Effector T-cell infiltration positively impacts survival ... · 4/8/2011 · 3 ABSTRACT Purpose:...

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Effector T-cell infiltration positively impacts survival of glioblastoma

patients and is impaired by tumor-derived transforming growth factor-

betas

1Jennifer Lohr, 1Thomas Ratliff, 1Andrea Huppertz, 2Yingzi Ge, 1Christine Dictus, 1Rezvan

Ahmadi, 3Stefan Grau, 4Nobuyoshi Hiraoka, 5Volker Eckstein, 6Rupert C. Ecker, 7Thomas

Korff, 8Andreas von Deimling, 1Andreas Unterberg, 2Philipp Beckhove, 1Christel Herold-

Mende

1Division of Neurosurgical Research, Dpt. Neurosurgery, University of Heidelberg, INF400,

Heidelberg, Germany; 2Translational Immunology Unit, DKFZ, INF460, Heidelberg,

Germany; 3Dpt. Neurosurgery, Klinikum Grosshadern, University of Munich,

Marchioninistrasse 15, 81377 Munich, Germany; 4Pathology Division, National Cancer

Center Research Institute, 5-1-1 Tsukji, Chuo-ku, Tokyo 104-0045, Japan; 5Dpt. Internal

Medicine V, University of Heidelberg, INF 410, Heidelberg, Germany; 6TissueGnostics

GmbH, Taborstrasse 10/2/8, A-1020, Vienna, Austria; 7Institute of Physiology and

Pathophysiology, Div. Cardiovascular Physiology, University of Heidelberg, INF307

Heidelberg, Germany; 8Dpt. Neuropathology, University of Heidelberg, INF220/221,

Heidelberg, Germany;

Running Title: T-cell infiltration improves survival

Key Words: glioma, T-cell infiltration, survival, adhesion molecule expression, TGF-ß

Funding: This work has been supported by the “Tumorzentrum Heidelberg/Mannheim”

[CHM].

Correspondence:

Prof. Dr. Christel Herold-Mende

Sektion Neurochirurgische Forschung

Neurochirurgische Universitätsklinik

INF400, 69120 Heidelberg, Germany

Tel.: 49 6221-566405

Fax: 49 6221-5633979

Email: [email protected]

Research. on February 4, 2021. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 8, 2011; DOI: 10.1158/1078-0432.CCR-10-2557

http://clincancerres.aacrjournals.org/

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TRANSLATIONAL RELEVANCE In most cancers, the type and density of intratumoral T-cells have been shown to have strong

prognostic significance. However, in glioma, studies of the clinical relevance of T-cell

infiltration have yielded incongruent results. We have discovered that T-effector (Teff) cell

infiltration positively affects glioblastoma (GBM) patient survival. Thus, Teff-cell infiltration

might serve as a tool to monitor the responses of patients to immunotherapy treatment. We

found that the glioma cytokine TGF-ß is an important negative regulator of T-cell

transmigration through GBM-derived endothelium. Further, we identified a novel

immunosuppressive mechanism of TGF-ß in glioma: the downregulation of CAM expression,

which consequently reduces T-cell transmigration. Accordingly, our results predict that the

effectiveness of glioma immunotherapy could be improved by simultaneously inhibiting

TGF-ß signaling within tumors.




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ABSTRACT Purpose: In glioma–in contrast to various other cancers–the impact of T-lymphocytes on

clinical outcome is not clear. We investigated the clinical relevance and regulation of T-cell

infiltration in glioma.

Experimental Design: T-cell subpopulations from entire sections of 93 WHO°II–IV gliomas

were computationally identified using markers CD3, CD8 and Foxp3; survival analysis was

then performed on primary glioblastomas (pGBM). Endothelial cells expressing cellular

adhesion molecules (CAMs) were similarly computationally quantified from the same glioma

tissues. Influence of prominent cytokines (as measured by ELISA from 53 WHO°II–IV

glioma lysates) on CAM-expression in GBM-isolated endothelial cells was determined using

flow cytometry. The functional relevance of the cytokine-mediated CAM regulation was

tested in a transmigration assay using GBM-derived endothelial cells and autologous T-cells.

Results: Infiltration of all T-cell subsets increased in high-grade tumors. Most strikingly,

within pGBM, elevated numbers of intratumoral effector T-cells (Teff, cytotoxic and helper)

significantly correlated with a better survival; regulatory T-cells were infrequently present and

not associated with GBM patient outcome. Interestingly, increased infiltration of Teff -cells

was related to the expression of ICAM-1 on the vessel surface. Transmigration of autologous

T-cells in vitro was markedly reduced in the presence of CAM-blocking antibodies. We found

that TGF-ß molecules impeded transmigration and downregulated CAM-expression on GBM-

isolated endothelial cells; blocking TGF-ß receptor signaling increased transmigration.

Conclusions: This study provides comprehensive and novel insights into occurrence and

regulation of T-cell infiltration in glioma. Specifically, targeting TGF-ß1 and TGF-ß2 might

improve intratumoral T-cell infiltration and thus enhance effectiveness of immunotherapeutic

approaches.




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INTRODUCTION

Glioblastoma (GBM) is the most malignant type of brain tumor. More than 90% of GBMs are

diagnosed de novo (primary GBM, pGBM) while others are thought to arise from malignant

transformation of lower-grade astrocytic and oligodendroglial tumors (secondary GBM,

sGBM, ref.1). Despite intensive therapy including surgery followed by radio- and

chemotherapy, GBMs are still incurable. This is mainly due to the high propensity of GBM

cells to invade the surrounding normal brain which prevents a complete tumor resection (2).

Therefore investigation of complementary approaches is urgently needed in order to eliminate

persisting glioma cells.

Triggering the immune response is theoretically an attractive treatment method since even

scattered tumor cells can be selectively targeted without damaging the normal brain (3). That

immunotherapy can be efficient and safe was shown by a number of clinical trials aiming at a

specific anti-glioma T-cell activation. These approaches include adoptive transfer of tumor-

reactive T-cells, ex vivo tumor antigen-primed dendritic cells, or vaccination with peptides,

inactivated autologous tumor cells and gene-modified tumor cells (4). However, despite

beneficial observations patients were not cured and some of them even did not respond to

anti-glioma immunotherapy. This is largely due to the location and special properties of all

glial brain tumors.

