Specific Aqueous Humor Factors Induce Activation of Regulatory T Cells

Abstract

purpose. To examine the possibility that aqueous humor–induced regulatory T cells could function in vivo, these T cells were examined for their ability to suppress adoptive transfer of delayed-type hypersensitivity (DTH). To begin to understand the mechanisms by which aqueous humor induces activation of regulatory T cells, α-melanocyte stimulating hormone (MSH) and transforming growth factor (TGF)-β2 were examined for ability to induce regulatory T cells.

methods. Primed T cells were treated with aqueous humor and assayed for regulatory activity by injecting them intravenously along with DTH-mediating T cells into syngeneic mice. Antigen-pulsed antigen-presenting cells (APCs) were injected into the pinna of the mouse ear, and swelling was measured 24 hours later. Primed T cells were also activated in vitro in the presence of α-MSH, TGF-β1, or TGF-β2 and were assayed for proliferation and TGF-β production along with suppressing DTH.

results. Aqueous humor–treated T cells suppressed inflammation mediated by DTH T cells. Maximum regulatory T cell activity was induced when primed T cells were activated in vitro in the presence of α-MSH followed 4 hours later with active TGF-β2. Such T cells proliferated, produced TGF-β, and suppressed DTH, suggesting that α-MSH and TGF-β2 induce activation of regulatory T cells. No regulatory T cell activity could be induced in the presence of TGF-β1.

conclusions. The ocular microenvironment constitutively produces immunoregulatory factors that suppress the induction of inflammatory activity and promotes regulatory T cell activity. Such regulatory T cells can further contribute to maintaining the normal immunosuppressive ocular microenvironment through their ability to suppress activation of other inflammatory T cells.

Over the past 30 years, research into the mechanisms of ocular immune privilege has led to the understanding that it is an active process mediated in part by the constitutive production of immunosuppressive factors within the ocular microenvironment. ^{1 2 3} Immune privilege involves mechanisms that suppress induction of an inflammatory immune response within the eye, and immunosuppressive factors found in aqueous humor. ^{3 4 5 6 7} The constitutive expression of these immunosuppressive factors regulates systemic and regional immune responses to antigen within the ocular microenvironment. 

The immunosuppressive mediators in aqueous humor were first demonstrated by Kaiser et al. ³ who suppressed various in vitro T-cell assays with normal aqueous humor. Streilein and Cousins ² showed that when T cells primed for inflammatory T cell activity were pretreated with aqueous humor they did not mediate the expected inflammatory response in a local adoptive transfer of delayed-type hypersensitivity (DTH) assay in skin. Recently, we have found that primed T cells activated in the presence of aqueous humor were deflected from an expected inflammatory T-(Th1) cell response to a regulatory T cell (Th3-like) response. ⁸ Such aqueous humor-induced effector T cells produce transforming growth fator (TGF)-β and suppressed interferon (IFN)-γ production by other inflammatory T cells; however, it has not been shown whether these aqueous humor–induced T cells can suppress DTH. 

There are several factors in aqueous humor that have the potential to influence effector T cell activities. ¹ Of the factors in aqueous humor we have chosen to examine two that have the potential to induce regulatory T cells, α-melanocyte stimulating hormone (MSH) and TGF-β2. ^{8 9 10 11} Primed T cells activated in the presence ofα -MSH proliferate but are suppressed in IFN-γ production. ¹⁰ This effect of α-MSH, which is independent of IL-4, suggests that α-MSH mediates differential responses in T cells to T-cell receptor (TCR) stimulation. Recently, it has been found that TGF-β mediates its own production by T cells. ¹¹ It is not known whether these immunosuppressive factors have a role in aqueous humor induction of regulatory T cells. Our results suggest thatα -MSH and TGF-β2 can mediate induction of regulatory T cells that suppress DTH. It is possible that these regulatory T cells can contribute further to the normal immunosuppressive microenvironment of the eye by their TGF-β production and suppression of inflammatory T cell activity. Our findings suggest that within the normal ocular microenvironment there is the potential to induce regulatory T cells that support the immunosuppressive, anti-inflammatory microenvironment of the eye and possibly mediate peripheral tolerance to ocular autoantigens. 

