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Basic and Experimental Research

Impact of Immunosuppressants on the Therapeutic Efficacy of In Vitro-Expanded CD4+CD25+Foxp3+ Regulatory T Cells in Allotransplantation

Lim, Dong-Gyun1,4; Koo, Sun-Kyung2; Park, Youn-Hee2; Kim, Youngji2; Kim, Hye-Mi2; Park, Chan-Sik2,3; Kim, Song-Cheol1,2; Han, Duck-Jong1,2

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doi: 10.1097/TP.0b013e3181d3c9d4
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Abstract

Successful organ transplantation has been made possible by the introduction of powerful, but nonspecific, immunosuppressive drugs that efficiently control acute graft rejection. However, the lifelong use of those drugs has several clinical limitations, including adverse effects and the inability to prevent chronic rejection (1). Therefore, the ultimate goal in transplantation is to induce immune tolerance to donor alloantigens. Among the several mechanisms that exist for immune tolerance acquisition, active regulation by regulatory T lymphocytes (Tregs) has recently been highlighted as crucial for inducing and maintaining tolerance to donor alloantigens in vivo (2–6). There are several types of Tregs in the body. Of these, natural regulatory T cells Tregs (nTregs) have been the most thoroughly studied because they can be isolated based on their surface expression of CD25 molecules and expanded in vitro in an antigen- specific or -nonspecific manner (7–9). Furthermore, these in vitro-expanded nTregs maintain their suppressive activity. Thus, a substantial effort is underway to use nTregs as a therapeutic agent in preventing rejection of allografts (10–15). We have also recently established nTreg cell therapy in a murine skin allograft model (manuscript in preparation). Although Treg cell therapy has been shown capable of inducing a modest prolongation of allograft survival when combined with transient T-cell depletion, it fails to induce immune tolerance to alloantigens. A similar outcome of Treg cell therapy has been observed in other experimental models using wild-type animals as recipients (10). Thus, supplemental measures are required to make Treg cell therapy a practical tool for preventing allograft rejection. Furthermore, it would be medically inappropriate to use Tregs in clinical transplantation in the absence of concurrent immunosuppressive therapy. These considerations led us to study the in vivo effects of various immunosuppressants on Tregs expanded in vitro and adoptively transferred into the recipient.

MATERIALS AND METHODS

Mice

Adult male C57BL/6 (H-2b), BDF1 (C57BL/6× DBA/2; H-2b/d), and B6C3F1 (C57BL/6× C3H; H-2b/k) mice were purchased from Japan SLC Inc. (Shizuoka, Japan). B6.PL (Thy1.1-congenic) mice were from the Jackson Laboratory (Bar Harbor, ME). All mice were housed in specific pathogen-free conditions and underwent the experimental procedure at 8–12 weeks of age in accordance with the protocols for animal experiments approved by the Animal Use and Care Committee of Ulsan University.

Antibodies and Immunosuppressive Agents

Fluorochrome-conjugated monoclonal antibodies (mAbs) to CD4 (RM-4–5), CD25 (PC61), CD62L (MEL-14), CD90.1 (HIS51), and Foxp3 (FJK-16s) were purchased from BD Pharmingen (San Diego, CA) or eBioscience (San Diego, CA). An Alexa Fluor 647-conjugated mAb to H-2Dd was from BioLegend (San Diego, CA). Depleting anti-CD4 (GK1.5) (16) and anti-CD8 (2.43) (17) mAbs were obtained from Bio Express Inc. (West Lebanon, NH), as were an anti-CD40L mAb (MR-1) and human CTLA4Ig (human CTLA4-human IgG1 Fc, wild type, fusion protein) (18). Rapamycin was purchased from LC Laboratories (Woburn, MA) and dissolved in ethanol at a concentration of 1 mg/mL. Cyclosporine A (CsA; Sandimmune, Novartis Pharma, Basel, Switzerland), mycophenolate mofetil (MMF; CellCept, Roche Pharma, Nutley, NJ), and methylprednisolone (mPD; Methysol, Kunwha Pharma, Seoul, Korea) were obtained from the Asan Medical Center Pharmacy.

