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TGF--Mediated Suppression by CD4⁺CD25⁺ T Cells Is Facilitated by CTLA-4 Signaling

Takatoku Oida^1,*, LiLi Xu^1,*, Howard L. Weiner, Atsushi Kitani^* and Warren Strober^2,*

^* Mucosal Immunity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
CD4⁺CD25⁺ T cells play a pivotal role in immunological homeostasis by their capacity to exert immunosuppressive activity. However, the mechanism by which these cells function is still a subject for debate. We previously reported that surface (membrane) TGF- produced by CD4⁺CD25⁺ T cells was an effector molecule mediating suppressor function. We now support this finding by imaging surface TGF- on Foxp3⁺CD4⁺CD25⁺ T cells in confocal fluorescence microscopy. Then, using a TGF--sensitive mink lung epithelial cell (luciferase) reporter system, we show that surface TGF- can be activated to signal upon cell-cell contact. Moreover, if such TGF- signaling is blocked in an in vitro assay of CD4⁺CD25⁺ T cell suppression by a specific inhibitor of TGF-RI, suppressor function is also blocked. Finally, we address the role of CTLA-4 in CD4⁺CD25⁺ T cell suppression, showing first that whereas anti-CTLA-4 does not block in vitro suppressor function, it does complement the blocking activity of anti-TGF-. We then show with confocal fluorescence microscopy that incubation of CD4⁺CD25⁺ T cells with anti-CTLA-4- and rB7-1/Fc-coated beads results in accumulation of TGF- at the cell-bead contact site. This suggests that CTLA-4 signaling facilitates TGF--mediated suppression by intensifying the TGF- signal at the point of suppressor cell-target cell interaction.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The mechanism of the strong suppressive activity exerted by CD4⁺CD25⁺ regulatory T (Treg)³ cells on both in vitro and in vivo immune responses is still unclear. In initial studies using in vitro assay systems, it was shown that such suppression requires cell-cell contact (1, 2), and it was thus considered likely that the suppression was dependent on ligand-receptor interactions at the cell surface. In later studies conducted in our laboratory and again involving in vitro suppressor assays, we showed that CD25⁺ Treg cells express membrane-bound TGF- associated with latency-associated protein (LAP) and that the suppressor function of the regulatory cells could be blocked in vitro by addition of rLAP (3). These in vitro data supporting the role of TGF- in the mechanism of CD25⁺ suppressor function correlated with in vivo data showing that mice bearing a transgene expressing a dominant-negative TGF-RII receptor, and thus incapable of responding to TGF-, could not be protected from inflammation by the provision of regulatory CD25⁺ cells (4, 5).

Despite these data, doubts concerning TGF- as a mediator of CD25⁺ suppression have persisted because CD25⁺ cells from TGF- knockout (KO) mice still have the capacity to suppress both in vitro and, in some but not all studies, in vivo (3, 5, 6). However, these studies are subject to the caveat that activated and/or anergic T cells such as those present in TGF- KO mice can also manifest suppressor activity in in vitro assays by as yet unknown mechanisms (7, 8, 9, 10) and thus may be mediating an independent and separate form of suppressor activity.

Another potential effector molecule in CD25⁺ Treg cells arises from the observation that suppressor function by these cells could be blocked, both in vitro and in vivo, by anti-CTLA-4 mAb (11, 12). However, the role of CTLA-4 has also been challenged (13, 14, 15) because it does not explain the fact that CD25⁺ suppressor activity can occur in the absence of APC by T cell–T cell interaction alone (13, 16) and is observed in CD25⁺ T cells from CTLA-4 KO mice (17). These latter data suggest that CTLA-4 plays, at best, a secondary role in the mechanism of CD25⁺ T cell regulation.

In the present study, we first show by confocal microscopy that CD25⁺Foxp3⁺ Treg cells but not CD25⁺Foxp3^– T cells display TGF- on their surface and thus verify and expand on previous flow cytometric studies (3, 18). We then show, using a TGF--sensitive mink lung epithelial cell (luciferase) reporter system, that CD25⁺ Treg cells deliver a functional TGF- signal via cell-cell contact in the absence of soluble TGF-. In addition, we show in an assay system in which cells are either activated by anti-CD3-coated beads or by APC and anti-CD3 that suppression of CD4⁺CD25^– T cell proliferation does not occur in the presence of a specific TGF-RI inhibitor. Then, in a parallel series of studies in which the role of CTLA-4 in suppression is addressed, we show using confocal microscopy that exposure of CD25⁺ Treg cells to anti-CTLA-4- or rB7-1/Fc-coated beads leads to concentration of TGF- on the cell membrane at the point of cell-bead contact. Taken together, these studies indicate that, while the main mechanism of CD25⁺ T cell suppressor activity is via the expression of surface TGF-, CTLA-4 signaling of the CD25⁺ T cell enhances the suppressive signal by concentrating the TGF- at the point of cell-cell contact.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice

Specific pathogen-free, female BALB/c mice were purchased from the National Cancer Institute or The Jackson Laboratory. The mice were studied at 6–14 wk of age. Animal use adhered to National Institutes of Health Laboratory Animal Care Guidelines.

