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

Cells and Antibodies

This study was performed with the approval of the Institutional Review Board for human investigation at the Brigham and Women’s Hospital. Blood from healthy volunteers were obtained in heparinized tubes, and peripheral blood lymphocytes (PBL) were isolated by standard Ficoll-density-gradient centrifugation. Two types of ATG were used: A rabbit polyclonal serum raised against human thymocytes (Thymoglobulin; Genzyme, Cambridge MA) or a rabbit polyclonal serum raised against the lymphoblastic Jurkat T cell line (Fresenius, Bad Homburg, Germany). Purified rabbit polyclonal IgG was used as control. To compare the effects of ATG with agents that block the IL-2 receptor α-chain (IL-2Rα) on the surface of activated T lymphocytes, we used either Basiliximab (Simulect, Novartis, NJ), a chimeric (murine/human) mAb (IgG_1k), or Daclizumab (Zenapax, Roche, NJ), a humanized IgG1 mAb. In addition, we used alemtuzumab (Campath1-H, Genzyme, Cambridge, MA), a recombinant DNA-derived humanized mAb that is directed against the cell surface glycoprotein CD52, for comparison studies with ATG.

Generation of Treg

PBL (1 × 10⁶ per ml) from 10 healthy donors were incubated with ATG (Treg) or rabbit IgG (TControl) in RPMI 1640 medium (Cambrex Bioscience, Walkersville, MD) supplemented with 10% heat-inactivated human serum at 37°C with a Pco₂ 5% for various durations (6 to 96 h). All above agents were used in vitro at concentrations that ranged from 1 to 100 μg/ml.

These cultures are called “generating cultures.” For assessment of the role of cytokines in the expansion of Treg, anti–IL-4, anti–IL-13, and anti–IL-10 mAb each at a concentration of 10 μg/ml were added separately to the generating cultures (17). All of these antibodies were purchased from BD Bioscience (San Jose, CA).

In Vitro Suppression Assay

To test the suppression function of cells that were generated under conditions explained (Treg), we set up a mixed lymphocyte reaction (MLR) assay: 1 × 10⁵ cells that were obtained from the generating cultures above were co-cultured (1:1 ratio) with fresh responder (autologous or third-party PBL) and irradiated stimulator cells in a 96-well plate (96-well Cell Culture Cluster, round-bottom culture plate; COSTAR, New York, NY) for 120 h. The cultures were labeled with ³H-thymidine during the last 8 h of culture (Amersham Pharmacia Biotech, Piscataway, NJ). Cells then were harvested, and radionuclide uptake was measured by a scintillation counting machine. In a similar manner, we also tested whether Treg could suppress recall responses to mumps antigens.

Flow Cytometry

Cells that were harvested from generating cultures (Treg or TControl) were analyzed using flow cytometric analysis. A total of 2 × 10⁵ cells per sample were stained with anti-human CD4-allophycocyanin (APC), CD25-phycoerythrin (PE), and glucocorticoid-induced TNF receptor (GITR)-FITC, CD8-APC (BD Bioscience; eBioscience, San Diego, CA). For the intracellular cytotoxic T lymphocyte–associated antigen-4 (CTLA-4) staining, cells were permeabilized with Perm Buffer (BD Biosciences) for 20 min at 4°C and labeled with CTLA-4 for 30 min at 4°C. For flow cytometric analysis of Foxp3, 1 × 10⁶ cells first were stained with anti-human CD4-APC and CD25-PE. After washing, cells were resuspended in 1 ml of cold Fix/Perm Buffer (eBioscience) and incubated at 4°C overnight in the dark. After washing twice with 2 ml of permeabilization buffer, cells were blocked with 2% normal rat serum for 15 min. Anti-human Foxp3-FITC (PCH101; eBioscience) then was added, and cells were incubated at 4°C for another 30 min in the dark. Finally, cells were washed with 2 ml of permeabilization buffer and analyzed on a FACSCalibur flow cytometer using CellQuest software (Becton Dickinson, San Jose, CA). For evaluation of the induction of apoptosis/death of CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells, PBL that were incubated with ATG or rabbit IgG were stained with mAb against CD4, CD25, annexin-V, and 7-amino-actinomycin D as per the manufacturer’s instructions (BD Bioscience).

