International Immunopharmacology 11 (2011) 618–629
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International Immunopharmacology
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Adoptive transfer of CD4+CD25+ regulatory cells combined with low-dose sirolimusand anti-thymocyte globulin delays acute rejection of renal allografts inCynomolgus monkeys☆
Anlun Ma a, Shijie Qi a, Lijun Song a, Yanxin Hu a, Hao Dun a, Eric Massicotte b, Martine Dupuis b,Pierre Daloze c, Huifang Chen a,⁎a Laboratory of Experimental Surgery, Research Centre, Center hospitalier de l'Université de Montréal (CHUM)-Hôpital Notre-Dame, Department of Surgery, University of Montreal,Montréal, Québec, Canadab Institut de recherches cliniques de Montréal, Montréal, Québec, Canadac Department of Surgery, Hôpital Notre-Dame, University of Montréal, Montreal, Canada
☆ This work was supported in part by Fondation de laQuébec, Canada.⁎ Corresponding author. Laboratory of Experiment
Although donor alloantigen specific Treg cells play an important role in transplant tolerance, therapeuticapplications are limited by their low frequency. In this study, isolated Tregs from Cynomolgus monkeyswere efficiently expanded by a co-culture system, andmaintained suppressive function on the proliferation ofCD4+ effector cells in vitro. Adoptive transfer of expanded donor alloantigen specific Tregs without anyimmunosuppressants could prolong survival of MHC-mismatched allografts in Cynomolgus monkeys.To reach the feasibility of clinical transplantation, our objectives focused on whether exposure of monkeyTregs to immunosuppressants could preserve suppressive function in vitro and in vivo. The results showedthat low-dose sirolimus selectively expanded Tregs, increased the expression of CD25bright and Foxp3markers, and suppressed TCR- or allo-antigens induced CD4+ T cell proliferation in vitro. In vivo, after pre-treated with anti-thymocyte globulin (ATG) for consecutive 3 days, a 14-day therapy of adoptive infusion ofdonor alloantigen-specific Tregs combined with low-dose sirolimus delayed acute rejection of renal allograftsin Cynomolgus monkeys, showing an MST of 48.5 days as compared with those of untreated and sirolimus-treated monkeys (7 days and 22 days). The frequencies of CD4+CD25bright T cells were significantly elevatedin mesenteric lymph nodes vs. those in inguino lymph nodes and peripheral blood.In summary, expanded donor alloantigen specific Tregs exposed to sirolimus can preserve inhibition in vitroand in vivo. Tregs are more resistant to sirolimus than other T cells. Expanded donor alloantigen specific Tregscombined with sirolimus and ATG prolongs renal allograft survival in monkeys, suggesting that sirolimusmight be one of the best synergists for Tregs therapy.
Immunosuppressive therapy has been required for the reductionof long-term immunosuppressant administration because of itscomplications in transplantation. The ultimate goal in transplantationis the induction of a sustained state of specific tolerance to donoralloantigens with minimization or complete withdrawal of immuno-suppressants. Many studies have indicated that activation of T cell-mediated immune regulation is an important mechanism of periph-
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eral immune tolerance to self-antigens and alloantigens. Severalregulatory T (Treg) cell subsets have been identified to contribute toimmunological tolerance, including naturally arising CD4+CD25+
Forkhead box protein 3 (FoxP3)+ Tregs [1], T-regulatory type 1 (Tr1)cells which produce high amount of interleukin-10 (IL-10) [2], andCD4+TGF-β1+ Th3 cells [3]. The studies in rodents and humans havedemonstrated that CD4+CD25+ Tregs, distinct from CD4+CD25− Tcells, exhibit an activated and memory phenotype, and constitutivelyexpress high levels of IL-2 receptor-chain (CD25) [4], forkhead/wingedhelix transcription factor Foxp3 [5] and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) [6]. In vitro, functional assays indicatethat the suppressor activity of CD4+CD25+ Tregs is mediated by a cellcontact-dependent, cytokine-independent mechanism [7].
Several studies in experimental and clinical transplantation haveimplied that Treg mediated-regulation can control alloreactive CD4+
pathogenic effector cells and result in transplant tolerance [8–11].Thus, adoptive transfer of CD4+CD25+ Tregs represents an interesting
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therapeutic approach in transplantation and autoimmune diseases.Recognizing the low frequency of Tregs and their high potential foradoptive immunotherapy, some protocols of Treg expansion havebeen developed in rodents and humans [12–15].
Despite functional CD4+CD25+ Tregs can be expanded ex vivo, thereare some limitations for expansion in vitro. First, polyclonal activationand expansion of CD4+CD25+ Tregs bear risk of co-expanding effectorT cells in vitro. In humans, CD4+CD25+ T cells contain approximately50% of activated T cells, which ultimately overgrow the Tregs uponprolonged culture [16]. Secondly, recent strategies are to expand thedonor alloantigen specific Tregs in large number in laboratories beforeclinical application.However,withoutprior donor antigenexposure, thefrequency of alloantigen cross-reactive Tregswill be lowor not be foundin patientswhoarewaiting for organ to transplant. In vitro, expansion ofCD4+CD25+ Tregs requires at least 2–3 weeks to achieve enough cellnumber for CD4+CD25+ Treg therapy, but the source of deceased donororgans is usually on an emergency basis. Thirdly, it does not seemfeasible for patients with some diseases, such as in autoimmunediseases, cancer, and particularly in patients with leukemia orlymphoma, whose cells might be in fewer numbers and have lessresponse for expansion of Tregs in vitro [17].
