increase in tumor size following intratumoral injection of immunostimulatory cpg-containing...

ORIGINAL ARTICLE

Increase in tumor size following intratumoral injectionof immunostimulatory CpG-containing oligonucleotidesin a rat glioma model

Christian Ginzkey • Sven O. Eicker • Matthias Marget • Joerg Krause •

Stephan Brecht • Manfred Westphal • Hans H. Hugo • H. M. Mehdorn •

Joerg Steinmann • Wolfgang Hamel

Received: 25 July 2009 / Accepted: 9 September 2009 / Published online: 2 October 2009

� Springer-Verlag 2009

Abstract The immunosuppressive environment of

malignant gliomas is likely to suppress the anti-tumor

activity of infiltrating microglial cells and lymphocytes.

Macrophages and microglial cells may be activated by

oligonucleotides containing unmethylated CpG-motifs,

although their value in cancer immunotherapy has

remained controversial. Following injection of CpG-con-

taining oligonucleotides (ODN) into normal rat brain, we

observed a local inflammatory response with CD8? T cell

infiltration, upregulation of MHC 2, and ED1 expression

proving the immunogenic capacity of the CpG-ODN used.

This was not observed with a control ODN mutated in the

immunostimulatory sequence (m-CpG). To study their

effect in a syngeneic tumor model, we implanted rat 9L

gliosarcoma cells into the striatum of Fisher 344 rats. After

3 days, immunostimulatory CpG-ODN, control m-CpG-

ODN, or saline was injected stereotactically into the tumors

(day 3 group). In another group of animals (day 0 group),

CpG-ODN were mixed with 9L cells prior to implantation

without further treatment on day 3. After 3 weeks, the

animals were killed and the brains and spleens were

removed. Rather unexpectedly, the tumors in several of the

animals treated with CpG-ODN (both day 0 and day 3

group) were larger than in saline or m-CpG-ODN treated

control animals. The tumor size in CpG-ODN-treated

animals was more variable than in both control groups.

This was associated with inflammatory responses and

necrosis which was observed in most tumors following

CpG treatment. This, however, did not prevent excessive

growth of solid tumor masses in the CpG-treated animals

similar to the control-treated animals. Dense infiltration

with microglial cells resembling ramified microglia was

observed within the solid tumor masses of control- and

CpG-treated animals. In necrotic areas (phagocytic), acti-

vation of microglial cells was suggested by ED1 expression

and a more macrophage-like morphology. Dense lympho-

cytic infiltrates consisting predominantly of CD8? T cells

and fewer NK cells were detected in all tumors including

the control-treated animals. Expression of perforin serving

as a marker for T cell or NK cell activation was detected

only on isolated cells in all treatment groups. Tumors of all

treatment groups revealed CD25 expression indicating T

cells presumed to maintain peripheral tolerance to self-

antigens. Cytotoxic T cell assays with in vitro restimulated

lymphocytes (51chromium release assay) as well as inter-

feron-gamma production by fresh splenocytes (Elispot

assay) revealed specific responses to 9L cells but not

C. Ginzkey � S. O. Eicker � J. Krause � H. H. Hugo �H. M. Mehdorn � W. Hamel

Klinik fur Neurochirurgie,

Universitatsklinikum Schleswig-Holstein,

Campus Kiel, Germany

M. Marget � J. Steinmann

Institut fur Immunologie,



S. Brecht

Institut fur Pharmakologie,



C. Ginzkey

Klinik fur Hals-, Nasen- und Ohrenkrankheiten der Universitat

Wurzburg, University of Wurzburg, Wurzburg, Germany

M. Westphal � W. Hamel (&)

Neurochirurgische Klinik,

Universitatsklinikum Hamburg-Eppendorf,

Martinistr. 52, 20246 Hamburg, Germany

e-mail: [email protected]

123

Cancer Immunol Immunother (2010) 59:541–551

DOI 10.1007/s00262-009-0771-y

another syngeneic cell line (MADB 106 adenocarcinoma).

Surprisingly, the lysis rates with lymphocytes from CpG-

ODN-treated animals were lower compared to control-

treated animals. The tumor size of individual animals did

not correlate with the response in both immune assays.

Taken together, our data support the immunostimulatory

capacity of CpG-ODN in normal brain. However, intratu-

moral application proved ineffective in a rat glioma model.

CpG-ODN treatment may not yield beneficial effects in

glioma patients.

Keywords CpG � Glioma � Rat � Oligonucleotides �Stereotactic

Introduction

Tumor cell dissemination beyond the margins of resec-

tion and resistance to conventional adjuvant therapies

lead to the inevitable recurrence of malignant gliomas.

This results in the incurability of this disease, and despite

enormous research efforts the extremely poor survival

rates have not increased substantially over several

decades.

In principle, the immune system may be apt to trace and

destroy the diffusely infiltrating tumor cells. Dense lym-

phocytic and microglial infiltrates have been observed in

brain tumors indicating that they are recognized by the

immune system [7, 10, 16, 32]. On the other hand,

malignant brain tumors are renowned for their immuno-

suppressive capacity and effective immunosuppression is

regarded crucial for tumor maintenance and growth.

In a rat glioma model we observed that DNA vacci-

nation against a model antigen (E. coli lacZ) resulted in

a significant decrease in tumor size [15]. Nonetheless,

complete protection from intracerebral tumor formation

was not observed even after the addition of interleukin-

12 or flt3 ligand to the vaccine. Immunohistochemical

staining revealed dense infiltrates with microglial cells

which rather support tumor growth than being involved

in tumor destruction [16, 32]. In order to activate mi-

croglial cells and augment the anti-tumor effects of DNA

vaccination we administered immunostimulatory CpG-

oligonucleotides (CpG-ODN) resembling bacterial DNA

sequences [23, 29]. Rather unexpectedly, intratumoral

application of CpG-ODN did not elicit adjuvant anti-

tumor effects in conjunction with DNA vaccination.

Instead, we observed an increase in tumor size. Since

intratumoral CpG-ODN administration is pursued in

clinical trials [6, 22, 23], we studied their sole effect in

the syngeneic rat gliosarcoma model (9L) in Fisher 344

rats.

Materials and methods

Cell lines and in vitro cell proliferation

9L rat gliosarcoma cells (obtained from the Brain Tumor

Research Center, University of California, San Francisco,

USA) and MADB 106 rat adenocarcinoma cells (kindly

provided by Dr. Thomas von Horsten, Medizinische Ho-

chschule Hannover, Germany) were cultured in DMEM

(9L) or RPMI (MADB 106; both cell culture media

obtained from GIBCO BRL Life Technologies, Karlsruhe,

Germany) containing 10% heat-inactivated fetal calf serum

and antibiotics (penicillin/streptomycin, Sigma-Aldrich, St.

