Induction of CD4 CD25 Foxp3 Regulatory T Cells

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A Novel Mechanism of Action for Anti-Thymocyte Globulin: ؉ ؉ ؉ Induction of CD4 CD25 Foxp3 Regulatory T Cells

Marta Lopez, Michael R. Clarkson, Monica Albin, Mohamed H. Sayegh, and Nader Najafian Transplantation Research Center, Brigham and Women’s Hospital, and Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts

T cell–depleting agents are being tested as part of clinical tolerance strategies in humans with autoimmunity and transplantation. The immunosuppressive activity of anti-thymocyte globulin (ATG) has been thought to result primarily from depletion of peripheral lymphocytes. Herein is reported for the first time that ATG but not anti-CD52 mAb (alemtuzumab) or the IL-2R antagonists causes rapid and sustained expansion of CD4؉CD25؉ T cells when cultured with human peripheral blood lymphocytes. These cells display enhanced expression of the regulatory markers glucocorticoid-induced TNF receptor, cytotoxic T lymphocyte–associated antigen-4 (CTLA-4), and forkhead box P3 and efficiently suppress a direct alloimmune response of the original responder lymphocytes. It is interesting that the cells do not suppress memory responses to the recall antigen mumps. Ex vivo expansion of regulatory T cells is due mainly to conversion of CD4؉CD25؊ into CD4؉CD25؉ T cells and to a lesser degree to proliferation of natural CD4؉CD25؉ T cells. The induction of regulatory T cells depends on production of Th2 cytokines in the generating cultures. These novel data suggest that ATG not only may promote expansion/ generation of regulatory T cells but also may be useful in future ex vivo expansion of these cells for cellular therapy in autoimmunity and clinical transplantation. J Am Soc Nephrol 17: 2844–2853, 2006. doi: 10.1681/ASN.2006050422

olyclonal anti-thymocyte globulin (ATG) is the purified tolerance toward autoantigens (8) and alloantigens (10). Treg IgG fraction of sera from rabbits, horses, or, more also may play a role in preventing human renal autoimmune P rarely, goats that are immunized with human thymo- diseases such as Goodpasture’s disease (11). Our group previ- cytes or T cell lines. ATG is used for treatment of various ously demonstrated that active regulation of the alloimmune clinical conditions, including prevention or rescue treatment of responses by Treg may function to maintain hyporesponsive- acute rejection in organ transplantation (1), conditioning for ness to alloantigens in renal transplant patients (12,13). In pre- hematopoietic stem cell transplantation, treatment of severe clinical animal models, ex vivo expanded Treg protect mice aplastic anemia, various autoimmune diseases, and more re- from lethal graft-versus-host disease (14). In fact, a clinical trial cently graft-versus-host disease (2). The immunosuppressive was proposed recently to use ex vivo expanded Treg at the time activity of ATG has been thought to result primarily from the of hematopoietic stem cell transplantation (15,16). Therefore, depletion of peripheral lymphocytes from the circulating pool understanding the conditions that are required for the genera- through complement-dependent lysis or activation-associated tion and propagation of Treg would allow development of apoptosis (1,3–5). Other potential mechanisms of action include novel therapeutic strategies for inducing immunologic toler- modulation of surface adhesion molecules or chemokine recep- ance in various immune-mediated diseases. tor expression (6). Here we report the novel finding that ATG-mediated immu- Regulatory T cells (Treg) are specialized T cell subsets that nosuppression is delivered in part via immunologically specific play important roles in maintaining immune homeostasis (7,8). actions involving the generation of Treg, particularly ϩ ϩ Treg are characterized by the expression of the IL-2 receptor CD4 CD25highFoxp3 cells. This is due mainly to ATG’s ϩ Ϫ ␣-chain (CD25) and the transcription factor forkhead box P3 unique ability to convert the CD4 CD25 T cells into ϩ ϩ (Foxp3) (9). There exists emerging evidence in both rodents and CD4 CD25 T cells. More important, in a mixed lymphocyte humans to support important immunoregulatory functions of reaction, the regulatory function is restricted to autologous ϩ ϩ CD4 CD25 T Treg in maintaining both self-tolerance and responder cells from which Treg originally were generated and does not affect the memory response to recall antigens. Our findings have relevant clinical implications for designing new Received May 1, 2006. Accepted June 19, 2006. immunomodulatory protocols for immune-mediated diseases.

Published online ahead of print. Publication date available at www.jasn.org. Materials and Methods Address correspondence to: Dr. Nader Najafian, Brigham & Women’s Hospital, Transplantation Research Center, EBRC, 221 Longwood Avenue, 3rd Floor, Bos- Cells and Antibodies ton, MA 02115. Phone: 617-732-5259; Fax: 617-732-5254; E-mail: This study was performed with the approval of the Institutional [email protected] Review Board for human investigation at the Brigham and Women’s

