- Email: [email protected]
The limited capacity of malignant glioma-derived exosomes to suppress peripheral immune effectors
Journal of Neuroimmunology 290 (2016) 103–108
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
The limited capacity of malignant glioma-derived exosomes to suppress peripheral immune effectors J. Bryan Iorgulescu a,b,1, Michael E. Ivan a,⁎,2, Michael Safaee a,b, Andrew T. Parsa a,3,4 a b
Department of Neurological Surgery, UCSF, San Francisco, CA, USA Clinical and Translational Science Institute, UCSF, San Francisco, CA, USA
a r t i c l e
i n f o
Article history: Received 13 October 2015 Received in revised form 17 November 2015 Accepted 28 November 2015 Keywords: Glioma Exosome Microvesicle Immunosuppression Immunotherapy
a b s t r a c t Tumor-derived microvesicular exosomes permit intercellular communication both locally and systemically by delivering a snapshot of the tumor cell's constituents. We thus investigated whether exosomes mediate malignant glioma's facility for inducing peripheral immunosuppression. In Western blot and RT-PCR analyses, glioma-derived exosomes displayed exosome-specific markers, but failed to recapitulate the antigenpresentation machinery, surface co-modulatory signals, or immunosuppressive mediator status of their parent tumor cells. Treatment with glioma-derived exosomes promoted immunosuppressive HLA-DRlow monocytic phenotypes, but failed to induce monocytic PD-L1 expression or alter the activation of cytotoxic T-cells from patients' peripheral blood by FACS and RT-PCR analyses. Our results suggest that malignant glioma-derived exosomes are restricted in their capacity to directly prime peripheral immunosuppression. © 2015 Elsevier B.V. All rights reserved.
Author disclosure statement: The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.
1. Introduction Malignant gliomas, which universally portend a median survival less than 15 months, exhibit a tenacious propensity to subdue immune responses both locally and systemically (Stupp et al., 2005). In the local tumor environment, gliomas readily release anti-inflammatory cytokines, employ immunosuppressive surface ligands, and down-regulate their surface antigen-presentation machinery in order to successfully evade immune surveillance (Albesiano et al., 2010; Han et al., 2012; Waziri, 2010). Simultaneously, malignant gliomas secrete as of yet unidentified soluble factors that coax circulating monocytes and T lymphocytes into infiltrating the tumor as their anti-inflammatory phenotypes: M2 macrophages that up-regulate IL-10 and programmed
⁎ Corresponding author at: Department of Neurological Surgery, University of Miami Miller School of Medicine, Lois Pope Life Center, 1095 NW 14th Ter, Miami, FL 33136, USA. E-mail address: [email protected] (M.E. Ivan). 1 Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. 2 Department of Neurological Surgery, University of Miami, Miami, FL, USA. 3 Department of Neurological Surgery, Northwestern Feinberg School of Medicine, Chicago, IL, USA. 4 Deceased April 13, 2015.
http://dx.doi.org/10.1016/j.jneuroim.2015.11.025 0165-5728/© 2015 Elsevier B.V. All rights reserved.
death ligand 1 (PD-L1) and regulatory T lymphocytes, which together culminate in the apoptosis of anti-tumoral T lymphocytes (Bloch et al., 2013; Crane et al., 2012; Parsa et al., 2014; Zou et al., 1999). There is growing evidence suggesting that gliomas can influence cells both nearby and beyond the blood–brain barrier via the carefully regulated excretion of exosomes: 40–100 nm microvesicles formed during the normal cellular membrane sorting cycle by the inward budding of late endosomes (Ostrowski et al., 2010). These multi-vesicular endosomes are typically destined for recycling by lysosomes, but occasionally fuse with the plasma membrane and thereby release their microvesicle contents into the extracellular milieu. Exosomes contain a representative profile of the proteins, mRNAs, and microRNAs of their parent T-cells; in glioblastoma (GBM) this exosomal content reflects the tumor's unique signature of EGFR amplification, EGFRvIII mutation, IDH1 mutation R132H, TGF-β, and/or podoplanin status (Al-Nedawi et al., 2008; Graner et al., 2009; Henriksen et al., 2014; Li et al., 2013; Manterola et al., 2014; Noerholm et al., 2012; Shao et al., 2012). GBM-derived exosomes also may beckon angiogenesis during hypoxia, by delivery of proteins (e.g. metalloproteinase 8, IL-8, and PDGF AA/AB) and mRNA transcripts (e.g. IGF-binding protein, BCL-2, and N-myc downstream regulator 1) to vascular endothelial cells and pericytes (Kucharzewska et al., 2013; Svensson et al., 2011). MiR-1 microRNA dysregulation in GBMs, in addition to unleashing the oncogenic JNK, MET, and EGFR signaling pathways, has also been shown to uncheck the high levels of Annexin A2 packaged in GBM-derived exosomes, thus driving more aggressive GBM growth, neovascularization, and invasion (Bronisz et al., 2014). Tumor-derived exosomes have also been shown to contribute to immunosuppression in a number of other tumor types; notably, exosomes distilled from the sera of
104
J.B. Iorgulescu et al. / Journal of Neuroimmunology 290 (2016) 103–108
patients with advanced melanoma or colon carcinoma not only hindered monocyte precursor differentiation into dendritic cells, but in fact programmed the precursors into myeloid suppressive cells characterized by down-regulation of surface MHC class II expression, persistent CD14 pattern recognition receptor positivity, and suppression of T lymphocytes by way of TGF-β secretion; whereas exosomes derived from normal control patients' sera encouraged myeloid maturation and T lymphocyte stimulation (Valenti et al., 2006; Zhang and Grizzle, 2011). With the growing body of evidence implicating exosomes as an intercellular mediator of GBM pathophysiology, we hypothesized that they may comprise the unidentified soluble factors that exit the blood–brain barrier and reprogram distant immune effectors into their immunosuppressive phenotypes. In order to directly prime T lymphocytes responses, glioma-derived exosomes would require both surface antigen presentation machinery (including either MHC class I or class II molecules) and requisite co-modulatory signals. Alternatively, immune effectors can also be activated by the direct uptake of antigens bound to heat shock protein chaperones (HSP) released during cell necrosis (Bendz et al., 2007; Cho et al., 2009). Gliomas robustly express a number of constitutive and inducible HSPs, particularly HSP70 and HSP90 families, which chaperone the folding of several drivers of oncogenesis (Bloch et al., 2014; Graner et al., 2007; Yang et al., 2010). HSP70 has also been observed on the glioma cell surface, where it directly interacts with natural killer cells and γδ T lymphocytes (Wachstein et al., 2012; Zhang et al., 2005). HSP72 was identified on exosomes derived from a number of tumor types, which enabled the tumor-derived exosomes to trigger immunosuppressive Stat3 pathways in myeloidderived cells (Chalmin et al., 2010). Herein we investigated whether glioma-derived exosomes have the capacity to promote immunosuppressive phenotypes of effectors from myeloid or lymphoid lineages.