Gliomas develop in an organ that is normally shielded from the immune system by the blood

brain barrier (BBB), thereby limiting the transmigration of immune cells (5). However,

T-cells can cross an intact BBB if they are in an activated state (6). Moreover, relative to low

grade gliomas, GBMs are characterized by a compromised BBB due to breaks in tight

junctions, augmented fenestrations and decreases in BBB-associated pericytes (7, 8). Hence

the success of immunotherapy for the treatment of gliomas might be limited by the BBB, but

to a variable extent.

Anti-glioma immune responses are impaired by the tumor itself through expression of

transforming growth factor-beta (TGF-ß) and Interleukin-10 (IL-10) (9). For example, TGF-ß

inhibits T-cell activation and proliferation, represses production of lytic enzymes and drives

development of naive T-cells into regulatory T-cells (Treg) (10, 11). Under normal

physiological conditions Treg-cells are necessary to protect against autoimmune diseases but in

GBM patients they were suggested as a major contributor to depressed cellular immunity

(12). Moreover, tumor-secreted cytokines that are involved in the angiogenic process may add

to the immunosuppressive microenvironment. Basic fibroblast growth factor (bFGF) and

vascular endothelial growth factor (VEGF), considered being key mediators of glioma




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angiogenesis, act as inhibitors of cell adhesion molecule (CAM) expression in non-glioma

animal models and in experiments on endothelial cells derived from normal tissues (13-19).

Among these CAMs, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion

molecule 1 (VCAM-1) are regarded as the most important anchorage molecules for T-cells on

the blood vessel surface during the transmigration process (20-22).

Despite all these hurdles for T-lymphocytes to reach the tumor site, many investigators have

observed the presence of glioma T-cell infiltrates in patient tissues, implying the generation of

spontaneous antitumor immune responses (23). Whether these T-cell responses in therapy-

naive patients are linked to their clinical outcome is still not clear. Investigations of

intratumoral T-cells were performed over 20 years ago and identified immune infiltrates by

leukocyte cuffing, a morphological criterium that does not discriminate among leukocyte

types; their results are inconsistent however, reporting a positive correlation to clinical

outcome (24, 25), a negative correlation (26) and no correlation (27). By this method T-cells

were not clearly identified by specific markers and no discrimination was made among the

contents of immunosuppressing and effector T-cell (Teff) subpopulations. More recent studies

have not analyzed Teff populations or failed to find a WHO grade-independent correlation of

intratumoral Treg-cells in glioma patients (28-30). And to date, a comprehensive analysis of

different T-cell subsets in glioma has not been performed on native tumor tissues.

The role of intratumoral T-cell subsets in gliomas may be of critical importance for the design

of future immunotherapies. Therefore we investigated T-cell infiltrates in gliomas of different

WHO grades with a special emphasis on the quantitative analysis of effector and inhibitory

T-cell subsets by costaining of several lymphocyte markers in complete sections of native

tumor tissues. By using a novel FACS-like quantitative method, we found that Teff-cells

particularly had a positive influence on GBM patient survival. We then established a

functional model using GBM-derived endothelial cells and Teff-cells isolated from the

peripheral blood of the same patient. With this assay we investigated the influence of tumor-

expressed cytokines on the T-cell transmigration process. We found that transmigration is

mediated by both ICAM-1 and VCAM-1 and reduced in the presence of TGF-ß1 and TGF-ß2.

Strikingly, on GBM-derived endothelial cells in culture, we observed a strong downregulation

of ICAM-1 and VCAM-1 by recombinant TGF-ß1 and TGF-ß2.

Our data indicates that blocking both TGF-ß1 and TGF-ß2 promotes T-cell infiltration by

restoring CAM expression. It suggests that targeting TGF-ßs is a potentially useful strategy to

boost effectiveness of multi-modal glioma treatments by overcoming immune resistance

mechanisms.




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MATERIAL AND METHODS

Tumor material

Glioma specimens of different WHO grades were gathered from patients at the Department of

Neurosurgery at Heidelberg University, Germany. Informed consent was obtained from each

patient according to the research proposals approved by the Institutional Review Board at the

Heidelberg Medical Faculty. Samples used for immunostainings were immediately snap

frozen after surgery and stored at -80°C until processing. Intratumoral localization and WHO

grading was confirmed by an experienced neuropathologist. Clinical data of the respective

patients are summarized in Supplementary Table 1. A total of 93 glioma samples representing

WHO°II-IV were examined (study sample A). For survival analysis (study sample B, n=44,

newly diagnosed WHO°IV gliomas) only those patients were included who had received

current standard therapies and experienced a tumor-related death.

Multicolor Immunostainings and evaluation

Acetone-fixed cryostat sections (5-7µm) were used for the double and triple immunostaining

techniques. T-cell subpopulations were distinguished by combined antibody staining against

CD3, CD8 and Foxp3. In all staining series human tonsil were used as internal positive

control for comparable identification of T-cell subpopulations. Expression of ICAM-1 and

VCAM-1 was determined together with endothelial marker CD31. Evaluation was performed

by TissueFAXS. Advantages of this method compared to manual evaluation of native tissue

are the computerized quantification of multiple markers in large tissue areas. Leakiness of

blood vessels was examined by staining of fibrinogen together with CD31 and CD8. For

staining procedures, quality controls and image analysis see Supplementary Methods and

Supplementary Fig.1.

Growth factor ELISA

Frozen tumor pieces of approximately 0.5mm3 size were homogenized in 1ml PBS containing

25µl protease inhibitor (Roche). Lysates were purified from cell debris by a 13,000rpm

centrifugation for 30min at 4°C and filtering through a 22-µm sterile filter. VEGF, HGF,

bFGF, TGF-ß1, TGF-ß2, PDGF-AB, PDGF-BB, G-CSF and GM-CSF in the homogenates

were measured by a commercial sandwich ELISA (R&D Systems) as described (33).

Assessed cytokine levels were normalized to total protein content as determined by Bradford

assay (BioRad).