Hospital. Blood from healthy volunteers were obtained in heparinized ware (Becton Dickinson, San Jose, CA). For evaluation of the induction ϩ Ϫ ϩ ϩ tubes, and peripheral blood lymphocytes (PBL) were isolated by stan- of apoptosis/death of CD4 CD25 and CD4 CD25 T cells, PBL that dard Ficoll-density-gradient centrifugation. Two types of ATG were were incubated with ATG or rabbit IgG were stained with mAb against used: A rabbit polyclonal serum raised against human thymocytes CD4, CD25, annexin-V, and 7-amino-actinomycin D as per the manu- (Thymoglobulin; Genzyme, Cambridge MA) or a rabbit polyclonal facturer’s instructions (BD Bioscience). serum raised against the lymphoblastic Jurkat T cell line (Fresenius, Bad Homburg, Germany). Purified rabbit polyclonal IgG was used as ELISPOT Assay control. To compare the effects of ATG with agents that block the IL-2 To evaluate the frequency of cytokine-producing cells in the gener- ␣ ␣ receptor -chain (IL-2R ) on the surface of activated T lymphocytes, we ating culture, we made use of the ELISPOT assay as described previ- used either Basiliximab (Simulect, Novartis, NJ), a chimeric (murine/ ously (12). The resulting spots were counted on a computer-assisted human) mAb (IgG1k), or Daclizumab (Zenapax, Roche, NJ), a human- ELISASpot Image Analyzer (Cellular Technology Limited, Cleveland, ized IgG1 mAb. In addition, we used alemtuzumab (Campath1-H, OH). Cells were tested in triplicate wells. The frequencies then were Genzyme, Cambridge, MA), a recombinant DNA-derived humanized expressed as the number of spots per million PBL. mAb that is directed against the cell surface glycoprotein CD52, for comparison studies with ATG. Luminex Assay Supernatants from generating cultures (serum-free medium) were Generation of Treg tested for the presence of TGF-␤ by Luminex 100 system (Luminex PBL (1 ϫ 106 per ml) from 10 healthy donors were incubated with Corp., Austin, TX). Beadlyte Human Multi-Cytokine Beadmaster Kit ATG (Treg) or rabbit IgG (TControl) in RPMI 1640 medium (Cambrex and Beadlyte Human TGF-␤1/␤2 Detection system (Upstate, Char- Bioscience, Walkersville, MD) supplemented with 10% heat-inactivated lottesville, VA) were used as per protocol provided by the manufac- human serum at 37°C with a Pco2 5% for various durations (6 to 96 h). turer. Plates were analyzed using Multiplex Data Analysis software All above agents were used in vitro at concentrations that ranged from version 1 (Luminex, Austin, TX). 1to100␮g/ml. These cultures are called “generating cultures.” For assessment of the Carboxy-Fluorescein Diacetates Succinimidyl Ester Staining role of cytokines in the expansion of Treg, anti–IL-4, anti–IL-13, and To test whether the expansion of Treg by ATG is due to proliferation ␮ ϩ ϩ anti–IL-10 mAb each at a concentration of 10 g/ml were added of preexisting naturally occurring CD4 CD25 T cells, we incubated separately to the generating cultures (17). All of these antibodies were PBL with carboxy-fluorescein diacetates succinimidyl ester (CFSE) in purchased from BD Bioscience (San Jose, CA). the form of 5 mM stock solution in DMSO at a final concentration of 1 ␮M for 6 min at room temperature. CFSE-labeled cells were cultured in In Vitro Suppression Assay vitro with phytohemagglutinin (positive control), ATG, and rabbit IgG To test the suppression function of cells that were generated under for 72 h at 37°C. Cells then were stained with anti-human CD4-APC, conditions explained (Treg), we set up a mixed lymphocyte reaction CD8-PE, and CD25-PE. 7-Amino-actinomycin D was used to exclude (MLR) assay: 1 ϫ 105 cells that were obtained from the generating death cells. cultures above were co-cultured (1:1 ratio) with fresh responder (au- ϩ Ϫ ϩ ϩ tologous or third-party PBL) and irradiated stimulator cells in a 96-well Conversion of CD4 CD25 to CD4 CD25 T Cells plate (96-well Cell Culture Cluster, round-bottom culture plate; For testing whether Treg that are generated by ATG are induced ϩ Ϫ COSTAR, New York, NY) for 120 h. The cultures were labeled with from CD4 CD25 T cells, PBL were depleted of CD25 by magnetic cell 3H-thymidine during the last8hofculture (Amersham Pharmacia sorting using MACS columns and MACS separators (Miltenyi Biotec, ϩ Biotech, Piscataway, NJ). Cells then were harvested, and radionuclide Auburn, CA). CD25-depleted CD4 T cells then were incubated with uptake was measured by a scintillation counting machine. In a similar ATG or rabbit IgG for 24 h. The cells then were harvested and stained manner, we also tested whether Treg could suppress recall responses to for CD25 and regulatory markers. Similarly, their suppressor activity mumps antigens. was assessed as described previously.