Isolation of Cells

Single-cell suspensions were prepared from the spleen and lymph nodes (LNs) of mice. After lysis of red blood cells, CD3+ T cells or CD4+ T cells were purified by negative selection using a magnetically activated cell sorter (MACS) CD3+ or CD4+ T-cell isolation kit, respectively, according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). An enriched population of nTregs was prepared by staining CD4+ T cells with fluorescein isothiocyanate (FITC)-anti-CD4, PE-anti-CD25, and PE-Cy5-anti-CD62L mAbs and then isolating CD4+CD25h+CD62L+ T cells using a FACSVantage cell sorter (BD Immunocytometry Systems, San Jose, CA). CD25-positive and -negative CD3+ T cells were isolated using MACS by sequentially incubating CD3+ T cells with PE-anti-CD25 and anti-PE mAb-conjugated magnetic beads (Miltenyi Biotec). In some experiments, CD3+CD25+ T cells or adoptively transferred nTregs were isolated from recipient mice by FACSorting. Allostimulator cells were prepared from the splenocytes of BDF1 or B6C3F1 mice by depletion of T cells using anti-CD90 mAb-conjugated magnetic beads (Miltenyi Biotec). The purity of the isolated cells, as assessed by flow cytometry, was routinely greater than 90% (data not shown).

Bone Marrow-Derived Dendritic Cells

Bone marrow-derived dendritic cells (BM-dendritic cells [DCs]) were generated as described by Inaba et al. (19) with some modification. Briefly, a total of 1×107 bone marrow cells from BDF1 mice were cultured in 10 mL complete medium (Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS [Gibco, Invitrogen, Grand Island, NY], 4 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, 100 μM nonessential amino acids [all from Invitrogen], and 50 μM 2-mercaptoethanol [Sigma-Aldrich, St Louis, MO]) containing 20 ng/mL recombinant murine granulocyte macrophage-colony stimulating factor (GM-CSF, PeproTech Inc, Rocky Hill, NJ). On culture day 3, nonadherent cells were removed, and fresh medium with GM-CSF was added. On day 6, nonadherent and loosely adherent cells were collected, transferred to new dishes, and cultured for an additional 2 days in fresh medium supplemented with GM-CSF. On day 8, nonadherent cells were harvested and used as the stimulators in nTreg cultures.

In Vitro Expansion of nTregs

Purified CD4+CD25h+CD62L+ T cells were cultured with irradiated (25 Gy) donor BM-DCs at a 1:1 ratio in the presence of 500 U/mL recombinant human IL-2 (Chiron, Amsterdam, the Netherlands). On culture day 3, half the medium was replaced with fresh medium supplemented with IL-2. Cells were subjected to three rounds of weekly stimulation with donor alloantigens. Starting at the second round of stimulation, 2 ng/mL recombinant transforming growth factor (TGF)-β1 (PeproTech) was added to the medium. At the end of three cycles of culture stimulation, viable cells were harvested and used for subsequent experiments. Foxp3 expression rate in expanded nTreg cells, as examined by intracellular immunofluorescent staining using specific antibody, was routinely greater than 95% (data not shown).

Effect of Immunosuppressive Agents on In Vivo Proliferation of Adoptively Transferred nTregs

A total of 1×107 in vitro expanded nTregs (B6.PL, Thy1.1) were labeled with 5 μM CFSE (Molecular Probes, Leiden, the Netherlands) and intravenously administered into a BDF1 mouse. At the same time, 5×106 freshly isolated CD3+ T cells (B6, Thy1.2), prelabeled with 4 μM PKH26 (Sigma-Aldrich), were also transfused into the same host. As indicated in Figure 1(B), the recipient mice received various immunosuppressive agents at doses that had been used in previous studies (20–24). Four days later, spleen and LN cells were prepared from transfused mice and stained with anti-CD90.1-PerCp and anti-H-2Dd-Alexa Fluor 647 mAbs. The adoptively transferred nTregs were detected as CD90.1+H-2Dd− cells, and adoptively transferred CD3+ T cells were detected as CD90.1H-2Dd− cells. CD90.1+H-2Dd− and CD90.1H-2Dd− cell divisions were analyzed by CFSE and PKH26 dilution, respectively, using flow cytometry.