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Flow cytometry

Single cell suspensions of glioma-derived endothelial cells stained with monoclonal mouse

antibodies ICAM-1 (1:1,000; Acris) and VCAM-1 (1:100; Acris) in PBS containing 0.5%

BSA and 2mM EDTA for 1h at 4°C, then washed and stained with PE-conjugated anti-mouse

IgG mAb (1:100; Dianova). Flow-cytometric analyses were performed using FACSCalibur

(BD Biosciences). All samples were evaluated with appropriate isotype controls and analyzed

using FlowJo software (TreeStar).

In vitro transmigration assay

Isolated GBM-derived endothelial cells were allowed to grow as a monolayer on gelatin-

coated culture plate inserts (ThinCerts, 24-well, pore size 3.0 µm, Greiner Bio-One) in MV2

medium (Promocell) for 4 to 6 days. Tightness of the cell layer was confirmed by Evans blue

dye exclusion. 10µl of 0.25% Evans blue dye (Sigma) were added to the medium of the upper

chamber. Wells that were leaky were excluded from the assay. Transwell chambers were

washed twice with cytokine-free MV2 basal medium (MV2 Kit except supplement

components VEGF, IGF, bFGF and EGF; Promocell) and placed into new 24-well plates. To

prove influence of tumor-derived factors on transmigration of T-cells, transwells were

incubated with VEGF, HGF, bFGF, TGF-ß1 or TGF-ß2 (ReliaTech), 50µg/ml autologous

tumor lysate, 50µg/ml autologous lysate plus 1µM SD-208 (kindly provided by W. Wick,

University Hospital Heidelberg, Germany), or basal medium as a control. After 48 hours,

medium was renewed and supplemented with 100ng/ml SDF-1α at the lower chamber as a

chemoattractant. CD25-negative- or CD25-enriched T-cells (3x104/well) were added to the

upper chamber. The number of transmigrated T-cells was measured after 24h in duplicates

and detected by characteristic T-cell scatter profiles using FACSArray (BD Biosciences). For

detailed descriptions of isolation, cultivation and characterization of glioma-derived

endothelial cells and T-cells see Supplementary Methods and Supplementary Fig.8 and 10.

Statistical analysis

Data were analyzed with two-sided Student´s t-test, using GraphPad Prism 5.02 software. A

P-value <0.05 was defined as statistically significant. Kaplan Meier estimates were used to

visualize the survival curves and log-rank test was performed to compare overall survival

between patients of different groups. Cut-offs for CD3+ T-cell populations were set at the

median T-cell infiltration, cut-offs for CD8- and CD8+ T-cell populations were set according




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to their mean proportion on the CD3+ population. The Spearman´s correlation test was used to

determine the strength of correlation between different parameters analyzed within the same

patient tissue.




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RESULTS

Tumoral T-cell infiltration increases during malignant progression but correlates with

better survival in GBM patients

T-lymphocyte infiltration was determined in 93 gliomas of different WHO grade

(Supplementary Table 1) by staining with CD3 and CD8 antibodies (Fig.1A). Direct

identification of the T-helper subset by CD4 was impractical because of high background

staining in cryopreserved tissue produced by all CD4 antibodies tested (n=3). Therefore, CD3

and CD8 served as indirect marker combination to distinguish between putative T-helper

(CD3+CD8-) and cytotoxic (CD3+CD8+) subpopulations. Suitability of this approach was

confirmed by FACS analysis on freshly dissociated glioma samples where the CD8-negative

T-cell population was CD4-positive in >95% of CD3+CD8- cells (Supplementary Fig. 2A).

Infiltrates of CD3+ cells (T-cells), CD3+/CD8- cells (T-helper cells) and CD3+/CD8+ cells

(cytotoxic T-cells) were observed in all samples; however, measured numbers of T-cells

varied markedly between samples analyzed. In WHO°IV mean counts of all T-cell

populations increased 9-10-fold as compared to WHO°II and III tumors, whereas between

pGBMs and sGBMs no significant difference was measurable (Fig.1B). Nevertheless, even

within the subgroup of WHO°IV tumors considerable variances in T-cell counts were

observed.

Next we addressed whether the sharp increase of T-cell infiltration from WHO°II and III to

WHO°IV tumors might arise from a disturbed BBB. Therefore we stained tissues of different

WHO grade for the plasma protein fibrinogen, which can diffuse into the tumor stroma only if

the vessel wall becomes leaky. Indeed, significant staining of fibrinogen around leaky tumor-

supplying blood vessels was only found in WHO°IV (Supplementary Fig.3). Further,

infiltrating T-cells were most commonly seen in WHO°IV fibrinogen-positive tumor areas.

This supports our hypothesis that leaky vessels, which typically occur in GBM with the onset

of angiogenesis, facilitate T-cell transmigration as compared to lower grade tumors.

In summary, we found a WHO-grade-dependent increased T-cell infiltration. Second, in

WHO°IV tumors, elevated T-cell numbers were associated with BBB leakiness.

Occurrence of effector T-cells is associated with GBM patient survival

Next, we asked if T-cell subtypes vary among tumor grades and if the observed survival

differences could be attributed to the preferential infiltration of a specific T-cell subtype. This

is especially important because some T-cell subtypes have been implicated in the suppression




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of immune-mediated destruction of tumor cells (12). CD4+ and CD8+ regulatory T-cells are

considered the most prominent and tumor-relevant subtypes suppressing the activity of Teff-

cells (34). Therefore, our investigation of T-cell infiltration of gliomas was extended using the

Treg marker Foxp3. Because Foxp3 was shown to be also expressed by tumor cells (35), we

additionally stained lymphocyte markers CD3 and CD8 (Supplementary Fig.4). To prove the

validity of this marker combination to distinguish Teff and Treg subpopulations, we performed

flow cytometry analysis of glioma single cell suspensions with markers CD3, CD4, CD8,

Foxp3, CD25, CD127 according to a recent publication on gliomas, which defined Tregs as

Foxp3-CD25highCD127low (30) (Supplementary Methods and Supplementary Fig. 2B). This

analysis confirmed that Foxp3 was not present on CD127+- but on CD25+- cells. Thus

CD3/CD8/Foxp3 is a suitable marker to distinguish Teff and Treg cells. Further, nearly all

CD8- cells are CD4+ (see Supplementary Fig. 2A), constituting to define CD3+CD8-Foxp3-

cells as T-helper cells and CD3+CD8+Foxp3- cells as cytotoxic T-cells in our study.

