decreased antigen presentation by dendritic cells in ...decreased antigen presentation by dendritic...

Vol. 3, 483-490, Marc/i 1997 Clinical Cancer Research 483

Decreased Antigen Presentation by Dendritic Cells in Patients with

Breast Cancer’

Dmitry I. Gabrilovich,2 Jadranco Corak,

Ilja F. Ciernik, Denise Kavanaugh, and

David P. Carbone

The Vanderbilt Cancer Center, Vanderbilt University School of

Medicine, Nashville, Tennessee 37232-6838 ID. I. G., D. P. Cl:

Hamon Center for Therapeutic Oncology Research, University of

Texas Southwestern Medical Center, Dallas, Texas [J. C., D. K.l: andDepartement fuer Innere Medizin, Universitaetsspital Zurich

Raemistrasse 100, 8091 Zurich, Switzerland [I. F. C.l

ABSTRACT

We evaluated T-cell responses to mitogens and to de-fined antigens in breast cancer patients. Significant defectsin responses to tetanus toxoid and influenza virus were

observed in patients with advanced-stage breast cancer. To

define whether these defects were associated with a defect inantigen presentation [dendritic cells (DCs)1 or effector func-

tion (T cells), these cells were studied separately Purified

DCs from 32 patients with breast cancer demonstrated a

significantly decreased ability to stimulate control allogeneicT cells, but stimulation of patient T cells with either controlallogeneic DCs or immobilized anti-CD3 antibody resulted

in normal T-cell responses, even in patients with stage IV

tumors. These data suggest that reduced DC function couldhe one of the major causes of the observed defect in cellularimmunity in patients with advanced breast cancer. We then

tested whether stem cells from these patients could give riseto functional DCs after in vitro growth with granulocyte/

macrophage colony-stimulating factor and interleukin 4.

Normal levels of control allogeneic and tetanus toxoid-

dependent T-cell proliferation were observed when DCs

obtained from precursors were used as stimulators. Those

cells also induced substantially higher levels of influenzavirus-specific CTL responses than mature DCs from theperipheral blood of these patients, although responses didnot quite reach control values. Thus, defective T-cell func-tion in patients with advanced breast cancer can be over-

come by stimulation with DCs generated from precursors,suggesting that these cells may better serve as autologous

antigen carriers for cancer immunotherapy than mature

peripheral blood DCs.

Received 9/12/96; revised 1 1/27/96: accepted I 2/I 3/96.

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.

I This work was supported by Grant RO1-CA61242 from the National

Cancer Institute. Part of this work was presented at the Thirty-Second

Annual Meeting of the American Society of Clinical Oncology, May

1996.2 To whom requests for reprints should be addressed, at The VanderbiltCancer Center, 648 Medical Research Building II, Nashville. TN 37232-

6838. Phone: (615) 936-3320: Fax: (615) 936-1790.

INTRODUCTION

Defective function ofthe host immune system is thought to

be a major mechanism by which tumors escape from immune

control. Numerous studies have described defective function of

T lymphocytes, including tumor-infiltrating cells, in tumor-

bearing hosts (reviewed in Ref. 1). The precise mechanism of

this dysfunction is not known, but it has been reported that it can

be reversed if sufficient stimulation with antigen-presenting

cells and support with cytokines (such as 1L3-2) is provided

(2-4). Because host professional APCs are one of the most

important elements in the induction of specific antitumor im-

mune responses (5, 6), APC could be partly responsible for the

observed defective immune responses in tumor-bearing hosts.

This group of cells includes dendritic cells, macrophage/mono-

cytes, and B cells. DCs are thought to be the most potent APCs,

and only these cells are capable of inducing primary immune

responses and, therefore, are actively being studied for use as

vehicles for delivery of tumor-specific antigens in both animal

experimental systems and in patients with cancer (7-9). In our

previous study, we showed that the nonresponsiveness ofT cells

from tumor-bearing mice was reversed after stimulation with

antigen-pulsed control DCs, even without additional cytokine

support (4). It is not known, however, whether DCs obtained

from patients with cancer have sufficient functional activity for

use as autologous antigen carriers. In particular, the ability of

these cells to present soluble antigens and induce cytotoxic

immune responses in patients with cancer is not clear. The

clarification of this question is important not only for under-

standing the mechanisms of tumor progression but also for

the development of effective cancer immmunotherapeutic

strategies.

