International Immunology

International Immunology Advance Access originally published online on January 13, 2006
International Immunology 2006 18(2):279-289; doi:10.1093/intimm/dxh368

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Transfer of regulatory T cells generated ex vivo modifies graft rejection through induction of tolerogenic CD4+CD25+ cells in the recipient

Song Guo Zheng¹, Lingzhong Meng¹, Ju Hua Wang¹, Meguru Watanabe², Mark L. Barr², Donald V. Cramer², J. Dixon Gray¹ and David A. Horwitz¹

¹ Division of Rheumatology and Immunology, Department of Medicine, 2011 Zonal Avenue, HMR 711 and ² Department of Cardiothoracic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA

Correspondence to: D. A. Horwitz; E-mail: dhorwitz{at}usc.edu

	Abstract

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Certain CD4+CD25+ T cells can induce and maintain T-cell non-responsiveness to donor alloantigens and have therapeutic potential in solid organ transplantation. Peripheral CD4+CD25– cells alloactivated with IL-2 and transforming growth factor ß (TGF-ß) ex vivo express the transcription factor FoxP3, and become potent antigen-specific CD4+CD25– suppressor cells. Here we report that the transfer of TGF-ß-induced regulatory CD4+ and CD8+ T cells (Tregs) co-incident with transplantation of a histoincompatible heart resulted in extended allograft survival. To account for this result, we injected non-transplanted mice with a single dose of CD4+ and CD8+ Tregs and transferred donor cells every 2 weeks to mimic the continuous stimulation of a transplant. We observed increased splenic CD4+CD25+ cells that were of recipient origin. These cells rendered the animals non-responsive to donor alloantigens by an antigen-specific and cytokine-dependent mechanism of action. Both the increased number of CD4+CD25+ cells and their tolerogenic effect were dependent on continued donor antigen boosting. Thus, Tregs generated ex vivo can act like a vaccine that generates host suppressor cells with the potential to protect MHC-mismatched organ grafts from rejection.

Keywords: cardiac allografts, TGF-ß, tolerance, transplantation, regulatory T cells

	Introduction

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Experimental autoimmune and transplant models have shown that several mechanistic approaches that include clonal deletion, anergy and effector cell regulation can alter T-cell alloreactivity and drive the immune system towards one of unresponsiveness (1). There is increasing evidence that CD4+ cells that constitutively express CD25, the alpha chain of the IL-2 receptor, not only have an important role in preventing autoimmunity but can also prevent graft rejection (2–4). CD4+CD25+ cells with a typical phenotype and suppressive effects occur naturally (2, 3), or can be induced peripherally (5, 6). Endogenous CD4+CD25+ cells can be expanded (7) so that they can be used in clinical trials. Prior studies have shown that peripheral CD4+CD25+ cells that prevent graft rejection can be induced indirectly using non-depleting CD4 and CD8 mAbs, co-stimulatory inhibitors or immunosuppressive drugs (8–11).

We have previously reported that the combination of IL-2 and transforming growth factor ß (TGF-ß) can induce both CD4+ and CD8+ cells to develop potent immunosuppressive activity (5, 12–16). These cytokines induced naive human, alloantigen-stimulated, peripheral blood CD4+ cells to become CD25+ regulatory cells with a surface phenotype and cytokine-independent suppressive effects indistinguishable from natural CD4+CD25+ cells (5). Moreover, these CD4+CD25+ regulatory T cells (Tregs) were able to induce other CD4+ cells to develop cytokine-dependent suppressive activity in vitro (16).

In this study, we have generated H-2^d anti-H-2^b Tregs in the presence of IL-2 and TGF-ß ex vivo and used them without any other immunosuppression to prevent rejection of H-2^b heart transplants. This work follows up previous experiments where we generated CD4+ and CD8+ Tregs by stimulating DBA/2 (H-2^d) mouse T cells with C57BL/6 (H-2^b) alloantigens in the presence of IL-2 and TGF-ß. These Tregs were antigen specific and prevented a chronic graft-versus-host disease with features of systemic lupus erythematosus in (DBA/2 x C57BL/6) F1 mice. Moreover, a single injection of these cells in mice with established disease doubled their survival (15).

In this study, we report that these Tregs induced ex vivo can substantially delay rejection of heart allografts in non-lymphopenic mice using allogeneic spleen cell immunization, and investigated the mechanisms involved in these protective effects. We report that the transfer of TGF-ß-induced Tregs has antigen-specific tolerogenic effects in these mice. These cells induced recipient CD4+ cells to become CD4+CD25+ cells that are responsible for the T-cell non-responsiveness. In order to sustain these CD4+CD25+ cells and their tolerogenic effects, continuous boosting of allogeneic donor cells was required.

