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ntigen-Specific Suppression by Induced CD4�CD25high RegulatoryCells in Kidney Recipients

. Wang, X.-Y. Fu, B.-Y. Shi, Y.-Y. Qian, L. Xiao, and H.-W. Bai

ABSTRACT

The biology and function of induced CD4�CD25high regulatory T (Treg) cells have notbeen clarified for their specificity to a foreign antigen. To test whether the regulatoryfunctions of the induced CD4�CD25high Treg cells after transplantation require antigen-specific triggering, we analyzed the capacity of induced CD4�CD25high Treg cells to inhibitthe proliferation of conventional CD4�CD25� T cells in response to T-cell receptorstimulation using donor cells or HLA-mismatched third-party cells in vitro. CD4�CD25high

Treg cells did not proliferate in response to allogeneic stimulation and suppressedproliferation of the co-cultured autologous CD4�CD25� populations in a dose-dependentmanner. The proliferation of CD4�CD25�T cells from the same donor in mixedlymphocyte reactions was significantly inhibited at a 1:8 ratio of conventional T cells:Tregcells: 14,404 � 673 cpm without CD4�CD25high Treg cells versus 10,781 � 539 cpm withCD4�CD25high Treg cells P � .01). At the same 1:8 ratio, the proliferation ofCD4�CD25� cells derived from major histocompatibility complex–mismatched patientswas not significantly inhibited: 14,404 � 673 cpm without CD4�CD25high Treg cells versus12,471 � 709 cpm with CD4�CD25high Treg cells (P � .06). Antigen specificity of theinduced CD4�CD25high Treg cells was demonstrated, after transplantation, supporting the

use of antigen-specific Treg cells as a therapeutic strategy.

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ECENT STUDIES have hinted that peripheralCD4�CD25� regulatory T (Treg) have therapeutic

otential to induce tolerance to alloantigens after organransplantation.1–3 However, much of the data supportinghis view has been obtained using in vitro and small animalodel systems. Many questions remain regarding the rele-

ance of these findings to humans. Are the inducedoxP3�CD4�CD25� Treg cells antigen specific? In thistudy, we addressed question to facilitate our understandingf potential use of CD4�CD25� Treg-based therapy inransplantation.

ATERIALS AND METHODSatients and Samples

his prospective study was conducted from January, 2006, toecember, 2007, and included 9 subjects. All recipients received

mmunosuppressive therapy (cyclosporine or tacrolimus, and ste-oids, with or without mycophenolate mofetil). Peripheral bloodamples were collected from kidney recipients at posttransplant day8 and living donors. The study was approved by the Medicalthical Committee, and informed consent was obtained from all

atients. 1

041-1345/09/$–see front matteroi:10.1016/j.transproceed.2009.01.093

574

LA Genotyping

enomic DNA was extracted from whole venous blood using aIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany).LA-A, -B and -DRB1 genotyping was performed using the

olymerase chain reaction sequencing-based typing method withpplied Biosystems kits (Applied Biosystems, Foster City, Calif).

-Cell Isolation

eripheral blood mononuclear cells (PBMCs) were isolated frombout 15 mL of whole blood of kidney recipients using Ficoll-Hypaqueensity gradient centrifugation. CD4�CD25� Treg cells were isolatedy CD4-negative selection and CD25-positive selection, using aD4�25� T-cell isolation kit (Miltenyi Biotec) with a MiniMACS

From The Organ Transplant Center (Z.W., B.-Y.S., Y.-Y.Q.,.X., H.-W.B.) and Department of Gynaecology and Obstetrics

X.-Y.F.), The Chinese People’s Liberation Army General Hos-ital, Beijing, China.Z. Wang and X.-Y. Fu contributed equally to this work.Address reprint requests to B.-Y. Shi, The Organ Transplantenter, The Second Affiliated Hospital of the Chinese Liberationrmy general Hospital, No. 17A Heishanhu Road, Beijing

00091, China; E-mail: shibingyi@medmail.com.cn

© 2009 by Elsevier Inc. All rights reserved.360 Park Avenue South, New York, NY 10010-1710

Transplantation Proceedings, 41, 1574–1576 (2009)

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ANTIGEN-SPECIFIC SUPPRESSION 1575

eparator unit, according to the manufacturer’s instructions. Threeractions were sorted from PBMCs: non-CD4� cells, CD4�CD25�

ells, and CD4�CD25� cells. The purity of the resulting populationas more than 85% for CD4�CD8� cells, 90% for CD4�CD25� cells,nd 95% for CD4�CD25� (thymocytes) T cells.