Previously, we have shown that defects in T-cell function

in tumor-bearing mice could be reversed by stimulation with

DCs generated from stem cells from the bone marrow of tumor-

bearing mice (10). Here we investigated the function of DCs

from patients with breast cancer and found that these cells had

a considerably lower than normal ability to stimulate allogeneic

T cells as well as to present soluble antigen to autologous T

cells. To test the hypothesis that sufficient antigen presentation

may reverse observed defects in T-cell function, we generated

DCs from peripheral blood precursors and showed that those

cells were able to elicit an almost normal level of immune

response.

3 The abbreviations used are: IL, interleukin: APC, antigen-presentingcell; DC, dendritic cell: CCM, complete culture medium; GM-CSF,

granulocyte/macrophage colony-stimulating factor; NK. natural killer:

FLU. influenza virus: Ti’, tetanus toxoid: MLR. mixed leukocyte reac-tion.

Research. on October 7, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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484 Decreased Antigen Presentation by DC in Cancer

PATIENTS AND METHODS

Patients. Thirty-two patients aged 33-69 years with his-

tologically confirmed breast cancer were enrolled into the study.

All patients were newly diagnosed with no prior therapy. Stag-

ing was performed in accordance with American Joint Commit-

tee on Cancer criteria. Four patients had stage I tumors, 10 had

stage II, 13 had stage III, and S had stage IV. Fourteen healthy

volunteers were used as controls. Informed consent was ob-

tamed from all individuals.

Reagents. CCM included RPMI 1640 (Life Technolo-

gies, Inc., Gaithersburg, MD) supplemented with 10% FCS and

antibiotics. For the generation of DCs from progenitors, medium

was additionally supplemented with 5 X l0� 2-mercaptoeth-

anol. Ficoll-paque was purchased from Pharmacia Biotech, Inc.

(Uppsala, Sweden). GM-CSF and IL-4 were obtained from

R&D Systems (Minneapolis, MN). Influenza virus strain PR8

(A/Puerto Rico/8/34) was obtained from the American Type

Culture Collection (Rockville, MD) and was propagated in the

Laboratory of Virology at the University of Texas Southwestern

Medical Center (Dr. J. Luby). Anti-HLA DR. CD3, CD14, and

CDI9 antibodies were purchased from Sigma Chemical Co. (St.

Louis, MO). Anti-HLA ABC, B7-l, B7-2, and CD56 antibod-

ies were purchased from PharMingen (San Diego, CA). Anti-

CD34 antibody was obtained from Becton Dickinson (Mountain

View, CA). Purified anti-CD3 antibody (64.1 hybridorna cell

lines produce an IgG2a monoclonal antibody) was a generous

gift from The Cancer Immunobiology Center, University of

Texas-Southwestern (Dr. E. S. Vitetta). Goat antimouse IgO

antibody conjugated with magnetic beads was purchased from

Dynal (Lake Success, NY).

Cell Isolation. DCs and T cells were isolated from the

peripheral blood of patients or control donors as described (11)