Louis, MO, USA) at 37�C in a humid atmosphere with 5%

CO2. Both cell lines are syngeneic to Fisher 344 rats. To

assess the effects of immunostimulatory oligonucleotides

(ODN) on tumor cell growth in culture, 9L cells grown in

96 wells (2,000 cells per well) were incubated with two

different concentrations of ODN (10-5 and 10-6 M) con-

taining immunostimulatory CpG-motifs (50-TGACTGT

GAACGTTCGAGATGA-30) or ODN mutated in the CpG-

motif (m-CpG-ODN; 50-TGACTGTGAAGGTTAGAGAT

GA-30) [8, 27]. The oligonucleotides were synthesized with

a phosphorothioate backbone to increase their stability [18]

and purchased from Proligo (Paris, France). Viable cells

were counted by standard trypan blue staining after 2 and

4 days.

Tumor cell implantation and inoculation

of oligonucleotides into 9L tumors

and normal rat brain

Male Fisher 344 rats (250–275 g; Charles River, Sulzfeld,

Germany) were housed according to German Animal

Protection Regulations and permission had been obtained

for these experiments. For ODN or tumor cell implantation,

animals were anesthetized with 4% chloral hydrate (1 ml

per 100 g). Heads were fixed in a stereotactic frame (TSE

Systems, Michigan, MI, USA) and a burr hole was placed

2 mm lateral and 2 mm anterior to the bregma.

To assess the effects of ODN in normal brain, the

immunostimulatory CpG-ODN (50 lg/3 ll isotonic saline)

were injected into the brain of tumor-free Fisher rats

(implantation procedure described below). In each animal

the contralateral striatum was injected with the same

amount of ODN containing a mutation in the CpG-motif

(m-CpG; the CpG- and mCpG-ODN sequences are iden-

tical to those described above). Animals were killed at 2, 5

and 8 days after injection and the brains were removed for

immunohistochemistry as described below.

To investigate the effects of ODN treatment on 9L

tumor growth in vivo, 5 9 104 9L cells suspended in 5 ll

542 Cancer Immunol Immunother (2010) 59:541–551

123

DMEM without supplements were slowly injected into the

right striatum with a 10 ll Hamilton syringe. The needle

was carefully retracted and the burr hole was sealed with

bone wax. Treatment with ODN (50 lg CpG/4 ll saline)

was performed on day 3 following tumor cell implantation

except for a group of animals in which the cell suspension

was mixed with the CpG-ODN prior to tumor cell inocu-

lation. The latter group of animals received no further

treatment on day 3. The intratumoral injections of CpG- or

m-CpG-ODN or saline (vehicle control) were performed in

anesthesia with the same stereotactic coordinates used

previously for tumor cell implantation.

After 3 weeks, animals were anesthetized with diethyl-

ether and a lethal dose of chloral hydrate. Spleens were

removed under sterile conditions and kept in ice-cold RPMI

containing 10% FCS until lymphocyte preparation. Blood

was collected from the right atrium, and brains were removed

without prior fixation and transferred in anti-freeze medium,

shock-frozen in liquid nitrogen, and stored at -20�C.

Histology and immunohistochemistry

Hemalaun–eosin (HE)-stained sections (10 lm) were cut in

intervals of 200 lm with a cryostat 2800 Frigocut (Reic-

hert-Jung, Nußloch, Germany). Tumor volume was calcu-

lated with the following formula for an rotation ellipsoid:

V = 4/3 9 p 9 0.125 (length 9 height 9 width). For

immunohistochemistry and X-gal staining, brain slices

from non-fixed brains were mounted on coated slides

(Marienfeld GmbH, Lauda-Konigshofen, Germany), air-

dried, and stored at -20�C in aluminum foil.

Staining was performed according to Barclay et al. [4].

In brief, slices were fixed in acetone at -20�C for 10 min

and rinsed in tap water for 1 min. To eliminate endogenous

peroxidase activity, slices were transferred in PBS/metha-

nol (1:1) containing 0.3% H2O2 and washed thrice with

PBS. Immunohistochemistry was performed with the fol-

lowing mouse anti-rat monoclonal antibodies (obtained

from Serotec, Oxford, UK; all antibodies were diluted

1:400): W3/25 (CD4), MRC OX-8 (CD8), MRC OX-39

(CD25), R73 (T cell receptor alpha and beta), 10/78 (NKR-

P1), and ED1 (CD68). In addition, the following rabbit

anti-rat polyclonal antibodies were used: anti-perforin

(Torrey Pine Biolabs, San Diego, CA; dilution 1:400) and

Iba1 directed against an ionized calcium-binding adaptor

molecule-1 expressed in microglia and macrophages (kind

gift from Y. Imai, Department of Neurochemistry, National

Institute of Neuroscience, Tokyo; dilution 1:1,500). Incu-

bation with primary antibodies in PBS with 1% bovine

serum albumin was performed at 4�C overnight. Slices

were washed three times with PBS, and the secondary

biotinylated rabbit anti-mouse or swine anti-rabbit anti-

body (Dako, Hamburg, Germany; diluted 1:400 in PBS

with 10% heat-inactivated rat serum) was added at 37�C

for 1 h. After incubation for 45 min at 37�C with a strep-

tavidin-complex (Dako, Hamburg, Germany), slices were

stained with the chromogene substrate diaminobenzidine

(DAB) (Dako, Hamburg, Germany) followed by nuclear

staining with hemalaun.

Cytotoxic T lymphocyte assay

Spleens removed under sterile conditions were minced in a

Petri dish and passed through a 70 lm pore filter (Becton–

Dickinson Labware, Franklin Lakes, NJ, USA). This sus-

pension was layered onto Lympholyte M (Fa. Cedar Lane,

Ontario, Canada) and separated by density-gradient cen-

trifugation at 2,500 U/min for 20 min. The phase above the

lympholyte consisting of mononuclear cells was removed

and washed thrice with RPMI without supplement. This

cell suspension was transferred to a Petri dish and the cells

were allowed to adhere to the bottom at 37�C for 2 h. In

contrast to monocytes/macrophages and B cells, which all

adhere to plastic T cells, which can be collected by aspi-

ration off the culture media after gentle shaking [33]. Cells

were washed twice with RPMI containing 10% FCS and

100 U/ml penicillin/100 lg/ml streptomycin and counted.

Usually 1–2 9 108 mononuclear cells per spleen were

obtained.

