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Year: 2009
Site-specific anti-tumor immunity: differences in DC function,
TGF-beta production and numbers of intratumoral Foxp3+ Treg
Biollaz, G; Bernasconi, L; Cretton, C; Püntener, U; Frei, K; Fontana, A; Suter, T
Biollaz, G; Bernasconi, L; Cretton, C; Püntener, U; Frei, K; Fontana, A; Suter, T (2009). Site-specific anti-tumor
immunity: differences in DC function, TGF-beta production and numbers of intratumoral Foxp3+ Treg. European
Journal of Immunology, 39(5):1323-1333.
Postprint available at:
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Posted at the Zurich Open Repository and Archive, University of Zurich.
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Originally published at:
European Journal of Immunology 2009, 39(5):1323-1333.
Site-specific anti-tumor immunity: differences in DC function,
TGF-beta production and numbers of intratumoral Foxp3+ Treg
Abstract
Gliomas localized within the CNS are generally not rejected by the immune system despite being
immunogenic. This failure of the immune system has been associated both with glioma-derived
immunosuppressive molecules and the immune-privileged state of the CNS. However, the relative
contribution of tumor location to the glioma-mediated immunosuppression, as well as the immune
mechanisms involved in the failure of glioma rejection are not fully defined. We report here that
syngeneic GL261 gliomas growing either intracranially or subcutaneously in mice are infiltrated by DC
and T cells. However, only subcutaneous gliomas elicit an effective anti-tumor immune response. In
contrast to DC infiltrating subcutaneously grown GL261 gliomas, tumor-infiltrating DC from
intracranial gliomas do not activate antigen-dependent T-cell proliferation in vitro. In addition,
brain-localized GL261 gliomas are characterized by significantly higher numbers of Foxp3(+) Treg and
higher levels of TGF-beta1 mRNA and protein expression when compared with GL261 gliomas in the
skin. Our data show that gliomas in the CNS, but not in the skin, give rise to TGF-beta production and
accumulation of both Treg and functionally impaired DC. Thus, not the tumor itself, but its location
dictates the efficiency of the anti-tumor immune response.
Site-specific anti-tumor immunity: differences in DC
function, TGF- production and numbers of intratumoral
Foxp3+ regulatory T cells
Gregoire Biollaz1, Luca Bernasconi1, Christine Cretton1, Ursula Püntener1, Karl
Frei2, Adriano Fontana1,* and Tobias Suter1,*
1
Division of Clinical Immunology, University Hospital of Zurich, Haeldeliweg 4,
CH-8044 Zurich
2
Department of Neurosurgery, University Hospital of Zurich, Frauenklinikstr. 10,
CH-8091 Zurich
Keywords: tumor immunity, dendritic cell, regulatory T cells, immune privilege
Corresponding author: Tobias Suter, Division of Clinical Immunology, University
Hospital, Moussonstrasse 13, CH-8044 Zurich, Switzerland
Phone: +41 44 6343815; Fax: +41 44 6342902; email: [email protected]
Abbreviations used in this paper: GBM: glioblastoma multiforme; TIDC: tumor
infiltrating dendritic cell; TIL: tumor infiltrating lymphocyte.
*
These authors have contributed equally to this work as last authors.
1
Summary
Gliomas localized within the CNS are generally not rejected by the
immune system despite being immunogenic. This failure of the immune system
has been associated both with glioma-derived immunosuppressive molecules and
the immune privileged state of the CNS. However, the relative contribution of
tumor localization to the glioma-mediated immunosuppression and the immune
mechanisms involved in the failure in glioma rejection are not fully defined. We
report here that syngeneic GL261 gliomas growing either intracerebrally or
subcutaneously in mice both are infiltrated by dendritic cells and T cells.
However, only subcutaneous gliomas elicit an effective anti-tumor immune
response. In contrast to dendritic cells infiltrating subcutaneously grown GL261
gliomas, tumor infiltrating dendritic cells from intracerebral gliomas do not
activate antigen-dependent T cell proliferation in vitro. In addition, brainlocalized GL261 gliomas are characterized by significantly higher numbers of
Foxp3+ regulatory T cells and higher levels of TGF-1 mRNA and protein
expression when compared to GL261 gliomas in the skin. Our data show that
gliomas in the CNS, but not in the skin, give rise to TGF- production and
accumulation of regulatory T cells and functionally impaired dendritic cells. Thus,
not the tumor but its localization dictates the efficiency of the anti-tumor immune
response.
2
Introduction
Malignant gliomas, especially their most frequent and aggressive form, the
glioblastoma multiforme (GBM), are among the most fatal tumor species. The
best standard of care for GBM, consisting in surgery followed by radiotherapy
and chemotherapy with temozolomide, is associated with a median overall
survival of 14.6 months following diagnosis [1]. Gliomas were shown to lead to
reduced peripheral T cell responses [2], to down modulate functions of circulating
APCs [3] and particularly to suppress the maturation of peripheral DCs [4]. In
addition, the localization within the immune privileged CNS combined with
tumor-derived immunosuppressive molecules including TGF- [5-7], IL-10 [8],
VEGF [9] and prostaglandins [10] are suggested to account for the absence of a
successful anti-tumor immune response. As a consequence, glioma patients fail to
generate an effective immune response against the intracranially growing tumors.
Although rarely, gliomas metastasize outside of the brain [11]. The low frequency
of peripheral glioma metastases might be due to an efficient recognition of tumor
cells outside of the CNS. Gliomas express tumor-associated antigens such as
HER-2, gp100, and MAGE-1, which can be recognized by cytotoxic T
lymphocyte clones [12]. Furthermore, T cell clonal expansion was detected in
glioma patients [13] and vaccination of patients with autologous tumor lysatepulsed DCs or autologous tumor peptide-pulsed DCs [14] elicited tumor-specific
T cell responses and intratumoral cytotoxic and memory T cell infiltration.
