Lymphadenectomy exacerbates tumor growth while lymphadenectomy plus the adoptive transfer of...

ORIGINAL ARTICLE

Lymphadenectomy exacerbates tumor growthwhile lymphadenectomy plus the adoptive transferof autologous cytotoxic cells and low-dose cyclophosphamideinduces regression of an established murine fibrosarcoma

Andrea Maglioco • Damian Machuca • Juliana Mundinano • Gabriel Cabrera •

Gabriela Camicia • Juan Bruzzo • Gabriela Camerano • Hector Costa •

Raul A. Ruggiero • Graciela I. Dran

Received: 18 August 2010 / Accepted: 19 November 2010 / Published online: 14 December 2010

� Springer-Verlag 2010

Abstract Tumor-draining lymph node (TDLN) ablation is

routinely performed in the management of cancer; never-

theless, its usefulness is at present a matter of debate. TDLN

are central sites where T cell priming to tumor antigens and

onset of the antitumor immune response occur. However,

tumor-induced immunosuppression has been demonstrated

at TDLN, leading to downregulation of antitumor reaction

and tolerance induction. Tolerance in turn is a main

impairment for immunotherapy trials. We used a murine

immunogenic fibrosarcoma that evolves to a tolerogenic

state, to study the cellular and molecular mechanisms

underlying tolerance induction at the level of TDLN and to

design an appropriate immunotherapy. We determined that

following a transient activation, the established tumor

induces signs of immunosuppression at TDLN that coexist

with local and systemic evidences of antitumor response.

Therefore, we evaluated the feasibility of removing TDLN

in order to eliminate a focus of immunosuppression and

favor tumor rejection; but instead, a marked exacerbation of

tumor growth was induced. Combining TDLN ablation with

the in vivo depletion of regulatory cells by low-dose

cyclophosphamide and the restoring of the TDLN-derived

cells into the donor mouse by adoptive transference, resulted

in lowered tumor growth, enhanced survival and a consid-

erable degree of tumor regression. Our results demonstrate

that important antitumor elements can be eliminated by

lymphadenectomy and proved that the concurrent adminis-

tration of low-dose chemotherapy along with the reinocu-

lation of autologous cytotoxic cells provides protection. We

suggest that this protocol may be useful, especially in the

cases where lymphadenectomy is mandatory.

Keywords Tumor immunity � Tolerance �Immunotherapy � Tumor-draining lymph node �Lymphadenectomy

Introduction

Overall cancer remission rates have remained essentially

unchanged for the past decades [1]. Increasing evidence in

patients and animal models indicating that malignant tissue

is potentially immunogenic has led to immunological

therapies (IT) to control and eradicate cancer [2, 3].

However, even in the case of proven antigenicity and

significant lymphocyte infiltration, therapeutically induced

rejection of established tumors is a very unusual event.

Consensus exists that an active process of tolerization of

the immune system is mounted and maintained by tumor

cells [4–6]. In advanced disease, the state of tolerance is

not merely the absence of immune response but a wide-

spread immunosuppression that constitutes a major obsta-

cle against the efficacy of immunological trials [5, 6].

Therefore, new insights into the mechanisms governing the

establishment of tolerance in tumor hosts are necessary

before IT can be successfully applied.

The experimental murine fibrosarcoma MCC is a highly

immunogenic tumor induced by 3-methylcholanthrene in a

BALB/c mouse [7, 8]. The specific immune response

elicited by MCC is not strong enough to reject the growing

tumor, and after a certain volume it declines and disappears

A. Maglioco � D. Machuca � J. Mundinano � G. Cabrera �G. Camicia � J. Bruzzo � G. Camerano � H. Costa �R. A. Ruggiero � G. I. Dran (&)

Division Medicina Experimental,

Academia Nacional de Medicina de Buenos Aires,

ILEX-CONICET, Buenos Aires, Argentina

e-mail: [email protected]

123

Cancer Immunol Immunother (2011) 60:389–399

DOI 10.1007/s00262-010-0949-3

creating a state of tolerance [7, 9]. The immunological

characteristics of MCC, including its rapid growth in

vivo, make it a suitable model to study mechanisms

underlying early tumor immunity and tumor-mediated

immunosuppression.

Among tumor host lymph nodes (LN), those which drain

the lymphatic vessels from the tumor site (tumor-draining

lymph nodes or TDLN) have been associated with the

malignant dissemination, and partial or vast lymphade-

nectomy is routinely performed for diagnosis, staging and

treating cancer [10]. At present, however, the helpfulness

of TDLN removal or keeping is a matter of debate [11, 12],

and these organs are given greater biological than anatomic

relevance since they constitute the physical spot where

both the initiation of antitumor immune response [13] and

tolerization [14] may occur.

The aim of this work was to study the immunological

changes that underlie tumor-generated tolerance at the

level of TDLN in order to design an appropriate immu-

nological treatment.