ELISPOT Assay

To evaluate the frequency of cytokine-producing cells in the generating culture, we made use of the ELISPOT assay as described previously (12). The resulting spots were counted on a computer-assisted ELISASpot Image Analyzer (Cellular Technology Limited, Cleveland, OH). Cells were tested in triplicate wells. The frequencies then were expressed as the number of spots per million PBL.

Luminex Assay

Supernatants from generating cultures (serum-free medium) were tested for the presence of TGF-β by Luminex 100 system (Luminex Corp., Austin, TX). Beadlyte Human Multi-Cytokine Beadmaster Kit and Beadlyte Human TGF-β1/β2 Detection system (Upstate, Charlottesville, VA) were used as per protocol provided by the manufacturer. Plates were analyzed using Multiplex Data Analysis software version 1 (Luminex, Austin, TX).

Carboxy-Fluorescein Diacetates Succinimidyl Ester Staining

To test whether the expansion of Treg by ATG is due to proliferation of preexisting naturally occurring CD4⁺CD25⁺ T cells, we incubated PBL with carboxy-fluorescein diacetates succinimidyl ester (CFSE) in the form of 5 mM stock solution in DMSO at a final concentration of 1 μM for 6 min at room temperature. CFSE-labeled cells were cultured in vitro with phytohemagglutinin (positive control), ATG, and rabbit IgG for 72 h at 37°C. Cells then were stained with anti-human CD4-APC, CD8-PE, and CD25-PE. 7-Amino-actinomycin D was used to exclude death cells.

Conversion of CD4⁺CD25⁻ to CD4⁺CD25⁺ T Cells

For testing whether Treg that are generated by ATG are induced from CD4⁺CD25⁻ T cells, PBL were depleted of CD25 by magnetic cell sorting using MACS columns and MACS separators (Miltenyi Biotec, Auburn, CA). CD25-depleted CD4⁺ T cells then were incubated with ATG or rabbit IgG for 24 h. The cells then were harvested and stained for CD25 and regulatory markers. Similarly, their suppressor activity was assessed as described previously.

Statistical Analyses

The t test was used for comparison of means between experimental groups examined by flow cytometry and ELISPOT assay. Differences were considered to be significant at P < 0.05.

ATG Expands CD4⁺CD25⁺Foxp3⁺ T Cells Ex Vivo

PBL that were derived from healthy volunteers were incubated with Thymoglobulin or rabbit IgG (10 μg/ml) for various time periods (0, 6, 18, 24, 48, 72, and 96 h). Flow cytometric analysis of harvested cells demonstrated a significant upregulation of CD25 expression that began at 18 h and was maintained beyond 96 h, with peak expression of CD25 at 24 h (percentage of CD4⁺CD25⁺ T cells gated on all viable lymphocytes 20.5 ± 7.8 versus 4.5 ± 1.6; P = 0.002; n = 7; Figures 1A and 2A).

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Figure 1.

Anti-thymocyte globulin (ATG) expands CD4⁺CD25⁺ Foxp3⁺ T cells ex vivo. (A) A representative example of seven 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 of 10 μg/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 ± 12 versus 6.6 ± 4%; n = 5), intracellular cytotoxic T lymphocyte–associated antigen-4 (CTLA-4) (41.3 ± 19.5 versus 7 ± 1.8%; n = 4), and forkhead box P3 (Foxp3; 65.3 ± 21.5 versus 43.8 ± 12.3; n = 5) as compared with control cells (percentage expression of regulatory markers calculated when gating 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 ± 2.9 versus 0.7 ± 0.5%; n = 8; P = 0.001). More important, representative examples of two color flow staining (CD25 and each of regulatory markers GITR, CTLA-4, and Foxp3) are shown to compare better the expression of regulatory markers on CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells after incubation with ATG or rabbit IgG. Among all CD4⁺ T cells (see gate), the percentage of cells that expressed CD25 and GITR (ATG 18.3 ± 6.9, rabbit IgG 0.5 ± 0.4; n = 6; P = 0.001) or CD25 and CTLA-4 (ATG 19.5 ± 6.2, rabbit IgG 1.5 ± 0.4; n = 4; P = 0.008) was markedly enhanced when cells were incubated with ATG versus rabbit IgG. Similarly, gating on all CD4⁺ T cells, the percentage of cells that expressed CD25 and Foxp3 (%CD4⁺CD25⁺Foxp3⁺ T cells) was significantly increased when cells were treated with ATG versus rabbit IgG (ATG 10.4 ± 2.5 versus rabbit IgG 2.2 ± 0.5; n = 8; P < 0.0001). In contrast, the percentage of CD25⁻ T cells that expressed GITR (ATG 1.8 ± 1.5%, rabbit IgG 0.3 ± 0.2; n = 6; NS) or CTLA-4 (ATG 5.4 ± 3.7, rabbit IgG 1.3 ± 0.7; n = 4; NS) was minimal. In addition, the percentage of CD4⁺CD25⁻Foxp3⁺ T cells (gated on CD4⁺ T cells) was minimal after incubation with ATG versus rabbit IgG (1.1 ± 0.6 versus 0.7 ± 0.4), indicating that ATG did not induce CD4⁺CD25⁻Foxp3⁺ regulatory T cells (Treg).

