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The Journal of Immunology, 2006, 177: 6091-6097.
Copyright © 2006 by The American Association of Immunologists, Inc.

TGFbeta Protein Processing and Activity through TCR Triggering of Primary CD8+ T Regulatory Cells1

Antoine Ménoret*, Lara M. Myers*, Seung-Joo Lee*, Robert S. Mittler{dagger}, Robert J. Rossi* and Anthony T. Vella2,*

* Department of Immunology, University of Connecticut Health Center, Farmington, CT 06032; and {dagger} Department of Surgery and Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30329


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In general, TGFbeta is synthesized as a procytokine that requires proteolytic activation, release of the mature cytokine from its noncovalently associated latent-associated peptide, and binding to TGFbetaRII to mediate suppressive activity. We tracked this process in mice containing primed CD8 regulatory T cells (Tregs) by immunoblotting in primary whole cell lysates for pro-TGFbeta, latent-associated peptide and mature TGFbeta. Generation of CD8 Tregs promoted processing of the 50 kDa pro-TGFbeta protein into a 12.5 kDa mature TGFbeta species in vivo. Despite the inability to detect mature TGFbeta in the sera of mice with primed CD8 Tregs and in the synthetic culture medium of stimulated CD8 Tregs, we demonstrated engagement of TGFbetaRII through immunoblotting for Smad2 phosphorylation. This process relied on continual TCR triggering, which also induced Smad3 phosphorylation. To understand the movement of mature TGFbeta, we showed that in contrast to IFN-{gamma}, mature TGFbeta does not remain a soluble cytokine but is likely to be rapidly adsorbed by neighboring cells. These data show the exquisite local control directed toward TGFbeta by the immune system and underscore the fine specificity involved in its detection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The TGFbetas are multifunctional small cytokines (25 kDa) with pleiotropic effects on cell growth and differentiation. Experiments in animal models, as well as studies in human diseases, have implicated TGFbeta in the regulation of important physiological and pathophysiological processes. TGFbeta has been involved in regulation of wound healing, bone formation, angiogenesis, lactation, embryogenesis, and apoptosis (1, 2, 3, 4, 5, 6). The role of TGFbeta in immunity is diverse as illustrated by its regulation of isotype switching, inflammation, and suppression (7, 8, 9, 10). The physiological effect of TGFbeta has also been associated with remission of autoimmune diseases, like experimental autoimmune encephalomyelitis, autoimmune diabetes, collagen-induced arthritis, and relapsing experimental allergic encephalomyelitis (11, 12, 13).

The emerging role of T regulatory cells (Tregs)3 in these diseases has brought a new but controversial role for TGFbeta. Several authors have shown that CD4 (10, 14) and CD8 Tregs (15, 16) can mediate immunosuppression through TGFbeta. However, other investigators have shown that CD4 Tregs from TGFbeta–/– mice retain the ability to prevent autoimmunity through a mechanism that can be blocked with anti-active TGFbeta Ab (17, 18), indicating that TGFbeta remains central to Tregs even if they do not synthesize it for themselves. Considering the ubiquitous expression of pro-TGFbeta, these data argued for a fine TGFbeta control mechanism by Tregs. We have recently described an example of such regulation in a new CD8 Treg model (19). It was shown that T cell immunotherapy with anti-CD137 and TLR ligand induced a population of CD8 Tregs dependent on IFN-{gamma} stimulation for elaboration of TGFbeta activity (20). Importantly, neutralization by anti-active TGFbeta mAb reduced the suppressive action exerted by CD8 Tregs on primed T cells (19) and on proliferation of stimulated naive T cells (20). Interestingly, surface detection of latent-associated peptide (LAP) on CD8 Tregs did not track with their suppressive function. Nevertheless, these results show an integral role for TGFbeta in immune-based suppression and complement a compendium of data establishing a powerful role for TGFbeta on cellular immune responses. Ironically, however, physiologically defined control mechanisms of TGFbeta protein activation in the immune system are not understood, but studies on nonimmunological systems have revealed general principles (2). Pro-TGFbeta is the product of a single gene consisting of the LAP and mature TGFbeta (21). LAP and mature TGFbeta combine into a complex that forms the basis of TGFbeta presence and activity (22).