Materials and methods

Mice

Two- to three-month-old male BALB/c and C57BL/6 mice

inbred in the animal facilities of the Instituto de Investi-

gaciones Hematologicas, Buenos Aires, and housed

according to the policies of the NIH Guide for the Care and

Use of Laboratory Animals, were used.

Tumor and tumor lysate

The MCC fibrosarcoma was induced in a BALB/c male

by s.c. implantation of a methylcholanthrene pellet. It is

maintained by syngeneic transplantation, giving rise to a

solid non-metastatic tumor. Studies were performed

between passages 20 and 25 in two successive stages of

MCC growth: IMM (immunogenic established tumor,

volume 100–400 mm3) and ADV (tolerogenic advanced

tumor, volume 800–1,200 mm3). In some experiments an

early time point (2 days post-tumor cell inoculation

‘‘2 days p.i.’’) is also shown. Proximal and distal LN

remain free from tumor cells along MCC development

[8]. Tumor size and volume were assessed every 2 days

according to the Attia and Weiss formula. For tumor

lysate preparation, MCC cells (1 9 106/ml) were lysed by

six freezing–thawing cycles, sonicated for 10 min in a

Branson Digital Sonifier and passed through a 0.2 lm

filter.

Cells and culture conditions

LN or tumor tissue were aseptically excised and mechan-

ically disaggregated in PBS. Single cell suspensions were

cultured in complete medium (CM: RPMI1640, Gibco;

10% fetal bovine serum, Natocor, Argentina; L-glutamine,

penicillin/streptomycin/amphotericin B and 2-mercap-

toethanol; Gibco), at 37�C in 5% CO2 atmosphere. Single

spleen cell suspensions were obtained by mechanical

dissociation followed by sedimentation over Ficoll

(1.09 g/cm3).

Flow cytometry

LN cells (1 9 106) were incubated with appropriate con-

centration of the following mAb (Pharmingen, San Diego,

CA): FITC or PE anti-CD8a (clone 53-6.7), FITC anti-

CD80 (clone 16-10A1), FITC anti-CD40 (clone HM40-3),

FITC anti-I-Ad (clone AMS-32.1), FITC or PE anti-CD11c

(clone HL3), FITC anti-CD4 (clone H129.19), PE

anti-CD19 (clone 1D3), PE anti-IFN-c, PE anti-IL-10,

PE-Cy5.5 anti-CD45R/B220 (clone RA3-6B2), PE-Cy5.5

anti-CD25 (clone PC61). Cells were analyzed using a

FACS flow cytometer (BD Biosciences) and Winmdi

software. Irrelevant isotype-matched antibodies were used

as controls. Intracellular presence of Foxp3 was detected

with PE anti-Foxp3 and the Foxp3 staining buffer set

(e-Bioscience); intracellular staining of IL-10 and IFN-cwere detected with above-mentioned antibodies and

Cytofix/Cytoperm and Perm/Wash buffer (Pharmingen)

according to the manufacturer’s protocol.

Cytokine production

Commercial ELISA kits (e-Bioscience) were used follow-

ing the manufacturer’s instructions. Secreted cytokines

were determined in serum or in cell-free supernatants from

tumor or LN cell cultures exposed to tumor lysate (1:1).

Cells (2–5 9 106 cells/ml) were cultured for 24 h for

determination of TGF-b and IFN-c or 72 h for IL-10 and

IL-12.

Contact sensitivity response (CSR)

A modification of a previous protocol was used [15]. At

day 0 mice were painted on TDLN or nTDLN skin areas

with 50 ll 1% FITC in acetone–dibutylphthalate (DBF)

solution (1:1 v/v) or vehicle. At day 6, animals were

challenged with 10 ll 1% FITC onto both sides of one ear.

Ear swelling was measured by micrometer before and 48 h

after the second challenge.

390 Cancer Immunol Immunother (2011) 60:389–399

123

Mixed lymphocyte reaction (MLR)

C57BL/6 mouse LN cells (2 9 105) were incubated with

5 9 104 mitomycin C (Sigma)-treated tumor bearing

mouse LN cells for 96 h, with a final 18 h pulse of 1 lCi/

well [3H]-thymidine (Dupont, NEN Res Products, Boston,

MA). Incorporated radioactivity was measured in a liquid

scintillation Beta counter (Beckman). 5 lg/ml IL-10 neu-

tralizing antibody (Pharmingen) was added or not at the

beginning of the incubation. Proliferation rate standard

deviation was calculated by the error propagation method.

Cytotoxicity assays

In vitro cytotoxicity was evaluated through the JAM test

[16]. Briefly, tumor-bearing mice LN cells (1 9 106) were

incubated with [3H]-thymidine-labeled MCC cells (2.5 9

104) for 8 h. Specific killing was calculated as % =

100 9 (S - E)/S, where S is the spontaneous killing and

E is the experimental killing. Normal [3H]-thymidine-

labeled fibroblasts (target) were used as controls.