pellets were resuspended in either PBS for cell treatment or lysis buffer for Western blot analysis. 2.3. Western blot analysis Cells and exosomes were lysed by radioimmunoprecipitation assay buffer (RIPA; UCSF Cell Culture Facility) supplemented with protease inhibitors (Roche Diagnostics) on a 4 °C shaker for 45 min. Protein concentrations were determined by BCA Protein Assay Kit (Pierce) and loaded in equal amounts for polyacrylamide gel electrophoresis, with either SDS or non-reducing conditions as specified by the antibody manufacturer, then transferred to PVDF membranes for blocking and subsequent probing with primary antibodies against: CD81 (Santa Cruz Biotech), CD9 (Abcam), HSP70 (Santa Cruz Biotech), HSP90 (Abcam), GAPDH (Cell Signal), CTLA4 (Abcam), CD80 (Abcam), CD86 (Abcam), PD-L1 (Abcam), HLA-ABC (Biolegend), and HLA-DR (Biolegend). Secondary antibodies conjugated to horseradish peroxidase visualized the proteins by way chemilluminescence (ECL Western blotting substrate; Pierce). 2.4. Cell sorting As previously described, peripheral blood leukocytes (PBL) were isolated from primary GBM patients' and healthy naïve donors' whole blood samples by Ficoll-Paque Plus (GE Healthcare) centrifugation.(Bloch et al., 2013; Crane et al., 2012) Monocytes were extracted by CD14+ selection using magnetic nanoparticles (EasySep, Stem Cell Technologies) and T lymphocytes by negative magnetic nanoparticle selection, then suspended in RPMI-1640 media supplemented with 1% penicillin–streptomycin, 1 mmol/L sodium pyruvate, 10 mmol/L nonessential amino acids, and 2.5% exosome-depleted FBS.
2. Methods
2.5. Treatment of cells
2.1. Cell culture
Monocytes were plated at 2 × 105 cells per well in a 24 well plate and incubated at 37 °C for 24 h with escalating doses of exosomes from established or primary glioma lines, as compared to positive controls treated with glioma conditioned media and untreated controls, in triplicate. T lymphocytes were plated at 3.1 × 105 per well in a 96 well plate and incubated at 37 °C for 24 h with Golgi plug protein transport inhibitor (BD Bioscience) and either exosomes from established or primary glioma lines, compared to treatment with NHA-derived exosomes or untreated controls.
Established glioma cell lines U87 and U251 were obtained through the UCSF Brain Tumor Research Center and cultured in Dulbecco's modified Eagle medium H21 (UCSF Cell Culture Facility). Primary GBM lines from two patients, denoted as GBM1 and GBM2, were derived from patients' fresh tumor tissue collected during initial resection and cultured for no more than 15 passages in RPMI-1640 media supplemented with 10 mmol/L non-essential amino acids. All glioma cultures were additionally supplemented with and 1% penicillin–streptomycin and 5% fetal bovine serum (FBS) depleted of exosomes by ultracentrifugation at 110,000 g at 4 °C for 4 h and careful collection of the resulting supernatant. Normal human astrocytes (NHA) were acquired from Sciencell Laboratories and cultured in Lonza proprietary media with exosomedepleted FBS. 2.2. Exosome isolation Conditioned media were harvested after 48 h from cultures at 80– 90% confluence and centrifuged at 600 g at 4 °C for 10 min to precipitate cell debris. The supernatant was then centrifuged at 16,500 g at 4 °C for 20 min and passed through a 0.22 μL filter (Milipore). The resulting supernatant was ultracentrifuged at 110,000 g at 4 °C for 1 h to pellet the exosomes, which were then resuspended in a phosphate-buffered saline (PBS) wash and ultracentrifuged again at 110,000 g at 4 °C for 1 h. In order to maximize detection of the effects of exosomes in our experiments, concentrations many magnitudes higher than found in circulation were used. Additionally, protein was precipitated from supernatant fractions of exosome-depleted conditioned media by using 20% trichloroacetic acid on ice, thrice washed with 1:1 acetone and ethanol, and pelleted by 16,500 g at 4 °C for 20 min. Exosome and protein
2.6. Flow cytometry analysis Monocytes were stained extracellularly with CD45 FITC (clone HI30, eBioscience), CD11b PeCy (clone ICRF44, eBioscience), HLA-DR APC (clone LN3, eBioscience), and PD-L1 PE (clone MIH1, eBioscience) or isotype control (eBioscience) and T lymphocytes extracellularly with CD3 PerCP (eBioscience), CD4 PE (eBioscience), and CD8 FITC (BD Pharmingen), and intracellularly with IFNγ APC (eBioscience) in saponin-containing buffer (PermWash, BD Pharmingen), all in PBS with 2% bovine serum albumin on ice for 30 min, washed, fixed with 2% paraformaldehyde, and read using a BD FACScaliber flow cytometer with CellQuest Software (Beckton Dickinson), as previously described (Bloch et al., 2013; Crane et al., 2012). PBLs were treated in triplicate. The gate strategy for identifying monocytes included forward vs side scatter gates for size, then CD45 vs CD11b gates for monocytic phenotype, with CD11b vs PDL-1 to assess PDL-1 expression levels, and CD11b vs HLA-DR to evaluate monocyte phenotype changes. CD8+ Tcells were identified by forward vs side scatter for size, and forward scatter vs CD3 to confirm T-lymphocytic phenotype, with CD8 vs CD4 for identifying CD8+ T-cells and CD8 vs IFNγ for assessing changes in CD8+ T-cell activation levels.
J.B. Iorgulescu et al. / Journal of Neuroimmunology 290 (2016) 103–108
105
the media protein precipitate for exosome-specific markers CD9 and CD81. 3.2. Glioma-derived exosomes lacked antigen-presentation machinery Glioma-derived exosomes demonstrated no appreciable content of MHC class I (HLA-ABC) or class II (HLA-DR) molecules, which respectively prime CD8+ and CD4+ T-cell responses; with the exception of U87 samples, which selectively concentrated HLA-DR into exosomal fractions (Fig. 1B). As in prior reports, astrocytes from normal central nervous tissue (NHA) were devoid of HLA-ABC (Traugott, 1987). Additionally, we found robust expression of HSP70 and HSP90 in all glioma lines, but with limited content within exosome fractions (Fig. 1C). 3.3. Glioma-derived exosomes lacked surface co-modulatory molecules
Fig. 1. Protein content of respective cellular and exosomal fractions. (A) Exosome isolation was confirmed by blotting for exosome-specific tetraspanins CD9 and CD81, with cellular fractions identified by GAPDH. Regardless of expression in parent cellular fractions, glioma-derived exosomes lacked appreciable (B) HLA-ABC or HLA-DR antigen presentation machinery, (C) heat shock protein 70 or 90 families of antigen chaperones, or (D) requisite co-modulatory signals CD80, CD86, and CTLA-4.