Similarly, using the murine glioma model GL261, vaccination with DCs loaded
3
with tumor RNA [15] or tumor extract [16] prolonged the survival of mice
bearing intracranial tumors. However, to which extent the localization of the
tumor modulates the glioma-mediated immunosuppression is not known.
Likewise, the precise mechanisms accounting for the failure of the immune
system in rejection of intracranial glioma are not fully defined.
DCs are highly effective APCs which have the ability to initiate primary
immune responses but also to induce T cell tolerance [17]. Their role in the
immune response against tumors is not yet fully understood. Although infiltration
by DCs has been reported for different tumor types, no absolute correlation
between the presence of tumor infiltrating DCs (TIDCs) and the outcome for the
patients can be found. Indeed, DC infiltration of lung tumors, gastric cancer,
melanoma or papillary thyroid carcinoma [18] is associated with a positive
outcome. In contrast, DC infiltration does not influence survival in bronchioalveolar carcinoma [19]. Furthermore, despite the presence of TIDCs, immunemediated tumor rejection is only seldom successful.
Regulatory T cells (Tregs) represent a subset of peripheral CD4+ cells,
which mitigate the immune response. Tregs play a key role in prevention of T cell
responses to self-antigens. However, tumors can take advantage of Tregs by
attracting them and manipulating Treg-mediated immunosuppression in order to
escape recognition and elimination by the host [20]. Their role in tumor
progression is highlighted by the observation that systemic depletion of Tregs
results in reduced tumor growth and increased survival in several models [21].
While Tregs were recently found within gliomas [22-24], to the best of our
4
knowledge there is no report on TIDCs in gliomas and their function during tumor
progression.
We report here for the first time on phenotypical and functional
characterization of TIDCs in experimental gliomas. We found the function of
TIDCs to depend on the site of tumor growth. GL261 gliomas grown in the skin
express low levels of TGF-, harbor DCs which activate T cells, and induce a
productive anti-tumor immune response. In contrast, intracranial GL261 tumor
tissue expresses higher levels of TGF-, accumulates Tregs, and contains DCs
which are unable to stimulate T cells but promote the development of Tregs.
These findings suggest that gliomas do not suppress per se immune surveillance,
but do so only when growing in their natural microenvironment.
Results
Tumor localization influences the anti-glioma immune response
We first sought to determine whether the growth of a glioma outside of the
immune privileged CNS would elicit an immune response different to the one
against an intracranial glioma. To this end, wild-type (C57BL/6) and Rag1deficient (Rag1-/-) mice, which are devoid of both T and B lymphocytes, were
inoculated with GL261 cells either intracranially or subcutaneously, and tumor
appearance was monitored daily. Kaplan-Meier survival analysis of animals
intracranially injected with GL261 glioma cells revealed similar growth rates in
wild-type and Rag1-/- mice (Fig. 1A). In contrast, peripheral glioma cell
5
inoculation resulted in a significantly delayed tumor development in wild-type
mice compared to Rag1-/- mice (Fig. 1B; P = 0.0074). Thus, a glioma grown in the
periphery of an immunocompetent mouse induces a response leading to reduced
tumor growth, while intracranial growth is not retarded in wild-type animals
compared to Rag1-/-.
In contrast to TIDCs, tumor infiltration by T cells decreases with time in
intracranial gliomas, but not in gliomas in the skin
Since the anti-tumor response relies on accessibility of immune cells to the
tumor, we investigated whether the difference in tumor immune surveillance was
due to differences in the number of DCs and T cells seen in intracranial gliomas
and skin gliomas. Brain and subcutaneous tumors were isolated at various time
points and sections were stained for T cell and DC markers. CD4+ and CD8+ T
cells as well as CD11c+ cells with typical DC morphology were found to infiltrate
intracranial gliomas at day 9 post inoculation (data not shown). The numbers of
CD11c+ TIDCs further increased until peak clinical stage, which was reached
after 4 weeks of tumor growth (Fig. 2A). DCs were only found within the tumor
but not in the surrounding healthy brain parenchyma (data not shown). Only low
numbers of CD4+ and CD8+ T cells could be identified 2 weeks post injection
(Fig. 2A). Contrary to DC infiltration, intracranial tumor infiltration by T cells
decreased further over time (Fig. 2A and C). In the subcutaneously grown GL261
tumors, DCs were present 2 weeks after inoculation as well as after 4 weeks of
6
tumor growth, representing the time point when mice with intracranial tumors had
to be sacrificed due to neurological symptoms and weight loss (Fig. 2B). Both
CD4+ and CD8+ T cells were detected in subcutaneous tumors 2 weeks after
inoculation (Fig. 2B). In contrast to the dynamics of tumor infiltrating
lymphocytes (TILs) in intracranial tumors, the numbers of TILs in peripheral
tumors were not decreased after 4 weeks of tumor growth (Fig. 2B and C).
Interestingly, infiltration by DCs in both locations does not require DC-T cell
interaction, since TIDCs were also found in Rag1-/- mice (data not shown). Thus,
immune cells were able to reach both tumor sites. However, differences in T cell
persistence might explain the differences in tumor growth. Hence, we addressed
whether differences in TIDC phenotype and function were responsible for the
disappearance of T cells in the brain tumor.