Flow Cytometry Statistical Analyses Cells that were harvested from generating cultures (Treg or TCon- The t test was used for comparison of means between experimental trol) were analyzed using flow cytometric analysis. A total of 2 ϫ 105 groups examined by flow cytometry and ELISPOT assay. Differences cells per sample were stained with anti-human CD4-allophycocyanin were considered to be significant at P Ͻ 0.05. (APC), CD25-phycoerythrin (PE), and glucocorticoid-induced TNF receptor (GITR)-FITC, CD8-APC (BD Bioscience; eBioscience, San Diego, Results ϩ ϩ ϩ CA). For the intracellular cytotoxic T lymphocyte–associated antigen-4 ATG Expands CD4 CD25 Foxp3 T Cells Ex Vivo (CTLA-4) staining, cells were permeabilized with Perm Buffer (BD PBL that were derived from healthy volunteers were incu- Biosciences) for 20 min at 4°C and labeled with CTLA-4 for 30 min at bated with Thymoglobulin or rabbit IgG (10 ␮g/ml) for various ϫ 6 4°C. For flow cytometric analysis of Foxp3, 1 10 cells first were time periods (0, 6, 18, 24, 48, 72, and 96 h). Flow cytometric stained with anti-human CD4-APC and CD25-PE. After washing, cells analysis of harvested cells demonstrated a significant upregu- were resuspended in 1 ml of cold Fix/Perm Buffer (eBioscience) and lation of CD25 expression that began at 18 h and was main- incubated at 4°C overnight in the dark. After washing twice with 2 ml tained beyond 96 h, with peak expression of CD25 at 24 h of permeabilization buffer, cells were blocked with 2% normal rat ϩ ϩ serum for 15 min. Anti-human Foxp3-FITC (PCH101; eBioscience) then (percentage of CD4 CD25 T cells gated on all viable lympho- Ϯ Ϯ ϭ ϭ was added, and cells were incubated at 4°C for another 30 min in the cytes 20.5 7.8 versus 4.5 1.6; P 0.002; n 7; Figures 1A dark. Finally, cells were washed with 2 ml of permeabilization buffer and 2A). and analyzed on a FACSCalibur flow cytometer using CellQuest soft- To evaluate the dose-response of ATG and expansion of 2846 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 2844–2853, 2006

ϩ ϩ ϩ Figure 1. Anti-thymocyte globulin (ATG) expands CD4 CD25 Foxp3 T cells ex vivo. (A) A representative example of seven ϩ ϩ experiments demonstrating enrichment of a CD4 CD25 T cell population at 24 h (peak expression): Cells incubated with Thymoglobulin showed an increase in CD25 expression as compared with cells incubated with rabbit IgG (each at a concentration ϩ ϩ of 10 ␮g/ml). Such expanded CD4 CD25 T cells generated after incubation with Thymoglobulin had significantly enhanced expression of regulatory markers glucocorticoid-induced TNF receptor (GITR; 32 Ϯ 12 versus 6.6 Ϯ 4%; n ϭ 5), intracellular cytotoxic T lymphocyte–associated antigen-4 (CTLA-4) (41.3 Ϯ 19.5 versus 7 Ϯ 1.8%; n ϭ 4), and forkhead box P3 (Foxp3; 65.3 Ϯ 21.5 versus 43.8 Ϯ 12.3; n ϭ 5) as compared with control cells (percentage expression of regulatory markers calculated when gating ϩ ϩ ϩ ϩ on CD4 CD25 T cells). (B) A representative example showing a significant increase in the frequency of CD4 CD25 high population in ATG-treated versus rabbit IgG–treated cells (6.5 Ϯ 2.9 versus 0.7 Ϯ 0.5%; n ϭ 8; P ϭ 0.001). More important, representative examples of two color flow staining (CD25 and each of regulatory markers GITR, CTLA-4, and Foxp3) are shown ϩ ϩ ϩ Ϫ to compare better the expression of regulatory markers on CD4 CD25 and CD4 CD25 T cells after incubation with ATG or ϩ rabbit IgG. Among all CD4 T cells (see gate), the percentage of cells that expressed CD25 and GITR (ATG 18.3 Ϯ 6.9, rabbit IgG 0.5 Ϯ 0.4; n ϭ 6; P ϭ 0.001) or CD25 and CTLA-4 (ATG 19.5 Ϯ 6.2, rabbit IgG 1.5 Ϯ 0.4; n ϭ 4; P ϭ 0.008) was markedly enhanced ϩ when cells were incubated with ATG versus rabbit IgG. Similarly, gating on all CD4 T cells, the percentage of cells that expressed ϩ ϩ ϩ CD25 and Foxp3 (%CD4 CD25 Foxp3 T cells) was significantly increased when cells were treated with ATG versus rabbit IgG Ϫ (ATG 10.4 Ϯ 2.5 versus rabbit IgG 2.2 Ϯ 0.5; n ϭ 8; P Ͻ 0.0001). In contrast, the percentage of CD25 T cells that expressed GITR (ATG 1.8 Ϯ 1.5%, rabbit IgG 0.3 Ϯ 0.2; n ϭ 6; NS) or CTLA-4 (ATG 5.4 Ϯ 3.7, rabbit IgG 1.3 Ϯ 0.7; n ϭ 4; NS) was minimal. In ϩ Ϫ ϩ ϩ addition, the percentage of CD4 CD25 Foxp3 T cells (gated on CD4 T cells) was minimal after incubation with ATG versus ϩ Ϫ ϩ rabbit IgG (1.1 Ϯ 0.6 versus 0.7 Ϯ 0.4), indicating that ATG did not induce CD4 CD25 Foxp3 regulatory T cells (Treg).

ϩ ϩ CD4 CD25 T cells, we incubated PBL with ATG or rabbit IgG 50 ␮g/ml 21.7 Ϯ 5, and 100 ␮g/ml 21 Ϯ 6.7; percentage of ϩ ϩ at various concentrations (1, 5, 10, 50, and 100 ␮g/ml) for 24 h CD4 CD25 T cells with various concentrations of rabbit Ig is ϩ ϩ (percentage of CD4 CD25 T cells gated on all viable lympho- between 3 and 5). We found a dose-dependent increase in ϩ ϩ cytes: 1 ␮g/ml 6.3 Ϯ 0.5, 5 ␮g/ml 12 Ϯ 4.9, 10 ␮g/ml 20 Ϯ 6.5, percentage of CD4 CD25 T cells using between 1 and 10 J Am Soc Nephrol 17: 2844–2853, 2006 Ex Vivo Expansion of Regulatory T Cells by ATG 2847

ϩ ϩ Figure 2. Expansion of CD4 CD25 T cells is unique to polyclonal ATG. (A) Peripheral blood lymphocytes (PBL) that were derived from healthy human volunteers were incubated with Thymoglobulin (10 ␮g/ml) for various time periods (0, 6, 18, 24, 48, 72, and 96 h). Cells then were harvested and underwent flow cytometric analysis. There was significant upregulation of CD25 expression that started at 18 h and was maintained beyond 96 h. (B) T cell depletive agent Campath1-H and anti–IL-2 receptor agents ϩ ϩ (basiliximab and daclizumab) did not expand CD4 CD25 T cells (representative experiment of n ϭ 3).