	Methods

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Animals
Male C57BL/6 (B6, H-2^b), DBA/2 (D2, H-2^d) and C3H (H-2^k) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Animals 8–10 weeks of age were used as graft donors, recipients and controls. All mice were housed in conventional facilities at the University of Southern California using animal care protocols approved by the Institutional Animal Care and Use Committee of the University of Southern California.

Antibodies and reagents
The following antibodies were obtained from eBioscience (San Diego, CA, USA): Anti-CD3–PE (145-2011), anti-CD4–FITC (RM4-5), anti-CD4–PE (GK1.5), anti-CD8–PE (53-6.7), anti-CD25–PE (PC61), anti-CTLA-4–PE (UC10-4B9), anti-CD122–PE (51-14), anti-CD103–FITC (2E7), anti-IFN- (XMG1.2), anti-FoxP3 (FJK-16S), anti-Thy1.1–PE (A20) and anti-Thy1.2–FITC (104). The anti-H-2^d–FITC (SF1-1.1) and anti-H-2^b (AF6-88.5) came from BD Pharmingen (San Diego, CA, USA). Isotype control antibodies were also obtained from eBioscience and BD Pharmingen. Anti-glucocorticoid-induced TNFR family-related protein-biotin (GITR, BAF524), anti-IL-10 (mAb417), anti-TGF-ß (mAb240) and matched isotype control antibodies were obtained from R&D Systems (Minneapolis, MN, USA).

Cell preparation and adoptive transfer
T cells were prepared from D2 spleen cells by collecting nylon wool column non-adherent cells (15). The T-enriched cells (1.5 x 10⁶ ml^–1) were stimulated with similar numbers of irradiated (2000 rad) B6 nylon adherent, non-T cells for 5–6 days in 24-well plates (2 ml per well) (Becton Dickinson Labware, Franklin Lakes, NJ, USA) in AIM V (Invitrogen, Carlsbad, CA, USA) serum-free medium with additives (15). Some wells contained TGF-ß1 (2 ng ml^–1) and rhuIL-2 (15–20 units ml^–1) (R&D Systems) or IL-2 only. Groups of six D2 mice were injected intravenously 1 day before and 5 days after receiving B6 heart allograft with 10 million viable alloactivated T cells primed with IL-2 and TGF-ß (regulatory T cells, Treg) and others with IL-2 only (control T cells, Tcon) or with Treg depleted of CD25+ cells with immunomagnetic beads (Miltenyi). These preparations contained 10% residual B6 stimulator cells.

Heterotopic heart transplantation
Abdominal vascularized heterotopic heart transplants were performed essentially as previously described (17). Rejection was defined as complete cessation of a palpable cardiac contraction and confirmed by visualization after laparotomy. Recipients with grafts surviving >100 days were considered as permanent and were sacrificed for in vitro experiments.

Assays of T-cell function
The proliferative activity of T cells to alloantigens was measured using a standard one-way mixed-lymphocyte culture with 2 x 10⁵ T cells and an equal number of irradiated allogeneic non-T cells in a 96-well flat-bottom plate using RPMI 1640 culture medium and 10% FCS with additives as described previously (15). Proliferation was measured after 4–5 days as uptake of [³H]thymidine in triplicate cultures. In order to analyze the IFN--producing cells, intracellular cytokine staining was performed as described previously (16). In cultures used to assess the suppressive activity of CD4+CD25+ cells, the ratio of primed cells to CD4+CD25– responder cells was 1:6. T-cell cytotoxic activity was assessed using various ratios of effector cells to target cells (chromium-labeled Con A blasts) in a standard 4-h assay as described previously. Values indicate the mean ± SEM of triplicate cultures and in some experiments expressed as the lytic units per 10⁶ cells (5). Lytic units were based on the number of effector cells required to kill 30% of the target cells.

FoxP3 expression by real-time–PCR
Total RNA was prepared with TRIzol LS reagent (Invitrogen). First-strand cDNA was synthesized using Omniscript TR kit (Qiagen, Valencia, CA, USA) with random hexamer primers (Invitrogen). Real-time PCR was performed with a LightCycler (Roche, Mannheim, Germany), and message levels were quantified using the LightCycler Fast Start DNA Master SYBR Green I Kit (Roche), according to the manufacturer's instructions. Amplification was conducted for 45 cycles. The recovered PCR product and amplicon were checked by agarose gel electrophoresis for a single band of the expected size. The samples were run in triplicate and the relative expression of FoxP3 was determined by normalizing the expression of each target to hypoxanthine guanine phosphoribosyl transferase (HPRT). Primer sequences were as follows: HPRT 5'-TGA AGA GCT ACT GTA ATG ATC AGT CAA C-3' and 5'-AGC AAG CTT GCA ACC TTA ACC A-3'; FoxP3 primers: 5'-CCC AGG AAA GAC AGC AAC CTT-3' and 5'-TTC TCA CAA CCA GGC CAC TTG-3' (18).