FIGURE 1.
FIGURE 1.:
Effect of immunosuppressive agents on the survival and proliferation of adoptively transferred, in vitro-expanded natural regulatory T lymphocytes (nTregs). (A) Experimental scheme. nTregs (Thy1.1 C57BL/6 origin), expanded by stimulation with BDF1 bone marrow-derived dendritic cells (BM-DCs) and labeled with CFSE, were adoptively transferred into semiallogeneic BDF1 mice (1×107 cells/mouse) together with freshly isolated, PKH26-labeled CD3+ T cells (Thy1.2 C57BL/6 origin; 5×106 cells/mouse). (B) Various immunosuppressive agents were administered to recipient mice. Four days after cell transfer, pooled spleen and lymph node (LN) cells were stained for CD90.1 and H-2Dd and analyzed for the presence and CFSE/PKH26 dilution of donor T cells. (C) Representative CFSE- and PKH26-dilution profiles of transferred T cells from each group. Adoptively transferred nTregs and CD3+ T cells were gated as CD90.1+H-2Dd− and CD90.1−H-2Dd− lymphocytes, respectively, and displayed in histograms after acquiring a total of 100,000 spleen/LN cells. (D) Total number of donor cells recovered from recipient mice after adoptive transfer and the percentage of transferred cells with more than three cycles of cell division were determined in each group of recipient mice. Data are presented as mean±SEM (n=3 mice/group).

Skin Grafting

Recipient T lymphocytes were transiently depleted by a single intraperitoneal injection of depleting anti-CD4 and anti-CD8 mAbs (200 μg each/mouse) 14 days before skin grafting. Some recipient mice received tail vein injections of in vitro-expanded nTregs (1×107/mouse) on the same day as skin grafting. Full-thickness donor tail skin was grafted onto the lateral thoracic area of anesthetized recipient mice. On day 2 or 3 after transplantation, nTregs were transferred once more into the recipient mice to minimize their potential removal by residual depleting Abs. The various immunosuppressive agents were also administered to the recipient mice as indicated in Figure 2(C). The grafts were monitored daily from day 7 by visual inspection. Graft rejection was defined as the complete destruction of the grafted skin.

FIGURE 2.
FIGURE 2.:
Impact of immunosuppressive agents on the therapeutic efficacy of in vitro-expanded natural regulatory T lymphocytes (nTregs). (A) Experimental scheme. Recipient C57BL/6 mice were treated with depleting anti-CD4 and anti-CD8 mAbs (200 μg each) 14 days before receiving a skin allograft (BDF1). nTregs expanded in vitro by three cycles of stimulation with donor (BDF1) bone marrow-derived dendritic cells (BM-DCs) were injected intravenously (1×107 cells/mouse) before the engraftment of skin on the same day and once more 3 days after transplantation. This manipulation of transient T-cell depletion and adoptive transfer of nTregs induces short-term prolongation of allograft survival, as shown in (B). (C) In this experimental model, immunosuppressive agents were administered to recipient mice according to the schedule and doses. (D) The influence of individual immunosuppressive agents on skin allograft survival was examined in recipient mice treated with T-cell depletion alone (upper graph) or together with Treg cell therapy (lower graph). Data are shown as a Kaplan-Meier graft survival.

Flow Cytometric Analysis

After incubating with Fc Block Ab (BD Pharmingen), single cells were stained for 30 min at 4°C with an antibody cocktail specific to cell-surface molecules. Intracellular FoxP3 was detected with an allophycocyanin (APC) anti-mouse FoxP3 staining set (eBioscience) according to the manufacturer's instructions. The stained cells were analyzed using a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson).

Histologic Examination

Skin samples were fixed in acetone at 4°C for 1 hr. Cryosections (4-μm thick) were hydrated in phosphate- buffered saline for 10 min, treated with protein blocking solution (DAKO) for 5 min, and incubated for 60 min at room temperature with biotin-conjugated anti-FoxP3 mAbs. After washing with phosphate-buffered saline, the sections were incubated with APC-conjugated anti-CD4, streptavidin-conjugated Alexa fluor 546 (Molecular Probes), and FITC-conjugated anti-CD90.1 mAbs. Images were collected on a Leica TCS-NT/SP confocal microscope (Leica, Korea) using a 40× objective (NA 0.75, zoom 1-4 X). Fluorochromes were excited using an argon laser at 488 nm for FITC, a Gre/Ne laser at 543 nm for Alexa fluor 546, and a Helium-Neon laser at 633 nm for APC. Images were processed using Leica TCS-NT/SP software (version LCS) and Adobe Photoshop 7.0.

