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University of Zurich Zurich Open Repository and Archive Winterthurerstr. 190 CH-8057 Zurich http://www.zora.uzh.ch 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: http://www.zora.uzh.ch Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch 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 References 1 Stupp, R., Mason, W. P., van den Bent, M. J., Weller, M., Fisher, B., Taphoorn, M. J., Belanger, K. et al., Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005. 352: 987996. 2 Chahlavi, A., Rayman, P., Richmond, A. L., Biswas, K., Zhang, R., Vogelbaum, M., Tannenbaum, C. et al., Glioblastomas induce Tlymphocyte death by two distinct pathways involving gangliosides and CD70. Cancer Res 2005. 65: 5428-5438. 3 Zou, J. P., Morford, L. A., Chougnet, C., Dix, A. R., Brooks, A. G., Torres, N., Shuman, J. D. et al., Human glioma-induced immunosuppression involves soluble factor(s) that alters monocyte cytokine profile and surface markers. J Immunol 1999. 162: 4882-4892. 4 Ogden, A. T., Horgan, D., Waziri, A., Anderson, D., Louca, J., McKhann, G. M., Sisti, M. B. et al., Defective receptor expression and dendritic cell differentiation of monocytes in glioblastomas. Neurosurgery 2006. 59: 902-909; discussion 909-910. 5 Wrann, M., Bodmer, S., de Martin, R., Siepl, C., Hofer-Warbinek, R., Frei, K., Hofer, E. and Fontana, A., T cell suppressor factor from human glioblastoma cells is a 12.5-kd protein closely related to transforming growth factor-beta. Embo J 1987. 6: 1633-1636. 26 6 Fontana, A., Hengartner, H., de Tribolet, N. and Weber, E., Glioblastoma cells release interleukin 1 and factors inhibiting interleukin 2-mediated effects. J Immunol 1984. 132: 1837-1844. 7 de Martin, R., Haendler, B., Hofer-Warbinek, R., Gaugitsch, H., Wrann, M., Schlusener, H., Seifert et al., Complementary DNA for human glioblastoma-derived T cell suppressor factor, a novel member of the transforming growth factor-beta gene family. Embo J 1987. 6: 36733677. 8 Naganuma, H., Sasaki, A., Satoh, E., Nagasaka, M., Nakano, S., Isoe, S. and Nukui, H., Down-regulation of transforming growth factor-beta and interleukin-10 secretion from malignant glioma cells by cytokines and anticancer drugs. J Neurooncol 1998. 39: 227-236. 9 Gabrilovich, D. I., Chen, H. L., Girgis, K. R., Cunningham, H. T., Meny, G. M., Nadaf, S., Kavanaugh, D. and Carbone, D. P., Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells. Nat Med 1996. 2: 1096-1103. 10 Kokoglu, E., Tuter, Y., Sandikci, K. S., Yazici, Z., Ulakoglu, E. Z., Sonmez, H. and Ozyurt, E., Prostaglandin E2 levels in human brain tumor tissues and arachidonic acid levels in the plasma membrane of human brain tumors. Cancer Lett 1998. 132: 17-21. 11 Frank, S., Muller, J., Bonk, C., Haroske, G., Schackert, H. K. and Schackert, G., Transmission of glioblastoma multiforme through liver transplantation. Lancet 1998. 352: 31. 27 12 Liu, G., Ying, H., Zeng, G., Wheeler, C. J., Black, K. L. and Yu, J. S., HER-2, gp100, and MAGE-1 are expressed in human glioblastoma and recognized by cytotoxic T cells. Cancer Res 2004. 64: 4980-4986. 13 Tang, J., Flomenberg, P., Harshyne, L., Kenyon, L. and Andrews, D. W., Glioblastoma patients exhibit circulating tumor-specific CD8+ T cells. Clin Cancer Res 2005. 11: 5292-5299. 14 Yu, J. S., Liu, G., Ying, H., Yong, W. H., Black, K. L. and Wheeler, C. J., Vaccination with tumor lysate-pulsed dendritic cells elicits antigenspecific, cytotoxic T-cells in patients with malignant glioma. Cancer Res 2004. 64: 4973-4979. 15 Insug, O., Ku, G., Ertl, H. C. and Blaszczyk-Thurin, M., A dendritic cell vaccine induces protective immunity to intracranial growth of glioma. Anticancer Res 2002. 