Materials and Methods

Mice

B10.A and BALB/c mice (6–8 weeks old) were obtained from Jackson Laboratories (Bar Harbor, ME). Animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the U.S. Animal Welfare Act. 

Antigens

Ovalbumin (OVA; Sigma, St. Louis, MO), Bovine serum albumin (BSA; Sigma), and desiccated Mycobacterium tuberculosis (MT-Ag; Difco, Detroit, MI) were used to immunize the mice. 

Aqueous Humor

Aqueous humor was obtained from New Zealand White rabbits (Pine Acres Rabbitry, West Brattleboro, VT) with no observed ocular and systemic disease. Aqueous humor was passively drained from the ocular anterior chamber by paracentesis through a 27-gauge perfusion set that ended in a siliconized microcentrifuge tube (Fisher Scientific, Pittsburgh, PA). Collected aqueous humor was used immediately in the assays. 

Ovalbumin-Specific DTH T Cells

B10.A mice were immunized with 1 mg/ml OVA in complete Freund’s adjuvant (Difco,). After 7 days, popliteal lymph nodes were collected, and T cells were isolated using a mouse CD3 enrichment column (R&D Systems, Minneapolis, MN). T cells were cultured with irradiated (2000R) spleen cells (5 × 10⁶ cells/well) from syngeneic B10.A mice in the presence of OVA (300 μg/ml) for 7 days. The T cells were seeded at 2 × 10⁶ cells/well in a 24-well plate (Corning, Corning, NY) in Dulbecco’s minimum essential medium (BioWhittaker, Walkerville, MD) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 0.05 mM 2-mercaptoethanol (Gibco, Grand Island, NY), 25 mM HEPES (BioWhittaker), 50 μg/ml gentamicin (Sigma), 5 μg/ml l-asparagine (Gibco), and 5 μg/ml l-arginine (Gibco). The T cells were collected and restimulated with OVA and syngeneic irradiated spleen cells in culture media containing 80 U/ml mouse recombinant interleukin (IL)-2 (R&D Systems) and 4000 U/ml mouse recombinant IFN-γ (R&D Systems). The T cells were restimulated with OVA once every 2 to 3 weeks in the presence of syngeneic spleen cells. Cultures of developing T cells were determined to be inflammatory Th1 cells when the T cells produced only IFN-γ with no detectable IL-4 production when stimulated by OVA antigen-presenting cells (APCs) in the absence of exogenous growth factors (IL-2, IFN-γ) and mediated inflammation in a standard assay of adoptive transfer of DTH. 

Sandwich Enzyme-Linked Immunosorbent Assay

The wells of a 96-well flexible microtiter plate (Falcon, Oxnard, CA) were coated with capturing monoclonal anti-IFN-γ antibody (Pharmingen, San Diego, CA). The plate was incubated overnight at 4°C; washed with a solution of phosphate buffered saline (PBS), 0.02% Tween-20, and 1% BSA (wash buffer); and blocked with PBS plus 1% BSA (PBS-BSA). The plate was incubated for 1 hour at room temperature and washed. Samples were added to the wells, and the plate was incubated for 3 hours at room temperature. The plate was washed three times, and into each well 100 μl of 1.0 μg/ml biotinylated detecting monoclonal anti-IFN-γ antibody (Pharmingen) was added. The plate was incubated for 1 hour and washed three times. Streptavidin-β-galactosidase (100 μl; Gibco) was added to the wells, and the plate was incubated for 30 minutes and washed five times. The substrate chlorophenyl-red-β-d-galactoside (Calbiochem, San Diego, CA) was added to the wells, and color was allowed to develop for 30 minutes The optical density of the converted chlorophenyl-red-β-d-galactoside was read on a standard enzyme-linked immunosorbent assay plate reader at a wavelength of 570 nm. The IFN-γ concentrations of the standard samples were plotted against their optic density (OD) to create a standard curve. Using this standard curve, the concentration of IFN-γ in the assayed culture supernatant was determined from the OD of the test well and sample dilution factor. 