Foxp3+CD3+ infiltrates were found in only 36% of the sGBMs and 43% of the pGBMs

analyzed (Fig.2A). Further, the proportion of the immunosuppressive T-cells was very low:

immunosuppressive T-cells represented <1% of the total T-cells identified in GBMs (Fig.2A).

This perhaps explains why we did not detect any immunosuppressive T cells in WHO°II

tissues, which generally had very low T-cell infiltration.

To confirm sensitivity of the Foxp3 antibody we used sections of human tonsil served as

positive control (Supplementary Fig.5). With a proportion of 3% Foxp3+ cells within the

CD3+CD8+ population, our data are in good agreement with a previous study reporting a

content of 1.5 % (36).

To determine the respective influences of effector and immunosuppressive T-cell

subpopulations on clinical outcome, we compared their amount to the overall survival time of

newly diagnosed WHO°IV glioma patients (study sample B, n=44). We observed a significant

survival correlation with helper T-cells (CD3+CD8-Foxp3-; p=0.01) and cytotoxic T-cells

(CD3+CD8+FoxP3-; p=0.02) as well as both effector T-cell subtypes together (CD3+Foxp3-;

p<0.01) (Fig.2B) but not for age, extent of resection and adjuvant treatment (Supplementary

Table 2). In contrast, we observed neither a correlation between survival and the infiltration

of Treg cells (Fig.2B) nor ratios of Treg/Teff populations (data not shown).

Collectively, our analysis suggests that infiltration of GBM tumors with effector T-cells in

particular has a positive impact on patient survival.




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T-cell infiltration is associated with CAM expression

Prior to transmigration, T-cells must attach to the luminal side of the tumor-supplying

endothelium. This process requires endothelial expression of adhesion molecules such as

ICAM-1 and VCAM-1 (21, 22). Indeed, we found that ICAM-1 and VCAM-1 colocalized

with the endothelial cell marker, CD31, in GBM tissues of study sample A (Supplementary

Table 1 and Fig.3A). Approximately 20-30% of CD31-positive endothelial cells from both

sGBM and pGBM coexpressed ICAM-1 and VCAM-1 (Fig.3B).

To study the relationship between T-cell infiltration and endothelial CAM expression, we

performed statistical correlation analysis on data obtained from 65 GBM tumor tissues. A

significant positive correlation was revealed for ICAM-1-expressing endothelial cells and all

T-cell subpopulations analyzed except Treg-cells (Fig.4). We note that even on low ICAM-1-

expressing endothelia remarkable T-cell infiltration was still observed, indicating that other

factors (e.g. leaky vessels) might also influence the infiltration process. Nonetheless, ICAM-1

negative endothelium did not correlate with T-cell infiltration (Supplementary Fig.6), pointing

to an influence of ICAM-1 on T-cell transmigration. Regarding VCAM-1-positive endothelial

cells no significant relationship with T-cell infiltration could be proven (Supplementary

Fig.7). That this is caused by the limited case number and the generally lower VCAM-1

expression as compared to ICAM-1-expression cannot be excluded.

In summary, we found that a substantial portion of tumor-derived endothelial cells expressed

CAMs and that their expression of ICAM-1, but not VCAM-1, was positively associated with

T-cell infiltration.

Pro-angiogenic cytokines are frequently present in glioma tissues

Gliomas secrete angiogenesis-promoting factors that are suspected of altering adhesion

molecule expression (14-18, 33). To determine the expression levels of nine known

angiogenic cytokines, we measured their respective concentrations relative to total protein in

WHO°II (n=14), III (n=16), and IV [sGBM (n=7); pGBM (n=16)] tumor lysates (Fig.5).

Except for bFGF, cytokine expression levels were significantly higher in WHO°IV gliomas

(p<0.05). Cytokines bFGF, VEGF and HGF were detected in all samples from WHO°IV

tumors (pGBM and sGBM) and were the most highly expressed cytokines in these tumors.

Cytokines TGF-ß1, TGF-ß2, PDGF-AB and PDGF-BB were also detected in all WHO°IV

tumor samples, but were expressed at lower concentrations than bFGF, VEGF and HGF.

G-CSF and GM-CSF showed an infrequent expression pattern.




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Our results indicate that, with few exceptions, angiogenic cytokine expression is ubiquitous

but WHO grade-dependent, peaking in grade IV gliomas.

TGF-ß1 and TGF-ß2 downregulate ICAM-1 and VCAM-1 expression on cultured GBM

endothelial cells

We then evaluated the capacity of glioma-derived cytokines investigated above to regulate

endothelial cell expression of ICAM-1 and VCAM-1. Similar experiments using umbilical

vein endothelial cells (HUVECs) previously found that VEGF and bFGF were inhibitory (16,

18). Yet endothelial cells from different sources have been known to respond differently to

the same stimulus (14, 16). Given our interests in and access to GBM, we used endothelial

cells extracted from fresh GBM tissues (n=3). For each sample, endothelial cells were

positively identified by their characteristic uptake of acetylated low density protein (AcLDL)

(Supplementary Fig.8). Cells were then incubated with the cytokines most consistently and

highly expressed in GBM samples: VEGF, HGF, bFGF, TGF-ß1 and TGF-ß2 (Fig.5). Flow

cytometry analysis revealed that VEGF, bFGF and HGF had only a slight effect on ICAM-1

and VCAM-1 expression (Fig.6A). In contrast, both TGF-ß1 and TGF-ß2 reduced ICAM-1

and VCAM-1 expression to approximately 42% and 8% of control levels, respectively

(Fig.6A). This result suggested that TGF-ß signaling plays an important role in regulating

adhesion molecule expression. To address whether TGF-ß signaling is biologically relevant to

GBM, we confirmed that TGF-ß receptors TGFßRI and TGF-ßRII were both expressed on

endothelial cells derived from each GBM sample (Supplementary Fig.9).

In summary, we found that treatment with TGF-ß1 and TGF-ß2, but not VEGF, HGF or

bFGF, markedly lowers adhesion molecule expression on cultured GBM endothelial cells.

Effector T-cell transmigration is reduced with exogenous TGF-ß1 and -ß2 treatment

and increased when TGF-ß receptor signaling is blocked

Finally, to study the effects of TGF-ß signaling on T-cell function, we established a

transmigration assay consisting of GBM endothelial cells and autologous T-cells (n=6).