To assess the feasibility in clinical transplantation, our hypothesisis that recipients would first receive a short-term course (2–4 weeks)of immunosuppressants after organ transplantation; at the same time,donor alloantigan-specific Tregs would be expanded in vitro so thatenough Treg cells would be harvested and transferred to patients. Toaddress this hypothesis, the key question is which immunosuppres-sant is the best synergist for Treg therapy. Our studies in rodents haveshown that isolated CD4+CD25+ regulatory T cells from mice wereefficiently expanded by a co-culture system in vitro [18]. In vivo, anadoptive infusion of donor-alloantigen-specific CD4+CD25+ Tregscombined with low-dose sirolimus, but not tacrolimus or Cyclospor-ine A, showed prolonging survival of heart allografts (N100 days) inBalb/c to C57 mice.
Served as surrogate tools for human allograft transplantation,nonhuman primates often provide the most accepted models inpreclinical transplantation. However, studies of CD4+CD25+ Tregs innonhuman primate lag behind those in humans and rodents [19,20].Understanding the safety and effectiveness of Tregs awaits preclinicalstudies in large outbred species animals. Thus, developing methods toexpand nonhuman primate Tregs have potential for adoptiveimmunotherapy in transplantation.
In this study, a co-culture system was used to expand donoralloantigen specific Cynomolgus monkey CD4+CD25+ Tregs in vitro.Three representative immunosuppressants, Cyclosporine A and tacro-limus of calcineurin inhibitor (CNI) family and sirolimus of mTORinhibitors were employed in vitro study. Our aims focus on whetherexposure of monkey CD4+CD25+ Tregs to different immunosuppres-sants could preserve suppressive function in vitro, and whether acombination therapy of donor alloantigen specific CD4+CD25+ Tregswith immunosuppressants could prolong the survival of renal allograftsin Cynomolgus monkeys.
2. Materials and methods
2.1. Animals
Outbred healthy, male Cynomolgus monkeys (Macaca fascicularis,aged 3–4 years old, weighing 3–3.5 kg) were purchased fromLaboratory Animal Center, Academy of Military Medical Sciences,China. Monkeys were randomly distributed over four groups:untreated, sirolimus-treated, Tregs-treated and Tregs plus sirolimus-treated groups. All procedures of the animals were in accordance withthe Principles for Animal Experiments Using Nonhuman Primates(Primate Society of China) and were approved by the LaboratoryAnimal Center, Academy of Military Medical Sciences. The monkeys
were bred and housed in individual cages and allowed free access towater, fruits and monkey chow.
2.2. ABO typing and MLR tests
Donor–recipient pairs were selected based on ABO blood typingand mixed lymphocyte reaction (MLR, stimulation index ≥3). Eachanimal was tested against all potential donors, so that donor–recipient pairs with high incompatibility for transplantation couldbe identified.
2.3. Reagents and Abs
Anti-human monoclonal antibodies (mAbs) demonstrated tocross-react with Cynomolgus monkeys were selected for this study.All mAbs were purchased from BD Biosciences-Pharmingen exceptFoxp3 mAbs (eBioscience). Matching isotype control mAbs wereincluded in all experiments. Nonhuman primate CD4+CD25+ TregsMACS isolation kit, Tregs detection kit and rhesus monkey CD1c(BDCA-1)+ dendritic cell isolation kit (cross-react with antigens ofCynomolgus monkeys) were products of Miltebyi Biotec. Humanrecombinant granulocyte macrophage-colony-stimulating factor(GM-CSF) was the product of Biosource. CFDA SE cell tracer kit, rIL-2, RPMI-1640, fetal calf serum (FCS), L-glutamine, penicillin, strepto-mycin were obtained from Invitrogen. Monkey IL-2, IL-4, IL-10 andIFN-gamma ELISA kits were obtained from Cell Sciences.
Sirolimus and Cyclosporine A were obtained from Sigma-Aldrich.Tacrolimus was product of Astellas. Rabbit anti-thymocyte globulin(ATG) was purchased from Genzyme.
2.4. Purification of CD4+CD25+ Treg cells
Freshly isolated CD4+CD25+ or CD4+CD25− T cells were purifiedfrom the spleens of Cynomolgus monkeys in a two-step MACSprocedure. Non-CD4+ T cells were first indirectly and magneticallylabeled with a cocktail of biotin-conjugated antibodies and anti-BiotinMicroBeads. The cell suspension was loaded onto MACS columns; themagnetically labeled non-CD4+ T cells were retained in the columns,while the unlabeled CD4+ T cells run through. The purity of thenegatively selected CD4+ population was greater than 96% on FACSanalysis. Afterwards, the CD25+ PE-labeled cells in the enriched CD4+
T cell fraction were magnetically labeled with anti-PE MicroBeads.CD25+ cells were positively selected from the enriched CD4+ T cellfraction and performed the second separated to achieve highestpurities. The positively selected CD4+CD25+ cells were greater than90% on FACS analysis.
2.5. Generation of bone marrow-DCs (BM-DCs) and spleen-DCs (S-DCs)
Monkey fresh BM cells were collected by gently flushing the bonemarrow cavities of humerus with ice-cold 0.9% NS and refluxing theexpelled cells plug through 18-gauge needle to form a single-cellsuspension. The BM cells were suspended in complete RPMI1640 medium containing 1% autologous serum, 25 mM HEPES,2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin,and cultured in the presence of 20 ng/ml GM-CSF at 37 °C in 5% CO2.The non-adherent cells were collected on day 7 of culture. After 3additional days of culture, non-adherent cells were collected.
Monkey S-DCs were isolated from splenic monocular cell suspen-sions. Single cells were cultured for 2 h at 37 °C in plastic cultureplates, followed by removal of non-adherent cells. Adherent cellswere collected and washed with PBS containing 2% FBS three times.Monkey DCs were purified with nonhuman primate CD1c (BDCA-1)+
DC isolation kit.