In addition to surface antigen-presentation machinery, concomitant binding of surface co-modulatory molecules is required to properly guide a T lymphocyte response against an antigen. CD80 (B7-1) and CD86 (B7-2) are co-modulatory surface molecules respectively found on activated monocytes and antigen-presenting cells; which can bind either stimulatory CD28 or inhibitory CTLA-4 cognate T lymphocyte receptors. The glioma lines exhibited CD86 and limited CD80 expression,
2.7. Reverse transcribed-quantitative PCR mRNA was extracted from cells using the RNeasy Mini Kit (Qiagen), transcribed to cDNA with the Superscript III Kit (Invitrogen), and subsequently quantified by CFX96 Real-Time System (Bio-Rad Laboratories) detection of transcript-incorporated SYBR Green (Applied Biosystems), as previously reported. Cycle thresholds for PD-L1 (5′-GCTGTT-GAAG GA-CCAGCT-CT/TGCTTG-TCCAGA-TGACTT-CG-3′) and IFNγ (5′-CGAG AT-GACTTC-GAAAAG-CTG/ATATTG-CAGGCA-GGACAA-CC-3′) were compared to 18S rRNA (5′-GTAACC-CGTTGA-ACCCCA-TT/CCATCCAATCGG-TAGTAG-CG-3′). 2.8. Statistical analysis Data were compared by one-way analysis of variance (ANOVA) with post-hoc Bonferroni corrections, using SPSS version 19 (IBM), and reported as mean ± standard error of the mean (SEM). Significance was defined as p b 0.05. 3. Results 3.1. Exosome isolation Exosomes were purified by serial ultracentrifugation and filtration of conditioned media from both established (U87 and U251) and patientderived primary (GBM1 and GBM2) glioma lines, with NHA lines as controls. Purification was confirmed by Western blot probing for the exosome-specific tetraspanins CD9 and CD81 (Fig. 1A). Whether house-keeping proteins are equally partitioned into exosomal and cellular compartments remains unclear, thus CD81was used as a loading control for exosome samples and GAPDH as a loading control for cell samples, with qualitative comparison of densitometry between corresponding exosomal and cellular fractions. Removal of all cellular fractions from exosome samples was confirmed by probing for GAPDH, one house-keeping protein found to be cellular-specific and not enriched in exosome samples. Likewise, complete extraction of exosome fractions from conditioned media was confirmed by probing
Fig. 2. Glioma-derived exosomes failed to promote PD-L1 expression in, or deliver PD-L1 to peripheral monocytes. (A) Monocytes were identified by flow cytometry for CD11b and CD45 positivity and (B) demonstrated no change in PD-L1 mean fluorescence intensity (MFI) when treated with glioma-derived exosomes (15.5 ± 0.3 from U87 exosomes, p = 1.00; and 15.5 ± 0.3 from GBM2 exosomes, p = 1.00), as compared to untreated controls (16.0 ± 0.3). Glioma conditioned media (CM) was a positive control of immunosuppression (20.8 ± 0.5, ***p b 0.001). Dotted black line denotes the isotype control. (C) Glioma-derived exosomes also lacked PD-L1 protein or mRNA content, regardless of expression in parent cell fractions. avg CT: average cycle threshold.
106
J.B. Iorgulescu et al. / Journal of Neuroimmunology 290 (2016) 103–108
cytometry after 24 h of culture and exhibited no significant change following treatment with glioma-derived exosomes, as compared to untreated controls (MFI 15.5 ± 0.3 from U87 exosomes, p = 1.00 and 15.5 ± 0.3 from GBM2 exosomes, p = 1.00; vs untreated controls 16.0 ± 0.3) (Fig. 2B). There was additionally no benefit to treatment with double the concentration of glioma-derived exosomes (15.2 ± 0.2 from U87 exosomes, p = 1.00; and 15.1 ± 0.3 from GBM2 exosomes, p = 0.88); whereas the conditioned media-treated controls expressed significantly more PD-L1 (20.8 ± 0.5) than all treated (p b 0.001) or untreated groups (p b 0.001). In addition, quantitative RT PCR revealed that exosomes derived from U87, U251, GBM1, and GBM2 cultures were devoid of PD-L1 transcripts (Fig. 2C). Similarly, despite variable expression of PD-L1 in parent glioma lines, no PD-L1 protein content was found in their respective exosome fractions.
3.5. Glioma-derived exosomes induced immunosuppressive surface HLA-DRlow expression on peripheral monocytes
Fig. 3. Glioma-derived exosomes promoted immunosuppressive HLA-DRlow phenotypes in peripheral monocytes. (A) Monocytes were identified by flow cytometry for CD11b and CD45 positivity and (B) displayed significant reductions in HLA-DR mean fluorescence intensity (MFI) when treated with glioma-derived exosomes (460.8 ± 7.1 from U87 exosomes, #p = 0.039; and 451.8 ± 9.5 from GBM2 exosomes, ##p = 0.009), as compared to untreated controls (502.4 ± 9.2). Glioma conditioned media (CM) was a positive control of immunosuppression (262.1 ± 7.9, ***p b 0.001). Dotted black line denotes the isotype control. avg CT: average cycle threshold.
although this expression was not reflected in their exosome fractions (Fig. 1D). CTLA-4 is posited to out-compete CD28 in binding CD80 and CD86, thereby preventing activation of T lymphocytes in a dosedependent fashion (Curran et al., 2010). Glioma-derived exosomes were thus examined for CTLA-4 content, which could either be delivered to T-cells or monopolize the binding of CD80 and CD86 ligands, and were found to be limited in their content of CTLA-4 regardless of expression levels in parent cells (Fig. 1D). 3.4. Glioma-derived exosomes failed to induce PD-L1 expression in peripheral monocytes To investigate whether gliomas employ exosomes in inducing PD-L1 expression in peripheral monocytes, monocytes from a naïve patient were treated with exosomes from both established (U87) and primary (GBM2) glioma lines. Treatment with concentrated conditioned media from GBM cell culture, previously shown to induce robust PD-L1 expression in peripheral monocytes, served as an immunosuppressive positive control (Bloch et al., 2013; Parsa et al., 2014). Flow cytometry experiments were conducted in triplicate and measured in mean fluorescence intensities (MFI). PD-L1 surface expression was assessed by flow
Surface levels of HLA-DR on peripheral monocytes, typically increased in pro-inflammatory phenotypes and reduced in immunosuppressive phenotypes, was examined following 24 h of treatment with glioma-derived exosomes, and exhibited significantly lower levels of monocyte HLA-DR following treatment with GBM2-derived exosomes (MFI 451.8 ± 9.5, p = 0.009) or U87-derived exosomes (460.8 ± 7.1, p = 0.039), as compared to untreated controls (502.4 ± 9.2), reflecting a switch from inactivated or pro-inflammatory monocytic phenotypes to HLA-DRlow immunosuppressive phenotypes (Fig. 3B). These trends were not shared by monocytes treated with twice the concentration of glioma-derived exosomes (470.1 ± 8.1 from GBM2 exosomes, p = 0.186; and 462.3 ± 3.3 from U87 exosomes, p = 0.005). Condition media-treated controls displayed significantly lower HLA-DR expression than all other groups (262.1 ± 7.9, p b 0.001).
3.6. Glioma-derived exosomes failed to alter the activation of peripheral CD8+ T lymphocytes Activation of peripheral CD8 + cytotoxic T lymphocytes following 24 h of treatment with glioma-derived exosomes was assessed by flow cytometry for intracellularly stained interferon-γ (IFNγ). There were no significant differences in IFNγ levels following treatment of GBM patient peripheral T-cells with autologous GBM-derived exosomes, as compared to either untreated or NHA exosome-treated controls (Table 1, Fig. 4). T-cells from a naïve non-glioma patient provided an additional control, and also failed to alter IFNγ levels. There appeared to be limited activation of T-cells when treated with nonautologous exosomes (either derived from U251 or NHA), which likely represents T-cell recognition of foreign surface-bound antigens. IFNγ mRNA levels remained below threshold for detection by quantitative RT PCR, despite moderate 18S levels, for both untreated and gliomaderived exosome treated groups of naïve patient T-cells. Additionally, there were no significant differences in the proportions of CD8 + or CD4+ T-cells following treatment with glioma-derived exosomes.
Table 1 Cytotoxic T lymphocyte IFNγ levels following exosome treatment. Naïve
GBM1
GBM2
Treatment
MFI
SEM
p-value
MFI
SEM
p-value
MFI
SEM
p-value
Untreated (reference) Autologous GBM exosomes⁎ NHA exosomes U251 exosomes
7.1 7.8 8.1 8.7
0 0.3 0 0.6
1 0.62 0.17
7.8 8.1 10.4 11.9
0.4 0.4 0.2 0.7
1 0.87 0.02
8.2 7.9 8.3 8.6
0.3 0.1 0.5 0.6
1 1 1
MFI: mean fluorescence intensity, SEM: standard error of the mean. ⁎ Naïve patient peripheral T-cells were treated with GBM2-derived exosomes.