T-cells were isolated from the peripheral blood of donors and then enriched for

subpopulations of Teff and Treg-cells using the Treg-cell surface marker, CD25 (Supplementary

Fig.10). However, remarkably, both T-cell populations were found to perform similarly in

control transmigration experiments (20% of T-cells transmigrated) (Fig.6B).

To test the functional importance of CAM molecules on T-cell transmigration, endothelial

cells were first incubated with neutralizing antibodies to ICAM-1 and VCAM-1. We observed




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a significant decrease in T-cell transmigration efficiency in the presence of either antibody, an

effect even more pronounced when they were used simultaneously (Fig.6C).

The transmigration assay provided a means to test whether TGF-ß-induced reduction of CAM

expression on GBM endothelial cells could affect T-cell function. We found that incubation

of the endothelial monolayer with TGF-ß1 and TGF-ß2, but not bFGF, VEGF or HGF,

markedly impaired T-cell transmigration efficiency (Fig.6D). The transmigration assay also

permitted us to address the converse experiment: whether inhibiting TGF-ß signaling in GBM

endothelial cells could positively effect T-cell transmigration. We used the TGF-ß receptor

kinase inhibitor, SD-208, for this purpose (37). We found that incubation of the endothelial

monolayer with SD-208 and GBM tumor lysate increased the efficiency of T-cell

transmigration 1.5-fold over incubation with tumor lysate alone (Fig.6E).

We conclude from these data that T-cell transmigration is directly mediated by CAM

molecules expressed on GBM endothelial cells and that T-cell transmigration is specifically

inhibited by TGF-ß signaling activity within GBM endothelial cells.




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DISCUSSION

The aims of this study were to determine whether T-cell infiltration of gliomas was clinically

relevant and to identify molecules that regulate glioma infiltration. Using a novel FACS-like

method for the objective quantification of T-cell subtypes in large tumor sections, we found

that, relative to low-grade gliomas, high-grade gliomas had significantly larger populations of

effector T-cells and immunosuppressive T-cells. Our studies revealed that Teff-cells in

particular were associated with increased GBM patient survival; immunosuppressive Treg-

cells were found to not affect GBM patient outcome. To investigate mechanisms regulating

T-cell infiltration, we first established a transmigration assay that more accurately models the

process in vivo by using glioma-derived endothelial cells and autologous T-cells. We

discovered that treating tumor tissue with the glioma cytokines TGF-ß1 and TGF-ß2 caused

suppressed T-cell transmigration. Further, treating with tumor lysates together with a TGF-ß-

signaling inhibitor caused enhanced transmigration. Considering these results, we hypothesize

that TGF-ß signaling antagonizes T-cell infiltration of gliomas.

Peculiar to glioma is the paradoxical relationship between T-cell infiltration and tumor grade.

Many reports describe a WHO grade-dependent increase of T-cells generally (this study, 29,

38, 39) and Treg-cells specifically (this study, 40, 29, 30). This relationship implies that T-cell

infiltration negatively correlates with patient survival. If true, this would contrast with the

positive correlation identified in various other cancers such as skin, breast, prostate and

colorectal cancers (41).

Studies of the relationship between glioma T-cell infiltration and patient survival have yielded

conflicting results (24-27). In recent studies, Treg-cells were found to correlate with a worse

patient outcome (29, 30). However, when GBM is considered separately from lower grade

gliomas, differences in patient survival have not correlated with Treg-cell infiltration (this

study, 29, 30). In this regard, one must consider the unique environment of gliomas. The brain

is normally shielded from inflammatory cells by the blood-brain barrier. However, as in other

cancers, tumor blood vessels of higher-grade gliomas likely become hyperpermeable, causing

a breach in the blood-brain barrier and providing an opening for inflammatory cells and

plasma blood proteins to more easily enter the tumor (42). Indeed, consistent with a grade-

dependent hyperpermeability in glioma, we detected the plasma protein fibrinogen in samples

of GBM tissue but not lower grade tissue. Given these considerations and observations, we

suggest that grade-dependent hyperpermeability provides a valid explanation for the grade-

dependent infiltration of T-cells in glioma. Therefore, we propose that the most accurate




15

method of evaluating the relationship between glioma T-cell infiltration and patient survival

will account for differences in tumor blood vessel permeability among the different WHO

grades. That is, it is important to maintain a distinction between tumor grades and restrict

comparisons of survival to patients falling within the same tumor grade category. By focusing

on GBM, we have identified a subpopulation of glioma-infiltrating T-cells, Teff-cells, that

positively affect patient survival.

Teff-cell subsets have not been specifically accounted for in previous glioma studies and

represent the first example of that T-cell subtype to have a role in glioma. This discovery is

complemented by earlier morphology-based analyses of T-cell-containing cuffs in GBM

tissues. These cuffs were predominantly found in long-term surviving GBM patients (43) and

were associated with an improved patient outcome (24, 44). Further support comes from a

very recent publication describing higher numbers of infiltrating CD8+ T-cells occurring in

GBM patients with a prolonged survival (31).

Regulatory T-cells represented less than one percent of the T-cells identified in our GBM

samples in contrast to a recent publication reporting a Treg content of 14% on total CD4+

T-cell population as analyzed by FACS on glioma single cell suspensions (30). To exclude

that this low Treg content is not due to low sensitivity of our staining procedure, human tonsil

served as internal positive control in all staining series confirming that observed frequencies

of Foxp3+ cells were in line with the current literature (36). To further investigate whether

these contrasting results might be caused by the use of different methods, we repeated their

FACS experiment on four WHO-grade III–IV single cell suspensions. We found Foxp3+ cells

represented 10.5%±5.8 of the total CD4+ population (see Supp. Fig. 2B), which was much

higher than we observed by TissueFAXS. This supports the idea that the cause of these

different results might be technical in nature. In the approach used by Jacobs et al. – the

analysis of suspensions – cells derived from peritumoral areas cannot be excluded. This is a

likely and important point because for other tumor entities like metastatic melanoma (45) or

hepatocellular carcinoma (46), it has been shown that T-cell levels are greater peritumorally

than intratumorally. Further, we have made the same observation in large paraffin sections

containing invasion zone/tumor border in addition to the tumor center (N. Höll and C. Herold-

Mende, unpublished).