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2.6. Cell co-culture with or without immunosuppressants
Freshly isolated CD4+CD25+ T cells were cultured with 10%syngeneic irradiated (30 Gy) BM-DCs or S-DCs pulsed 10% irradiated-(30 Gy) donor splenocytes in RPMI 1640 medium that was supple-mented with 0.1 μg/ml anti-CD3 mAb, 10 U/ml rIL-2, 100 IU/mlpenicillin and streptomycin, and 1% autologous monkey serum,incubated at 37 °C in a humidified atmosphere with 5% CO2. Oneweek after priming, T cells were collected and re-stimulated weeklywith irradiated donor splenocytes. In other culture system, in order toevaluate effects of immunosuppressants on expansion of Tregs,isolated CD4+CD25+ or CD4+CD25− T cells were co-cultured withsirolimus, or tacrolimus, or Cyclosporine A at the final concentrationof 1 nM, 10 nM, 100 nM and 1000 nM, respectively, and used forproliferation assays.
2.7. Cell proliferation assays
Proliferation assays were performed in triplicate in 96-well plates.CD4+CD25+ T cells (104) were co-cultured with syngeneic responderCD4+ T cells (104) stimulated by the TCR signaling of anti-CD3 (FN18,1 μg/ml)/anti-CD28 (CD28.2, 1 μg/ml), or irradiated donor allogeneicor third-party allogeneic CD4+T cells (SI N3, 5×104) in the presence ofirradiated syngeneic T cell-depleted splenocytes (antigen-presentingcells, APCs, 104) and with or without different immunosuppressants.Cell proliferationwasmeasured by 3H-thymidine incorporation (cpm)on day 5.
2.8. Inhibition assays
Expanded CD4+CD25+ T cells were treated with sirolimus, ortacrolimus, or Cyclosporine A at different concentrations for 3 days,then washed and rested for 2 days. On day 5, CD4+CD25+ T cellsderived from control (untreated), sirolimus-, or tacrolimus-,or Cyclosporine A-treated groups were co-cultured with syngeneicCD4+ T cells in the stimulation of irradiated allogeneic donor spleniccells, and examined for suppressive function by 3H-thymidineincorporation on day 10.
2.9. Cell surface and intracellular staining
All Tregs were stained with PE- or FITC-conjugated anti-CD25,CD4, CD62L, CD69, CD95 and CCR7mAbs to determine surface markerprofiles. Intracellular staining was performed on detection ofintracellular expression of Foxp3 and CTLA-4 antigens. Sampleswere first incubated with mAbs against surface markers CD4 andCD25. After subsequent washes, cells were fixed with PBS containing4% formaldehyde for 20 min at room temperature, washed with PBScontaining 0.5% BSA and 2 nM EDTA, permeabilized with PBScontaining 0.5% (w/v) BSA and 0.2% (v/v) saponin, and stained withanti-CTLA-4 and anti-Foxp3 mAbs for 30 min at room temperature.Cells were further washed twice with PBS containing 0.5% BSA and0.2% saponin, resuspended in FACS flow solution, and analyzed byFACS using Cell Quest software.
2.10. CFDA-SE labeling
Expanded Tregs were stained with Vybrant carboxyfluoresceindiacetate, succinimidyl ester (CFDA SE) at a final concentration of5 μM for 6 min at 37 °C. After staining, cells were immediately washedby ice-cold RPMI plus 10% FCS twice, resuspended in PBS, and countedbefore infusion in vivo. To evaluate cell expansion, stained cells werefurther washed twice with PBS containing 0.5% BSA and 0.2% saponin,resuspended in FACS flow solution, and analyzed by FACS. Cellexpansion was indicated by proliferation index in each histogram.
2.11. Splenectomy and renal transplantation
Before transplantation, donor and recipient animals were anes-thetized with 10 mg/kg of intramuscular ketamine HCL and 1 mg/kgof xylazine. Both donors and recipients were splenectomized forexpanding Tregs in vitro. Three weeks later, renal transplantation wasperformed following our previous methods with some modifications[21]. Briefly, donor left kidney with ureter was excised and perfusedby 4 °C UW solution, then transplanted into the recipient's original leftrenal capsule with end-to-side anastomoses of renal artery to aortaand renal vein to vena cava, and with end-to-end anastomosis ofdonor and recipient ureter. Rejection was diagnosed by enlargementof renal allografts and an increase of serum creatinine (≥10 mg/dL)which was finally confirmed by histopathology.
2.12. Adoptive infusion of Tregs and combined therapy withimmunosuppressants
Recipients were injected intravenously with ATG (2 mg/kg/day)for consecutive 3 days, on day -2, -1 and 0 of transplantation.Expanded CD4+CD25+ T cells (107/day) were injected intravenouslydaily to recipients consistent for 14 days, together with a 14-daytherapy of low-dose sirolimus (0.5 mg/kg/day) by gavage adminis-tration from the day of transplantation.
2.13. Statistical analysis
Statistical analysis was performed using the SPSS v16.0 software(SPSS Inc., Chicago, Illinois, USA).Datawere reportedasMean±StandardError. p value of less than 0.05 was considered to be significant.
3. Results
3.1. The expanded Cynomolgus CD4+CD25+ T cells are more potentsuppressors than fresh isolated CD4+CD25+ T cells
Toverifywhether freshly isolatedCynomolgusmonkeyCD4+CD25+Tcells canexpandeffectively in vitro, CD4+CD25+Tcellswere cultured inaco-culture system, Tregs plus irradiated donor splenocytes and autologusS-DCs or BM-DCs were cultured in the presence of low concentration ofrIL-2 and anti-CD3mAb. One week after priming, T cells were re-stimulated weekly with donor splenocytes plus DCs. The fold expansionof CD4+CD25+ T cells was counted after each round stimulation. Ourresults showed that a 12-fold expansion of cultured CD4+CD25+ T cellswas achieved in 4-week culture. There was no significant difference inthe presence of BM-DCs (Fig. 1A) or S-DCs (Fig. 1B) culture (pN0.05).These results suggested that Cynomolgus CD4+CD25+ T cells could begreatly expanded in vitro by repeated stimulation with donor allogeneicsplenocytes plus autologus BM-DCs or S-DCs in the presence of rIL-2 andanti-CD30 mAb.