In vivo cytotoxic T-cell activity
Groups of eight DBA/2 mice were injected intravenously with 10⁷ Tregs or Tcon cells generated ex vivo as described above. Another group was not injected. Three weeks later, four mice from each group were injected with 10⁷ C57BL/6 splenocytes (immunized) or served as controls. In vivo cytotoxic T-cell activity was assessed at week 4 using an assay modified from that described by Suvas et al. (19). Splenic target cells from C57BL/6 or C3H mice were labeled with high (2.5 mM) or low (0.25 mM) concentrations of carboxyfluorescein diacetate succinimidyl ester (CFSE). Equal numbers (10⁷) of donor-specific and third-party target cells were mixed together and adoptively transferred intravenously into control and immunized DBA/2 mice. Splenocytes were collected at 1, 2 or 4 h after adoptive transfer from recipient mice, erythyrocytes were lysed and cell suspensions were analyzed by flow cytometry. Each population could be distinguished by their respective fluorescence intensity. Assuming that the number of C57BL/6 target cells that migrated to the spleen in unimmunized mice is equivalent to the number of splenic C57BL/6 target cells injected in immunized mice, the percentage of killing of target cells in the immunized animals was determined as follows: percentage of killing = [(percentage of CFSE⁺ subset in the control mice – percentage of CFSE⁺ in the immunized mice)/percentage of CFSE⁺ in the control mice] x 100.

Statistical analysis
Analysis for statistically significant differences between groups of mice was performed by t test and Wilcoxon test survival curves with the log rank test using GraphPad PRISM software (GraphPad, San Diego, CA, USA).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Treatment with regulatory T cells generated ex vivo markedly prolongs the survival of heart allografts
Since we have shown that TGF-ß induces both CD4+ and CD8+ cells to become suppressor cells (5, 12), and others have described CD8+ regulatory cells that express FoxP3 with functional properties similar to CD4+CD25+ Tregs (20), we generated Tregs from unseparated T cells. Our objective was to learn whether a combination of CD4+ and CD8+ Tregs induced ex vivo with TGF-ß used as sole therapy could prolong survival of totally MHC-mismatched heart allografts. After culture of DBA/2 (H-2^d) T cells with irradiated C57BL/6 (H-2^b) spleen cells for 5–6 days with IL-2 and TGF-ß, we recovered approximately the starting number of T cells in cultures with TGF-ß, and 50% of T cells in cultures without TGF-ß. In cultures with IL-2 and TGF-ß, 60 ± 4.1% of CD4+ cells expressed CD25 and 55 ± 4.8% of CD8+ cells expressed this marker. These cells are called Treg. In cultures without TGF-ß, these values were 45 ± 3.4% and 49 ± 4.1%. These cells are called Tcon. Of the 10 million cells injected into recipient mice, Treg preparations contained 3.4 ± 0.3 x 10⁶ CD4+CD25+ cells and 2.1 ± 0.2 x 10⁶ CD8+CD25+ cells. Tcon preparations contained 2.1 ± 0.2 x 10⁶ CD4+CD25+ cells and 1.6 ± 0.15 x 10⁶ CD8+CD25+ cells.

All hearts from B6 mice that were transplanted into D/2 recipients were rejected within 11 days of transplantation. Transfer of 10 million Treg at days –1 and +5 resulted in extended survival of B6 heterotopic heart transplants up to 100 days, at which point the experiment was terminated. By contrast, rejection was accelerated in D2 mice that received similar numbers of Tcon (Fig. 1). The extended survival was dependent on CD25+ cells, since depletion of this subset completely abolished all suppressive effects.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Donor anti-H-2b-specific Tregs induced ex vivo with TGF-ß cause long-term survival of mismatched allogeneic heart transplants. Tregs were generated by stimulating DBA/2 (D2, H-2d) T cells with irradiated C57BL/6 (B6, H-2b) non-T cells and IL-2 in the presence of TGF-ß (2 ng ml–1) for 5–6 days. T cells stimulated with IL-2 only served as controls (Tcon). D2 mice that received B6 heart transplants were injected with 10 x 106 Treg, Tcon or Treg depleted CD25+ cells intravenously on days –1 and +5. Six mice were in each group.