Regulatory T cell inhibitory function

T-reg suppressive function was evaluated through a pre-

viously described protocol [17]. CD4?CD25? (T-reg),

CD4?CD25- (T-eff) and CD4- were isolated from ingui-

nal lymph node (Normal, IMM, Cy-treated mice) by cell

sorting (FACSARIA II). CD4- were irradiated (3,300 rad)

and plated onto 96-well plates (105 cells/well). T-eff cells

(104cells/well) were then added with or without T-reg

(104cells/well). Cells were incubated in CM with a com-

bination of 1 lg/ml of soluble anti-CD3 and 1 lg/ml of

soluble anti-CD28 to provide the polyclonal stimulus for

proliferation. At day 4, 1 lCi of 3H-thymidine was added

for 18 h to assess proliferation.

Ablation of TDLN (LNx)

Inguinal TDLN were surgically removed during IMM or

ADV.

Ex vivo activation and expansion of TDLN-derived

cells

A previously described method was used [18]. Briefly,

TDLN cells were activated with 1 lg/ml immobilized anti-

CD3 (clone 145-2C11, BD Pharmigen) on 24-well culture

plates at 2 9 106/ml CM for 2 days. Harvested cells were

further cultured at 2 9 105/ml CM containing 5U/ml rIL-2

(BD biosciences). Three days later cells were resuspended

in PBS for adoptive transference or cytometry, or in CM

for the JAM test.

Immunotherapeutic protocol: low-dose

cyclophosphamide (Cy) ? TDLN ablation

(LNx) ? adoptive transference of TDLN-derived

cells (AT)

At day 0, mice carrying IMM tumors were i.p. inoculated

with 50 mg/kg Cy (Kampel Martian) and 4 days later,

TDLN were removed and cells were cultured with anti-

CD3 and IL-2 as explained above. At day 9, donor mice

were i.v. injected with 5 9 106 cultured cells/ml PBS.

Untreated and partially treated animals were used as con-

trols. Subsequent tumor growth and survival were assessed

daily for a period of 4 months.

Statistics

Differences in phenotype were evaluated by Student’s

t test. Cytokines levels and tumor growth curves were

compared by one-way ANOVA and Turkey’s multiple

comparison test. Survival data were analyzed by the

log-rank test. Differences in JAM test were analyzed by

two-way ANOVA and Bonferroni’s multiple comparison

test. All tests were interpreted in a two-tailed fashion and

p \ 0.05 was considered significant (*p \ 0.05,

**p \ 0.01, ***p \ 0.001, compared to control non-

tumor-bearing mice; ap \ 0.05, bp \ 0.01, cp \ 0.001,

compared to other experimental groups).

Results

Tumor growth induces changes in the cellular

composition of tumor-draining (TDLN) and distal

non-tumor-draining lymph nodes (nTDLN)

Studies were performed at two separate phases of MCC

progression, 15 and 30 days after tumor inoculum, repre-

senting (a) an intermediate-volume tumor, which has been

proven to elicit specific immunity, ‘‘IMM’’ and (b) an

advanced high-volume tumor, to which the host is habit-

ually tolerant, ‘‘ADV’’ [7]. The presence of MCC induced

increased total nucleated cell number in the TDLN. When

individual immune cell populations were analyzed,

CD11c? dendritic cells (DC), CD8? and CD4? T cells and

more distinctly, CD19?B220? B lymphocytes, augmented

compared with non-tumor-bearing mice LN (normal). In

contrast, no variations occurred in nTDLN until the ADV

stage, when every cell type decreased below normal values,

except for B cells which augmented (Fig. 1). Interestingly,

during ADV, not only the contralateral inguinal nTDLN,

but also every LN distant to the tumor site had small size

and low cell number compared to normal LN (not shown).

Variations in the percentual composition were seen at both

Cancer Immunol Immunother (2011) 60:389–399 391

123

LN and comprised increased proportion of B cells and

decreased proportion of T cells.

Tumor growth induces changes in the phenotype

and function of LN-derived antigen presenting cells

(APC)

Two days after tumor cells inoculation (2 days p.i.) there was

a per cell increase in the expression of the costimulatory

molecules CD40 and CD80 on DC and CD40 on B cells at

TDLN, denoting an initial, probably non-specific APC acti-

vation at the site of tumor implantation. Once the tumor is

established, at IMM and ADV stages, APC showed enhanced

expression of MHC class II molecules without variation in

CD40 or CD80, both in TDLN and nTDLN, suggesting

immature/suppressor phenotype [19] (Fig. 2a, b). Among

suppressor DC described in mouse LN, a subset of plasma-

cytoid DC (pDC) that constitutively expresses immunosup-

pressive molecules and B lineage markers was described [20].