Abs and reagents

Anti-CD3 mAb (145-2C11), anti-CD28 mAb (37.51), purified hamster anti-CTLA-4 mAb (UC10–4F10-11), normal hamster IgG, FITC-streptavidin, PE-Cy5-streptavidin, and anti-CD16/CD32 (FcBlock) were purchased from BD Pharmingen. Mouse anti-TGF-1, -2, and -3 mAb (1D11), rLAP, biotinylated goat anti-LAP Ab, biotinylated goat IgG, and biotinylated chicken anti-TGF- Ab were purchased from R&D Systems. Normal mouse IgG and biotinylated chicken IgY were purchased from Jackson ImmunoResearch Laboratories. FITC-anti-mouse Foxp3 (clone FJK-16s) and FITC-Rat IgG2a isotype control were purchased from eBioscience. ALK5 (TGF-RI) inhibitor SB431542 (19, 20, 21) was a gift from Dr. N. Laping (GlaxoSmithKline Pharmaceuticals, King of Prussia, PA). Furin inhibitor I (Dec-RVKR-CMK) was obtained from Calbiochem. [methyl-³H]Thymidine was purchased from Amersham Biosciences.

Cell lines

For use in a TGF- bioassay, a mink lung epithelial cell line transfected with the Smad-responsive plasminogen activator inhibitor-1 promoter driving a luciferase reporter gene (MLEC-PAI-1-Luc) (originally developed by Abe et al. (22)) was obtained from Dr. L. van de Water (Massachusetts General Hospital, Boston, MA). A subclone was selected for use on the basis of responsiveness in the assays described in the text. For use as an APC with reproducible properties, a I-A^k- and I-A^d-expressing B cell hybridoma, TA3 (23, 24), was obtained from Dr. L. Glimcher (Harvard School of Public Health, Boston, MA). A subclone of this hybridoma was maintained in 10% FCS (Invitrogen Life Technologies) containing complete DMEM (Invitrogen Life Technologies) until use.

Coating of polystyrene microspheres (beads)

Polystyrene microspheres (beads) (4.5 µm in diameter) were purchased from Polysciences and coated with anti-CD3 mAb and anti-CD28 mAb, according to the manufacturer’s instructions. In brief, 7.5 x 10⁷ beads were mixed with 15 µg of anti-CD3 and 5 µg of anti-CD28 in 300 µl of 0.1 M borate buffer (pH 8.5) for 5–8 h at room temperature (RT). For TGF- bioassays, the anti-CD3/anti-CD28-coated beads were further incubated in 400 µl of poly-L-lysine (250 µg/ml) (Sigma-Aldrich) for 1–2 h, washed with 10% FCS DMEM three times, suspended in 10% FCS DMEM, and stored at 4°C until use. For studies of the effects of coated beads on CD25⁺ T cells, anti-CTLA-4, rB7-1/Fc, and hamster IgG (50 µg) that had been dialysed in borate buffer overnight were mixed with 7.5 x 10⁷ beads and then subjected to end-over-end rotation overnight. The anti-CTLA-4-, rB7-1/Fc-, and hamster IgG-coated beads were then incubated with 400 µl of poly-L-lysine (250 µg/ml) (Sigma-Aldrich) for 1–2 h, washed with borate buffer three times, and, finally, suspended in PBS containing 10 mg/ml BSA, 5% glycerol. They were then stored at 4°C until use.

T cell purification

CD4⁺ T cells were purified from total splenocytes using CD4 T cell enrichment columns (R&D Systems). CD25⁺ T cells were purified by MACS with anti-CD25-FITC (7D4; BD Pharmingen) or anti-CD25-PE (Miltenyi Biotec), then further incubated with anti-FITC or anti-PE microbeads and selected through mini-separation columns (Miltenyi Biotec) as described previously (18). The purity of CD25⁺ T cells was always >90%, and the flow-through was shown to be >95% CD4⁺CD25^– T cells by flow cytometric analysis.

In vitro suppressor assays

For assays in which bead stimulation was used, 2.5 x 10⁴ CD25^– T cells mixed with various ratios of CD25⁺ T cells were cultured with 2.5 x 10⁴ anti-CD3/anti-CD28-coated beads in round-bottom 96-well plates for 72 h. 1 µCi/well [³H]thymidine was added to the wells during the last 8 h of culture. Alternatively, CD25^– T cells labeled with fluorescent dye CFSE (Molecular Probes) by incubation in PBS containing 1 µM CFSE for 15 min at 37°C were used, and the fluorescence was detected at 64 h by a FACScan analyzer (BD Biosciences). For suppressor assays in which APC stimulation was used, T cells were stimulated with 5 x 10³ 3000-rad irradiated TA3 cells plus 0.5 µg/ml anti-CD3 in 96-well flat-bottom plates.

For studies of inhibition of suppression in suppressor assays, freshly isolated CD25⁺ cells were incubated with 50 µg/ml anti-CTLA-4 (UC10-4F10-11) (BD Pharmingen) for 2 h or 50 µg/ml anti-TGF-1, -2, and -3 (1D11) (R&D Systems) for 1 h or with both Abs for 2 h at RT. Alternatively, CD25⁺ cells were preactivated with plate-bound anti-CD3 (10 µg/ml), soluble anti-CD28 (2 µg/ml), and IL-2 (50 U/ml) for 48 h, after which they were rested in IL-2 (50 U/ml) for another 12 h. The preactivated cells were then collected, extensively washed, and recounted, and then incubated with 50 µg/ml anti-CTLA-4, 50 µg/ml anti-TGF-1, -2, and -3 or 50 µg/ml rLAP for 2 h at RT. Following this incubation, 2.5 x 10⁴ Ab-treated CD25⁺ T cells, or serial dilution of preactivated Treg cells, 2.5 x 10⁴ CD25^– T cells, and 1 x 10⁵ irradiated syngenic splenocytes (3000 rad) were cocultured with anti-CD3 mAb (5 µg/ml) at 37°C for 72 h and pulsed with 1 µCi of [³H]thymidine for the last 6–8 h in flat-bottom 96-well plates (0.2 ml). The cells were then harvested and assessed for thymidine incorporation in a liquid scintillation counter.