To assess whether expanded Cynomolgus CD4+CD25+ T cells retainsuppressive function in vitro, expanded and freshly isolated CD4+CD25+
cells in different ratios to responder CD4+CD25− T cells were used tomeasure their inhibition on the proliferation of syngeneic CD4+CD25−
cells in the response to TCR stimulation. The results showed that 4-weekexpanded CD4+CD25+ T cells were able to suppress the proliferation ofCD4+CD25−Tcells, andweremorepotent suppressors than those in freshCD4+CD25+Tcells, inducinga77% inhibitionat1:4 ratioofCD4+CD25+Tcells to CD4+CD25− cells as compared with 50% inhibition at the sameratio of fresh CD4+CD25+ T cells to CD4+CD25− cells (pb0.05, Fig. 1C).
To the stimulation of specific donor alloantigens (SIN3 with recipientalloantigens), expanded CD4+CD25+ T cells displayed and preservedsuppressive functionon theproliferationof syngeneicCD4+CD25−Tcells.Their inhibition is more efficient than that of fresh CD4+CD25+ T cells.Although freshly purified CD4+CD25+ T cells showed 50% suppression ata ratio of 1:4 of CD4+CD25+ suppressive cell to CD4+CD25− responder
Fig. 1. The expanded Cynomolgus CD4+CD25+ T cells are more potent suppressors than fresh isolated CD4+CD25+ T cells. Freshly isolated CD4+CD25+ T cells were cultured with10% syngeneic irradiated (30 Gy) BM-DCs or S-DCs plus 10% irradiated (30 Gy) donor splenocytes in RPMI-1640 medium that was supplemented with 10 U/ml rIL-2, 0.1 μg/ml anti-CD3, 100 IU/ml penicillin, 100 μg/ml streptomycin, 25 mM HEPES and 1% autologous monkey serum, incubated at 37 °C in a humidified atmosphere with 5% CO2. One week afterpriming, T cells were re-stimulated weekly with irradiated donor splenocytes. The fold expansion of CD4+CD25+ T cells was counted after each round stimulation in the presence ofautologus BM-DCs (A) or S-DCs (B). Freshly isolated or expanded CD4+CD25+ T cells (104) were co-cultured with syngeneic responder CD4+ T cells with different ratios (1:8, 1:4,1:2, 1:1) stimulated by anti-CD3 (1 μg/ml)/anti-CD28 (1 μg/ml) (C), or irradiated specific donor allogeneic CD4+ T cells (104) that SI wasN3 with recipient alloantigens, (D) in thepresence of S-DC, and or stimulated by third-party allogeneic CD4+ T cells (SIN3, 104) (E) in the presence of S-DC and low concentration of rIL-2. Proliferation was measured by 3 H-thymidine incorporation (cpm) on day 5. Results were representative of 3 independent experiments.
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T cells, expanded Treg cells displayed similar inhibition at a ratio of 1:8(Fig. 1D). To response to the stimulation of non-specific third-partyallogeneic CD4+ T cells, their inhibitive function was less efficient thanthose in the response to the specific donor allogeneic CD4+ T cells,showing 50% suppression at 1:4 ratio compared to similar inhibition tospecific donor alloantigens at 1:8 ratio (Fig. 1E).
Taken together, expanded CD4+CD25+ T cells were able to retainTreg suppressive function, and more suppressed the proliferation ofautologous CD4+ T cells than fresh isolated CD4+CD25+ T cells underthe stimulation of either TCR or alloantigens in a cell dose-dependentfashion in vitro. SpleenDCs, like BM-DCs,were effective to differentiateCD4+CD25+ Tregs in vitro. Thus, replacement of BM-DCs with spleenDCs was thought to be the better choice to minimum trauma andreduced surgical complications in monkey transplant model.
3.2. Phenotypic profiles of expanded Cynomolgus monkey CD4+CD25+
Tregs
To evaluate phenotypic profiles of Cynomolgusmonkey CD4+CD25+
T cells, expanded Treg cells were analyzed by flow cytometry after3-consecutive-week induction. The data showed that expandedTregs expressed higher levels of Foxp3, CTLA-4, CD62L and CD95markers. One representative experiment was presented in Fig. 2.The results showed that expanded cells expressed double positivefor CD4+CD25+ markers (85%±3%), and CD95 marker (45%±7%),indicating a memory phenotype; Tregs positively expressed CD69(25.1%±7%), demonstrating an activated memory phenotype. Theexpression of CD62L, a secondary lymphoid organ homing receptor,was retained on the 45.7%±8.3% of expanded CD4+CD25+ Tregs;
Fig. 2. Phenotypes of expand CD4+CD25+ T cells by a co-culture system. Freshly isolated CD4+CD25+ T cells were expanded by co-culture system for 3-consecutive-week, and stainedwith PE- or FITC-conjugated anti-CD25, CD4, CD62L, CD69, CD95 and CCR7 mAbs to determine surface marker profiles. Intracellular staining was performed on detection of intracellularexpression of Foxp3 and CTLA-4. Results were presented as mean±SD cpm values of 3 independent experiments. One representative experiment was presented at Fig. 2.
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CCR7, another secondary lymphoid organ homing chemokinereceptor, was detected on 11%±1.9% of expanded Tregs. In addition,intracellular CTLA-4 protein expressed on Tregs of 36%±7%.
Foxp3has been considered to beamajor phenotypicmarkerof Tregs.Therefore, its expression was detected in fresh isolated CD4+CD25+
and expanded Treg cells. In our study, there were 39–78% of expandedCD4+CD25+ cells expressed Foxp3 marker by co-culture system. Thesedata indicated that expanded Cynomolgus CD4+CD25+ cells differen-tially expressed and retained regulatory, memory, inhibitory markersand homing chemokine receptors after 3-consecutive-week inductionin vitro.