We found that the proportion of these potentially tolerogenic

cells increased in IMM and ADV at TDLN and nTDLN

(% B220?CD19? in CD11c? cells ± SD, normal, 19.2 ±

2.6; IMMTDLN, 44.6 ± 3.5***; IMMnTDLN, 33.0 ± 0.8***;

ADVTDLN, 46.2 ± 4.3***; ADVnTDLN, 35.5 ± 1.7***; data

from one representative experiment, n = 4 mice/group).

The function of LN-derived APC was studied by two

different assays: first, their ability to induce allogeneic

proliferation in a MLR. Despite the high membrane

expression of MHCII on DC, LN cells from mice carrying

IMM and ADV tumors induced lower allogeneic prolifer-

ation than their normal counterparts, suggesting that an

inhibitory factor or cellular type might be interfering with

proliferation. Since IL-10 production is a widespread

inhibitory mechanism for B lymphocytes and pDC in

tumors [21–23], we added a blocking concentration of anti-

IL10 mAb, and obtained restored MLR response (Fig. 2c).

Secondly, we studied the ability of skin Langerhans cells

(LC) to uptake a surface antigen, migrate to the nodes and

stimulate a T cell response; this method was previously

proposed as a way to study the effect of tumor presence on

the APC function [15]. We observed enhanced antigen

uptake and migration during IMM and ADV, not only

when the antigen was applied over the TDLN area, but also

distantly. Unexpectedly, most (65–78%) of the FITC-loa-

ded APC that got the LN draining the painted area were

B220? B cells. The contact sensitivity response (CSR) was

measured 6 days later. Whereas normal mice showed a

strong response, it was largely inhibited in IMM and ADV

tumor-bearing mice, first at tumor proximity (IMM) and

then systemically (ADV) (Fig. 2d).

Tumor growth induces changes in the phenotype

and cytotoxic capacity of LN-derived T cells

Phenotypically activated T cells (CD4?CD25?Foxp3-)

increased among the CD4? population during IMM and

4500

5000

5500

6000DCs (CD11c+)

T CD8+

B CD19+B220+

T CD4+

Total LN cells

***

*

3000

3500

4000of

cel

ls (

x10

3 )/ L

N

1000

1500

2000

2500

Num

ber o

**

*** ***

***

****

***

0

500

NORMAL IMM TDLN ADV TDLN ADV nTDLN

*** *

***

******

Fig. 1 The growth of MCC induces an increase in the number of total

cells, DC, T and B lymphocytes within the TDLN, and a late decrease

of total cells, DC and T cells within nTDLN. Lymph node cells were

isolated from inguinal TDLN and contralateral nTDLN and total cell

number was determined by hemocytometer. Cell suspensions were

then stained with the specific monoclonal antibodies for flow

cytometry. Absolute cell number for different cell types at the

indicated stages of tumor growth was calculated into the nucleated-

viable cell gate. The graphic shows mean number of cells ± SD from

a representative experiment, n = 4 mice per group. For total LN cells,

cumulative data from 9–24 mice per group is shown


123

decreased in ADV, both in TDLN and nTDLN. Whereas

activated T cells decreased, the proportion of

CD4?CD25?Foxp3? regulatory T cells (T-reg) aug-

mented, reaching significance in ADV, as did the

CD4?CD25-Foxp3? subset, which has been described as a

reservoir [24] or dividing T-reg [25] (Fig. 3a). Although in

low percentage, CD4?CD25? T cells isolated from the

TDLN during IMM were able to inhibit proliferation of

conventional T cells (Fig. 3b).

On the other hand, the number of CD8? T lymphocytes

expressing CD25 increased during IMM suggesting

activation (no. CD25?CD8? T cells 9 103: normal,

21.4 ± 5.6; IMM, 62.4 ± 21.6*; ADV, 28.1 ± 6.6). The

expression of Foxp3 in CD8? T cells was null.

Anti-MCC cytotoxicity was evaluated by studying the

ability of LN-derived cells to kill MCC cells in vitro [16].

Cultured TDLN and nTDLN cells from IMM and ADV

exhibited enhanced percentage of specific cytotoxicity com-

pared with normal mice (% mean ± SD: normal, 12.3 ± 0.7;

IMMTDLN, 29.8 ± 0.7***; IMMnTDLN, 27.8 ± 4.3**;

ADVTDLN, 28.5 ± 0.9***; ADVnTDLN, 29.4 ± 3.6**; data

from a reproducible experiment, n = 3 mice/group).

ba

700

800

900 CD40

CD80

MHCII

*700

800

900 CD40

MHCII

200

300

400

500

600

700

**

***

200

300

400

500

600

700 ***

** **

c

0

100

Normal 2 days pi IMM ADVMea

n f

luo

resc

ence

inte

nsi

ty (

MF

I)

Mea

n f

luo

resc

ence

inte

nsi

ty (

MF

I)

0

100

Normal 2 days pi IMM ADV

180

200 No anti-IL-10

Anti-IL10

100

120

140

160

180

b b Normal allogeneic

proliferation

b c

0

20

40

60

80

% P

rolif

erat

ion

of

C57

cel

ls

**

**

***

d

ADV TDLN

ADV nTDLN

IMM TDLN IMM nTDLN ADV TDLN ADV nTDLN

Normal

IMM TDLN

IMM nTDLN

0 20 40 60 80

Δ Ear thickness (x 10-2 mm)

*

*

#**

Fig. 2 MCC growth alters APC

phenotype and impairs APC

function at TDLN and nTDLN.