Isolation of Graft-Infiltrating Cells

Skin grafts were cut into small pieces and digested in RPMI1640 medium containing 1 mg/mL collagenase type I, 0.5 mg/mL hyaluronidase type IV-S (both from Sigma-Aldrich), and 50 μg/mL DNase I (Roche Diagnostics, Mannheim, Germany) in shaking incubator at 37°C for 30 min. The remaining pieces were collected and digested once more. After filtered through 70-μm nylon meshes, the cells were washed twice with RPMI media and used for flow cytometric analysis.

Mixed Lymphocyte Reaction

T cells (1×105/well) were stimulated with irradiated (25 Gy), allogeneic T-cell-depleted splenocytes (2×105/well) in a 96-well round-bottomed plate. After incubating for 60 hr, culture supernatants were harvested for the detection of secreted cytokines. T-cell proliferation was measured by incubating cell mixtures for 4 days and pulsing with 3H-thymidine (1 μCi/well; Amersham, Buckinghamshire, UK) over the final 8 hr. For mixed lymphocyte reaction suppression assays, various numbers of endogenous host CD3+CD25+ T cells or adoptively transferred Tregs isolated from manipulated recipient mice were added to the above cultures containing normal-responder CD3+CD25 T cells. The cells were harvested on filter plates, and 3H-thymidine incorporation was measured with a TopCount NXT beta counter (PerkinElmer, Downers Glove, IL).

Measurement of Cytokines

The levels of interferon (IFN)-γ, IL-10, and IL-4 present in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA, BD Biosciences). TGF-β and IL-17 were quantified using a sandwich ELISA (R&D Systems).

Statistics

Statistical analyses were performed using GraphPad Prism 4.0. Allograft survival between two groups was analyzed using the Kaplan-Meier method, and survival curves were compared using the log-rank test. A Student's t test was used to compare cell numbers between two groups.

RESULTS

Effects of Various Immunosuppressive Agents on the Survival and Proliferation of Adoptively Transferred nTregs

To determine the direct effect of immunosuppressive agents on the response of in vitro-expanded nTregs to alloantigens in vivo, we took advantage of the graft-versus-host reaction (GVHR). CFSE-labeled nTregs, originated from B6.PL mice (Thy1.1) and expanded in vitro by stimulation with allogeneic (BDF1) BM-DCs, were transferred into BDF1 recipient mice together with conventional CD3+ T cells freshly isolated from C57BL/6 mice (Thy1.2) and labeled with PKH26. Various immunosuppressive agents were administered to the recipient mice, as indicated in Figure 1(B). Four days after cell transfer, spleen and LN cells of the recipient mice were analyzed for the survival and proliferation of transferred cells (Fig. 1A). Short-term treatment with costimulation blockers (MR-1 and CTLA4Ig) or with rapamycin reduced the total number of adoptively transferred cells regardless of whether they were nTregs or conventional T cells (Tconvs). The division of nTregs was more severely inhibited by CTLA4Ig and rapamycin compared with Tconvs, whereas both the cell types were similarly affected by MR-1. The other three immunosuppressive drugs, MMF, CsA, and mPD, did not substantially inhibit either nTregs or Tconvs at the doses and treatment schedules used; under these conditions, CsA was the only drug that had a slightly lesser inhibitory effect on nTreg survival than on Tconv survival (Fig. 1C and D). This pattern of cell susceptibility was retained at higher doses of drugs (data not shown). Based on these data, all six immunosuppressive agents tested had a similar (or greater) effect on in vitro-expanded nTregs compared with Tconvs.