After liberation from LAP, the mature or active TGFbeta can activate its signaling pathway by binding to a heterodimeric receptor composed of TGFbeta type I (activin receptor-like kinase 5) and type II serine/theronine kinase receptor subunits (23). TGFbeta binding leads to phosphorylation of activin receptor-like kinase 5, and recruitment and phosphorylation of receptor-regulated Smad2 and Smad3. Once phosphorylated, Smad2 and 3 associate with Smad4 and translocate into the nucleus to regulate gene transcription (24). In view of the ubiquitous expression of TGFbeta (25) and its signaling receptor (26) and the multitude of processes in which TGFbeta is involved, it stands to reason that activation is tightly regulated and potentially specific for perhaps each physiological system.

To date, only a small number of studies have used T cell lines, even fewer with primary T cells to study processing of mature TGFbeta protein. The lack of progress concerning the maturation of TGFbeta in the immune system is partly due to the difficulty of handling a cytokine with a highly positive charge at normal pH (27, 28) and the deficiency in a direct functional readout for its in vivo activity. Moreover, besides binding its signaling receptor, active TGFbeta binds several soluble carriers (29, 30) and cell type-specific receptors that have not all been identified (31). Therefore, a pivotal step for the studies of TGFbeta in Tregs will be to detect the production of active TGFbeta protein, and to monitor its activity in vivo by ways more accurate than ELISA of acid-treated sera.

Our data examined the state of TGFbeta in naive mice compared with mice containing primed CD8 Tregs. Priming naive mice to generate CD8 Tregs induced substantial processing of TGFbeta into its active form, which did not correlate to TGFbeta levels in serum as measured by ELISA. Maintenance of TGFbeta activity, as measured by Smad2 and Smad3 phosphorylation, required TCR triggering as did processing of mature TGFbeta into its active form. Finally, after processing mature TGFbeta appeared to rapidly bind cells suggesting that its mode of action is local as opposed to systemic. Therefore, inducing the processing of TGFbeta protein and exertion of its activity in the immune system relies on specific priming conditions, TCR triggering and refined detection methods.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, reagents, and Abs

C57BL/6 mice were purchased from the National Cancer Institute or The Jackson Laboratory. SM1 (32), DNTGFbetaRII transgenic mice were a gift from Dr. R. Flavell (Yale University, New Haven, CT; see Ref. 33), and OT-I mice (34) were bred at The University of Connecticut Health Center, and all mice were maintained under pathogen-free conditions and handled in accordance to National Institutes of Health federal guidelines. Polyinosinic-polycytidylic acid (poly(IC)) was purchased from Sigma-Aldrich. Recombinant mature and latent TGFbeta were purchased from GeneCopoeia. The goat polyclonal anti-LAP Ab (AF-246-NA) was purchased from R&D Systems, and the rat anti-TGFbeta mAb (555052) was purchased from BD Pharmingen. All secondary chicken HRP-conjugated Abs were adsorbed on mouse and human cells, and anti-Samd2/3 Ab were purchased from Santa Cruz Biotechnology. Anti-IFN-{gamma} Ab (AB2110P) was purchased from Chemicon International. The agonist anti-CD137 mAb was purified from 3H3 hybridoma culture supernatant using protein G-agarose (Invitrogen Life Technologies). Anti-Smad 2 (51-1300) and anti-Smad3 (51-1500) were purchased from Zymed; a second anti-Smad2 (L16d3) was purchased from Cell Signaling, as was the anti-pSmad2 (3101 S). The anti-pSmad3 rabbit antisera (35) was a gift from Dr. E. B. Leof (Mayo Clinic, Rochester, MN). The anti-CD4, -CD8, -CD103, and -CD69 staining mAbs were purchased from BD Pharmingen.

