than did unseparated spleen cells, suggesting that most if not all GFP+ cells arise from GFP– precursor cells.


Figure 2
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  		FIGURE 2. Source of GFP+ TCR75 cells activated by TGF-beta. FoxP3gfp.TCR75 spleen cells from naive mice were sorted for GFP– cells and, along with unsorted TCR75 cells, were cultured with irradiated B6 filler cells, 0.3 µM Kd54–68 peptide, 100 U/ml IL-2, or 3 ng/ml TGF-beta as described in Fig. 1. Naive TCR spleen cells and those cultured for 6 days were stained with anti-CD4+ and anti-Vbeta8.3. After gating on CD4+ T cells, Vbeta8.3 was plotted against GFP. Values shown represent percentage of GFP+ and GFP– cells expressed.

 
Before testing the regulatory activity of GFP+ and GFP– T cells from FoxP3gfp.TCR75 mice, conditions that optimized the yield of GFP+ T cells were established. First, the yield of Vbeta8.3+ and GFP+ Vbeta8.3+ cells was determined 6 days after stimulation with various doses of Kd peptide in the presence or absence of 3 ng/ml TGF-beta, with or without 100 U/ml IL-2. The yield of TCR75 T cells showed a broad peak between 0.1 and 3.0 µM Kd peptide in the absence of cytokines (Fig. 3A). The addition of IL-2 enhanced the yield of TCR75 cells at low doses of Kd peptide, whereas TGF-beta slightly decreased the yield at low doses, but greatly enhanced the yield at high doses, of Kd peptide. The mixture of IL-2 and TGF-beta enhanced the yield at all doses of the Kd peptide. GFP+ TCR75+ T cells were not induced by any dose of Kd peptide unless TGF-beta was present, in which case 0.1–3 µM Kd peptide stimulated 17 to 28% GFP+ TCR75 cells (Fig. 3B). IL-2 did not induce GFP expression at any dose of Kd in the absence of TGF-beta, but it significantly enhanced the effect of TGF-beta at doses of Kd below 1.0 µM Kd (Fig. 3B). It is also clear from this experiment, that activation of Treg is more susceptible to inhibition by high doses of Kd peptide (Fig. 3B) than is the activation of TCR75 (Fig. 3A).


Figure 3
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  		FIGURE 3. Optimal growth and expression of GFP in cultured spleen cells from FoxP3gfp.TCR75 mice. Spleen cells from FoxP3gfp.TCR75 mice were incubated with various doses of Kd54–68, with or without 3 ng/ml TGF-beta and with or without 100 U/ml recombinant IL-2. Cells were harvested after 6 days. After gating on CD4+ T cells, the samples were analyzed for expression of Vbeta8.3 and GFP. The number of TCR75+ (A) and GFP+TCR75+ (B) cells, determined by flow cytometry by adding 10-µm diameter fluorescent beads as a volume calibrator, were plotted vs the dose of Kd. In a separate experiment, spleen cells from FoxP3gfp.TCR75 mice were incubated with 0.3 µM Kd54–68, the indicated concentrations of TGF-beta, with or without 100 U/ml recombinant IL-2, harvested, and stained. The number of TCR75+ (C) and the percentage of GFP+TCR75+ (D) cells was determined.

 
In a separate experiment, the dose of TGF-beta was titrated in the presence or absence of 100 U/ml IL-2 at a constant dose of 0.3 µM Kd peptide. TGF-beta had no effect on the yield of TCR75 that was stimulated by Kd peptide except at the highest dose, which marginally reduced the yield of TCR75+ cells unless IL-2 was present (Fig. 3C). TGF-beta induced the up-regulation of GFP+ cells up to a maximum of ~35% a dose of ≥1 ng/ml and IL-2 only modestly increased GFP+ cells to 40% at 3 and 10 ng/ml TGF-beta (Fig. 3D). In the presence of TGF-beta, IL-2 induced growth and induction of GFP+ expression in a dose-dependent manner with the optimal dose between 30 and 100 U/ml (data not shown).