ixed Lymphocyte Reactions

o analyze Treg-mediated suppression of T-cell proliferation initro, various numbers of CD4�CD25� cells isolated fromidney recipients were cultured with viable responder cells (105 cellser well) from the same recipient in complete RPMI 1640 medium10% heat-inactivated pooled human serum, 2 mmol/L L-glutamine,00 U/mL penicillin, and 100 �g/mL streptomycin). We used 5 � 104

rradiated PBMCs from the living donors as stimulators (37 Gy) andere added for a 1-way mixed lymphocyte reaction (MLR). Prolifer-tion was measured by adding 1 �Ci of [3H]thymidine during the final6 hours of a 5-day assay. Cells were then analyzed using a scintillationounter (PerkinElmer Life Sciences, Norwalk, Conn).

tatistical Analysis

o analyze differences between paired groups, we performed a-tailed Mann-Whitney nonparametric U test P � .05 was consid-red significant. Both analyses and graphics used the GraphPadrism Version 4.0 (San Diego, Calif).

ESULTS

o ascertain whether the induced CD25high cells had sup-ressive activity, the function of CD4�CD25high T cells wasnalyzed in an allogeneic MLR. Isolated non-CD4� cells,D4�CD25� cells and CD4�CD25� cells (105 cells/well)

rom 3 recipients with stable kidney function were culturedith or without fresh CD4�CD25� cells from the same

ecipient at various ratios upon stimulation with allogeneic,rradiated PBMCs derived from the living-related donor. Ashown in Figure 1, CD4�CD25high Treg cells did not

ig 1. Non-CD4� cells and CD4�CD25� cells proliferated mark-dly, whereas CD4�CD25� cells only showed minimal proliferation.

nhibition of the proliferation of CD4�CD25� T and non-CD4� cells by

gD4�CD25high Treg cells at varying ratios (responders:suppressors).

roliferate in response to allogeneic stimulation, whereasD4� and CD4�CD25� fractions showed strong prolifer-tion under the same conditions.

CD4�CD25high Treg cells suppressed the proliferation ofo-cultured autologous CD4�CD25� and CD4� popula-ions in a dose-dependent manner (Fig 2). The titrationurves (Fig 2) revealed a break point at a suppressor:esponder ratio of more than 1:8 (12,500 suppressorso 100,000 responders) for the CD4�CD25� fraction.D4�CD25high Treg cells showed clear suppressive effects

about 65% inhibition of proliferation) at a suppressor:esponder ratio of 1:2 (Fig 2).

To test whether the regulatory functions of the inducedD4�CD25high Treg cells required antigen-specific trigger-

ng, we examined whether the CD4�CD25high Treg cellsere activated by an unrelated antigen or mediated sup-ression in response to that antigen. Freshly isolated re-ponder T cells derived from recipients were culturedith irradiated PBMCs derived from donors or HLA-ismatched third-party patients as the stimulating antigen

Table 1). The proliferation of CD4�CD25�T cells from theame donor in MLRs was significantly inhibited at a 1:8 ratiof conventional T cells:Treg cells: 14,404 � 673 cpm withoutD4�CD25high Treg cells versus 10,781 � 539 cpm withD4�CD25high Treg cells (P � .01) (Fig 3). At the same 1:8

atio, the proliferation of CD4�CD25� cells derived fromHC-mismatched patients was not significantly inhibited:

4,404 � 673 cpm without CD4�CD25high Treg cellsersus 12,471 � 709 cpm with CD4�CD25high Treg cells,P � .06).

ISCUSSION

key question that has emerged from cell-based tolero-

ig 2. Up to a 1:8 dilution (suppressor:responder) for theD4�CD25� fraction markedly impaired the MLR. Results areresented as the mean cpm of 3 trials with error bars represent-

ng standard deviations. CD4�CD25� responder cells wereultured with or without fresh CD25� cells (left) from the sameonor at a 1:8 ratio and stimulated with 5 � 104 stimulator cells

rom the organ donor (middle) or from an HLA-mismatchedonor (right).