Cell processing, culturing, and staining

For all experiments we transferred 1–5 x 105 OT-I cells i.v. into C57BL/6 mice, and 1 day later mice were injected i.p. with SIINFEKL (100 µg) or OVA (1 mg), anti-CD137 (3H3 mAb at 50–100 µg), and poly(IC) (150 µg). On day 7 after stimulation, spleens were crushed through Falcon nylon mesh cell strainers (BD Biosciences), RBC were lysed with ammonium chloride, and cells were enumerated using a Z1 particle counter (Beckman Coulter). For the suppression assay in Fig. 1, CD8 OT-I cells were purified using MACS bead separation and lymphoid cells from naive SM1 transgenic mice were labeled with CFSE as performed in the past. Purified CD8 OT-I and 10,000 CFSE labeled SM1 cells were placed into a culture containing ~830,000 APCs with filler CD4 cells. The cultures were stimulated with nothing, 5 µg/ml SIINFEKL peptide and/or 1000 nM flagellin peptide427–441 (Invitrogen Life Technologies). At 48 h, CFSE dilution analysis was performed using flow cytometry. Additionally, some day 7 mice were restimulated in vivo with 100 µg of SIINFEKL peptide and were assayed for CD103 expression 1 day later. Briefly, cells were placed into staining buffer (balanced salt solution, 3% FBS, and 0.1% sodium azide), followed by blocking with a mixture of normal mouse serum, anti-FcR supernatant from the 2.4.G.2 hybridoma (36), and human {gamma}-globulin. Incubation with labeled primary Abs and flow cytometry analysis was conducted as before (37).


Figure 1
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  		FIGURE 1. CD8+ Tregs require specific TCR stimulation to suppress CD4+ T cell division. a, After OT-I transfer, C57BL/6 mice were immunized with SIINFEKL, anti-CD137 mAb and poly(IC). On day 7, OT-I CD8+ Tregs were purified from spleen, and naive SM1 CD4+ TCR transgenic T cells were labeled with CFSE. Both populations were cultured with either the CD4-specific peptide (upper panel) or the CD4- and CD8-specific peptide (lower panel). After 48 h, CFSE dilution of the CD4+ T cells was analyzed by flow cytometry. b, C57BL/6 mice were immunized as described in a, and 7 days later mice received either 100 µg of SIINFEKL peptide or nothing. One day after recall, viable OT-I T cells were gated and analyzed for CD69 and CD103 expression. The percentage of CD103+CD69+ cells is given in the upper right quadrant. Data are representative of three identical experiments. Cont., Control.

 
For biochemical analysis, lymph node (LN) and spleen tissue were taken from OVA, anti-CD137 and poly(IC)-treated mice on day 6 after priming. These lymphoid populations, designated Imm lysate, were destined for SDS-PAGE and immunoblotting analysis, either before or after in vitro culture. For in vitro culture, we used AIM V synthetic medium (Invitrogen Life Technologies). The AIM V synthetic medium is serum free, and we did not detect appreciable levels of TGFbeta as measured by immunoblotting and ELISA. Cells were used fresh or frozen at –80°C as a dried pellet. Cell lysis was performed by incubating 20–25 x 106 of cells in 100 µl of 50 mM Tris, 0.1% SDS (pH 6.8), in the presence of DNase I (40 µg/ml), RNase A (20 µg/ml), MgCl2 (2 mM) for 5 min at room temperature, followed by incubation in 0.5 M Tris, 150 mM NaCl, 0.5% Triton X-100, 0.5% octyl-beta-D-glucopyranoside, 10 mM NaF, 5 mM EDTA (pH 6.8) for 5 min at room temperature (all chemicals were purchased from Sigma-Aldrich). Cell lysates were spun down at 16,000 x g at 4°C for 10 min, and supernatants were directly used for SDS analysis or frozen at –80°C.

SDS-PAGE, immunoblotting, and immunoprecipitation

Protein samples were heated at 100°C for 10 min in nonreducing or reducing SDS sample buffer with excess 2-ME. Samples were resolved on 4–15% SDS-PAGE at 150 V for 45–60 min. After electrophoresis, proteins were either stained by Coomassie blue or transferred onto an 0.2-µm nitrocellulose pore size membrane (Bio-Rad) and probed with appropriate Abs. Because TGFbeta adheres to nonsiliconized glassware and plastic, especially under neutral conditions, all the tubes and tips used to prepare and handle the cell lysates were siliconized. Western blot detection was performed using ECL plus Western blotting detection kit from Amersham.

For immunoprecipitation, cell lysates were incubated at 4°C for 1 h under constant shaking with protein G-agarose beads (Invitrogen Life Technologies) coated with either anti-LAP goat polyclonal Ab (R&D Systems) or control goat Ig (Santa Cruz Biotechnology). The supernatant and beads were separated by centrifugation at 5000 x g for 10 min. An aliquot of the supernatant was frozen for further analysis, whereas the rest of the supernatants were transferred to fresh protein G-agarose coated with the same Abs as before except that the incubation was performed for 16 h at 4°C. The supernatants separated from the beads were analyzed by SDS-PAGE and immunoblotting. The beads were washed three times in phosphate buffer. Bound material was then eluted from the beads by boiling for 5 min in reducing SDS-PAGE sample buffer and loaded on SDS-PAGE.