Adoptive Transfer and DTH

T cells from the draining lymph nodes of B10.A mice immunized 7 days previously with OVA, BSA, or MT-Ag were isolated using a CD3-enrichment column (R&D Systems). The enriched primed T cells (4 × 10⁵ cells) were added to cultures containing aqueous humor (50% diluted in culture media) and antigen-pulsed APCs. The antigen-pulsed APCs were adherent spleen cells (1 × 10⁶ cells) from syngeneic naive mice pulsed overnight with antigen (OVA, BSA, or MT-Ag) and washed with media before adding T cells and aqueous humor. The cultures were incubated for 24 hours at 37°C, 5% CO₂. In some experiments, instead of aqueous humor the T cells were added to cultures containing antigen-pulsed APCs and 30 pg/ml α-MSH. After a 4-hour incubation, TGF-β (5 ng/ml) was added, and the cultures were incubated for the remaining 20 hours. The cells were collected and assayed for regulatory activity in the adoptive transfer of DTH. The culture media was serum free containing RPMI 1640, 1 mg/ml BSA, and a 1:500 dilution of ITS+ solution (Collaborative Biomedical Products/Becton Dickinson Labware, Bedford, MA). 

T cells (2 × 10⁵ cells) from the inflammatory OVA T-cell cultures were injected along with aqueous humor or α-MSH and TGF-β–treated T cells (2 × 10⁵ cells) into the tail veins of syngeneic (B10.A) mice in a volume of 200 μl. Within 1 hour antigen-pulsed syngeneic APCs (1 × 10⁵ cells) were injected into the right ear pinna of the mouse and ear swelling was measured with a micrometer (Mitsutoyo, Japan) at 24 and 48 hours. Maximum ear swelling occurred at 24 hours, and these data are presented as the mean ± SEM of the difference between ear thickness of the APC-injected ear and the opposite ear injected with PBS alone, minus their respective original ear thicknesses. Significance was determined by a Student’s t-test with an overall confidence level of 5% (P ≤ 0.05). 

Primed T-Cell In Vitro Assays

From draining lymph nodes of BALB/c mice immunized 7 days previously with MT-Ag, primed T cells were isolated using CD3-enrichment columns (R&D Systems). T-cells (4 × 10⁵ cells) suspended in serum-free media were added to the wells of a 96-well, round bottom plate (Corning). To the wells were added α-MSH (30 pg/ml) and anti-TCR antibody (2C11; 1μ g/ml) diluted in serum-free culture media. Various concentrations of TGF-β1 or TGF-β2 in media were added. In a second set of experiments TGF-β1 or TGF-β2 at a fixed concentration of 5 ng/ml were added to the wells at various times (0, 2, 4, and 6 hours) after addition of anti-TCR antibody. The cultures were incubated for 24 hours, 0.5 μCi [³H]thymidine (NEN Life Sciences Products, Boston, MA) was added to the wells, and the cultures were incubated for an additional 24 hours. The cells were collected, and incorporated radiolabel was measured by scintillation counting. Production of TGF-β by the treated primed T cells was accomplished by centrifuging the culture plates 24 hours after the addition of anti-TCR antibody, removing the supernatant, washing the cultures once, and adding fresh media. The cultures were incubated for an additional 24 hours, and the culture supernatant was assayed for TGF-β using the standard CCL-64 bioassay for TGF-β activity, as we have previously described. ⁸  

Assay for Regulatory T-Cell Activities In Vitro

T cells from peripheral lymph nodes were enriched and cultured as described above, except the T cells were treated with α-MSH (30 pg/ml) and anti-TCR. After 4 hours of incubation, TGF-β1 or TGF-β2 at 5 ng/ml was added, and the cultures were incubated for an additional 44 hours. The plate was spun down at 250g for 10 minutes, all the supernatant was removed, and cells were washed once with media. Freshly isolated primed T cells (4 × 10⁵ cells) were added to all the wells along with anti-TCRs (1 μg/ml), and the cultures were incubated for 48 hours. The culture supernatant was assayed for IFN-γ by sandwich enzyme-linked immunosorbent assay. 

