May 2000
Volume 41, Issue 6
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Immunology and Microbiology  |   May 2000
Analysis of In Vivo Regulatory Properties of T Cells Activated In Vitro by TGFβ2-Treated Antigen Presenting Cells
Author Affiliations
  • Takeshi Kezuka
    From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
  • J. Wayne Streilein
    From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
Investigative Ophthalmology & Visual Science May 2000, Vol.41, 1410-1421. doi:
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      Takeshi Kezuka, J. Wayne Streilein; Analysis of In Vivo Regulatory Properties of T Cells Activated In Vitro by TGFβ2-Treated Antigen Presenting Cells. Invest. Ophthalmol. Vis. Sci. 2000;41(6):1410-1421.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To determine whether naive T cells activated in vitro by antigen-pulsed, transforming growth factorβ (TGFβ)–treated antigen presenting cells (APCs) acquire the capacity to suppress the induction and expression of delayed hypersensitivity in vivo.

methods. Naive ovalbumin (OVA)-specific T cells from DO11.10 Tcr transgenic mice were stimulated in vitro with OVA-pulsed TGFβ2-treated APCs. The cultured cells were harvested and assayed for in vitro production of mature TGFβ. Similar cells were coinjected with primed OVA-specific BALB/c T cells plus OVA-pulsed APCs into ear pinnae of normal BALB/c mice (assay for delayed hypersensitivity expression) or coinjected with OVA-pulsed APCs into footpads of naive DO11.10 mice whose draining lymph node cells were harvested 4 days later and assayed in vitro for capacity to secrete interferon-γ (IFN-γ) and interleukin-4 (IL-4) when stimulated with OVA (assay for induction of delayed hypersensitivity).

results. DO11.10 T cells activated in vitro by OVA-pulsed TGFβ2-treated APCs secreted large amounts of mature TGFβ and suppressed the expression of delayed hypersensitivity in a local adoptive transfer assay. Suppression was reversed in the presence of neutralizing anti-TGFβ antibodies. In addition, in vitro generated regulatory T cells influenced naive T cells in DO11.10 mice that were responding to an initial immunization with OVA to secrete IL-4, rather than IFN-γ. This influence was independent of TGFβ.

conclusions. OVA-pulsed APCs, pretreated in vitro with TGFβ2, activate DO11.10 T cells in a manner that endows the responding cells with the capacity to suppress the induction and then the expression of delayed hypersensitivity in vivo. In certain ways, these properties of in vitro–activated DO11.10 T cells resemble the properties of afferent and efferent regulatory T cells typically found in the spleens of animals with anterior chamber–associated immune deviation.