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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 μg/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 (basiliximab and daclizumab) did not expand CD4⁺CD25⁺ T cells (representative experiment of n = 3).

To evaluate the dose-response of ATG and expansion of CD4⁺CD25⁺ T cells, we incubated PBL with ATG or rabbit IgG at various concentrations (1, 5, 10, 50, and 100 μg/ml) for 24 h (percentage of CD4⁺CD25⁺ T cells gated on all viable lymphocytes: 1 μg/ml 6.3 ± 0.5, 5 μg/ml 12 ± 4.9, 10 μg/ml 20 ± 6.5, 50 μg/ml 21.7 ± 5, and 100 μg/ml 21 ± 6.7; percentage of CD4⁺CD25⁺ T cells with various concentrations of rabbit Ig is between 3 and 5). We found a dose-dependent increase in percentage of CD4⁺CD25⁺ T cells using between 1 and 10 μg/ml ATG. In contrast, the percentage of CD4⁺CD25⁺ T cells does not seem to increase much further when PBL are incubated with 50 to 100 μg/ml ATG, and we observed increased activation of CD4⁺ T cells (percentage of CD4⁺CD69⁺ T cells: 1 μg/ml 4.4 ± 4.8, 5 μg/ml 15.6 ± 7.7, 10 μg/ml 12 ± 7.2, 50 μg/ml 21 ± 5.6 and 100 μg/ml 22 ± 5; percentage of CD4⁺CD69⁺ T cells with various concentration of rabbit Ig is <1%), a change in cell size, and increasingly poor viability of cells. These data suggest increased activation of CD4⁺ T cells and a decline in expansion of CD4⁺CD25⁺ T at higher concentrations of ATG.

Because the regulatory function in humans is attributed mainly to the CD25^high subset (18), we also compared the frequency of CD4⁺CD25⁺ high population in ATG-treated versus rabbit IgG–treated cells and found it to be significantly increased in the former group (6.5 ± 2.9 versus 0.7 ± 0.5%; P = 0.001; n = 8; Figure 1B). These results also could be duplicated using a second ATG preparation (Fresenius; at 10 μg/ml for 24 h) that was generated from rabbit serum against the lymphoblastic Jurkat T cell line (percentage of CD4⁺CD25⁺ T cells: 16.7 ± 4.2 versus 4.5 ± 1.6, P = 0.04, n = 3; percentage of CD25^high subset of T cells: 4.7 ± 1 versus 0.7 ± 0.5, P = 0.02, n = 3).

Because alemtuzumab (anti-CD52 mAb; Campath-1H) and IL-2R antagonists are commonly used as induction agents in organ transplantation and are currently being tested in various autoimmune diseases, we next explored whether they can expand CD4⁺CD25⁺ T cells similar to ATG. It is interesting that neither of these agents could expand CD4⁺CD25⁺ T cells in vitro (Figure 2B). Therefore, the expansion observed is unique to polyclonal ATG.

