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 Womens Hospital, and Childrens Hospital Boston, Harvard Medical
School, Boston, Massachusetts
T celldepleting agents are being tested as part of clinical tolerance strategies in humans with autoimmunity and transplan-
tation. 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 lymphocyteassociated 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: 28442853, 2006. doi: 10.1681/ASN.2006050422
P
olyclonal anti-thymocyte globulin (ATG) is the purified
IgG fraction of sera from rabbits, horses, or, more
rarely, goats that are immunized with human thymo-
cytes or T cell lines. ATG is used for treatment of various
clinical conditions, including prevention or rescue treatment of
acute rejection in organ transplantation (1), conditioning for
hematopoietic stem cell transplantation, treatment of severe
aplastic anemia, various autoimmune diseases, and more re-
cently graft-versus-host disease (2). The immunosuppressive
activity of ATG has been thought to result primarily from the
depletion of peripheral lymphocytes from the circulating pool
through complement-dependent lysis or activation-associated
apoptosis (1,35). Other potential mechanisms of action include
modulation of surface adhesion molecules or chemokine recep-
tor expression (6).
Regulatory T cells (Treg) are specialized T cell subsets that
play important roles in maintaining immune homeostasis (7,8).
Treg are characterized by the expression of the IL-2 receptor
-chain (CD25) and the transcription factor forkhead box P3
(Foxp3) (9). There exists emerging evidence in both rodents and
humans to support important immunoregulatory functions of
CD4

CD25

T Treg in maintaining both self-tolerance and

tolerance toward autoantigens (8) and alloantigens (10). Treg
also may play a role in preventing human renal autoimmune
diseases such as Goodpastures disease (11). Our group previ-
ously demonstrated that active regulation of the alloimmune
responses by Treg may function to maintain hyporesponsive-
ness to alloantigens in renal transplant patients (12,13). In pre-
clinical animal models, ex vivo expanded Treg protect mice
from lethal graft-versus-host disease (14). In fact, a clinical trial
was proposed recently to use ex vivo expanded Treg at the time
of hematopoietic stem cell transplantation (15,16). Therefore,
understanding the conditions that are required for the genera-
tion and propagation of Treg would allow development of
novel therapeutic strategies for inducing immunologic toler-
ance in various immune-mediated diseases.
Here we report the novel finding that ATG-mediated immu-
nosuppression is delivered in part via immunologically specific
actions involving the generation of Treg, particularly
CD4

CD25
high
Foxp3

cells. This is due mainly to ATGs

unique ability to convert the CD4

CD25

T cells into
CD4

CD25

T cells. More important, in a mixed lymphocyte

reaction, the regulatory function is restricted to autologous
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
immunomodulatory protocols for immune-mediated diseases.
Materials and Methods
Cells and Antibodies
This study was performed with the approval of the Institutional
Review Board for human investigation at the Brigham and Womens
Received May 1, 2006. Accepted June 19, 2006.
Published online ahead of print. Publication date available at www.jasn.org.
Address correspondence to: Dr. Nader Najafian, Brigham & Womens Hospital,
Transplantation Research Center, EBRC, 221 Longwood Avenue, 3rd Floor, Bos-
ton, MA 02115. Phone: 617-732-5259; Fax: 617-732-5254; E-mail:
nnajafian@rics.bwh.harvard.edu
Copyright 2006 by the American Society of Nephrology ISSN: 1046-6673/1710-2844
Hospital. Blood from healthy volunteers were obtained in heparinized
tubes, and peripheral blood lymphocytes (PBL) were isolated by stan-
dard 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 -chain (IL-2R) 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 human-
ized 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 10
6
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 37C with a Pco
2
5% for various durations (6 to 96 h).
All above agents were used in vitro at concentrations that ranged from
1 to 100 g/ml.
These cultures are called generating cultures. For assessment of the
role of cytokines in the expansion of Treg, antiIL-4, antiIL-13, and
antiIL-10 mAb each at a concentration of 10 g/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 10
5
cells that were obtained from the generating
cultures above were co-cultured (1:1 ratio) with fresh responder (au-
tologous 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
3
H-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 TCon-
trol) were analyzed using flow cytometric analysis. A total of 2 10
5
cells per sample were stained with anti-human CD4-allophycocyanin
(APC), CD25-phycoerythrin (PE), and glucocorticoid-induced TNF re-
ceptor (GITR)-FITC, CD8-APC (BD Bioscience; eBioscience, San Diego,
CA). For the intracellular cytotoxic T lymphocyteassociated antigen-4
(CTLA-4) staining, cells were permeabilized with Perm Buffer (BD
Biosciences) for 20 min at 4C and labeled with CTLA-4 for 30 min at
4C. For flow cytometric analysis of Foxp3, 1 10
6
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 4C 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 4C 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 soft-
ware (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 manu-
facturers instructions (BD Bioscience).
ELISPOT Assay
To evaluate the frequency of cytokine-producing cells in the gener-
ating culture, we made use of the ELISPOT assay as described previ-
ously (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- by Luminex 100 system (Luminex
Corp., Austin, TX). Beadlyte Human Multi-Cytokine Beadmaster Kit
and Beadlyte Human TGF-1/2 Detection system (Upstate, Char-
lottesville, VA) were used as per protocol provided by the manufac-
turer. 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
M 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 37C. 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.
Results
ATG Expands CD4