Generation of CTL was performed according to Stein-

mann et al. [31]. In brief, 9L cells serving as stimulator

cells were seeded in 6-well plates and irradiated with a

lethal dose of 40 Gy. T cell-enriched mononuclear cells

(5 9 106) were added at a ratio of 1:10 which proved

optimal in preliminary experiments and co-cultures were

incubated for 7 days. On days 3 and 5, fresh RPMI (with

10% FCS and penicillin G/streptomycin) containing 30 U/ml

human recombinant Il-2 (Sigma-Aldrich, St. Louis, MO,

USA) was added.

On day 7, the target cells (TC) (5 9 105) were labeled

with 200 lCi Na51CrO4 (Amersham-Buchler, Braun-

schweig, Germany) in 1 ml RPMI containing 10% FCS in

a shaking water bath at 37�C for 1 h. Labeled TC (9L or

MADB 106) were washed thrice to eliminate non-incor-

porated Na51CrO4 and their viability was determined by

trypan blue exclusion. Cell viability was [90% in all

experiments. TC (3,000 cells/well) were distributed in 96-

well round-bottom plates (Corning Incorporated, Corning,

NY, USA) in RPMI containing 10% FCS and centrifuged

to the bottom at 1,000 U/min for 1 min. Cells were let to

adhere for 2–3 h before effector cells (EC) were added.

These were collected, counted, and added in triplicates to

labeled cells in fresh RPMI medium in different ratios

(10:1, 20:1, 40:1, 80:1). The plates were centrifuged

for 3 min to assure a close contact between TC and EC.

Plates were incubated at 37�C for 4 h. The radioactive

Cancer Immunol Immunother (2010) 59:541–551 543

123

supernatant was harvested with a harvesting system

(Scatron, Suffolk, UK) and transferred to a Gamma counter

(Fa. Canberra-Packard, Frankfurt, Germany). Spontaneous

release of 51Cr was determined from six wells devoid of EC

(equivalent to 0% specific release). Maximum (100%)

release of 51Cr was determined after incubation of six wells

with 10% Triton-X detergent. Specific lysis was calculated

as follows:

Specific lysis ð%Þ ¼ 100� ½ðexperimental cpm

� spontaneous cpm=maximum cpm

� spontaneous cpmÞ�:

Intracellular IFN-c synthesis (Elispot assay)

Intracellular IFN-c synthesis was determined with a com-

mercially available Elispot kit (Diaclone, Besancon,

France) following the provided protocol with minor

modifications according to Heiser et al. [19]. In brief,

PVDF-96-well plates were incubated with an anti-rat IFN-cantibody (capture antibody) at 4�C overnight. The next

day, freshly isolated spleen cells (5 9 105 responder cells

per well) were washed and resuspended in RPMI con-

taining 10% FCS. These cells were co-incubated with 9L or

MADB106 (105 stimulator cells per well) in the IFN-cantibody precoated 96-well plates. After incubation at 37�C

for 20 h, a biotinylated anti-rat IFN-c antibody (detection

antibody) was added and detected with streptavidin-con-

jugated alkaline phosphatase converting the substrate

BCIP/NBT to a blue dye. Dots were counted with the

Bioreader�-System (BIO-SYS GmbH, Karben, Germany).

Statistical analysis

For multiple group comparisons, ANOVA was performed

followed by the Student–Newman–Keuls test for between-

group analysis.

Results

The immunostimulatory activity of CpG-ODN was asses-

sed first in the brain of tumor-free rats. Oligonucleotides

mutated in the immunostimulatory CpG-motif (m-CpG)

were injected into the contralateral hemisphere serving as

an internal control (Fig. 1). After 2 days, the CpG-treated

hemispheres revealed focal staining for ED1 at the injec-

tion site as well as in the leptomeninges (Fig. 1a). ED1

staining was even more pronounced when investigated

after 5 days (Fig. 1b). No such ED1 induction was

observed with m-CpG-ODN (Fig. 1a, b). Similarly, CpG

but not m-CpG treatment resulted in MHC 2 induction

5 days after ODN administration (Fig. 1d, f). The mor-

phology of the MHC 2 positive cells resembled microglial

cells. After CpG treatment, CD8? cells could also be

detected, predominantly in the vicinity of vessels forming

perivascular cuffs (Fig. 1e). These cells may represent

microglia, macrophages, or lymphocytes [21]. As descri-

bed for ED1, CD8? and MHC 2 positive cells were also

found in the leptomeninges of the CpG-ODN-treated

hemisphere (Fig. 1g, h).

Prior to intracerebral tumor cell inoculation, we inves-

tigated direct effects of ODN treatment on tumor cell

growth in culture. CpG-ODN added at concentrations

of 10-6 M as well as m-CpG-ODN (10-5 and 10-6 M)

m-CpG CpG m-CpG CpG

ED1 ED1(day 2) (day 5)

C D

ED1 ED1

m-CpG (day 5) CpG (day 5)

2CHM2CHM

CpG (day 5)CpG (day 2)

A B

E F

G H

2CHM8DC

CpG (day 2) CpG (day 2)

2CHM8DC

Fig. 1 Effect of immunostimulatory CpG-ODN or CpG-ODN

mutated in the immunostimulatory sequence (m-CpG) in normal rat

brain. a After 2 days, focal staining for ED1 was observed at the

injection site and in the leptomeninges (magnified in the inserted

image) of the CpG-ODN-treated hemisphere. b After 5 days, ED1

staining at the inoculation site is more pronounced (the ED1 positive

area is magnified in the inserted image). Similarly, CpG-ODN (d, f)but not mCpG-ODN (c) treatment resulted in MHC 2 induction

5 days after ODN administration. Magnification revealed MHC 2

positive microglial cells or macrophages (f) and perivascular inflam-

mation with CD8 positive cells representing microglia, macrophages,

or lymphocytes (e). Similarly, in the leptomeninges of the CpG-ODN-

treated hemisphere CD8 positive (g) and MHC 2 positive (h) cells

were detected


123

inhibited exponential growth of 9L cells (Fig. 2). CpG-

ODN administered at higher concentrations (10-5 M)

resulted in a slight decrease in viable cells indicating

cytotoxic effects (Fig. 2).