Results

Suppression of DTH by Aqueous Humor–Treated Primed T Cells

Previously, we have demonstrated that primed T cells activated in the presence of aqueous humor suppress in vitro IFN-γ produced by other inflammatory T cells. ⁸ This suggests that these aqueous humor–treated, primed T cells should also suppress in vivo induction of DTH-meditating T cells. To examine this possibility, aqueous humor–treated T cells, primed to OVA, were injected intravenously along with OVA-specific DTH-mediating T cells. The aqueous humor–treated T cells significantly suppressed the inflammation mediated by the DTH T cells in response to OVA-pulsed APCs that were injected into the pinna of the mouse ear (Fig. 1 A). Therefore, aqueous humor–induced regulatory T cells suppressed in vivo induction of DTH. 

To examine whether the aqueous humor–induced regulatory T cells were antigen specific, aqueous humor–treated T cells primed to MT-Ag were injected instead of the aqueous humor–treated OVA-specific T cells (Fig. 1B) . When the aqueous humor–treated primed T cells did not share the same antigen-specificity as the DTH-mediating T cells and were not reactive to the antigens presented, there was no suppression of inflammation mediated by the DTH T cells. However, if the aqueous humor–treated primed T cells were reactive to an antigen presented in the ear, they suppressed the inflammation mediated by the DTH T cells, even though the two T-cell populations did not share the same antigen specificity (Fig. 1C) . Therefore, aqueous humor–induced regulatory T cells are antigen specific in their activation, but not in their suppressive activity. 

Regulation of TCR-Stimulated Proliferation of Primed T Cells by Factors of Aqueous Humor

Because primed T cells activated in the presence of aqueous humor produce TGF-β, we examined whether the constitutive levels of TGF-β2 and α-MSH in aqueous humor can mediate induction of TGF-β–producing T cells. To examine the effects of TGF-β2 in the presence of α-MSH on TCR-stimulated T-cell proliferation, primed T cells were TCR stimulated in the presence of α-MSH and active TGF-β1 or TGF-β2. Regardless of the presence of α-MSH, increasing concentrations of either TGF-β1 and TGF-β2 suppressed T-cell proliferation (Fig. 2) . It is interesting to find that low concentrations of TGF-β1 either had no effect or enhanced T-cell proliferation (Fig. 2) . 

Previously, we have shown that under serum-free conditions aqueous humor does not suppress proliferation of TCR-stimulated primed T cells. ⁸ The suppression of proliferation seen in Figure 2 could have resulted from being able to add only active TGF-β to the cultures. It is possible that the effects of TGF-β2 on the T cells activated in the presence of whole aqueous humor was related to activation over time of increasing levels of latent TGF-β2 in the cultures. This can be simulated by adding active TGF-β2 at various times after TCR stimulation in the presence of α-MSH. Primed T cells were TCR stimulated in the presence of α-MSH and at various times afterward with active TGF-β1 or TGF-β2. Here, not only was it important whether α-MSH was present but also that there was a difference between the effects of TGF-β1 and TGF-β2 (Fig. 3) . Both TGF-β1 and TGF-β2 added from the start of TCR stimulation through 6 hours suppressed T-cell proliferation. However, if α-MSH was present, proliferation recovered only with the addition of TGF-β2 at or later than 4 hours after TCR stimulation (Fig. 3) . Therefore, it is possible that in aqueous humor the presence of α-MSH antagonizes the antiproliferative activity mediated by activated TGF-β2. 