Finally, the kinetics of induction of GFP+ TCR75 T cells by various doses of Kd peptide in the presence of 3 ng/ml TGF-beta with or without 100 U/ml IL-2 was examined (Fig. 4). No significant number of GFP+ cells was stimulated at 10 µM Kd in the presence of TGF-beta at any day whether IL-2 was present or not, which verifies the inhibition of the induction of FoxP3 that was observed with high doses of peptide in Fig. 3B. Day 5 was the peak response of GFP+ cells at the two intermediate doses of Kd. IL-2 enhanced the yield of GFP+ cells at doses of 0.3 and 0.1 µM Kd and at the 0.1 µM dose of Kd the peak response shifted to day 7. These results demonstrate that the induction of FoxP3 is not only dose dependent but also that it is inhibited by high doses of Ag and this inhibitory effect cannot be rescued by IL-2.


Figure 4
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  		FIGURE 4. Kinetics of GFP expression stimulated by different doses of Kd peptide. Spleen cells from FoxP3gfp.TCR75 mice were incubated with the indicated doses of Kd54–68, 3 ng/ml TGF-beta, with or without 100 U/ml recombinant IL-2, and harvested on days 3, 5, and 7. The cultured cells were analyzed by flow cytometry, adding 10-µm diameter fluorescent beads as a volume calibrator, and plotted vs the dose of Kd as described in Fig. 3.

 
Collectively, these results indicate that the relationship of the dose of Ag, TGF-beta, and IL-2 that induce expression of GFP is complex but that in the presence of 3 ng/ml TGF-beta and 100 U/ml IL-2, there is a range from 0.1 to 1 µM Kd peptide and a range from 5 to 7 days in which the maximum number of GFP+ cells can be recovered. In the remainder of the experiments in this study, cultures of spleen cells from FoxP3gfp.TCR75 mice were harvested on day 6 after stimulation with 0.3 µM Kd peptide, 3 ng/ml TGF-beta, and 100 U/ml IL-2, unless otherwise stated.

Functional activities of GFP+ and GFP– TCR75 T cells activated in the presence of TGF-beta

Natural Tregs are universally reported to have little or no capacity to proliferate in vitro when challenged with Ag or polyclonal activators such as anti-CD3. Thus, the ability of TCR75 from naive mice to proliferate upon stimulation with Kd peptide was compared with that of TCR75 cells incubated for 6 days with Kd peptide with or without TGF-beta. TGF-beta-activated but unsorted TCR75 T cells proliferated upon restimulation with Kd peptide in a dose-dependent fashion albeit at a lower level than freshly isolated spleen cells from naive TCR75 mice (Fig. 5A). GFP– TCR75 cells also proliferated in a dose-dependent manner upon restimulation, but the response fell off at high peptide concentrations. GFP+ TCR75 responded only marginally and at much higher doses of Kd peptide than GFP– TCR75 cells. Presumably the proliferation of the unseparated TCR75 cells, which did not fall off at high concentrations, results from the additive effects of the GFP+ and GFP– T cells. Like natural Tregs, the proliferative responses of GFP+ TCR75 T cells could be reconstituted by the addition of recombinant IL-2 (Fig. 5B).


Figure 5
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  		FIGURE 5. Analysis of proliferative responses of TGF-beta-activated FoxP3gfp.TCR75 spleen cells. Spleen cells from FoxP3gfp.TCR75 mice were activated with irradiated B6 filler cells, 0.3 µM Kd54–68 peptide, 100 U/ml IL-2, or 3 ng/ml TGF-beta as described in Fig. 1. After 6 days, unsorted, GFP+ and GFP– populations and freshly prepared spleen cells from naive FoxP3gfp.TCR75 mice were cultured with irradiated B6 filler cells and various doses of Kd peptide in 96-well plates. A, After 3 days, 1 µCi [3H]thymidine/well was added and the cells harvested and counted on day 4. B, The ability of IL-2 (20 U/ml) to reconstitute the proliferative response of anergic GFP+ TCR75 T cells was also examined.