with minor modifications. Briefly, mononuclear cells obtained

after centrifugation of peripheral blood over Ficoll-paque gra-

dient were incubated with neurominidase-treated sheep red cells

(R). Cells that adhered to the red cells (R�) and those that did

not (R �) were separated on Ficoll-paque gradient. R cells were

then incubated for 18 h in CCM at 37#{176}C,followed by incubation

of nonadherent cells for 60 mm on Petri dishes coated with

human -y-globuhin to remove remaining FcR� rnonocytes. Fi-

nally, cells were centrifuged over a metrizamide gradient (14.5

g of metrizamide; Neyegaard, Oslo, Norway, in 100 ml of

culture medium). Cells collected from the interface were 50-

70% DCs, as estimated morphologically or by flow cytornetry

after labeling a mixture of monoclonal antibodies (CD3, CD 14,

CDL9, CD56, and HLA-DR). Contaminants were B cells, NK

cells, and few T cells. R� cells were further processed to obtain

an enriched T-cell fraction by overnight incubation in CCM at

37#{176}C,followed by osmotic lysis of the red cells. Nonadherent

cells were >90% T cells, as estimated by flow cytometry. In

several experiments, the DC fraction was additionally purified

using a magnetic bead separation technique. Briefly, the DC

fraction isolated as described above was treated at 4#{176}Cwith a

cocktail of monoclonal antibodies: anti-CD3, anti-CD19, anti-

CD14, and anti-CD57/HNK. After a 30-mm incubation, cells

were washed and labeled for 30 mm with goat antimouse IgO

antibody conjugated to magnetic beads (Dynal), followed by

washing and magnetic separation. Unlabeled cells were more

than 95% DCs, as estimated by flow cytometry.

DCs were generated from progenitors in peripheral blood

as described previously (12). Briefly, peripheral blood mononu-

clear cells were incubated for 2 h in six-well plates, nonadherent

cells were removed, and adherent cells were incubated with

GM-CSF (5 ng/ml) and IL-4 (5 ng/mJ) for 6-7 days. After that

time, cells were collected, washed, and analyzed. The purity of

the DCs was more than 70% with >95% viability.

MLR. The ability of DCs to stimulate allogeneic T cells

was tested in MLRs. To minimize the effect of differences in

HLA between individuals on allogeneic T-cell proliferation,

DCs from each of 14 normal controls were tested independently

against allogeneic T cells obtained from another three control

individuals, and the pairs giving maximal responses were used.

Cells from the patients were similarly tested against the panel of

normals, and those giving maximal responses were used. For

each experiment, T cells from patients were tested against DCs

from three control volunteers. DCs and T cells were cultured at

different ratios for 5 days in 96-well plates. Eighteen h prior to

harvesting the cells, 1 p.Ci of [3H}thymidine was added to each

well. [3H]Thymidine uptake was counted in a liquid scintillation

counter (Beckman, Palo Alto, CA).

Tetanus Toxoid-specific T-Cell Proliferation. DCs

were cultured with autologous T cells in the presence of 0.5

p.g/ml tetanus toxoid (this concentration was selected after

preliminary testing). [3H]Thymidine uptake was measured 5

days later. The background level of T-cell proliferation stimu-

lated by autologous DCs was subtracted from all experimental

values.

FLU-specific CTL Responses. DCs were cultured in

serum-free medium with influenza virus strain PR8 (A/Puerto

Rico/8/34) for 2 h, washed, and cultured with autologous T cells

at a ratio of 1:5 in 24-well plates (Costar, Cambridge, MA).

Seven days later, the cells were harvested and distributed at

various dilutions to 96-well plates. CTL activity was measured

against FLU-infected and uninfected 5tCr-labeled autologous

phytohemagglutinin blasts in a standard 6-h assay. At the ter-

mination of the assay, � ‘Cr release into the supernatants was

measured using absorption cartridges (Skatron Instruments, Inc.,

Sterling, VA) and counted in a gamma counter. The percentage

of specific 51Cr release was calculated from the following

formula:

% specific 51Cr release =

Release by CTL - spontaneous release

Total release - spontaneous release X 1

Spontaneous release was equal to or less than 25% of the total

release.