TGF-β2 Enhancement of TGF-β Production by Primed T Cells Activated in the Presence of α-MSH

Another characteristic of primed T cells activated in the presence of aqueous humor is their TGF-β production. ⁸ Primed T cells TCR stimulated in the presence of α-MSH produced enhanced levels of TGF-β (Fig. 4) . Addition of TGF-β1 at various times after TCR stimulation did not change the level of α-MSH–induced TGF-β production (Fig. 4) . However, the addition of TGF-β2 at various times after TCR stimulation enhanced α-MSH–induced TGF-β production by the primed T cells (Fig. 4) . Therefore, the aqueous humor factors α-MSH and TGF-β2 mediated induction of TGF-β–producing T cells, which are potential regulatory T cells. 

Mediation of Induction of Regulatory T Cells by Aqueous Humor Factors α-MSH and TGF-β2

Because TGF-β2, when added 4 hours after TCR stimulation in the presence of α-MSH enhanced TGF-β production by the treated T cells, we thought it possible that α-MSH and TGF-β2 induces activation of regulatory T cells. If regulatory T cells are activated, they should suppress IFN-γ production by other inflammatory T cells. Primed T cells TCR stimulated in the presence of α-MSH and with TGF-β2 4 hours later suppressed IFN-γ production by other T cells when the treated T cells and activated primed T cells were mixed into the same culture (Fig. 5) . Individually, α-MSH, more so than TGF-β2, induced regulatory T cell activity, but the addition of TGF-β2 enhanced the regulatory activity (Fig. 5) . In contrast, TGF-β1 alone or with α-MSH could not induce regulatory T cells. Moreover, it appears that TGF-β1 antagonized α-MSH-mediated induction of regulatory T cells (Fig. 5) . The induction of regulatory T cells by α-MSH with TGF-β2 corresponds with the recovered proliferation and enhanced levels of TGF-β production by the treated T cells seen in Figures 3 and 4 . 

To demonstrate that these factor-induced regulatory T cells could, like aqueous humor–induced regulatory T cells, suppress DTH, primed T cells treated with α-MSH and TGF-β2 were injected intravenously with OVA-specific DTH T cells. The α-MSH– and TGF-β2–treated primed T cells suppressed the inflammation mediated by the DTH T cells (Fig. 6) . Therefore, the aqueous humor immunoregulatory factors α-MSH with TGF-β2 mediated activation of functional regulatory T cells. 

Discussion

Aqueous humor, possibly through its immunoregulatory factorsα -MSH and TGF-β2, mediated induction of TGF-β–producing T cells that require antigen specificity for activation but suppress induction of inflammation by DTH-mediating T cells through non–antigen-specific mechanisms. Our results suggest that the ocular microenvironment has the potential to divert locally activated primed T cells from their programmed inflammatory response to a regulatory response. The results also report that specific, physiologically relevant ocular factors can mediate induction of regulatory T cells. The ability of the ocular microenvironment and of α-MSH and TGF-β2 to mediate induction of regulatory T cells has implications for the manner by which the regional immune response within the eye prevents induction of inflammatory T-cell activity. 

TGF-β–producing T cells have been described in oral tolerance models of experimental autoimmune uveitis and experimental autoimmune encephalomyelitis. ^{12 13} In the oral tolerance models, low doses of orally administered autoantigens induce, through the gut-associated lymphoid tissues, activation of TGF-β–producing T cells that actively suppress autoimmune disease. ¹⁴ These regulatory T cells, also known as Th3 cells, suppress the activity of other disease-mediating T cells through their secretion of anti-inflammatory mediators. ^{12 13 14} It is possible that the ocular microenvironment, through its constitutive production of immunosuppressive factors, mediates induction of Th3 cells that further maintain and contribute to the normal anti-inflammatory microenvironment of the eye. In addition, there is the potential for the induction of ocular antigen-specific regulatory T cells that mediate peripheral tolerance to antigens within the eye. ^{15 16}  

The results indicate that α-MSH is sufficient for induction of regulatory T cells. But, aqueous humor also contains TGF-β2. TGF-β2 alone can induce activation of regulatory T cells; however, T-cell proliferation is relatively suppressed. When TGF-β2 was added to the cultures 4 hours after primed T cells were TCR stimulated in the presence of α-MSH, there was significant proliferation and TGF-β production by the T cells. Because our primed T cells are normally DTH mediating when activated, the results suggest that α-MSH suppressed their inflammatory programming while promoting regulatory activity, which was enhanced by TGF-β2. 