The effect of intratumoral CpG-ODN treatment on

tumor size was determined 3 weeks after intracerebral

implantation of rat 9L cells. Treatment with the mutated

ODN (m-CpG-ODN) resulted in tumors similar in size to

the saline-treated controls. Rather surprisingly, CpG-ODN

(applied both on day 3 or day 0) led to a higher variability

in tumor size, and several CpG-treated animals revealed

tumors exceeding the largest tumors in both control groups

(Fig. 3). The overall difference between all treatment

groups, however, did not prove statistically significant

(ANOVA; p [ 0.05). One animal in the group having

received the CpG-ODN together with the tumor cell sus-

pension on day 0 had died prior to killing. At autopsy

excessive tumor growth with a tumor diameter of[10 mm

representing the likely cause of death was found. Since the

tumor volume could not be determined with accuracy, this

animal had to be excluded from further analysis.

All tumors were investigated by standard HE histology

and immunohistochemistry (Fig. 4). In the majority of the

CpG-treated tumors histology revealed more abundant

necrosis which contributed to the higher variation in tumor

size in the CpG-ODN-treated animals compared to controls

(Fig. 4l, m). In the saline-treated control animals only

small areas of necrosis were observed. Although the

necrotic areas contributed to the increase in tumor size in

some of the CpG-treated animals, most of the tumors

consisted of a solid tumor mass. Notably, tumor mor-

phology was not different between animals which had

received intratumoral CpG injections (day 3) or tumor cell

suspensions mixed with CpG-ODN (day 0).

Both in CpG-treated and control-treated animals, dense

infiltrations with lymphocytes could be observed, in

particular, in perivascular areas (Fig. 4a; T cell receptor).

Most of these cells were CD8? T cells and only few NK

cells could be detected (Fig. 5c, e). CD4 staining revealed

both lymphocytes and microglial cells [26] (Fig. 4b).

Irrespective of prior CpG treatment only few cells stained

positive for perforin serving as a marker for T cell or NK

cell activation (Fig. 4f). Lymphocytes staining positive for

CD25 were detected throughout the tumors of all treatment

groups indicating T cells playing a role in the maintenance

of tolerance to self-antigens. The extent of lymphocytic

infiltration within individual tumors showed regional

heterogeneity (e.g., perivascular accumulation) and we

observed some variation between animals of the same

treatment group. Notably, the treatment groups could not

be distinguished based on a characteristic lymphocytic

staining pattern except for the presence of necrotic areas

which were more prevalent in the CpG-treated animals.

The tumors of all treatment groups were densely infil-

trated with microglial cells. Two types of microglial cells

could be distinguished. Microglia resembling macrophages

and with high expression of ED1 (regarded as a marker for

phagocytic activity) but also Iba1 staining was predomi-

nantly found in the CpG-treated animals, in particular, in

the vicinity of necrotic areas observed predominantly in

this treatment group (Fig. 4l, m, p, q). However, focal ED1

expression was also seen in some of the control-treated

animals (Fig. 4j). Microglial cells of the ramified pheno-

type were detected by staining for MHC 2 or Iba1 and

3000

4000

coun

ts

saline

m-CpG (10-6 M)

CpG (10 6 M)

1000

2000

cell

c CpG (10-6 M)

m-CpG (10-5 M)

CpG (10-5 M )

00 48 96 time (h)

Fig. 2 Effect of CpG-ODN on 9L cells grown in culture. Although

exponential cell proliferation was observed in vehicle-treated cul-

tures, the addition of CpG-ODN (10-6 M) and CpG-ODN mutated in

the immunostimulatory sequence (m-CpG; 10-6 M) resulted in

growth inhibition. A decrease in cell numbers indicating cytotoxicity

was observed with CpG-ODN when added at a concentration of

10-5 M

400

500

600

mm

3 )

J

H

200

300

400

Tu

mo

r vo

lum

e (m

CB

GF

LK

N

OE

A

M

0

100

saline m-CpG CpG CpG (day 0)

T DL M

Fig. 3 Effect of intratumoral CpG-ODN treatment on tumor volumes

3 weeks after intracerebral implantation of rat 9L cells. On day 3 after

tumor cell implantation, intratumoral injections were performed with

saline (control), a control oligonucleotide mutated in the immuno-

stimulatory sequence (m-CpG), or a CpG-containing immunostimu-

latory ODN (CpG). In treatment group ‘CpG (day 0)’, the 9L cells

were mixed with immunostimulatory CpG-ODN prior to tumor cell

inoculation to simulate an optimal distribution of CpG-ODN within

the tumor. These animals received no additional CpG treatment on

day 3. In this group, one animal had died prior to killing because of

excessive tumor growth (not included). No statistically significant

differences were detected between the treatment groups (p [ 0.05;

ANOVA). The letters on top of the bars indicate the plot in Fig. 5

depicting the CTL response of the same animal, e.g., the CTL

response of the saline-treated animal labeled with an a is shown in

panel a of Fig. 5


123

occurred as dense and rather evenly distributed cell popu-

lation within the solid, non-necrotic tumor masses as well

as the peritumoral brain parenchyma of control but also

CpG-treated animals (Fig. 4g, h, n, o).

To assess the induction of cytotoxic T cell responses

directed against the implanted 9L tumor cells, cytotoxic

lymphocytes were generated from in vitro restimulated

spleen cell preparations. Specific lysis of [25% was

observed with 9L cells but not with another syngeneic

adenocarcinoma cell line (MADB 106) serving as target

cells (Fig. 5a–o). Lymphocyte preparations from naive rats

which had not been implanted with 9L cells did not lyse

syngeneic 51Cr-labeled 9L or MADB 106 target cells (lysis

\5%; not shown). Rather unexpectedly, more effective

lysis was observed with lymphocytes generated from

control-treated animals. Although high lysis rates ([80–

90%) could be observed with effector cells from saline

(Fig. 5a–d) and m-CpG-treated animals (Fig. 5e–g), four of

five animals treated by intratumoral CpG injections (day 3)

revealed markedly reduced lysis rates (Fig. 5h–m). This,

however, was not observed with effector cells derived from

two animals treated with CpG-ODN on day 0 in conjunc-

tion with tumor grafting (Fig. 5n, o). In these animals, lysis

rates were achieved similar to both control groups (saline

and m-CpG). The tumor volumes of all the animals studied

by CTL analysis are indicated in Fig. 3, e.g., the CTL

response depicted in Fig. 5 panel A refers to the tumor

volume of animal ‘‘a’’ as indicated on top of the bar graph

in Fig. 3. The CTL response of individual animals did not

reflect the size of the tumors.