Lymph node cells were isolated

from tumor-bearing mice and

the expression of MHCII, CD40

and CD80 on CD11c? DC

(a) and MHCII and CD40 on

CD19? B cells (b) was assessed

by flow cytometry. Bars show

the mean fluorescence intensity

(MFI) – SD from a

representative experiment

(n = 3–5). c The ability of

LN-derived cells to induce

allogeneic proliferation was

evaluated by MLR using

C57BL/6 mice LN cells as a

target. The bars show C57BL/6

proliferation (cpm) obtained in

the presence of LN cells from

tumor-bearing mice, related to

that obtained in the presence of

LN cells from normal mice

(100%). Gray and black barsrepresent C57 proliferation

in the presence/absence of

anti-IL-10 neutralizing

antibody, respectively. One

representative experiment is

shown (bp \ 0.01 andcp \ 0.001 vs. non-IL-10-

treated wells). d The contact

sensitivity reaction (CSR)

against the surface antigen

DBF-FITC was measured

6 days after the first exposure.

Ear thickness was measured

before and 48 h after the second

challenge. Values represent the

mean change in ear

thickness ± SD (vehicle,

0–10 9 10-2 mm) from a

reproducible experiment,

n = 3–5 mice/group (#p \ 0.05

IMMTDLN vs. IMMnTDLN)


123

Tumor growth induces changes in the cytokine profile

of lymph nodes and sera

The local cytokine milieu has a significant impact on

immune cell maturation and function (i.e., immune sup-

pressive [21, 22, 26] or stimulatory [27]). To study the

involvement of most important cytokines in the immune

response to MCC, sera from tumor-bearing mice, and

tumor and LN culture supernatants, were examined for the

presence of IL-10, IFN-c, IL-12 and TGF-b. No stimula-

tory cytokines IL-12 or IFN-c were detected in tumor

bearers’ sera or supernatants at any time point. With

respect to suppressive cytokines, significant amounts of

IL-10 were secreted by LN cells obtained during IMM and

ADV stimulated in vitro with tumor lysate (normal,

235.3 ± 25; IMMTDLN, 367.2 ± 45***; IMMnTDLN,

354 ± 41***; ADVTDLN, 367.2 ± 17***; ADVnTDLN,

310.7 ± 12** pg/ml IL-10), whereas TGF-b was detected

in IMM and ADV sera and in MCC culture supernatant

(2,883 ± 536; 2,838 ± 610 and 1,988 ± 604 pg/ml TGF-

b, respectively) and was undetectable in sera from normal

mice (\62 pg/ml TGF-b). Data were obtained from two

separate experiments with 5–6 mice per group.

In another set of experiments, intracellular presence of

IFN-c and IL-10 was evaluated in LN-derived T, B and

DC. The number of CD8? and CD4? T lymphocytes

expressing IFN-c augmented during IMM at both LN

(Fig. 4a), suggesting that a potential cytotoxic activity

involving effector CD8? T and helper CD4? T cells exists

during the IMM phase, both near and distant to the tumor

site. At the same time, the number of B lymphocytes

expressing IL-10 markedly increased in every phase

(Fig. 4a, b), probably indicating inhibitory roles for this

cell population. No expression of IL-10 by DC and T-reg

or IFN-c by DC was detected at any time point.

The ablation of the TDLN (LNx) during the IMM phase

exacerbates tumor growth

The above data indicated that immunosuppressive elements

are present at the LN of mice bearing established IMM

tumors. Since TDLN is considered a central place where

immunosuppression and tolerance to tumor antigens are

originated, and given the fact that systemic anti-MCC

immunity is manifested in this stage [7–9], we evaluated

the hypothesis that the surgical removal of TDLN (LNx) at

IMM would negatively affect tumor progression. On the

contrary, LNx induced a marked exacerbation of tumor

growth (Fig. 5a), while no differences were obtained in the

growth of tumors from intact, sham-operated and mice that

underwent LNx during ADV (not shown).