Although we did not find Treg-cells to influence the survival of GBM patients, in animal

models Treg-cell depletion has been associated with enhanced immune responses and

improved survival (12, 47, 48). Therefore, in an immunotherapeutic setting it might also be

important to control immune-inhibitory T-cell subsets. Nevertheless, our data supports the




16

hypothesis that to improve survival of GBM patients, it would be beneficial to increase

infiltration of Teff-cells.

T-cell infiltration depends on the expression of several adhesion molecules on the endothelial

cell surface, including ICAM-1 and VCAM-1 (21, 22). Blocking these molecules can

substantially decrease adhesion and transmigration of T-cells (22). Indeed, our pretreatment of

GBM-derived endothelial cells with neutralizing antibodies against both CAMs reduced

transmigration, providing evidence of their functional relevance to T-cell infiltration.

However, that adhesion molecules were only found to be expressed on ~20-30% of GBM-

derived endothelial cells and with a large intertumoral heterogeneity points to the need to

learn more about their regulation in tumor-derived vessels.

We hypothesized that the low frequency of CAM-expressing endothelial cells in GBM tissue

might be due to the presence of tumor-secreted cytokines that are known to downregulate

these adhesion molecules during neoangiogenesis (13-19). Quantification of nine well-known

angiogenic cytokines in our study sample revealed that five of them were constitutively

present in glioma lysates; these included: VEGF, HGF, bFGF, TGF-ß1 and TGF-ß2.

Furthermore, except for bFGF, the cytokines were detected in highest amounts in GBM

tissues. It has been reported that VEGF and bFGF can downregulate ICAM-1 and VCAM-1 in

extracranial tumors (16, 17). However, in GBM-derived endothelial cells, we found that only

TGF-ß1 and TGF-ß2 resulted in a strong and reliable decrease of CAM expression. This

suggests that glioma-derived blood vessels regulate CAM expression by a mechanism distinct

from the endothelium in other organs.

In glioma, TGF-ßs have a multifaceted role in tumor promotion. They function as

proliferative, pro-angiogenic and pro-invasive factors by inducing the expression of VEGF,

matrix metalloproteinases and integrins, especially in high-grade gliomas (49). TGF-ßs also

stifle the host immune response to glioma, for example, by inhibiting Teff-cell proliferation

and inducing their apoptosis, and by facilitating the transition of naive T-cells into

immunosuppressive Treg-cells (9-11). Thus, blocking TGF-ß signaling would appear to be a

useful strategy in glioma treatment. In animal models of glioma, treatment with the TGF-ß

receptor small molecule inhibitor, SD-208, caused increased animal survival and increased

immune cell infiltration (37, 50). Consistent with these results, we found that treating glioma-

derived endothelial cells with SD-208 significantly enhanced the transmigration of autologous

T-cells. Thus, collectively, our data suggests an additional mechanism by which TGF-ß

asserts its immunosuppressive effect in glioma: the downregulation of CAM expression on




17

tumor-supplying endothelium which thereby hinders T-cells from overcoming the vessel

barrier.

There are immediate applications of our discovery that Teff-cell infiltration positively affects

GBM patient survival and that the glioma cytokine TGF-ß is an important negative regulator

of T-cell transmigration through GBM-derived endothelium. For example, as a prognostic

marker for patient outcome, Teff-cell infiltration might serve as a tool to monitor the responses

of patients to immunotherapy treatment. Our results also predict that the effectiveness of

glioma immunotherapy could be improved by simultaneously inhibiting TGF-ß signaling

within tumors.




18

References

1. Ohgaki H, Dessen P, Jourde B, Horstmann S, Nishikawa T, Di Patre PL, et al. Genetic pathways to glioblastoma: a population-based study. Cancer Res 2004;64:6892-9.

2. Lamszus K, Kunkel P, Westphal M. Invasion as limitation to anti-angiogenic glioma therapy. Acta Neurochir Suppl 2003;88:169-77.

3. Cabarrocas J, Bauer J, Piaggio E, Liblau R, Lassmann H. Effective and selective immune surveillance of the brain by MHC class I-restricted cytotoxic T lymphocytes.

Eur J Immunol 2003;33(5):1174-82. 4. Grauer OM, Wesseling P, Adema GJ. Immunotherapy of diffuse glioma: biological

background, current status and future developments. Brain Pathol. 2009;19:674-93. 5. Walker PK, Calzascia T, Dietrich PY. All in the head: obstacles for immune rejection

of brain tumors. Immunology 2002;107:28-38. 6. Engelhardt B, Ransohoff RM. The ins and outs of T-lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms. Trends Immunol 2005;26:485-95. 7. Davies DC. Blood-brain barrier breakdown in septic encephalopathy and brain

tumours. J Anat 2002;200:639-46. 8. Rascher G, Fischmann A, Kröger S, Duffner F, Grote EH, Wolburg H. Extracellular

matrix and the blood-brain-barrier in glioblastoma multiforme: spatial segregation of tenascin and agrin. Acta Neuropathol 2002;104:85-91

9. Prins RM, Liau LM. Cellular immunity and immunotherapy of brain tumors. Front Biosc 2004;9:3124-36.

10. Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA. Transforming growth factor-beta regulation of immune responses. Annu Rev Immunol. 2006;24:99-146.

11. Fantini MC, Becker C, Montelione G, Pallone F, Galle PR, Neurath NL. Cutting edge: TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7. J Immunol 2004;172:5149-53.

12. Fecci PE, Mitchell DA, Whitesides JF, Xie W, Friedman AH, Archer GE, et al. Increased regulatory T-cell fraction amidst a diminished CD4 compartment explains immune defects in patients with malignant glioma. Cancer Res 2006;66:3294-3302.

13. Tate MC, Aghi MK. Biology of angiogenesis and invasion in glioma. Neurotherapeutics 2009;6:447-57.

14. Dirkx AE, Oude Egbrink MG, Kuijpers MJ, van der Niet ST, Heijnen VV, Bouma-ter Steege JC, et al. Tumor angiogenesis modulates leukocyte-vessel wall interactions in vivo by reducing endothelial adhesion molecule expression. Cancer Res 2003;63:2322-39.

15. Tromp SC, oude Egbrink MG, Dings RP, van Velzen S, Slaaf DW, Hillen HF, et al. Tumor angiogenesis factors reduce leukocyte adhesion in vivo. Int Immunol 2000;12(5):671-6.