22: 613-621. 16 Ni, H. T., Spellman, S. R., Jean, W. C., Hall, W. A. and Low, W. C., Immunization with dendritic cells pulsed with tumor extract increases survival of mice bearing intracranial gliomas. J Neurooncol 2001. 51: 1-9. 17 Steinman, R. M. and Nussenzweig, M. C., Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proc Natl Acad Sci U S A 2002. 99: 351-358. 18 Vicari, A. P., Caux, C. and Trinchieri, G., Tumour escape from immune surveillance through dendritic cell inactivation. Semin Cancer Biol 2002. 12: 33-42. 28 19 Tosi, P., Sforza, V., Santopietro, R., Lio, R., Gotti, G., Paladini, P., Cevenini, G. and Barbini, P., Bronchiolo-alveolar carcinoma: an analysis of survival predictors. Eur J Cancer 1992. 28A: 1365-1370. 20 Curiel, T. J., Coukos, G., Zou, L., Alvarez, X., Cheng, P., Mottram, P., Evdemon-Hogan, M. et al., Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 2004. 10: 942-949. 21 Zou, W., Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol 2006. 6: 295-307. 22 Grauer, O. M., Nierkens, S., Bennink, E., Toonen, L. W., Boon, L., Wesseling, P., Sutmuller, R. P. and Adema, G. J., 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. 23 El Andaloussi, A., Han, Y. and Lesniak, M. S., Prolongation of survival following depletion of CD4+CD25+ regulatory T cells in mice with experimental brain tumors. J Neurosurg 2006. 105: 430-437. 24 El Andaloussi, A. and Lesniak, M. S., An increase in CD4+CD25+FOXP3+ regulatory T cells in tumor-infiltrating lymphocytes of human glioblastoma multiforme. Neuro-oncol 2006. 8: 234-243. 25 Chaux, P., Favre, N., Martin, M. and Martin, F., Tumor-infiltrating dendritic cells are defective in their antigen-presenting function and inducible B7 expression in rats. Int J Cancer 1997. 72: 619-624. 29 26 Fischer, H. G., Bonifas, U. and Reichmann, G., Phenotype and functions of brain dendritic cells emerging during chronic infection of mice with Toxoplasma gondii. J Immunol 2000. 164: 4826-4834. 27 Ponomarev, E. D., Shriver, L. P., Maresz, K. and Dittel, B. N., Microglial cell activation and proliferation precedes the onset of CNS autoimmunity. J Neurosci Res 2005. 81: 374-389. 28 Enk, A. H., Jonuleit, H., Saloga, J. and Knop, J., Dendritic cells as mediators of tumor-induced tolerance in metastatic melanoma. Int J Cancer 1997. 73: 309-316. 29 Chen, W., Jin, W., Hardegen, N., Lei, K. J., Li, L., Marinos, N., McGrady, G. and Wahl, S. M., Conversion of peripheral CD4+CD25naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med 2003. 198: 1875-1886. 30 Scarpino, S., Stoppacciaro, A., Ballerini, F., Marchesi, M., Prat, M., Stella, M. C., Sozzani, S. et al., Papillary carcinoma of the thyroid: hepatocyte growth factor (HGF) stimulates tumor cells to release chemokines active in recruiting dendritic cells. Am J Pathol 2000. 156: 831-837. 31 Bell, D., Chomarat, P., Broyles, D., Netto, G., Harb, G. M., Lebecque, S., Valladeau, J. et al., In breast carcinoma tissue, immature dendritic cells reside within the tumor, whereas mature dendritic cells are located in peritumoral areas. J Exp Med 1999. 190: 1417-1426. 30 32 Suter, T., Biollaz, G., Gatto, D., Bernasconi, L., Herren, T., Reith, W. and Fontana, A., The brain as an immune privileged site: dendritic cells of the central nervous system inhibit T cell activation. Eur J Immunol 2003. 33: 2998-3006. 33 McMahon, E. J., Bailey, S. L., Castenada, C. V., Waldner, H. and Miller, S. D., Epitope spreading initiates in the CNS in two mouse models of multiple sclerosis. Nat Med 2005. 11: 335-339. 34 Guiducci, C., Vicari, A. P., Sangaletti, S., Trinchieri, G. and Colombo, M. P., Redirecting in vivo elicited tumor infiltrating macrophages and dendritic cells towards tumor rejection. Cancer Res 2005. 