Administration of low-dose cyclophosphamide (Cy)

followed by the adoptive transference (AT)

of ex vivo-treated TDLN cells induces tumor regression

and improves survival

In view of the LNx-induced tumor exacerbation, we evalu-

ated the helpfulness of supplementing LNx with two pro-

cedures: the reduction of endogenous regulatory cells by

low-dose Cy and the restoration of cytotoxic cells from the

excised TDLN, which had been previously activated and

expanded ex vivo with anti-CD3 and IL-2. Remarkably, 19

out of 30 (63%) fully treated mice (Cy ? LNx ? AT)

20000

30000b

0

10000***

Co

un

ts p

er M

inu

te

CD4+CD25- + +CD4+CD25+ - +

a

Normal

8.1 ± 1.2

6.1 ± 1.4

1.0 ± 0.3

IMM ADV

TDLN

nTDLN

9.3 ± 1.5

6.6 ± 1.6

3.1 ± 1.3***

13.3 ± 2.5

10.1 ± 1.5*

1.2 ± 0.3

14.5 ± 0.9***

10.5 ± 1.3***

1.3 ± 0.5

9.2 ± 1.1

7.6 ± 1.1

2.5 ± 0.5***

Foxp3

CD

25

8.1 ± 1.2

6.1 ± 1.4

1.0 ± 0.3

9.3 ± 1.5

6.6 ± 1.6

3.1 ± 1.3***

13.3 ± 2.5

10.1 ± 1.5*

1.2 ± 0.3

14.5 ± 0.9***

10.5 ± 1.3***

1.3 ± 0.5

9.2 ± 1.1

7.6 ± 1.1

2.5 ± 0.5***

Fig. 3 The LN proportion of activated CD4?CD25?Foxp3- T cells

increases during IMM, while CD4?CD25?Foxp3? regulatory T cells

increase during ADV. Regulatory T cells isolated from tumor-bearing

mice LN exhibit inhibitory function. a Dot plots from one reproduc-

ible experiment show CD25 versus Foxp3 positive cells, gated within

CD4? cells. Rectangles show increased proportion of activated T

cells (CD25?Foxp3-/CD4?) in IMM and T-reg (Foxp3?/CD4?) in

ADV. b Bars show the inhibitory effect of T-reg obtained from IMM

TDLN on the proliferation of CD4?CD25- T cells in the presence of

anti-CD3 and anti-CD28 stimulation. Representative data from one of

two experiments is shown (n = 6)


123

underwent complete tumor regression with no recurrences,

while no regression was obtained either in untreated or

partially treated animals (not shown). Additionally, the

growth of those tumors that did not regress (11/30) exhibited

significantly lower growth compared to the untreated group,

as shown in Fig. 5b. It is worthy to mention that neither Cy

alone nor adoptive transference of cytotoxic T cells induces

regression. Survival rates shown in Fig. 5c indicated that

treatments with Cy ? LNx ? AT and Cy ? LNx signifi-

cantly enhanced survival.

In vivo administration of Cy reduces B and T-reg cell

number, while ex vivo exposition to anti-CD3 and IL-2

changes the composition and cytotoxic ability

of cultured TDLN cells

To elucidate the mechanisms operating in the treatment, we

evaluated the effects of in vivo administration of 50 mg/kg

Cy on day 4 (corresponding to the time point previous to

LNx; see methods) and on day 9 (5 days after LNx), on the

number of potentially suppressive cells, B and T-reg, present

at contralateral nTDLN. A non-significant reduction in the

content of total B cells, IL-10-expressing B cells and T-reg

was induced by Cy on day 4 (not shown), whereas decreases

were significant on day 9 (Fig. 6a). Moreover, isolated T-reg

cells from Cy-treated mice did not show inhibitory capacity

upon CD4?CD25- T cell proliferation (not shown).

We analyzed the effect of anti-CD3 and IL-2 on cultured

TDLN cells before their AT. While freshly harvested cells

from Cy-treated mice were mostly composed of B, CD4?T

and CD8?T cells, ex vivo exposition to anti-CD3 and IL-2

increased the proportion of CD4? and CD8?T and strongly

decrease B cells (% cells before versus after the culture:

B220?CD19?, 44 ± 3 vs 7.3 ± 2***; CD4?, 37 ± 1 vs

74 ± 7***; CD8?, 17.3 ± 0.5 vs 26.8 ± 6**; one repre-

sentative experiment, n = 6). Additionally, the culture

raised the proportion of T cells expressing intracellular

IFN-c (not shown). The anti-MCC cytotoxicity of these

cells is shown in Fig. 6b. Lymph node cells from

Cy-administered mice showed higher in vitro cytotoxicity

than PBS-treated and non-tumor-bearing mice. In turn,

culture with anti-CD3 and IL-2 improved the cytotoxicity

of cells from PBS-treated mice but did not change the

already high cytotoxicity of cells from Cy-treated animals.

80

100

120

/LN

CD8+IFN +

CD4+IFN +

CD19+IL10+ **

**

** *

a

20

40

60

Cel

ls (

x10

3)

* *

**

** **

b VDAlamroN IMM

0Normal IMM TDLN IMM nTDLN ADV TDLN ADV nTDLN

IL-1

0

0.1 ± 0.01 1.8 ± 0.2**1.2 ± 0.4*

17.1 ± 2.7 **6.0±8.13*6.0±7.22

CD19

Fig. 4 Tumor induces changes in the intracellular cytokine expres-

sion in LN-derived cells. a Figure shows a representative experiment

(n = 3–5) showing the number of CD4? and CD8? T cells expressing

intracellular IFN-c and B cells expressing IL-10 within LN.

b Representative dot plots showing the increase in LN-derived B

cells expressing IL-10. Numbers next to the dot plots indicate the

percentage of cells positive for B220 and IL-10 within the whole LN


123

Discussion

Tumor progression in normal individuals may reflect a

failure of the innate and adaptive immune responses.