TGF- bioassays

TGF- bioassays were performed on preactivated cells or supernatants of cultures of these and other cells. Preactivated cells were obtained by stimulating 2.5 x 10⁵ CD25⁺ T or CD25^– T cells with 5 x 10⁴ irradiated (3000 rad) TA3 cells, 0.5 µg/ml anti-CD3, and 100 U/ml recombinant human (rh) IL-2 in 10% FCS DMEM for 4 days, after which the cells were recovered, washed, and recounted. The bioassay used MLEC-PAI-1-Luc cell reporter cell monolayers; the latter were constructed by seeding MLEC-PAI-Luc cells at 2.5–3 x 10⁴ cells/well in flat-bottom 96-well plates and incubating the seeded cells at 37°C for 4–6 h to let them attach to the surface of the wells; in assays of preactivated cells, 2.5 x 10⁵ anti-CD3/anti-CD28/poly-L-lysine-coated beads were incubated with the formed monolayers for 1.5 h, after which extra beads were washed away with 3 x 100 µl of PBS. In subsequent assays of TGF- on the surface of preactivated T cells, the MLEC-PAI-1-Luc cell monolayers/adherent anti-CD3/anti-CD28/poly-L-lysine-coated beads were overlaid with 1 x 10⁵ preactivated T cells, and the cell mixture was incubated for 14 h; the cell mixture was then lysed, and any luciferase signal was measured by Bright-Glo luciferase reagent (Promega) using a luminometer (Turner Design). The specificity of the TGF- assay was confirmed by the loss of luciferase signal in the presence of anti-TGF- mAb. rhTGF- (R&D Systems) was used for conversion of luciferase units to TGF- picograms.

Immunofluorescence staining

Freshly isolated CD25⁺ and CD25^– T cells were blocked with anti-CD16/anti-CD32 and stained with biotinylated anti-LAP or biotinylated goat IgG, followed by streptavidin-PE-Cy5. After two washes, cells were resuspended in Fix/Perm buffer for 1 h, followed by two washes in permeabilization buffer, then stained with FITC-anti-mouse Foxp3 Ab or FITC-Rat IgG2a isotype control for 30 min. After two washes, cells were subjected to confocal fluorescence microscopy or flow cytometry.

For studies of the effects of coated beads on surface TGF- expression, freshly isolated CD25⁺ and CD25^– T cells were stimulated with plate-bound anti-CD3 (10 µg/ml) and IL-2 (50 U/L) in serum-free medium X-Vivo 15 and cultured for 3640 h, then harvested and incubated with anti-CTLA-4-beads, rB7-1/Fc-beads, hamster-IgG-beads, or soluble anti-CTLA-4 in a ratio of 1:2 (cell:bead), respectively, for 1 h at RT. After washing and Fc block, cells were stained with biotinylated chicken anti-TGF- or biotinylated chicken IgY, followed by streptavidin-FITC. After two washes, cells were resuspended in staining buffer (PBS-1% BSA-0.02% sodium azide) and subjected to confocal fluorescence microscopy.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
CD25⁺Foxp3⁺ but not CD25^–Foxp3^– T cells express surface TGF-

In a previous study, we showed by flow cytometric analysis using both chicken anti-TGF- polyclonal Ab and anti-LAP (anti-TGF- LAP) mAb that CD25⁺ T cells express cell surface TGF- (3, 18). While such expression was noted in resting cells, it was particularly evident in activated CD4⁺CD25⁺ T cells; in contrast, CD4⁺CD25^– T cells expressed little or no surface TGF- in resting cells and only marginal amounts in activated cells. In the present study, we sought to verify and expand on these findings with confocal fluorescence microscopy. Accordingly, we prepared purified populations of both CD4⁺CD25⁺ and CD4⁺CD25^– T cells from mouse spleens and then subjected the cells to surface and intracellular staining with anti-LAP and anti-Foxp3 Abs (see Materials and Methods) to detect both membrane-bound TGF- and intracellular Foxp3. As shown in Fig. 1A, surface and intracellular staining of CD4⁺CD25⁺ T cells with anti-LAP and anti-Foxp3 revealed that LAP⁺ cells were also Foxp3⁺, whereas CD4⁺CD25^– T cells were both surface LAP^– and Foxp3^– (the few LAP⁺CD25^– seen were only weakly LAP⁺). In addition, as shown in Fig. 1A, activated CD25⁺ cells (see Materials and Methods) were also LAP⁺ and Foxp3⁺, whereas in data not shown, activated CD25^– cells were LAP^– and Foxp3^–. Finally, as shown in Fig. 1B, flow cytometric analysis of the same cell population revealed that >80% of CD4⁺CD25⁺ T cells are Foxp3⁺ and the majority of these Foxp3⁺ cells are also LAP⁺, whereas <3% of CD4⁺CD25^– T cells are Foxp3⁺. These studies corroborate earlier studies showing that CD25⁺ T cells bear surface TGF-; in addition, they show that these TGF-⁺ cells are indeed regulatory cells that express Foxp3.