In addition, we found that the effects of TGF-β1and TGF-β2 were different. Regulatory T cell induction did not occur in the presence of TGF-β1. It appears that TGF-β1 clearly suppressed α-MSH induction of regulatory T-cell activity. TGF-β1 could be considered immunosuppressive under the experimental conditions, whereas TGF-β2 is immunomodulating. Only the different receptor requirements for binding the TGF-β isoforms and the different affinities for TGF-β1 and TGF-β2 by the type II receptor can explain the observed differential response to TGF-β1 and TGF-β2. ^{17 18} Therefore, changes in TGF-β receptor signals, possibly influenced byα -MSH, may mediate the different responses seen by activated primed T cells treated with TGF-β1 and TGF-β2 in the presence of α-MSH. 

The finding that TGF-β2 and not TGF-β1 could induce activation of regulatory T cells is consistent with finding only TGF-β2 protein in aqueous humor. ^{2 5 7} Under uveitic conditions in which the blood–ocular barrier is compromised, entry of TGF-β1 from plasma into the aqueous humor could antagonize α-MSH and TGF-β2 induction of regulatory T cells. This could promote activation and, if active TGF-β1 is at a low concentration, enhance proliferation of activated uveitis-mediating T cells. Recently, it has been found that IL-4 and TGF-β can mediate development of TGF-β–producing T cells from a population of naive T cells. ¹¹ Previously, we have shown that the effects of α-MSH on primed T cells are similar to the effects of IL-4 on primed T-cell activation. ¹⁰ Therefore, it is possible that we were observing a similar cytokine-mediated mechanism by aqueous humor in the induction of regulatory T cells, withα -MSH inducing IL-4–like signals followed by the effects of TGF-β2 in the T cells. This would be similar to primed or memory T cells entering the ocular microenvironment, being influenced immediately byα -MSH, and then, in time, encountering cells that produce and activate TGF-β2. 

Our results demonstrate that aqueous humor, and therefore the ocular microenvironment, possibly through α-MSH and TGF-β2, goes beyond suppressing activation of DTH-mediating T cells ^{2 19} in that it also promotes activation of regulatory T cells. Therefore, only specific types of immunologic responses are activated within the normal ocular microenvironment. The induction of regulatory T cells could reinforce the immunosuppressive ocular microenvironment of the eye by contributing immunosuppressive lymphokines and suppressing inflammatory T-cell activity. 

The ability of the ocular microenvironment to produce constitutive levels of immunosuppressive cytokines that also mediate induction of regulatory T cells is an example of how a regional tissue site can manipulate, mold, and coerce an immune response that is tailored to the needs of the tissue. The eye’s use of cytokines to regulate the immune response allows for examining the possibility whether these specific immunoregulating factors are neutralized, antagonized, or no longer produced in eyes susceptible to or affected by uveitis. It may even be possible to use the same factors to suppress, systemically and locally, immune-mediated inflammatory diseases. The finding that the ocular microenvironment may induce activation of regulatory T cells indicates that such T cells may be a normal physiological occurrence within the eye, and that failure of the ocular microenvironment to maintain induction of autoreactive regulatory T cells could contribute to making the eye susceptible to immunogenic inflammation. 

 Supported by Grant EY10752 from the United States Public Health Service.

 Submitted for publication August 25, 1998; revised April 30, 1999; accepted May 20, 1999.

 Proprietary interest category: N.

