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Development in a B Cell Lymphoma Model
Early but Not Late Phases of Tumor
Multiple Immune Evasion Mechanisms in
T Regulatory Cells Dominate
S. Yolcu and Haval Shirwan
Kutlu G. Elpek, Chantale Lacelle, Narendra P. Singh, Esma
2007; 178:6840-6848; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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Copyright © 2007 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month by
The Journal of Immunology
by guest on October 18, 2015
by guest on October 18, 2015
CD4?CD25?T Regulatory Cells Dominate Multiple Immune
Evasion Mechanisms in Early but Not Late Phases of Tumor
Development in a B Cell Lymphoma Model1
Kutlu G. Elpek, Chantale Lacelle, Narendra P. Singh, Esma S. Yolcu, and Haval Shirwan2
Tumors use a complex set of direct and indirect mechanisms to evade the immune system. Naturally arising CD4?CD25?FoxP3?
T regulatory (Treg) cells have been implicated recently in tumor immune escape mechanism, but the relative contribution of these
cells to overall tumor progression compared with other immune evasion mechanisms remains to be elucidated. Using the A20 B
cell lymphoma as a transplantable tumor model, we demonstrate that this tumor employs multiple direct (expression of immu-
noinhibitory molecule PD-L1, IDO, and IL-10, and lack of expression of CD80 costimulatory molecule) and indirect (down-
regulation of APC function and induction of Treg cells) immune evasion mechanisms. Importantly, Treg cells served as the
dominant immune escape mechanism early in tumor progression because the physical elimination of these cells before tumor
challenge resulted in tumor-free survival in 70% of mice, whereas their depletion in animals with established tumors had no
therapeutic effect. Therefore, our data suggest that Treg cells may serve as an important therapeutic target for patients with early
stages of cancer and that more vigorous combinatorial approaches simultaneously targeting multiple immune evasion as well as
immunosurveillance mechanisms for the generation of a productive immune response against tumor may be required for effective
immunotherapy in patients with advanced disease. The Journal of Immunology, 2007, 178: 6840–6848.
expressing tumor-associated Ags, which are either mutated or
over/aberrantly expressed self proteins or proteins derived from
oncogenic viruses (2). However, the recognition of tumor-associ-
ated Ags by TCRs (signal 1) is not sufficient for the generation of
a productive T cell response against cancer. Costimulation via
CD28/B7 or other costimulatory receptor/ligand interactions (sig-
nal 2) is also required for the initiation of the response (3). After
initiation, a productive immune response is maintained by signal 3
in the form of cytokines and chemokines produced as a result of
reciprocal T cell and APC activation (4).
Tumors have evolved various direct and indirect mechanisms to
evade the immune system (1, 5). These mechanisms include the
following: 1) lack of signal 1, arising from either the inefficient
display of MHC/tumor Ag bimolecular complexes on tumor cells,
defects in the transduction of this signal, or expression of MHC
homologues, MHC class I chain-related, that inhibit NK cells ex-
pressing NKG2 inhibitory receptors; 2) absence of signal 2 orig-
inating from the lack of costimulatory molecules on tumor cells or
tumor expression of coinhibitory molecules; 3) tumor-mediated
suppression of immune responses through the secretion of anti-
daptive immune responses mediated by T cells serve as
critical components of immunosurveillance mechanisms
against cancer (1). T cells respond to cancerous cells
inflammatory molecules, such as TGF-? and IL-10, induction of
anergy in tumor-reactive T cells, physical elimination of effector T
CD4?CD25?FoxP3?T regulatory (Treg)3cells and tolerogenic
APCs; and 4) regulation of immunity by the tumor stroma. Accu-
mulating evidence suggests that multiple immune evasion mecha-
nisms may simultaneously operate in patients with advanced disease
evasion and their temporal cross-regulation in the course of tumor
progression remain to be elucidated.
CD4?CD25?FoxP3?Treg cells have recently been the focus of
intense studies due to their critical roles in tolerance to self Ags
and regulation of immune responses to infections and transplan-
tation Ags (7). A series of recent studies also provided evidence for
the involvement of these cells in immune evasion mechanisms
used by tumors (8–11). A positive correlation between the in-
creased numbers of Treg cells and tumor progression in experi-
mental as well as clinical settings provided the first indirect evi-
dence that these cells may play an important role in tumor immune
evasion (8, 9, 11). Direct evidence for such a role has been pro-
vided recently by studies demonstrating that physical elimination
of Treg cells before tumor challenge resulted in tumor rejection in
some animal tumor models (12–15). However, it is unknown
whether elimination of Treg cells in established tumor models has
any therapeutic efficacy and whether Treg cells serve as the common
denominator of immune escape mechanisms used by all tumors or
they may play a more dominant role in tumor settings, in which a
limited number of immune evasion mechanisms are operative.
Treg cells have been shown to mediate their suppressive func-
tion through cell-to-cell contact as well as via soluble mediators,
such as anti-inflammatory cytokines IL-10 and TGF-? (16–18).
Institute for Cellular Therapeutics and Department of Microbiology and Immunology,
University of Louisville, Louisville, KY 40202
Received for publication August 2, 2006. Accepted for publication March 19, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was funded in parts by grants from Kentucky Lung Cancer Research
Program, National Institutes of Health/National Cancer Institute Grant R43
CA109866, and the Commonwealth of Kentucky Research Challenge Trust Fund.