TCR - CpG CD25 - CpGCD8 - CpGCD4 - CpG

MHC 2 - controlPerforin - controlNKR-P1 - CpG MHC 2 - control

ED1 - controlED1 - control ED1 - CpG ED1 - CpG

A B C D

E F G H

MLKJ

PN QO

ED1 - controlED1 - control ED1 - CpG ED1 - CpG

GpC-1abIlortnoc-1abI GpC-1abIlortnoc-1abI

Fig. 4 Immunohistochemical staining of tumor sections following

intratumoral application of CpG-ODN (CpG) or saline (control). a–ethe same region revealing the tumor margin and the adjacent normal

brain was stained. In particular, in perivascular areas, dense infiltrates

with TCR? cells (a) were observed which consisted predominantly of

CD8? lymphocytes (c). The tumors contained numerous CD25

positive cells indicating CD4? (or CD8?) regulatory T cells

implicated in peripheral tolerance to self-antigen (d). The same

region also contained NK cells which were less abundant (e). Perforin

serving as a marker for cytotoxic effector activity of NK and CD8?

cells was only detected on few cells within CpG- and saline-treated (f)tumors. Staining for CD4 revealed dense infiltrates (b) consisting of

lymphocytes and microglia. More specific staining (Iba1 and ED1)

distinguished two types of microglial cells (g–q). In particular, around

necrotic areas which were more prevalent in CpG-treated tumors,

patchy areas were found revealing cells which resembled macro-

phages and which showed strong expression of ED1, a marker for

phagocytic activity (l, m, p, q). To a lesser extent, focal expression of

ED1 was also observed in saline-treated control animals (j, k). An

abundance of more evenly distributed microglial cells with a ramified

morphology was found within the tumors and peritumoral brain

parenchyma (g, h, n, o). Such ramified microglia was predominant in

the non-necrotic areas of both control-treated or CpG-treated animals


123

To further assess Th1-related lymphocytic reactivity,

Elispot assays were performed with splenocytes from a few

of the animals selected for 51chromium release assays

(Fig. 5) IFN-c synthesis. The letters on the x-axis refer to

the corresponding CTL plot of the same animal, e.g., ‘‘c’’

refers to a saline-treated control animal for which the CTL

response is indicated in panel C and for which the tumor

size can be derived from Fig. 3. Exposure of freshly iso-

lated spleen cells to 9L cells induced specific IFN-c syn-

thesis in all treatment groups. IFN-c production appeared

specific for 9L cells since lower levels of IFN-c were

detected upon stimulation with the syngeneic rat adeno-

carcinoma cell line MADB 106. In most animals, the

amount of IFN-c measured compared well to the lysis rates

detected in CTL assays performed with lymphocytes which

had been restimulated in culture for 1 week (Fig. 5). In one

animal lower levels of IFN-c were detected, although the

CTL assay revealed a strong cytotoxic response (Fig. 5,

animal ‘‘o’’). Two animals treated intratumorally with

CpG-ODN on day 3 were investigated with both assays

(Fig. 5, animal ‘‘l’’ and ‘‘k’’). Both assays do not suggest an

immunostimulatory effect of this treatment since both the

CTL response and the amount of IFN-c synthesized were

lower than in the control-treated animals.

Discussion

Despite clear immunostimulatory effects elicited by CpG-

ODN in normal rat brain, intratumoral CpG treatment did

not inhibit the formation of syngeneic gliomas. The tumor

size in the CpG-treated groups was more variable and most

of the CpG-treated tumors were larger than control-treated

tumors. Although inflammatory responses associated with

necrosis contributed to this increase in tumor size, most of

the tumors consisted of solid masses similar to the control-

treated animals. This was observed both in animals injected

with CpG-ODN 3 days after tumor cell implantation and in

animals having receiving tumor cells mixed with CpG-

ODN prior to grafting. Thus, inhomogenous distribution of

the CpG-ODN injected on day 3 possibly causing necrosis

in CpG-spilled areas and tumor growth in regions not

exposed to the ODN is unlikely.

Since tumor size was assessed 3 weeks after grafting, it

remains unresolved whether some of the tumors had

shrinked or even disappeared after longer observation

periods. Increased survival has been reported after intra-

tumoral CpG treatment in a similar intracranial model

using CNS-1 glioma cells in Lewis rats as well as the

murine GL261 glioma model [8, 12, 17]. With 9L glio-

sarcoma cells, Auf et al. [1] observed reduced tumor

growth in a subcutaneous model in Fischer rats after

repetitive intratumoral CpG injections. It has not been

mentioned whether repetitive administration was required

for successful treatment in this subcutaneous 9L model

since repetitive administration was not applied in other

studies of this laboratory. We seeked the permission to

investigate animal survival which, however, could not been

obtained from the local authorities and could not be

investigated. During killing, we observed excessive tumor

growth in most animals resulting in general slowing and

moderate weight loss. This argues against the speculation

that tumor rejections had possibly occurred with longer

observation periods. In fact, killing at later stage had to be

regarded unethical in several animals, and one of the ani-

mals having received the optimized treatment (CpG-ODN

mixed homogeneously with the tumor cells before grafting)

died from excessive tumor growth before killing.

Our findings are supported by other studies reporting

inconsistent anti-tumor effects following CpG-ODN

administration. CpG-ODN treatment had differential

effects in normal brain and in tumors on the baseline

expression and induction of MHC 2 and of co-stimulatory

molecules, such as B7.1 and B7.2 [2, 29]. Furthermore,

differences have been observed between different brain

tumor models. Interestingly, in their discussion Schartner

et al. [29] mentioned that they were not able to detect

inhibition of tumor growth or extended survival in intra-

cranial RG2 rat gliomas treated intratumorally with CpG-

ODN. One explanation is that the therapeutic effects

depend on the tumor model studied. On the other hand, the

actual immune responses may vary within the same tumor

model due to factors which cannot be easily controlled. For

example, it is almost impracticable to rule out changes in

the immunogenicity of cell lines which could arise during

routine maintenance in culture. In addition, the immuno-

logical capabilities of the same inbred rat strain may vary

between different animal cohorts purchased or bred at an

institution.

The immunostimulatory CpG-ODN did not induce

proliferation of tumor cells in cell culture arguing against

direct promotion of tumor growth. We rather observed

dose-dependent cytotoxic effects of CpG-ODN which,

however, were moderate because only slowing of expo-

nential cell proliferation or slight cytotoxicity in higher

concentrations was detected. Although human and rodent

glioma cell lines have been reported to express Toll-like

receptor 9 [12] such expression had not been detected in 9L

cells [24] arguing in favor of other (unspecific) mecha-

nisms mediating this moderate cytotoxicity. Although the

in vivo relevance of these cytotoxic effects is unclear, it

appears more likely that this inhibits tumor growth and this

does not explain an increase in tumor size as observed in

several of our CpG-treated animals. Since necrotic areas

had been observed in some of the large CpG-ODN-treated

tumors, one may speculate that this reflects direct cytotoxic


123

effects of CpG-ODN at the site of injection. This, however,

is unlikely since necrosis had not been observed in normal

brain treated with the same amount of CpG-ODN. Thus,

necrosis rather reflects the excessive tumor growth

observed in some of the CpG-ODN-treated animals or late

inflammatory responses induced by CpG-ODN.