J.B. Iorgulescu et al. / Journal of Neuroimmunology 290 (2016) 103–108
107
Fig. 4. Cytotoxic T lymphocyte activation was not altered by glioma-derived exosomes. Treatment with glioma-derived exosomes demonstrated no change in IFNγ levels in CD8+ T-cells from (A) naïve non-glioma, (B) GBM1, and (C) GBM2 patients' PBLs, as compared to untreated and NHA-derived exosome-treated controls. * Naïve patient peripheral T-cells were treated with GBM2-derived exosomes.
4. Discussion GBM's proficiency at subverting local antitumoral immunity and globally suppressing immune effectors poses a key obstacle to the efficacy of immunotherapies (Crane et al., 2013; Han et al., 2012; Sayegh et al., 2014). We have previously demonstrated that dysregulation of PTEN and PI(3)K pathways in glioma leads to robust PD-L1 surface expression, which widely subdues the activation of tumor-infiltrating immune effectors upon binding of its cognate PD-1 receptor, as well as outcompetes T lymphocytes for the affinity of CD80 costimulatory signals (Han et al., 2009; Parsa et al., 2007; Waldron et al., 2010). We subsequently found that gliomas also secrete unidentified soluble factors which promote PD-L1 expression by monocytes circulating in the periphery (Bloch et al., 2013; Crane et al., 2012). With growing evidence that tumor-released exosomes contribute to immunosuppression in other tumor types and may play a role in glioma pathogenesis, we hypothesized that glioma-derived exosomes may comprise glioma's secreted soluble factors which promote immunosuppressive phenotypes (Valenti et al., 2006; Zhang and Grizzle, 2011). However, gliomaderived exosomes lacked the necessary antigen presentation machinery for priming immune responses and, other than promoting significant but limited immunosuppressive HLA-DRlow expression in peripheral monocytes, failed to directly induce phenotypic changes in peripheral blood immune effectors in vitro. It is likely that antigens carried by glioma-derived exosomes first require processing by antigen presenting cells (APCs), in order to appropriately prime an antigen-specific immune response, as observed in other tumor models (Wolfers et al., 2001; Yang et al., 2012). In fact, several in vitro experiments have harnessed APCs' capacity for processing antigens from glioma-derived and tumor-derived exosomes to successfully prime antigen-specific antitumoral immune responses (André et al., 2004; Bu et al., 2011; Mignot et al., 2006). Prophylactic vaccination of mouse glioma models with glioma-derived exosomes resulted in robust antitumoral immunity, complete rejection of subsequent glioma implantation, and increased overall survival (Graner et al., 2009). However, these successes were not mirrored by mice with pre-existing orthotopic gliomas, in which glioma-derived exosome-stimulated vaccines failed to induce tumor rejection or improve survival, partly as a consequence of the intact immunosuppression wrought by malignant gliomas. 5. Conclusions Our results suggest that malignant glioma-derived exosomes are restricted in their ability to directly prime peripheral immunosuppression. Although glioma-derived exosomes may play a limited direct role in immunosuppression, there is burgeoning promise in their diagnostic utility as a non-invasive means of typing gliomas by only using tumor exosomes from blood samples (Chen et al., 2010; Shao et al., 2012;
Skog et al., 2008). Likewise, as a circulating reservoir of tumor antigens, glioma-derived exosomes may help overcome a key hurdle in vaccine therapies for malignant glioma: the need for repeat tissue biopsies of recurrences in order to re-stimulate the vaccine against the newly selected-for glioma phenotypes (Bloch et al., 2014; Crane et al., 2013; Han et al., 2012; Sayegh et al., 2014). Analysis of circulating gliomaderived exosomes could obviate our reliance on delayed clinical or radiographic signs of recurrence, by permitting the real-time monitoring of glioma antigens. Exosomes are an exciting facet of cellular biology, one in which further inquiry is necessary for teasing apart what roles exosomes play in cancer pathophysiology versus those instances where exosomes are merely physiologic byproducts of cellular garbage disposal systems. Acknowledgments This work was graciously supported by Doris Duke Clinical Research Fellowships (JBI and MS), the Clinical and Translational Science Institute at UCSF (JBI and MS), the National Research and Education Foundation (MEI), and the Michael J. Marchese Professor and Chair at Northwestern University (ATP). We thank O. Bloch for technical guidance and R. Kaur for technical assistance. We are incomparably grateful for Andew Parsa's mentorship, supervision, and stewardship of this study. References Albesiano, E., Han, J.E., Lim, M., 2010. Mechanisms of local immunoresistance in glioma. Neurosurg. Clin. N. Am. 21, 17–29. Al-Nedawi, K., Meehan, B., Micallef, J., Lhotak, V., May, L., Guha, A., Rak, J., 2008. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat. Cell Biol. 10, 619–624. André, F., Chaput, N., Schartz, N.E.C., Flament, C., Aubert, N., Bernard, J., Lemonnier, F., Raposo, G., Escudier, B., Hsu, D.-H., Tursz, T., Amigorena, S., Angevin, E., Zitvogel, L., 2004. Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells. J. Immunol. 1950 (172), 2126–2136. Bendz, H., Ruhland, S.C., Pandya, M.J., Hainzl, O., Riegelsberger, S., Braüchle, C., Mayer, M.P., Buchner, J., Issels, R.D., Noessner, E., 2007. Human heat shock protein 70 enhances tumor antigen presentation through complex formation and intracellular antigen delivery without innate immune signaling. J. Biol. Chem. 282, 31688–31702. Bloch, O., Crane, C.A., Kaur, R., Safaee, M., Rutkowski, M.J., Parsa, A.T., 2013. Gliomas promote immunosuppression through induction of B7-H1 expression in tumorassociated macrophages. Clin. Cancer Res. 19, 3165–3175. Bloch, O., Crane, C.A., Fuks, Y., Kaur, R., Aghi, M.K., Berger, M.S., Butowski, N.A., Chang, S.M., Clarke, J.L., McDermott, M.W., Prados, M.D., Sloan, A.E., Bruce, J.N., Parsa, A.T., 2014. Heat-shock protein peptide complex-96 vaccination for recurrent glioblastoma: a phase II, single-arm trial. Neuro-Oncology 16, 274–279. Bronisz, A., Wang, Y., Nowicki, M.O., Peruzzi, P., Ansari, K.I., Ogawa, D., Balaj, L., De Rienzo, G., Mineo, M., Nakano, I., Ostrowski, M.C., Hochberg, F., Weissleder, R., Lawler, S.E., Chiocca, E.A., Godlewski, J., 2014. Extracellular vesicles modulate the glioblastoma microenvironment via a tumor suppression signaling network directed by miR-1. Cancer Res. 74, 738–750. Bu, N., Wu, H., Sun, B., Zhang, G., Zhan, S., Zhang, R., Zhou, L., 2011. Exosome-loaded dendritic cells elicit tumor-specific CD8 + cytotoxic T cells in patients with glioma. J. Neuro-Oncol. 104, 659–667.