2Address correspondence and reprint requests to Dr. Haval Shirwan, Institute for
Cellular Therapeutics, 570 South Preston Street, Donald Baxter Biomedical Building,
Suite 404E, University of Louisville, Louisville, KY 40202. E-mail address:
3Abbreviations used in this paper: Treg, T regulatory; 1-MT, 1-methyl-D-tryptophan;
DC, dendritic cell; DP, double positive; HPRT, hypoxanthine phosphoribosyltrans-
ferase; SP, single positive; Teff, T effector.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
by guest on October 18, 2015
These cytokines further provide a positive feedback loop for immune
suppressive mechanisms by generating inducible Treg cells from
CD4?CD25?T effector (Teff) cells (19). This positive feedback loop
in a tumor setting may further be amplified by CTLA-4 on Treg cells
engaging CD80/86 costimulatory molecules on dendritic cells (DCs),
leading to the functional differentiation of DCs into suppressive cells
expressing IDO (20, 21). IDO?DCs may in turn perpetuate the sup-
pressive milieu by further contributing to the generation of inducible
or naturally occurring Treg cells. Recruitment and/or induction of
Treg cells, as a bridge between anti-inflammatory cytokines and
tolerogenic APCs, may be one of the major immune evasion mech-
anisms used by tumor cells. Therefore, elucidating the role of Treg
cells in tumor progression, particularly their relative contribution to
immune evasion mechanisms, may have important implications in the
design of effective immunotherapeutic approaches against cancer.
A20 B cell lymphoma was used as an aggressive tumor model
to delineate the role of Treg cells in tumor development and pro-
gression. In this study, we demonstrate that tumor growth in this
model was associated with a series of simultaneously operating
direct and indirect immune evasion mechanisms. In particular, an-
imals with tumors had increased percentages of intratumoral and
systemic Treg cells, high levels of intratumoral and systemic IL-
10, moderate level of intratumoral TGF-?, and APCs with altered
function. Furthermore, A20 cells expressed the coinhibitory mol-
ecule PD-L1, anti-inflammatory cytokine IL-10, and immuno-
modulatory IDO, and lacked the expression of the costimulatory
molecule CD80. Importantly, Treg cells played a dominant role in
early tumor progression because in vivo depletion of these cells
using an Ab to CD25 before tumor challenge resulted in tumor-
free survival in 70% of the animals. In marked contrast, depletion
of these cells in tumor-bearing animals had no beneficial effect.
These findings were further corroborated in an adoptive transfer
model in which the protective effect of ex vivo expanded tumor-
specific Teff cells was blunted by ex vivo expanded Treg cells
from tumor-bearing animals. The implication of these findings for
strategies targeting Treg cells for immunotherapy in cancer pa-
tients is discussed.
Materials and Methods
Male BALB/c and C57BL/6 mice aged 6–8 wk were purchased from The
Jackson Laboratory or bred in our animal facility at the University of
Louisville. Animals were provided food and water ad libitum and housed
under specific pathogen-free conditions. Animals bearing tumors were eutha-
nized when tumors reached a size of 20 mm in diameter or earlier if tumors
ulcerated or animal showed sign of discomfort. All animals were cared for in
accordance with institutional and National Institutes of Health guidelines.
Abs and reagents
Fluorochrome-conjugated Abs (CD4 allophycocyanin, CD8 PerCP, CD25
PE, CD19 allophycocyanin, IL-10 PE, H2Kd PE, I-A/I-E PE, PD-L1 PE,
CD80 FITC, CD40 FITC, and CD86 allophycocyanin) and isotype controls
were purchased from BD Pharmingen and eBioscience. Intracellular FoxP3
staining kit was purchased from eBioscience. Mouse IL-10 ELISA kit was
purchased from BioSource International, and the assay was performed ac-
cording to the manufacturer’s protocol. Hybridoma for anti-mouse CD25
(PC61) and anti-mouse CD4 (GK1.5) Ab were gifts from S. Ildstad (In-
stitute for Cellular Therapeutics and Department of Microbiology and Im-
munology, University of Louisville, Louisville, KY). The PC61 Ab was
produced and purified in the laboratory of N. Egilmez (Brown Cancer
Center, University of Louisville). Anti-mouse IL-10 (JES5-2A5) and isotype
controls were purchased from Bioexpress. The 1-methyl-D-tryptophan (1-MT)
was purchased from Sigma-Aldrich and prepared in 0.1 N NaOH (pH 8).
Flow cytometry and cell sorting
For phenotyping and sorting, spleens, lymph nodes, and tumors were pro-
cessed into single-cell suspension, and cells were labeled with fluoro-
chrome-conjugated Abs. Intracellular cytokine staining was performed as
described (22). Briefly, single-cell suspensions of tumor cells were cultured
in MLR medium supplemented with 1 ?l/ml GolgiPlug (BD Pharmingen)
for 2 h. Cells were then stained with allophycocyanin-conjugated anti-
mouse CD19 Ab, fixed with 4% paraformaldehyde, and stained with PE-
conjugated anti-mouse IL-10 or isotype control in permeabilization buffer
containing saponin. Intracellular FoxP3 staining was performed using the
anti-mouse/rat FoxP3 staining kit, according to the manufacturer’s protocol
(eBioscience). Flow cytometry was performed using a FACSCalibur flow
cytometer (BD Biosciences), and data were analyzed using CellQuest (BD
Biosciences) and FlowJo (Tree Star) softwares. CD4?, CD4?CD25?, and
CD4?CD25?cells were sorted using a FACSVantage cell sorter (BD Bio-
sciences). Sorted cells were reanalyzed by flow cytometry and found to be
Tumor models and depletion studies
A20 B lymphoma cell line was purchased from the American Type Culture
Collection and cultured in DMEM supplemented with 10% FBS, 2 mM
L-glutamine, 100 U/ml penicillin, and 100 ?g/ml streptomycin at 37°C in
a humidified 5% CO2incubator. To establish tumors, 1 ? 106live A20
cells were resuspended in 200 ?l of PBS and s.c. injected into the right
back flank of naive syngeneic BALB/c mice. Tumor growth was monitored
three times per week using calipers. Average tumor size was calculated by
measuring two perpendicular diameters. For depletion studies, tumor-
bearing animals were injected with 50 ?g of anti-mouse CD4 (GK1.5)
intratumorally or 300 ?g of anti-mouse CD25 (PC61) i.v. twice at a 7-day
interval as soon as the tumors were palpable. A third group of animals was
treated with concurrent injection of 300 ?g of PC61 i.v. and 50 ?g of
GK1.5 intratumorally when tumors were palpable, followed by intra-
tumoral injection of the same dose of GK1.5 mAb 7 days later. For
predepletion studies, 300 ?g of PC61 was injected i.v. 4 days before the
tumor challenge. PBS was used as a control. Depletion of regulatory T
cells was confirmed by flow cytometry analysis of blood samples at
various times after the administration of the Ab using the FoxP3 stain-
ing kit (eBioscience). For IL-10 neutralization, one group of animals
received 1 mg of anti-mouse IL-10 (JES5-2A5) mAb i.p. twice with a
1-wk interval starting 1 day after tumor challenge. This treatment reg-
imen was adopted from a previously published study in tumor setting
demonstrating therapeutic utility of blocking IL-10 in vivo (23–25). In
addition to anti-IL-10 Ab, a second group of animals also received anti-
CD25 mAb i.v. when tumors were palpable. Isotype Abs were used as
RNA was isolated from the spleen, lymph nodes, and/or tumors using TRI
reagent (Molecular Research Center) and cDNA generated by reverse tran-
scription. PCR for cytokines and hypoxanthine phosphoribosyltransferase
(HPRT) were performed, as previously described (26). PCR for FoxP3 (35
cycles) and IDO (40 cycles) were performed using primers for FoxP3 (for-
ward, 5?-CAG CTG CCT ACA GTG CCC CTA G; reverse, 5?-CAT TTG
CCA GCA GTG GGT AG) and IDO (forward, 5?-GAT GTG GGC TTT
GCT CTA CC; reverse, 5?-TTC TTC CAG TTT GCC AGG AC).