Effect of Various Immunosuppressive Agents on the Therapeutic Efficacy of nTregs

The functional activity of T lymphocytes is not always closely related to their proliferative status. Moreover, it is difficult to discern indirect and long-term effects of immunosuppressive agents on nTregs in our GVHR model. Accordingly, we assessed the impact of various immunosuppressive agents on the therapeutic effectiveness of nTregs using a more informative model (Fig. 2A). Our previous studies have shown that this model, which combines transient lymphodepletion and cell therapy using ex vivo-expanded nTregs, yields a short-term prolongation of major histocompatibility complex-mismatched skin allograft survival (Fig. 2B, manuscript in preparation). We used the same doses of individual immunosuppressive agents as in the GVHR model but modified the treatment schedule as indicated in Figure 2(C). Importantly, we administered immunosuppressive agents to recipient mice for only a short time; this is desirable for clinical application regimens while allowing us to clearly visualize the effect of these agents on nTreg cell therapy. Without nTreg cell therapy, short-term treatment with individual immunosuppressive agents did not significantly extend graft survival in lympho-depleted mice (Fig. 2D). When combined with nTreg cell therapy, MR-1 treatment significantly extended graft survival (P=0.0122), increasing the median survival time (MST) from 21 days (nTreg cell therapy alone) to 60.5 days; 2 of 10 mice even retained intact grafted skin for more than 100 days after transplantation (Fig. 2D). Rapamycin marginally prolonged graft survival (MST=30 days), whereas CTLA4Ig and mPD provided no additional benefit. By contrast, MMF and CsA adversely affected the therapeutic effectiveness of nTregs, reducing the MST of allografts in recipient mice from 21 days (untreated controls) to 16 and 14 days, respectively. Although MR-1 and rapamycin individually gave a beneficial effect on Treg cell therapy, no further improvement in graft survival was achieved by the combined use of these two immunosuppressants (Fig. 3A).

FIGURE 3.
FIGURE 3.:
Effect of combined use of MR-1 and rapamycin or low doses of individual immunosuppressants on skin allograft survival. Transiently lympho-depleted C57BL/6 mice were grafted with allogeneic skin and transferred with natural regulatory T lymphocytes (nTregs) as described in Figure 2. The recipient mice were administered with MR-1 together with rapamycin (A) or low doses of individual immunosuppressive agents (B). The doses used in this study are rapamycin, 0.3 mg/kg per day; MMF, 20 mg/kg per day; CsA, 5 mg/kg per day; and mPD, 5 mg/kg per day.

All the above experiments were performed using the full therapeutic doses of individual immunosuppressants, which had been commonly used as a single regimen in murine transplantation studies (20–24). To know whether low doses of these drugs that can be applicable to combination therapy also produce similar effect, we performed the same graft experiments but using a low dose of drugs. Different from their high dose effect, MMF and CsA did not show any antagonistic effect on Treg cell therapy; the former exhibited a slightly beneficial effect. Nevertheless, the concomitant use of all these drugs at low doses in Treg cell therapy failed to induce long-term graft survival (Fig. 3B). These data clearly demonstrate that MR-1 treatment and nTreg cell therapy act synergistically to prevent allograft rejection. They further indicate that MR-1 (and probably rapamycin) is unlikely to severely compromise the regulatory activity of adoptively transferred nTregs.

Cellular Mechanism Underlying the Synergistic Effect of nTreg Cell Therapy and Anti-CD40L Ab (MR-1) Treatment

To examine the effects of MR-1 on the long-term survival and proliferation of transferred nTregs at the graft site and in peripheral lymphoid tissues, we adoptively transferred in vitro-expanded nTregs originated from Thy1.1 congenic mice into transiently lympho-depleted recipient mice, which were then grafted with allogeneic skin and treated with MR-1. After 3 weeks, grafted skin, spleen, and LNs were evaluated for the presence of adoptively transferred nTregs and host CD4+ T cells. In agreement with results obtained with the GVHR model, fewer transferred nTregs were recovered from both grafted skin and draining LNs of MR-1-treated mice than from untreated recipient mice (Fig. 4A and B; P<0.05). Both fractions of Foxp3+ and Foxp3 CD4+ T cells of recipient origin were also recovered at lower numbers at these sites on treatment with MR-1. However, the numbers of adoptively transferred nTregs and host Foxp3CD4+ T cells did not decrease in nondraining LNs or spleens on MR-1 treatment, at this time point (Fig. 4B). Interestingly, in contrast to its significant decrease in LNs and spleen, the percentage of recipient Foxp3+CD4+ T cells was slightly increased in grafted skin of MR-1-treated mice (Fig. 4B). Thus, these data suggest that MR-1 inhibits the antigen-specific proliferation of adoptively transferred nTregs and host CD4+ T cells and may specifically induce the accumulation of host Foxp3+CD4+ Tregs at the graft site.