View larger version (40K): [in this window] [in a new window]   FIGURE 1. CD4/CD25+Foxp3+ but not CD4/CD25–Foxp3– cells express surface TGF-. Freshly isolated CD4/CD25+ and CD4/CD25– splenocytes were blocked with anti-CD16/CD32 and incubated with biotinylated goat anti-LAP Ab or biotinylated goat IgG (15 min), followed by streptavidin-PE-Cy5 (15 min). After two washes, the cells were incubated in Fix/Perm buffer for 1 h and washed twice in permeabilization buffer and reincubated with FITC-anti-mouse Foxp3 Ab or FITC-rat IgG2a isotype control (30 min). Finally, after two additional washes, the cells were examined by confocal fluorescence microscopy or flow cytometry. A, Cells were viewed under the confocal fluorescence microscope (x63). Left panel, The overlay of intracellular staining of Foxp3 (green) and cell surface LAP (red). Right panel, Differential interference contrast (DIC) view of cells. B, Flow cytometry of the cells stained with isotype control Ig for anti-LAP and anti-Foxp3 (left panel) and with both Abs according to the protocol given above (middle and right panels).

CD25⁺ T cells present active TGF- upon cell-cell contact

In further studies, we sought to verify that the surface TGF- detected above was biologically active. Thus, while in previous studies we showed that exposure of CD25^– T cells to CD25⁺ cells led to Smad activation (i.e., activation of the intracellular TGF- signaling pathway), these studies fell short of unequivocally demonstrating that such biological activity was actually due to cell surface TGF- and not secreted TGF- and did not demonstrate that activated Smad induced by surface TGF- actually had transcriptional activity.

To address these questions, we turned to a bioassay system for the detection of surface TGF- activity on activated CD4⁺ T cells that used indicator cells that respond to active TGF- by generating a luciferase signal. The latter consisted of TGF-R-bearing mink lung epithelial cells containing a TGF--sensitive plasminogen activator inhibitor-1 promoter driving a luciferase gene (MLEC-PAI-1-Luc cells) (22). As shown in the schematic diagram depicted in Fig. 2A, CD25⁺ and CD25^– T cells isolated from BALB/c spleen cells were preactivated with APC/soluble anti-CD3/IL-2 for 4 days, after which the cells were recovered and counted. To measure surface TGF- activity of the preactivated T cells, the cells were seeded onto a monolayer of MLEC-PAI-1-Luc reporter cells with attached anti-CD3/anti-CD28/poly-L-lysine-coated beads, the latter to link the T cells to the reporter cells and, at the same time, to stimulate the T cells. After 14 h of culture, cells were lysed, and luciferase activity induced by the surface TGF-⁺ cells was measured. To measure secreted TGF- activity by the same preactivated cells, the latter were cultured with plate-bound anti-CD3/anti-CD28 for 14 h, after which the culture supernatants were collected and assayed for the presence of active TGF- in the MLEC-PAI-1-Luc reporter cell assay described above. Finally, the luciferase activity was converted to TGF- equivalent units using a dose-response curve generated by adding increasing amounts of rhTGF- to the MLEC-PAI-1-Luc assay system. The specificity of the assay for TGF- was confirmed by adding anti-TGF- mAb into the assay.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. CD4/CD25+ T cells signal MLEC-PAI-1-Luc reporter cells via surface TGF- upon cell-cell contact. CD4/CD25+ or CD4/CD25– T cells were prestimulated with anti-CD3+APC+IL-2 for 4 days. They were then recovered and cocultured for 14 h with a MLEC-PAI-1-Luc reporter cell monolayer with adherent anti-CD3/anti-CD28-coated beads. The latter served to both restimulate the T cells and to bring them into close contact with the reporter cells. Luciferase activity was measured by a luminometer. A, Schematic diagram of the TGF- bioassay. B, TGF- activity detected in the T cell-reporter cell coculture assay from preactivated CD4/CD25+ or CD4/CD25– T cells. The specificity of the assay for TGF- was confirmed by addition of anti-TGF- mAb.

CD25⁺ T cell-mediated suppression via cell-cell contact occurs mainly via TGF-

In previous studies, we sought to establish the role of TGF- in CD25⁺ T cell-mediated suppression by conducting in vitro suppressor assays in the presence and absence of anti-TGF- or LAP (3, 18). We did indeed obtain inhibition of suppression in these studies (as well as in similar studies described below), but such inhibition required the use of relatively high concentrations of anti-TGF- or LAP. To overcome any uncertainty that may have therefore resulted from these previous observations, we turned to an independent method of assessing the role of TGF- on CD25⁺ T cell suppressor activity based on the use of a specific inhibitor of TGF- signaling. In particular, we determined the ability of CD4⁺CD25⁺ T cells to mediate suppression in the presence of a specific inhibitor of TGF- signaling, i.e., an inhibitor of the kinase activity of TGF-RI (ALK5) known as ALK5 inhibitor (SB4315420; GlaxoSmithKline) (19, 20, 21).