3.3. Suppressive function of expanded Tregs treated withimmunosuppressants in vitro
Todeterminewhether addition of CD4+CD25+Treg cells can interferesuppressive effect on the CD4+CD25− effector T-cells, CD4+CD25−
effector T cells were co-cultured with expanded CD4+CD25+ Tregs (1:8)with or without immunosuppressants at different concentration in thepresence of allo-antigens stimulation. The study found that neitherimmunosuppressant can abrogate the suppressive effect by the additionof CD4+CD25+ Tregs. In the culture with sirolimus of 1 nM, 10 nMconcentration, addition of CD4+CD25+ T cells led to an enhancedinhibition on CD4+CD25− effector T-cell proliferation, as comparedwith CD4+CD25− T cell control groups (pb0.05, Fig. 3A). However, theseenhanced suppressive activitieswere not found in the groupof tacrolimusor Cyclosporine A of the same concentration.
To evaluate whether Tregs could preserve their suppressivefunctions when exposed to immunosuppressants, treated CD4+CD25+
Tregs were added to syngeneic CD4+ T cell culture, stimulated byirradiated allogeneic splenocytes for 5 days, and examined for suppres-
sive capacity by 3H-thymidine incorporation. As shown in Fig. 3B,sirolimus-exposed Treg cells maintained suppressive ability of 64.89%(1 nM), 58.22% (10 nM), and 43% (100 nM) in comparison to untreatedcontrols of 42.22%. In contrast, exposure of Treg cell to tacrolimusmarkedly reduced suppressor potential to 36% (1 nM), 22.22% (10 nM),and 11.11% (100 nM). Similarly, pre-culture with cyclosporine reducedsuppressive capacity to 55.56% (1 nM), 33.33% (10 nM) and 15.56%(100 nM). (pb0.05). As comparedwith these immunosuppressants, ourdata showed that Treg cells are less sensitive to the pre-treatment ofsirolimus than those of tacrolimus or Cyclosporine A. Thus, itdemonstrated that biologically active concentration of sirolimus(10 nM) preserves suppressor function of CD4+CD25+ T cells in awide range of concentrations.
In order to verify the effects of immunosuppressants on the cellgrowth of Tregs, fold expansion of CD4+CD25+ T cells was countedafter each round stimulation in the presence of anti-CD3/CD28antibodies with or without sirolimus, or tacrolimus, and or Cyclo-sporine A of 10 nM. Our results showed that sirolimus-treated Tregshad a delayed cell proliferation kinetic as compared with medium-treated Tregs. The number of Tregs was recovered at the end of thefourth week of culture (Fig. 3C). However, Tacrolimus or CyclosporineA did not permit the expansion of CD4+CD25+ T cells in vitro, therewas no obvious increase of cell number within 3 consecutive weeks.
3.4. Regulatory phenotypes of expanded Tregs treated withimmunosuppressants in vitro
To define the effects of immunosuppressants on Tregs, phenotypesof expanded CD4+CD25+ T cells were determined after exposure toimmunosuppressants for 3 consecutive weeks. The data showed thatexpanded Treg cells retained higher levels of CD25bright, Foxp3, CD69
Fig. 3. Sirolimus allows theactivation and regulatory function of CD4+CD25+T cells in vitro.In co-culture assay, expandedCD4+CD25+T cellswere culturedwith CD4+CD25− T cells ina suppressor-to-effector ratio of 1:8, and stimulated by irradiated (30 Gy) donor splenic cell.Sirolimus, or tacrolimus, and or CyclosporineAwere added at thebeginning of primaryMLRat indicated concentrations(xaxis). Proliferationon theday5asmeasuredby 3 H-thymidineincorporationwas shown on the y axis (A). Expanded CD4+CD25+ T cellswere pre-treatedwith sirolimus, or tacrolimus, and or Cyclosporine A at various final concentrations (1 nM,10 nM, 100 nM and 1000 nM) for 3 days, then washed and rested for 2 days. On the day 5,CD4+CD25+ T cells co-cultured with syngeneic responder CD4+ T cells (1:8) in thestimulation of irradiated donor allogenetic splenic cells, and examined for sup-pressive function by 3 H-thymAidine incorporation on the day 10. Results were presentedas mean±SD cpm values of triplicate wells, and were representative of 3 independentexperiments. * pb0.05 as compared to responder cells alone (B). CD4+CD25+ T cells wereweekly stimulated by irradiated (30 Gy) donor splenic cells with or without sirolimus, ortacrolimus, orCyclosporineAof 10 nMinthepresenceof rIL-2 (10 IU/ml) andanti-CD3mAb(0.1 μg/ml) for 4 weeks, fold expansion of CD4+CD25+T cellswas counted after each roundstimulation. Results were representative of 3 independent experiments (C).
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and CD62L markers when exposed to sirolimus, compared with thoseexposed to tacrolimus, or Cyclosporine A (pb0.05, Fig. 4A). Onerepresentative experiment was shown at Fig. 4B, which revealed that
the percentages of CD25bright cells were 45%, 0.6% and 1.1%, respectivelyin the presence of sirolimus, tacrolimus, or Cyclosporine A of 10 nMconcentration, compared with those of 29% in medium control group.
Foxp3has been considered to be amajorphenotypicmarker of Tregs.Therefore, its expression was detected in expanded CD4+CD25+Tregs,as well as Tregs plus immunosuppressants after 3-week culture. WhenTregs was co-cultured with sirolimus of 10 nM, the expression ofFoxp3 was significantly higher than that in tacrolimus- or CyclosporineA-treated Tregs at the same concentration (pb0.05). It showed onerepresentative experiment of Foxp3 expression in Fig. 4C. Sirolimus-treated Tregs contained 73.34% of Foxp3+ cells compared with those ofno-treated Tregs (60.86%). In contrast, when exposed to tacrolimus orCyclosporine A, the expression of Foxp3 was significantly decreased as73.34% vs. 3.06% and 8.79%, respectively. Our results suggested thatsirolimusmay be one of the best synergists for Treg immune-therapy intransplantation.