16. Griffioen AW, Damen CA, Martinotti S, Blijham GH, Groenewegen G. Endothelial intercellular adhesion molecule-1 expression is suppressed in human malignancies: the role of angiogenic growth factors. Cancer Res 1996;56:1111-7.

17. Griffioen AW, Damen CA, Blijham GH, Groenewegen G. Tumor angiogenesis is accompanied by a decreased inflammatory response of tumor-associated endothelium. Blood 1996;88:667-73.

18. Bouma-ter Steege J, Baeten C, Thijssen V, Satijn SA, Verhoeven IC, Hillen HF, et al. Angiogenic profile of breast carcinoma determines leukocyte infiltration. Clin Cancer Res 2004;10:7171-8.

19. Kitayama J, Nagawa H, Yasuhara H, Tsuno N, Kimura W, Shibata Y, et al. Suppressive effect of basic fibroblast growth factor on transendothelial emigration of CD4+ T-lymphocyte. Cancer Res 1994;54:4729-33.




19

20. Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 1994;76:301-14.

21. Lyck R, Reiss Y, Gerwin N, Greenwood J, Adamson P, Engelhardt B. T-cell interaction with ICAM-1/ICAM-2 double-deficient brain endothelium in vitro: the cytoplasmic tail of endothelial ICAM-1 is necessary for transendothelial migration of T cells. Blood 2003;102:3675-83.

22. Mazzolini G, Narvaiza I, Bustos M, Duarte M, Tirapu I, Bilbao R, et al. ανβ3 Integrin-mediated adenoviral transfer of interleukin-12 at the periphery of hepatic colon cancer metastases induces VCAM-1 expression and T-cell recruitment. Mol Ther 2001;3:665-72.

23. Ueda R, Low KL, Zhu X, Fujita M, Sasaki K, Whiteside TL, et al. Spontaneous immune responses against glioma-associated antigens in a long term survivor with malignant glioma. J Transl Med 2007;5:68.

24. Palma L, Di Lorenzo N, Guidetti B. Lymphocytic infiltrates in primary glioblastomas and recidivous gliomas. Incidence, fate, and relevance to prognosis in 228 operated cases. J Neurosurg. 1978;49:854-61.

25. Brooks WH, Markesbery WR, Gupta GD, Roszman TL. Relationship of lymphocyte invasion and survival of brain tumor patients. Ann. Neurol. 1978;4:219-24.

26. Safdari H, Hochberg FH, Richardson EP Jr. Prognostic value of round cell (lymphocyte) infiltration in malignant gliomas. Surg Neurol 1985;23:221-6.

27. Schiffer D, Cavicchioli D, Giordana MT, Palmucci L, Piazza A. Analysis of some factors effecting survival in malignant gliomas. Tumori 1979;65:119-25.

28. Mittelbronn M, Simon P, Löffler C, Capper D, Bunz B. Elevated HLA-E levels in human glioblastomas but not in grade I to III astrocytomas correlate with infiltrating CD8+ cells. J Neuroimmunol 2007;189:50-8.

29. Heimberger AB, Abou-Ghazal M, Reina-Ortiz C, Yang DS, Sun W. Incidence and prognostic impact of FoxP3+ regulatory T cells in human gliomas. Clin Cancer Res 2008;14:5166-72.

30. Jacobs JF, Idema AJ, Bol KF, Grotenhuis JA, de Vries IJ, Wesseling P, et al. Prognostic significance and mechanism of Treg infiltration in human brain tumors. J Neuroimmunol. 2010;225:195-9.

31. Yang I, Tihan T, Han SJ, Wrensch MR, Wiencke J, Sughrue ME, et al. CD8+ T-cell infiltrate in newly diagnosed glioblastoma is associated with long-term survival. J Clin Neurosci 2010;17:1381-85.

32. Maurer MH, Thomas C, Bürgers HF, Kuschinsky W. Transplantation of adult neural progenitor cells transfected with vascular endothelial growth factor rescues grafted cells in the rat brain. Int J Bio Sci 2007;4:1-7.

33. Karcher S, Steiner HH, Ahmadi R, Zoubaa S, Vasvari G, Bauer H, et al. Different angiogenic phenotypes in primary and secondary glioblastomas. Int J Cancer 2006;118:2182-9.

34. Wang RF. CD8+ regulatory T cells, their suppressive mechanisms, and regulation in cancer. Hum Immunol 2008;69:811-14.

35. Ebert AM, Tan BS, Browning J, Svobodova S, Russell SE, Kirkpatrick N, et al. The regulatory T cell-associated transcription factor FoxP3 is expressed by tumor cells. Cancer Res 2008;68:3001-9.

36. Siegmund S, Rückert B, Ouaked N, Bürgler S, Speiser A, Akdis CA, et al. Unique phenotype of human tonsillar and in vitro-induced Foxp3+CD8+ T cells. J Immunol 2009;182:2124-30.

37. Uhl M, Aulwurm S, Wischhusen J, Weiler M, Ma JY, Almirez R, et al. SD-208, a novel transforming growth factor beta receptor I kinase inhibitor, inhibits growth and




20

invasiveness and enhances immunogenicity of murine and human glioma cells in vitro and in vivo. Cancer Res 2004;64:7954-61.

38. Stevens A, Klöter I, Roggendorf W. Inflammatory infiltrates and natural killer cell presence in human brain tumors. Cancer 1988;61:738-43.

39. Rossi ML, Jones NR, Candy E, Nicoll JA, Compton JS, Hughes JT, et al. The mononuclear cell infiltrate compared with survival in high-grade astrocytomas. Acta Neuropath 1989;78:189-93.

40. El Andaloussi A, Lesniak MS. CD4+ CD25+ FoxP3+ T-cell infiltration and heme oxygenase-1 expression correlate with tumor grade in human gliomas. J Neurooncol 2007;83:145-52.

41. Pages F, Galon J, Dieu-Nosjean MC, Tartour E, Sautès-Fridman C, Fridman WH. Immune infiltration in human tumors: a prognostic factor that should not be ignored. Oncogene 2010;29:1093-102.

42. Senger DR, Van De Water L, Brown LF, Nagy JA, Yeo KT, Yeo TK, et al. Vascular permeability factor (VPF, VEGF) in tumor biology. Cancer Met Res 1993;12:303-24.