CD25

Foxp3

T Cells Ex Vivo
PBL that were derived from healthy volunteers were incu-
bated with Thymoglobulin or rabbit IgG (10 g/ml) for various
time periods (0, 6, 18, 24, 48, 72, and 96 h). Flow cytometric
analysis of harvested cells demonstrated a significant upregu-
lation of CD25 expression that began at 18 h and was main-
tained beyond 96 h, with peak expression of CD25 at 24 h
(percentage of CD4

CD25

T cells gated on all viable lympho-

cytes 20.5 7.8 versus 4.5 1.6; P 0.002; n 7; Figures 1A
and 2A).
To evaluate the dose-response of ATG and expansion of
J Am Soc Nephrol 17: 28442853, 2006 Ex Vivo Expansion of Regulatory T Cells by ATG 2845
CD4

CD25

T cells, we incubated PBL with ATG or rabbit IgG

at various concentrations (1, 5, 10, 50, and 100 g/ml) for 24 h
(percentage of CD4

CD25

T cells gated on all viable lympho-

cytes: 1 g/ml 6.3 0.5, 5 g/ml 12 4.9, 10 g/ml 20 6.5,
50 g/ml 21.7 5, and 100 g/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

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 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 12 versus 6.6 4%; n 5), intracellular
cytotoxic T lymphocyteassociated 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 IgGtreated 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).

2846 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 28442853, 2006
g/ml ATG. In contrast, the percentage of CD4

CD25

T cells
does not seem to increase much further when PBL are incu-
bated with 50 to 100 g/ml ATG, and we observed increased
activation of CD4

T cells (percentage of CD4

CD69

T cells:
1 g/ml 4.4 4.8, 5 g/ml 15.6 7.7, 10 g/ml 12 7.2, 50
g/ml 21 5.6 and 100 g/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 concen-
trations 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 ver-

sus rabbit IgGtreated 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 g/ml for
24 h) that was generated from rabbit serum against the lym-
phoblastic 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 ex-
pand 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- (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 ex-
pression of regulatory markers GITR and CTLA-4 on the ex-
panded CD4

CD25

T cells (Figure 1A). GITR showed en-

hanced 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 g/ml) showed higher
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 antiIL-2 receptor agents
(basiliximab and daclizumab) did not expand CD4

CD25

T cells (representative experiment of n 3).

J Am Soc Nephrol 17: 28442853, 2006 Ex Vivo Expansion of Regulatory T Cells by ATG 2847
expression of Foxp3 as compared with those that were incu-
bated 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 g/ml 13 4.2, 100

g/ml 15.2 0.35). Importantly, gating on all CD4

T cells, the
percentage of cells that expressed both CD25 and Foxp3 (per-
centage of CD4

CD25

Foxp3

T cells) was significantly in-

creased 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 incuba-

tion 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

(Fig-
ure 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 de-
scribed 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 ex-

panded 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).
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 comple-
mentary 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 dem-
onstrating 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 (gat-
ing 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 g/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
g/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 prolifera-

tion 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).
To determine whether ATG is capable of converting
CD4