RESULTS

Defective Responses to Mitogenic and Antigemc Stim-ulation in Patients with Breast Cancer. We first examined

antigen- and mitogen-driven T-cell proliferation in patients with

breast cancer. Because this required the presence of APCs, DCs

and T cells were isolated from the peripheral blood of breast

cancer patients and mixed at different concentrations with var-



A 140000

120000

100000

80000

060000

40000

20000

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B 20000

18000

16000

14000

12000

� 100000

8000

6000

4000

2000

�- --

ConA

stimulation

++

C �60000

50000 �-40000

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D 70000

60000

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40000

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.�jj�_jI �[�1. ri�Stage Stage Stage Stage

1 (n=3) 2 (n=7) 3 (n=4) 4anti-CD3

stimulation

Control Stage Stage Stage Stage(n=14) 1(n=3) 2 (n=7) 3 (n=4) 4 (n=4)

Control Stage Stage Stage Stage(n=14) 1(n=3) 2 (n=7) 3 (n=4) 4 (n=4)

Fig. I Function of DCs and I cells in patients with breast cancer. A, T-cell responses are decreased in response to ConA but maintained for anti-CD3

stimulation. For ConA stimulation, DCs and T cells were isolated as described in “Patients and Methods” and cultured together at a ratio of 1:50 (2 X

lO� DCs and lO� I cells) in the presence of 10 p.g/ml ConA. Averages (bars, SE) of 3H uptake on day 3 are shown. EJ, statistically significant

differences from control (P < 0.05). For anti-CD3 stimulation, 96-well plates were coated with S pg/mI anti-CD3 antibody and washed; then lO� T

cells were added to each well. 3H uptake in triplicates was measured on day 3. �, control (ii = I I); �, stage 1-2 (,z = 5); E, stage 3-4 (n = 4).

B, U-stimulated T-cell proliferation is decreased in patients with breast cancer. DCs and T cells were isolated as described in “Patients and Methods”

and mixed at different ratios (from 1:20 to 1: 160). iT-driven T-cell proliferation was tested as described in “Patients and Methods.” Averages (bars,

SE) of [3H]thymidine at a 1 :40 DC:T cell ratio are shown. Similar patterns of the results were found for all DC:T cell ratios. �, statistically significant

differences from control (P < 0.05). C, DCs from breast cancer patients show decreased stimulation of control allogeneic T cells. Patients’ DCs and

donors’ T cells were isolated, and allogeneic MLR was performed as described in “Patients and Methods.” The means (bars, SE) of [3H]thymidineuptake at a 1 :40 DC:T cell ratio are shown. Similar patterns of the results were found for all DC:T cell ratios. �, statistically significant differences

from control (P < 0.05). D, DCs from control individuals effectively stimulate breast cancer patients’ T cells. Experimental protocol was as describedin “Patients and Methods.” Means (bars, SE) of [3H]thymidine uptake at a 1 :40 DC:T cell ratio are shown. Similar patterns of the results were found

for all DC:T cell ratios.

Clinical Cancer Research 485

ious stimulators. Interestingly, patients with advanced breast

cancer demonstrated a substantially reduced yield and purity of

DCs, as assessed by flow cytometry and light microscopy. The

proportion of DCs in the fractions isolated from patients with

stages I and II was the same as that from control individuals

(55-70%), but samples from patients with stages III and IV

tumors yielded a substantially lower DC purity than control

(25-35%). Because of these differences in the purity of DCs,

DC numbers were always carefully adjusted to equal concen-

trations before any assays were performed. Significant decreases

in the ability of T cells to respond to ConA stimulation were

seen in patients with progressively advanced stages of breast

cancer (Fig. hA). This was accompanied by a dramatic reduction

in the response to the soluble antigen ‘FT (Fig. 1B). Defects in

‘VT-dependent T-cell proliferation were seen in patients at ear-

her stages (stage II) than the defects in responses to mitogens

(stage III; Fig. 1B). The majority of patients with stage II breast

cancer demonstrated normal or only slightly decreased levels of

influenza virus-specific CTL responses. However, a significant

decrease in this response was observed in patients with more

advanced disease (Fig. 2).

Defective Function of DCs but not T Cells in Breast

Cancer Patients. These data are consistent with defective

function of either T cells or APCs. To characterize the mecha-



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5.

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Control Stage Stagedonors I-Il lll-IV

Fig. 2 Anti-FLU-specific CU responses are decreased in patients with

breast cancer. CTL assays were performed as described in “Patients and

Methods” at an E:T ratio of 40: 1 . Each point represents the cytotoxicityassay results from one individual. Bar, average.

nism of the observed effects, we purified DCs and T cells from

patients and controls and tested the ability of both patient and

control DCs to stimulate allogeneic T cells. As shown in Fig.