 
TCR75 T cells that had be cultured with Kd peptide but no TGF-beta expressed high levels of intracellular IFN-{gamma} and TNF-{alpha} but low levels of IL-4, and IL-10 upon restimulation with Kd in a 4 h assay (Fig. 6). By contrast, GFP+ TCR75 T cells that had been activated with Kd in the presence of TGF-beta developed only very low levels of IFN-{gamma}, IL-4, IL-10 and modest levels of TNF-{alpha} upon restimulation, which correlates with their inability to proliferate upon restimulation. GFP– TCR75 T cells, which had been activated with Kd in the presence of TGF-beta, also developed only very low levels of IFN-{gamma}, IL-4, and IL-10 and modest levels of TNF-{alpha} upon restimulation, despite the fact that they proliferated upon restimulation, as shown in Fig. 5. This observation suggests that the developmental pathway of all the TCR75 T cells was altered by activation in the presence of TGF-beta even though only a subset up-regulated FoxP3. Because TGF-beta, in the presence of anti-IFN-{gamma} (48) or IL-6 (49, 50), inhibits the development of Th1 and Th2 cells while inducing the development of Th17 cells, activated TCR75 T cells were also tested for IL-17 expression. Neither the activated TCR75 cells nor the TGF-beta-activated GFP+ or GFP– T cells expressed IL-17 upon restimulation (Fig. 6), although TCR75 T cells could produce IL-17 if activated with TGF-beta and IL-6 (data not shown). These results rule out the possibility that the TGF-beta-activated FoxP3-TCR75 T cells were activated into the Th17 pathway by TGF-beta. This ruling is not particularly surprising because these culture conditions would not be expected to activate production of IL-6.


Figure 6
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  		FIGURE 6. ICC responses upon restimulation of FoxP3gfp.TCR75 spleen cells activated with or without TGF-beta. Spleen cells from FoxP3gfp.TCR75 mice were activated with irradiated B6 filler cells, 0.3 µM Kd54–68 peptide, 100 U/ml IL-2, or 3 ng/ml TGF-beta as described Fig. 1. After 6 days, cultures were harvested and restimulated with irradiated spleen cells from B6.CD901/1 mice as a source of APC and 10 µM Kd54–68 and brefeldin A. After 4 h, the cells were harvested and stained with Ab to CD4 and anti-CD90.2 followed by fixation, permeabilization, and staining with and Abs for various cytokines. After gating on CD4+, CD90.2 T cells, the samples were analyzed for expression of ICC and GFP. A, The histograms in the upper panel display the isotype control (thin line) and Ab to the cytokine (thick line) for TCR75 activated with Kd54–55, which are all GFP–. The numbers represent the percentage of positive indicated by the horizontal lines. The lower panel displays the response stimulated with Kd54–55 and TGF-b, in which GFP– cells (thick line) and GFP+ (shaded) and the percentage of positive for GFP– are shown on top and GFP+ underneath the line. B, The number of cytokine-positive cells determined in A were calculated and plotted as a percentage of positive cells.

 
Suppressive activity of GFP+ TCR75 T cells activated in the presence of TGF-beta

The suppressive activity of the TCR75 cells, activated in the presence of TGF-beta was assessed on responses by spleen cells from naive TCR75 mice stimulated by irradiated spleen cells from B6 Tg mice expressing Kd. Proliferation of TCR75 T cells, as measured by dilution of CFSE and the mean cycle number, was inhibited in a dose-dependent fashion by GFP+ but not GFP– TCR75 cells activated in the presence of TGF-beta (Fig. 7A). GFP+ TCR75 cells also inhibited the ability of the target TCR75 T cells to produce either IL-10 or IFN-{gamma} upon restimulation with B6 spleen cells and Kd54–68 (Fig. 7B). In contrast, GFP– TCR75 cells did not inhibit the IL-10 response at any dose tested, although they partially inhibited the IFN-{gamma} response. However, the effect on IFN-{gamma} production was variable but not titratable and hence probably not specific. These results demonstrate that Ag-induced responses can be suppressed by GFP+ TCR75 cells, similar to the activity of natural Tregs, which will typically inhibit proliferative responses and effector T cell functions.


Figure 7
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  		FIGURE 7. Suppressive activity of GFP+TCR75 cells on responses by naive TCR75 spleen cells. Spleen cells from FoxP3gfp.TCR75 mice were activated with irradiated B6 filler cells, 0.3 µM Kd54–68 peptide, 100 U/ml IL-2, or 3 ng/ml TGF-beta as described in Fig. 1. After 6 days, cultured cells were harvested, flow sorted into GFP+ and GFP– TCR75 cells, and added at the indicated ratios to CFSE-labeled spleen cells from TCR75 mice and stimulated with 3 x 105 irradiated spleen cells from B6.Kd mice. After 4 days, the cultures were harvested, and a portion were stained and analyzed by flow cytometry. A, Data are displayed as dilution of CFSE by gating on CD4+ cells as shown by the histogram and the mean cycle number indicated. B, Another portion of the cultures was stained for ICC expression of IFN-{gamma} and IL-10. Tr, Treg cells; Te, T effector cells.