CD25

cells into CD4

CD25

cells with regulatory prop-

erties, 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-
2848 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 28442853, 2006
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).
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 34.1
versus 13 9.5; P 0.01), IL-5 (137 19.7 versus 45.8 46.7;
P 0.004), and IL-10producing 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-1 (131.9 39.6 versus
307 112 pg/ml; n 7; NS) and TGF-2 (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-
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
antiIL-4, antiIL-13, and antiIL-10 antibodies to the generat-
ing cultures (each at 10 g/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
(antiIL-4 4.2 0.4%, antiIL-13 4.1 1.2%, antiIL-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).
Discussion
We and others previously reported that active regulation of
the alloimmune responses by Treg may function to maintain
hyporesponsiveness to alloantigens in stable renal transplant
patients (12,13). A recent study of renal transplant patients who
were experiencing acute rejection demonstrated increased lev-
els not only of mRNA of CD25 and perforin (markers of acti-
vated and cytotoxic T cells) but also of Foxp3 (functional
marker of Treg) in samples of urinary lymphocytes (25). More
interesting, higher levels of Foxp3 mRNA were associated with
improved probability of reversibility of acute rejection and
lower risk for graft failure 6 mo after rejection episode. These
Figure 3. Suppressor function of Treg generated by ATG is re-
stricted 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 sup-
pression of a direct alloimmune response of autologous respond-
ers (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 donorrecipient combination.
The proliferative response to recall antigen mumps was not inhib-
ited 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).
J Am Soc Nephrol 17: 28442853, 2006 Ex Vivo Expansion of Regulatory T Cells by ATG 2849
findings are consistent with the hypothesis that Treg also can
serve to limit anti-graft immunity during acute rejection. Taken
together, the above studies suggest that drugs that enhance the
generation of Treg or administration of Treg themselves may
improve the outcome of both acute rejection episodes and the
long-term survival of allografts overall. In this article, we report
the novel finding that ATG can lead to expansion of
CD4

CD25

Foxp3

Treg from na ve CD4

CD25

T cells. Our
data are in agreement with several other studies showing gen-
eration of Foxp3-expressing CD4

CD25

T cells from
CD4

CD25

precursors in the periphery (24,26). In contrast to

mouse, in which Foxp3 could be induced on Foxp3

cells under
specialized conditions (e.g., activation plus TGF-) (24), human
CD25

T cells seemed to upregulate both CD25 and Foxp3 (26).

Therefore, activation of human CD4

CD25

T cells led to
generation of regulatory CD4

CD25

Foxp3

T cells (26), rais-

ing the possibility that ATG in a similar manner may expand
the Treg by initially activating the CD4

CD25

T cells. Al-
though further studies are needed to elucidate the exact mech-
anism of the conversion, our dose-response experiments using
ATG at higher concentration in the generating culture sug-
gested increased activation but a decline in Foxp3 expression
by CD4

T cells. Although deletion of T cells by apoptosis has

been demonstrated to promote immunoregulation (27), we did
not observe significant apoptosis after 24 h of incubation of PBL
with ATG at the concentration of 10 g/ml, suggesting that
apoptosis is unlikely to contribute. Furthermore, whether the
original CD4

CD25

T cells are truly na ve T cells or regula-

tory cells that were generated initially in thymus and later lost
the CD25 expression and then recovered their suppressive ac-
tivity again upon activation remains unclear (9). It is interesting
not only that this process is accompanied by an increase in the
Th2 cytokines in the generating culture but also that in fact
neutralization of each of the Th2 cytokines can significantly
hamper the Treg-expanding effects of ATG. While Th2 cyto-
kines have the capacity to promote Treg by various mecha-
nisms such as altering T cell activation, affecting apoptosis or
cell proliferation, it is likely that they promote the conversion of
CD4

CD25

T cells into Treg, as conversion is established

clearly as the main contributor to Treg expansion. These find-
ings are very intriguing and supported by recent published
data demonstrating that IL-4R -chainbinding cytokines, such
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
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).
2850 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 28442853, 2006
as IL-4 and IL-13, play a crucial role in generating
Foxp3