1C, a reduced activity of DCs (not statistically significant, P =

0.07) was observed in patients with stages I and II breast cancer.

A significant decrease in stimulation was observed in patients in

stages III and IV. In contrast, no differences were found in the

isolated T cells of these patients, for stimulation of patient T

cells with either control allogeneic DCs or immobilized anti-

CD3 antibody resulted in normal T-cell proliferation, even in

patients in stage IV (Fig. 1, A and D). These data indicate that

defects in the functional activity of DCs could be one of the

major reasons for the observed decreased cellular immune re-

sponses in patients with advanced breast cancer. Because the

purity of the DC preparation obtained from patients with ad-

vanced disease was lower than that from control individuals,

there is always the possibility that described effects could be due

to contamination of DC fractions by other cells. To evaluate

this, several experiments were performed using highly purified

DCs. In these experiments, DCs were additionally purified using

magnetic bead separation, and their ability to stimulate control

allogeneic T cells was compared with that of the partially

purified DCs described previously. Even the highly purified

DCs isolated from patients with stage III tumors showed the

same low level of activity as those from partially purified

fractions (Fig. 3).

In the next group of experiments, we tested the hypothesis

that changes in the level of expression of molecules on the

surface of DCs might contribute to their defective function. DCs

were isolated from four control donors and five patients with

stage III breast cancer. Cells were double labeled with a cocktail

of monoclonal antibodies against T, B, and NK cells and mono-

cytes and with antibodies against MHC class I, class II, and B-7

molecules. Only DC (“cocktail-negative” cells) were analyzed.

As shown in Fig. 4, no differences were observed in the level of

Ea_ 30000C-)

50 100 200 400

T cell:DC ratio

Fig. 3 Additional purification of DCs did not improve their specific

function. DCs were isolated from patients with stage III breast cancer asdescribed in “Patients and Methods”. One-half of the cells were addi-

tionally purified using monoclonal antibodies against T, B, and NK cells

and monocytes using magnetic beads as described in “Patients andMethods.” Control allogeneic T cells were stimulated with DCs fromanother donor, and equal numbers of partially and highly purified DCswere obtained from the patient. Allogeneic MLR was performed intriplicate as described in “Patients and Methods”. Means (bars, SD) areshown. #{149}, control; �, pure DCs; A, DC fraction.

expression of MHC class I molecules (HLA A, B, and C),

whereas a substantial decrease in the expression of MHC class

II (HLA DR and HLA DQ) was seen. A decline in the expres-

sion of B7-l and B7-2 was also notable, albeit less profound

than that of MHC class II molecules.

Function of DCs Generated from Progenitors. Theabove data demonstrate serious defects in the function of pe-

ripheral blood DCs in patients with advanced breast cancer. We,

therefore, investigated the mechanisms of this dysfunction. Pre-

viously, we have shown that soluble factors released by animal

and human tumor cells affected the maturation of DCs but did

not impair the function of mature DCs from peripheral blood

(4). We, therefore, hypothesized that DCs generated in vitro

from precursors from advanced cancer patients, without the

influence of any tumor-produced factors, could be functionally

intact. To test this hypothesis, peripheral blood from patients

with stage III and control donors was processed to obtain T cells

and DCs as described before. In parallel, DCs were generated

from precursors using OM-CSF and IL-4. Generation of DCs

from precursors requires at least 7 days of in vitro culture.

Mature DCs isolated from peripheral blood are optimally func-

tional on the day after harvest. To use autologous T cells and to

standardize experimental conditions, T cells from each patient

were cryopreserved in ahiquots immediately after isolation and

thawed before testing. DCs generated from precursors showed

high levels of expression of MHC class II and costimulatory

molecules consistent with phenotype of relatively mature DCs.