 
To ascertain whether the pattern of suppression by GFP+ and GFP– TCR75 T cells was representative of polyclonal T cells, the cells were added to cultures of B6 splenic T cells that were stimulated with anti-CD3. Anti-CD3 Ab stimulated CFSE-labeled CD4+ and CD8+ B6 spleen cells to enter cell cycle giving mean cycle numbers of 4.52 and 6.22, respectively (Fig. 8). GFP+ TCR75 cells inhibited entry into cell cycle and mean cycle numbers of CD4+ at all doses tested. GFP+ TCR75 cells also inhibited CD8+ T cells, in a dose-dependent fashion, but they were not as sensitive to suppression as CD4+ cells. GFP– TCR75 did not inhibit cell cycle progression of CD4+ T cells, although there was a modest reduction in the number of cells that entered cell cycle indicated by an increase in the number of cells in C = 0 at the highest dose of GFP– TCR75 cells. GFP– TCR75 cells inhibited neither the entry of CD8+ T cells into cell cycle nor their cell cycle progression.


Figure 8
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  		FIGURE 8. Suppressive activity of GFP+TCR75 cells on responses by naive B6 spleen cells stimulated with anti-CD3. Spleen cells from FoxP3gfp.TCR75 mice were activated with irradiated B6 filler cells, 0.3 µM Kd54–68 peptide, 100 U/ml IL-2, or 3 ng/ml TGF-beta as described Fig. 1. After 6 days, they were harvested, flow sorted into GFP+ and GFP– TCR75 cells, and added at the indicated Treg to T effector (Tr:Te) cell ratios to CFSE-labeled CD4+ or CD8+ spleen cells from B6 mice and 1 µg/ml anti-CD3 Ab. Cells were assayed by CFSE dilution as described in Fig. 7A, and data are shown by the histogram and the mean cycle number indicated.

 
The observation that polyclonal CD8+ T cells were inhibited by GFP+ TCR75 afforded an opportunity to address the Ag specificity of suppression by assessing their cytolytic response. Responses by B6 T cells stimulated by irradiated spleen cells from B6.Kd Tg mice were inhibited by GFP+ TCR75 cells (Fig. 9A). By contrast, GFP– TCR75 cells did not inhibit Kd-specific CTL responses, but routinely enhanced their lytic activity. Because both the TCR75 Tregs and the effector B6 CD8+ T cells recognize Kd peptides presented by B6.Kd APC, determining the specificity of suppression required an alternative strategy. Thus, we tested a mixture of I-Abeta–/–.Kd and beta2M–/–.Kd spleen cells that can be individually recognized only by CD8+ B6 responder T cells and TCR75 Tregs, respectively. Neither GFP+ nor GFP– TCR75 cells inhibited cultures stimulated with the mixture of I-Abeta–/–.Kd and beta2M–/–.Kd spleen cells, demonstrating that GFP+ cells display linked suppression. In addition, anti-TGF-beta did not significantly inhibit the suppressive activity of the GFP+ TCR75 cells in cultures stimulated with B6.Kd, demonstrating that the specific suppressive activity of GFP+ TCR75 cells does not involve either carryover of exogenous TGF-beta or synthesis of endogenous TGF-beta in the secondary culture in this system. The extremely small amount of suppression that was observed in cultures containing the mixture of I-Abeta–/–.Kd and beta2M–/–.Kd spleen cells (Fig. 9A) was reversed by inclusion of anti-TGF-beta in the cultures (Fig. 9B), but it is not clear that this result is biologically significant.


Figure 9
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  		FIGURE 9. Suppressive activity of GFP+TCR75 T cells on Ag-specific effector function of spleen cells from B6 mice. FoxP3gfp.TCR75 spleen cells were activated for 6 days as described in Fig. 1, added to CD8+ splenic T cells from B6 mice in a 0.4:1 ratio, and stimulated with irradiated spleen cells (SpLx) from B6.Kd mice or a mixture of I-Abeta–/–.Kd and beta2M–/–.Kd spleen cells, as a source of APC. Similar cultures were incubated with neutralizing Ab to TGF-beta. A, After 5 days, cultures were harvested and tested for CTL activity at the indicated E:T ratios using P815 targets and EL4 control targets. B, This experiment was repeated using Rag.FoxP3gfp.TCR75 spleen cells as a source of Tregs. They were added to spleen cells from B6.Kd mice or a mixture of I-Abeta–/–.Kd and beta2M–/–.Kd spleen cells, as a source of APC and assayed for CTL activity.