Antigens injected into the anterior chamber of the eye induce a stereotypic deviant systemic immune response termed anterior chamber–associated immune deviation (ACAID). 1 The key antigen-specific characteristics of this response are failed induction and expression of delayed hypersensitivity (DH), 2 intact, and sometimes even enhanced, production of antibodies, 3 4 5 and primed and clonally expanded cytotoxic T cell precursors. 4 6 In immunologically naive mice, it is believed that indigenous intraocular antigen presenting cells (APCs) capture injected antigen, migrate across the trabecular meshwork to escape from the eye, and traffic via the blood to the spleen. 7 8 9 10 In this secondary lymphoid organ, the regulatory T cells that dictate the unique features of ACAID are activated. Two types of T cells that regulate DH have been described: one acts on the afferent limb of naive mice, suppressing initial activation of antigen-specific T cells after injection of antigen plus adjuvant; the other acts on the efferent limb, suppressing the expression of DH. 11 It is important to understand the processes by which these disparate regulatory cells are generated, because the deviant immune response of ACAID plays a central role in the phenomenon of ocular immune privilege and serves as a model for regulation of immunopathogenic responses at other sites in the body. 
Experimental analysis of the APCs responsible for ACAID induction has been materially advanced by the discovery that conventional APCs (harvested from peritoneal exudate, or from peripheral blood) that are pulsed with antigen in the presence of transforming growth factorβ (TGFβ) and are then injected intravenously into naive mice induce a systemic immune response similar to ACAID. 12 In the recent past, Takeuchi et al. reported that peritoneal macrophages treated in vitro with TGFβ2 secrete reduced amounts of interleukin-12 (IL-12), express low levels of CD40, and produce enhanced amounts of mature TGFβ compared with untreated APCs. 13 When pulsed with ovalbumin (OVA), TGFβ2-treated APCs resemble untreated APCs in their ability to activate naive T cells harvested from Tcr transgenic DO11.10 mice. 14 However, OVA-specific transgenic T cells stimulated with OVA-pulsed APCs untreated with TGFβ-2 secrete T helper 1 (Th1)–like cytokines, especially IL-2 and interferon-γ (IFN-γ), whereas transgenic T cells activated by OVA-pulsed, TGFβ2-treated APCs secrete large amounts of IL-4 but little IFN-γ and IL-2. 15 Thus, TGFβ2 alters the functional properties of APCs in a manner that enables the cells to induce T-cell differentiation down a pathway that leads away from immunogenic inflammation of the DH type and toward suppression of this type of reactivity. 
We have now conducted a series of experiments designed to evaluate in vivo the regulatory properties of DO11.10 T cells activated in vitro by antigen-pulsed, TGFβ2-treated APCs. The results reveal that transgenic T cells activated in this manner display the ability to suppress both the induction and expression of OVA-specific DH. Moreover, TGFβ mediates suppression of DH expression by in vitro–activated T cells. 
Methods
Mice
Female BALB/c mice, between 6 and 8 weeks of age, were purchased from Taconic Farms (Germantown, NY). These mice were used as a source of peritoneal exudate cells (PECs). DO11.10 Tcr transgenic mice were maintained in our colony (original parents were a kind gift of Dennis Loh, Washington University, St. Louis, MO) on an H-2d background. These mice were used as the source of T cells and as experimental subjects for in vivo studies of induction and expression of DH. DO11.10 mice express the DO11.10 Tcr that is specific for the peptide fragment of OVA, 323-339, in the context of I-Ad. 14 16 All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Culture Medium
Serum-free medium was used for all cell cultures. This medium was composed of RPMI 1640, 10 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin (all from Biowhitaker, Walksville, MD), 1 × 10−5 M 2-ME (Sigma Chemical, St. Louis, MO), supplemented with 0.1% bovine serum albumin (Sigma Chemical), ITS+ culture supplement [1 μg/ml iron-free transferrin, 10 ng/ml linoleic acid, 0.3 ng/ml Na2Se, and 0.2 μg/ml Fe(NO3)3; Collaborative Biochemical Products, Bedford, MA]. 
Reagents
Porcine TGFβ2, anti-TGFβ2 neutralizing antibody, and nonspecific goat antibody were purchased from R&D Systems (Minneapolis, MN). Anti-TGFβ2 neutralizing antibody was used at a concentration 10-fold higher than the 50% neutralizing dose stated by the manufacturer. Ovalbumin was obtained from Sigma. 
Preparation of Pure DO11.10 T Cells
Spleens were removed from DO11.10 mice and pressed through nylon mesh to produce a single-cell suspension. Red blood cells were lysed with Tris–NH4Cl. The remaining cells were washed three times with RPMI 1640 and passed through T-cell enrichment column (R&D Systems). The resultant cell suspension contained >95% CD3+ cells. 
Preparation of PECs
PECs were harvested from normal BALB/c mice that received 2 ml of thioglycolate (Sigma) intraperitoneally 3 days earlier. The cells were washed and resuspended, placed in 24-well culture plates (1 × 106/well), and treated with or without 5 ng/ml of porcine TGFβ2 in serum-free medium at 37°C in an atmosphere of 5% CO2. After overnight culture, plates were washed three times with culture medium to remove TGFβ2 and nonadherent cells. Adherent cells were retained in the wells for use in all subsequent experiments. More than 90% of these adherent cells were F4/80+ dendritic cells or macrophages. 
In Vitro Preparation of Regulatory T Cells
DO11.10 T cells (3 × 105) were cultured in 24-well plates containing TGFβ2-treated, or -untreated, PECs and 100 μg/ml OVA. After 48 hours, nonadherent DO11.10 T cells were harvested, washed three times, and then suspended at appropriate concentrations for in vivo experiments. 
Proliferation Assay of T Cells Activated In Vivo
Naive DO11.10 mice received hind footpad inoculations of DO11.10 T cells (1 × 106) that had been activated in vitro with OVA-pulsed PECs pretreated (or not) with TGFβ2. In some experiment, these cultured DO11.10 T cells were exposed to X-irradiation (2000 R) before injections. Thirty minutes later, OVA (0.5 μg/injection) was injected into the same hind footpads. In control experiments, mice that received cultured DO11.10 T cells followed by OVA injected into the hind footpads were exposed to X-irradiation (800 R/mouse) immediately postinjection. After 4 days, the draining popliteal lymph nodes were harvested, rendered into single-cell suspensions, added (3 × 105/well) to 96-well plates, and cultured with (or without) various concentrations of OVA in serum-free medium for 3 days at 37°C in an atmosphere of 5% CO2. The cultures were pulsed with 0.5 μCi[ 3H]thymidine 8 hours before termination, then harvested onto glass filters using an automated cell harvester (Tomtec, Orange, CT). Radioactivity was assessed by liquid scintillation spectrometry, and the amount expressed as counts per minute. 
Assays of IL-4 and IFN-γ Production by T Cells Activated In Vivo
T cells harvested from draining nodes (as described above) were cultured with OVA in serum-free medium for 24, 48, and 72 hours. At each point, supernatants were collected and analyzed by quantitative capture enzyme-linked immunosorbent assay, according to the manufacturer’s instructions (PharMingen, San Diego, CA). Rat monoclonal antibodies (mAbs) to mouse cytokine IL-4 (BVD4-1D11), IFN-γ (R4-6A2) were purchased from PharMingen and used as coating antibodies. Biotinylated rat mAbs to mouse cytokines IL-4 (BVD6-24G2) and IFN-γ (XMG1.2; PharMingen) were used as detecting antibodies. 
Assay of TGFβ Production by T Cells Activated In Vitro by TGFβ2-Treated PECs
PECs (1 × 106/well), pretreated with or without TGFβ2 at 5 ng/ml in 24-well plates, were cultured in 0.5 ml serum-free medium. After overnight culture, the plates were washed three times with culture medium to remove TGFβ2 and nonadherent cells. DO11.10 T cells (3 × 105) were added in 24-well plates containing TGFβ2-treated, or -untreated PECs and 100 μg/ml OVA. After 24 hours, nonadherent cells (>98% T cells) were collected and cultured (1 × 106/well) in serum-free medium in 24-well plates for an additional 24 hours. Supernatants were collected, and biologically active TGFβ was measured using Mv1Lu cells (ATCC, Rockville, MD). To detect mature TGFβ, the supernatants were diluted 1:4 with Eagle’s minimum essential medium (EMEM) (Biowhitaker), which consisted of 2 mM l-glutamine, 10 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.5% fetal calf serum. Diluted supernatants (100 μl) were added to 96-well flat-bottomed plates. To measure total TGF-β, supernatants were pretreated with 1N HCl (1:10) for 1 hour, then neutralized with a mixture of 1N NaOH:1 M HEPES (1:5). These acidified supernatants were diluted 1:10 with complete EMEM containing 0.5% fetal calf serum, then 100 μl was added to 96-well flat-bottomed plates. Mv1Lu cells (1 × 105 cells/100 μl) were added to each well and cultured for 24 hours at 37°C in 5% CO2. Cultures were pulsed with 1 μCi [3H]thymidine 6 hours before termination and harvested onto glass filters using an automated cell harvester. Radioactivity was assessed as described above. Half-maximal inhibition was determined by polynomial regression on log–log transformation of standard curves and experimental samples. The results were expressed as picograms per milliliter. 
Local Adoptive Transfer Assay of DH
This assay, as described previously, 17 was used to detect the capacity of in vitro-activated DO11.10 T cells to suppress the expression of DH. Briefly, DO11.10 T cells were cultured for 48 hours with OVA-pulsed PECs pretreated (or not) with TGFβ2. Nonadherent cells removed from these cultures (as regulators) were added (5 × 105/inoculum) to suspensions of OVA-pulsed PECs (as stimulators, 1 × 106/inoculum) and responder T cells (5 × 106/inoculum). Responder T cells were obtained as splenocytes from normal BALB/c mice primed 7 days previously with OVA plus complete Freund’s adjuvant (CFA). The mixtures of responders, stimulators, and regulators were injected (10 μl/injection) into the ear pinnae of naive BALB/c mice. Ear swelling responses were assessed with an engineer’s micrometer (Mitsutoyo; MTI Corporation, Paramus, NJ) at 24 and 48 hours. In some experiments neutralizing anti-TGFβ2 antibodies or nonspecific goat antibodies (100 μg/mouse) were mixed with the cells and injected into ear pinnae. 
Statistical Analysis