TGFbeta ELISA

Sera from mice were collected in siliconized tubes, diluted 1/50 in ELISA sample buffer and assayed according to the manufacturer’s recommendations (Promega). This ELISA was designed to measure only the bioactive TGFbeta species. Therefore, we measured the naturally processed TGFbeta in the sera by directly testing the sample without treatment. To assay total TGFbeta, we performed an acid treatment with 1.0 N acetic acid followed by neutralization with 1.0 N sodium hydroxide before proceeding with the ELISA protocol. A linear dilution of mature TGFbeta was used as standard positive control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
In many cases, regulatory or suppressor T cells are linked to the immunosuppressive activity of TGFbeta. Recently, we defined a CD8 Treg population that utilizes IFN-{gamma} to elaborate TGFbeta suppressive activity (20). Although much is known about the cellular consequences of TGFbeta-based immunosuppression (39, 40), relatively little is known about the biochemical manifestation of TGFbeta activity in the immune system (41). Using biochemical methodology on primary lymphoid populations containing CD8 Tregs, we carefully delineated TGFbeta protein processing, characterization and exertion of TGFbeta activity.

To study this process, we generated OT-I CD8 Tregs by priming mice with OVA, poly (IC), and anti-CD137 costimulation, similar to previous studies (19). After 7 days, it is shown that purified OT-I CD8 T cells profoundly inhibited peptide-specific CD4 T cell proliferation as measured by CFSE dilution (Fig. 1a). Importantly, the SIINFEKL-stimulated OT-I CD8 Tregs did not kill the CFSE-labeled responder CD4 T cells, and in vivo recall of the OT-I cells mediated rapid induction of the TGFbeta-dependent integrin CD103 (Fig. 1b) (42). Our goal was to address the physical processing of TGFbeta protein in lymphoid tissue containing this Treg population, and, secondly, biochemically delineate exertion of TGFbeta activity on the immune system.

We explored this issue by examining TGFbeta protein expression in mice containing a primed CD8 Treg population through protein analysis. Under reducing conditions using a TGFbeta immunoblot, we detected two main molecular species of 50 and 12.5 kDa (Fig. 2a). The preeminent 50 kDa band corresponded to the recombinant pro-TGFbeta, whereas the 12.5 kDa band comigrated with the recombinant mature TGFbeta (see Fig. 2c for theoretical TGFbeta molecular species). The band migrating at 25 kDa was either a degradation product of the pro-TGFbeta or a homodimer of mature TGFbeta resistant to reduction by 2-ME. Under nonreducing conditions, TGFbeta detection was greatly reduced (Fig. 2a). Mature homodimeric TGFbeta was detected at ~20 kDa, whereas pro-TGFbeta was undetectable under these conditions, suggesting that the epitope recognized by the Ab was masked in the pro form in a nonreduced state. Therefore, the bands of 50 and 160–170 kDa detected in the nonreduced cell lysate are unlikely to be pro-TGFbeta but may be autoaggregates of mature TGFbeta or association of TGFbeta with endogenous carrier protein.


Figure 2
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  		FIGURE 2. Biochemical TGFbeta detection in mice with primed CD8+ Tregs. a, Spleen and LN cells were harvested from C57BL/6 mice 6 days after immunization with OVA, anti-CD137, and poly(IC) and plated for 3 h in AIM V synthetic medium in the presence of SIINFEKL. Lysates (Imm lysate) obtained from 3.5 x 106 cells were resolved by 4–15% gradient SDS-PAGE under reducing and nonreducing conditions, transferred to nitrocellulose membrane, and probed with anti-TGFbeta Ab as described in Materials and Methods. Recombinant pro and mature TGFbeta (20 ng) were loaded in parallel to serve as positive controls. b, Imm lysate was depleted of its mouse Ig component by incubation with protein G-agarose and immunoblotted with Abs specific for TGFbeta and mouse IgG (top panels). Protein G-agarose beads used for the three serial depletions (depl.) were boiled in SDS-PAGE sample buffer and immunoblotted with Abs specific for TGFbeta and mouse IgG (two bottom panels). c, Schematic representation of theoretical precursor and mature TGFbeta molecular species. The monomers of 50 kDa and 12.5 kDa correspond respectively to the reduced pro and mature TGFbeta, whereas the homodimers of 100 kDa and 25 kDa represent their nonreduced counterparts. Data are representative of three identical experiments. MWM, Molecular mass.