enic therapy is whether the suppressive functions of in-

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1576 WANG, FU, SHI ET AL

uced Treg cells are specific for particular peptide–MHComplexes. In contrast to their in vitro hyporesponsivenesso TCR stimulation, CD4�CD25� Treg cells were potentlyuppressive when assayed in MLR systems.4,5 However, theuppressive potency of CD4�CD25� Treg cells in co-ultures, where PBMCs derived from the same donor or ahird person were used as the stimulating antigens, wasurprisingly different. Our in vitro experiments suggestedhat the suppressive function of these Treg cells requiredctivation of their TCR, indicating that the in vivo activa-ion and function of CD4�CD25� Treg cells were con-rolled by the specificity of the TCR. Animal studies havelearly shown that transfer of Treg cells expressing a TCRpecific for the pathogenic antigen, rather than polyclonalild-type Treg cells, was efficacious to reverse active dis-ase.6,7 In the nonobese diabetic mouse model of type 1iabetes, islet-specific Treg cells from TCR-transgenic miceere markedly more effective at disease suppression thanolyclonal Treg cells expanded using the same protocol.8

Naturally occurring CD4�CD25� Treg cells are consid-red to be specific for antigens. The precursor frequency ofD4�T cells to any specific peptide can range from 1/2,000

o 1/200,000 or greater.9 Growth of a small portion ofntigen-specific CD4�CD25� Treg cells triggered throughn antigen-specific TCR with a specific peptide–MHComplex could be a preferable alternative to polyclonalxpansion. The idea that induction of CD4�CD25� Tregells occurred through antigenic stimulation might explainheir antigen specificity. This expansion should allow forelection against MHC and self-specificities. More recentata have shown that Treg cells specific for particulareptide–MHC complexes were also generated from humanD4�CD25� T cells.10

It can now be suggested that induced CD4�CD25high

reg cells are alloantigen specific. They require activation

Table 1. HLA-A, -B, and -DRB1 Genotyping

Recipient HLA-A HLA-B HLA-DR1

Donor *0201/*11011 *07021/*2702 *04031/*15021Recipient *2501/*11011 *07021/*4701 *04031/*1404Third party *24021/*31012 *3502/*44031 *01021/*03011

Donor *8011/*3001 *4101/*4601 *013021/*15011Recipient *02011/*0217 *4601/*1501 *01301/*16011Third party *2501/*03011 *0801/*1302 *08041/*11011

Donor *0205/*3201 *4001/*4001 *0101/*1310Recipient *0207/*3201 *4001/*4001 *0101/*07011

hThird party *3301/*6901 *5501/*5609 *16011/*0404

hrough their TCR to exert suppressive functions afterransplantation.

EFERENCES

1. Roncarolo MG, Battaglia M: Regulatory T-cell immunother-py for tolerance to self antigens and alloantigens in humans. Natev Immunol 7:585, 20072. Shevach EM: From vanilla to 28 flavors: multiple varieties ofregulatory cells. Immunity 25:195, 20063. Chai JG, Xue SA, Coe D, et al: Regulatory T cells, derived

rom naïve CD4�CD25� T cells by in vitro Foxp3 gene transfer,an induce transplantation tolerance. Transplantation 79:1310,0054. Xu D, Fu J, Jin L, et al: Circulating and liver resident

D4�CD25� regulatory T cells actively influence the antiviralmmune response and disease progression in patients with hepatitis. J Immunol 177:739, 20065. Balandina A, Lécart S, Dartevelle P, et al: Functional defect

f regulatory CD4�CD25� T cells in the thymus of patients withutoimmune myasthenia gravis. Blood 105:735, 2005

6. Mottet C, Uhlig HH, Powrie F: Cutting edge: cure of colitis byD4�CD25� regulatory T cells. J Immunol 170:3939, 20037. Tang Q, Henriksen KJ, Bi M, et al: In vitro-expanded

ntigen-specific regulatory T cells suppress autoimmune diabe-es. J Exp Med 199:1455, 2004

8. Tarbell KV, Petit L, Zuo X, et al: Dendritic cell-expanded,slet-specific CD4� CD25� CD62L� regulatory T cells restoreormoglycemia in diabetic NOD mice. J Exp Med 204:191, 20079. Novak EJ, Masewicz SA, Liu AW, et al: Activated human

pitope-specific T cells identified by class II tetramers reside withinCD4high, proliferating subset. Int Immunol 13:799, 200110. Walker MR, Carson BD, Nepom GT, et al: De novo

eneration of antigen-specific CD4�CD25� regulatory T cells from

ig 3. Proliferation of responder cells. Data collected from 3eparate recipients. P values are based on a 1-tailed Mann–hitney test.

uman CD4�CD25 cells. Proc Natl Acad Sci U S A 102:4103, 2005