Mixed lymphocyte reactions
Standard 5-day MLR assays were performed using lymph node cells from
naive BALB/c mice (1 ? 105/well) as responders and irradiated (2000
cGy) splenocytes from naive C57BL/6 mice (1 ? 105/well) as stimulators.
To test the suppressive effect of sera from tumor-bearing animals on MLR,
cultures were supplemented with various amounts (1–10 ?l/well) of sera
collected from naive, tumor-bearing, and tumor-free animals. Cells were
pulsed with [3H]thymidine during the last 16 h of the 5-day culture, and
harvested on a Tomtec Harvester 96 (Tomtec) for quantification of incor-
porated thymidine. Results were expressed as mean cpm of triplicate wells.
In some experiments, splenocytes from naive C57BL/6 mice were labeled
with CFSE (Molecular Probes), and used as responder against APCs from
naive and tumor-bearing animals. Briefly, cells were washed with PBS, and
then incubated in 4 ml of 2.5 ?M CFSE/1 ? 108cells for 7 min at room
temperature. Cells were incubated with FBS for 1 min, and washed twice
To test the function of APCs, splenocytes from naive and tumor-bearing
animals were processed into single cells and panned on plastic dishes.
Irradiated (2,000 cGy) adherent cells (1 ? 105/well) were then used as
stimulators in MLR assays, whereas splenocytes (1 ? 105/well) from naive
C57BL/6 were used as responders. In some assays, irradiated (10,000 cGy)
A20 cells were used as stimulators in 4-day cultures against CFSE-labeled
C57BL/6 splenocytes at a 1:4 ratio in the presence or absence of 200 ?M
1-MT. Proliferation was assessed using flow cytometry.
6841The Journal of Immunology
by guest on October 18, 2015
Suppression and adoptive transfer assays
For ex vivo activation, spleen and/or lymph nodes were harvested from
naive, tumor-bearing, and tumor-free animals; processed into single-cell
suspensions; and cultured in a 2:1 ratio with irradiated (10,000 cGy) A20
cells in MLR medium supplemented with 50 U/ml IL-2 (Roche) for 4–5
days. For suppression assays, CD4?CD25?(single-positive (SP)) and
CD4?CD25?(double-positive (DP)) cells were sorted by flow cytometry. DP
cells from naive, tumor-bearing, and tumor-free cultures were used as sup-
pressors for SP responder cells from naive BALB/c mice. The 3-day suppres-
sion assay was performed in the presence of 2.5 ? 104SP and/or DP cells/
well, 0.5 ?g/ml anti-mouse CD3 (clone 145-2C11) Ab (BD Pharmingen), and
1 ? 105irradiated (2,000 cGy) splenocytes from naive BALB/c mice.
For adoptive transfer studies, lymphocytes from tumor-free animals
were cultured for 4 days in the presence of 50 U/ml IL-2 and irradiated A20
(10,000 cGy), and then purified using Lympholyte-M, according to the
manufacturer’s protocol (Cedarlane Laboratories). Cells from tumor-free
animal cultures (1 ? 106) were mixed with 5 ? 105live A20 cells and
injected s.c. with or without DP cells (3–7 ? 105) sorted from ex vivo
activation cultures of tumor-bearing animals. Tumor growth was moni-
tored, as described above.
Increased percentages of CD4?CD25?FoxP3?Treg cells in
CD4?CD25?FoxP3?Treg cells are critical to peripheral tolerance
to self Ags and have been implicated in immune evasion mecha-
nisms by tumors (7, 10). To study the contribution of Treg cells in
tumor progression, we choose A20 B lymphoma as a model sys-
tem. This tumor cell line is derived from a spontaneous reticulum
cell neoplasm in BALB/c mice and is highly tumorogenic and
poorly immunogenic (27, 28). Four groups of mice were used in
this study, as follows: naive animals (n ? 3) and animals with
small tumors (8.6 ? 1.0 mm, n ? 4), medium tumors (13.3 ? 1.0
mm, n ? 6), and large tumors (19.2 ? 1.3 mm, n ? 5). Spleen,
draining lymph nodes, and tumors were harvested and analyzed for
the presence of Treg cells using Abs to various cell surface mark-
ers in flow cytometry. There was a decrease in the percentages of
CD4?and CD8?cells in the spleen, lymph nodes, and tumors as
tumor sized increased (Fig. 1A, top and center panels). These dif-
ferences were significant when the spleen and lymph node cells
from large tumor-bearing animals were compared with those of
naive and small tumor-bearing animals.