Results of experiments were analyzed by either Student’s t-test or ANOVA with Scheffé’s test. Mean values were considered to be significantly different when P < 0.05. 
Results
Effects of T Cells Activated In Vitro by TGFβ2-Treated APCs on Antigen-Induced T-Cell Activation In Vivo
To evaluate the effect of in vitro generated regulatory T cells on the earliest phase of T-cell activation in vivo, advantage was taken of the fact that a single injection of OVA into hind footpads of naive DO11.10 mice results in proliferation of T cells in the draining popliteal lymph nodes. In preliminary experiments we determined that the dose of OVA required to induce an optimal proliferative response was 0.5 μg and that the optimal time interval to assess T-cell proliferation was 4 days postinoculation of antigen. We used these conditions to conduct the following experiments. Putative OVA-specific regulatory T cells were generated in vitro by culturing naive DO11.10 T cells with peritoneal macrophages that had been treated with TGFβ2 plus OVA. Control regulatory T cells were similarly generated in cultures with OVA-pulsed peritoneal macrophages that had not been exposed to TGFβ2. Nonadherent T cells were removed from these cultures after 48 hours, washed, and injected (1 × 106) into both hind footpads of panels (five animals each) of naive DO11.10 mice. Within 30 minutes, each hind footpad received a second inoculation of OVA (0.5 μg). Four days later the mice were killed, the popliteal lymph node excised, and single-cell suspensions derived thereof were cultured with various concentrations of OVA for 72 hours. In some experiments, 0.5 μCi[ 3H]thymidine was added 8 hours before termination of culture to assess proliferation. In other experiments, supernatants were harvested from cultures after 24, 48, and 72 hours and assayed for content of IFN-γ and IL-4. The results of one of three similar experiments are summarized in Figure 1 . Popliteal lymph node cells harvested from mice that received footpad injections of control regulatory T cells followed by OVA, proliferated in vitro in a conventional response fashion to increasing doses of OVA. Popliteal lymph node cells from mice that received regulatory T cells exposed to TGFβ2-treated APCs in vitro also proliferated in response to OVA, and the extent of [3H]thymidine incorporation was similar to that of the positive control (see Fig. 1A ). However, the supernatants of cultures containing popliteal lymph node cells from mice that received control regulatory cells produced large amounts of IFN-γ and only background levels of IL-4, whereas lymph node cells from mice that received regulatory T cells incubated in vitro with TGFβ2-treated APCs secreted high levels of IL-4 but only trivial levels of IFN-γ. These findings indicate that OVA-specific transgenic T cells that are activated in vitro by OVA-pulsed APCs in the presence of TGFβ2 acquire regulatory properties that enable these cells to alter the response of naive OVA-specific T cells encountering OVA for the first time in vivo. Although in vitro generated regulatory T cells had no global inhibitory effect on responding recipient T cells, they did impair the capacity of naive DO11.10 T cells to secrete IFN-γ. In addition, in vitro generated regulatory cells promoted the responding T cells’ capacity to produce IL-4. 
Effects of X-Irradiation on Regulatory T Cells Activated In Vitro by TGFβ2-Treated APCs
Lymphoid cells injected into the hind footpad can migrate via lymphatics to the regional popliteal lymph node. Therefore, the lymph node cell suspensions used in the previous experiment provably contained injected T cells that had been cultured in vitro before being injected into the footpad. Therefore, it was important to determine the extent to which T cells injected into the footpad contributed to the proliferative responses and cytokine secretions observed. To examine this point, regulatory T cells generated in vitro (plus control cells incubated with APCs not exposed to TGFβ2) were removed from culture and exposed to 2000 R X-irradiation before injection into the hind footpad. As described above, OVA (0.5 μg) was injected into the same footpads within 30 minutes, and popliteal lymph nodes were removed from these mice 4 days later. Lymph node cell suspensions were prepared and cultured with various concentrations of OVA as described above. The cultures were evaluated after 72 hours for proliferation and for cytokine content in the supernatants. As revealed by results displayed in Figure 2 , irradiation of in vitro generated regulatory T cells injected into the footpads had no discernible effect on either proliferative responses or cytokine secretions displayed by in vitro cultured lymph node cells. These results suggest two conclusions: First, in vitro generated T cells make little or no contribution to the proliferative responses revealed when cells from lymph node–draining injection sites are stimulated with OVA in vitro. Second, X-irradiation does not rob regulatory T cells of their capacity to shift the cytokine secretory profile of responding T cells away from IFN-γ and toward IL-4. 
Effects of T Cells Activated In Vitro by TGFβ2-Treated APCs on Antigen-Induced T-Cell Activation In Vivo in X-Irradiated DO11.10 Mice
The results described above do not completely exclude the possibility that in vitro–activated T cells injected into the footpad contributed to the observed proliferation and/or cytokine secretion by lymph node cells harvested 4 days later. To resolve this point, we used X-irradiated DO11.10 mice as recipients. Regulatory T cells that were generated in vitro by stimulation with OVA-pulsed APCs exposed to TGFβ2 were injected (1 × 106) into the hind footpads of normal DO11.10 mice. Within 30 minutes, OVA (0.5 μg) was injected into the same footpads. Immediately thereafter the mice were exposed to 800 R X-irradiation. Four days later, popliteal lymph nodes were removed from these mice. Lymph node cell suspensions were prepared and cultured with various concentrations of OVA as described above. The cultures were evaluated after 72 hours for proliferation and for cytokine content in the supernatants. As revealed by results displayed in Figure 3 , T cells taken from X-irradiated DO11.10 mice that received regulatory T cells failed to proliferate or secrete IFN-γ and IL-4. These results indicate that recipient, rather than injected, T cells were responsible for the proliferation, and cytokines were secreted by popliteal lymph node cells harvested at 4 days and stimulated in vitro with OVA. In aggregate, these finding indicate that T cells activated in vitro by OVA-pulsed TGFβ2-treated APCs acquired the capacity to regulate the manner in which naive DO11.10 T cells responded to initial encounter with antigen in vivo (i.e., they acted as afferent regulatory cells). Under the influence of in vitro generated afferent regulatory T cells, naive T cells proliferated in vivo in response to OVA but produced Th2-like, rather than Th1-like, cytokines. 
Effects of T Cells Activated In Vitro by TGFβ2-Treated APCs on Expression of OVA-Specific DH In Vivo
We next turned our attention to the possibility that regulatory T cells activated in vitro by TGFβ2-treated APCs could suppress the expression of DH. In these experiments, a local adoptive transfer protocol was used in which T cells (responders) from normal BALB/c mice immunized with OVA plus CFA were mixed with OVA-pulsed APCs (stimulators). We have previously shown that injection of this cell mixture into the ear pinnae of naive BALB/c mice generates a delayed-in-time swelling that reflects the expression of DH. If regulatory cells are added to the mixture of responders and stimulators, the extent of inhibition of ear swelling reflects suppression. Accordingly, regulatory T cells were prepared as before by incubating DO11.10 T cells with OVA-pulsed TGFβ2-treated APCs for 48 hours. The nonadherent T cells were removed from these cultures and added as regulators (5 × 105/injection) to cell suspensions containing OVA-specific responders (5 × 106/injection) and OVA-pulsed stimulators (1 × 106/injection). These cell mixtures were injected into the pinnae of naive BALB/c mice, and the ear swelling responses were assessed 24 and 48 hours later. The results of a representative experiment (of three) are presented in Figure 4 . When the injection mixture contained T cells exposed in vitro to APCs that were not treated with TGFβ2 (positive control), ear swelling responses were intense. By contrast, cell mixtures that contained regulatory T cells exposed in vitro to APCs treated with TGFβ2 elicited ear swelling responses significantly less often than positive controls. These results indicate that DO11.10 T cells activated in vitro by OVA-pulsed TGFβ2-treated APCs acquired the capacity to suppress the expression of DH in vivo (i.e., they functioned as efferent regulators). 
Capacity of T Cells Activated In Vitro by TGFβ2-Treated APCs to Secrete TGFβ
TGFβ can act as an autocrine agent. Peritoneal macrophages that are exposed to TGFβ2 in vitro are stimulated first to enhance their production of the growth factor and second to convert a higher fraction of the latent molecule into its active, or mature, form. In the previous experiments DO11.10 T cells that were cultured with OVA-pulsed TGFβ2-pretreated APCs were actually placed in an environment rich in active TGFβ. Because TGFβ has been shown to possess immunosuppressive properties, we wondered whether T cells cultured in this manner acquired the capacity to secrete their own TGFβ. To test this possibility, peritoneal macrophages were treated by overnight culture with TGFβ2. The next day, the cells were washed free of TGFβ, pulsed with OVA, and used as stimulators for secondary cultures to which DO11.10 T cells were added. In positive control cultures, peritoneal macrophages that were cultured overnight in the absence of TGFβ were pulsed with OVA and used to stimulate DO11.10 T cells. After 24 hours, the nonadherent T cells were removed and recultured with medium alone for an additional 24 hours. The supernatants of these cultures were harvested, and the content of mature, as well as total, TGFβ was assessed, using a bioassay based on inhibition of proliferation of mink lung cells. Total TGFβ was measured after the supernatants were acidified with HCl, then neutralized with NaOH. The results of one such experiment are presented in Figure 5 . Little if any mature TGFβ was present in supernatants of T cells cultured previously with untreated OVA-pulsed APCs. By contrast, supernatants of T cells previously stimulated with OVA-pulsed TGFβ2-treated APCs contained approximately 150 pg/ml TGFβ. The latter cultures also contained large amounts of total TGFβ. Supernatants from T cells exposed to OVA-pulsed APCs untreated with TGFβ contained more total TGFβ than did the supernatants of unstimulated T cells but less than supernatants of T cells exposed to TGFβ-treated APCs. These results indicate that regulatory T cells generated by in vitro exposure to TGFβ2-treated APCs acquired the capacity to secrete significant quantities of mature TGFβ. 