 
The data shown in Fig. 2b formally test whether or not the 50 kDa band is an endogenous mouse Ig H chain, that may have been mistaken as pro-TGFbeta. Serial depletions with protein-G agarose beads entirely depleted mouse Ig H chain present in the sample (Fig. 2b, upper panel), but not the 50 kDa band recognized by the anti-TGFbeta mAb (Fig. 2b, lower panel). One possibility to explain the 50 kDa band is the concatemerization of the 12.5 kDa active TGFbeta. Alternatively, it may represent the theoretical pro or latent TGFbeta, which contains LAP linked to mature TGFbeta (Fig. 2c). Because the 50 kDa band was not an Ig H chain, we tested whether it was the presence of LAP. Immunoblotting for LAP detection revealed a 50 kDa band that comigrated with the one detected by the anti-TGFbeta Ab (Fig. 3a). Importantly, the 37 kDa free LAP chain was detected in the lane containing r-pro TGFbeta (Fig. 3a, middle panel), but we rarely detected this species in primary cell lysates. To further confirm the identification of LAP, we used immunodepletion with anti-LAP Ab on protein G beads and successfully eliminated detection of the 50 kDa band as determined by blotting with the anti-TGFbeta Ab (Fig. 3b, lanes 4 and 6). Therefore, the 50 kDa polypeptide is made of the mature and latent TGFbeta moieties, whereas the 12.5 kDa species corresponds to a processed mature TGFbeta. Altogether, these immunoblotting data demonstrated specific detection of both pro and mature TGFbeta, a notoriously difficult task. This improvement is particularly important because TGFbeta is synthesized as a procytokine and requires maturation by proteolysis or some other process before its activity is manifested.


Figure 3
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  		FIGURE 3. Latent and mature TGFbeta molecular species are present in lymphoid tissue from CD8+ Treg-containing mice. a, Imm lysate and recombinant pro-TGFbeta (positive control) were analyzed by reducing SDS-PAGE and immunoblotted with isotype control (goat Ig, left panel), anti-LAP Ab (central panel), and TGFbeta Ab (right panel) as described in Fig. 2a. b, Pro-TGFbeta immunoprecipitated with anti-LAP Ab is detected with anti-mature TGFbeta Ab. Imm lysates (lanes 2–6) were analyzed by reducing SDS-PAGE before (lane 2) and after (lanes 3–6) depletion, using protein G-agarose coated with either anti-LAP Ab (lanes 4 and 6) or negative control goat Ig (lanes 3 and 5). Protein G-agarose beads used for depletion were washed, boiled in reducing SDS-PAGE sample buffer and run on SDS-PAGE (lanes 7 and 8). Recombinant pro-TGFbeta is used as positive control (lane 1). All samples were immunoblotted with anti-TGFbeta Ab as described in Fig. 2a. Data are representative of two identical experiments. MWM, Molecular mass.

 
Because specific proteolysis of pro-TGFbeta is the first step required for its activation, we hypothesized that CD8 Treg priming induces the generation of mature TGFbeta. Therefore, we explored the biochemistry governing the mechanism of TGFbeta activation in mice with primed CD8 Tregs. Using immunoblotting, we showed that lymphoid cells from naive mice express the ubiquitous pro-TGFbeta (Fig. 4a, lanes 1–3); whereas 6 days postpriming, lymphoid cells expressed both pro and mature TGFbeta (Fig. 4a, lanes 4–7). Consistent with these results, a cell lysate derived from lymphoid tissue after an OTI transfer followed by no immunization yielded comparable results compared with the naive control (data not shown). Interestingly, the sera taken from the mice reported in Fig. 4a had undetectable levels of mature TGFbeta (data not shown). Acid treatment of the sera activated a molecular form of TGFbeta, a latent form and/or an active form bound to a molecular partner, that was present at the same level in all the mice (Fig. 4b). Therefore, priming CD8 Tregs induces the appearance of mature TGFbeta, which was not readily observed in naive mice even though naive mice contain CD4+CD25+ Tregs. Secondly, the data show that serum ELISA results did not correspond to mature TGFbeta levels detected in lymphoid tissue.