65: 3437-3446. 35 Vicari, A. P., Chiodoni, C., Vaure, C., Ait-Yahia, S., Dercamp, C., Matsos, F., Reynard, O. et al., Reversal of tumor-induced dendritic cell paralysis by CpG immunostimulatory oligonucleotide and anti-interleukin 10 receptor antibody. J Exp Med 2002. 196: 541-549. 36 Preynat-Seauve, O., Schuler, P., Contassot, E., Beermann, F., Huard, B. and French, L. E., Tumor-infiltrating dendritic cells are potent antigen-presenting cells able to activate T cells and mediate tumor rejection. J Immunol 2006. 176: 61-67. 37 Facoetti, A., Nano, R., Zelini, P., Morbini, P., Benericetti, E., Ceroni, M., Campoli, M. and Ferrone, S., Human leukocyte antigen and antigen processing machinery component defects in astrocytic tumors. Clin Cancer Res 2005. 11: 8304-8311. 31 38 Frei, K., Siepl, C., Groscurth, P., Bodmer, S., Schwerdel, C. and Fontana, A., Antigen presentation and tumor cytotoxicity by interferongamma-treated microglial cells. Eur J Immunol 1987. 17: 1271-1278. 39 Flugel, A., Labeur, M. S., Grasbon-Frodl, E. M., Kreutzberg, G. W. and Graeber, M. B., Microglia only weakly present glioma antigen to cytotoxic T cells. Int J Dev Neurosci 1999. 17: 547-556. 40 Boissonnas, A., Fetler, L., Zeelenberg, I. S., Hugues, S. and Amigorena, S., In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor. J Exp Med 2007. 19: 345-356. 41 Flugel, A., Berkowicz, T., Ritter, T., Labeur, M., Jenne, D. E., Li, Z., Ellwart, J. W. et al., Migratory activity and functional changes of green fluorescent effector cells before and during experimental autoimmune encephalomyelitis. Immunity 2001. 14: 547-560. 42 Bauer, J., Bradl, M., Hickley, W. F., Forss-Petter, S., Breitschopf, H., Linington, C., Wekerle, H. and Lassmann, H., T-cell apoptosis in inflammatory brain lesions: destruction of T cells does not depend on antigen recognition. Am J Pathol 1998. 153: 715-724. 43 Didenko, V. V., Ngo, H. N., Minchew, C. and Baskin, D. S., Apoptosis of T lymphocytes invading glioblastomas multiforme: a possible tumor defense mechanism. J Neurosurg 2002. 96: 580-584. 44 Hollenbaugh, J. A. and Dutton, R. W., IFN-gamma regulates donor CD8 T cell expansion, migration, and leads to apoptosis of cells of a solid tumor. J Immunol 2006. 177: 3004-3011. 32 45 Jonuleit, H., Schmitt, E., Schuler, G., Knop, J. and Enk, A. H., Induction of interleukin 10-producing, nonproliferating CD4(+) T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J Exp Med 2000. 192: 1213-1222. 46 Li, M. O., Wan, Y. Y., Sanjabi, S., Robertson, A. K. and Flavell, R. A., Transforming growth factor-beta regulation of immune responses. Annu Rev Immunol 2006. 24: 99-146. 47 Fakhrai, H., Dorigo, O., Shawler, D. L., Lin, H., Mercola, D., Black, K. L., Royston, I. and Sobol, R. E., Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy. Proc Natl Acad Sci U S A 1996. 93: 2909-2914. 48 Fontana, A., Frei, K., Bodmer, S., Hofer, E., Schreier, M. H., Palladino, M. A., Jr. and Zinkernagel, R. M., Transforming growth factor-beta inhibits the generation of cytotoxic T cells in virus-infected mice. J Immunol 1989. 143: 3230-3234. 49 Nagashima, G., Suzuki, R., Asai, J. and Fujimoto, T., Immunohistochemical analysis of reactive astrocytes around glioblastoma: an immunohistochemical study of postmortem glioblastoma cases. Clin Neurol Neurosurg 2002. 104: 125-131. 50 Liu, Y., Teige, I., Birnir, B. and Issazadeh-Navikas, S., Neuronmediated generation of regulatory T cells from encephalitogenic T cells suppresses EAE. Nat Med 2006. 12: 518-525. 33 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 TGF1/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