In marked contrast, we found an increase in the percentage of
CD4?CD25?T cells in the spleen, lymph nodes, and tumors as
tumor size increased; animals with large tumors had significantly
higher percentages of Treg cells in their spleen and lymph nodes as
compared with naive and small tumor-bearing animals (Fig. 1A,
bottom panel). The regulatory nature of these cells was confirmed
by two independent lines of evidence. First, these cells expressed
the signature transcriptional factor FoxP3 as determined by intra-
cellular staining with FoxP3 Ab (Fig. 1B) and RT-PCR (data not
shown). Second, CD4?CD25?T cells from tumor-bearing ani-
mals suppressed the proliferation of CD4?CD25?Teff cells in
vitro. In this assay, lymphocytes were harvested from medium size
tumor-bearing animals or tumor-free animals that had undergone
successful immunotherapy with CD80-decorated cells (29). These
cells were then cocultured with irradiated A20 cells as the relevant
source of tumor-associated Ags in the presence of exogenous IL-2
for 4–5 days. CD4?CD25?DP T cells were sorted from both
cultures using flow cytometry, and their regulatory function was
tested in coculture experiments with sorted naive CD4?CD25?SP
T cells in a standard CD3 stimulation assay. DP T cells sorted from
cultures of tumor-bearing animals were anergic, and as such failed
to respond to CD3 stimulation. However, these cells potently in-
hibited the proliferation of SP naive T cells (Fig. 1C). In marked
contrast, DP cells from cultures of tumor-free animals responded
to CD3 stimulation and did not inhibit the proliferative response of SP
T cells. Taken together, these data demonstrate that CD4?CD25?T
cells expanded in lymphocyte cultures of tumor-bearing animals are
Treg cells, but not Teff cells that up-regulated CD25 in response to
irradiated A20 cells.
CD4?CD25?Treg cells facilitate the growth of A20 tumors
We next tested the role of these ex vivo expanded Treg cells in tumor
immune evasion mechanisms using an adoptive transfer model.
function of tumor size. BALB/c mice were inocu-
lated s.c. in the right flank with 1 ? 106live A20
cells. Cells harvested from tumor, spleen, and drain-
ing lymph nodes of animals with small (8.6 ? 1.0
mm, n ? 4), medium (13.3 ? 1.0 mm, n ? 6), and
large (19.2 ? 1.3 mm, n ? 5) tumors analyzed for
phenotype and function. Cells from naive animals
(n ? 3) served as controls. A, Percentage of CD4?
(top panel), CD8?(middle panel), and CD4?CD25?
T cells (bottom panel) in various tissues. ?, p ? 0.05
as compared with percentages of respective groups.
B, FoxP3 expression in intratumoral CD4?CD25?T
cells from a medium size tumor using anti-FoxP3 Ab
in flow cytometry (gray filled histogram represents
the isotype control). C, Suppression assay. Spleen
and lymph node cells of tumor-bearing (Tumor-DP)
and tumor-free (TFree-DP) animals were cultured
with irradiated A20 cells in the presence of IL-2 for
4–5 days. CD4?CD25?DP T cells were then sorted
by flow cytometry and used in coculture experiments
with sorted CD4?CD25?SP T cells from naive an-
imals at 1:1 ratio in a standard anti-CD3 stimula-
tion and [3H]thymidine incorporation-based assay.
Sorted DP T cells from naive BALB/c mice (DP)
were used as controls.
CD4?CD25?Treg cells increase as a
6842 ROLE OF CD4?CD25?Treg CELLS IN CANCER IMMUNE EVASION
by guest on October 18, 2015
Adoptive transfer of 1 ? 106total cells from ex vivo stimulated lym-
phocytes from cultures of mice that had undergone successful immu-
notherapy with CD80-decorated A20 cells (29) into mice simulta-
neously challenged with a lethal dose of A20 cells resulted in 50%
tumor-free survival during a 100-day observation period (Fig. 2).
However, this protective effect was totally abolished by coadoptive
transfer of 3–7 ? 105flow-sorted CD4?CD25?T cells from cultures
of tumor-bearing animals. These results further confirm that
CD4?CD25?T cells accumulating in tumor-bearing animals as a
function of tumor growth are Treg cells, and that these cells play a
critical role in tumor evasion mechanisms in this model when present
at early stages of tumor progression.
Early, but not late, depletion of CD4?CD25?Treg cells during
tumor progression results in tumor-free survival
It was demonstrated recently by Yu et al. (30) that systemic de-
pletion of Treg cells using the PC61 Ab against CD25 or intratu-
moral depletion using a low dose anti-CD4 Ab resulted in com-
plete eradication of large tumors in a fibrosarcoma model. We,
therefore, adapted this protocol and demonstrated that i.v. injection
of 300 ?g of PC61 Ab twice at a 7-day interval into mice with
palpable tumors had no effect on tumor growth (Fig. 3A), albeit
successful depletion of Treg cells in the periphery and within the
tumor, as confirmed using Abs to FoxP3 and CD25 (Fig. 3B).
Indeed, Treg cells were absent in the blood of these animals for 7
days and only recovered to one-half of their original levels by day
14 following Ab treatment.
mors in an adoptive transfer model. Splenocytes and lymph node cells were
harvested from BALB/c mice bearing medium to large tumors and tumor-
free animals having undergone successful immunotherapy with irradiated
cells decorated with chimeric CD80 costimulatory molecule. Cells were
stimulated in vitro with irradiated A20 cells as the source of tumor Ags in
the presence of IL-2 for 5 days. Live cells harvested from cultures of
tumor-free animals using Lympholyte-M were directly used in adoptive
transfer experiments, whereas those from cultures of tumor-bearing ani-
mals were sorted by flow cytometry to isolate CD4?CD25?T cells. Adop-
tive transfer of 1 ? 106ex vivo activated lymphocytes from tumor-free
(TFree) animals into naive secondary animals simultaneously challenged
with a lethal dose of live A20 cells s.c. (n ? 6). One group of animals was
coadoptively transferred with 3–7 ? 105CD4?CD25?T cells (Tumor-DP)
sorted from cultures of tumor-bearing animals (n ? 6). Animals inoculated
with live A20 alone served as controls (n ? 5).