Figure 4
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  		FIGURE 4. CD8+ Treg priming induces the generation of mature TGFbeta protein as detected by immunoblotting, but not by ELISA. a, Spleen and LN cells were harvested from naive and immunized C57BL/6 mice as described in Fig. 1. Cell lysates were analyzed by reducing SDS-PAGE and immunoblotting using Ab specific to TGFbeta and beta-actin as described in Fig. 2a (top two panels). b, Sera from the same mice were analyzed by ELISA for presence of TGFbeta. Data are representative of three identical experiments, and four other similar experiments with fewer mice.

 
Our hypothesis suggested that TCR stimulation of the primed CD8 Tregs was directly linked to TGFbeta maturation. Therefore, lymphoid cells from primed mice were cultured in the presence or absence of the MHC I SIINFEKL peptide for 3 h. We observed that specific TCR stimulation of CD8+ Tregs was required for activation of pro-TGFbeta into mature TGFbeta given that exclusion of SIINFEKL peptide resulted in undetectable levels of mature TGFbeta (Fig. 5). Therefore, under these conditions, our data in Figs. 4 and 5 show that even if the activation of CD8+ Tregs resulted in elaboration of mature TGFbeta, it did not result in detectable active TGFbeta in the serum. This could be due either to a lack of released mature TGFbeta from the cells that elaborate TGFbeta or by a very short half-life of active TGFbeta in vivo.


Figure 5
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  		FIGURE 5. CD8+ Tregs rely on TCR triggering in vivo to maintain activation of pro-TGFbeta. Spleen and LN cells were harvested from C57BL/6 mice 6 days after immunization and plated for 3 h in synthetic medium in the absence (no peptide) or presence of MHC-I restricted SIINFEKL peptide, washed, and analyzed by reducing SDS-PAGE and immunoblotted as described in Fig. 2a (top panels). Acrylamide gels were stained with Coomassie blue to control for equivalent loading of the samples (bottom panels). Three sets of data are representative of five identical experiments.

 
To assess whether active TGFbeta was released and its activity exerted, we measured phosphorylation of the signal transduction protein Smad2, which is a reliable marker for TGFbeta potency (42). We confirm in vitro that TGFbeta stimulation of enriched T cells induced phosphorylation of Smad2 in wild-type mice, but not very well in T cells from dominant negative TGFbetaRII transgenic mice (33) (Fig. 6a). Using the same Smad2 and pSmad2 immunoblotting strategy, we showed that 6 days after immunization, primary cells from lymphoid tissue expressed phosphorylated Smad2, whereas lymphoid cells from naive mice had very little (Fig. 6b). Similar to the experiment in Fig. 4, the sera of these mice contained undetectable levels of active TGFbeta (data not shown) and unchanged levels of acid sensitive TGFbeta (Fig. 6c). Finally, we observed that primed lymphoid cells restimulated with SIINFEKL peptide in vitro, as described in Fig. 5, maintained phosphorylation of Smad2 and also Smad3 (Fig. 6d). Therefore, we conclude that TCR triggering of CD8+ Tregs indeed resulted in physiologically active TGFbeta that was not traceable by ELISA.


Figure 6
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  		FIGURE 6. Accurate measurement of TGFbeta activity in vivo as determined by Smad phosphorylation, but not by TGFbeta ELISA. a, Spleen cells from C57BL/6 mice and from DNTGFbetaRII transgenic mice were harvested and stimulated in vitro in the absence or presence of active TGFbeta (10 ng/ml). After 3 h, cells were washed, lysed, and analyzed by immunoblotting using an Ab specific for phosphorylated Smad2 (pSmad2). Expression level of unphosphorylated Smad2 and Smad3 were controlled by immunoblotting using a mixture of anti-Smad2 and anti-Smad2/3 Abs. Additionally, the anti-phosphorylated Smad2 blot was stripped and reincubated with an anti-beta-actin Ab. b, Spleen and LN cells were harvested from C57BL/6 mice 6 days after immunization as described in Fig. 1. Cell lysates were obtained and analyzed by immunoblotting for expression of phosphorylated Smad2, Smad2, and beta-actin as described in Fig. 6a (top panel). These data are representative of three experiments. c, Sera obtained from the same mice 6 days after immunization were analyzed by ELISA for presence of TGFbeta as described in Fig. 4b. These data are representative of three experiments. d, Spleen and LN cells were harvested from C57BL/6 mice 6 days after immunization, plated for 3 h in synthetic medium in the presence of MHC I-restricted SIINFEKL peptide or with no peptide, and immunoblotted for phosphorylated Smad2, Smad2, phosphorylated Smad3, Smad3, Smad2/3, and beta-actin. These data are representative at least five comparable experiments.