 
The transfer of Tregs results in the antigen-specific tolerance in the recipients
We next developed a model designed to investigate the mechanism of action of the long-term suppressive effects. D2 mice were given a single injection of 10⁷ Treg or Tcon cells. One month later they were tested for T-cell responsiveness to donor alloantigen. Figure 2 shows that animals injected with Tcon proliferated vigorously to H-2^b antigen. By contrast, animals injected with Treg cells were non-responsive. They were unable to proliferate when challenged with alloantigen (Fig. 2A). CD8+ cells were unable to produce IFN- (Fig. 2B and C), and were unable to kill H-2^b target cells even after further stimulation in vitro (Fig. 2D). This T-cell non-responsiveness was antigen specific. D2 T cells proliferated strongly in response to third-party C3H H-2^k stimulator cells (Fig. 2A).

View larger version (33K): [in this window] [in a new window]   Fig. 2. Transferred anti-H-2b Tregs induce alloantigen-specific T-cell non-responsiveness. Groups of four naive DBA/2 mice were injected intravenously with or without 10 x 106 D2 Tcon and Treg cells generated as described in Fig. 1. Another non-injected group served as additional controls. One month later the animals were sacrificed and splenic T cells were alloactivated with B6 or third-party, C3H (H-2k) stimulator cells in vitro for 4 days. (A) Proliferative activity [mean counts per minute (CPM) ± SEM]. P-values indicate significant differences between mice that received Tregs and mice that received Tcon cells or no transfer (Nil). (B) Example of percentages of IFN--producing CD8+ cells in response to H-2b antigen determined by flow cytometry. (C) Number of IFN--producing splenic CD8+ cells against H-2b and H-2k antigens. P-values were determined as described above. The experiment was repeated with similar results. (D) DBA/2 mice were immunized with 10 x 106 B6 splenocytes injected intravenously with or without 10 x 106 D2 Treg cells. Unimmunized mice served as controls. One month later, fresh splenic T cells were tested for anti-H-2b CTL activity or alloactivated with B6 stimulator cells. The cells from the mixed lymphocyte reaction cultures were re-counted and assayed for CTL activity at the indicated effector to target ratio. Values indicate the mean ± SEM of six mice. The experiment was repeated with similar results.

 
In addition to documenting T-cell non-responsiveness in vitro effects, we observed similar effects in vivo. Following transfer of Treg previously primed with H-2^b alloantigen, and then boosted with donor cells, mice were injected with CFSE-labeled donor and third-party target cells and examined for the presence of these cells in the spleen. Pilot studies revealed that following immunization, there was a marked reduction of donor, but not third-party target cells within 2 h of injection (Fig. 3A). However, in mice that had received Treg, similar numbers of CFSE-labeled donor target cells were observed in control and immunized mice. By contrast, in mice that had received Tcon, numbers of both donor and third-party targets were markedly reduced. The reduction of third-party target cells probably reflects the non-specific CTL activity associated with the vigorous CTL response to donor alloantigen. Table 1 indicates that the effects we observed were very similar in the four mice of each group. Since the in vivo CTL assay does not require the in vitro expansion, this approach is considered to be direct evidence of Treg function in vivo (19).

View larger version (20K): [in this window] [in a new window]   Fig. 3. Transferred anti-H-2b Tregs result in the reduced cytotoxicity activity in vivo. The experimental design is similar to that described in Fig. 1. Naive DBA/2 mice were injected intravenously with or without 10 x 106 D2 Tcon or Treg cells or no cells (N = 8 per group). On the third week, four mice (one half) of each group were injected with 10 x 106 B6 splenocytes. To assess the immune response to B6 cells, 1 week later all mice received 10 x 106 B6 splenocytes brightly labeled with CFSE and a similar number of dimly CFSE-labeled C3H splenocytes. The animals were sacrificed 2 h later and splenic cells examined for intensity of CFSE staining by flow cytometry. Results are expressed as the percentage of killing B6 or third-party C3H CFSE-stained cells in immunized animals compared with that in unimmunized animals.