Immunosuppressive mechanisms elicited by many cancers

not only dampen endogenous antitumor responses but also

weaken the efficacy of current immunotherapies (IT).

Thus, a better understanding of interactions between the

immune system and developing tumors is needed in order

to reverse tumor-mediated immunosuppression and

improve IT for cancer. We used an experimental tumor,

MCC, which has been shown to evolve from a state of

immunogenicity (IMM) to a tolerance condition (ADV)

[7]. During the IMM phase, MCC generates a strong sys-

temic antitumor response, which does not impede tumor

progression and progressively declines and disappears

[7–9]. Yet, the main aspects referring the cellular and

molecular mechanisms responsible for the changes in

tumor immunogenicity remain still unknown. Given that

the proximal lymphatic nodes are judged relevant organs in

cancer immunity, those draining the tumor zone (TDLN)

were chosen to study the tumor-induced changes in the

main immune cell components. As a distal counterpart,

non-tumor-draining contralateral inguinal lymph nodes

(nTDLN) were used.

After transient phenotypic signs of immune activation

mainly at tumor proximities (Figs. 3, 4), once the tumor is

established, immunosuppression prevails in both LN. Signs

of immunosuppression at LN comprised, firstly, variations

in APC subsets. The low expression of costimulatory

molecules by DC and B cells and the plasmacytoid phe-

notype of most DC, suggested impaired APC function. In

effect, both the ability to induce allogeneic proliferation

and evoke a T response to a non-related antigen was

deficient in LN cells from mice carrying IMM and ADV

tumors. In the second place, there was altered ratio between

effector and regulatory cells. Previous studies have shown

that immature and plasmacytoid DC generate tolerance to

ba

2000

2500

3000

um

e (m

m3)

LNx

SHAM LNx

b c

c c

20002500300035004000

um

e (m

m3)

Cy+LNx+AT

Untreated

D 0

0

500

1000

1500

0 5 10 15Days after LNx

Tu

mo

r vo

l

0500

100015002000

0 5 10 15 20 25 30 35Days post-inoculation

Tum

or

vol ay

treatment b

c

c

80

100Cy+LNx+AT (p=0.0004)

Cy+LNx (p=0.0014)

LNx+AT (p=0.5510)

Untreated

LNx (p=0 1564)

20

40

60

Per

cen

t su

rviv

al LNx .

Cy (p=0.7271)

00 10 20 30 40 50 60 70 80 90

Days post-inoculation

Fig. 5 Effect of lymphadenectomy, cyclophosphamide and adoptive

transference on the tumor growth and host survival. a Mice carrying

tumors IMM were subjected to ablation of the TDLN (LNx) or sham-

operated. Tumor growth was assessed daily, and experiments were

ended at a tumor volume of 2,500 mm3. Representative data from one

of four experiments is shown (n = 7–9, b p \ 0.01 and c p \ 0.001

for LNx vs. Sham). b MCC-bearing mice were administered 50 mg/kg

Cy and subjected to LNx or Sham-operation. The excised TDLN-

derived cells were ex vivo activated with anti-CD3 and expanded with

IL-2, and subsequently re-injected in the donor mouse by i.v. AT. The

curves show the growth of tumors that did not undergo complete

regression, over a period of 30 days (b p \ 0.01 and c p \ 0.001 for

Cy ? LNx ? AT vs. untreated). c Survival of 15–32 mice per group

was monitored over a period of 80 days. Numbers next to the graphreference indicate the statistical significance versus the untreated

group


123

tumors through the induction of T-reg [5, 20], which in

turn are a potent suppressor of cytotoxic lymphocytes [28–

30]. In our model, the proportion of T-reg increases as

activated T cells decrease with tumor progression; more-

over, T-reg isolated from TDLN showed inhibitory effect

upon conventional T cell proliferation (Fig. 3). In addition

to T-reg, an elevated number of IL-10-expressing B cells in

TDLN and nTDLN parallels MCC growth in every tumor

stage, suggesting an inhibitory role for B cells as well.

Accordingly, previous studies in human [31] and animal

[21, 22] cancer provided evidence of a regulatory role for B

cells, mostly through IL-10 production.