In preliminary studies, we determined the dose range of the inhibitory effect of the ALK5 inhibitor on TGF- signaling using the MLEC-PAI-1-Luc reporter cell assay described above. Accordingly, MLEC-PAI-1-Luc cells were cultured with rhTGF- in the presence of varying doses of ALK5 inhibitor or in the latter’s absence for 12 h, and the luciferase activities obtained were converted to TGF- units as described above. As shown in Fig. 3A, 10 µM ALK5 inhibitor completely blocked rhTGF- activity and was the maximum inhibitory concentration of ALK5 inhibitor used in the suppressor assays described below. It should be noted that cell viability and growth were quite normal even when the reporter cells were cultured in the presence of 20 µM ALK5 inhibitor for 1 wk. Thus, luciferase production in this assay was not influenced by nonspecific cell cytotoxicity.

View larger version (35K): [in this window] [in a new window]   FIGURE 3. TGF-RI (ALK5) inhibitor reverses CD25+ T suppression. A, MLEC-PAI-1-Luc reporter cells were stimulated with 640 pg/ml rhTGF- in the presence of the indicated concentrations of ALK5 inhibitor for 12 h. At this point, luciferase activity was measured, and the values obtained were converted to equivalent TGF- concentrations by reference to a previously constructed TGF- standard curve. B, A total of 2.5 x 104 CFSE-labeled CD4/CD25– T cells was mixed with unlabeled CD4/CD25+ T cells at the indicated ratios and stimulated with 2.5 x 104 anti-CD3/anti-CD28-coated beads in the presence of the indicated concentrations of ALK5 inhibitor for 64 h in round-bottom plates. CFSE+ cells were then analyzed by flow cytometry. C, A total of 2.5 x 104 CFSE-labeled CD25– T cells was mixed with unlabeled CD25+ T cells at the indicated ratios and stimulated with 5 x 103 3000-rad irradiated TA3 cells plus 0.5 µg/ml anti-CD3 in the presence of the indicated concentrations of ALK5 inhibitor for 64 h in flat-bottom plates. CFSE+ cells were then analyzed by flow cytometry.

Blocking TGF- processing reverses CD25⁺-mediated suppression

To examine further the involvement of TGF- in the CD25⁺ T cell-mediated suppression, we determined the effect of an inhibitor of TGF- processing, furin inhibitor I, on the suppressor activity of these cells. TGF- is synthesized as a precursor peptide that is cleaved by furin protease to form TGF- peptide and LAP (26, 27). Thus, blocking furin protease activity with a furin protease inhibitor should affect surface TGF- activity. As shown in Fig. 4A, using the MLEC-PAI-1-Luc reporter coculture assay as described above, we first confirmed that furin inhibitor I (at a concentration of 100 µM) reduced the cell surface-TGF- activity exhibited by preactivated CD25⁺ T cells to one-third the activity observed in the absence of inhibitor. Secretion of total (mostly latent) TGF- to the culture supernatant was also inhibited (data not shown). We then tested the effect of furin inhibitor I in an in vitro CD25⁺ T cell suppression assay stimulated by anti-CD3/anti-CD28-coated beads. As shown in Fig. 4B, the furin inhibitor I (at 100 µM) inhibited suppressor activity to an extent in keeping with its effect on TGF- expression and, in any case, caused a major inhibition of suppressor activity at lower CD25^–/CD25⁺ T cell ratios. It should be noted that the effect of a higher concentration of the inhibitor (200 µM) could not be explored because at this higher concentration the inhibitor exhibited a significant cytotoxic effect (data not shown), possibly due to the fact that furin and furin-like proteases process other important molecules necessary for cell growth (28).

View larger version (14K): [in this window] [in a new window]   FIGURE 4. Furin inhibitor I reverses CD4/CD25+ T suppression. A, Preactivated CD4/CD25+ T cells were cocultured with MLEC-PAI-1-Luc cells (as described in the legend to Fig. 2) in the presence or absence of the furin inhibitor I. The luciferase activities obtained were converted to TGF- concentrations by reference to a TGF- standard curve. B, A total of 2.5 x 104 CD25– T cells was mixed with CD25+ T cells at the indicated ratios and stimulated with anti-CD3/anti-CD28-coated beads in the presence of 100 µM furin inhibitor I in round-bottom plates for 72 h. Proliferation was measured by [3H]thymidine uptake during the last 8 h of culture. *, p < 0.05; **, p < 0.01; and ***, p < 0.001, compared with CD25– alone.

Anti-TGF- and anti-CTLA-4 bring about complete inhibition of the in vitro suppressor activity of CD25⁺ T cells when added in combination

The above data provide strong new support for the idea that surface TGF- is present on the surface of CD4⁺CD25⁺ suppressor T cells, that such TGF- is functionally active, and that inhibition of its signaling function inhibits suppressor function. However, these data do not exclude the possibility that TGF--mediated suppression requires additional factors to be fully functional. This possibility arises, at least in part, from in vivo studies in which it has been shown that administration of anti-CTLA-4 Ab abolishes the protective effect of Treg cells in a cell-transfer colitis and diabetes model (11, 29). In addition, there are reports from some, but by no means all authors, that anti-CTLA-4 Ab capable of blocking CTLA-4 function reverses CD25⁺ T cell-mediated suppression in in vitro assay systems (12, 13, 14, 17).