 
Because recombinase sufficient TCR Tg T cells can be expected to express host derived TCR {alpha}-chains, we wanted to determine whether they contributed to the suppressive activity observed in the GFP+ Vbeta8.3+ T cells. We could not verify that they expressed the TCR V{alpha}1.1 for lack of an appropriate Ab, so we tested this by breeding the FoxP3gfp.TCR75 mice to Rag–/–.B6 mice, backcrossing the F1 hybrids to B6.Rag–/–, and testing whether the homozygous Rag–/–.FoxP3+.TCR75 mice exhibited the same activity as recombinase sufficient mice. The results demonstrated that GFP+ Rag–/–.FoxP3+.TCR75 inhibited the B6 CTL response stimulated by B6.Kd APC and suppression required that both peptides be expressed by the same APC (Fig. 9B). These observations suggest that all of the suppressive activity of the GFP+ cells can be attributed to cells expressing the TCR Tg Vbeta8.3 V{alpha}1.1 pair.

Finally, to determine whether the linked suppression could be generalized to CD8 T cells of another specificity, GFP+ and GFP– TCR75 cells were tested on T cells from Rag.OT1 mice (Fig. 10). GFP+ TCR75 T cells inhibited the CTL response by OT1 T cells stimulated by APC expressing if they expressed both Kd and OVA (Fig. 10, left) but not when stimulated with a mixture of Kd plus OVA expressing APC (Fig. 10, middle). GFP– TCR75 T cells routinely enhanced the CTL responses to both types of APC. Suppression by GFP+ TCR75 did not involve either carry over of exogenous TGF-beta or synthesis of endogenous TGF-beta (Fig. 10, right).


Figure 10
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  		FIGURE 10. Suppressive activity of GFP+TCR75 T cells on Ag-specific effector function of spleen cells from OT1 mice. FoxP3gfp.TCR75 spleen cells were activated for 6 days as described in Fig. 1, sorted into GFP+ and GFP– subsets, and added to OT1.CD90.1 spleen cells in a 3:1 ratio. Cells were stimulated with irradiated B6.Kd.OVA (left), a mixture of B6.Kd and B6.OVA spleen cells (middle), as a source of APC, or with B6.Kd.OVA and neutralizing Ab specific for TGF-beta (right). After 4 days, the cultures were harvested and assayed for CTL activity at the indicated E:T ratios using E.G7-OVA as targets.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Few if any CD4+ T cells from naive FoxP3gfp.TCR75 Tg mice expressed both the Vbeta8.3 transgene and FoxP3+ (GFP), which is in contrast to reports for other TCR Tg mouse strains such as: OVA-specific DO11.10 (51), myelin basic protein-specific (52), and I-Abm12 alloantigen-specific anti-bm12 ABM mice (53), in which ~5% of the TCR Tg T cells express CD25 and/or FoxP3. However, the natural Tregs observed in the latter TCR Tg systems are dependent upon the expression of nontransgenic TCR {alpha}-chains because they are not detected in recombinase-deficient mice (51, 54, 55). Why TCR75 mice fail to develop significant levels of natural Tregs expressing nontransgenic TCR {alpha}-chains has not been determined, but this phenomenon is not limited to the TCR75 Tg mice because we have observed a similar pattern in Tg mice expressing the OT2 TCR (our unpublished observations). The variability in the level of expression of nontransgenic TCR {alpha}-chains in different TCR Tg strains of mice might be accounted for by various mechanisms, including the rate of positive selection of the Tg TCR, which controls the likelihood of endogenous {alpha}-chain expression mediating Treg differentiation, or the ability of the Tg TCR beta-chain to be selected by self-peptide/MHC epitopes when combined with permissible endogenous {alpha}-chains.

The failure of T cells expressing TCR transgenes to differentiate into Tregs in Rag–/– mice is not entirely unexpected because most were derived from T cell clones originally stimulated by exogenous Ags, which are not present in the normal mice. However, if the selecting epitopes are provided by introduction of an appropriate Ag as a transgene, there is no inherent problem in the selection of a Tg TCR into the Treg lineage (56, 57, 58). The ability of TGF-beta to alter the developmental pathway of Ag-activated TCR Tg T cells (23, 24) into the Treg pathway provides an opportunity to study the biology of Tregs using TCR Tg T cells of various defined specificities. However, results obtained from this approach must be interpreted cautiously because the TCR Tg T cells that are acutely activated in vitro by TGF-beta may not share all the properties of natural Tregs, which are activated in the thymus and chronically stimulated by autoantigens in the periphery (59).