Role of TGFβ Secreted by In Vitro–Activated T Cells in Mediating In Vivo Regulatory Functions
The results described above indicate that T cells activated in vitro by OVA-pulsed TGFβ2-treated APCs acquired the capacity to regulate both the induction and the expression of OVA-specific cell–mediated immunity. Because these regulatory cells secreted mature TGFβ, we examined whether the ability to regulate was mediated by this cytokine. In the first of these experiments, regulatory cells were generated by culturing DO11.10 T cells for 48 hours with OVA-pulsed TGFβ2 treated (or not) APCs. The nonadherent cells were collected from these cultures, mixed with anti-TGFβ2 antibodies, and injected (5 × 105) into the hind footpad of naive DO11.10 mice. Within 30 minutes, OVA (5 μg) was injected into the same footpads. Four days later, the draining popliteal lymph nodes were harvested, and cell suspensions prepared from these nodes were cultured with OVA and assayed for proliferation and cytokine content in the supernatant as described above. The results of a representative experiment are presented in Figure 6 . Neutralizing anti-TGFβ2 antibodies failed to alter the regulatory effects of in vitro–activated DO11.10 T cells. Lymph node cell–draining footpad sites that received injections of regulatory T cells with, or without, anti-TGFβ2 antibodies proliferated comparably well. Moreover, lymph node cells of mice that received regulatory T cells produced enhanced amounts of IL-4 and reduced levels of IFN-γ whether anti-TGFβ2 antibodies were present or not. These findings indicate that DO11.10 T cells activated in vitro by TGFβ2-treated APCs alter the phenotype of responding T cells during immune induction in vivo by a TGFβ-independent mechanism. 
In the second set of experiments, regulatory DO11.10 T cells were activated in vitro as described above. These cells (as regulators) were added to local adoptive transfers containing responders (primed T cells obtained from OVA-primed normal BALB/c mice) and OVA-pulsed APCs (as stimulators). Neutralizing anti-TGFβ2 antibodies (or isotype controls) were added to this cell mixture. The cell mixtures were then injected into the ear pinnae of naive BALB/c mice, and ear swelling responses were assessed 24 and 48 hours later. The results of a representative experiment are presented in Figure 7 . When the injection mixture contained in vitroactivated regulatory T cells plus anti-TGFβ2 antibodies, ear swelling responses were intense and comparable to positive controls. Similar cell mixtures that contained anti-goat IgG antibodies suppressed expression of ear swelling responses. Based on these findings we conclude that in vitro–activated regulatory T cells suppressed DH expression in vivo via their secretion of TGFβ2. 
Discussion
OVA-specific DO11.10 T cells that are activated by exposure in vitro to OVA-pulsed APCs pretreated with TGFβ2 possess a novel set of characteristics that may be pertinent to ACAID. We have previously reported that T cells activated in this manner preferentially secrete IL-4 rather than IFN-γ when restimulated in vitro with OVA-pulsed conventional APCs. 13 15 In the current communication, we present evidence that DO11.10 T cells activated by OVA-pulsed TGFβ2-treated APCs display the capacity to regulate activation of OVA-specific T cells in vivo. On the one hand, naive DO11.10 T cells that encountered OVA in popliteal lymph nodes draining footpads in which in vitro–activated regulatory T cells had been injected responded to a subsequent stimulation of OVA-pulsed APCs in vitro by producing IL-4, rather than IFN-γ. On the other hand, OVA-pulsed BALB/c T cells failed to elicit DH when mixed with in vitro–activated regulatory T cells and injected into the ear pinnae of naive BALB/c mice. Together these results indicate that DO11.10 T cells first activated in vitro by OVA-pulsed TGFβ2-treated APCs acquired the ability to modify OVA-specific immunity at both the afferent and efferent limbs of the response. In this regard, these in vitro–activated T cells resemble the afferent and efferent regulatory T cells that have been described in ACAID. 
The prominent role played by TGFβ2 in generating regulatory T cells in vitro from naive DO11.10 T cells reflects a similarly prominent role for this cytokine in ACAID. Ocular APCs, which capture antigen locally and carry it to the spleen for presentation to T cells, reside in a microenvironment rich in TGFβ2. 18 Aqueous humor, the clear liquid that fills the anterior chamber, not only contains TGFβ2 19 but has been demonstrated to confer ACAID-inducing properties on conventional APCs. 12 PECs that are pulsed with antigen in vitro in the presence of aqueous humor induce ACAID when injected intravenously in naive mice, and this property of aqueous humor is nullified if neutralizing anti-TGFβ antibodies are present during the in vitro incubation. 20 In addition, Kosiewicz et al. have recently reported that the spleens of mice that received an intraocular injection of OVA 1 week previously contain a unique population of OVA-specific CD4+ T cells that secrete TGFβ (but not IL-2, IFN-γ, IL-4, or IL-10) when stimulated in vitro with OVA plus conventional APCs. 21 Thus, OVA-specific T cells that encounter antigen-bearing APCs exposed to TGFβ—whether in vivo or in vitro—acquire the capacity to secrete TGFβ. 
The results of our present experiments further indicate a role for TGFβ in suppressing the expression of OVA-specific DH. In vitro activated DO11.10 regulatory cells were no longer able to suppress DH expression in local adoptive transfers to which neutralizing anti-TGFβ antibodies had been added. Efferent suppressors are one of the most reliable indicators of the existence ACAID, and these results suggest the possibility that impaired DH after an anterior chamber injection of antigen may be mediated by antigen-specific T cells that secrete TGFβ. However, when ACAID is induced in normal mice, the efferent suppressor T cells are CD8+, 11 whereas the DO11.10 T cells that acquire regulatory properties in vitro are CD4+. Therefore, we cannot be absolutely confident that the regulatory cells generated in vitro are equivalent to the regulatory cells generated in vivo. It is relevant that when ACAID is induced in BALB/c mice by an intracameral injection of OVA peptide 323-339, a population of CD4+ efferent suppressor cells is indeed generated. 22 Thus, the CD4/CD8 phenotype of efferent regulator T cells in ACAID may depend on whether the immunogenic peptides generated by ocular APCs bind preferentially to class II or class I molecules. 
In vitro–activated DO11.10 T cells that acquire the capacity to regulate the induction of immunity to OVA in vivo (afferent suppressors) express CD4 on their surface, just as do the afferent suppressor T cells induced by injection of antigen into the anterior chamber in vivo. Based on our current results, the mechanism by which in vitro–activated cells regulate immune induction does not appear to be TGFβ-dependent. Moreover, the regulator cells generated in vitro have a different effect on naive T cells responding to antigen in vivo than do in vivo generated regulatory cells. In the latter instance, footpad injection of afferent suppressor T cells prevented recipient T cells from proliferating in the popliteal lymph node draining the site of antigen injection. By contrast, when in vitro generated regulatory DO11.10 T cells were injected into the footpad of DO11.10 mice followed by an injection of OVA, recipient T cells harvested 4 days later retained their capacity to proliferate when exposed in vitro to OVA. Moreover, the responding T cells in these cultures secreted IL-4, rather than IFN-γ. There is no evidence that CD4+ afferent suppressors with this phenotype exist in mice with ACAID. 
T and B lymphocytes, with their distinctive antigen recognition structures, provide the adaptive immune response with its exquisite specificity for antigen. However, forces well beyond antigen-specific lymphocytes shape the qualities of immune responses to antigens. Our evidence supports the view that APCs play a key role in determining the functional properties of T cells that they stimulate. 13 Our evidence further indicates that the modifying role of APCs on T-cell function is dictated by a spectrum of costimulatory molecules expressed by APCs. So-called conventional APCs, such as adherent cells harvested from the peritoneal cavity and peripheral blood of BALB/c mice, present immunogenic peptide fragments derived from OVA with an array of costimulatory molecules, such as B7.1 and B7.2, intercellular adhesion molecule-1, and heat-stable antigen, that induce responding T cells to proliferate. In addition, conventional APCs, through expression of CD40 and secretion of IL-12, guide responding T cells to secrete an array of cytokines enriched for IL-2 and IFN-γ. 23 24 T cells of this phenotype provide help for B cells that will switch to IgG isoforms, which fix complement efficiently. 25 Moreover, T cells of this type are the primary effectors of cell-mediated immunity of the DH type. Similarly, when conventional APCs are pretreated with TGFβ2 and pulsed with OVA, they induce clonal expansion of DO11.10 T cells. However, these APCs secrete high levels of mature TGFβ, but only low amounts of IL-12, and express little CD40, and the responding T cells differentiate down pathways that limit, rather than trigger, immunogenic inflammation. 13 Considerable circumstantial evidence supports the hypothesis that immune privilege in the eye exists to prevent inflammation from disrupting the visual axis and causing blindness. We believe that ACAID is one dimension of ocular immune privilege and that eye-derived APCs shape the systemic immune response to ocular antigens, in part through their production of TGFβ. A systemic immune response shaped through the prism of TGFβ is deficient in the mediators of immunogenic inflammation, in part because antigen-specific T cells respond to antigen encounters by secreting their own TGFβ. 
 