FIGURE 4.
FIGURE 4.:
Reduction in the number of adoptively transferred natural regulatory T lymphocytes (nTregs) and host CD4+ T cells at graft sites and in draining lymph nodes (LNs) of recipient mice treated with anti-CD40L Abs. Transiently lymphodepleted C57BL/6 mice received allogeneic (BDF1) skin grafts. On days 0 and 3 after skin grafting, recipient mice were adoptively transferred twice with nTregs originated from congenic (Thy1.1) C57BL/6 mice and expanded in vitro by stimulation with donor bone marrow-derived dendritic cells (BM-DCs). One group of recipient mice was additionally treated with MR-1, as indicated in Figure 2(C). Three weeks after transplantation, recipient mice were killed, and the grafted skin, spleens, and LNs were sampled. (A) Cryosections of grafted skin were stained for CD4 (blue), FoxP3 (red), and CD90.1 (green), as described in Materials and Methods, and examined by confocal microscopy. Larger numbers of host CD4+ T cells (blue) and adaptively transferred nTregs (blue and green costained, arrow) are evident in the control sample (upper) compared with the MR-1-treated sample (lower). A representative staining profile from three experiments is shown (left: ×200; right: ×600). (B) The presence of adoptively transferred nTregs and host CD4+ T cells was assessed by flow cytometric analysis of graft-infiltrating, LNs, and spleen cells after staining with antibodies to CD4, CD90.1, and Foxp3. Each symbol represents an individual mouse; small horizontal lines indicate the mean. Data are combined from three independent experiments with a total of six (for graft-infiltrating cells) or nine mice (for spleen/LN cells). Significant P values are indicated above the respective data symbols (*P<0.05 and **P<0.005).

Next, we examined the reactivity of T cells isolated from recipient mice 4 weeks after allogeneic skin grafting. CD3+ T cells recovered from recipient nTreg/MR-1-treated mice that retained intact grafted skin exhibited a decrease in proliferation and did not secrete detectable levels of the cytokines IFN-γ, IL-4, IL-17, IL-10, or TGF-β in response to donor alloantigens (Fig. 5). Although removal of the CD25+ cell fraction from CD3+ T cells produced some increase in T-cell proliferation, it failed to induce effector cytokine secretion or completely restore T-cell proliferation. By contrast, T cells from recipient mice that had rejected their skin graft despite receiving the combination therapy exhibited a full proliferative and IFN-γ secretory response to donor antigens after removal of CD3+CD25+ T cells (Fig. 5). In an in vitro mixed leukocyte reaction suppression assay, adoptively transferred nTregs recovered from nTreg/MR-1-treated recipient mice exhibited strong inhibitory activity that higher than 50% inhibition of responder T cell (Tresp) proliferation was detected even at a nTreg:Tresp ratio as low as 1:128. In contrast, host CD3+CD25+ T-cell population exhibited suppressive activity approximately 20-fold weaker than adoptively transferred Tregs, but a similar level of suppression was observed in CD3+CD25+ T cells from unmanipulated normal mice. These data indicate that both active suppression by Tregs, especially contributed by adoptively transferred nTregs, and hyporesponsiveness of effector T cells are involved in the efficacy of combined nTreg/MR-1 therapy in controlling the immune response against allograft. Interestingly, the hyporesponsiveness of the T-cell compartment and the active suppression by regulatory T cells observed in recipient mice on treatment with nTregs and MR-1 seem to be antigen nonspecific or linked suppression; this is because a similar, although somewhat less robust, pattern of reactivity was observed in response to the third-party alloantigen, H-2b/k. Moreover, this pattern of T-cell reactivity was also observed in recipient mice grafted with syngeneic skin under the cover of the same combination therapy (Fig. 5).