The study of Treg is complicated by a paucity of phenotypic markers to distinguish activated effector T cells from Treg. Expression of molecules such as CTLA-4 and GITR (10) and secretion of regulatory cytokines such as TGF-β (19) and IL-10 (20,21) each have been linked with but not proved to be entirely specific for Treg. Therefore, we first analyzed the surface expression of regulatory markers GITR and CTLA-4 on the expanded CD4⁺CD25⁺ T cells (Figure 1A). GITR showed enhanced expression in ATG-induced CD4⁺CD25⁺ T cells as compared with CD4⁺CD25⁺ T cells that were incubated with rabbit IgG (32 ± 12 versus 6.6 ± 4%; P = 0.005; n = 5). Similar results were observed for intracellular CTLA-4 expression (41.3 ± 19.5 versus 7 ± 1.8%; P = 0.04; n = 4). When gating on all CD4⁺ T cells, the percentage of cells that expressed both CD25 and GITR (ATG 18.3 ± 6.9, rabbit IgG 0.5 ± 0.4; n = 6; P = 0.001) or CD25 and CTLA-4 (ATG 19.5 ± 6.2, rabbit IgG 1.5 ± 0.4; n = 4; P = 0.008) was markedly enhanced when cells were incubated with ATG versus rabbit IgG (Figure 1B).

In contrast to GITR and CTLA-4, Foxp3, a gene that encodes a forkhead winged helix transcription factor scurfin, is required for Treg development and function (22,23). CD4⁺CD25⁺ T cells that were expanded by ATG (at 10 μg/ml) showed higher expression of Foxp3 as compared with those that were incubated with rabbit IgG (63.4 ± 12 versus 47 ± 8.3%; P = 0.03; n = 5; Figure 1A). It is interesting that contrary to CD69 expression (see above), the Foxp3 expression declined with increasing dosage of ATG in the generating culture (percentage of Foxp3 expression in CD4⁺CD25⁺ T cells: 50 μg/ml 13 ± 4.2, 100 μg/ml 15.2 ± 0.35). Importantly, gating on all CD4⁺ T cells, the percentage of cells that expressed both CD25 and Foxp3 (percentage of CD4⁺CD25⁺Foxp3⁺ T cells) was significantly increased when cells were treated with ATG versus rabbit IgG (ATG 10.4 ± 2.5 versus rabbit IgG 2.2 ± 0.5; P < 0.0001; n = 8; Figure 1B). In addition, expression of all of the regulatory markers was even markedly more enhanced in the CD4⁺CD25⁺ high population that was expanded after incubation with ATG (GITR 49.4 ± 15.9%, n = 7; CTLA-4 55 ± 24.4%, n = 6; Foxp3 71 ± 14.7%, n = 5).

Previous work indicated that Foxp3 also can be induced in CD4⁺CD25⁻ T cells and that these cells can function as Treg (24). However, in contrast to CD4⁺CD25⁺ T cells, CD4⁺CD25⁻ T cells that were incubated with ATG or rabbit IgG showed only minimal increase in GITR (5.6 ± 4.4 versus 0) and CTLA-4 (11.5 ± 4.2 versus 0). Similarly, gating on all CD4⁺ T cells, the percentage of CD25⁻ T cells that expressed GITR (ATG 1.8 ± 1.5% rabbit IgG 0.3 ± 0.2; n = 6; NS) or CTLA-4 (ATG 5.4 ± 3.7, rabbit IgG 1.3 ± 0.7; n = 4; NS) was minimal (Figure 1B). In addition, the percentage of CD4⁺CD25⁻Foxp3⁺ T cells (gated on CD4⁺ T cells) was minimal after incubation with ATG versus rabbit IgG (1.1 ± 0.6 versus 0.7 ± 0.4), indicating that after incubation with ATG, CD4⁺CD25⁻ T cells remain Foxp3⁻ (Figure 1B). Although overall there was a slight decrease in the CD8⁺ T cells after incubation with ATG as compared with rabbit IgG (21.96 ± 4.5 versus 25.6 ± 5.1; n = 8; P = 0.02), we found no significant difference in the percentage of CD8⁺CD28⁻ T cells (11.3 ± 5.6 versus 14.9 ± 6; n = 5). In addition, we found no evidence of CD8⁺Foxp3⁺ T cells before or after treatment with ATG or rabbit IgG. Taken together, these data do not indicate expansion of other previously described Treg populations in our model.

Suppressor Function of CD4⁺CD25⁺ T Cells Generated by ATG Is Restricted to Responder Cells

After demonstrating that CD4⁺CD25⁺ T cells that are expanded by ATG can maintain their regulatory phenotype, we next set out to study the actual suppressor function of these cells in vitro. The in vitro suppressive activity of the cells was evaluated by examination of their ability to suppress an MLR to donor alloantigens. We found significant suppression of direct alloimmune responses of the original responders (PBL from which the Treg initially were generated) to stimulator cells by Treg but not by TControl (1:1 ratio, 61.3 ± 7.4 inhibition versus 20.2 ± 18.4%; n = 4; P = 0.01; Figure 3A). It is interesting that the same Treg were unable to suppress an MLR of responder cells that were not autologous to the original responder cells (Figure 3B). We next tested the ability of these Treg to suppress a recall memory response of original responders to mumps antigen. It is interesting that the proliferative response to recall antigen mumps was not inhibited by Treg, indicating that the expanded Treg did not block memory cells to the antigen (mumps alone 4848 ± 969, Treg 5214 ± 900, control 9595 ± 1231; Figure 3C).