ϩ ϩ ϩ ϩ ␮g/ml ATG. In contrast, the percentage of CD4 CD25 T cells pand CD4 CD25 T cells similar to ATG. It is interesting that ϩ ϩ does not seem to increase much further when PBL are incu- neither of these agents could expand CD4 CD25 T cells in bated with 50 to 100 ␮g/ml ATG, and we observed increased vitro (Figure 2B). Therefore, the expansion observed is unique ϩ ϩ ϩ activation of CD4 T cells (percentage of CD4 CD69 T cells: to polyclonal ATG. 1 ␮g/ml 4.4 Ϯ 4.8, 5 ␮g/ml 15.6 Ϯ 7.7, 10 ␮g/ml 12 Ϯ 7.2, 50 The study of Treg is complicated by a paucity of phenotypic ␮g/ml 21 Ϯ 5.6 and 100 ␮g/ml 22 Ϯ 5; percentage of markers to distinguish activated effector T cells from Treg. ϩ ϩ CD4 CD69 T cells with various concentration of rabbit Ig is Expression of molecules such as CTLA-4 and GITR (10) and Ͻ1%), a change in cell size, and increasingly poor viability of secretion of regulatory cytokines such as TGF-␤ (19) and IL-10 ϩ cells. These data suggest increased activation of CD4 T cells (20,21) each have been linked with but not proved to be entirely ϩ ϩ and a decline in expansion of CD4 CD25 T at higher concen- specific for Treg. Therefore, we first analyzed the surface ex- trations of ATG. pression of regulatory markers GITR and CTLA-4 on the ex- ϩ ϩ Because the regulatory function in humans is attributed panded CD4 CD25 T cells (Figure 1A). GITR showed en- ϩ ϩ mainly to the CD25high subset (18), we also compared the hanced expression in ATG-induced CD4 CD25 T cells as ϩ ϩ ϩ ϩ frequency of CD4 CD25 high population in ATG-treated ver- compared with CD4 CD25 T cells that were incubated with sus rabbit IgG–treated cells and found it to be significantly rabbit IgG (32 Ϯ 12 versus 6.6 Ϯ 4%; P ϭ 0.005; n ϭ 5). Similar increased in the former group (6.5 Ϯ 2.9 versus 0.7 Ϯ 0.5%; P ϭ results were observed for intracellular CTLA-4 expression 0.001; n ϭ 8; Figure 1B). These results also could be duplicated (41.3 Ϯ 19.5 versus 7 Ϯ 1.8%; P ϭ 0.04; n ϭ 4). When gating on ϩ using a second ATG preparation (Fresenius; at 10 ␮g/ml for all CD4 T cells, the percentage of cells that expressed both 24 h) that was generated from rabbit serum against the lym- CD25 and GITR (ATG 18.3 Ϯ 6.9, rabbit IgG 0.5 Ϯ 0.4; n ϭ 6; ϩ ϩ phoblastic Jurkat T cell line (percentage of CD4 CD25 T cells: P ϭ 0.001) or CD25 and CTLA-4 (ATG 19.5 Ϯ 6.2, rabbit IgG 16.7 Ϯ 4.2 versus 4.5 Ϯ 1.6, P ϭ 0.04, n ϭ 3; percentage of CD25high 1.5 Ϯ 0.4; n ϭ 4; P ϭ 0.008) was markedly enhanced when cells subset of T cells: 4.7 Ϯ 1 versus 0.7 Ϯ 0.5, P ϭ 0.02, n ϭ 3). were incubated with ATG versus rabbit IgG (Figure 1B). Because alemtuzumab (anti-CD52 mAb; Campath-1H) and In contrast to GITR and CTLA-4, Foxp3, a gene that encodes IL-2R antagonists are commonly used as induction agents in a forkhead winged helix transcription factor scurfin, is required ϩ ϩ organ transplantation and are currently being tested in various for Treg development and function (22,23). CD4 CD25 T cells autoimmune diseases, we next explored whether they can ex- that were expanded by ATG (at 10 ␮g/ml) showed higher 2848 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 2844–2853, 2006 expression of Foxp3 as compared with those that were incu- expanded Treg did not block memory cells to the antigen bated with rabbit IgG (63.4 Ϯ 12 versus 47 Ϯ 8.3%; P ϭ 0.03; n ϭ (mumps alone 4848 Ϯ 969, Treg 5214 Ϯ 900, control 9595 Ϯ 5; Figure 1A). It is interesting that contrary to CD69 expression 1231; Figure 3C). (see above), the Foxp3 expression declined with increasing dosage of ATG in the generating culture (percentage of Foxp3 ϩ Ϫ ϩ ϩ ϩ ϩ Conversion of CD4 CD25 into CD4 CD25 T Cells Is expression in CD4 CD25 T cells: 50 ␮g/ml 13 Ϯ 4.2, 100 the Main Mechanism of Expansion of Treg by ATG ϩ ϩ ϩ ␮g/ml 15.2 Ϯ 0.35). Importantly, gating on all CD4 T cells, the The absolute number of CD4 CD25 T cells (mean values of percentage of cells that expressed both CD25 and Foxp3 (per- cell numbers counted in wells) that were incubated with ATG ϩ ϩ ϩ centage of CD4 CD25 Foxp3 T cells) was significantly in- (before 75,833 Ϯ 31,051, after 639,167 Ϯ 249,448; n ϭ 6; P ϭ creased when cells were treated with ATG versus rabbit IgG 0.002) but not rabbit IgG was dramatically increased (before (ATG 10.4 Ϯ 2.5 versus rabbit IgG 2.2 Ϯ 0.5; P Ͻ 0.0001; n ϭ 8; 90,833 Ϯ 33,229, after 113,333 Ϯ 54,283; NS) after 24 h of Figure 1B). In addition, expression of all of the regulatory incubation. In contrast, there was much more decrease in the ϩ Ϫ markers was even markedly more enhanced in the number of CD4 CD25 T cells that were treated with ATG ϩ ϩ CD4 CD25 high population that was expanded after incuba- (before 1878,300 Ϯ 322,020, after 1082,500 Ϯ 301,027; P ϭ 0.005) tion with ATG (GITR 49.4 Ϯ 15.9%, n ϭ 7; CTLA-4 55 Ϯ 24.4%, as compared with rabbit IgG (1991,700 Ϯ 379,548 versus n ϭ 6; Foxp3 71 Ϯ 14.7%, n ϭ 5). 