CD4?CD25?Treg cells facilitate the growth of A20 tu-
A20 cells s.c. and injected with 300 ?g of anti-CD25 Ab PC61 (n ? 5) or PBS (n ? 5) i.v. twice at a 7-day interval when tumors were palpable. B, Confirmation
of Treg cell depletion in mice injected i.v. with a single dose of 300 ?g of PC61 Ab. Depletion was shown in peripheral blood using FoxP3 Ab (top panel) and
within tumor using an anti-CD25 mAb (clone 7D4) that recognizes a different epitope than PC61 Ab used for CD25 depletion (bottom panel). C, Tumor growth
in mice subjected to various Ab treatments. A group of animals (n ? 5) was injected intratumorally with 50 ?g of anti-CD4 Ab GK1.5 when tumors were palpable,
followed by a second injection 7 days later (GK1.5). A second group of animals (n ? 5) was injected i.v. with 300 ?g and intratumorally with 50 ?g of PC61
when tumors were palpable. Seven days later, these animals received one dose of intratumoral injection of 50 ?g of GK1.5 (PC61 ? GK1.5). Arrows indicate
the time points at which PC61 and GK1.5 were injected. D, Tumor growth in BALB/c mice (n ? 10) subjected to systemic depletion by a single injection of 300
?g of PC61 Ab i.v. 4 days before A20 challenge. Tumor growth in animals injected with PBS served as controls (n ? 5).
CD4?CD25?Treg cells are critical to the initial stages of tumor progression. A, Tumor growth in BALB/c mice inoculated with 1 ? 106of live
6843The Journal of Immunology
by guest on October 18, 2015
Because the lack of a protective response may be due to the
elimination of activated CD25?Teff cells rather than the depletion
of Treg cells, we followed the protocol of Yu et al. (30), who
demonstrated that intratumoral injection of one dose of 40 ?g of
anti-CD4 Ab (GK1.5) resulted in the eradication of established
fibrosarcoma tumors. In marked contrast, intratumoral injection of
50 ?g of GK1.5 Ab twice at a 7-day interval had no effect on tumor
growth in our A20 model (Fig. 3C). Furthermore, concurrent sys-
temic and intratumoral injection of PC61 when tumors were pal-
pable, followed by an intratumoral GK1.5 injection 7 days later did
not change the kinetics of tumor growth (Fig. 3C).
We next assessed whether systemic pretreatment of mice with
PC61 has any effect on the growth of A20 tumors. Intravenous
injection of 300 ?g of PC61 Ab 4 days before tumor inoculation
resulted in tumor-free survival in 70% of mice over a 60-day ob-
servation period (Fig. 3D). In contrast, all animals without Ab
treatment were euthanized within 35 days due to large tumor bur-
den. Importantly, tumor-free animals did not develop tumors when
rechallenged with a lethal dose of live A20 60 days after the initial
tumor inoculation (data not shown), suggesting that elimination of
Treg cells not only leads to the rejection of tumors, but also allows
for the generation of a memory response that protects against re-
currences. Taken together, these data demonstrate that physical
elimination of Treg cells early in tumor progression has a thera-
peutic effect, whereas depletion of these cells in animals with es-
tablished tumors has no effect on tumor growth.
A20 B lymphoma cells express an altered pattern of
Our data demonstrating that Treg cells serve as an important im-
mune evasion mechanism early in the progression of A20 tumors
suggested that other immune evasion mechanisms either induced
by Treg cells or independent of these cells may operate in late
stages of tumor growth. We, therefore, characterized A20 cells for
the expression of various cell surface molecules involved in im-
mune responses. This tumor cell line expressed normal levels of
both MHC class I and class II involved in the transduction of
signal 1 (Fig. 4A) and high levels of CD40 and CD86 and low
levels of 4-1BBL costimulatory molecules involved in the trans-
duction of signal 2 (data not shown). Consistent with our previous
observation, the CD80 costimulatory molecule was not detected on
the surface of A20 cells. The lack of CD80 is highly significant
because we and others have demonstrated that expression of this
molecule via genetic manipulations or cell surface protein displays
results in high immunogenicity and tumor cell rejection in synge-
neic BALB/c mice (29, 31, 32). Importantly, A20 cells expressed
high levels of coinhibitory molecule PD-L1, which has been im-
plicated in tumor immune evasion mechanisms as well as tolerance
in various animal models (33). This pattern of MHC and costimu-
latory molecule expression was similar between cultured A20 cells
and cells extracted from tumors (data not shown).
Intratumoral and systemic expression of IL-10
Cytokines play important roles in the regulation of adaptive as well
as innate immune responses to tumors. In particular, Treg cell
development and function are regulated by cytokines, such as IL-2,
IL-10, and TGF-? (18, 34). We, therefore, characterized the cy-
tokine expression pattern within A20 tumors using RT-PCR with
particular focus on Th1 (IL-2, IFN-?) and Th2/Treg (IL-2, IL-10,
TGF-?) cytokines (26). A20 tumors were found to express low
levels of IL-2, IL-4, and IFN-?; moderate levels of TGF-?; and
high levels of IL-10 (Fig. 5A).