 

View this table: [in this window] [in a new window]   Table 1. Mean value ± SEM of four mice in each group and P-values are indicated

 
T-cell non-responsiveness depends on CD4+CD25+ cells that require continuous specific antigen stimulation
The next series of experiments confirmed the requirement of CD4+CD25+ cells for the suppressive effects and revealed that continuous stimulation of specific antigen was needed to sustain T-cell non-responsiveness. Groups of mice received a single injection of Treg or Tcon, or no cells. Some mice received booster injections of donor alloantigen every 2 weeks and others not injected served as controls. In animals that had received the booster injections, we observed a progressive increase in the splenic CD4+CD25+ cells during the next 3 months in those that had received Tregs, but not in those that had received Tcon cells (Fig. 4A). Since these mice were not lymphopenic, the increase could not be attributed to the homeostatic expansion of CD4+CD25+ cells described by others (21). This expansion was dependent on continuous boosting with donor alloantigen. If at 2 months the mice received splenic cells from H-2^k C3H mice instead of H-2^b B6 cells, the numbers of CD4+CD25+ cells decreased to baseline values within 1 month (Fig. 4B). Splenic CD8+CD25+ cells probably did not play a significant role since they comprised <1% of CD8+ cells in mice that had received Treg.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Continuous antigen-stimulation results in a progressive increase in CD4+CD25+ cells and maintenance of tolerogenic effects. (A) Groups of six mice received a single injection of 10 x 106 D2 Treg (circles), Tcon (squares) or no cells (triangles). Those with filled symbols were also injected with 10 x 106 H-2b B6 irradiated splenocytes every 2 weeks. Those with empty symbols were not boosted with alloantigen. Splenic CD4+CD25+ cell numbers were determined each month by cell counts and flow cytometry in mice. Note the antigen-dependent increase in CD4+CD25+ cells in mice that received alloantigen. (B) Two months after the single dose of Tcon or Tregs, some groups continued to receive specific antigens, but others were injected with third-party H-2k (C3H) antigen followed by another injection 2 weeks later. Note that the increased numbers of CD4+CD25+ cells in mice given Tregs and boosted with H-2b cells decreased to baseline levels when H-2k cells were given instead. (C) D2 mice received a single injection intravenously of 10 x 106 syngeneic Tcon or Tregs, and 10 x 106 irradiated B6 splenocytes every 2 weeks to provide a continuous source of antigen. One, two and three months post-injection, splenic T cells were tested for CTL activity in an allo-mixed lymphocyte reaction with results in lytic units expressed as the mean ± SEM. One lytic unit is the number of lymphocytes required to give 30% lysis. Six mice per group were examined at each time point. (D) The tolerogenic response was antigen dependent. Using the protocol described in (B), H-2k cells were substituted for H-2b cells at 2 months and the animals were tested for anti-B6 CTL activity 1 month later. Note the loss of CTL activity at this time that is associated with the cessation of vH-2b antigen stimulation.

 
Continuous stimulation with specific antigen was required for Treg to sustain blockade of CTL activity. Figure 4(C) shows that the animals that had received Tregs and three to five subsequent booster immunizations of donor alloantigen for 2–3 months were unable to develop anti-H-2^b CTL activity. However, if injections of third-party H-2^k cells instead of donor cells were given, the mice demonstrated strong anti-H-2^b CTL activity within 1 month (Fig. 4D).

We next obtained evidence that the increased numbers of CD4+CD25+ cells in mice given Treg followed by booster immunizations of donor alloantigen expressed FoxP3 and were required for T-cell non-responsiveness. Mice that received Treg, Tcon or no cells followed by booster immunizations every 2 weeks were sacrificed at 2 months. Although the total numbers of splenic CD4+ cells were similar in each of the groups, the CD4+CD25+ subset was significantly increased in mice that had received Treg (Table 2). Examination of CD4+CD25+ and CD4+CD25– cells revealed that the CD25+ subset expressed significantly higher levels of FoxP3 mRNA by real-time PCR (Fig. 5A). Moreover, the number of CD4+CD25+FoxP3+ cells quantified by flow cytometry was significantly increased in mice that had received Treg compared with those that received Tcon (Fig. 5B and C).

View this table: [in this window] [in a new window]   Table 2. CD4+CD25+ cells at two months following cell transfer

 

View larger version (18K): [in this window] [in a new window]   Fig. 5. CD4+CD25+ cells express the increased levels of FoxP3 mRNA and protein. The experimental design is similar to that shown in Fig. 4. Groups of four D2 mice received a single injection of 10 x 106 syngeneic Treg or Tcon. Another two mice received no T cells. All mice shown were injected with 10 x 106 H-2b B6 irradiated splenocytes every 2 weeks. Splenic CD4+CD25+ cell numbers of each mouse was determined at 2 months by cell counts and FACS staining. (A) Splenic CD4+CD25+ cells were positively selected from individual mice by immunomagnetic beads, and FoxP3 mRNA was quantified by real-time PCR. The numbers shown are the mean ± SEM of each group. (B) A representative example of FoxP3 protein expression in these CD4+CD25+ cells was determined by staining with anti-mouse FoxP3 antibody. (C) The numbers shown indicate the mean ± SEM of total CD4+CD25+FoxP3+ cells of each group.