1861,666 Ϯ 238 362; NS). On the basis of these findings, we Previous work indicated that Foxp3 also can be induced in hypothesized that the expansion of Treg by ATG may result ϩ Ϫ CD4 CD25 T cells and that these cells can function as Treg from one or more mutually nonexclusive and possibly comple- ϩ ϩ ϩ Ϫ (24). However, in contrast to CD4 CD25 T cells, CD4 CD25 mentary mechanisms: First, ATG may preferentially promote ϩ Ϫ T cells that were incubated with ATG or rabbit IgG showed death/apoptosis of CD4 CD25 T cells as compared with ϩ ϩ only minimal increase in GITR (5.6 Ϯ 4.4 versus 0) and CTLA-4 CD4 CD25 T cells, creating a new balance in favor of the ϩ (11.5 Ϯ 4.2 versus 0). Similarly, gating on all CD4 T cells, the latter cells. Second, ATG may promote the proliferation of Ϫ ϩ ϩ percentage of CD25 T cells that expressed GITR (ATG 1.8 Ϯ already existing, naturally occurring CD4 CD25 T cells. ϩ Ϫ 1.5% rabbit IgG 0.3 Ϯ 0.2; n ϭ 6; NS) or CTLA-4 (ATG 5.4 Ϯ 3.7, Third, ATG may be able to convert CD4 CD25 T cells into ϩ ϩ rabbit IgG 1.3 Ϯ 0.7; n ϭ 4; NS) was minimal (Figure 1B). In CD4 CD25 T cells with regulatory functions (induction of ϩ Ϫ ϩ addition, the percentage of CD4 CD25 Foxp3 T cells (gated Treg). The first possibility is supported by published data dem- ϩ on CD4 T cells) was minimal after incubation with ATG versus onstrating that ATG in fact can bind to multiple epitopes on the rabbit IgG (1.1 Ϯ 0.6 versus 0.7 Ϯ 0.4), indicating that after T cell surface and induce apoptosis in T lymphocytes through ϩ Ϫ Ϫ incubation with ATG, CD4 CD25 T cells remain Foxp3 (Fig- Fas-L (CD95L) (3,5). Nevertheless, we found no difference (gat- ure 1B). Although overall there was a slight decrease in the ing on all lymphocytes without excluding the dead cells) in ϩ ϩ ϩ ϩ Ϫ CD8 T cells after incubation with ATG as compared with apoptosis of CD4 CD25 T cells and CD4 CD25 T cells that rabbit IgG (21.96 Ϯ 4.5 versus 25.6 Ϯ 5.1; n ϭ 8; P ϭ 0.02), we were incubated for 24 h with 10 ␮g/ml ATG (6.7 Ϯ 3.1 versus found no significant difference in the percentage of 5 Ϯ 4.7%) or rabbit IgG (5 Ϯ 3 versus 3.2 Ϯ 2.5%). In fact, the ϩ Ϫ CD8 CD28 T cells (11.3 Ϯ 5.6 versus 14.9 Ϯ 6; n ϭ 5). In overall viability of the cells was Ͼ95% regardless of incubation ϩ ϩ addition, we found no evidence of CD8 Foxp3 T cells before with ATG or rabbit IgG. or after treatment with ATG or rabbit IgG. Taken together, To address the possibility of the proliferative effect of ATG these data do not indicate expansion of other previously de- on already existing, naturally occurring Treg, we cultured in scribed Treg populations in our model. vitro CFSE-labeled PBL with ATG or rabbit IgG for 72 h (10 ␮g/ml). We found three to four discrete division cycles in ϩ ϩ ϩ ϩ Suppressor Function of CD4 CD25 T Cells Generated by CD4 CD25 cells (proliferative cells 16 Ϯ 9%; n ϭ 3; P ϭ 0.01) ϩ Ϫ ATG Is Restricted to Responder Cells but only one cycle of division in a CD4 CD25 subpopulation ϩ ϩ After demonstrating that CD4 CD25 T cells that are ex- that was incubated with ATG (7.4 Ϯ 7.7%) and none at all with panded by ATG can maintain their regulatory phenotype, we rabbit IgG, suggesting a moderate degree of expansion of ϩ ϩ next set out to study the actual suppressor function of these CD4 CD25 T cells. It is possible that some of the proliferating ϩ ϩ cells in vitro. The in vitro suppressive activity of the cells was CD4 CD25 T cells simply may be activated T cells rather than evaluated by examination of their ability to suppress an MLR to proliferating naturally occurring Treg. Nevertheless, whereas ϩ ϩ donor alloantigens. We found significant suppression of direct there is an eight-fold expansion of CD4 CD25 T cells at 24 h alloimmune responses of the original responders (PBL from with ATG, proliferating cells make up only approximately 16% ϩ which the Treg initially were generated) to stimulator cells by of the CD25 population at 72 h. This indicates that prolifera- Treg but not by TControl (1:1 ratio, 61.3 Ϯ 7.4 inhibition versus tion is unlikely to contribute significantly to the expansion of ϩ 20.2 Ϯ 18.4%; n ϭ 4; P ϭ 0.01; Figure 3A). It is interesting that the cells. In contrast, the CD8 T cells that were incubated with the same Treg were unable to suppress an MLR of responder ATG did not show proliferation at all (Figure 4). cells that were not autologous to the original responder cells To determine whether ATG is capable of converting ϩ Ϫ ϩ ϩ (Figure 3B). We next tested the ability of these Treg to suppress CD4 CD25 cells into CD4 CD25 cells with regulatory prop- ϩ a recall memory response of original responders to mumps erties, we first depleted naturally occurring CD25 T cells from antigen. It is interesting that the proliferative response to recall PBL ex vivo with magnetic bead separation (before 5.9 Ϯ 2.9 antigen mumps was not inhibited by Treg, indicating that the versus after depletion 0.6 Ϯ 0.9%; Figure 5A). Such CD25- J Am Soc Nephrol 17: 2844–2853, 2006 Ex Vivo Expansion of Regulatory T Cells by ATG 2849