FIGURE 5.
FIGURE 5.:
Involvement of active suppression by natural regulatory T lymphocytes (nTregs) and hyporesponsiveness of effector T cell in the long-term skin allograft survival induced by combined therapy with nTregs and MR-1. Transiently lymphodepleted C57BL/6 mice were grafted with allogeneic BDF1 (“Tol” or “Rej” group) or syngeneic B6 (“Sham” group) skin and administered combined nTreg/MR-1 treatment as described in Figure 2. Four weeks later, spleens and lymph nodes (LNs) were sampled from recipient mice with intact or rejected skin grafts. Spleens and LNs collected at the same time from unmanipulated B6 mice served as a control group (designated as “Nor”). CD3+ T lymphocytes were isolated from the pooled spleen and LN cells using magnetic beads and further divided into CD25+ and CD25− T cells by magnetically activated cell sorter (MACS) or FACSorting. Adoptively transferred Tregs were separated from host CD3+CD25+ T cells by FACSorting of congenic marker (Thy1.1)-positive cells. Proliferation and cytokine production of these isolated CD3+ T cells and CD3+CD25− T cell were measured by 3H-thymindine incorporation assays and enzyme-linked immunosorbent assays (ELISAs), respectively, after stimulation with T-depleted splenocytes derived from donor (BDF1) or third-party (B6C3F1) mice. The suppressive activity of isolated host CD3+CD25+ T cells or adoptively transferred nTregs was measured in a mixed lymphocyte reaction (MLR) suppression assay using CD3+CD25− T cells isolated from unmanipulated B6 mice as responders and T-depleted splenocytes from BDF1 or B6C3F1 mice as stimulators. Data are combined from four independent experiments with a total of three (for Rej group), ten (for Tol group), or seven mice (each for Sham and Nor group).

DISCUSSION

Harnessing the potential of Tregs for clinical transplantation applications requires a thorough understanding of the effects of immunosuppressive agents on Treg cell therapy. Our experiments investigating this question highlight several important points. First, blocking CD40-CD40L interactions (with MR-1) and treatment with rapamycin could be successfully combined with Treg cell therapy to prevent allograft rejection, whereas the concomitant use of high dose of MMF and CsA should be avoided when considering Treg cell therapy using in vitro-expanded nTregs. Second, the effect of immunosuppressive agents on the in vivo proliferation of adoptively transferred nTregs alone is not predictive of the therapeutic efficacy of their combined use in transplantation. Third, a short-term course of immunosuppressive agents, which is clearly advisable given the adverse effects of chronic immunosuppressant use, might be sufficient to promote a synergistic effect in combined therapy with nTregs. Finally, the active regulation established by combined therapy with nTregs and anti-CD40L Ab might not be sufficiently robust to stably maintain tolerance to the allograft.

Recent studies have shown that the preventative effect of costimulation blockers, including anti-CD40L Abs and CTLA4Ig (25), MMF, and rapamycin (21, 22) on allograft rejection is partially dependent on the suppressive activity of Tregs. These observations indicate that these immunosuppressive agents are not harmful to Tregs and may have beneficial effects. One study that examined the effects of immunosuppressants on in vivo tolerance induced by intratracheal delivery of alloantigens also revealed that rapamycin and MMF did not inhibit the induction of tolerance (26). By contrast, several studies have shown that CsA antagonizes the induction of tolerance (21, 26, 27). The fact that the survival and suppressive activity of Tregs depend on an exogenous supply of IL-2 helps to explain the inhibitory effect of CsA, which inhibits IL-2 production from T cells by inhibition of the calcineurin pathway (28–30). Although the results of many previous studies provide indirect evidence for the effects of immunosuppressive drugs on Tregs, one study performed using a murine GVHR model addressed this point directly (24). In this study, the effect of immunosuppressants on tolerance established by the administration of freshly isolated Tregs was examined. The results demonstrated that rapamycin and MMF did not significantly inhibit Treg expansion or suppressive function, whereas CsA diminished the suppressive function of Tregs. Although these data collectively provide some insight into the effects of individual immunosuppressive drugs on the function of preexisting Tregs or on the generation of inducible Tregs in vivo, they may not necessarily reflect the impact of immunosuppressants on the expansion and function of ex vivo-expanded nTregs after adoptive transfer into the recipient. Our in vivo experiments using the GVHR model clearly showed that all six immunosuppressive agents tested, including rapamycin and MR-1, inhibited the expansion of adoptively transferred nTregs as efficiently as that of Tconvs. Notably, our results with rapamycin are inconsistent with those reported by other investigators who showed that rapamycin inhibited the proliferation of T effector cells while allowing the proliferation of nTregs (31). A reduction in the activity of the mTOR pathway (32) and the constitutive expression of Pim 2 induced by Foxp3 (33) have been proposed to be involved in mechanism responsible for the resistance of Tregs to rapamycin. Additional studies will be necessary to determine whether rapamycin susceptibility differs between freshly isolated and in vitro-expanded nTregs, including studies on the signaling characteristics of expanded nTregs.