Data in this communication demonstrate that Kd peptide plus TGF-beta activates FoxP3 expression in FoxP3– precursor cells in vitro and that the proportion of cells that become FoxP3+ depends upon the dose of peptide, TGF-beta, and IL-2. Although the TGF-beta-activated FoxP3– TCR75 T cells proliferated to a broad range of Kd peptide upon restimulation, the FoxP3+ T cells proliferated modestly and only at high doses of Ag. Like natural Tregs, the proliferative response of FoxP3+ T cells could be rescued by addition of IL-2 to the culture. By contrast, activation with Kd peptide plus TGF-beta significantly reduced the ability of both FoxP3+ and FoxP3– TCR75 cells to produce cytokines upon restimulation in comparison to TCR75 T cells activated by Kd peptide alone, although only a subset develop into FoxP3+ cells. Nevertheless, FoxP3+ TCR75 cells suppressed proliferation as measured by dilution of CFSE and ICC responses by naive TCR75 target cells, whereas the FoxP3– cells did not. GFP+, but not GFP–, TCR75 T cells also inhibited proliferation as measured by cell cycle progression in polyclonal responses by CD4+ and CD8+ B6 spleen cells. Thus, TGF-beta-activated, FoxP3+ TCR75 T cells are similar to natural Tregs in their ability to inhibit polyclonal proliferation and effector responses by naive T cells.

Inhibition of TCR75 responses to Kd by FoxP3+ TCR75 T cells cannot be used to address the specificity of suppression because both the responder and the regulatory cells recognize the same peptide and I-A restricting element. Suppression of polyclonal responses by GFP+ TCR75 also cannot be used to address the specificity issue because anti-CD3 reacts with the TCR of both the responder and the regulatory cells. However, the observation that GFP+ TCR75 cells inhibited Kd-specific CTL responses of B6 spleen cells stimulated with Kd-expressing Tg spleen cells afforded us the opportunity to address the issue of linked recognition because the CD4+ Treg and the responder CD8+ T cells recognize Kd peptides presented by MHC class II and class I, respectively. Thus, a mixture of I-Abeta–/–.Kd and beta2M–/–.Kd Tg spleen cells that can be individually recognized only by CD8+ B6 responder T cells and TCR75 Tregs, respectively, was compared with B6.Kd spleen cells. If the GFP+ TCR75 cells would have inhibited B6 anti-Kd CTL in cultures stimulated with the mixture of I-Abeta–/–.Kd and beta2M–/–.Kd spleen cells as well as B6.Kd spleen cells, it would have been taken as evidence of unlinked, or bystander, suppression. However, GFP+ TCR75 cells inhibited the response stimulated with B6.Kd spleen cells but not the mixture of I-Abeta–/–. Kd and beta2M–/–.Kd spleen cells, a pattern that was originally termed "linked suppression" by Holan and Mitchison (60), which has been validated by others (61, 62, 63) and also recently reported in a TCR Tg model of transplant tolerance (53). APCs are thought to serve as a "bridge" to mediate functional communication between the two T cells in linked suppression, and this contact requires that the epitopes recognized by the Tregs and the responder T cells are both expressed by the same APC. Whether APCs facilitate direct cell contact between the regulatory cells and the effector cells or whether the contact is indirect and mediated by sequential interaction of the APC first with the regulatory and subsequently with the responder T cells, as has been shown for Th cells by Ridge et al. (64), is not yet known. Nevertheless, our observation that Tregs and responder T cells must recognize the same APC provides a mechanistic explanation for the often reported, but poorly understood, requirement that Tregs must be in direct contact with effector T cells to inhibit their responses.