500

400

300

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25000

20000

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10000

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40 60 ‘ 40 80160 * 40

T cells: DC ratio

Fig. 5 DCs grown from the peripheral blood precursors of patientswith breast cancer are capable of normal levels of allogeneic stimula-

tion. A, DCs isolated from peripheral blood; B, DCs generated fromprecursors in peripheral blood. The experimental protocol was as de-scribed in “Patients and Methods.” [3H]Thymidine uptake by T cells on

day S is shown. #{149},donor 2; 0, donor 4; U, patient B; A, patient K; V.patient L.


Fig. 4 Expression of MHC class Iand class II and costimulatory mole-cules on the surface of DCs isolated

from control individuals and patients

with breast cancer. DCs were directlyisolated from peripheral blood or gen-

crated from precursors in peripheralblood as described in “Patients andMethods”. Blood was obtained fromcontrol individuals and patients withstage III breast cancer. Cells were Ia-beled with a cocktail of phycoerythrin-labeled antibodies (CD3, CD19, CD56,and CD14) and FITC-labeled antibod-ies against MHC class I, MHC class II,

B7-l, and B7-2 molecules. Expres-sion of the FITC-labeled markers was

analyzed in phycoerythrin-negative

cells (DCs). The means of fluorescence

in each experiment are shown. 0, con-

trol individuals; #{149},patients. 0, cellsgenerated using OM-CSF and IL-4from precursors in peripheral bloodfrom control donors; U, DCs generatedfrom precursors in peripheral bloodfrom cancer patients.

Direct comparison of the levels of expression of surface mole-

cules between DCs generated from precursors from control

individuals and from the patients showed no differences (Fig. 4).

DCs generated from precursors in the peripheral blood of pa-

tients with breast cancer had a normal ability to stimulate

control allogeneic T cells, whereas DCs from peripheral blood

from the same patients had more than 2-fold lower stimulatory

capacity (Fig. 5). Normal levels of U-dependent T-cell stimu-

lation were observed when DCs obtained from precursors were

used as stimulators (Fig. 6). Those cells also induced a substan-

tially higher level of FLU-specific Cli responses than DCs

from peripheral blood. However, that response did not reach the

control figures (Fig. 7).

DISCUSSION

The effective function of APCs is an integral part of

immune responses in general, and antiturnor immune responses

in particular. Defects in antigen presentation would make the

development of specific immune responses almost impossible.

However, until now it was not clear whether the function of

APCs was normal or suppressed in tumor-bearing hosts, and if

suppressed, whether methods could be developed to restore

function. The few reports in this field are controversial and

incomplete. It has been shown, for example, that a subset of

I-At epidermal APCs may be capable of inducing tolerance to

tumor antigens and that activated rnacrophages might induce

structural abnormalities of the T-cell receptor/CD3 complex (13,

14). Zou et a!. (15) have reported norma] and even increased

function of APCs (mostly macrophages) in tumor-bearing mice

(15). However, defective function of macrophages in patients

with cancer has also been described in several reports ( 16, 17).

Dendritic cells are the most potent APCs; therefore, we

focused on investigation of their ability to present antigens and



Control donors Patients

A B A

Donor 1 Donor 5 Patient W Patient P Patient T

Fig. 7 Improved stimulation of influenza virus-specific CTLs by breastcancer patient DCs grown from peripheral blood precursors. T:E ratio1 :40, experimental protocol as described in “Patients and Methods.”�,

peripheral blood DCs; 0, DCs from precursors. Bars. SE.


40000�

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10000

5000

0. #{149}, , #{149}� I I I I I I I I I 1

40 80160 * 40 80160 #{149}40 80160 #{149}40 80160

T cells:DC ratio

Fig. 6 DCs grown from the peripheral blood precursors of patientswith breast cancer are capable of improved induction of U-dependentautologous T-cell proliferation. A, DCs isolated from peripheral blood.B, DCs isolated from precursors in peripheral blood. The experimentalprotocol was as described in “Patients and Methods.” #{149},donor 1 ; 0,donor 2; U, donor 3; A, patient D; V. patient N: I, patient R. Bars, SE.

induce CTL responses in patients with breast cancer. Previously,

we reported defective functioning of DCs in tumor-bearing mice

(4), and we speculated that the same defects may occur in

patients with cancer. Several other reports have suggested pos-

sible DC dysfunction in patients with cancer (16, 18). However,

these observations did not answer the main question of whether

DCs from patients with cancer are able to stimulate specific

immune responses.