3.5. The trafficking of CD4+CD25+ Treg cells in vivo
To confirm whether inoculated CD4+CD25+ Treg cells can surviveand expand in recipient monkeys, intracellular dye CFDA SEwas used toobserve the trafficking of the in vitro-expanded CD4+CD25+ T cells invivo. CFDA SE-labelled autologus CD4+CD25+ T cells were injectedintravenously to recipientswhoreceivedorun-received the treatmentofsirolimus. The trafficking of labeled CD4+CD25+ T cells frommesentericlymph nodes (mLN) and inguino lymph nodes (iLN) was analyzedby FACS on day 7 after cell infusion. As illustrated in Fig. 5, although theCD4+CD25+T cellswere hyporesponsive in vitro, they could be detectedin mLNs and iLNs in the recipients which received Tregs alone. Theproliferation of Tregs in mLNs was higher than those in distant iLNs,showing proliferation index 53.41%±4.3% vs. 25.20%±2.1%, respec-tively (pb0.05). However, there was no difference was found in therecipients received Tregs combined with sirolimus, showing prolifera-tion index 35.15%±5.9% vs. 32.60%±6.2% in mLNs and iLNs, respec-tively. (pN0.05). These studies suggested that after adoptive transfer,CD4+CD25+ T cells could migrate and home preferentially to LNs, andlow-dose sirolimus combined with Treg cell therapy preserve theproliferate function of Treg cells in vivo.
3.6. Combined therapy of expanded Tregs with immunosuppressantsprolonged the allograft survival in vivo
To investigatewhether Treg cells combinedwith low-dose sirolimuscould preserve their inhibitive function in vivo, renal transplantationwasperformed indonor–recipient pair Cynomolgusmonkeys. ExpandedTregs (107/day) were injected intravenously to recipient monkeysconsistently for 14 days, together with a 14-day therapy of low-dosesirolimus (0.5 mg/kg/day) from the day of transplantation, while ATG(20 mg/kg/day) was consistently given for 3 days before transplanta-tion. As shown in Fig. 6A, all recipients in untreated group rejected theirrenal allografts within 7 days of median survival time (MST). Threemonkeys treatedwith sirolimus, but not inoculatedwith Treg cells, diedof acute rejection between postoperative day (POD) 20 and 32 (MST22 days). When Treg cells were transferred to 3 monkeys in group 3,whichdidnot received sirolimus, delay acute rejectionof renal allograftswas observed as comparedwith those in untreated group (MST, 32 daysvs. 7 days) and in sirolimus group (MST, 32 days vs. 22 days). One of 3monkeys in group 3 survived up to 106 days, died of chronic allograftnephropathy. Infusion of Tregs with low-dose sirolimus represented anallograft prolongation ofMST of 48.5 days comparedwithMST of 7 daysin group 1, 22 days in group 2 and 32 days in group 3.
In order to monitor the function of renal allografts, an appropriatemonitoring of serum creatinine (sCr) was performed during thesurvival period of themonkeys. Level of sCr in un-treated (Fig. 6B) andlow-dose sirolimus-treated (Fig. 6C) groups were increased on 7 daysafter transplantation (N2 mg/dl), and reached high level of N10 mg/dl
Fig. 4. Regulatory phenotypes of expanded Tregs treated with immunosuppressants in vitro. The phenotypes of expanded CD4+CD25+ T cells were determined by FACS assay afterexposure to immunosuppressants for 3 consecutive weeks. All Tregs were stained with PE- or FITC-conjugated anti-CD25, CD62L and CD69 mAbs to determine surface markerprofiles. Intracellular staining was performed on detection of intracellular expression of Foxp3 antigen. Samples were analyzed by FACS using Cell Quest software. The FACS datashowed expression of CD25bright, CD62L, CD69 and Foxp3 antigens on Tregs when exposed to sirolimus, or tacrolimus, and or Cyclosporine A (A). One representative experiment (B)showed that the percentage of CD25bright cells in the presence of 10 nM of sirolimus, or tacrolimus, or Cyclosporine A and medium. Another representative FACS result (C) showeddifferent expression of Foxp3 on the expanded Treg cells with or without 10 nM of sirolimus, or tacrolimus, or Cyclosporine A and medium, respectively.
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at end points. Interestingly, in the group of Tregs (Fig. 6D) orcombined with sirolimus (Fig. 6E), sCr levels slightly elevated withinthree weeks after transplantation, and were kept at smooth levels ofb4 mg/dl until end points. One of the recipients was found an abruptincreased level of sCr (8.8 mg/dl) on day 35 postoperatively due toleakage of urine at the region of end-to-end ureteral anastomosis.After second ureterorrhaphy, the level of sCr decreased immediatelyto 0.8 mg/dl. In this study, no unanticipated complications were foundin Treg cell therapy.
3.7. Immunological and pathological profiles in recipients receivedregulatory T cells with immunosuppressants in vivo
To examinewhether immunosuppressants could interferewith Tregfunction in vivo, the percentages of CD4+CD25+ and CD4+CD25bright
cells were evaluated on the peripheral blood mononuclear cells(BMCs) of the recipient monkeys on POD 21. The mean percentage ofCD4+CD25+ T cells was higher in the monkeys received Tregs or Tregsplus sirolimus than those in sirolimus-treated or tacrolimus-treatedmonkeys (Fig. 7A). When considering the frequency of CD4+CD25bright
T cells in the total PBMCs, they were found increase in the groups ofTregs and Tregs plus sirolimus. There were a 17-fold increase ofCD4+CD25bright T cells compared with those in tacrolimus-treatedmonkeys, and 2-fold higher than those in sirolimus-treated monkeys(pb0.05, Fig. 7B). There was no significant change to be found in theproportion of CD4+IL-10+ Tr1 cells in all groups at POD 21 (pN0.05,Fig. 7C). When the levels of CD4+CD25bright and CD4+IL-10+ cells wereevaluated on secondary lymphoid organs, inguino lymph nodes (iLN)andmesenteric lymph nodes (mLN), the frequencies of CD4+CD25bright
T cells were shown to be significantly higher inmLNs than those in iLNs
Fig. 5. The trafficking of the inoculated CD4+CD25+ T cells in vivo. CFDA SE-labelled syngeneic expanded CD4+CD25+ T cells (107) were injected intravenously to recipient monkeyson the day 0 of transplantation. The survival trafficking of CFDA SE-labelled CD4+CD25+ T cells in the mesenteric lymph nodes (mLN) and inguino lymph nodes (iLN) was analyzedby FACS on the day 7 after the treatment of Tregs alone and Tregs plus sirolimus. The histograms correspond to a minimum of 30,000 acquired events in a lymphocyte gate ofCD4+CD25+ T cells. One representative experiment was presented, and proliferation index was indicated in each histogram.