Although the anti-CD40L mAb, MR-1, and rapamycin severely compromised the expansion of adoptively transferred nTregs at the doses used, the synergy between these two immunosuppressants and nTreg cell therapy on graft survival was detected. Thus, these data indicate that differences in the suppression of nTreg and Tconv division by immunosuppressants alone cannot predict the outcome of combination therapy. Our results also support the previous reports that these two immunosuppressants do not compromise the suppressive function of adoptively transferred, residual Tregs (34, 35). By contrast, because coadministration of MMF or CsA reduced the prolongation of graft survival induced by nTreg cell therapy in our study, it is probable that MMF and CsA incapacitate adoptively transferred nTregs by blocking cell division, either alone or together with suppression of nTreg function. However, it should be of note that the combined use of these drugs at low doses does not greatly affect the therapeutic efficacy of Treg cell therapy.

The precise mechanisms by which nTregs or anti-CD40L Abs exert their immunosuppressive actions are poorly understood. Tregs have been shown to act directly on effector T cells to suppress their function and to act indirectly by modulating antigen-presenting cells (36, 37). We also observed that in vitro-expanded nTregs retain this antigen-presenting cell modulating activity (unpublished observation). Although blockade of CD40-CD40L interaction with anti-CD40L treatment has also been reported to inhibit DC maturation and activation (38), we were unable to detect this activity (unpublished observation). Instead, Fc-dependent depletion of activated T cells (39) or anergy of donor-reactive T cells (40), which has been proposed as an immunosuppressive mechanism of anti-CD40L treatment, is likely to be related to the T-cell alloantigen hyporesponsiveness observed in our recipient mice after combined treatment with MR-1 and nTregs. Especially, our observation that the number of adoptively transferred nTregs and host CD4+ T cells reduced at graft site and in draining LN but not in spleen and nondraining LNs of recipient mice strongly favor the former mechanism. Active suppression by adoptively transferred nTregs was revealed as an important component of graft survival prolongation produced by combined nTreg/MR-1 treatment. Although both nTregs and anti-CD40L Abs can induce the de novo generation of inducible Treg (iTreg) cells (41, 42), our study suggests that iTregs, if generated, may not have a dominant role in regulation of alloimmune T cell responses or they could be included in CD25 T cell population in our experimental model.

Our results recommend the concomitant use of anti-CD40L Ab to potentiate the efficacy of Treg cell therapy using in vitro-expanded nTreg cells. However, anti-CD40L Abs, such as MR-1, cause a serious side effect—disseminated thromboembolism—that prevent them from being used for clinical transplantation (43). Thus, studies designed to determine whether alternative approaches for blocking CD40-CD40L interactions, such as the use of anti-CD40 Abs, can also synergize with Treg cell therapy, could prove invaluable.

In summary, our study shows that blocking CD40-CD40L interactions with the anti-CD40L mAb MR-1 or, less efficiently, the administration of rapamycin could be successfully combined with cell therapy using ex vivo-expanded nTregs to prevent allograft rejection. Importantly, the combined use of MMF or CsA should be carefully considered in this type of Treg cell therapy because they can antagonize the therapeutic effectiveness of Treg cell therapy in their high-dose administration. We believe that the approaches established in this study could be successfully applied to screen existing and newly developed immunosuppressive agents for efficacy in combined therapy with Tregs.

ACKNOWLEDGMENTS

The authors thank Hae-Jin Jung for her cell-sorting expertise.

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Keywords:

Regulatory T lymphocyte; Immunosuppressant; Cell therapy; Skin transplantation; Anti-CD40L antibody

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