TIDCs from both intracranial and subcutaneous gliomas display an
immature myeloid phenotype
Tumors have been described to interfere with DC maturation and
upregulation of proteins involved in antigen presentation and T cell stimulation
[25]. Thus, we decided to quantify the expression of MHC class II as well as B7
and CD40 costimulatory molecules and the co-inhibitory molecules PD-1, PD-L1
and PD-L2 on TIDCs isolated from gliomas which grew in the skin and the brain.
We found TIDCs isolated from intracranial tumors to express intermediate levels
of MHC class II molecules, but only low levels of B7.1, and almost no B7.2 and
7
CD40 costimulatory molecules could be detected (Fig. 3A). This expression
pattern suggests an immature phenotype as seen with in vitro generated immature
bone marrow-derived DCs (BM-DCs) (Fig. 3A and Table I). BM-DCs matured
with LPS expressed by far higher levels of MHC class II, B7-1, B7-2 and CD40
(Fig. 3A). No differences between intracranial and subcutaneous TIDCs were
found in the expression of the co-inhibitory molecules PD-1, PD-L1 and PD-L2
(supplementary figure 1). TIDCs isolated from gliomas in the skin show an
immature phenotype resembling TIDCs in intracranial gliomas and immature
BM-DC (Fig. 3A and Table I). The weak expression of B7.1 and the absence of
B7.2 was confirmed by immunohistochemistry performed on frozen brain sections
(data not shown), indicating that the phenotype of the TIDCs was not influenced
by the isolation method.
We also did not find phenotypic differences between intracranial and
subcutaneous TIDCs when analyzing other characteristic DC surface molecules
(Fig. 3B). MHC class I was expressed to a similar extent by both populations of
infiltrating DCs. The finding that both DC types stained positively for CD11b and
negatively for CD8 (Fig. 3B) strongly suggests that they are of myeloid origin.
Notably, negative CD4 as well as CD19 stainings ensured that no T cells or B
cells contaminated the DC populations. Furthermore, plasmacytoid DCs and
myeloid suppressor cells would potentially make up only a marginal fraction
among the TIDCs, since staining for Gr-1 and B220 were low-negative
(supplementary figure 2).
8
It has been shown that DCs recruited to the CNS during autoimmune or
infectious diseases may originate from microglia cells [26, 27]. In order to address
whether intracranial TIDCs originate from CNS-resident precursors and thus
would differ from subcutaneous TIDCs by their origin, intracranial and
subcutaneous TIDCs in CD45-congenic bone marrow chimeric mice were studied.
These experiments clearly show that virtually all TIDCs (>95%) in CNS gliomas
are blood-derived and thus do not originate from microglia (Fig. 3C). Moreover,
the data suggests that at both tumor sites, TIDCs originate from the same bloodderived precursors.
The capacity of infiltrating DCs to stimulate T cells depends on the site of
tumor growth
As we found no differences between intracranial and subcutaneous TIDCs
with respect to the expression of population and activation markers, we aimed at
characterizing the functional properties of these cells. First, we examined the T
cell priming capacities of TIDCs in an allogeneic proliferation assay. In vitro
generated LPS-matured BM-DCs and DCs isolated from subcutaneous tumors
were able to induce a strong proliferative response of allogeneic CD4+ T cells
(Fig. 4A). In contrast, intracranial TIDCs failed to induce allogeneic T cell
proliferation, even at high APC:T ratio (Fig. 4A). Since TIDCs were reported to
exert immunosuppressive functions on T cells [25, 28], we tested the suppressive
potential of purified TIDCs in an inhibition assay (Fig. 4B). Constant numbers of
9
mature BM-DCs and allogeneic CD4+ T cells were cultured at a ratio of 1:10
(base line stimulation index set to 100%). To evaluate the respective potential of
each type of TIDC to enhance or inhibit the reference T cell proliferation induced
by the mature BM-DCs, increasing numbers of TIDCs or mature BM-DCs (as
control cells) were added. As expected from the proliferation assay, subcutaneous
TIDCs as well as control mature BM-DCs further increased T cell proliferation
compared to base line (Fig. 4B). Intracranial TIDCs on the other hand did neither
increase nor inhibit base line proliferation (Fig. 4B). Thus, intracranial TIDCs fail
to act as stimulators in an allogeneic T cell proliferation system and do not
suppress mature BM-DC induced proliferation. We then confirmed the difference
in TIDC function in an antigen-specific system. We found ovalbumin-specific
TCR-transgenic OT-II responder T cells to proliferate in the presence of
subcutaneous TIDCs with either the OVA323-339 peptide (Fig. 4C) or whole OVA
protein (Fig. 4D) as antigen, while they were not responding in the presence of
intracranial TIDCs. Taken together, in contrast to subcutaneous TIDCs,
intracranial TIDCs are not able to induce allogeneic or syngeneic T cell
proliferation. Thus, despite no differences in surface marker expression, TIDCs
isolated from the same tumor grown at different sites exhibit opposite stimulatory
capacities.