Figure 1.
 
Effects of in vitro–activated DO11.10 T cells on priming of DO11.10 T cells in vivo. DO11.10 T cells, activated in vitro by OVA-pulsed TGFβ2-treated APCs, were injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. One half hour later, OVA (0.5 μg/mouse) was injected into the same footpads. Positive controls received only OVA injection into footpads. Four days later, popliteal lymph node cells were harvested, cultured in vitro with OVA (100 μg/ml; total volume per well, 2 ml), and assayed for T cell proliferation ([3H]thymidine incorporation) after 72 hours (A) and IFN-γ content (B), and IL-4 content (C) of culture supernatants at 24, 48, and 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 1.
 
Effects of in vitro–activated DO11.10 T cells on priming of DO11.10 T cells in vivo. DO11.10 T cells, activated in vitro by OVA-pulsed TGFβ2-treated APCs, were injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. One half hour later, OVA (0.5 μg/mouse) was injected into the same footpads. Positive controls received only OVA injection into footpads. Four days later, popliteal lymph node cells were harvested, cultured in vitro with OVA (100 μg/ml; total volume per well, 2 ml), and assayed for T cell proliferation ([3H]thymidine incorporation) after 72 hours (A) and IFN-γ content (B), and IL-4 content (C) of culture supernatants at 24, 48, and 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 2.
 
Effects of X-irradiation on regulatory T cells activated in vitro by TGFβ2-treated APCs. DO11.10 T cells were activated in vitro by OVA-pulsed TGFβ2-treated APCs. The responding cells were then exposed to X-irradiation (2000 R) and injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. One half hour later, OVA (0.5 μg/mouse) was injected into the same footpads. Positive controls received only OVA injection into footpads. Four days later, popliteal lymph node cells were harvested, cultured in vitro with various concentration of OVA (total volume per well, 1 ml), and assayed for T cell proliferation ([3H]thymidine incorporation; A) after 72 hours and IFN-γ content (B) and IL-4 content (C) of culture supernatants at 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 2.
 