Treatment of tumors with CpG-ODN has been shown to

promote invasion [20, 25]. In principle, CpG-ODN at the

time of their administration may have induced 9L tumor

cell infiltration. Although we did not observe increased

tumor cell infiltration in CpG-ODN-treated animals, early

tumor cell dissemination upon CpG-ODN administration

may have occurred and promoted tumor growth. None-

theless, this is unlikely since CpG-ODN-induced invasion

is dependent on TLR9 [20, 25] which is not expressed by

9L cells.

In normal rat brains we observed clear and widespread

activation of microglial cells (ED1 and MHC 2 staining)

following CpG-ODN treatment proving that the CpG-ODN

sequence and the batch of CpG-ODN employed is bioac-

tive. This was evident after 2 days with a further increase

after 5 days. The immune responses were specific for the

CpG-ODN since no such activation was observed in the

contralateral hemispheres injected with a mutated CpG-

ODN. CD8 staining was most pronounced in perivascular

areas. These cells may represent not only lymphocytes but

also a population of microglia or macrophages accumu-

lating early in CNS lesions, as described following ische-

mia [21].

In tumor-bearing animals, all tumors showed strong

MHC 2 and Iba1 expression in and around the tumors

indicating microglial cells. This staining was independent

of prior CpG treatment and this is in agreement with the

well-known prevalence of microglia in this glioma model

[2]. Compared to control-treated animals (saline and

m-CpG) in the CpG-treated tumors (phagocytic), activation

of microglial cells was observed more frequently. In par-

ticular, in necrotic areas, we detected intense staining for

ED1, a microglial activation marker found on lysosomal

membranes and representing phagocytic activity [9].

However, ED1 was also detected in control-treated tumors,

and, to a large extent, the overall increased ED1 expression

in CpG-treated tumors (days 3 and 0) reflected the fact that

necrotic areas were more prevalent in the CpG-group.

Activation of microglial cells by CpG-ODN appears to be a

transient and self-limiting process [30]. Thus, ED1

expression is unlikely to represent a direct consequence of

CpG administration (as observed in normal brain) but

rather reflects (phagocytic) microglial activity present at

the day of killing, in particular, in necrotic areas. It cannot

be ruled out that the necrotic areas indicate focal anti-

tumor activity elicited by CpG-ODN. However, this effect

was not strong enough to result in regression or even

rejection of the tumors. Furthermore, all CpG-treated

tumors (including those which had been treated with CpG-

ODN on the day of tumor grafting) consisted of large,

necrosis-free areas revealing solid tumor growth not dis-

tinguishable from control-treated rats.

In all tumors we observed dense infiltrates with T cells

and NK cells representing a common feature of gliomas.

However, the extent of T cell or NK cell infiltration and the

activation status was not altered by CpG-ODN treatment.

T cell and NK cell infiltrates varied more among different

animals of the same treatment group (and within individual

tumors, e.g., necrosis vs. areas of solid tumor growth) than

between animals of different treatment groups. In particu-

lar, staining with perforin, a marker for cytolytic activity of

NK cells and T cells, was restricted to single cells. Notably,

the number of perforin-positive cells was not increased in

the CpG-treated animals. This indicates that the lympho-

cytic infiltrates are rendered anergic or self-tolerant a

phenomenon commonly observed in gliomas and thought

to represent one of the obstacles for effective immuno-

therapy. This may be mediated by the numerous CD25

positive cells detected in all tumors. These cells are likely

to represent CD4? (or CD8?) regulatory T cells which

have been implicated in the maintenance of peripheral

tolerance to self-antigens [11, 14, 28]. Similar to what has

been found in glioma patients, one should consider that

rather than the absolute number the fraction of the CD25

positive cells among the CD4 positive T cell population

may be relevant [13].

Quantification of lymphocytes is hampered by the fact

that cells staining CD8 or CD4 positive may also represent

Fig. 5 Cytotoxic T cell responses assessed by 51chromium release

(a–l) and IFN-c synthesis quantified by the ELISPOT assay (m). a–l51Cr release lymphocytes were restimulated with 9L cells for 1 week

and added to 51Cr-labeled 9L target cells or another syngeneic

adenocarcinoma cell line (MADB 106) in different effector to target

ratios (E:T ratio). In all animals (a–l), specific lysis was observed with

9L cells but not with syngeneic MADB 106 cells. In both control

groups (saline, a–d, and m-CpG, e–g), high lysis rates ([80–90%)

were detected. In CpG-treated animals similar levels were measured

following CpG-ODN treatment on day 0 concomitant with tumor cell

grafting (n, o). Rather surprisingly, intratumoral application of

immunostimulatory CpG-ODN 3 days after tumor cell grafting

resulted in markedly lower lysis rates in 4 of 5 animals (h–m).

IFN-c synthesis in freshly isolated splenocytes following exposure to

9L cells or syngeneic MADB 106 cells was determined by Elispot

assays. The letters on the x-axis indicate the panel showing the51chromium release response of the same animal. For example, ‘‘c’’

corresponds to panel c depicting the CTL response of the same

animal. This represents a saline-treated animal, and the tumor volume

measured in the same animal can be determined from Fig. 3. IFN-csynthesis was observed in all treatment groups following exposure to

9L cells. Reduced levels of IFN-c synthesis were observed upon

stimulation with the syngeneic rat adenocarcinoma cell line MADB

106 suggesting a 9L specific response. In most animals (c, f, g, k, l, n,

o) the amount of IFN-c synthesis detected was similar to the CTL

response obtained after restimulation of lymphocytes in culture

c


123

lysi

s (%

)

40

60

80

100A saline

40

60

80

100B saline C saline

40

60

80

100

0

20

10:1 20:1 40:1 80:10

20

10:1 20:1 40:1 80:10

20

10:1 20:1 40:1 80:1

9L

MADB

80

100D saline

80

100E m-CpG

80

100F m-CpG

lysi

s (%

)