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Figure 3.

Suppressor function of Treg generated by ATG is restricted by responder cells in a mixed lymphocyte reaction (MLR) but does not affect the memory response to recall antigen mumps. PBL were incubated with Thymoglobulin (Treg) or rabbit IgG (TControl) for 24 h in the generating cultures. The cells then were collected and washed twice with PBS and added into an MLR assay. After 5 d of incubation, the proliferative response was measured by thymidine incorporation. There was significant suppression of a direct alloimmune response of autologous responders (R) to donor antigens (S) (A; Treg 61.3 ± 7.4 versus TControl 20.2 ± 18.4; n = 4; P = 0.01). However, when the responder cells that were used originated from individuals other than the ones who were used to generate the Treg (third-party responders), there was no suppression of MLR (B). TControl did not exhibit any inhibition of MLR regardless of donor–recipient combination. The proliferative response to recall antigen mumps was not inhibited by Treg, indicating that the expanded Treg did not block memory cells to the antigen (C; mumps alone 4848 ± 969, Treg 5214 ± 900, control 9595 ± 1231).

Conversion of CD4⁺CD25⁻ into CD4⁺CD25⁺ T Cells Is the Main Mechanism of Expansion of Treg by ATG

The absolute number of CD4⁺CD25⁺ T cells (mean values of cell numbers counted in wells) that were incubated with ATG (before 75,833 ± 31,051, after 639,167 ± 249,448; n = 6; P = 0.002) but not rabbit IgG was dramatically increased (before 90,833 ± 33,229, after 113,333 ± 54,283; NS) after 24 h of incubation. In contrast, there was much more decrease in the number of CD4⁺CD25⁻ T cells that were treated with ATG (before 1878,300 ± 322,020, after 1082,500 ± 301,027; P = 0.005) as compared with rabbit IgG (1991,700 ± 379,548 versus 1861,666 ± 238 362; NS). On the basis of these findings, we hypothesized that the expansion of Treg by ATG may result from one or more mutually nonexclusive and possibly complementary mechanisms: First, ATG may preferentially promote death/apoptosis of CD4⁺CD25⁻ T cells as compared with CD4⁺CD25⁺ T cells, creating a new balance in favor of the latter cells. Second, ATG may promote the proliferation of already existing, naturally occurring CD4⁺CD25⁺ T cells. Third, ATG may be able to convert CD4⁺CD25⁻ T cells into CD4⁺CD25⁺ T cells with regulatory functions (induction of Treg). The first possibility is supported by published data demonstrating that ATG in fact can bind to multiple epitopes on the T cell surface and induce apoptosis in T lymphocytes through Fas-L (CD95L) (3,5). Nevertheless, we found no difference (gating on all lymphocytes without excluding the dead cells) in apoptosis of CD4⁺CD25⁺ T cells and CD4⁺CD25⁻ T cells that were incubated for 24 h with 10 μg/ml ATG (6.7 ± 3.1 versus 5 ± 4.7%) or rabbit IgG (5 ± 3 versus 3.2 ± 2.5%). In fact, the overall viability of the cells was >95% regardless of incubation with ATG or rabbit IgG.

To address the possibility of the proliferative effect of ATG on already existing, naturally occurring Treg, we cultured in vitro CFSE-labeled PBL with ATG or rabbit IgG for 72 h (10 μg/ml). We found three to four discrete division cycles in CD4⁺CD25⁺ cells (proliferative cells 16 ± 9%; n = 3; P = 0.01) but only one cycle of division in a CD4⁺CD25⁻ subpopulation that was incubated with ATG (7.4 ± 7.7%) and none at all with rabbit IgG, suggesting a moderate degree of expansion of CD4⁺CD25⁺ T cells. It is possible that some of the proliferating CD4⁺CD25⁺ T cells simply may be activated T cells rather than proliferating naturally occurring Treg. Nevertheless, whereas there is an eight-fold expansion of CD4⁺CD25⁺ T cells at 24 h with ATG, proliferating cells make up only approximately 16% of the CD25⁺ population at 72 h. This indicates that proliferation is unlikely to contribute significantly to the expansion of the cells. In contrast, the CD8⁺ T cells that were incubated with ATG did not show proliferation at all (Figure 4).