The high level of intratumoral IL-10 expression was rather strik-
ing and suggested that it may be expressed by the tumor itself in
addition to tumor-infiltrating lymphocytes. To test this hypothesis,
total RNA isolated from cultured A20 cells was subjected to RT-
PCR analysis. A20 cells expressed high levels of IL-10 transcripts
(Fig. 5B, top panel) with minimal or undetectable levels of tran-
scripts for other cytokines (data not shown). The expression of
IL-10 was further confirmed at the protein level by intracellular
cytokine staining (Fig. 5B, bottom panel). Importantly, A20 cells
freshly extracted from tumors expressed higher levels of IL-10
protein than A20 cell line maintained in culture (44 vs 10%; Fig.
5B, bottom panel). These data suggest the existence of positive
feedback mechanism(s) within the tumor microenvironment con-
tributing to the higher expression of IL-10.
IL-10 secretion was systemic, as determined using ELISA.
There was significantly higher level of circulating IL-10 in the sera
of tumor-bearing animals as compared with that of naive and tu-
mor-free animals vaccinated with CD80-decorated A20 cells (Fig.
5C). IL-10 was also present in the culture medium of A20 cells,
suggesting that these cells are likely to be the main source of IL-10
and use this molecule as a means of immune evasion. Indeed, sera
from tumor-bearing animals inhibited BALB/c T cell response to
irradiated C57BL/6 splenocytes in in vitro proliferative responses.
There was a significant inhibition of T cell proliferation in cultures
supplemented with 1 ?l of sera from tumor-bearing animals as
compared with cultures containing sera from naive or tumor-free
animals (Fig. 5D). The proliferative response was significantly re-
covered using a blocking Ab (JES5-2A5) against IL-10 (data not
To demonstrate whether IL-10 neutralization in vivo has any
therapeutic effect, a group of animals was challenged with A20
cells and subjected to i.v. treatment twice with 1 mg of clone
JES5-2A5 at weekly intervals starting 1 day posttumor challenge.
This treatment regimen had no effect on tumor growth (Fig. 5E). A
combination treatment that involved both anti-IL-10 and anti-
CD25 Abs also had no effect on the normal kinetics of tumor
growth. Although the in vivo neutralization experiments do not
provide a direct role for IL-10 in the progression of A20 tumor,
they cannot rule out a possible role for this cytokine in tumor
immune evasion. This is consistent with our in vitro experiments
as well as various reports implicating IL-10 in tumor immune eva-
sion (24, 35, 36). A20 cells express high levels of IL-10 and may
use this cytokine as a means of immune evasion mechanisms by
affecting various arms of the immune system, including Treg cells
and DCs (16, 18).
latory molecules. A20 cells were stained with Abs against various mole-
cules involved in the transduction of signal 1 (A) and signal 2 (B), and
analyzed using multiparameter flow cytometry.
A20 B lymphoma cells show altered expression of costimu-
6844 ROLE OF CD4?CD25?Treg CELLS IN CANCER IMMUNE EVASION
by guest on October 18, 2015
A20 cells and APCs from tumor-bearing animals serve as poor
stimulators of allogeneic responses
Altered function of APCs has been shown to be an important tu-
mor immune evasion mechanism (37, 38). In particular, it has been
demonstrated recently that a subpopulation of DCs expressing IDO
plays a critical role in tumor escape from immune destruction (19–
21). IDO may also be expressed by tumor cells and exhibit a direct
effect on T cells (39). We, therefore, tested the expression of IDO
in A20 cells and within the tumor using RT-PCR. A20 cell line as
well as freshly extracted tumors expressed high levels of IDO tran-
scripts (Fig. 6A). To test whether IDO expression plays a role in
the Ag-presenting function of A20 cells, we performed CFSE-
based MLR using C57BL/6 lymphocytes as responders against
irradiated A20 cells. A20 cells generated a moderate alloreactive
proliferative response, which was further enhanced by the addition
of the IDO inhibitor 1-MT into the culture medium (49 vs 61%;
To test whether A20 tumor-bearing animals have APCs with
altered function, irradiated splenocytes from naive and tumor-bear-
ing animals were used as stimulators for CFSE-labeled splenocytes
from naive C57BL/6 mice. There was minimal T cell proliferative
response to splenocytes from tumor-bearing animals as compared
with those from naive animals (9.3 vs 54.4%; Fig. 6C). Unlike A20
cultures, the addition of 1-MT did not restore the proliferative
response. This observation suggests that either IDO does not play
a major role or this effect is subdominant to other more tolerogenic
mechanisms regulating APC function in tumor-bearing animals.
Taken together, these data demonstrate that A20 tumors may evade
the immune system by directly expressing IDO and/or indirectly
via regulating the function of host APCs.
Tumor development and progression is a complex process that
involves various genetic alterations in the tumor as well as tumor
immune evasion via a complex set of molecular and cellular mech-
anisms that may operate simultaneously or sequentially in the
course of tumor progression. The critical role of the immune sys-
tem in immunosurveillance against tumors served as the basis of
intense efforts over the last few decades to develop effective im-
munotherapeutic approaches against cancer. Although some of
these approaches demonstrated efficacy in preclinical transplant-
able tumor models in preventive as well as therapeutic settings,
their translation to clinical settings was met with little or no suc-
cess (40). This may be due to the existence of various immune
evasion mechanisms in individuals with advanced tumors and the
inability of current therapeutic approaches to override these im-
munoregulatory mechanisms while generating new antitumor re-
sponses and/or up-regulating the existing ones.
CD4?CD25?FoxP3?Treg cells have emerged recently as im-
portant players in tumor immune evasion mechanisms. Increase in
the number of Treg cells as a function of tumor growth has been
shown in various animal models as well as clinical settings (8, 9,
11, 30), suggesting that these cells may serve as a common im-
mune evasion mechanism. Treg cells may, therefore, serve as an
tumor cells using primer sets specific for the indicated cytokines and HPRT as an internal control. Data are presented as the ratio of cytokines to HPRT. B,
on CD19-gated cells in flow cytometry (bottom panel; gray filled, isotype control; black line, IL-10). C, ELISA showing IL-10 levels in sera from naive,
tumor-bearing (Tumor), tumor-free (TFree) immunotherapy animals, and A20 culture supernatants. D, Effect of sera from the same groups of animals (C) on
allogeneic proliferative responses. Naive BALB/c lymph node cells were used as responders to irradiated C57BL/6 splenocytes, and cultures were supplemented
with 1 ?l/well serum from the indicated animals (top panel). E, Tumor growth in mice treated with 1 mg of neutralizing anti-IL-10 Ab (JES5-2A5)/animal injected
twice at weekly intervals (n ? 6) starting 1 day after tumor challenge. Another group of animals (n ? 5) was treated with both anti-IL-10 and anti-CD25 Abs.