0

20

40

60

80

0

20

40

60

80

0

20

40

60

80

10:1 20:1 40:1 80:1 10:1 20:1 40:1 80:1 10:1 20:1 40:1 80:1

sis

(%) 60

80

100

G m-CpG

60

80

100J CpG

60

80

100H CpG

lys

0

20

40

10:1 20:1 40:1 80:10

20

40

10:1 20:1 40:1 80:1

100

K CpG100

L CpG100

M CpG

0

20

40

10:1 20:1 40:1 80:1

20

40

60

80

20

40

60

80

20

40

60

80

lysi

s (%

)

010:1 20:1 40:1 80:1

010:1 20:1 40:1 80:1

E:T ratio

010:1 20:1 40:1 80:1

s (%

) 60

80

100N CpG(day 0)

60

80

100O CpG

(day 0)

100

150

200

ots

/wel

l

IFN-γγ synthesis

lysi

s

0

20

40

10:1 20:1 40:1 80:1

E:T ratio

0

20

40

10:1 20:1 40:1 80:1

E:T ratio

0

50

100

spo

C G F K N OL

saline m-CpG CpG CpG (day 0)


123

microglia or macrophages [21]. With respect to the time

point (3 weeks following CpG treatment) and their mor-

phology, most of the CD8-positive cells are likely to rep-

resent lymphocytes. This differs from the early response to

CpG treatment of normal brain revealing CD8-positive

cells resembling microglia or macrophages (cf. Fig. 1;

[21]). On the other hand, staining of the tumors for the CD4

antigen revealed a great amount of microglia or macro-

phages as suggested by cell morphology. This is in

accordance with the occurrence of CD4 positive microglia

or macrophages in a later phase after brain lesioning [21].

The microglia or macrophages detected in rodent and

human gliomas amount to a significant portion of the tumor

mass and meanwhile their tumor promoting and immuno-

suppressive character has been exposed [16, 32]. This is

mediated by various mechanisms, e.g., by the production of

immunosuppressive cytokines such as IL-10 and PGE-2

and by altering the cytokine environment shifting T helper

cell responses from Th1 to Th2. In addition, T cells may be

rendered anergic or apoptotic by FasL or other molecules

expressed by microglia which has not been studied in our

model [3].

Functional characterization of lymphocytes in CTL

assays demonstrated that all animals exposed to 9L cells

developed a strong cytolytic response to 9L cells reflecting

the intrinsic immunogenicity of this model. Both CTL lysis

and IFN-c production appeared specific since it was

restricted to stimulation with 9L cells and not observed

with another syngeneic tumor cell line (MADB 106). This

response occurred irrespective of prior treatment with

immunostimulatory CpG-ODN, and rather surprisingly, the

average lysis with lymphocytic effector cells generated

from CpG-ODN-treated animals was lower than with

effector cells derived from control-treated animals (saline

and m-CpG). Although this apparent suppression of cyto-

toxic response has to remain unexplained, a similar

response was observed in Elispot assays measuring IFN-csynthesis in fresh splenocyte preparations exposed to 9L

cells. This supports the conception that, at least in our

model, intratumoral application of CpG-ODN may inhibit

cytotoxic immune responses. It may even be speculated

that this inhibitory response contributes to the formation of

larger tumors as observed in some of the CpG-treated

animals. However, the interpretation of these data is lim-

ited by the fact that even in the tumor models in which

CpG-ODN have been used successfully the exact mecha-

nisms have not been resolved. Although activation of

microglia/macrophages appears to play a crucial role, B or

T cells may only be involved at a later stage maintaining an

anti-tumor response [1, 5].

Taken together, CpG-ODN with proven immunostimu-

latory activity in vivo (in normal rat brain) do not elicit

reliable anti-tumor effects. Our data are in line with the

well-known immunosuppressive features of gliomas and

their resistance to all therapeutic approaches investigated to

date. Despite the intrinsic immunogenicity of the 9L gli-

oma model in which complete tumor rejection can be

achieved, CpG-ODN were not sufficient to induce anti-

tumor effects. This appears to reflect the systemic and local

immunosuppressive features of gliomas going along with

anergic or tolerant lymphocytes found within the tumors

and an abundance of intratumoral microglial cells supposed

to serve the purpose of the tumor rather than being

involved in anti-tumor defense mechanisms [16, 32]. The

clinical use of CpG-ODN may not increase the survival of

glioma patients.

Acknowledgments We are indebted to Dr. Axel Heiser for his

support in performing the Elispot assays. We thank Dr. T. von Hor-

sten (Medizinische Hochschule Hannover, Germany) for supplying us

with the MADB 106 adenocarcinoma cell line. The Iba1 antibody was

a kind gift from Dr. Y. Imai (Department of Neurochemistry, National

Institute of Neuroscience, Tokyo). The technical support by Mrs.

Barbel Hufnagel is appreciated. This work has been supported by the

‘Interdisziplinares Zentrum fur Krebsforschung der Christian-

Albrechts Universitat zu Kiel’ and by the ‘Hensel Stiftung’.

References

1. Auf G, Carpentier AF, Chen L, Le Clanche C, Delattre JY (2001)

Implication of macrophages in tumor rejection induced by CpG-

oligodeoxynucleotides without antigen. Clin Cancer Res 7:3540–

3543

2. Badie B, Bartley B, Schartner J (2002) Differential expression of

MHC class II and B7 costimulatory molecules by microglia in

rodent gliomas. J Neuroimmunol 133:39–45

3. Badie B, Schartner J, Prabakaran S, Paul J, Vorpahl J (2001)

Expression of Fas ligand by microglia: possible role in glioma

immune evasion. J Neuroimmunol 120:19–24

4. Barclay AN (1981) The localization of populations of lympho-

cytes defined by monoclonal antibodies in rat lymphoid tissues.