Tumor infiltrating T cells in intracerebral and subcutaneous gliomas display
no differences in activation markers
10
In order to determine whether the differences observed in T cell infiltration
at peak clinical stage were paralleled by changes in the activation state of the
TILs, we characterized the phenotype of TILs for both tumor locations. CD4- and
CD8-positive TILs were stained for L-selectin (CD62L), the very early activation
antigen CD69, the adhesion receptor CD44 and the IL-2 receptor alpha chain
CD25 (Fig. 5 and Table I). Most of the TILs had downregulated CD62L, except
for a minority of the CD8+ T cells. CD69+ cells were detected among the CD4+ as
well as among the CD8+ TILs from both locations to similar extent. All CD4+ and
CD8+ T cells expressed CD44, an indication of the presence of effector-memory
(both activated and long-lived) T cells in both tumor locations. For all markers
analyzed, no statistically significant differences were found. Strikingly, while only
few CD8+ T cells expressed CD25 (10%), more than 40% of the CD4+ TILs did,
and this regardless of tumor localization. This suggests that the CD4+ TILs
contain a population of either activated effector or regulatory T cells. To find out
whether, in parallel to DCs, tumor infiltrating T cells also showed a functional
difference when comparing both tumor locations, we monitored the capacity of
isolated tumor infiltrating T cells to produce IFN- upon restimulation with PMA
and ionomycin. Intracellular cytokine staining showed that subcutaneous tumor
infiltrating T cells secrete higher amounts of IFN- compared to intracranial tumor
infiltrating T cells, albeit not reaching statistical significance (data not shown).
Furthermore, we found no difference in the expression of FasL by CD8+ tumor
infiltrating T cells (data not shown).
11
Tregs and the expression of TGF- are prominent in intracerebral gliomas,
but not in subcutaneous gliomas
Since our flow cytometric analysis of TILs showed a large population of
CD4 positive T cells expressing CD25 (Fig. 5A), we aimed at defining the
proportion of Tregs in each tumor type. Staining for the transcription factor
Foxp3, a specific marker of Tregs in mice, revealed that intracranial TILs
contained a significantly higher proportion of Tregs than the TILs of
subcutaneous tumors (Fig. 6A and B). This difference was only seen for TILs and
not for peripheral T cells, since similar percentages of Tregs were detected in the
blood and in the spleen of animals bearing intracranial or subcutaneous tumors.
Notably, intracranial Tregs proved to be functionally suppressive in vitro (Fig.
6D) to the same extent as splenic Tregs isolated from the same animals (Fig. 6C).
Moreover, in vivo depletion of CD25+ Tregs before intracranial tumor inoculation
significantly prolonged the mean survival (data not shown) as previously
published [22, 23].
Since CD25+Foxp3+ Tregs can be generated upon exposure of naive T
cells to TGF- and also secrete TGF- themselves to suppress T cell priming and
T cell effector mechanisms [29], we determined the expression of TGF-1 and
TGF-2 in GL261 tumors resected from the two inoculation sites. Both TGF-1
and TGF-2 isoforms were expressed at higher levels by intracranial tumors
compared to subcutaneous tumors (Fig. 6E). In contrast to TGF-, no significant
differences in the expression of IL-4, IL-6 and IL-10 were detected in gliomas of
12
the brain and the skin (Fig. 6E). Furthermore, increased levels of TGF-1 protein
were detected in intracranial tumors (Fig. 6F). Taken collectively, these data show
increased TGF- expression in GL261 tumors growing within the brain, its
natural environment, compared to peripheral tumors.
DCs from intracranial gliomas are more efficient in Treg induction than
subcutaneous TIDCs
Since intracranial TIDCs were neither able to induce T cell priming nor
inhibited T cell proliferation triggered by stimulatory DCs (Fig. 4) we assessed
whether they had the potential to induce Treg cells from naïve CD25-CD4+ T
cells. CD25-depleted naive CD4+ T cells were cultured for 5 days in presence or
absence of either intracranial or subcutaneous TIDCs. Intracellular Foxp3 staining
revealed the induction of significantly more Tregs in the presence of intracranial
TIDCs compared to subcutaneous TIDCs or no DCs (Fig. 6G).
13
Discussion
The most aggressive forms of malignant gliomas are generally associated
with a median overall survival of around one year after diagnosis. On one hand,
this has been attributed to their growth within the immune privileged CNS. On the
other hand, gliomas were shown to release immunosuppressive molecules
including TGF-. Here, we provide evidence that the fatality of gliomas does not
only reside in features of the tumor itself but in the specific cohabitance of the
glioma in its “natural” environment, the CNS. We show that the intracerebral
glioma GL261 is infiltrated by DCs which are not able to prime T cells, whereas
the same tumor growing in the skin is infiltrated by immuno-competent DCs (Fig.
2 and 4). DC infiltration has been reported in several tumor types, such as lung
tumors, colon carcinoma, gastric cancer and papillary carcinoma [18], but as seen
by the still devastating effects of cancer, their presence does not necessarily
correlate with a positive outcome for patients. Phenotypically, DCs usually found
in tumors display an immature state of activation [30, 31]. This is in line with our
findings in gliomas. Both intracranial and subcutaneous TIDCs only express
intermediate levels of MHC class II, and low levels of B7.1, and almost no B7.2
and CD40 (Fig. 3A). Moreover, when analyzing the production of IL-12p35, IL12p40, IL-23p19, IL-10, IL-1, IL-6 and TNF- by isolated TIDCs, we found no
differences between subcutaneous and intracranial TIDCs (data not shown).
However, we found that, depending on the site of tumor growth, glioma TIDCs
differ functionally. TIDCs from gliomas of the skin are able to induce T cell
14
proliferation, whereas TIDCs harvested from gliomas of the brain fail to do so
(Fig. 4). Furthermore, intracranial TIDCs induce higher numbers of Treg cells
than subcutaneous TIDCs (Fig. 6G).
To the best of our knowledge, this is the first report presenting functional
data on glioma-infiltrating DCs. The function of CNS-derived DCs has been
analyzed in autoimmune and infectious mouse models, with a variety of
phenotypes described, ranging from DCs inhibiting T cell activation [32] to DCs
contributing to disease spreading [33]. Likewise, TIDCs in non-glioma tumors
have been assigned several roles in the anti-tumor response. In hepatocarcinoma
and colon carcinoma, functional impairment of TIDCs can be reversed by IL-10
neutralization combined with CpG stimulation [34, 35]. In contrast, melanomaderived TIDCs are potent APCs and can mediate tumor rejection without prior
restimulation [36]. Our results add to the complexity by showing site-specific
differences of DC function.