Effects of X-irradiation on regulatory T cells activated in vitro by TGFβ2-treated APCs. DO11.10 T cells were activated in vitro by OVA-pulsed TGFβ2-treated APCs. The responding cells were then exposed to X-irradiation (2000 R) and injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. One half hour later, OVA (0.5 μg/mouse) was injected into the same footpads. Positive controls received only OVA injection into footpads. Four days later, popliteal lymph node cells were harvested, cultured in vitro with various concentration of OVA (total volume per well, 1 ml), and assayed for T cell proliferation ([3H]thymidine incorporation; A) after 72 hours and IFN-γ content (B) and IL-4 content (C) of culture supernatants at 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 3.
 
Effects of X-irradiation of DO11.10 recipients on regulatory T cells activated in vitro by TGFβ2-treated APCs. DO11.10 T cells stimulated in vitro with OVA-pulsed TGFβ2-treated APCs were injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. One half hour later, OVA (0.5 μg/mouse) was injected into the same footpads. Immediately after injection, the mice were exposed to X-irradiation (800 R). Positive controls received only OVA injection into footpads and were not exposed to X-irradiation. Four days later, popliteal lymph node cells were harvested, cultured in vitro with various concentrations of OVA for T cell proliferation ([3H]thymidine incorporation; A) after 72 hours, and cultured in vitro with OVA (100 μg/ml) for IFN-γ content (B) and IL-4 content (C) of culture supernatants at 24, 48, and 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 3.
 
Effects of X-irradiation of DO11.10 recipients on regulatory T cells activated in vitro by TGFβ2-treated APCs. DO11.10 T cells stimulated in vitro with OVA-pulsed TGFβ2-treated APCs were injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. One half hour later, OVA (0.5 μg/mouse) was injected into the same footpads. Immediately after injection, the mice were exposed to X-irradiation (800 R). Positive controls received only OVA injection into footpads and were not exposed to X-irradiation. Four days later, popliteal lymph node cells were harvested, cultured in vitro with various concentrations of OVA for T cell proliferation ([3H]thymidine incorporation; A) after 72 hours, and cultured in vitro with OVA (100 μg/ml) for IFN-γ content (B) and IL-4 content (C) of culture supernatants at 24, 48, and 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 4.
 
Effects of in vitro–activated DO11.10 T cells on expression of DH in vivo. DO11.10 T cells were stimulated in vitro with OVA-pulsed TGFβ2-pretreated APCs. T cells for inclusion in positive controls were stimulated with OVA-pulsed APCs in the absence of TGFβ2. In vitro–activated cells were then added (5 × 105/injection) as “regulators” to cell mixtures comprised of “responders” (T cells from BALB/c mice primed in vivo with OVA plus CFA, 5 × 106/injection) and“ stimulators” (OVA-pulsed APCs, 1 × 106/injection), and the cell mixtures were injected (10μ l) into the ear pinnae of normal BALB/c mice. T cells from naive BALB/c mice were used as “responders” in negative controls. Ear swelling responses at 24 hours were assessed, expressed as mean ± SEM (n = 5), and compared with positive control. Asterisk indicates values significantly less than positive controls, P < 0.05.
Figure 4.
 
Effects of in vitro–activated DO11.10 T cells on expression of DH in vivo. DO11.10 T cells were stimulated in vitro with OVA-pulsed TGFβ2-pretreated APCs. T cells for inclusion in positive controls were stimulated with OVA-pulsed APCs in the absence of TGFβ2. In vitro–activated cells were then added (5 × 105/injection) as “regulators” to cell mixtures comprised of “responders” (T cells from BALB/c mice primed in vivo with OVA plus CFA, 5 × 106/injection) and“ stimulators” (OVA-pulsed APCs, 1 × 106/injection), and the cell mixtures were injected (10μ l) into the ear pinnae of normal BALB/c mice. T cells from naive BALB/c mice were used as “responders” in negative controls. Ear swelling responses at 24 hours were assessed, expressed as mean ± SEM (n = 5), and compared with positive control. Asterisk indicates values significantly less than positive controls, P < 0.05.
Figure 5.
 
Production of TGFβ by DO11.10 T cells activated in vitro by OVA-pulsed TGFβ2-treated APCs. DO11.10 T cells were cultured for 24 hours with OVA and TGFβ2-pretreated or untreated APCs in a 24-well culture plate, washed, and resuspended in serum-free medium without antigen. After 24 hours, supernatants were collected and assayed for mature (A) and total (latent and mature) TGFβ (B) by bioassay. Results represent mean ± SEM of triplicate cultures. Asterisks indicate values significantly less than amount of TGFβ in supernatants of cultures of T cells activated in vitro in the presence of TGFβ2. *P < 0.01, **P < 0.05.
Figure 5.
 
Production of TGFβ by DO11.10 T cells activated in vitro by OVA-pulsed TGFβ2-treated APCs. DO11.10 T cells were cultured for 24 hours with OVA and TGFβ2-pretreated or untreated APCs in a 24-well culture plate, washed, and resuspended in serum-free medium without antigen. After 24 hours, supernatants were collected and assayed for mature (A) and total (latent and mature) TGFβ (B) by bioassay. Results represent mean ± SEM of triplicate cultures. Asterisks indicate values significantly less than amount of TGFβ in supernatants of cultures of T cells activated in vitro in the presence of TGFβ2. *P < 0.01, **P < 0.05.
Figure 6.
 
Capacity of anti-TGFβ antibodies to restore in vivo priming of DO11.10 T cells suppressed by in vitro–activated DO11.10 T cells. DO11.10 T cells, activated in vitro by OVA-pulsed TGFβ2-treated APCs, were suspended in medium containing anti-TGFβ2 antibodies and injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. Control mice received in T cells activated in vitro in the absence of anti-TGFβ. One half hour later, OVA (0.5μ g/mouse) was injected into the same footpads. Positive controls received only OVA injection into footpads. Four days later, popliteal lymph node cells were harvested, cultured in vitro with various concentration of OVA for T cell proliferation ([3H]thymidine incorporation; A) after 72 hours, and cultured in vitro with OVA (100 μg/ml; total volume per well, 1 ml) for IFN-γ content (B) and IL-4 content (C) of culture supernatants at 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 6.
 
Capacity of anti-TGFβ antibodies to restore in vivo priming of DO11.10 T cells suppressed by in vitro–activated DO11.10 T cells. DO11.10 T cells, activated in vitro by OVA-pulsed TGFβ2-treated APCs, were suspended in medium containing anti-TGFβ2 antibodies and injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. Control mice received in T cells activated in vitro in the absence of anti-TGFβ. One half hour later, OVA (0.5μ g/mouse) was injected into the same footpads. Positive controls received only OVA injection into footpads. Four days later, popliteal lymph node cells were harvested, cultured in vitro with various concentration of OVA for T cell proliferation ([3H]thymidine incorporation; A) after 72 hours, and cultured in vitro with OVA (100 μg/ml; total volume per well, 1 ml) for IFN-γ content (B) and IL-4 content (C) of culture supernatants at 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 7.
 