43. Di Lorenzo N, Palma L, Nicole S. Lymphocytic infiltration in long-survival glioblastomas: possible host's resistance. Acta Neurochir (Wien) 1977;39:27-33.

44. Böker DK, Kalff R, Gullotta F, Weekes-Seifert S, Möhrer U. Mononuclear infiltrates in human intracranial tumors as a prognostic factor. Influence of preoperative steroid treatment. I. Glioblastoma. Clin Neuropathol 1984;3:143-7.

45. Ahmadzadeh M, Johnson LA, Heemskerk B, Wunderlich JR, Dudley ME, White DE, et al. Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. Blood 2009;114:1537-44.

46. Kasper HU, Drebber U, Stippel DL, Dienes HP, Gillessen A. Liver tumor infiltrating lymphocytes: comparison of hepatocellular and cholangiolar carcinoma. World J Gastroenterol. 2009;15:5053-7.

47. Grauer OM, Nierkens S, Bennink E, Toonen LW, Boon L, Wesseling P, et al. CD4+FoxP3+ regulatory T cells gradually accumulate in gliomas during tumor growth and efficiently suppress antiglioma immune responses in vivo. Int J Cancer 2007;121:95-105.

48. El Andaloussi A, Lesniak MS. An increase in CD4+CD25+FOXP3+ regulatory T cells in tumor-infiltrating lymphocytes of human glioblastoma multiforme. J Neurooncol 2006;8:234-43.

49. Platten M, Wick W, Weller M. Malignant glioma biology: role for TGF-beta in growth, motility, angiogenesis, and immune escape. Microsc Res Tech 2001;52:401-10.

50. Tran TT, Uhl M, Ma JY, Janssen L, Sriram V, Aulwurm S, et al. Inhibiting TGF-ß signaling restores immune surveillance in the SMA-560 glioma model. Neuro-Oncol 2007;9:259-70.




21

Acknowledgements

The authors would like to thank R. Warta and B. Campos for critically reading the manuscript

as well as H. Discher, M. Greibich, F. Kashfi, I. Hearn and D. Zito for excellent technical

assistance.




22

FIGURE LEGENDS

Figure 1: Glioma T-cell infiltration increased with malignancy

(A) Representative epifluorescence images and overlay of pGBM tissue stained for CD3 (red)

and CD8 (green) and DAPI (blue). Arrows identify cytotoxic T-cells (CD3+CD8+). Scale bar:

100µm.

(B) Computational quantification of T-cell infiltration among gliomas of different WHO

grade (n=93, Study Sample A). Data are expressed as mean ±SEM. Asterisks label significant

differences (**p<0.01; *p<0.05).

Figure 2: Effector T-cells, but not immune-inhibitory T-cells, were associated with

patient survival.

(A) Quantification of infiltration by immune-inhibitory (Foxp3+) and effector (Foxp3-) T-cells

among gliomas of different WHO grade (n=93, Study Sample A). Asterisks label significant

differences (**p<0.01; *p<0.05) (upper row). CD8- and CD8+ regulatory T-cells were

detected infrequently and not at significantly different levels between tumor grades (lower

row).

(B) Comparison of infiltration by different T-cell populations with GBM patient survival

(n=44).

Figure 3: VCAM-1 and ICAM-1 were expressed in similar numbers of GBM tumor-

supplying endothelial cells.

(A) Representative epifluorescence images and overlays of GBM patient tissue stained with

for ICAM-1 (green in left column), VCAM-1 (green in right column) and endothelial cell

marker CD31 (red) and DAPI (blue). Scale bars: 50µm.

(B) Quantification of endothelial cells from GBM tissue expressing ICAM-1 and VCAM-1

(n=65, GBM subset of Study Sample A).

Figure 4: In GBM, effector T-cells positively correlated with endothelial cells expressing

ICAM-1

Spearman rank correlation coefficient was determined for the respective types of T-cells and

ICAM-1-expressing endothelial cells (n=65, GBM subset of Study Sample A).

Significant correlation was found between T-cells and endothelia expressing ICAM-1 with the

exception of immune-inhibitory T-cells.




23

Figure 5: Angiogenic growth factor concentrations generally increase with glioma

tumor grade.

A significant WHO grade-dependent concentration increase was found for VEGF, HGF,

TGF-ß1, TGF-ß2, PDGF-AB, PDGF-BB, G-CSF and GM-CSF. Values are mean

concentrations of growth factors in tumor lysates of gliomas of different WHO grade (n=52).

Asterisks depict significant differences (p<0.05) between grades. t.p. = total protein.

Figure 6: TGF-ß1 and TGF-ß2 negatively regulate CAM protein expression in GBM-

derived endothelial cells and transmigration of Teff-cells in an autologous functional

assay.

(A) Flow cytometric quantification of ICAM-1 and VCAM-1 expression in GBM-derived

endothelial cells after 72h incubation with 100ng/ml HGF, VEGF and bFGF, respectively, or

10ng/ml TGF-ß1 and TGF-ß2, respectively. Asterisks indicate significant inhibition of

ICAM-1 and VCAM-1 protein expression (p<0.05).

(B) Comparison of effector (Teff) and inhibitory (Treg) T-cell transmigration through

autologous, GBM-derived endothelial cells (n=4).

(C) Mean ratios of transmigrated Teff-cells in the presence of blocking antibodies to ICAM-1,

VCAM-1 or both.

(D) Transmigration of Teff-cells in the presence of 100ng/ml HGF, VEGF and bFGF,

respectively, or 10ng/ml TGF-ß1 and TGF-ß2, respectively.

(E) Effect of SD-208 (TGF-ßRI kinase inhibitor, 1µM) on transmigration of Teff-cells in the

presence of autologous GBM lysate (50µg/ml).

Data in graphs C, D and E present mean ratios of transmigrated T-cells to total number of

added T-cells ± SEM (n=6); asterisks indicate significant differences (p<0.05).




Published OnlineFirst April 8, 2011.Clin Cancer Res Jennifer Lohr, Thomas Ratliff, Andrea Huppertz, et al. transforming growth factor-betasglioblastoma patients and is impaired by tumor-derived Effector T-cell infiltration positively impacts survival of

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