The non-stimulatory but Treg-inducing DCs in the GL261 intracranial
gliomas may impair the anti-glioma T cell response and thereby tumor immune
surveillance. It is unlikely that other types of cells compensate for the lack of APC
functions of TIDCs. Glioma cells work poorly as APCs due to the low expression
of MHC molecules and the lack of costimulatory molecules needed to reactivate
the primed T cells [37]. Microglia, which have been described to function as
APCs in vitro [38], were shown to be unable to present glioma cell antigens to
cytotoxic T lymphocytes [39]. Moreover, using congenic bone marrow chimeras,
we did not see any involvement of microglia in the anti-tumor response. The
15
absence of priming functions by TIDCs in brain gliomas strongly suggests that
they are unable to reactivate glioma infiltrating lymphocytes in situ, which could
explain the reduction in T cell numbers as observed in the course of tumor
development. Indeed, the extent of the T cell activation level is pivotal for T cell
infiltration deep within the tumor tissue [40], and T cells which do not reencounter their antigen disappear from the CNS, either via migration back to the
periphery [41], or via apoptosis [42]. Interestingly, in glioblastoma patients,
invading T cells were reported to undergo apoptosis [43]. The re-activation of T
cells within the tumor is not only required for survival and maintenance of
cytotoxic activity, but also for the attraction of other effector cells such as
neutrophils, macrophages and NK cells [44]. Thus, the lack of stimulatory DCs in
intracranial gliomas is very likely to have a broad negative impact on the antitumor response.
DCs unable to prime normal T cells were shown to have the potential to
induce suppressive regulatory T cells [45]. This is in line with our detection of an
increase of Foxp3 expressing regulatory T cells within the intracranial tumor
compared to the subcutaneous tumor (Fig. 6A and 6B) and the fact that
intracranial TIDCs are superior to subcutaneous TIDCs in inducing Treg cells
without exogenous TGF- (Fig 6G). The presence of Tregs in human
glioblastomas has recently been reported [24] and the function of TIDCs in human
glioblastomas is under investigation (KF, personal communication).
Bone marrow chimera experiments allowed us to determine the origin of
the myeloid DCs detected in gliomas. In experimental infectious and autoimmune
16
diseases, subpopulations of CNS-DCs were shown to be derived from microglia
[26, 27]. This is in contrast to intracranial GL261 tumors where we found almost
all the DCs to be derived from donor bone marrow cells (Fig. 3C). Thus, the
glioma infiltrating DCs migrate from the blood into the tumor parenchyma in the
CNS and the functional differences between subcutaneous and intracranial TIDCs
cannot be attributed to different origins of the cells.
The increased levels of TGF- in intracranially grown gliomas compared
to gliomas grown in the skin could contribute to the different immune responses
observed (Fig. 6E and 6F). TGF- and TGF-have pleiotropic functions (for
review see [46]) and have been described to be very strongly expressed by glioma
cells [5-7]. The importance of TGF- in supporting glioma growth is attested by
experiments blocking TGF- production, which positively influenced the survival
of rats with established gliomas [47]. TGF- regulates T cell proliferation and T
helper cell differentiation and it interferes with the generation of cytotoxic T cells
[48], an effect which is central in tumor surveillance as CD8+ T cells are
necessary for efficient anti-tumor host responses. Moreover, TGF- converts
CD4+CD25- T cells into functional Tregs [29] and blocks the maturation of DCs,
which then display diminished T cell activation potential. 
Based on the site-specific differences observed when analyzing
experimental GL261 gliomas in the CNS and the skin, and taking into account the
present knowledge of DC function, Tregs and TGF-, our results propose that the
high levels of TGF-in intracranial gliomas lead to accumulation of both Tregs
and immature DCs having the potential to induce Tregs. This milieu prevents T
17
cell priming and restimulation and interferes with T cell effector function,
resulting in an impaired anti-tumor immune response. We suspected that the
programmed death signaling pathway involving the molecules PD-1, PD-L1 and
PD-L2 could be involved in the pronounced Treg induction in intracranial
gliomas. However, we found no differences in the expression of these molecules
on the respective DCs (supplementary figure 1). The reasons for this site-specific
anti-tumor response are still unknown, but are likely to rely on the surrounding
tissue and/or the tumor vasculature. Indeed, astrocytes, the type of glial cells
which encircle intracranial gliomas, were shown to strongly produce VEGF as
well as matrix metalloproteinases and could thus promote tumor growth and
invasion [49]. Moreover, neurons have been shown to promote Treg induction via
TGF- secretion, thus neurons could be at least in part responsible for the relative
increase of Tregs in intracranial gliomas [50]. We show here that a glioma
residing in the CNS is not ignored by the immune system, and that the immunomodulatory capacity of GL261 glioma is not purely tumor cell inherent. However,
the fatal nature of brain tumors might be due to the production of immunomodulatory factors, such as TGF-which is potentiated when the glioma grows
within the CNS. Our results suggest to consider the increased Treg infiltration,
impaired TIDC function and high TGF- production associated with intracranial
gliomas as the targets in the development of new therapies or in the improvement
of existing treatments.