 
CD4+CD25+ cells were probably responsible for antigen-specific non-responsiveness to B6 alloantigens. As shown in Fig. 6(A), depletion of CD25+ cells abolished the tolerogenic effect and adding back this subset restored the suppression. As with CTL activity, depletion of these CD25+ cells increased allo-CTL activity to levels similar to animals that had received Tcon. Again, adding back CD25+ Tregs in a 1:10 ratio restored suppressive activity (Fig. 6B). Since CD8+CD25+ cells comprised only 1% of total CD25+ cells, this suppressive effect was presumably due to CD4+CD25+ cells. These experiments, however, do not exclude an effect of CD8+ suppressor cells.

View larger version (30K): [in this window] [in a new window]   Fig. 6. CD4+CD25+ cells are responsible for tolerance to donor alloantigens. (A) Splenic T cells, T cells depleted of CD25 prior to the culture and CD25-depleted T cells with 10% of these CD25+ cells added back were prepared from mice that had received a single injection of Tcon or Tregs or no injection (No transfer) 3 months previously. These D2 T cells were alloactivated with B6 stimulator cells and tested for proliferative ability. Note that CD4+CD25+ cells were responsible for the suppressive effects. (B) Each T-cell preparation was also tested for anti-B6 CTL activity and these suppressive effects were also dependent on CD25+ cells. Values shown are representative of the six mice in each group.

 
Donor Tregs educate recipient T cells to become tolerogenic CD4+CD25+ cells in vivo
To learn whether the increased CD4+CD25+ suppressor cells were the progeny of donor Tregs or derived from the recipient, the experiment was repeated using the protocol described above, with Thy1.1 B6 mice serving as the source of the Tregs and congenic Thy1.2 mice as recipients. Here we again noted a progressive increase in CD4+CD25+ cells in B6 mice during the 3 months following a single injection of 8 million anti-H-2^d Treg and almost all cells were of recipient Thy1.2 origin (Fig. 7A). At 1 month, only 1% of splenic T cells were stained by anti-Thy1.1 (results not shown). Thy1.2-negative T cells were <2% and these cells did not express CD25 (Fig. 7B). In comparison with Tcon, Tregs were enriched in cells expressing CD25, CD122 (IL-2R chain), CD103 (alpha E integrin) and GITR (Fig. 7C and Table 3), and most of the CD122 and CD103 cells also expressed CD25 (Fig. 7C). Others have shown that TGF-ß up-regulates CD103 expression (22).

View larger version (49K): [in this window] [in a new window]   Fig. 7. The transferred Tregs increase recipient CD4+CD25+ cells that express CD103, CD122 and GITR. To distinguish transferred T cells from recipient T cells, anti-H-2d Tregs and Tcon were prepared from cells from B6 Thy1.1 mice and 8 x 106 cells transferred to congenic Thy1.2 mice. Using the repeated stimulation protocol described above, the numbers of CD4+CD25+ cells and phenotype were assessed sequentially for 3 months. (A) Total numbers of recipient Thy1.2 CD4+CD25+ cells each month. (B) Flow cytometry profile at 1 and 3 months of splenic cells stained with CD4, Thy1.2 and CD25. The cells shown were gated on CD4+ cells. (C) Percentage of CD4 cells expressing CD25, CD122 and CD103 in the Thy1.2 gate.

 

View this table: [in this window] [in a new window]   Table 3. Phenotypic characterization of CD4+ cells at 3 months following T-cell transfer

 
The functional properties of the educated mouse splenic CD4+CD25+ cells were similar to educated human peripheral blood CD4+ cells reported previously (16). While natural CD4+CD25+ cells and human natural-like CD4+CD25+ cells induced with TGF-ß have suppressive activity that is not diminished by anti-TGF-ß or anti-IL-10 (2, 3, 5), the suppressive activity of our educated CD4+CD25+ cells was abolished by either anti-TGF-ß or anti-IL-10 (Fig. 8A). Of great interest, the anti-H-2^d suppressive activity of CD4+CD25+ cells from mice that had received Tregs was significantly greater than CD4+CD25+ cells from mice that had received Tcon. This effect was antigen specific since anti-H-2^d CD4+CD25+ cells had minimal suppressive activity against H-2^k stimulator cells (Fig. 8B). These experiments were performed with a single source of CD25– responder cells and a ratio of CD4regs to CD4 responders of 1:6. At this ratio, the antigen non-specific suppressive activity of endogenous CD4+CD25+ regulatory cells on the response of CD4+ cells to allogeneic stimulator cells is diluted out (Fig. 8C).