Animals treated with isotype Abs served as controls (n ? 5). Downward arrows show the injection time for anti-IL-10 mAb, whereas the upward arrow shows
the time of injection for anti-CD25 Ab.
A20 cells express high levels of intratumoral and systemic IL-10. A, Expression of intratumoral cytokines. RT-PCR of total RNA obtained from
6845The Journal of Immunology
by guest on October 18, 2015
important target for immunotherapeutic approaches against cancer.
However, before such approaches are developed, it is paramount to
delineate the relative contribution of these cells to overall tumor
progression. In this study, we used A20 B cell lymphoma as an
aggressive transplantable tumor model to delineate the role of Treg
cells in tumor growth at various stages of tumor progression. Tu-
mor-bearing animals showed increased percentages of Treg cells
within the tumor, spleen, and tumor-draining lymph nodes as a
function of tumor size. Importantly, almost all CD4?CD25?T
cells within the tumor expressed FoxP3, suggesting that these were
Treg cells, not newly activated Teff cells. It is presently unknown
whether these cells were derived from the preferential infiltration
of naturally occurring Treg cells into the tumor and/or from
CD4?CD25?Teff cells converted into Treg cells in response to
various cues within the tumor microenvironment. The latter notion
is consistent with a recent study demonstrating that the majority of
Treg cells in tumor-bearing mice were derived from CD4?CD25?
Teff cells without a requirement for thymus or proliferation (41).
The presence of high levels of IL-10 and TGF-? within the tumor
in our model may drive this process. It has been shown recently
that Gr-1?CD115?immature myeloid suppressor cells accu-
mulate within tumors and secrete IL-10 and TGF-?, which play
critical roles in the conversion of CD4?CD25?Teff cells into
Treg cells (42).
Direct evidence for the role of Treg cells in A20 evasion of the
immune system in our model was provided by three lines of evi-
dence. First, CD4?CD25?T cells sorted from cultures of lym-
phocytes harvested from tumor-bearing animals and stimulated ex
vivo against A20 cells in the presence of IL-2 suppressed the pro-
liferation of CD4?Teff cells in a CD3 stimulation assay. Second,
these cells blocked the therapeutic efficacy of Teff cells, harvested
from animals vaccinated with CD80-decorated A20 cells (29),
when used in coadoptive transfer experiments. Third, consistent
with several published studies (12–15), systemic treatment of mice
with one dose of a depleting anti-CD25 mAb 4 days before tumor
challenge resulted in tumor-free survival in 70% of animals. Taken
together, these data provide direct evidence for the critical role of
Treg cells in tumor immune evasion mechanisms early in tumor
progression and are consistent with a series of recent findings im-
plicating these cells in tumor growth (10, 30, 41, 42).
However, the physical elimination of Treg cells in established
tumors had no therapeutic efficacy. Animals with palpable tumors
injected with two doses of PC61 Ab i.v. at a 1-wk interval devel-
oped tumors at a similar rate to control animals. The lack of a
therapeutic effect is not due to the inability of the Ab to deplete
Treg cells, because we demonstrated that one dose of PC61 was
sufficient to deplete Treg cells to background levels and these cells
could only recover to 50% of their initial value in 2 wk. This is
further consistent with other studies (12–15) and our results dem-
onstrating that a single injection of this dose of Ab was effective in
preventing tumor growth in 70% of the animals when used for
pretreatment. The lack of a PC61 Ab therapeutic effect in animals
with established tumors may be attributable to the fact that this Ab
not only depletes Treg cells, but also newly activated Teff cells
expressing CD25. However, this notion is inconsistent with the
findings of Yu et al. (30), showing that systemic administration of
one dose of PC61 Ab resulted in eradication of established tumors
in a fibrosarcoma model, suggesting that this Ab may not deplete
Teff cells. This may be due to the lack of sustained expression of
CD25 on Teff cells in contrast to Treg cells that express high and
sustained levels of CD25 (43).
A more stringent regimen involving intratumoral treatment with
depleting doses of an anti-CD4 mAb singly or in combination with
systemic and intratumoral injection of depleting doses of anti-
CD25 mAb also failed to alter the rate of tumor growth. In contrast
to our study, this regimen resulted in the eradication of established
tumors in the aforementioned fibrosarcoma model (30), providing
further evidence for the lack of a therapeutic effect of Treg cell
depletion in late stage tumors in our A20 model. This fibrosarcoma
tumor, to our knowledge, is the only model in which the depletion
of Treg cells using CD25 Ab alone could result in the eradication
of established tumors.