Materials and Methods
18
Cell lines and mice. The GL261 glioma cell line, originally induced by
implantation of 3-methylcholanthrene pellets in the brain of a C57BL/6 mouse,
was kindly provided by Dr. Paul R. Walker (Geneva University Hospital,
Switzerland) and grown in DMEM containing 4500 mg/l D-glucose (Invitrogen,
Basel, Switzerland) supplemented with 10% FCS, 2 mM N-acetyl-L-alanyl-Lglutamine (Biochrom AG, Berlin, Germany), and 20 μg/ml gentamycin
(Invitrogen). C57BL/6 (H-2b) and BALB/c (H-2d) mice were purchased from
Harlan (Horst, The Netherlands) or RCC (Füllinsdorf, Switzerland). B6.SJLPtprca Pep3b/BoyJ and Rag1-/- (H-2b) mice were kindly provided by Prof.
Burkhard Becher (University of Zurich, Switzerland). OT-II mice (H-2b) were
kindly provided by Prof. Hans Acha-Orbea (University of Lausanne,
Switzerland). Bone marrow chimeras were generated by irradiation with 1100 rad
(split dose) and i.v. reconstitution with 5 Mio. bone marrow cells.
Reagents. Antibodies to CD4 (RM4-5), CD8α (53-6.7), CD11b (M1/70), CD25
(PC61), CD40 (3/23), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD62L (MEL14), CD69 (H1.2F3), CD80 (16-10A1), CD86 (GL1), MHC I (AF6-88.5) and
MHC II (2G9) were purchased from BD Biosciences (Allschwil, Switzerland);
anti-Foxp3 (FJK-16s) and anti-PD-1, PDL1 and PD-L2 were from e-Bioscience
(San Diego, CA); anti-CD11c (N418) was from Caltag (Burlingame, CA). 7-AAD
was from Sigma (Buchs, Switzerland). rmGM-CSF was purchased from
Immunotools (Friesoythe, Germany), OVA protein was purchased from Sigma,
and OVA323-339 peptide from NeoMPS (Strasbourg, France).
19
GL261 glioma tumor model. 5x104 GL261 cells were inoculated either in the
frontal cortex (2 mm lateral to bregma and 2 mm deep) in 10 l PBS using a 27G
syringe or in 2 ul using a Hamilton syringe or subcutaneously in the right flank of
anesthetized C57BL/6 or Rag1-/- males. Tumor appearance was checked daily by
weight measurement and assessment of neurological symptoms associated with an
intracranial tumor. Endpoint for intracranial tumor was determined by a weight
loss of more than 15% of maximal weight (= peak clinical stage reached 25-28
days after tumor inoculation). Mice injected subcutaneously were considered
tumor free as long as no tumor could be detected by palpation and had to be
sacrificed when the tumor volume was >1cm3. All animal experiments were
performed in compliance with the law and approved by the Veterinary Office of
the Canton of Zurich.
Histology. Brains and subcutaneous tumors were embedded in Jung tissue
freezing medium (Leica Instruments GmbH, Nussloch, Germany) and frozen on a
metal plate chilled by liquid nitrogen. Tissue sections of 6 m thickness were cut
in a cryostat and fixed in 2% paraformaldehyde/PBS for 5 min. Thereafter,
sections were incubated with antibodies against CD4, CD8, CD11c or the
respective isotype control for 1 hour. Binding of primary antibodies was revealed
by the following means: primary rat anti-murine CD4 and CD8 antibodies were
detected by the alkaline phosphatase anti-alkaline phosphatase (APPAP) system
(DAKO, Zug, Switzerland) and hamster anti-murine CD11c antibodies by goat
anti-hamster followed by donkey anti-goat alkaline phosphatase antibodies
20
(Jackson ImmunoResearch, West Growe, Pennsylvania). Color development was
performed with Fast Red (DAKO) and counterstaining with Mayer’s Hemalaun.
Preparation of tumor infiltrating mononuclear cells. Intracranial and
subcutaneous tumors of animals perfused with Ringer solution (Braun Medical,
Emmenbrücke, Switzerland) were minced with a scalpel blade and digested for 30
min at 37°C in HBSS containing 50 g/ml DNase I and 0.1 mg/ml
Collagenase/Dispase (both from Roche, Rotkreuz, Switzerland). The digestion
was quenched on ice after addition of 10% FCS. The digested tissue was passed
through a 100 m nylon mesh (Falcon, BD Biosciences). The suspension was
pelleted, resuspended in 30% Percoll (GE Healthcare, Uppsala, Sweden) and
underlayed with 70% Percoll in HBSS. The gradient was centrifuged at 500 g for
20 min at RT without brakes. Thereafter, if applicable, the myelin was removed
by aspiration, and the interphase and about 80% of the 30% Percoll was collected,
and cells were washed with ice cold HBSS and stained for FACS analysis or
sorting.
Flow cytometry. Tumor infiltrating mononuclear cells resuspended in PBS
containing 2% FCS, 10 mM EDTA and 0.01% NaN3 were first incubated with Fcblock (BD Biosciences) and subsequently with specific antibodies. For Foxp3
intracellular staining, mononuclear cells were enriched after the Percoll gradient
by subsequent purification over a Lympholyte M (Cedarlane Labs, Burlington,
Canada) gradient according to manufacturer’s protocol. Then the cells were
stained for CD4 and thereafter intracellularly for Foxp3 according to the
manufacturer’s instructions. All analyses were done on a CyFlow Space (Partec,
21
Münster, Germany). Post-acquisition analysis was done with WinMDI 2.8
software (Scripps-Research Institute, La Jolla, CA).
For flow cytometric sorting, tumor infiltrating mononuclear cells were stained in
sterile PBS containing 2% FCS and 10 mM EDTA. TIDCs, CD4+ CD25– T cells
and CD4+ CD25+ Tregs were sorted simultaneously on a FACSAria (BD
Biosciences). Dead cells were excluded by 7-AAD staining.