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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 μg/ml) or rabbit IgG (10 μg/ml) for 72 h. The proportion of CFSE cells proliferating were calculated as described in the literature. We found three to four discrete division cycles in CD4⁺CD25⁺ cells (proliferative cells 16 ± 9%; n = 3; P = 0.01) but only one cycle of division in a CD4⁺CD25⁻ subpopulation that was incubated with ATG (7.4 ± 7.7%) and none at all with rabbit IgG, suggesting a moderate 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).

To determine whether ATG is capable of converting CD4⁺CD25⁻ cells into CD4⁺CD25⁺ cells with regulatory properties, we first depleted naturally occurring CD25⁺ T cells from PBL ex vivo with magnetic bead separation (before 5.9 ± 2.9 versus after depletion 0.6 ± 0.9%; Figure 5A). Such CD25-depleted T cells then were incubated for 24 h with ATG or rabbit IgG, after which the induction of CD25⁺ T cells was evaluated by flow cytometry. We found not only significant upregulation of CD25 expression on CD4⁺ T cells that were incubated with ATG but not rabbit IgG (18.7 ± 4 versus 3.4 ± 1.8%; n = 5; P = 0.02) but also clear expansion of the CD4⁺CD25⁺Foxp3⁺ T cells (gating on CD4⁺ T cells: ATG 8.2 ± 0.3 versus rabbit IgG 0.6 ± 1.1%; n = 3; P = 0.003; Figure 5). These newly induced Treg showed high expression of Foxp3 similar to naturally occurring CD4⁺CD25⁺ T cells that were expanded by ATG (gating on CD4⁺CD25⁺ T cells 52.6 ± 13.8 versus 63.4 ± 12%; n = 3; P = 0.57) and also were equally capable of suppressing the proliferative response in an MLR (44 ± 14 versus 5 ± 7%; n = 3; P = 0.01).

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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 CD4⁺ T cells (representative example of three independent experiments shown). The two-color staining demonstrated also clear expansion of the CD4⁺CD25⁺Foxp3⁺ T cells (ATG 8.2 ± 0.3 versus rabbit IgG 0.6 ± 1.1%; n = 3; P = 0.003, gated on all CD4⁺ T cells).

Th2 Cytokines Play a Critical Role in the Expansion of Treg by ATG

Next, to evaluate the role of regulatory cytokines, we determined the frequency of cytokine-producing cells (IL-4, IL-5, IL-10, IL-13, and IFN-γ) 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 ± 34.1 versus 13 ± 9.5; P = 0.01), IL-5–(137 ± 19.7 versus 45.8 ± 46.7; P = 0.004), and IL-10–producing PBL (247.8 ± 65.9 versus 30.2 ± 20.5; n = 3; P = 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-β1 (131.9 ± 39.6 versus 307 ± 112 pg/ml; n = 7; NS) and TGF-β2 (28.6 ± 7.2 versus 38.3 ± 9.5; n = 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-γ–producing cells overall was low, it still was slightly more in ATG-incubated cells as compared with controls (28.7 ± 20.22 versus 14.6 ± 10.3; n = 4; P = 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 μg/ml) and analyzed the percentage of CD4⁺CD25⁺Foxp3⁺ T cells (Figure 6B). Neutralization of each of the cytokines resulted in reversal of Treg expansion by ATG (anti–IL-4 4.2 ± 0.4%, anti–IL-13 4.1 ± 1.2%, anti–IL-10: 3.8 ± 0.3%, n = 2, P < 0.01 for all versus ATG alone 10.4 ± 2.5%, rabbit Ig alone 2.2 ± 0.5, P < 0.0001).

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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 μg/ml). Cells then were harvested after 24 h, and %CD4⁺CD25⁺Foxp3⁺ T cells (gated on CD4⁺ lymphocytes) were measured by flow cytometry. The neutralization led to a significant decline in percentage of CD4⁺ T cells that expressed CD25 and Foxp3. Mean values of two independent experiments are shown here.