Although the source of the discrepancy between our study and
that of Yu et al. (30) is presently unknown, the aggressive nature
of the A20 tumor model and the use of multiple immune evasion
mechanisms by A20 may provide an explanation. Indeed, our data
imply that A20 tumors may use several direct (lack of CD80 co-
inflammatory cytokine IL-10, and immunoregulatory IDO en-
zyme) as well as indirect immune evasion mechanisms, such as
Treg cells and APCs with altered immune functions, to evade im-
The importance of the lack of CD80 costimulation as an impor-
tant immune evasion mechanism has already been demonstrated
by studies using genetically manipulated A20 cells expressing
CD80 as vaccine (29, 31, 32). We have also shown that A20 cells
stimulators of allogeneic responses. A, RT-PCR analysis of IDO expression
in total tumor and A20 cells. B, Effect of IDO inhibitor 1-MT on the
proliferative response of CFSE-labeled C57BL/6 T cell response against
irradiated A20 cells. C, APCs from naive or tumor-bearing BALB/c mice
were irradiated and used as stimulators for CFSE-labeled C57BL/6 lymph
node cells, as in B. The 1-MT did not restore the proliferative response of
naive C57BL/6 T cells against APCs harvested from tumor-bearing
A20 cells and APCs from tumor-bearing animals are poor
6846ROLE OF CD4?CD25?Treg CELLS IN CANCER IMMUNE EVASION
by guest on October 18, 2015
decorated with a chimeric CD80 protein served as effective vac-
cine for the prevention of tumor growth (29). Although we did not
specifically probe the role of PD-L1 coinhibitory molecule ex-
pressed by A20 in tumor growth, this molecule has been impli-
cated in peripheral tolerance in various experimental settings, in-
cluding tumors (44). Many tumors express PD-L1 on their surface,
and blockade of this molecule results in susceptibility to immune
destruction (33). PD-L1 interaction with PD-1 receptor may play
an important role in the generation of Treg cells and/or regulation
of their function in the A20 model. Signaling through PD-1 in Teff
cells results in IL-10 production, and IL-10 has been shown to
have a plethora of anti-inflammatory effects on the immune sys-
tem, including generation of tolerogenic APCs as well as Treg
cells or modulation of their function (45). Consistent with this
notion is our observation that serum from tumor-bearing animals
had significant levels of IL-10 and inhibited alloreactive responses
ex vivo. The suppressive effect of serum could be neutralized using
a mAb against IL-10. However, we failed to demonstrate a signif-
icant role for IL-10 in tumor immune evasion in vivo because the
treatment of tumor-challenged animals with the IL-10 mAb singly
or in combination with anti-CD25 mAb had no effect on tumor
growth. These data suggest that IL-10 in the A20 model may serve
a complementary role to other immune evasion mechanisms, such
as IDO, PD-L1, and Treg cells, rather than playing a dominant one.
Alternatively, the in vivo blocking conditions tested in our study
may not be sufficient to neutralize all the available IL-10 for a
detectable effect. Although the amount of anti-IL-10 mAb used in
this study was comparable to that used for the same clone in sev-
eral studies with efficacy (24, 46, 47), the high levels of IL-10
secretion by A20 cells in our model may require a more aggressive
There was an inverse correlation between the tumor size and the
percentages of CD4?and CD8?T cells within the spleen, lymph
nodes, and tumor. Although exact mechanisms are unknown, ap-
optotic molecules, such as PD-L1 (48) expressed by A20 cells or
NO released by host cells, such as tumor-infiltrating macrophages
(49), may play a role. Furthermore, we have observed metastases/
migration of A20 tumor cells into peripheral lymph tissues as a
function of tumor growth (data not shown) that may also be re-
sponsible for the observed decrease in the percentages of T cells in
These various immune evasion mechanisms may complement
and/or augment the role of Treg cells in tumor progression. It has
been shown that various immunoregulatory molecules, such as
PD-L1, B7-H4, IL-10, TGF-?, and IDO, mediate immune sup-
pression in tumor environment (20, 21, 24, 33, 48, 50–53). These
molecules may be constitutively expressed by tumor cells or be
induced by tumor microenvironment for expression in tumor cells
or APCs (20, 21, 24, 33, 48, 50–53). The coordinated expression
of these immunoregulatory molecules within the tumor may set in
motion a suppressive circuit involving various immune cells with
regulatory functions. This notion is consistent with recent studies
demonstrating the role of some of these immunoregulatory mole-
cules in the generation, expansion, and/or function of CD11b?IL-
4R??or Gr-1?CD115?immature myeloid suppressor cells (42,
54) and CD4?CD25?FoxP3?, CD4?CD25?FoxP3?, or CD8?
Treg cells (55). Therefore, tumors that use fewer of these immune
evasion mechanisms may primarily rely on Treg cells for progres-
sion. However, those that use multiple immune evasion mecha-
nisms, such as A20, may rely on Treg cells early when some of
these immune evasion mechanisms are not fully developed. Once
other mechanisms are induced and fully functional late in tumor
progression, the lack/elimination of any one immune evasion
mechanism, including Treg cells, may be compensated by others,
leading to undetectable/minimal effect on tumor growth. This no-
tion is consistent with our findings that in vivo blocking of IL-10
and physical depletion of Treg cells in established A20 tumors had
no detectable effect on tumor growth.
Our findings are further supported by clinical studies demon-
strating that the rIL-2 diphtheria toxin conjugate (ONTAK) is ef-
fective in reducing the number of Treg cells in the peripheral blood
of patients with metastatic renal cell carcinoma without therapeutic
efficacy (56). In addition, the therapeutic efficacy of Treg cell de-
pletion in various preclinical models was shown to depend on the
use of other adjuvant immunotherapeutic regimens, such as IL-12
gene therapy or DC vaccines (57, 58). These observations from
animal studies and clinical trials reveal that therapeutic approaches
only targeting Treg cells in cancer patients may fail or not be
sufficient to reverse tumor growth depending on the cancer type.
Treg cells may only be critical at early stages of tumor progression,
and other immune evasion mechanisms that are related or unre-
lated to Treg cells may be the dominant factors at late stages of
tumor progression. Therefore, immunotherapeutic approaches tar-
geting Treg cells for physical and functional elimination in cancer
patients need to consider the type and stage of tumor, and its as-
sociated immune evasion mechanisms for success. Furthermore,
the efficacy of immunotherapeutic approaches will depend on their
ability not only to effectively overcome various immune evasion
mechanisms, but also generate new antitumor responses and/or
up-regulate the existing ones.
The authors have no financial conflict of interest.
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6848 ROLE OF CD4?CD25?Treg CELLS IN CANCER IMMUNE EVASION
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