Proliferation assays. Proliferation and inhibition assays were performed as
described previously [32]. Briefly, CD4+ T cells were isolated from BALB/c or
OT-II spleens using anti-mouse CD4-biotin (BD Bioscience) and CELLection
biotin binder beads (Dynal/Invitrogen, Basel, Switzerland). For proliferation
assays, 105 T cells per well were cultured in a 96-well U-bottom plate (Falcon,
BD) in the presence of titrating numbers of various DC preparations in 200 l XVIVO 20 medium (BioWhittaker, Walkersville, MD), supplemented with 2 mM
acetyl-alanyl-glutamine, 1 × MEM nonessential amino acids (Invitrogen), 1 mM
sodium pyruvate (MP Biomedicals, Solon, Ohio), 50 mM -mercaptoethanol
(Sigma) and 25 g/ml gentamycin. Mature BM-DCs were generated by
cultivation of BM cells in GM-CSF and induction of maturation by LPS as
previously described [32]. For the inhibition assays, 105 CD4+ T cells and 104
mature BM-DCs were cultured in the presence of titrating numbers of various DC
preparations. Trypan blue staining before co-cultivation of the cells for
proliferation experiments ensured that only viable sorted cells were used. For all
assays DCs were irradiated (2000 rad) before incubation with the T cells. After 2
days, proliferation was assessed by measurement of 3H-thymidine incorporation.
22
In vitro Treg induction. Naive splenic CD4+ T cells were CD25-depleted by
MACS (Miltenyi Biotech, Bergisch Gladbach, Germany). CD4+CD25- T cells
were then cultured for 5 days in the presence or absence of flow cytometry-sorted
CD11c+ DCs from either intracranial or subcutaneous tumors at a ratio of 10:1 (T
cell: TIDC). The development of Treg cells was determined by intracellular
staining for Foxp3 and flow cytometry analysis.
Real Time PCR. Total RNA was prepared from resected intracranial or
subcutaneous tumor tissue using Trizol (Invitrogen) according to manufacturer’s
instructions. cDNA was synthesized from total RNA using random hexamers and
M-MuLV reverse transcriptase (Roche). Quantitative PCR was performed using
an ABI PRISM 7700 detection system (Applied Biosystems, Foster City, CA) and
the TaqMan Universal Master Mix (Applied Biosystems). Primer–probe sets were
purchased from Applied Biosystems. The relative levels of each RNA were
calculated by the 2-CT method, with 18s rRNA used as housekeeping gene
control. Results were normalized with respect to the internal control, and
expressed relative to the levels found in intracranial GL261.
TGF-1 ELISA. Resected tumors were homogenized in isotonic buffer. TGF-1
in the homogenates was measured by a commercial ELISA (R&D Systems,
Minneapolis, MN). TGF-1 levels were normalized to total protein content that
was determined by BCA (Pierce/Thermo Scientific, Rockford, IL).
Statistical analysis. Survival analysis was computed by the Kaplan-Meier
method. Significances by Wilcoxon rank test (for survival) and t-tests were
calculated using StatView (SAS Institute, Inc., Cary, NC).
23
24
Acknowledgements
The authors would like to thank Eva Niederer and Oralea Büchi (Institute for
Biomedical Techniques) and Seppoei Muljana for technical assistance. The
authors further thank Jan A. Ehses for critical reading of the manuscript. This
study was supported by the Swiss National Science Foundation (grant 3161136.00), the National Competence Centre (NCCR) Neuronal Plasticity and
Repair, the Roche Research Foundation (to C.C.) and the Gianni Rubatto Stiftung.
Conflict of interest
The authors declare that they have no competing financial interest.
25
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Figure Legends
Figure 1: Difference in tumor growth comparing intracranial with
-/-
subcutaneous tumor localization. Wild-type or Rag1 mice were injected with
GL261 cells either intracranially (A, n = 8 for each strain) or subcutaneously (B, n
= 10 for each strain) and monitored for survival or tumor appearance,
respectively. Statistically significant difference is reached for subcutaneous
tumors (P = 0.0074).
Figure 2: Infiltration of intracranial and subcutaneous tumors by immune
cells. Cryosections of either intracranial (A) or subcutaneous (B) tumors were
+
+
+
stained to detect the infiltration of CD11c DCs, CD4 T cells and CD8 T cells
during tumor development. Magnification, x200. (C) quantitative representation
of (A) and (B) depicting the number of stained dendritic cells and CD4+ or CD8+
T cells per field during tumor development (P < 0.001). Error bars represent SEM.
3 mice per time point were analyzed and 10 fields per section were counted.
Figure 3: Phenotypic characterization of TIDCs. (A) TIDCs display a
phenotype similar to immature BM-DCs. Tumor infiltrating mononuclear cells
were isolated at peak clinical stage as described in Materials and Methods and
then stained for CD11c in combination with antibodies to MHC class II or
costimulatory molecules. 7-AAD was used to exclude dead cells. Histograms are
gated on live CD11c+ DCs (for TIDCs: black line, specific staining; grey line,
34
isotype control. For BM-DCs: black line, mature BM-DCs; dark grey line,
immature BM-DCs; light grey line, isotype control). Representative histograms of
at least three experiments are shown. (B) both intracranial and subcutaneous
+
TIDCs are of myeloid origin. Histograms are gated on live CD11c DCs (black
line, specific staining, grey line, isotype control). Data are representative of at
least three experiments. Numbers indicate the mean fluorescence intensity of the
marker analyzed. (C), TIDCs isolated from intracranial gliomas as well as TIDCs
isolated from gliomas in the skin are blood-derived. Tumor cells were inoculated
+
in CD45.2 mice that were lethally irradiated and reconstituted with bone marrow
+
cells from CD45.1 congenic C57BL6/J six weeks previous to tumor inoculation.