In this study, we investigated the immune function of

patients with breast cancer and particularly the ability of DCs to

present antigens to T cells. Unlike animal experimental models,

it is much more difficult to analyze the function of APCs in

patients because antigen-specific T-cell lines and clones with

the same haplotype are not readily available. Therefore, we

chose two antigens (iT and influenza virus) with prevalent

immune responses in the general population. We studied a

sufficient number of control individuals and patients to reach

statistically significant conclusions. A clear inverse correlation

between the stage of breast cancer and the level of antitetanus

toxoid or FLU immune responses was found. In almost all

patients with stages III and IV breast cancer, no FLU-specific

CTL responses were observed. These patients’ cells also showed

a dramatic decrease in T-cell response to mitogeneic stimulation

with ConA. These data are consistent with defects in either T

cells or DCs because in these experimental systems, the pres-

ence of APCs is important to getting a maximal T-cell response.

To characterize the contribution of each of these types of

cells to the observed decreased function, we studied allogeneic

MLR reactions using purified cell fractions. In this system,

control allogeneic T cells were stimulated with DCs from pa-

tients and vice versa. To minimize the effect of chance MHC

matching in the DC-T cell pairs, DCs from each patient were

tested against T cells from at least three different control mdi-

viduals, and the maximum responses were scored. These studies

showed that the ability of DCs from patients with advanced

breast cancer to stimulate control allogeneic T cells was signif-

icantly impaired. Initially, we used partially purified fractions of

DCs in all of our experiments. In control individuals and pa-

tients with stages I and II, it provided 50-70% pure DCs.

However, we found that the purity of identically prepared cells

from patients with advanced disease was significantly lower.

Because we always adjusted the cell concentration so that the

DC numbers were equal, fractions from patients with stages III

and IV contained more “non DC cells” than in controls. It was

possible that these contaminating cells produced factors or di-

rectly interfered with DC function, rather than the defect being

an intrinsic property of the DC. To evaluate this possibility, we

additionally purified DCs using a magnetic bead separation

technique, effectively eliminating all T, B, and NK cells and

rnonocytes from this fraction. The remaining cells had the same

low level of activity as those from the partially purified frac-

tions, indicating that the differences we observed were not due

to possible contamination of the final DC fractions with lym-

phocytes or monocytes. However, this approach does not rule

out interference from immature cells lacking the markers spe-

cific for T, B, and NK cells and monocytes. We have shown

previously that supernatants from breast cancer cells inhibited

the functional maturation of DCs in vitro and resulted in the

production of as yet incompletely characterized immature cells

of rnyelomonocytic lineage (19). These in vitro findings could

provide an explanation for the in vivo observations reported

here. Decreased expression of MHC class II and some costimu-

latory molecules on the surface of DCs isolated from the pe-

ripheral blood of cancer patients might be one of the possible

causes of the observed impaired ability of these cells to stimu-

late allogeneic MLR and antigen-specific T-cehl responses.

These molecules are known to be directly involved in T-cell

stimulation (20, 21). CD4� cells engaged through MHC class II

molecules play an important role in induction of the CD8� -




specific CTL responses. Depletion of CD4� cells dramatically

decreased CD8� cell-mediated cytotoxicity against tumor-spe-

cific antigens (4).

A normal level of proliferation was observed when T cells

from patients were stimulated with DCs from control individu-

als. To confirm the normal proliferative capacity of the T cells

from cancer patients, we tested their responses to immobilized

anti-CD3 antibody, which is able to stimulate T-cehl prolifera-

tion almost independently of the presence of APCs through

direct triggering of the CD3/T-cell receptor complex (22). Using

this approach, we did not find any differences in the level of

proliferative responses between control individuals and breast

cancer patients. These data indicate that peripheral blood T cells

from these patients are capable of normal responses to these

stimuli.