depleted T cells then were incubated for 24 h with ATG or ϩ rabbit IgG, after which the induction of CD25 T cells was evaluated by flow cytometry. We found not only significant ϩ upregulation of CD25 expression on CD4 T cells that were incubated with ATG but not rabbit IgG (18.7 Ϯ 4 versus 3.4 Ϯ 1.8%; n ϭ 5; P ϭ 0.02) but also clear expansion of the ϩ ϩ ϩ ϩ CD4 CD25 Foxp3 T cells (gating on CD4 T cells: ATG 8.2 Ϯ 0.3 versus rabbit IgG 0.6 Ϯ 1.1%; n ϭ 3; P ϭ 0.003; Figure 5). These newly induced Treg showed high expression of Foxp3 ϩ ϩ similar to naturally occurring CD4 CD25 T cells that were ϩ ϩ expanded by ATG (gating on CD4 CD25 T cells 52.6 Ϯ 13.8 versus 63.4 Ϯ 12%; n ϭ 3; P ϭ 0.57) and also were equally capable of suppressing the proliferative response in an MLR (44 Ϯ 14 versus 5 Ϯ 7%; n ϭ 3; P ϭ 0.01).

Th2 Cytokines Play a Critical Role in the Expansion of Treg by ATG Next, to evaluate the role of regulatory cytokines, we deter- mined the frequency of cytokine-producing cells (IL-4, IL-5, IL-10, IL-13, and IFN-␥) by incubating PBL that were isolated from healthy volunteers with either ATG or rabbit IgG in ELISPOT plates for 48 h (Figure 6A). Expansion of Treg by ATG was accompanied by significant increase in IL-4– (64.5 Ϯ 34.1 versus 13 Ϯ 9.5; P ϭ 0.01), IL-5– (137 Ϯ 19.7 versus 45.8 Ϯ 46.7; P ϭ 0.004), and IL-10–producing PBL (247.8 Ϯ 65.9 versus 30.2 Ϯ 20.5; n ϭ 3; P ϭ 0.0003). Although the IL-13 production also was higher in the presence of ATG as compared with rabbit IgG, the difference did not reach statistical significance (data not shown). The analysis of TGF-␤1 (131.9 Ϯ 39.6 versus 307 Ϯ 112 pg/ml; n ϭ 7; NS) and TGF-␤2 (28.6 Ϯ 7.2 versus 38.3 Ϯ 9.5; n ϭ 6; NS) in the culture supernatants of PBL that were incubated with ATG or rabbit IgG did not reveal any significant difference. Even though the frequency of IFN-␥– producing cells overall was low, it still was slightly more in ATG-incubated cells as compared with controls (28.7 Ϯ 20.22 versus 14.6 Ϯ 10.3; n ϭ 4; P ϭ 0.003). To confirm the functional role of Th2 cytokines in expansion of Treg, we added each anti–IL-4, anti–IL-13, and anti–IL-10 antibodies to the generating cultures (each at 10 ␮g/ml) and analyzed the percentage of ϩ ϩ ϩ CD4 CD25 Foxp3 T cells (Figure 6B). Neutralization of each Figure 3. Suppressor function of Treg generated by ATG is re- of the cytokines resulted in reversal of Treg expansion by ATG stricted by responder cells in a mixed lymphocyte reaction (MLR) (anti–IL-4 4.2 Ϯ 0.4%, anti–IL-13 4.1 Ϯ 1.2%, anti–IL-10: 3.8 Ϯ but does not affect the memory response to recall antigen mumps. 0.3%, n ϭ 2, P Ͻ 0.01 for all versus ATG alone 10.4 Ϯ 2.5%, PBL were incubated with Thymoglobulin (Treg) or rabbit IgG Ϯ Ͻ (TControl) for 24 h in the generating cultures. The cells then were rabbit Ig alone 2.2 0.5, P 0.0001). collected and washed twice with PBS and added into an MLR assay. After5dofincubation, the proliferative response was Discussion measured by thymidine incorporation. There was significant sup- We and others previously reported that active regulation of pression of a direct alloimmune response of autologous respond- the alloimmune responses by Treg may function to maintain Ϯ ers (R) to donor antigens (S) (A; Treg 61.3 7.4 versus TControl hyporesponsiveness to alloantigens in stable renal transplant Ϯ ϭ ϭ 20.2 18.4; n 4; P 0.01). However, when the responder cells patients (12,13). A recent study of renal transplant patients who that were used originated from individuals other than the ones were experiencing acute rejection demonstrated increased lev- who were used to generate the Treg (third-party responders), els not only of mRNA of CD25 and perforin (markers of acti- there was no suppression of MLR (B). TControl did not exhibit vated and cytotoxic T cells) but also of Foxp3 (functional any inhibition of MLR regardless of donor–recipient combination. The proliferative response to recall antigen mumps was not inhib- marker of Treg) in samples of urinary lymphocytes (25). More ited by Treg, indicating that the expanded Treg did not block interesting, higher levels of Foxp3 mRNA were associated with memory cells to the antigen (C; mumps alone 4848 Ϯ 969, Treg improved probability of reversibility of acute rejection and 5214 Ϯ 900, control 9595 Ϯ 1231). lower risk for graft failure 6 mo after rejection episode. These 2850 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 2844–2853, 2006