Immunology 42:593–600

5. Buhtoiarov IN, Sondel PM, Eickhoff JC, Rakhmilevich AL

(2007) Macrophages are essential for antitumour effects against

weakly immunogenic murine tumours induced by class B CpG-

oligodeoxynucleotides. Immunology 120:412–423

6. Carpentier A, Laigle-Donadey F, Zohar S, Capelle L, Behin A,

Tibi A, Martin-Duverneuil N, Sanson M, Lacomblez L, Taillibert

S, Puybasset L, Van Effenterre R, Delattre JY, Carpentier AF

(2006) Phase 1 trial of a CpG oligodeoxynucleotide for patients

with recurrent glioblastoma. Neuro Oncol 8:60–66

7. Carpentier AF, Meng Y (2006) Recent advances in immuno-

therapy for human glioma. Curr Opin Oncol 18:631–636

8. Carpentier AF, Xie J, Mokhtari K, Delattre JY (2000) Successful

treatment of intracranial gliomas in rat by oligodeoxynucleotides

containing CpG motifs. Clin Cancer Res 6:2469–2473

9. Damoiseaux JG, Dopp EA, Calame W, Chao D, MacPherson GG,

Dijkstra CD (1994) Rat macrophage lysosomal membrane anti-

gen recognized by monoclonal antibody ED1. Immunology

83:140–147

10. Ehtesham M, Black KL, Yu JS (2004) Recent progress in

immunotherapy for malignant glioma: treatment strategies and

results from clinical trials. Cancer Control 11:192–207


123

11. El Andaloussi A, Han Y, Lesniak MS (2006) Prolongation of

survival following depletion of CD4?CD25? regulatory T cells

in mice with experimental brain tumors. J Neurosurg 105:430–

437

12. El Andaloussi A, Sonabend AM, Han Y, Lesniak MS (2006)

Stimulation of TLR9 with CpG ODN enhances apoptosis of

glioma and prolongs the survival of mice with experimental brain

tumors. Glia 54:526–535

13. Fecci PE, Mitchell DA, Whitesides JF, Xie W, Friedman AH,

Archer GE, Herndon JE 2nd, Bigner DD, Dranoff G, Sampson JH

(2006) Increased regulatory T-cell fraction amidst a diminished

CD4 compartment explains cellular immune defects in patients

with malignant glioma. Cancer Res 66:3294–3302

14. Fecci PE, Sweeney AE, Grossi PM, Nair SK, Learn CA, Mitchell

DA, Cui X, Cummings TJ, Bigner DD, Gilboa E, Sampson JH

(2006) Systemic anti-CD25 monoclonal antibody administration

safely enhances immunity in murine glioma without eliminating

regulatory T cells. Clin Cancer Res 12:4294–4305

15. Ginzkey C, Eicker SO, Krause J, Brecht S, Hugo HH, Marget M,

Mehdorn HM, Steinmann J, Hamel W (2009) DNA vaccination

against rat gliomas expressing the E. coli lacZ gene as a model

antigen (submitted)

16. Graeber MB, Scheithauer BW, Kreutzberg GW (2002) Microglia

in brain tumors. Glia 40:252–259

17. Grauer OM, Molling JW, Bennink E, Toonen LW, Sutmuller RP,

Nierkens S, Adema GJ (2008) TLR ligands in the local treatment

of established intracerebral murine gliomas. J Immunol

181:6720–6729

18. Hartmann G, Weeratna RD, Ballas ZK, Payette P, Blackwell S,

Suparto I, Rasmussen WL, Waldschmidt M, Sajuthi D, Purcell

RH, Davis HL, Krieg AM (2000) Delineation of a CpG phosp-

horothioate oligodeoxynucleotide for activating primate immune

responses in vitro and in vivo. J Immunol 164:1617–1624

19. Heiser A, Coleman D, Dannull J, Yancey D, Maurice MA, Lallas

CD, Dahm P, Niedzwiecki D, Gilboa E, Vieweg J (2002)

Autologous dendritic cells transfected with prostate-specific

antigen RNA stimulate CTL responses against metastatic prostate

tumors. J Clin Invest 109:409–417

20. Ilvesaro JM, Merrell MA, Swain TM, Davidson J, Zayzafoon M,

Harris KW, Selander KS (2007) Toll like receptor-9 agonists

stimulate prostate cancer invasion in vitro. Prostate 67:774–781

21. Jander S, Schroeter M, D’Urso D, Gillen C, Witte OW, Stoll G

(1998) Focal ischaemia of the rat brain elicits an unusual

inflammatory response: early appearance of CD8? macrophages/

microglia. Eur J Neurosci 10:680–688

22. Jurk M, Vollmer J (2007) Therapeutic applications of synthetic

CpG oligodeoxynucleotides as TLR9 agonists for immune mod-

ulation. BioDrugs 21:387–401

23. Krieg AM (2007) Development of TLR9 agonists for cancer

therapy. J Clin Invest 117:1184–1194

24. Meng Y, Carpentier AF, Chen L, Boisserie G, Simon JM,

Mazeron JJ, Delattre JY (2005) Successful combination of local

CpG-ODN and radiotherapy in malignant glioma. Int J Cancer

116:992–997

25. Merrell MA, Ilvesaro JM, Lehtonen N, Sorsa T, Gehrs B,

Rosenthal E, Chen D, Shackley B, Harris KW, Selander KS

(2006) Toll-like receptor 9 agonists promote cellular invasion by

increasing matrix metalloproteinase activity. Mol Cancer Res

4:437–447

26. Perry VH, Gordon S (1987) Modulation of CD4 antigen on

macrophages and microglia in rat brain. J Exp Med 166:1138–

1143

27. Roman M, Martin-Orozco E, Goodman JS, Nguyen MD, Sato Y,

Ronaghy A, Kornbluth RS, Richman DD, Carson DA, Raz E

(1997) Immunostimulatory DNA sequences function as T helper-

1-promoting adjuvants. Nat Med 3:849–854

28. Schabowsky RH, Madireddi S, Sharma R, Yolcu ES, Shirwan H

(2007) Targeting CD4?CD25?FoxP3? regulatory T-cells for

the augmentation of cancer immunotherapy. Curr Opin Investig

Drugs 8:1002–1008

29. Schartner JM, Hagar AR, Van Handel M, Zhang L, Nadkarni N,

Badie B (2005) Impaired capacity for upregulation of MHC class

II in tumor-associated microglia. Glia 51:279–285

30. Schluesener HJ, Seid K, Deininger M, Schwab J (2001) Transient

in vivo activation of rat brain macrophages/microglial cells and

astrocytes by immunostimulatory multiple CpG oligonucleotides.

J Neuroimmunol 113:89–94

31. Steinmann J, Kaden J, May G, Schroder K, Herwartz C, Muller-

Ruchholtz W (1994) Failure of in vitro T-cell assays to predict

clinical outcome after human kidney transplantation. J Clin Lab

Anal 8:157–162

32. Watters JJ, Schartner JM, Badie B (2005) Microglia function in

brain tumors. J Neurosci Res 81:447–455

33. Zeis M, Siegel S, Wagner A, Schmitz M, Marget M, Kuhl-

Burmeister R, Adamzik I, Kabelitz D, Dreger P, Schmitz N, Heiser A

(2003) Generation of cytotoxic responses in mice and human

individuals against hematological malignancies using survivin-

RNA-transfected dendritic cells. J Immunol 170:5391–5397


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