Tumor infiltrating mononuclear cells were stained with anti-CD11c, anti-CD45.1
and anti-CD45.2 antibodies. Dot blots are gated on live cells. Numbers in
quadrants indicate percentage of positive cells. The data are representative for 8
mice with intracranial tumors and for 3 mice with subcutaneous tumors analyzed.
Figure 4: Unlike subcutaneous TIDCs, TIDCs isolated from intracranial
tumors fail to induce allogeneic or antigen-specific T cell proliferation. TIDCs
from intracranial or subcutaneous tumors were sorted by flow cytometry at peak
clinical stage. As a control mature BM-DCs were used. (A) for allogeneic T cell
5
+
proliferation, 10 CD4 T cells from BALB/c mice were added to each well of
titrated DCs and the proliferation was measured by 3H-thymidine incorporation.
5
+
(B) for proliferation inhibition assays, 10 CD4 T cells from BALB/c mice as
4
well as 10 mature BM-DCs from C57BL/6 mice were added to each well. Then
35
TIDCs from intracranial or subcutaneous gliomas or alternatively BM-DCs as
control cells were added. The T cell proliferation stimulated by the mature BMDCs in the absence of TIDCs is set to 100%. (C and D) For antigen-specific
5
proliferation assays, 10 OT-II T cells were added with either OVA323-339 peptide
(C), or OVA protein (D). (*, P < 0.05, **, P < 0.01, ***, P < 0.005 comparing
intracranial with subcutaneous TIDCs). Experiments were performed at least 3
times.
Figure 5: TILs from intracranial or subcutaneous GL261 gliomas show
similar activation marker expression. (A) At peak clinical stage (days 25-28)
tumor infiltrating mononuclear cells were stained with either anti-CD4 antibody
or (B) anti-CD8 antibody in combination with antibodies directed against T cell
+
+
activation markers. Dot plots are gated on live CD4 T cells (A) or live CD8 T
cells (B) respectively, and numbers indicate the percentage of positive cells in the
respective quadrant. One representative experiment out of four is shown.
Figure 6: More Tregs and increased levels of TGF- are found in intracranial
+
gliomas. (A) CD4 T cells from spleen, blood and tumors were stained for Foxp3
expression. Numbers indicate the percentage of positive cells in the respective
quadrant. (B) Quantification of (A). Increased Treg levels are found in intracranial
(n = 4) compared to subcutaneous (n = 6) tumors (*, P < 0.005), while no
difference is seen in the blood or in the spleen of tumor bearing animals
(intracranial, n = 4; subcutaneous, n = 5). (C and D) Tregs isolated from
36
intracranial tumors are functionally suppressive. 105 CD4+CD25- T cells (Tn)
from either spleens (C) or tumors (D) were cultured on anti-CD3-coated plates in
the presence or absence of 5x104 Tn or CD4+CD25+ Treg cells isolated from the
same site. After three days, the T cell proliferation was assessed by 3H-thymidine
incorporation. The data is normalized to the proliferation of 105 Tn. The data in
(C) and (D) is representative of 3 experiments each. (E) increased expression of
TGF-1 and TGF-2 mRNA is found in intracranial tumors. Total RNA was
isolated from intracranial or subcutaneous GL261 tumors and cytokine expression
was quantified by real-time PCR. Results represent mean  SEM from 3 mice
each (*, P < 0.05, **, P < 0.01). (F) TGF-1 protein was determined in extracts of
subcutaneous and intracerebral gliomas by ELISA. The data is shown as pg TGF1/mg of total protein. n = 11 (i.c.) and 7 (s.c.). (G) TIDCs from the brain induce
more Tregs in vitro than subcutaneous TIDCs. CD25-depleted naive splenic CD4+
T cells were cultured in the presence or absence of TIDCs. After 5 days the
percentage of Foxp3+ Tregs was determined by flow cytometry. The bar graph
shows the relative induction of Tregs by TIDCs normalized to intracerebral
TIDCs (= 2.8% FoxP3+ among CD4+ cells). The data shows the mean of three
experiments with SEM. P(no DC – i.c. DC) = 0.035; P(i.c. DC – s.c. DC) = 0.002.
All analyses were done at peak clinical stage (days 25-28).
37
Table I. Summary of the activation state of the TIDCs and the CD4+ or CD8+
TILs in intracranial and subcutaneous gliomasa
TIDCs
CD4+ TILs
CD8+ TILs
a
b
intracranial
subcutaneous
69.4 ± 17.2b
82.7 ± 7.2
B7.1
13.8 ± 16.5
29.7 ± 15.0
B7.2
4.9 ± 3.1
4.5 ± 1.9
CD40
7.0 ± 7.9
6.9 ± 4.0
CD69
32.3 ± 10
21.0 ± 9.1
CD62L
18.5 ± 9.0
16.2 ± 16.7
CD25
48.5 ± 9.0
42.8 ± 15.9
CD44
92.0 ± 4.8
92.0 ± 10.3
CD69
33.0 ± 8.6
35.8 ± 16.5
CD62L
35.5 ± 8.5
20.2 ± 17.7
CD25
7.0 ± 3.4
6.6 ± 2.5
CD44
86.8 ± 7.1
89.8 ± 16.1
MHCII
Data represent the mean ± SEM of at least 3 different mice.
Percentage of marker-positive cells among the indicated cell type.
1