Capacity of anti-TGFβ antibodies to restore DH suppressed in vivo by in vitro–activated DO11.10 T cells. DO11.10 T cells were stimulated in vitro with OVA-pulsed TGFβ2-pretreated APCs. T cells for inclusion in positive controls were stimulated with OVA-pulsed APCs in the absence of TGFβ2. In vitro-activated T cells (with neutralizing anti-TGFβ antibodies or anti-goat Ig) were then added (5 × 105/injection) as “regulators” to cell mixtures comprised of “responders” (T cells from BALB/c mice primed in vivo with OVA plus CFA, 5 × 106/injection) and“ stimulators” (OVA-pulsed APCs, 1 × 106/injection). The antibody-containing cell mixtures were then injected (10 μl) into the ear pinnae of normal BALB/c mice. T cells from naive BALB/c mice were used as “responders” in negative controls. Ear swelling responses at 24 hours were assessed, expressed as mean ± SEM (n = 5), and compared with positive control. Asterisk indicates values significantly less than positive controls, P < 0.05.
Figure 7.
 
Capacity of anti-TGFβ antibodies to restore DH suppressed in vivo by in vitro–activated DO11.10 T cells. DO11.10 T cells were stimulated in vitro with OVA-pulsed TGFβ2-pretreated APCs. T cells for inclusion in positive controls were stimulated with OVA-pulsed APCs in the absence of TGFβ2. In vitro-activated T cells (with neutralizing anti-TGFβ antibodies or anti-goat Ig) were then added (5 × 105/injection) as “regulators” to cell mixtures comprised of “responders” (T cells from BALB/c mice primed in vivo with OVA plus CFA, 5 × 106/injection) and“ stimulators” (OVA-pulsed APCs, 1 × 106/injection). The antibody-containing cell mixtures were then injected (10 μl) into the ear pinnae of normal BALB/c mice. T cells from naive BALB/c mice were used as “responders” in negative controls. Ear swelling responses at 24 hours were assessed, expressed as mean ± SEM (n = 5), and compared with positive control. Asterisk indicates values significantly less than positive controls, P < 0.05.
We thank Koh–Hei Sonoda, Andrew Taylor, and Jun Yamada for helpful suggestions and Jacqueline Doherty and Marie Ortega for managerial assistance. 
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Figure 1.
 
Effects of in vitro–activated DO11.10 T cells on priming of DO11.10 T cells in vivo. DO11.10 T cells, activated in vitro by OVA-pulsed TGFβ2-treated APCs, were injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. One half hour later, OVA (0.5 μg/mouse) was injected into the same footpads. Positive controls received only OVA injection into footpads. Four days later, popliteal lymph node cells were harvested, cultured in vitro with OVA (100 μg/ml; total volume per well, 2 ml), and assayed for T cell proliferation ([3H]thymidine incorporation) after 72 hours (A) and IFN-γ content (B), and IL-4 content (C) of culture supernatants at 24, 48, and 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 1.
 
Effects of in vitro–activated DO11.10 T cells on priming of DO11.10 T cells in vivo. DO11.10 T cells, activated in vitro by OVA-pulsed TGFβ2-treated APCs, were injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. One half hour later, OVA (0.5 μg/mouse) was injected into the same footpads. Positive controls received only OVA injection into footpads. Four days later, popliteal lymph node cells were harvested, cultured in vitro with OVA (100 μg/ml; total volume per well, 2 ml), and assayed for T cell proliferation ([3H]thymidine incorporation) after 72 hours (A) and IFN-γ content (B), and IL-4 content (C) of culture supernatants at 24, 48, and 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 2.
 
Effects of X-irradiation on regulatory T cells activated in vitro by TGFβ2-treated APCs. DO11.10 T cells were activated in vitro by OVA-pulsed TGFβ2-treated APCs. The responding cells were then exposed to X-irradiation (2000 R) and injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. One half hour later, OVA (0.5 μg/mouse) was injected into the same footpads. Positive controls received only OVA injection into footpads. Four days later, popliteal lymph node cells were harvested, cultured in vitro with various concentration of OVA (total volume per well, 1 ml), and assayed for T cell proliferation ([3H]thymidine incorporation; A) after 72 hours and IFN-γ content (B) and IL-4 content (C) of culture supernatants at 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 2.
 
Effects of X-irradiation on regulatory T cells activated in vitro by TGFβ2-treated APCs. DO11.10 T cells were activated in vitro by OVA-pulsed TGFβ2-treated APCs. The responding cells were then exposed to X-irradiation (2000 R) and injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. One half hour later, OVA (0.5 μg/mouse) was injected into the same footpads. Positive controls received only OVA injection into footpads. Four days later, popliteal lymph node cells were harvested, cultured in vitro with various concentration of OVA (total volume per well, 1 ml), and assayed for T cell proliferation ([3H]thymidine incorporation; A) after 72 hours and IFN-γ content (B) and IL-4 content (C) of culture supernatants at 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 3.
 
Effects of X-irradiation of DO11.10 recipients on regulatory T cells activated in vitro by TGFβ2-treated APCs. DO11.10 T cells stimulated in vitro with OVA-pulsed TGFβ2-treated APCs were injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. One half hour later, OVA (0.5 μg/mouse) was injected into the same footpads. Immediately after injection, the mice were exposed to X-irradiation (800 R). Positive controls received only OVA injection into footpads and were not exposed to X-irradiation. Four days later, popliteal lymph node cells were harvested, cultured in vitro with various concentrations of OVA for T cell proliferation ([3H]thymidine incorporation; A) after 72 hours, and cultured in vitro with OVA (100 μg/ml) for IFN-γ content (B) and IL-4 content (C) of culture supernatants at 24, 48, and 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 3.
 
Effects of X-irradiation of DO11.10 recipients on regulatory T cells activated in vitro by TGFβ2-treated APCs. DO11.10 T cells stimulated in vitro with OVA-pulsed TGFβ2-treated APCs were injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. One half hour later, OVA (0.5 μg/mouse) was injected into the same footpads. Immediately after injection, the mice were exposed to X-irradiation (800 R). Positive controls received only OVA injection into footpads and were not exposed to X-irradiation. Four days later, popliteal lymph node cells were harvested, cultured in vitro with various concentrations of OVA for T cell proliferation ([3H]thymidine incorporation; A) after 72 hours, and cultured in vitro with OVA (100 μg/ml) for IFN-γ content (B) and IL-4 content (C) of culture supernatants at 24, 48, and 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 4.
 