View larger version (37K): [in this window] [in a new window]   Fig. 8. The transferred Tregs induce recipient CD4+ cells to become antigen-specific suppressor cells. (A) Three months after transfer of Tcon or Tregs, splenic CD4+CD25+ and CD4+CD25– cells were obtained by cell sorting and their suppressive effects on the allogeneic response of fresh, syngeneic CD4+CD25– cells to H-2d, indicated as baseline. The ratio of sorted CD4+ cells to responder CD4+CD25– cells was 1:6 to dilute out the non-specific suppressive activity of CD4+CD25+ cells (see panel B). The effect of neutralizing anti-IL-10 (10 µg ml–1) or TGF-ß (10 µg ml–1) antibodies on the suppressive activity of CD4+CD25+ cells is also shown. (B) Lack of suppression following stimulation by third-party (H-2k) cells. Results are expressed as mean counts per minute (CPM) ± SEM of triplicate wells (n = 6 mice per group). (C) Suppressive activity of naive CD4+CD25+ cells. CD4+CD25+ and CD25– cells from naive mice were prepared by cell sorting and assayed for their suppressive effects on the response of CD4+CD25– cells to H-2b stimulator cells.

 

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study we observed that Tregs induced with IL-2 and TGF-ß ex vivo can prolong the survival of heart allografts in completely MHC-mismatched mice without any additional immunosuppression. We also administered repeat allogeneic cell infusions in non-transplanted mice to investigate the mechanism of action. We observed that a single injection of T cells primed with allogeneic cells and TGF-ß (Treg) followed by continuous boosts of alloantigen could induce long-term antigen-specific non-responsiveness in the recipients. This tolerogenic effect appeared to be secondary to the ability of the transferred Tregs to educate donor CD4+ cells to become CD25+ cells.

It is recognized that TGF-ß can induce both CD4+ and CD8+ cells to become suppressor cells. In 1994 we reported that human CD8+ cells activated with IL-2 and TGF-ß became cytokine-dependent suppressors of T-cell-dependent antibody production (12). We subsequently observed that TGF-ß induced naive CD4+ cells to become CD4+CD25+ cells with a phenotype and suppressive activities indistinguishable from the natural CD4+CD25+ cells described by others (2, 3). These cells had a contact-dependent, mechanism of action not affected by anti-TGF-ß or anti-IL-10 and were potent inhibitors of CD8+ T-cell activation (5, 23). We observed that TGF-ß did not expand endogenous CD4+CD25+ cells, but induced CD4+CD25– cells to develop this function (16). Subsequently, Chen et al. (24) reported that TGF-ß induced mouse CD4+CD25– cells to become CD25+ suppressor cells that express FoxP3, and our laboratory and others have confirmed this finding (16, 25–28). Prior work by Blazar and co-workers found that CD4+CD25– cells tolerized with IL-2 and TGF-ß could increase survival in a model of alloantigen-induced graft-versus-host disease (6).

Since we had been able to induce CD8+ cells to become suppressor cells with TGF-ß, and others had shown that CD8+ regulatory cells could suppress the rejection of heart allografts (29), we utilized total T-cell preparations containing both CD4+ and CD8+ cells in our initial study. In preliminary work using the mouse graft-versus-host disease model that we had used previously (15), we found that the combination of CD4+ and CD8+ Tregs has more potent therapeutic effects than purified CD4+ Tregs (unpublished observations). In the present experiments, we observed only small numbers of CD8+CD25+ cells in the recipients, and <1% Thy1.1 congenic T cells remained in the recipients 1 month after transfer. Nonetheless, we cannot exclude the possibility that CD8+ Tregs induced with TGF-ß contributed to the observed therapeutic effects.

In this study the number of CD4+CD25+ cells of recipient origin progressively increased in response to biweekly booster immunizations with allogeneic cells. Although CD25 is a marker of activated T cells, it is unlikely that the cells we observed are allogeneic effector cells. These cells were non-responsive to donor alloantigen. They blocked the ability of recipient T cells to proliferate and produce cytokines in response to donor alloantigens and they prevented CD8+ cells from developing CTL activity. Furthermore, the persistence of donor target cells in the in vivo CTL assay provides additional evidence of Treg activity in vivo as stated above. Finally, the evidence that these CD4+CD25+ cells express both FoxP3 mRNA and protein strongly suggests that the booster immunizations were expanding CD4+CD25+ regulatory cells in vivo.