108
J.B. Iorgulescu et al. / Journal of Neuroimmunology 290 (2016) 103–108
Chalmin, F., Ladoire, S., Mignot, G., Vincent, J., Bruchard, M., Remy-Martin, J.-P., Boireau, W., Rouleau, A., Simon, B., Lanneau, D., De Thonel, A., Multhoff, G., Hamman, A., Martin, F., Chauffert, B., Solary, E., Zitvogel, L., Garrido, C., Ryffel, B., Borg, C., Apetoh, L., Rébé, C., Ghiringhelli, F., 2010. Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells. J. Clin. Invest. 120, 457–471. Chen, C., Skog, J., Hsu, C.-H., Lessard, R.T., Balaj, L., Wurdinger, T., Carter, B.S., Breakefield, X.O., Toner, M., Irimia, D., 2010. Microfluidic isolation and transcriptome analysis of serum microvesicles. Lab Chip 10, 505–511. Cho, J., Lee, Y.-S., Kim, S.-H., Ko, J.-K., Kim, C.-W., 2009. MHC independent anti-tumor immune responses induced by Hsp70-enriched exosomes generate tumor regression in murine models. Cancer Lett. 275, 256–265. Crane, C.A., Ahn, B.J., Han, S.J., Parsa, A.T., 2012. Soluble factors secreted by glioblastoma cell lines facilitate recruitment, survival, and expansion of regulatory T cells: implications for immunotherapy. Neuro-Oncology 14, 584–595. Crane, C.A., Han, S.J., Ahn, B., Oehlke, J., Kivett, V., Fedoroff, A., Butowski, N., Chang, S.M., Clarke, J., Berger, M.S., McDermott, M.W., Prados, M.D., Parsa, A.T., 2013. Individual patient-specific immunity against high-grade glioma after vaccination with autologous tumor derived peptides bound to the 96 KD chaperone protein. Clin. Cancer Res. 19, 205–214. Curran, M.A., Montalvo, W., Yagita, H., Allison, J.P., 2010. PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. Proc. Natl. Acad. Sci. U. S. A. 107, 4275–4280. Graner, M.W., Cumming, R.I., Bigner, D.D., 2007. The heat shock response and chaperones/ heat shock proteins in brain tumors: surface expression, release, and possible immune consequences. J. Neurosci. 27, 11214–11227. Graner, M.W., Alzate, O., Dechkovskaia, A.M., Keene, J.D., Sampson, J.H., Mitchell, D.A., Bigner, D.D., 2009. Proteomic and immunologic analyses of brain tumor exosomes. FASEB J. 23, 1541–1557. Han, S.J., Ahn, B.J., Waldron, J.S., Yang, I., Fang, S., Crane, C.A., Pieper, R.O., Parsa, A.T., 2009. Gamma interferon-mediated superinduction of B7-H1 in PTEN-deficient glioblastoma: a paradoxical mechanism of immune evasion. Neuroreport 20, 1597–1602. Han, S.J., Zygourakis, C., Lim, M., Parsa, A.T., 2012. Immunotherapy for glioma: promises and challenges. Neurosurg. Clin. N. Am. 23, 357–370. Henriksen, M., Johnsen, K.B., Olesen, P., Pilgaard, L., Duroux, M., 2014. MicroRNA expression signatures and their correlation with clinicopathological features in glioblastoma multiforme. Neruomol. Med. 16, 565–577. Kucharzewska, P., Christianson, H.C., Welch, J.E., Svensson, K.J., Fredlund, E., Ringnér, M., Mörgelin, M., Bourseau-Guilmain, E., Bengzon, J., Belting, M., 2013. Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development. Proc. Natl. Acad. Sci. U. S. A. 110, 7312–7317. Li, C.C.Y., Eaton, S.A., Young, P.E., Lee, M., Shuttleworth, R., Humphreys, D.T., Grau, G.E., Combes, V., Bebawy, M., Gong, J., Brammah, S., Buckland, M.E., Suter, C.M., 2013. Glioma microvesicles carry selectively packaged coding and non-coding RNAs which alter gene expression in recipient cells. RNA Biol. 10, 1333–1344. Manterola, L., Guruceaga, E., Gállego Pérez-Larraya, J., González-Huarriz, M., Jauregui, P., Tejada, S., Diez-Valle, R., Segura, V., Samprón, N., Barrena, C., Ruiz, I., Agirre, A., Ayuso, A., Rodríguez, J., González, A., Xipell, E., Matheu, A., López de Munain, A., Tuñón, T., Zazpe, I., García-Foncillas, J., Paris, S., Delattre, J.Y., Alonso, M.M., 2014. A small noncoding RNA signature found in exosomes of GBM patient serum as a diagnostic tool. Neuro-Oncology 16, 520–527. Mignot, G., Roux, S., Thery, C., Ségura, E., Zitvogel, L., 2006. Prospects for exosomes in immunotherapy of cancer. J. Cell. Mol. Med. 10, 376–388. Noerholm, M., Balaj, L., Limperg, T., Salehi, A., Zhu, L.D., Hochberg, F.H., Breakefield, X.O., Carter, B.S., Skog, J., 2012. RNA expression patterns in serum microvesicles from patients with glioblastoma multiforme and controls. BMC Cancer 12, 22. Ostrowski, M., Carmo, N.B., Krumeich, S., Fanget, I., Raposo, G., Savina, A., Moita, C.F., Schauer, K., Hume, A.N., Freitas, R.P., Goud, B., Benaroch, P., Hacohen, N., Fukuda, M., Desnos, C., Seabra, M.C., Darchen, F., Amigorena, S., Moita, L.F., Thery, C., 2010. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat. Cell Biol. 12, 19–30 (sup pp 1-13). Parsa, A.T., Waldron, J.S., Panner, A., Crane, C.A., Parney, I.F., Barry, J.J., Cachola, K.E., Murray, J.C., Tihan, T., Jensen, M.C., Mischel, P.S., Stokoe, D., Pieper, R.O., 2007.
Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in glioma. Nat. Med. 13, 84–88. Parsa, A.T., Bloch, O., Crane, C., Kaur, R., Safae, M., 2014. Gliomas promote induction of b7h1/pd-l1 expression on monocytes: clinical evidence of an immunosuppressive mechanism that can be targeted with antibody blockade. Neuro-Oncology 16 (Suppl. 3), iii41. Sayegh, E.T., Oh, T., Fakurnejad, S., Bloch, O., Parsa, A.T., 2014. Vaccine Therapies for Patients with Glioblastoma. J. Neurooncol. Shao, H., Chung, J., Balaj, L., Charest, A., Bigner, D.D., Carter, B.S., Hochberg, F.H., Breakefield, X.O., Weissleder, R., Lee, H., 2012. Protein typing of circulating microvesicles allows real-time monitoring of glioblastoma therapy. Nat. Med. 18, 1835–1840. Skog, J., Würdinger, T., van Rijn, S., Meijer, D.H., Gainche, L., Sena-Esteves, M., Curry, W.T., Carter, B.S., Krichevsky, A.M., Breakefield, X.O., 2008. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 10, 1470–1476. Stupp, R., Mason, W.P., van den Bent, M.J., Weller, M., Fisher, B., Taphoorn, M.J.B., Belanger, K., Brandes, A.A., Marosi, C., Bogdahn, U., Curschmann, J., Janzer, R.C., Ludwin, S.K., Gorlia, T., Allgeier, A., Lacombe, D., Cairncross, J.G., Eisenhauer, E., Mirimanoff, R.O., European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups, National Cancer Institute of Canada Clinical Trials Group, 2005. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N. Engl. J. Med. 352, 987–996. Svensson, K.J., Kucharzewska, P., Christianson, H.C., Sköld, S., Löfstedt, T., Johansson, M.C., Mörgelin, M., Bengzon, J., Ruf, W., Belting, M., 2011. Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells. Proc. Natl. Acad. Sci. U. S. A. 108, 13147–13152. Traugott, U., 1987. Multiple sclerosis: relevance of class I and class II MHC-expressing cells to lesion development. J. Neuroimmunol. 16, 283–302. Valenti, R., Huber, V., Filipazzi, P., Pilla, L., Sovena, G., Villa, A., Corbelli, A., Fais, S., Parmiani, G., Rivoltini, L., 2006. Human tumor-released microvesicles promote the differentiation of myeloid cells with transforming growth factor-beta-mediated suppressive activity on T lymphocytes. Cancer Res. 66, 9290–9298. Wachstein, J., Tischer, S., Figueiredo, C., Limbourg, A., Falk, C., Immenschuh, S., Blasczyk, R., Eiz-Vesper, B., 2012. HSP70 enhances immunosuppressive function of CD4(+)CD25(+)FoxP3(+) T regulatory cells and cytotoxicity in CD4(+)CD25(−) T cells. PLoS One 7, e51747. Waldron, J.S., Yang, I., Han, S., Tihan, T., Sughrue, M.E., Mills, S.A., Pieper, R.O., Parsa, A.T., 2010. Implications for immunotherapy of tumor-mediated T-cell apoptosis associated with loss of the tumor suppressor PTEN in glioblastoma. J. Clin. Neurosci. 17, 1543–1547. Waziri, A., 2010. Glioblastoma-derived mechanisms of systemic immunosuppression. Neurosurg. Clin. N. Am. 21, 31–42. Wolfers, J., Lozier, A., Raposo, G., Regnault, A., Théry, C., Masurier, C., Flament, C., Pouzieux, S., Faure, F., Tursz, T., Angevin, E., Amigorena, S., Zitvogel, L., 2001. Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat. Med. 7, 297–303. Yang, I., Fang, S., Parsa, A.T., 2010. Heat shock proteins in glioblastomas. Neurosurg. Clin. N. Am. 21, 111–123. Yang, C., Ruffner, M.A., Kim, S.-H., Robbins, P.D., 2012. Plasma-derived MHC class II+ exosomes from tumor-bearing mice suppress tumor antigen-specific immune responses. Eur. J. Immunol. 42, 1778–1784. Zhang, H.-G., Grizzle, W.E., 2011. Exosomes and cancer: a newly described pathway of immune suppression. Clin. Cancer Res. 17, 959–964. Zhang, H., Hu, H., Jiang, X., He, H., Cui, L., He, W., 2005. Membrane HSP70: the molecule triggering gammadelta T cells in the early stage of tumorigenesis. Immunol. Investig. 34, 453–468. Zou, J.P., Morford, L.A., Chougnet, C., Dix, A.R., Brooks, A.G., Torres, N., Shuman, J.D., Coligan, J.E., Brooks, W.H., Roszman, T.L., Shearer, G.M., 1999. Human gliomainduced immunosuppression involves soluble factor(s) that alters monocyte cytokine profile and surface markers. J. Immunol. 1950 (162), 4882–4892.
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
The limited capacity of malignant glioma-derived exosomes to suppress peripheral immune effectors J. Bryan Iorgulescu a,b,1, Michael E. Ivan a,⁎,2, Michael Safaee a,b, Andrew T. Parsa a,3,4 a b
Department of Neurological Surgery, UCSF, San Francisco, CA, USA Clinical and Translational Science Institute, UCSF, San Francisco, CA, USA
a r t i c l e
i n f o
Article history: Received 13 October 2015 Received in revised form 17 November 2015 Accepted 28 November 2015 Keywords: Glioma Exosome Microvesicle Immunosuppression Immunotherapy
a b s t r a c t Tumor-derived microvesicular exosomes permit intercellular communication both locally and systemically by delivering a snapshot of the tumor cell's constituents. We thus investigated whether exosomes mediate malignant glioma's facility for inducing peripheral immunosuppression. In Western blot and RT-PCR analyses, glioma-derived exosomes displayed exosome-specific markers, but failed to recapitulate the antigenpresentation machinery, surface co-modulatory signals, or immunosuppressive mediator status of their parent tumor cells. Treatment with glioma-derived exosomes promoted immunosuppressive HLA-DRlow monocytic phenotypes, but failed to induce monocytic PD-L1 expression or alter the activation of cytotoxic T-cells from patients' peripheral blood by FACS and RT-PCR analyses. Our results suggest that malignant glioma-derived exosomes are restricted in their capacity to directly prime peripheral immunosuppression. © 2015 Elsevier B.V. All rights reserved.
Author disclosure statement: The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.
1. Introduction Malignant gliomas, which universally portend a median survival less than 15 months, exhibit a tenacious propensity to subdue immune responses both locally and systemically (Stupp et al., 2005). In the local tumor environment, gliomas readily release anti-inflammatory cytokines, employ immunosuppressive surface ligands, and down-regulate their surface antigen-presentation machinery in order to successfully evade immune surveillance (Albesiano et al., 2010; Han et al., 2012; Waziri, 2010). Simultaneously, malignant gliomas secrete as of yet unidentified soluble factors that coax circulating monocytes and T lymphocytes into infiltrating the tumor as their anti-inflammatory phenotypes: M2 macrophages that up-regulate IL-10 and programmed
⁎ Corresponding author at: Department of Neurological Surgery, University of Miami Miller School of Medicine, Lois Pope Life Center, 1095 NW 14th Ter, Miami, FL 33136, USA. E-mail address: [email protected] (M.E. Ivan). 1 Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. 2 Department of Neurological Surgery, University of Miami, Miami, FL, USA. 3 Department of Neurological Surgery, Northwestern Feinberg School of Medicine, Chicago, IL, USA. 4 Deceased April 13, 2015.
http://dx.doi.org/10.1016/j.jneuroim.2015.11.025 0165-5728/© 2015 Elsevier B.V. All rights reserved.