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in all groups (pb0.05). However, no difference of population of CD4+IL-10+ Tr1 cells was found between iLNs and mLNs (pb0.05, Fig. 7D) atPOD 21.
Our histopathological studies indicated that the graft-infiltratinglymphocytes were mainly found in the untreated and sirolimus plusATG-treated allografts at an early stage after transplantation, showingintense interstitial inflammatory cell infiltrated characters, with typicalacute renal allograft rejection in the untreated recipients at post-transplant day 7 (Fig. 8A), and sirolimus plus ATG-treated recipients onPOD 14 (Fig. 8B), with moderate tubular atrophy and severe arteriolarhyalinosis. In Tregs plus ATG and Tregs plus sirolimus and ATG treatedmonkeys, Hematoxylin and Eosin staining indicated that renal allograftswere in almost normal histology on POD 7, and with minimallymphocyte infiltration on POD 14. Two representative renal allograftsshowed characteristics of chronic allograft nephropathy at the endpoint, Tregs plus ATG-treated renal allograft of POD 106 (Fig. 8C) andTregs plus sirolimus and ATG-treated allograft of POD 60 (Fig. 8D).
4. Discussion
Expansion of rare Tregs for adoptive cell immunotherapy has beenhighlighted in transplant models as well as by recent studies inautoimmune disease models [22–25]. However, some limitations,such as risk of co-expanding effector T cells, low frequency ofalloantigen cross-reactive Tregs in patients, interfere with strategiesof Tregs therapy. Recent many studies confirmed that Tregs could be
expanded in vitro, but these cells were isolated from naïve animals,and seems not feasible to patients with some diseases. In this study,we developed a cell co-culture system to expand Treg cells withindirect specificity for donor antigens, and achieved a large-scaleexpansion within 4-week culture. Expanded donor alloantigenspecific Tregs showed strong inhibitive capacity in the response tostimulation of either TCR or allogeneic antigens, and in the cell dosedependent fashion in vitro.
Although Tregs can be expanded in vitro, it seems not feasible inclinical transplantation. Usually, the expansion of Tregs requires at least2–3 weeks reaching enough cell number from patients. However, theproblem is that the source of deceased donor organ is usually unknown,and organ transplant is often on an emergency basis. To assess a feasiblestrategy in clinical, our hypothesis is that whether recipients couldreceive a short-term course of immunosuppressants after organtransplantation. During this period of immunosuppressive therapy,the donor alloantigan-specific Tregs could be expanded in vitro. Toaddress this hypothesis, we compared common applied immunosup-pressant drugs, Cyclosporine A, tacrolimus and sirolimus in this study.Our in vitro studies showed that Treg cells exposed to variousimmunosuppressants displayed different function on cell proliferationand cell inhibition. CD4+CD25+ Treg subsets could survive in thepresence of low-dose sirolimus, while the proliferation of CD4+CD25−
T cells was inhibited. After 3–4 weeks of expansion, Tregs exposed tosirolimus represented a high population of CD4+CD25brightFoxp3+
T cells, but not found in the Tregs treatedwith the sameconcentration of
Fig. 6. The median survival time and allograft renal function in combined therapy of expanded Tregs with immunosuppressants in vivo. Expanded Tregs (107/day) were injectedintravenously to recipient monkeys for 14 days consistently, together with a 14-day therapy of low-dose sirolimus (0.5 mg/kg/day) from the day of transplantation, while ATG(20 mg/kg/day) was consistently given for 3 days before transplantation. The recipient monkey survival time was observed in all groups (A). To evaluate the function of renalallografts, the changes of serum creatinine (sCr) were detected at different point during whole observation period in untreated (B), sirolimus-treated (C), Tregs-treated (D) andTregs plus sirolimus-treated monkeys (E).
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tacrolimus or Cyclosporine A. This differencemay account for the reasonwhy sirolimus permits apoptosis of alloreactive CD4+T cellsmore easilythan those treated by CNIs [34,35]. Recent studies have indicated thatcalcineurin inhibition byCNIsmay impair the function of Tregs,whereassirolimus was found to favor Tregs. Battaglia and colleagues demon-strated that sirolimus selectively expands CD4+CD25+FoxP3+ regula-tory T cells inmice [26]. Itmight be used as a tool for Treg cell expansion[27–31]. Compared to CNIs, Cyclosporine A has been observed to
antagonize the induction of tolerance in several experimental trans-plantation models [32,33]. It is also noted in our study, however,when the concentration of sirolimus was higher than 10 nM, the sup-pressive functionwas significantly decreased. This result suggested thatthere is a threshold for the concentrationof sirolimus in theexpansionofCD4+CD25+ T cells in vitro.