ϩ ϩ Figure 4. ATG can induce moderate proliferation of preexisting CD4 CD25 T cells. PBL were labeled with carboxy-fluorescein diacetates succinimidyl ester (CFSE) and cultured in the presence of mitogen phytohemagglutinin, ATG (10 ␮g/ml) or rabbit IgG (10 ␮g/ml) for 72 h. The proportion of CFSE cells proliferating were calculated as described in the literature. We found three to ϩ ϩ four discrete division cycles in CD4 CD25 cells (proliferative cells 16 Ϯ 9%; n ϭ 3; P ϭ 0.01) but only one cycle of division in ϩ Ϫ a CD4 CD25 subpopulation that was incubated with ATG (7.4 Ϯ 7.7%) and none at all with rabbit IgG, suggesting a moderate ϩ ϩ ϩ degree of expansion of CD4 CD25 T cells by ATG. In contrast, the CD8 T cells that were incubated with ATG did not show proliferation at all (this is a representative example of three independent experiments). findings are consistent with the hypothesis that Treg also can gested increased activation but a decline in Foxp3 expression ϩ serve to limit anti-graft immunity during acute rejection. Taken by CD4 T cells. Although deletion of T cells by apoptosis has together, the above studies suggest that drugs that enhance the been demonstrated to promote immunoregulation (27), we did generation of Treg or administration of Treg themselves may not observe significant apoptosis after 24 h of incubation of PBL improve the outcome of both acute rejection episodes and the with ATG at the concentration of 10 ␮g/ml, suggesting that long-term survival of allografts overall. In this article, we report apoptosis is unlikely to contribute. Furthermore, whether the ϩ Ϫ the novel finding that ATG can lead to expansion of original CD4 CD25 T cells are truly naı¨ve T cells or regula- ϩ ϩ ϩ ϩ Ϫ CD4 CD25 Foxp3 Treg from naı¨ve CD4 CD25 T cells. Our tory cells that were generated initially in thymus and later lost data are in agreement with several other studies showing gen- the CD25 expression and then recovered their suppressive ac- ϩ ϩ eration of Foxp3-expressing CD4 CD25 T cells from tivity again upon activation remains unclear (9). It is interesting ϩ Ϫ CD4 CD25 precursors in the periphery (24,26). In contrast to not only that this process is accompanied by an increase in the Ϫ mouse, in which Foxp3 could be induced on Foxp3 cells under Th2 cytokines in the generating culture but also that in fact specialized conditions (e.g., activation plus TGF-␤) (24), human neutralization of each of the Th2 cytokines can significantly Ϫ CD25 T cells seemed to upregulate both CD25 and Foxp3 (26). hamper the Treg-expanding effects of ATG. While Th2 cyto- ϩ Ϫ Therefore, activation of human CD4 CD25 T cells led to kines have the capacity to promote Treg by various mecha- ϩ ϩ ϩ generation of regulatory CD4 CD25 Foxp3 T cells (26), rais- nisms such as altering T cell activation, affecting apoptosis or ing the possibility that ATG in a similar manner may expand cell proliferation, it is likely that they promote the conversion of ϩ Ϫ ϩ Ϫ the Treg by initially activating the CD4 CD25 T cells. Al- CD4 CD25 T cells into Treg, as conversion is established though further studies are needed to elucidate the exact mech- clearly as the main contributor to Treg expansion. These find- anism of the conversion, our dose-response experiments using ings are very intriguing and supported by recent published ATG at higher concentration in the generating culture sug- data demonstrating that IL-4R ␣-chain–binding cytokines, such J Am Soc Nephrol 17: 2844–2853, 2006 Ex Vivo Expansion of Regulatory T Cells by ATG 2851