In initial studies to explore further the possible role of CTLA-4 in the function of CD25⁺ T cells and to discover the possible relationship between CTLA-4 and TGF- in such function, we performed CD25⁺ T cell suppressor assays in the absence and presence of blocking anti-CTLA-4 and anti-TGF- Abs alone or in combination using APC and anti-CD3 stimulation (see Materials and Methods). It is important to point out that in these studies the Abs used (anti-CTLA-4 (UC10-4F10-11) and anti-TGF-1, -2, and -3 (1D11)) were preincubated with the CD25⁺ T cells to let them interact with surface ligands on CD25⁺ T cells (B7 and TGF-, respectively) before the latter’s interaction with CD25^– T cells. We reasoned that this would avoid possible difficulties in Ab access to ligands following such interaction and would minimize interaction of the Abs with the CD25^– T cells. As shown in Fig. 5, A and B (depicting normalized data from four independent studies and a representative study respectively), anti-TGF- (50 µg/ml) alone inhibited suppression by CD25⁺ T cells by 80–90%, whereas anti-CTLA-4 (50 µg/ml) alone had no effect on suppression by these T cells. In addition, the combination of anti-TGF- and anti-CTLA-4 (at the same concentrations) completely inhibited suppression by CD25⁺ T cells. These results suggest that while TGF- is the main mediator of the suppressive effect, CTLA-4 also plays a role as a facilitator of the TGF- effect. In addition, the fact that preincubation of cell with anti-CTLA-4 alone did not have an inhibitory effect indicates that it was not acting in synergy with anti-TGF- by merely reducing CD25⁺ T cell activation, a possibility that might explain the inhibitory effects seen previously by some investigators.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Complete reversal of CD4/CD25+ T suppressor cell activity by anti-TGF- and anti-CTLA-4 in combination. Freshly isolated CD4/CD25+ cells were preincubated with anti-CTLA-4 (50 µg/ml) for 2 h, with anti-TGF-1, -2, and -3 (50 µg/ml) for 1 h or with both Abs for 2 h at RT. Following incubation in vitro suppressor cell assays were set up containing 2.5 x 104 CD4/CD25– cells, the same number of preincubated CD4/CD25+ cells and 1 x 105 irradiated syngenic splenocytes (3000 rad) in which cells were stimulated by anti-CD3 mAb (5 µg/ml) at 37°C. The cells were cultured for 72 h and pulsed with 1 µCi of [3H]thymidine for the last 6–8 h in flat-bottom 96-well plates (0.2 ml). Cells were harvested and assessed for thymidine incorporation in a liquid scintillation counter. A, Results of one representative assay of four independent assays. B, Normalized and combined data from all four assays showing relative proliferation in the different cell populations; data from four independent experiments. C, Alternatively, CD25+ cells were preactivated with plate-bound anti-CD3 (10 µg/ml), anti-CD28 (2 µg/ml), and IL-2 (50 U/ml) for 48 h then resting in IL-2 (50 U/ml) for another 12 h. After collection, extensive wash, and recounting, these preactivated Treg cells were incubated with 50 µg/ml anti-CTLA-4, 50 µg/ml anti-TGF-1, -2, and -3, or 50 µg/ml rLAP for 2 h at RT, and then added to a suppression assay as described above using the indicated ratio of preactivated Treg cells to effector T cells. CD25+(A), preactivated Treg cells. *, p < 0.05, and **, p < 0.01, compared with CD25+/CD25–.

CTLA-4 cross-linking mediates membrane-bound TGF- accumulation at the site of suppressor cell-target cell contact

To investigate further the role of CTLA-4 in CD25⁺ T cell suppression, we first considered the possibility that CTLA-4 signaling might induce increased membrane TGF- expression on CD25⁺ T cells. Therefore, we cultured CD25⁺ T cells with anti-CD3, anti-CD28, and IL-2 in the presence of anti-CTLA-4 (50 µg/ml) and subjected the cells to flow cytometric analysis. However, we found that CD25⁺ T cells did not express increased amounts of surface TGF- after such culture (data not shown). We then explored the possibility that cell surface CTLA-4 cross-linking could result in accumulation of TGF- at the site where suppressor cell-target contact and CTLA-4-B7 ligation occurs. To this end, we performed confocal microscopy on CD25⁺ and CD25^– T cells exposed to beads coated with either anti-CTLA-4, rB7-1/Fc, or anti-CD28 (for 1–2 h) to mimic interaction of these cells with target cells bearing B7 (APC). Control beads consisted of beads coated with isotype control IgG. To decrease nonspecific binding and nonspecific staining of the beads, anti-CTLA-4, rB7-1/Fc, and isotype IgG were subjected to dialysis before conjugation with beads, and the beads were blocked with BSA postconjugation. As shown in Fig. 6, CD25⁺ cells bound to anti-CTLA-4-, rB7-1/Fc-, and anti-CD28-coated beads, and the bound cells exhibited clumping or capping of their surface TGF- at or near the point of contact with the beads rather than their formerly diffuse TGF- staining. However, such capping was not seen in cultures of the CD25⁺ T cells containing beads coated with anti-ICAM-1, although these beads also bound to the cells; in addition, it was not seen in cultures containing only soluble anti-CTLA-4. To quantitate surface TGF- capping by the various coated beads, we determined the percentage of capped cells per hundred cells per sample. Approximately 80% of cells were capped by the anti-CTLA4-beads and the rB7-1/Fc-beads, and 64% of cells were capped by anti-CD28-beads; in addition, the capping induced by the anti-CD28-coated beads were somewhat less distinct. Finally, CD25^– T cells, in contrast to CD25⁺ T cells, were usually surface TGF-^– or only weakly positive and in any case did not exhibit TGF- capping when exposed to anti-CTLA-4- or rB7-1/Fc-coated beads. These findings were replicated in at least three different and independent studies. On the basis of these results, we conclude that interactions between CD25⁺ T cells and target cells bearing B7 result in concentration of TGF- at the point of contact between the cells and thus intensification of the negative TGF- signal.