To further confirm our in vitro finding, adoptive inoculation ofdonor–antigen specific Tregs combined with low-dose sirolimus were
Fig. 7. Immunological profiles in recipients received regulatory T cells with immunosuppressants in vivo. The percentages of CD4+CD25+ Tregs (A), CD4+CD25bright Tregs (B), andCD4+IL-10+ Tr1 cells (C) were evaluated in peripheral blood of recipient monkeys on day 21 after transplantation by FACS assay. The frequencies of CD4+CD25bright and CD4+IL-10+
cells were also examined on secondary lymphoid organs, inguino lymph nodes (iLN) and mesenteric lymph nodes (mLN) of all recipients on day 21 after transplantation (D).
Fig. 8. Histopathological profiles in recipients received regulatory T cells with immunosuppressants in vivo. The slides of renal allografts from the un-treated, Tregs-treated,sirolimus-treated, and Tregs plus sirolimus-treated groups were stained with hematoxylin-eosin. Severe cell infiltration of acute rejection was found in untreatedmonkeys on POD 7(A), and sirolimus plus ATG-treated monkeys on POD 14 (B), whereas allografts from Tregs- or Tregs plus sirolimus-treated recipients presented chronic allograft nephropathy at theend point. Two representative renal allografts were presented in Tregs-treated monkey on POD 60 (C) and Tregs plus sirolimus-treated monkey on POD 106 (D).
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administered in renal transplant recipient monkeys. The data showedthat treatment of Tregs combined sirolimus delayed acute rejection andprolonged the survival of allografts, compared with recipients receivedun-treated and tacrolimus-treated therapy [36–38]. Consistent withour in vitro results, sirolimus did not interfere with Treg function.The recipients received Tregs plus sirolimus showed higher levels ofCD4+CD25bright T cells in peripheral blood and lymph nodes. Our resultsare in linewith studies fromDr. Negrin andWeimar,who indicated thatcalcineurin inhibitors could reduce Foxp3 expression and antagonizethe induction of tolerance, whereas mTOR inhibitors have little or noimpact [39,40]. The mechanism might be that sirolimus, different fromCyclosporine A or tacrolimus, permits easily apoptosis of allo-reactiveCD4+ T cells, resulting in preservation of Treg function. The results ofStrauss et al. demonstrated that sirolimus-treated Tregs expressed asignificant higher level of CD95 molecule as compared with no-treatedTregs. However, no significant activation-induced-cell death was foundin sirolimus-treated Tregs in the response to TCR stimulation [41]. Wefully agreewith their findings, and suggest that Tregs aremore resistantto apoptosis despite high level of CD95 expression. In addition, otherimmunosuppressive drugs, such asMMFor steroids, have been recentlyshown to preserve murine Tregs function and Foxp3 expression afterallogeneic bone marrow transplantation [42]. Taken together, ourfindings suggest that the use of sirolimus instead of CNImightmaintainfunction of Tregs in renal transplantation.
Stable tolerance has been demonstrated to be related to high level ofIL-10 and high frequencies of CD4+CD25bright Tregs and CD4+IL-10+
Tr1 cells [43,44]. Our data showed the frequencies of CD4+CD25bright
Tregs were higher in the recipients received Tregs alone or combinedwith sirolimus, comparedwith those in untreated or tacrolimus-treatedmonkeys, but no change of CD4+IL-10+ Tr1 cells was found in themonkeys of all groups. The explanation may be due to the fact, thatresults of increased IL-10 or Tr1 cells from Thomas and Roncarolo labswere found in long-term survival recipients, different from our short-term survival monkeys. Thus, it suggests that CD4+CD25bright cellsmight be more sensitive than Tr1 cells in the early stage during theinduction of Tregs in vivo, and induction and maintenance of Tregspopulation are an important variable for long-term allograft survival.
The recent studies have indicated that the secondary lymphoidorgans are critical sites for the induction of tolerance [45–47].The results of Kawai et al. demonstrated that a relative increase ofCD4+CD25+ T cells in peripheral lymphoid organs was observed in ratstreated with sirolimus, [48]. In our in vivo study, trafficking of labeledCD4+CD25+ T cells was found in lymph nodes of recipients afterinfusion of Tregs plus sirolimus, suggesting that CD4+CD25+ T cellscould home andmigrate to secondary lymphoid organs in the presenceof sirolimus. One interesting finding was that the frequencies of Tregswere significantly higher in mesenteric lymph nodes than those indistant inguino lymph nodes and peripheral blood in all groups. Thisspecific recruitment of Tregs inmesenteric lymph nodesmay be relatedto 1) high expression of lymphoid-homing chemokine receptors onTregs, and 2) the location of mesenteric lymph nodes is close to the siteof renal allografts. Similar toour results, a recent study in amousemodelhave indicates that the recruitmentof Foxp3-expressingTregs to cardiacallograft tissue is dependent on the chemokine receptor, CCR4 [49]. Thishoming property is conducive to the appropriate migration andaccumulation of Tregs at the location where the alloantigens werecontinuous by present. The long-term maintenance of transplanttolerance is also associated to the continuous expansion of Treg cellsin recipients to respond to local allo-antigens.
As a T cell depletion agent, anti-thymocyte globulin has beendemonstrated to be able to induce increase of Tregs in vitro [50].Although Tregs are quite sensitive to elimination by depleting Abs, therelative resistance of Tregs can promote tolerance throughpreferentialdepletion of T-effector cells [51]. It is noted that, after the treatment ofTregs plus sirolimus, CD4+ T subset population was decreased inPBMCs and LNMCs (data not shown), but CD4+CD25bright subset
significantly increased in recipients. Thus, our combined therapymight beworkable via depleting T cells byATGand inhibiting T effectorcells by sirolimus, resulting in the out-growth of Foxp3+ Tregs.
In conclusion, we have shown that expanded CD4+CD25bright Tregcells can survive, proliferate, and preserve their suppressive functionin the presence of sirolimus in vitro and in vivo. The combinationtherapy of Tregs with sirolimus might lead to more favorableoutcomes in transplantation. It will be an important step towardsthe clinical application of immuno- or cell-therapy using expandedantigen specific Tregs to induce donor-specific transplant tolerance.
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