ϩ Ϫ Figure 5. ATG induces Treg from CD4 CD25 T cells. PBL were depleted of CD25 using MACS columns. Such CD25-depleted T cells then were incubated for 24 h with ATG or rabbit IgG. Flow cytometric analysis showed an increase in CD25 expression on ϩ CD4 T cells (representative example of three independent experiments shown). The two-color staining demonstrated also clear ϩ ϩ ϩ ϩ expansion of the CD4 CD25 Foxp3 T cells (ATG 8.2 Ϯ 0.3 versus rabbit IgG 0.6 Ϯ 1.1%; n ϭ 3; P ϭ 0.003, gated on all CD4 T cells).

as IL-4 and IL-13, play a crucial role in generating inhibit the memory responses. Given the great interest in cel- ϩ ϩ Foxp3 CD25 Treg extrathymically (17). In addition, these lular treatment with ex vivo expanded Treg as a means to data point to an intriguing link between the well-established modulate alloimmune responses without affecting memory re- immunoregulatory capacity of Th2 cells and the potent sponses to infectious agents, these findings are of crucial im- ϩ ϩ CD4 CD25 regulatory T cells. These results also are impor- portance. tant in light of data showing that cyclosporine and tacrolimus, Although the in vivo effects of ATG on the induction, expan- two commonly used immunosuppressive drugs, both inhibit sion, and function of Treg in allograft recipients remain to be ϩ the production of IL-2, an essential growth factor for Treg (28). characterized fully, recent data demonstrated that CD25 T Conversely, murine T cells that were activated in the presence cells are spared from anti-lymphocyte serum–mediated dele- ϩ ϩ ϩ of rapamycin were highly enriched in Foxp3 CD4 CD25 T tion in mice, suggesting the concomitant presence of both T cell cells in vitro (29). Nevertheless, our findings here are unique depletion and continuous regulatory T cell activity in an in vivo because we clearly demonstrate that ATG can rapidly expand animal model (30). Second, in studying the in vivo effects of human Treg by promotion of Th2 cytokine production ex vivo. ATG in humans, the achieved concentrations of the ATG in Although the identification of the exact component of ATG blood and lymphoid tissues should be taken carefully into (Genzyme; generated by using thymocytes as the immunogen) consideration. ATG is used at a dosage of 1 to 1.5 mg/kg per d that is responsible for the expansion of Treg will require further for3to5dasinduction immunosuppression and also for investigation, our ability to generate Treg using a different ATG treatment of steroid-resistant acute rejection, leading to serum ϩ (Fresenius; produced with purified and activated CD3 lym- levels of the drug between 50 and 100 ␮g/ml (2,5). This dosing phoblastic cells as immunogen) suggests a key role for antibod- regimen is based on ATG’s efficacy to deplete T cells in the ies that are directed against epitopes on T cells. Another unique peripheral blood rather than promotion of Treg. There are no finding of the study is that ATG-induced Treg significantly human data regarding the achieved concentrations in second- suppressed direct alloimmune responses to alloantigens by ary lymphoid tissues, a potentially important site for induc- autologous PBL from which Treg originally were generated. In tion/expansion of Treg, although the data in primates suggest contrast, nonautologous responders could not be regulated by good penetration into lymph nodes and spleen but minimal the Treg. Conversely, the memory response to recall antigen penetration into thymus (31). We were able to expand Treg in mumps is not affected. This may be due to lack of activation of vitro with a concentration of 10 ␮g/ml, a dosage that is signif- Treg by mumps antigen or the overall inability of Treg to icantly lower than the levels that currently are achieved in 2852 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 2844–2853, 2006

ϩ ϩ Figure 6. Th2 cytokines play a key role in the expansion of Treg. (A) Expansion of CD4 CD25 T cells by ATG in the generating culture is accompanied by Th2 cytokine production: PBL from healthy donors were incubated in an ELISPOT plate in the presence of ATG or rabbit IgG for 48 h (triplicate). The quantification of spots (mean of at least three independent experiments for each cytokine) revealed significant increase of IL-4–, IL-5–, and IL-10–producing PBL that were incubated with ATG. (B) Neutralization of Th2 cytokines decreases expansion of Treg: Anti–IL-4, anti–IL-10, and anti–IL-13 mAb or corresponding isotype controls each were added separately to PBL that were incubated with ATG or rabbit IgG (10 ␮g/ml). Cells then were harvested after 24 h, and ϩ ϩ ϩ ϩ %CD4 CD25 Foxp3 T cells (gated on CD4 lymphocytes) were measured by flow cytometry. The neutralization led to a ϩ significant decline in percentage of CD4 T cells that expressed CD25 and Foxp3. Mean values of two independent experiments are shown here.

blood. In fact, our in vitro data may suggest that lower dosages recipient of the American Society of Nephrology John Merrill Trans- of ATG may lead to expansion of Treg in vivo, but clearly plant Scholar Grant and the American Heart Association Scientist De- further investigation is needed and is currently ongoing. velopment Grant. M.H.S. is a consultant for Genzyme.

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See the related editorial, “Immunosuppression and Regulation: Cast in New Light,” on pages 2644–2646.