 
We set out to test one possibility for the discordant results between the direct blotting data vs the ELISA data. TGFbeta has a very short half-life in vivo (29), and this may be due to the ubiquitous expression of its high affinity receptor (44) and nonspecific binding to cell surfaces resulting from its high positive charge at physiological pH (28). To test this idea further, we cultured mouse spleen cells with either TGFbeta or control IFN-{gamma}. At various times, the supernatants were removed and assayed for remaining cytokine. We observed that TGFbeta was rapidly removed from the culture medium (Fig. 7, lanes 3–7), whereas IFN-{gamma} was present in culture medium even after 2 h of incubation. Thus, active TGFbeta may be rapidly absorbed by cells, making its mode of action more effective locally, rather than systemically.


Figure 7
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  		FIGURE 7. Mature TGFbeta, but not IFN-{gamma}, rapidly disappears from the liquid phase in cell culture. Spleen cells in synthetic culture medium were rotated with 120 ng of recombinant mature TGFbeta and 120 ng of recombinant IFN-{gamma} (cytokine control) for 1–120 min. Supernatants were obtained and analyzed by SDS-PAGE and immunoblotted for TGFbeta and IFN-{gamma} (lanes 2–7). Supernatant from spleen cells incubated for 120 min without cytokines (lane 8) were used as negative controls. Cytokines incubated in medium without cells for 120 min at 37°C (lane 9) and at –80°C (lane 1) served as positive controls. Data are representative of three identical experiments and of two other experiments containing fewer time points.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A rebirth of interest in TGFbeta-mediated suppressive function has recently emerged (7). Although the cellular effects of TGFbeta are well studied, the biochemical mechanism linked to its activation in the immune system is less clear (45). We previously demonstrated that immunization with a TLR-3 ligand and a CD137 agonist mAb primed a population of CD8 Tregs that mediated suppression through IFN-{gamma} stimulation leading to TGFbeta activity (20). Here, a biochemical approach was applied to evaluate the mechanism of TGFbeta activation in suppression mediated by CD8 Tregs. A 50 kDa TGFbeta protein was detected in naive and in suppressed lymphoid tissue, but a predominance of the active 12.5 kDa species was detected under suppressive conditions, which tracked with Smad2 phosphorylation. Our data showed that this activity was maintained only under conditions of TCR triggering and favored local action as opposed to systemic release.

To detect TGFbeta in immune cells, we screened and selected a mAb that recognized an epitope in mature TGFbeta (Figs. 2 and 3). Because LAP is covalently bound to mature TGFbeta before processing, we were able to detect pro-TGFbeta as a 50 kDa species using the same Ab. To our knowledge, there is no anti-TGFbeta Ab for immunoblot analysis that could uniquely bind mature or active TGFbeta without detecting its pro form (46, 47). This limitation has made it a challenge to understand the molecular mechanism by which mature TGFbeta mediates suppression. Moreover, it complicates the interpretation of ELISA data that require acid treatment of biological fluids to indirectly measure active TGFbeta protein.

Because a whole, primary population containing B cells was used, we made sure that our system was not detecting mouse Igs present in the sample. Depletion by protein G beads demonstrated that the 50 kDa protein detected by anti-TGFbeta was not an Ig H chain (Fig. 2b). Moreover, we formally showed by immunoprecipitation that the 50 kDa species detected under reducing conditions was a single polypeptide composed of LAP and mature TGFbeta. The importance of this clarification lies in the ubiquitous expression of pro-TGFbeta and the fact that immune cells did not process TGFbeta in the absence of primed CD8 Tregs. However, after CD8 Treg priming, a sometimes variable but consistent 12.5 kDa protein in the cell lysate of immunized mice was detected (Fig. 4a). On the contrary, active TGFbeta was not, or inconsistently, detected in the sera of these mice (data not shown), whereas pro or latent TGFbeta was present at similar levels regardless of CD8 Treg priming (Figs. 4b and 6c). Thus, in this in vivo system, high levels of circulating latent TGFbeta were largely irrelevant, but not mature intracellular TGFbeta, which was associated with suppressive conditions. Therefore, activation and detection of mature TGFbeta appear to be the limiting step for tracking TGFbeta-suppressive function.