death ligand 1 (PD-L1) and regulatory T lymphocytes, which together culminate in the apoptosis of anti-tumoral T lymphocytes (Bloch et al., 2013; Crane et al., 2012; Parsa et al., 2014; Zou et al., 1999). There is growing evidence suggesting that gliomas can influence cells both nearby and beyond the blood–brain barrier via the carefully regulated excretion of exosomes: 40–100 nm microvesicles formed during the normal cellular membrane sorting cycle by the inward budding of late endosomes (Ostrowski et al., 2010). These multi-vesicular endosomes are typically destined for recycling by lysosomes, but occasionally fuse with the plasma membrane and thereby release their microvesicle contents into the extracellular milieu. Exosomes contain a representative profile of the proteins, mRNAs, and microRNAs of their parent T-cells; in glioblastoma (GBM) this exosomal content reflects the tumor's unique signature of EGFR amplification, EGFRvIII mutation, IDH1 mutation R132H, TGF-β, and/or podoplanin status (Al-Nedawi et al., 2008; Graner et al., 2009; Henriksen et al., 2014; Li et al., 2013; Manterola et al., 2014; Noerholm et al., 2012; Shao et al., 2012). GBM-derived exosomes also may beckon angiogenesis during hypoxia, by delivery of proteins (e.g. metalloproteinase 8, IL-8, and PDGF AA/AB) and mRNA transcripts (e.g. IGF-binding protein, BCL-2, and N-myc downstream regulator 1) to vascular endothelial cells and pericytes (Kucharzewska et al., 2013; Svensson et al., 2011). MiR-1 microRNA dysregulation in GBMs, in addition to unleashing the oncogenic JNK, MET, and EGFR signaling pathways, has also been shown to uncheck the high levels of Annexin A2 packaged in GBM-derived exosomes, thus driving more aggressive GBM growth, neovascularization, and invasion (Bronisz et al., 2014). Tumor-derived exosomes have also been shown to contribute to immunosuppression in a number of other tumor types; notably, exosomes distilled from the sera of
104
J.B. Iorgulescu et al. / Journal of Neuroimmunology 290 (2016) 103–108
patients with advanced melanoma or colon carcinoma not only hindered monocyte precursor differentiation into dendritic cells, but in fact programmed the precursors into myeloid suppressive cells characterized by down-regulation of surface MHC class II expression, persistent CD14 pattern recognition receptor positivity, and suppression of T lymphocytes by way of TGF-β secretion; whereas exosomes derived from normal control patients' sera encouraged myeloid maturation and T lymphocyte stimulation (Valenti et al., 2006; Zhang and Grizzle, 2011). With the growing body of evidence implicating exosomes as an intercellular mediator of GBM pathophysiology, we hypothesized that they may comprise the unidentified soluble factors that exit the blood–brain barrier and reprogram distant immune effectors into their immunosuppressive phenotypes. In order to directly prime T lymphocytes responses, glioma-derived exosomes would require both surface antigen presentation machinery (including either MHC class I or class II molecules) and requisite co-modulatory signals. Alternatively, immune effectors can also be activated by the direct uptake of antigens bound to heat shock protein chaperones (HSP) released during cell necrosis (Bendz et al., 2007; Cho et al., 2009). Gliomas robustly express a number of constitutive and inducible HSPs, particularly HSP70 and HSP90 families, which chaperone the folding of several drivers of oncogenesis (Bloch et al., 2014; Graner et al., 2007; Yang et al., 2010). HSP70 has also been observed on the glioma cell surface, where it directly interacts with natural killer cells and γδ T lymphocytes (Wachstein et al., 2012; Zhang et al., 2005). HSP72 was identified on exosomes derived from a number of tumor types, which enabled the tumor-derived exosomes to trigger immunosuppressive Stat3 pathways in myeloidderived cells (Chalmin et al., 2010). Herein we investigated whether glioma-derived exosomes have the capacity to promote immunosuppressive phenotypes of effectors from myeloid or lymphoid lineages.
pellets were resuspended in either PBS for cell treatment or lysis buffer for Western blot analysis. 2.3. Western blot analysis Cells and exosomes were lysed by radioimmunoprecipitation assay buffer (RIPA; UCSF Cell Culture Facility) supplemented with protease inhibitors (Roche Diagnostics) on a 4 °C shaker for 45 min. Protein concentrations were determined by BCA Protein Assay Kit (Pierce) and loaded in equal amounts for polyacrylamide gel electrophoresis, with either SDS or non-reducing conditions as specified by the antibody manufacturer, then transferred to PVDF membranes for blocking and subsequent probing with primary antibodies against: CD81 (Santa Cruz Biotech), CD9 (Abcam), HSP70 (Santa Cruz Biotech), HSP90 (Abcam), GAPDH (Cell Signal), CTLA4 (Abcam), CD80 (Abcam), CD86 (Abcam), PD-L1 (Abcam), HLA-ABC (Biolegend), and HLA-DR (Biolegend). Secondary antibodies conjugated to horseradish peroxidase visualized the proteins by way chemilluminescence (ECL Western blotting substrate; Pierce). 2.4. Cell sorting As previously described, peripheral blood leukocytes (PBL) were isolated from primary GBM patients' and healthy naïve donors' whole blood samples by Ficoll-Paque Plus (GE Healthcare) centrifugation.(Bloch et al., 2013; Crane et al., 2012) Monocytes were extracted by CD14+ selection using magnetic nanoparticles (EasySep, Stem Cell Technologies) and T lymphocytes by negative magnetic nanoparticle selection, then suspended in RPMI-1640 media supplemented with 1% penicillin–streptomycin, 1 mmol/L sodium pyruvate, 10 mmol/L nonessential amino acids, and 2.5% exosome-depleted FBS.
2. Methods
2.5. Treatment of cells
2.1. Cell culture
Monocytes were plated at 2 × 105 cells per well in a 24 well plate and incubated at 37 °C for 24 h with escalating doses of exosomes from established or primary glioma lines, as compared to positive controls treated with glioma conditioned media and untreated controls, in triplicate. T lymphocytes were plated at 3.1 × 105 per well in a 96 well plate and incubated at 37 °C for 24 h with Golgi plug protein transport inhibitor (BD Bioscience) and either exosomes from established or primary glioma lines, compared to treatment with NHA-derived exosomes or untreated controls.
Established glioma cell lines U87 and U251 were obtained through the UCSF Brain Tumor Research Center and cultured in Dulbecco's modified Eagle medium H21 (UCSF Cell Culture Facility). Primary GBM lines from two patients, denoted as GBM1 and GBM2, were derived from patients' fresh tumor tissue collected during initial resection and cultured for no more than 15 passages in RPMI-1640 media supplemented with 10 mmol/L non-essential amino acids. All glioma cultures were additionally supplemented with and 1% penicillin–streptomycin and 5% fetal bovine serum (FBS) depleted of exosomes by ultracentrifugation at 110,000 g at 4 °C for 4 h and careful collection of the resulting supernatant. Normal human astrocytes (NHA) were acquired from Sciencell Laboratories and cultured in Lonza proprietary media with exosomedepleted FBS. 2.2. Exosome isolation Conditioned media were harvested after 48 h from cultures at 80– 90% confluence and centrifuged at 600 g at 4 °C for 10 min to precipitate cell debris. The supernatant was then centrifuged at 16,500 g at 4 °C for 20 min and passed through a 0.22 μL filter (Milipore). The resulting supernatant was ultracentrifuged at 110,000 g at 4 °C for 1 h to pellet the exosomes, which were then resuspended in a phosphate-buffered saline (PBS) wash and ultracentrifuged again at 110,000 g at 4 °C for 1 h. In order to maximize detection of the effects of exosomes in our experiments, concentrations many magnitudes higher than found in circulation were used. Additionally, protein was precipitated from supernatant fractions of exosome-depleted conditioned media by using 20% trichloroacetic acid on ice, thrice washed with 1:1 acetone and ethanol, and pelleted by 16,500 g at 4 °C for 20 min. Exosome and protein
2.6. Flow cytometry analysis Monocytes were stained extracellularly with CD45 FITC (clone HI30, eBioscience), CD11b PeCy (clone ICRF44, eBioscience), HLA-DR APC (clone LN3, eBioscience), and PD-L1 PE (clone MIH1, eBioscience) or isotype control (eBioscience) and T lymphocytes extracellularly with CD3 PerCP (eBioscience), CD4 PE (eBioscience), and CD8 FITC (BD Pharmingen), and intracellularly with IFNγ APC (eBioscience) in saponin-containing buffer (PermWash, BD Pharmingen), all in PBS with 2% bovine serum albumin on ice for 30 min, washed, fixed with 2% paraformaldehyde, and read using a BD FACScaliber flow cytometer with CellQuest Software (Beckton Dickinson), as previously described (Bloch et al., 2013; Crane et al., 2012). PBLs were treated in triplicate. The gate strategy for identifying monocytes included forward vs side scatter gates for size, then CD45 vs CD11b gates for monocytic phenotype, with CD11b vs PDL-1 to assess PDL-1 expression levels, and CD11b vs HLA-DR to evaluate monocyte phenotype changes. CD8+ Tcells were identified by forward vs side scatter for size, and forward scatter vs CD3 to confirm T-lymphocytic phenotype, with CD8 vs CD4 for identifying CD8+ T-cells and CD8 vs IFNγ for assessing changes in CD8+ T-cell activation levels.