Effects of in vitro–activated DO11.10 T cells on expression of DH in vivo. DO11.10 T cells were stimulated in vitro with OVA-pulsed TGFβ2-pretreated APCs. T cells for inclusion in positive controls were stimulated with OVA-pulsed APCs in the absence of TGFβ2. In vitro–activated cells were then added (5 × 105/injection) as “regulators” to cell mixtures comprised of “responders” (T cells from BALB/c mice primed in vivo with OVA plus CFA, 5 × 106/injection) and“ stimulators” (OVA-pulsed APCs, 1 × 106/injection), and the cell mixtures were injected (10μ l) into the ear pinnae of normal BALB/c mice. T cells from naive BALB/c mice were used as “responders” in negative controls. Ear swelling responses at 24 hours were assessed, expressed as mean ± SEM (n = 5), and compared with positive control. Asterisk indicates values significantly less than positive controls, P < 0.05.
Figure 4.
 
Effects of in vitro–activated DO11.10 T cells on expression of DH in vivo. DO11.10 T cells were stimulated in vitro with OVA-pulsed TGFβ2-pretreated APCs. T cells for inclusion in positive controls were stimulated with OVA-pulsed APCs in the absence of TGFβ2. In vitro–activated cells were then added (5 × 105/injection) as “regulators” to cell mixtures comprised of “responders” (T cells from BALB/c mice primed in vivo with OVA plus CFA, 5 × 106/injection) and“ stimulators” (OVA-pulsed APCs, 1 × 106/injection), and the cell mixtures were injected (10μ l) into the ear pinnae of normal BALB/c mice. T cells from naive BALB/c mice were used as “responders” in negative controls. Ear swelling responses at 24 hours were assessed, expressed as mean ± SEM (n = 5), and compared with positive control. Asterisk indicates values significantly less than positive controls, P < 0.05.
Figure 5.
 
Production of TGFβ by DO11.10 T cells activated in vitro by OVA-pulsed TGFβ2-treated APCs. DO11.10 T cells were cultured for 24 hours with OVA and TGFβ2-pretreated or untreated APCs in a 24-well culture plate, washed, and resuspended in serum-free medium without antigen. After 24 hours, supernatants were collected and assayed for mature (A) and total (latent and mature) TGFβ (B) by bioassay. Results represent mean ± SEM of triplicate cultures. Asterisks indicate values significantly less than amount of TGFβ in supernatants of cultures of T cells activated in vitro in the presence of TGFβ2. *P < 0.01, **P < 0.05.
Figure 5.
 
Production of TGFβ by DO11.10 T cells activated in vitro by OVA-pulsed TGFβ2-treated APCs. DO11.10 T cells were cultured for 24 hours with OVA and TGFβ2-pretreated or untreated APCs in a 24-well culture plate, washed, and resuspended in serum-free medium without antigen. After 24 hours, supernatants were collected and assayed for mature (A) and total (latent and mature) TGFβ (B) by bioassay. Results represent mean ± SEM of triplicate cultures. Asterisks indicate values significantly less than amount of TGFβ in supernatants of cultures of T cells activated in vitro in the presence of TGFβ2. *P < 0.01, **P < 0.05.
Figure 6.
 
Capacity of anti-TGFβ antibodies to restore in vivo priming of DO11.10 T cells suppressed by in vitro–activated DO11.10 T cells. DO11.10 T cells, activated in vitro by OVA-pulsed TGFβ2-treated APCs, were suspended in medium containing anti-TGFβ2 antibodies and injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. Control mice received in T cells activated in vitro in the absence of anti-TGFβ. One half hour later, OVA (0.5μ g/mouse) was injected into the same footpads. Positive controls received only OVA injection into footpads. Four days later, popliteal lymph node cells were harvested, cultured in vitro with various concentration of OVA for T cell proliferation ([3H]thymidine incorporation; A) after 72 hours, and cultured in vitro with OVA (100 μg/ml; total volume per well, 1 ml) for IFN-γ content (B) and IL-4 content (C) of culture supernatants at 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 6.
 
Capacity of anti-TGFβ antibodies to restore in vivo priming of DO11.10 T cells suppressed by in vitro–activated DO11.10 T cells. DO11.10 T cells, activated in vitro by OVA-pulsed TGFβ2-treated APCs, were suspended in medium containing anti-TGFβ2 antibodies and injected (1 × 106 cells/site) into both hind footpads of normal DO11.10 mice. Control mice received in T cells activated in vitro in the absence of anti-TGFβ. One half hour later, OVA (0.5μ g/mouse) was injected into the same footpads. Positive controls received only OVA injection into footpads. Four days later, popliteal lymph node cells were harvested, cultured in vitro with various concentration of OVA for T cell proliferation ([3H]thymidine incorporation; A) after 72 hours, and cultured in vitro with OVA (100 μg/ml; total volume per well, 1 ml) for IFN-γ content (B) and IL-4 content (C) of culture supernatants at 72 hours. Each data point represents the mean ± SD of triplicate (A) or duplicate (B, C) cultures. Asterisk indicates not detected.
Figure 7.
 
Capacity of anti-TGFβ antibodies to restore DH suppressed in vivo by in vitro–activated DO11.10 T cells. DO11.10 T cells were stimulated in vitro with OVA-pulsed TGFβ2-pretreated APCs. T cells for inclusion in positive controls were stimulated with OVA-pulsed APCs in the absence of TGFβ2. In vitro-activated T cells (with neutralizing anti-TGFβ antibodies or anti-goat Ig) were then added (5 × 105/injection) as “regulators” to cell mixtures comprised of “responders” (T cells from BALB/c mice primed in vivo with OVA plus CFA, 5 × 106/injection) and“ stimulators” (OVA-pulsed APCs, 1 × 106/injection). The antibody-containing cell mixtures were then injected (10 μl) into the ear pinnae of normal BALB/c mice. T cells from naive BALB/c mice were used as “responders” in negative controls. Ear swelling responses at 24 hours were assessed, expressed as mean ± SEM (n = 5), and compared with positive control. Asterisk indicates values significantly less than positive controls, P < 0.05.
Figure 7.
 
Capacity of anti-TGFβ antibodies to restore DH suppressed in vivo by in vitro–activated DO11.10 T cells. DO11.10 T cells were stimulated in vitro with OVA-pulsed TGFβ2-pretreated APCs. T cells for inclusion in positive controls were stimulated with OVA-pulsed APCs in the absence of TGFβ2. In vitro-activated T cells (with neutralizing anti-TGFβ antibodies or anti-goat Ig) were then added (5 × 105/injection) as “regulators” to cell mixtures comprised of “responders” (T cells from BALB/c mice primed in vivo with OVA plus CFA, 5 × 106/injection) and“ stimulators” (OVA-pulsed APCs, 1 × 106/injection). The antibody-containing cell mixtures were then injected (10 μl) into the ear pinnae of normal BALB/c mice. T cells from naive BALB/c mice were used as “responders” in negative controls. Ear swelling responses at 24 hours were assessed, expressed as mean ± SEM (n = 5), and compared with positive control. Asterisk indicates values significantly less than positive controls, P < 0.05.
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