 Corresponding author: Andrew W. Taylor, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114. E-mail: [email protected]

 

Figure 1.

 

Aqueous humor–treated effector T cells suppressed inflammation mediated by DTH T cells in an adoptive transfer assay. Ovalbumin-specific DTH T cells (responder T cells; OVA), were injected intravenously with aqueous humor–treated T cells (regulatory T cells) primed to (A) OVA (AqH/OVA), (B) MT-Ag (AqH/MT-Ag), or (C) BSA (AqH/BSA). Ovalbumin-pulsed APCs were injected into the ear pinna, except in (C), which depicts results of injection of OVA + BSA–pulsed APCs. Ear swelling was measured 24 hours later, and the data are representative of two experiments with similar results and are presented as difference (see the Methods section) in ear thickness ± SEM (n = 5). *P ≤ 0.05.

Figure 2.

 

There was a concentration-dependent effect of TGF-β1 and TGF-β2 on in vitro T-cell proliferation in the presence of α-MSH. Primed T cells were TCR stimulated in the presence or absence of 30 pg/mlα -MSH with TGF-β1 or TGF-β2 (0.005–5.0 ng/ml). Proliferation was measured as counts per minute of incorporated[ ³H]thymidine 48 hours after TCR stimulation. Data are presented as percentage proliferation ± SEM of eight independent experiments. Percentage counts per minute was calculated as counts per minute of sample divided by the counts per minute of untreated TCR-stimulated primed T cells (100% proliferation; 53,400 ± 2600 cpm), minus background (670 ± 100 cpm).

Figure 3.

 

There was a time-dependent effect of TGF-β1 and TGF-β2 on in vitro T-cell proliferation in the presence of α-MSH. Primed T cells were TCR stimulated in the presence or absence of 30 pg/ml α-MSH with TGF-β1 or TGF-β2 (5.0 ng/ml) added at different times after TCR stimulation. Proliferation was measured as counts per minute of incorporated [³H]thymidine 48 hours after TCR stimulation. Data are presented as percentage proliferation ± SEM of eight independent experiments as explained in Figure 2 .* Significantly (P ≤ 0.05) different from T cells treated only with TGF-β2.

Figure 4.

 

There was a time-dependent effect of TGF-β1 and TGF-β2 on TGF-β production by T cells in the presence of α-MSH. Primed T cells were TCR stimulated in the presence of 30 pg/ml α-MSH with TGF-β1 or TGF-β2 (5.0 ng/ml) added at different times after TCR stimulation. Culture supernatants were assayed for total TGF-β 48 hours after TCR stimulation. Data are presented as TGF-β (ng/ml) ± SEM of eight independent experiments. *Significantly (P ≤ 0.05) different from cultures with no added TGF-β. †The two controls were significantly different (P ≤ 0.05).

Figure 5.

 

TGF-β– and α-MSH–induced regulatory T cell activity. Primed T cells were TCR stimulated in the presence or absence of 30 pg/mlα -MSH with TGF-β1 or TGF-β2 (5.0 ng/ml) added 4 hours after TCR stimulation. The treated T cells (regulatory T cells) were added to cultures of freshly activated primed T cells. Culture supernatants were assayed for IFN-γ 48 hours after addition of treated T cells. Data are presented as percentage suppression ± SEM of IFN-γ produced by freshly activated T cells (560 ± 30 pg/ml) with no added regulatory cells from eight independent experiments.

Figure 6.

 

α-MSH+TGF-β2–treated T cells suppressed inflammation mediated by DTH T cells in an adoptive transfer assay. Activated OVA-primed T cells treated with α-MSH and TGF-β2 (regulatory T cells: α-MSH/TGF-β2 and OVA) were injected IV with DTH-mediating, OVA-specific T cells (responder T cells: OVA). OVA-pulsed APCs were injected into the ear pinna, and ear swelling was measured 24 hours later. The data are representative of two experiments with similar results and are presented as difference in ear thicknesses ± SEM (n = 5). *P ≤ 0.05.