CD25

Treg extrathymically (17). In addition, these

data point to an intriguing link between the well-established
immunoregulatory capacity of Th2 cells and the potent
CD4

CD25

regulatory T cells. These results also are impor-

tant in light of data showing that cyclosporine and tacrolimus,
two commonly used immunosuppressive drugs, both inhibit
the production of IL-2, an essential growth factor for Treg (28).
Conversely, murine T cells that were activated in the presence
of rapamycin were highly enriched in Foxp3

CD4

CD25

T
cells in vitro (29). Nevertheless, our findings here are unique
because we clearly demonstrate that ATG can rapidly expand
human Treg by promotion of Th2 cytokine production ex vivo.
Although the identification of the exact component of ATG
(Genzyme; generated by using thymocytes as the immunogen)
that is responsible for the expansion of Treg will require further
investigation, our ability to generate Treg using a different ATG
(Fresenius; produced with purified and activated CD3

lym-
phoblastic cells as immunogen) suggests a key role for antibod-
ies that are directed against epitopes on T cells. Another unique
finding of the study is that ATG-induced Treg significantly
suppressed direct alloimmune responses to alloantigens by
autologous PBL from which Treg originally were generated. In
contrast, nonautologous responders could not be regulated by
the Treg. Conversely, the memory response to recall antigen
mumps is not affected. This may be due to lack of activation of
Treg by mumps antigen or the overall inability of Treg to
inhibit the memory responses. Given the great interest in cel-
lular treatment with ex vivo expanded Treg as a means to
modulate alloimmune responses without affecting memory re-
sponses to infectious agents, these findings are of crucial im-
portance.
Although the in vivo effects of ATG on the induction, expan-
sion, and function of Treg in allograft recipients remain to be
characterized fully, recent data demonstrated that CD25

T
cells are spared from anti-lymphocyte serummediated dele-
tion in mice, suggesting the concomitant presence of both T cell
depletion and continuous regulatory T cell activity in an in vivo
animal model (30). Second, in studying the in vivo effects of
ATG in humans, the achieved concentrations of the ATG in
blood and lymphoid tissues should be taken carefully into
consideration. ATG is used at a dosage of 1 to 1.5 mg/kg per d
for 3 to 5 d as induction immunosuppression and also for
treatment of steroid-resistant acute rejection, leading to serum
levels of the drug between 50 and 100 g/ml (2,5). This dosing
regimen is based on ATGs efficacy to deplete T cells in the
peripheral blood rather than promotion of Treg. There are no
human data regarding the achieved concentrations in second-
ary lymphoid tissues, a potentially important site for induc-
tion/expansion of Treg, although the data in primates suggest
good penetration into lymph nodes and spleen but minimal
penetration into thymus (31). We were able to expand Treg in
vitro with a concentration of 10 g/ml, a dosage that is signif-
icantly lower than the levels that currently are achieved in
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).
J Am Soc Nephrol 17: 28442853, 2006 Ex Vivo Expansion of Regulatory T Cells by ATG 2851
blood. In fact, our in vitro data may suggest that lower dosages
of ATG may lead to expansion of Treg in vivo, but clearly
further investigation is needed and is currently ongoing.
Conclusion
We report a novel mechanism of action for ATG, namely its
ability to expand Treg ex vivo, mainly by inducing
CD4

CD25

Foxp3

T cells. The regulatory function clearly is

restricted by the responder cells but does not affect the memory
response to recall antigen. These findings are clinically impor-
tant for two important reasons: First, they may suggest that the
therapeutic effect of ATG may be due to both concomitant T cell
deletion and continuous regulatory T cell activity; second, they
may help us to develop strategies to expand these very rare
regulatory cells ex vivo for use as cellular treatment in trans-
plantation and autoimmunity.
Acknowledgments
This work was supported by National Institutes of Health grant PPG
PO1 AI-050157. Assay development was supported by National Insti-
tutes of Health grants UO1 AI-055801 and UO1 AI-063623. N.N. is a
recipient of the American Society of Nephrology John Merrill Trans-
plant Scholar Grant and the American Heart Association Scientist De-
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 26442646.
J Am Soc Nephrol 17: 28442853, 2006 Ex Vivo Expansion of Regulatory T Cells by ATG 2853