Cumulative data from the cytokine analysis indicate that

advanced MCC tumor evokes a late (ADV) suppressive

milieu with IL-10?B cells at both LN, while for interme-

diate tumor volumes (IMM), there were IL-10? B cells and

IFN-c? CD4 and CD8 T cells; these are potentially sup-

pressive and stimulatory elements exist simultaneously

inside both LN. Since many of the suppression signs were

first induced at TDLN, and given their markedly higher cell

content, TDLN constitute a more suitable initial and

functional focus of inhibition compared with neighboring

LN. As previously mentioned, a systemic anti-MCC

immune response had been shown to exist in MCC hosts

during IMM [7]; we therefore postulated that removing

TDLN (LNx) during IMM would impair MCC progression.

In contrast, LNx resulted in unexpected acceleration of

MCC growth, suggesting that suppressor mechanisms had

been activated and/or that important antitumor elements

had been subtracted by LNx.

It has been previously documented that low-dose Cy is

able to decrease the number and inhibit the function of

T-reg, thus enhancing the antitumor response and the

efficacy of some IT [32–35]. On the other hand, it is known

that high amounts of tumor-reactive CTL can be isolated

from TDLN cells cultured with anti-CD3 and IL-2 [18, 36].

In view of these antecedents, we attempted to restore some

of the primed cytotoxic cells through the AT of the excised

TDLN cells into Cy-administered tumor bearers. With such

protocol, decreased tumor growth, increased survival and

approximately 60% of tumor remission were achieved.

Additional observation of Cy and anti-CD3 plus IL-2 mode

of action suggested that the mechanisms accounting for the

protection are, at least in part, Cy-mediated T-reg and B

a

1500

2000

2500 B cells

CD4+ Foxp3+ cellsa

a

IL-10+ B cells

3 )PBS LN CY LN PBS LN CY LN PBS LN CY LN

0255075

500

a

a

N°

cells

/ n

TD

LN

(x

10

b

PBS+LNx CY+LNx PBS+LNx CY+LNx PBS+LNx CY+LNx

30

40

50

*

***

***

***

***

**

Non cultured anti-CD3 + IL-2 0

10

20

Naive vs MCC cells

PBS vs MCC cellsCy vs MCC cells

% D

eath

cel

ls

PBS vs normal fibroblasts

Fig. 6 In vivo effects of

cyclophosphamide and ex vivo

effects of anti-CD3?IL-2. a The

figure shows the number of total

B cells, T-reg and IL-10? B

cells at nTDLN at day 9 after

i.p. injection of 50 mg/kg Cy

from a representative

experiment; n = 4–6. b Tumor-

draining lymph node were

removed from MCC-bearing

mice 4 days after Cy or PBS

administration, and resulting

cell suspensions were cultured

with anti-CD3 and IL-2 for

5 days. In vitro specific anti-

MCC cytotoxicity was assessed

by JAM test before and after

exposure to anti-CD3 and IL-2.

Lymph node cells from naıve

mice and normal fibroblast were

used as controls. One

representative experiment is

shown (n = 3)


123

cell reduction, and anti-CD3 plus IL-2-induced increase of

IFN-c production and cytotoxicity of TDLN-derived cells.

In vivo effects of low-dose Cy have been referred in other

tumor models to selectively deplete/inhibit T-reg without

affecting conventional T cells. This specificity would rely

on the differential expression of foxp3 [37] and/or the

reduced intracellular ATP level [38] exhibited by T-reg. In

addition to T-reg, our present results suggesting an inhib-

itory role for B cells bring the possibility that Cy-mediated

B cell depletion constitutes another mechanism accounting

for low-dose Cy protective effect.

It is important to mention that even when regional

lymph node (RLN) adenectomy is routinely performed for

studying and treating cancer, the actual convenience of this

procedure is questioned by many facts. Firstly, the exis-

tence of lymphatic and venous shunts capable of bypassing

RLN, allow in many cases the persistence of dissemination

[39]. Secondly, RLN can constitute central priming sites

[40], act as effective barriers to tumor dissemination [13,

41] and harbor pre-effector cells capable of destroying

tumors through AT [34, 39]. Thirdly, clinical and experi-

mental data indicate that LNx not always improves prog-

nosis [42] and in some cases may decrease the

effectiveness of immunotherapeutic protocols [43]. In this

context, a more conservative behavior toward uninvolved

RLN that permits the maintenance of the integrity of the

immune system has been suggested to provide benefits.

We demonstrated that TDLN ablation can alter some

important endogenous antitumor mechanisms, and showed

that a therapeutic approach comprising the reinoculation of

activated autologous CTL, preceded by Cy-mediated B cell

and T-reg depletion, is able not only to counteract the

worsening impact of LNx, but also to improve the antitu-

mor immunity and induce tumor regression. We suggest

that such protocol may be useful especially in cases where

proximal lymphadenectomy is an inevitable procedure.

Acknowledgments This work was supported by grants from

Consejo Nacional de Investigaciones Cientıficas y Tecnicas (CONI-

CET), Agencia Nacional de Promocion Cientıfica y Tecnologica and

Fundacion ‘‘Albert J. Roemmers’’, Republica Argentina. The authors

are grateful to Dr. Christiane Dosne Pasqualini for critical discussion

of this article.

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