The molecular mechanism of this DC dysfunction is not

clear. Previously, we proposed that defects in DC maturation

mediated by soluble substances released by tumor cells could be

partly responsible ( 10). DCs generated from bone marrow pro-

genitors from tumor-bearing mice showed a normal level of

activity (presentation of the soluble antigen to CTL and stimu-

lation of allogeneic T cells). Recently, we found that vascular

endothelial growth factor released by tumor cells was one of the

factors responsible for inhibiting the functional maturation of

DCs, without affecting the function of the relatively mature cells

in peripheral blood ( 19). The data presented here suggest that

defective DC function consistent with the defects observed in

vitro can be observed in patients with breast cancer. They

further suggest that this defect may be caused by tumor-released

soluble factor(s) as seen in vitro.

To explore the latter hypothesis, we generated DCs from

peripheral blood precursors from these breast cancer patients

using OM-CSF and IL-4. This combination of cytokines pro-

motes the growth of DCs with high activity for taking up and

processing soluble antigens (12, 23). Several studies have dem-

onstrated that it is possible to generate DCs from progenitors in

patients with cancer using similar combinations of cytokines

(24, 25). The cells obtained after this procedure had the pheno-

type of DCs and were able to stimulate antigen-dependent T-cell

responses. However, in all of these cases, the patients were

pretreated with chemotherapy and granulocyte-CSF, which

could influence the function of the derived cells, and in addition,

the function of freshly isolated peripheral blood DCs from the

same patient was not investigated. Therefore, from these data it

is impossible to conclude whether those patients had any defects

in DC function and whether DCs generated from precursors had

any functional advantage over those obtained from peripheral

blood. To answer all of these questions, we compared the

function of DCs isolated from peripheral blood with cells gen-

crated from precursors from the same patients. We showed that

in control individuals, there were no significant differences

between the functional activity of DCs isolated from peripheral

blood and of those generated from precursors. U-specific T-

cell proliferation in patients with breast cancer was completely

restored when DCs generated from precursors were used as

APCs. Increased FLU-specific CU responses were also ob-

served, albeit not quite to normal levels. Why were DCs gen-

crated from precursors in peripheral blood functionally compe-

tent whereas those directly isolated from peripheral blood were

not? One of the possible explanations could be the fact that the

DCs we generated in the presence of OM-CSF and IL-4 were

derived not from CD34� stem cell precursors but from cells

which have the phenotypic features of monocytes (26-28).

Therefore, it is possible that tumor-derived factors specifically

affect the ability of CD34� cells to different into DCs while not

altering the production of these monocytoid cells or their ability

to be diverted to a DC phenotype in vitro. A second explanation

could relate to the fact that DCs isolated from blood are almost

terminally matured cells and do not undergo cell division,

whereas cells we generate in vitro in the presence of OM-CSF

and IL-4 actively divide (at least during the first 3-4 days in

culture). It is possible then that rather than a selective effect on

a different lineage of cells, several cycles of cell division in the

absence of the tumor-derived factors are required to produce

fully functional DCs, regardless of the lineage of the precursor.

These questions require additional clarification.

Several major conclusions can be drawn from these find-

ings: (a) cellular immune deficits in patients with advanced

cancer can at least partially be explained by a decreased number

and potency of DC; (b) defective DC function in patients with

advanced stage breast cancer can be at least partially overcome

when these cells are generated in vitro from precursors; (c)

T-cell hyporesponsiveness to soluble antigens can be overcome

by stimulation with functionally potent DCs generated from

precursors. These results not only suggest that the antigen pres-

entation function of DCs may be selectively targeted by cancer

cells as a means of immune escape but also that DCs generated

from precursors ex vivo may have a significant advantage over

DCs obtained directly from peripheral blood as vehicles for

antigen delivery in the immunotherapy of cancer.

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1997;3:483-490. Clin Cancer Res D I Gabrilovich, J Corak, I F Ciernik, et al. with breast cancer.Decreased antigen presentation by dendritic cells in patients

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