View larger version (60K): [in this window] [in a new window]   FIGURE 6. Beads coated with anti-CTLA-4 or rB7-1/Fc induce accumulation of TGF- near the point of contact with interacting CD4/CD25+ T cells. Freshly isolated CD4/CD25+ cells were stimulated with plate-bound anti-CD3 (10 µg/ml) and IL-2 (50 U/L) in serum-free medium (X-Vivo 15) cultured for 3640 h, then harvested and incubated with anti-CTLA-4 beads, rB7-1/Fc beads, anti-CD28 beads, or hamster-IgG-beads at a ratio of 1:2 (cell:bead, respectively) for 1–2 h at RT. Cells were incubated with biotinylated anti-TGF-1 followed by streptavidin-FITC and then examined by confocal fluorescence microscopy.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Further corroboration of the fact that TGF- is the major effector of CD25⁺ T cell suppressor function came from studies disclosing that surface TGF- is capable of signaling target cells and that blockade of such signaling inhibits suppressor function. Regarding surface TGF- signaling function, in this study, we developed a sensitive TGF- bioassay system to prove that CD25⁺ T cells bearing surface TGF- could deliver a TGF- signal to a target cell via cell-cell contact and in the absence of secreted TGF-. This system used mink lung epithelial cells bearing receptors for TGF- and carrying a construct expressing a TGF- promotor linked to a luciferase gene (MLEC-PAI-I-LUC cells). Using this assay, we showed that preactivated CD25⁺ T cells can induce a luciferase signal within a short time after admixture with the mink reporter cells, but only if the CD25⁺ T cells have direct contact with the reporter cells as a result of the presence of anti-CD3/anti-CD28-coated beads that bind nonspecifically to the mink cells. Furthermore, this luciferase signal occurred in the absence of detectable soluble TGF- activity in the culture supernatant, as determined by an assay using the same mink cell reporter system. These data provide unequivocal evidence that cell-cell contact is sufficient to lead to activation of CD25⁺ T cell surface TGF- and thus activation of the intracellular Smad signaling pathway. They thus add to previous, less rigorous evidence that while TGF- on the surface of CD4⁺CD25⁺ cells is present in an inactive form, it can be activated when CD25⁺ T cells are mixed with target cells (3).

It should also be noted that in this assay system the strength of the cell surface TGF- signal as determined by the luciferase readout was dependent on the extent of cell-cell contact achieved between the CD25⁺ cells and the mink epithelial cells. Since we had to rely on the ability of poly-L-lysine-coated beads to achieve such contact, the level of contact may have been relatively limited as compared with that achieved in vivo by T cell-T cell interactions involving CD25⁺ cells and CD25^– T cells or CD25⁺ T cells and APC. Indeed, in the in vitro suppression assay driven by anti-CD3/anti-CD28-coated bead stimulation, one sees T cell cluster formation that suggests a degree of cell-cell contact greater than that caused by the beads alone. That such contact is not simply random and transient is suggested by recent work showing that, in at least in one system, CD25⁺ T cell function was dependent on integrin-mediated binding (30). In addition, as discussed below, CTLA-4-B7 interactions determine binding of CD25⁺ T cells to APC because beads coated with B7-Fc bind avidly to CD25⁺ T cells, whereas beads coated with control Ig do not.

Consistent with the above verification of CD25⁺ T cell surface TGF- signaling function, it has been observed by some but not all investigators that CD25⁺ T cell suppressor activity in vitro can be at least partially inhibited by anti-TGF- Abs (29, 31, 32). However, it should be noted that high concentrations of anti-TGF- are necessary to achieve such inhibition, possibly because it is difficult for Ab to gain access to cell-cell interaction sites at which surface (latent) TGF- is activated and binds to TGF-R. In the present study, we used an approach to the inhibition of cell surface TGF- suppressor activity that avoids this potential problem. In particular, we determined if a specific inhibitor of TGF-RI (ALK5), SB431542, could inhibit CD4⁺CD25⁺ T cell-mediated suppression. The advantage of this inhibitor over anti-TGF- Ab lies in the fact that its low molecular mass (420 Da) allows ready access to the target cell and inhibition or the TGF-R regardless of whether the receptor is being engaged. We found that indeed this inhibitor caused virtually complete reversal of CD25⁺ T cell suppressor activity in both an anti-CD3/anti-CD28 bead-stimulated and in an anti-CD3/anti-CD28 APC-stimulated in vitro assay system. It should be noted that in the assay in which anti-CD3/anti-CD28 beads were used as a stimulant (and in which APC were not present) measurement of the extent of T cell proliferation by CFSE labeling revealed that the suppressive effect of CD25⁺ T cells is in fact on CD25^– T cell proliferation, which is consistent with the known activity of TGF- (33).