The direct implication of CD8 Tregs in TGFbeta processing was assessed in vitro by stimulating immune cells from primed mice with the SIINFEKL peptide. We observed that continuous Ag stimulation through TCR signaling was required for processing of TGFbeta. Interestingly, we were unable to detect active TGFbeta in the culture supernatant by either ELISA or immunoblotting (data not shown), suggesting that either mature TGFbeta was processed but not secreted or it was rapidly eliminated from the culture supernatant. Previous studies using tumor cell lines have shown that the 12.5 kDa TGFbeta active species was not detected after cell lysis, but rather in culture supernatant (48). In our case, the absence of mature TGFbeta in culture supernatant or sera (data not shown), and the presence of the 12.5 kDa protein in cell lysate suggest that, in contrast to tumor cells, intracellular processing happened before liberation of TGFbeta into the extracellular space. One possibility is that TCR triggering regulates furin convertase or a furin convertase-like protease to cleave pro-TGFbeta into its active species (49). This interpretation is compatible with the observation that anti-CD3 stimulation induces biologically active TGFbeta in Con A-timulated CD4 T cell populations (6). Altogether, our data are consistent with TCR triggering, inducing the expression of a specific protease responsible for TGFbeta processing and Treg activity, although this step has not been investigated in this Treg system.

It has been previously demonstrated that pro-TGFbeta is unable to bind its receptor (31), whereas mature TGFbeta does and mediates suppression specifically through Smad2 phosphorylation (44). Therefore, to investigate whether mature TGFbeta was indeed produced and secreted in our model, we sought evidence of signaling through the TGFbeta receptor in vivo. It is shown that immune cells from immunized mice, but not from naive mice, undergo Smad2 phosphorylation. Such activation strongly indicated that the cells have been signaled via TGFbeta receptor (Fig. 6). As with mature TGFbeta, Smad2 and Smad3 phosphorylation was also detected in vitro, but only with TCR triggering, demonstrating that CD8 Tregs initiate TGFbeta maturation and subsequent activity. However, in this model it is not known which cell population(s) is the actual source of active TGFbeta, and previously the possibility of cells other than the CD8 Tregs as the source of TGFbeta have been discussed (50).

Because mature TGFbeta was not detected in culture supernatant and in sera, the question therefore arose as to whether TGFbeta was rapidly absorbed. Compared with IFN-{gamma}, a classic systemic cytokine associated with TGFbeta activity, we observed that mature TGFbeta disappeared very quickly from the supernatant, suggesting that it may have been absorbed onto cells. This elimination from the milieu could be due to the ubiquitous expression of TGFbeta receptor combined with the nonspecific interaction of negatively charged cell surface with the positive charge of this extremely basic protein (pI = 9.5) (27, 28). Our data suggest that upon secretion, mature TGFbeta would bind the cells of its immediate environment, rather than diffuse systematically. This idea complements the well-documented studies showing TGFbeta release during cellular apoptosis (6). Thus, localized pockets of TGFbeta derived from Treg cells and apoptotic cells may formulate focal points of immunological tolerance.

Ultimately, these data speak to the difficulty in pinpointing or eliminating TGFbeta as a factor when studies rely on ELISA results as the assay for circulating protein. They also open the idea of using TGFbeta locally rather than systemically as in the case of treating multiple sclerosis.


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


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

1 This work was supported by National Institutes of Health Grants AI 142858 and AI 52108 (to A.T.V.). Back

2 Address correspondence and reprint requests to Dr. Anthony T. Vella, Department of Immunology-MC1319, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06032. E-mail address: vella{at}uchc.edu Back

3 Abbreviations used in this paper: Treg, T regulatory cell; LAP, latent-associated peptide; poly(IC), polyinosinic-polycytidylic acid; Imm lysate, lymphoid populations (as described in text); LN, lymph node. Back

Received for publication April 19, 2006. Accepted for publication August 8, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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