Linked suppression exhibited by FoxP3+ TCR75 T cells in this culture system agrees with the observations that natural Tregs must interact with Ag to inhibit autoimmune responses and that inhibition is specific in vivo (18) and, with the specificity exhibited in vivo and in vitro by FoxP3+ CD8+ T cells, activated by Ag and TGF-beta (47). By contrast, linked suppression was not observed in two previous in vitro studies specifically designed to address this issue (17, 19). It is, of course, possible that Tregs might express exquisite antigenic specificity under certain circumstances but nonspecific activity under others, as has long been appreciated for CD4+ Th cells. However, the lack of cognate or linked recognition by classical CD4+ T cells is associated with the secretion of cytokines that can act at a distance, whereas the Tregs studied by Takahashi et al. (17) and Thornton and Shevach (19) suppressed responses in a contact-dependent manner.

How to reconcile these dichotomous results is not completely clear, but the experiments supporting the nonspecific nature of Tregs are not unequivocal because of caveats in the experimental design. In the studies reported by Takahashi et al. (17), CD4+CD25+ T cells from BALB/c mice expressing either D011.10 or BOG1 TCR transgenes, which recognize distinct non-cross-reacting epitopes of OVA, were shown to cross-suppress responses to the other Ag when both peptides were included in the culture. Although these results provide evidence that the Tregs need to be activated by Ag to mediate suppression, they cannot be taken as evidence for nonspecific suppression because the APC in the test cultures could bind both peptides, thereby presenting Ag simultaneously to both the responder and Tregs. Hence, the results of Takahashi et al. (17) are no different from what we observed for TCR75 T cells stimulated with B6.Kd spleen cells except that they did not report results from cultures stimulated with mixtures of APC that had been individually pulsed with peptides, which might have clarified this issue.

In the study reported by Thornton and Shevach (19), suppression by Tregs from two different TCR Tg mice was observed not only when both Ags were present in culture but also when the Ags were presented by a mixture of APC that had been individually pulsed with the peptides. These results provide stronger evidence that suppression is nonspecific. However, this experiment is also not conclusive because the regulatory and effector T cells were obtained from recombinase-sufficient, TCR Tg mice and it is now appreciated that they are not likely to be monospecific because of the expression of endogenous TCR-{alpha} genes (51, 54, 55). This likelihood is especially important in the Thornton and Shevach study (19) because the TCR Tg Tregs were obtained from mice expressing different MHC haplotypes. Consequently, the test cultures contained APCs expressing two different MHC haplotypes. The TCR Tg Tregs potentially could recognize alloantigens as well as the nominal epitope specified by the TCR transgenes. Thus, suppression could be targeted to MHC-mismatched responder T cells by allorecognition of APC in the absence of the nominal Ag, thereby giving the appearance of nonspecific suppression.

Our data illustrate that FoxP3+ Tregs can exhibit more antigenic specificity in their suppressive activity in vitro than previously appreciated and suggest that data supporting nonspecific suppression by Tregs should be interpreted with caution. The finding that Tregs may be both specific in activation and in effector function in vitro correlates with data indicating that Tregs have exquisite functional specificity in vivo (18, 53, 65, 66). These observations that Tregs exhibit functional specificity provide support for the feasibility of using Tregs as immune modulators in the clinic, which would be severely compromised if they were nonspecific in their suppressive function. The capacity of Tregs to suppress effector T cell responses to distinct Ags if both Ags are presented by the same APC, as shown in this study and other reports (47, 53, 60, 61), promises to expand the utility of Tregs for treatment of diseases, such as type 1 diabetes and multiple sclerosis, in which pathogenic T cells with specificities for multiple Ags are involved, without the need to identify each epitope.


   Acknowledgments
 
We thank Dr. Marc Jenkins for the B6.OVA mice, Dr. Michael Bevan for the OT1 mice, and Dr. Casey Weaver for the Foxp3gfp mice that were provided by Dr. Alex Rudensky.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants HL50724 (to R.P.B.) and EY014877 (to J.A.K.) from the National Institutes of Health, from the Research to Prevent Blindness (to J.A.K.), and from the Foundation for Fighting Blindness (to J.A.K.). Back

2 Address correspondence and reprint requests to Dr. Judith A. Kapp, Room W287 Spain Wallace Building, University of Alabama at Birmingham, 619 South 19th Street, Birmingham, AL 35294-2170. E-mail address: jkapp{at}uab.edu Back

3 Abbreviations used in this paper: Treg, regulatory T cell; FoxP3, forkhead/winged helix transcription factor; ICC, intracellular cytokine; GITR, glucocorticoid-induced TNFR; beta2M, beta2-microglobulin; Tg, transgenic. Back

Received for publication May 8, 2007. Accepted for publication May 31, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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