CTLA-4 is another molecule highly expressed on the CD25⁺ T cell that has been said to play a role as an effector of CD25⁺ T cell suppressor activity. This possibility has been supported by in vivo studies of suppressor cell function in experimental autoimmune states as well as in in vitro studies showing that blocking (F(ab')₂) anti-CTLA-4 Ab neutralizes CD25⁺ T cell-mediated suppression when present in cultures at a high concentration (100 µg/ml) (12, 17). However, this inhibitory effect was not observed in studies by several other investigators using non-F(ab')₂ anti-CTLA-4 Abs (13, 14, 15). One possible cause of these disparate results is that highly potent blockade of CTLA-4 signaling can have several possible effects on cells in addition to its possible effect on suppression, including inhibition of CD25⁺ T cell activation and enhancement of CD25^– T cell proliferation, both of which would be read out as decreased suppressor function (18). In the present studies, we attempted to avoid some of these difficulties by evaluating the effect of anti-CTLA-4 (as well as anti-TGF-) on CD25⁺ T cell suppressor activity by exposing CD25⁺ T cells to anti-CTLA-4 Ab before coincubation with target CD25^– T cells. This approach also was advantageous because it allowed Ab to bind to ligands on the CD25⁺ T cell before the latter’s engagement with receptors on the CD25^– target cell when such binding might be difficult to achieve. We found that anti-CTLA-4 alone does not inhibit CD25⁺ T cell suppressor function in vitro and thus blocking of CTLA-4 signaling does not reverse such function. In addition, the fact that the anti-CTLA-4 Ab had no effect on suppression when present in the culture system alone strongly suggests that it is not impairing CD25⁺ T cell activation or CD25^– T cell proliferation. In contrast, we found that anti-TGF- caused strong but incomplete inhibition of CD25⁺ T cell suppressor activity and that the combination of anti-CTLA-4 and anti-TGF- caused complete inhibition of such suppressor activity. As already mentioned, this inhibitory effect of anti-TGF- alone is in accord with previous studies of the effect of TGF- inhibitors on in vitro assays of CD25⁺ T cell-mediated suppressor activity (18, 29, 32, 34), as well as the study reported here wherein TGF- signaling was blocked with the ALK5 inhibitor. Of perhaps greater interest to the present discussion, however, is that the combination of anti-TGF- and anti-CTLA-4 was synergistic in its effects on inhibition of CD25⁺ T cell suppressor activity, at least with suppressor activity manifested by nonactivated CD25⁺ T cells. This has also been noted previously (29, 32) and suggests that, while CTLA-4 signaling itself does not mediate the CD25⁺ T cell suppressor effect, it does in some way facilitate this effect. It should be noted, that while anti-TGF- could also inhibit activated cells, anti-CTLA-4 did not augment the inhibition in this case. We attribute this to the fact that activated CD25⁺ T cells are intrinsically more potent suppressor cells and thus do no require facilitation by CTLA-4.

Among the possible explanations for the above facilitation of TGF--mediated suppression by CTLA-4 is that CTLA-4 ligation causes a change in the way TGF- is displayed on the cell surface. Indeed, using anti-CTLA-4- and B7-1-coated beads to mimic responder cells (and which signal rather than block CTLA-4), we could in fact show by confocal fluorescence microscopy that surface TGF- is concentrated (capped) at the site where the cells bind to the beads and, moreover, that such capping was not nonspecific because it was not seen with anti-ICAM-1-coated beads. Therefore, it is reasonable to conclude that ligation of CTLA-4 on the surface of CD25⁺ T cells by B7 on APC causes TGF- molecules to move within the plane of the cell membrane and to concentrate at the site where cell-cell contact occurs so as to result in a stronger TGF- suppressor signal.

The above role of CTLA-4 in the facilitation of TGF- function fits with the fact that CD25⁺ T cells constitutively express high levels of CTLA-4 and such expression is further up-regulated by cell stimulation (11, 12). In addition, it is consonant with the fact that CTLA-4 has a much higher affinity for B7 than does CD28 (35) and interacts with B7 to form a highly ordered and stable lattice (36, 37, 38). These properties of CTLA-4 provide CD25^– T cells with an advantage over CD25^– T cells in the competition for interaction with B7 on the APC surface and suggest that CTLA-4, rather than CD28, is the major costimulatory molecule involved in the TGF- effect. However, it should be noted that signaling of CD28 via anti-CD28-coated beads also led to capping, albeit capping that was quantitatively and qualitatively less robust than that obtained with anti-CTLA-4-coated beads. Finally, the effect of CTLA-4 signaling on the display of TGF- may explain the finding that CD25⁺ T cells from CTLA-4 KO mice express more surface TGF- and exhibit a more TGF--dependent suppression than cells from wild-type mice (17), This would follow from the possibility that the population of regulatory cells that develops in CTLA-4-deficient mice express higher amounts of surface TGF- to compensate for their lack of ability to mediate a capping effect.

In summary, these findings demonstrate that CD25⁺ Treg cells mediate suppression upon cell-cell contact via negative TGF- signaling and that the latter is facilitated by CTLA-4- and CD28-induced effects on TGF- surface display.

	Acknowledgments

 
We thank Owen M. Schwartz and Meggan Czapiga (Biological Imaging Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health) for excellent technical supporting of confocal fluorescence microscopy.

	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 T.O. and L.X. contributed equally to this manuscript.

² Address correspondence and reprint requests to Dr. Warren Strober, Mucosal Immunity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Clinical Research Center, Room 5W3940, 10 Center Drive, Bethesda, MD 20892. E-mail address: wstrober{at}niaid.nih.gov

³ Abbreviations used in this paper: Treg, regulatory T; LAP, latency-associated peptide; KO, knockout; RT, room temperature; rh, recombinant human.

Received for publication October 7, 2005. Accepted for publication May 24, 2006.

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