Our results are consistent with other reports that CD4+CD25+ regulatory cells have a protective effect in transplant rejection. van Maurik et al. (8) have documented that CD4+CD25+ cells can markedly prolong survival of cardiac allografts, although these cells were induced by indirect methods. Benghiat et al. (30) have recently reported that natural CD25+ Tregs control T_h1- and T_h2-type allo-T_h responses. Both these groups have reported that the protective CD4+CD25+ cells require continuous antigen stimulation (31, 32). Schenk et al. (33) have reported that depletion of CD4+CD25+ cells markedly accelerates acute rejection of heart allografts. Because epitope spreading in alloreactive cells may contribute to chronic rejection (34), Salama et al. (35) have suggested that CD4+CD25+ cells may limit this effect and thus have a protective role.

While some research has shown that polyclonal CD4+CD25+ cells can educate other CD4+ cells to become suppressor cells in vitro (36, 37), others have used indirect methods to achieve infectious tolerance in vivo (38). This is the first demonstration that Tregs induced ex vivo can educate recipient CD4+ cells to become CD25+ cells that have similar suppressive activity. Thus, this TGF-ß-induced Tregs act more like a vaccine than a conventional adoptive therapy. They appear to prevent organ graft rejection by eliciting an active, protective immune response in the recipient.

Examination of the functional properties of CD4+CD25+ cells harvested from the tolerized recipients revealed that their mechanism of action could be blocked by either anti-TGF-ß or anti-IL-10. This result is consistent with a study of human CD4+CD25+ regulatory cells induced with TGF-ß. We reported that anti-TGF-ß was unable to block the suppressive effects of naive CD4+ cells induced ex vivo to become alloantigen-specific suppressor cells. Nonetheless, these cells produced both TGF-ß and IL-10 following restimulation, and both these cytokines were necessary for these CD4+CD25+ Tregs to induce other CD4+CD25– to become suppressor cells. Moreover, the suppressive effects of the secondary CD4+CD25+ Tregs were blocked by either anti-TGF-ß or anti-IL-10. Thus, the transfer of CD4+CD25+ Tregs with cytokine-independent suppressive effects in vitro may result in cytokine-dependent suppressive effects in vivo (16). In experimental models of immune-mediated disease in mice, the role of TGF-ß and IL-10 in supporting the suppressive effects of CD4+CD25+ cells has been established (39, 40).

In this study we observed long-term survival in some, but not all, of the allogeneic heterotopic heart transplants. Although we observed prolonged graft survival, only two of these six grafts survived to 100 days, making it highly unlikely that in vivo tolerance was achieved. By contrast, animals that received booster immunizations of donor alloantigen could not mount a response to donor cells. The results of these experiments infer that in the attempt to establish a tolerant state, there is a probable requirement for persistent antigenic stimulation to sustain the activity of the Tregs. After heart transplantation, there may not be sufficient donor antigen shed to sustain tolerogenic regulatory T cells. Histology of the hearts from animals sacrificed 100 days post-transplantation did not show classic pathologic evidence of acute or chronic rejection. However, despite intact function, the myocardium did have a moderate monocytic infiltrate. Unfortunately, we did not save frozen tissue at the time of the original experiments, so we cannot evaluate if there was FoxP3 mRNA displayed by these mononuclear cells, and therefore cannot exclude the possibility that this mononuclear infiltrate may be an atypical manifestation of chronic rejection. Further experiments in which animals receive additional injections of relevant donor MHC alloantigens may improve the graft survival results observed, as well as decrease the observed mononuclear infiltrates. The use of TGF-ß-treated T cells generated ex vivo to alter the recipient's immune system to develop a dominant regulatory response rather than an alloreactive one offers a novel therapeutic strategy for clinical organ transplantation.

	Acknowledgements

 
The authors wish to acknowledge Harold Soucier for his skilled support in flow cytometry and Erick Bonilla for his help in preparing the manuscript. This work was supported by grants from the National Institutes of Health (AI-41768, D.A.H.), the Nora Eccles Treadwell Foundation (D.A.H.), the Arthritis National Research Foundation (S.G.Z.), the Wright Foundation (S.G.Z.) and the Cystic Fibrosis Foundation (CFF-G965, M.L.B.).

	Abbreviations

 

HPRT	hypoxanthine guanine phosphoribosyl transferase
TGFß	transforming growth factor ß
Treg	regulatory T cell

	Notes

 
Transmitting editor: W. Strober

Received 12 September 2005, accepted 7 November 2005.

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