J.B. Iorgulescu et al. / Journal of Neuroimmunology 290 (2016) 103–108
105
the media protein precipitate for exosome-specific markers CD9 and CD81. 3.2. Glioma-derived exosomes lacked antigen-presentation machinery Glioma-derived exosomes demonstrated no appreciable content of MHC class I (HLA-ABC) or class II (HLA-DR) molecules, which respectively prime CD8+ and CD4+ T-cell responses; with the exception of U87 samples, which selectively concentrated HLA-DR into exosomal fractions (Fig. 1B). As in prior reports, astrocytes from normal central nervous tissue (NHA) were devoid of HLA-ABC (Traugott, 1987). Additionally, we found robust expression of HSP70 and HSP90 in all glioma lines, but with limited content within exosome fractions (Fig. 1C). 3.3. Glioma-derived exosomes lacked surface co-modulatory molecules
Fig. 1. Protein content of respective cellular and exosomal fractions. (A) Exosome isolation was confirmed by blotting for exosome-specific tetraspanins CD9 and CD81, with cellular fractions identified by GAPDH. Regardless of expression in parent cellular fractions, glioma-derived exosomes lacked appreciable (B) HLA-ABC or HLA-DR antigen presentation machinery, (C) heat shock protein 70 or 90 families of antigen chaperones, or (D) requisite co-modulatory signals CD80, CD86, and CTLA-4.
In addition to surface antigen-presentation machinery, concomitant binding of surface co-modulatory molecules is required to properly guide a T lymphocyte response against an antigen. CD80 (B7-1) and CD86 (B7-2) are co-modulatory surface molecules respectively found on activated monocytes and antigen-presenting cells; which can bind either stimulatory CD28 or inhibitory CTLA-4 cognate T lymphocyte receptors. The glioma lines exhibited CD86 and limited CD80 expression,
2.7. Reverse transcribed-quantitative PCR mRNA was extracted from cells using the RNeasy Mini Kit (Qiagen), transcribed to cDNA with the Superscript III Kit (Invitrogen), and subsequently quantified by CFX96 Real-Time System (Bio-Rad Laboratories) detection of transcript-incorporated SYBR Green (Applied Biosystems), as previously reported. Cycle thresholds for PD-L1 (5′-GCTGTT-GAAG GA-CCAGCT-CT/TGCTTG-TCCAGA-TGACTT-CG-3′) and IFNγ (5′-CGAG AT-GACTTC-GAAAAG-CTG/ATATTG-CAGGCA-GGACAA-CC-3′) were compared to 18S rRNA (5′-GTAACC-CGTTGA-ACCCCA-TT/CCATCCAATCGG-TAGTAG-CG-3′). 2.8. Statistical analysis Data were compared by one-way analysis of variance (ANOVA) with post-hoc Bonferroni corrections, using SPSS version 19 (IBM), and reported as mean ± standard error of the mean (SEM). Significance was defined as p b 0.05. 3. Results 3.1. Exosome isolation Exosomes were purified by serial ultracentrifugation and filtration of conditioned media from both established (U87 and U251) and patientderived primary (GBM1 and GBM2) glioma lines, with NHA lines as controls. Purification was confirmed by Western blot probing for the exosome-specific tetraspanins CD9 and CD81 (Fig. 1A). Whether house-keeping proteins are equally partitioned into exosomal and cellular compartments remains unclear, thus CD81was used as a loading control for exosome samples and GAPDH as a loading control for cell samples, with qualitative comparison of densitometry between corresponding exosomal and cellular fractions. Removal of all cellular fractions from exosome samples was confirmed by probing for GAPDH, one house-keeping protein found to be cellular-specific and not enriched in exosome samples. Likewise, complete extraction of exosome fractions from conditioned media was confirmed by probing
Fig. 2. Glioma-derived exosomes failed to promote PD-L1 expression in, or deliver PD-L1 to peripheral monocytes. (A) Monocytes were identified by flow cytometry for CD11b and CD45 positivity and (B) demonstrated no change in PD-L1 mean fluorescence intensity (MFI) when treated with glioma-derived exosomes (15.5 ± 0.3 from U87 exosomes, p = 1.00; and 15.5 ± 0.3 from GBM2 exosomes, p = 1.00), as compared to untreated controls (16.0 ± 0.3). Glioma conditioned media (CM) was a positive control of immunosuppression (20.8 ± 0.5, ***p b 0.001). Dotted black line denotes the isotype control. (C) Glioma-derived exosomes also lacked PD-L1 protein or mRNA content, regardless of expression in parent cell fractions. avg CT: average cycle threshold.
106
J.B. Iorgulescu et al. / Journal of Neuroimmunology 290 (2016) 103–108
cytometry after 24 h of culture and exhibited no significant change following treatment with glioma-derived exosomes, as compared to untreated controls (MFI 15.5 ± 0.3 from U87 exosomes, p = 1.00 and 15.5 ± 0.3 from GBM2 exosomes, p = 1.00; vs untreated controls 16.0 ± 0.3) (Fig. 2B). There was additionally no benefit to treatment with double the concentration of glioma-derived exosomes (15.2 ± 0.2 from U87 exosomes, p = 1.00; and 15.1 ± 0.3 from GBM2 exosomes, p = 0.88); whereas the conditioned media-treated controls expressed significantly more PD-L1 (20.8 ± 0.5) than all treated (p b 0.001) or untreated groups (p b 0.001). In addition, quantitative RT PCR revealed that exosomes derived from U87, U251, GBM1, and GBM2 cultures were devoid of PD-L1 transcripts (Fig. 2C). Similarly, despite variable expression of PD-L1 in parent glioma lines, no PD-L1 protein content was found in their respective exosome fractions.
3.5. Glioma-derived exosomes induced immunosuppressive surface HLA-DRlow expression on peripheral monocytes
Fig. 3. Glioma-derived exosomes promoted immunosuppressive HLA-DRlow phenotypes in peripheral monocytes. (A) Monocytes were identified by flow cytometry for CD11b and CD45 positivity and (B) displayed significant reductions in HLA-DR mean fluorescence intensity (MFI) when treated with glioma-derived exosomes (460.8 ± 7.1 from U87 exosomes, #p = 0.039; and 451.8 ± 9.5 from GBM2 exosomes, ##p = 0.009), as compared to untreated controls (502.4 ± 9.2). Glioma conditioned media (CM) was a positive control of immunosuppression (262.1 ± 7.9, ***p b 0.001). Dotted black line denotes the isotype control. avg CT: average cycle threshold.
although this expression was not reflected in their exosome fractions (Fig. 1D). CTLA-4 is posited to out-compete CD28 in binding CD80 and CD86, thereby preventing activation of T lymphocytes in a dosedependent fashion (Curran et al., 2010). Glioma-derived exosomes were thus examined for CTLA-4 content, which could either be delivered to T-cells or monopolize the binding of CD80 and CD86 ligands, and were found to be limited in their content of CTLA-4 regardless of expression levels in parent cells (Fig. 1D). 3.4. Glioma-derived exosomes failed to induce PD-L1 expression in peripheral monocytes To investigate whether gliomas employ exosomes in inducing PD-L1 expression in peripheral monocytes, monocytes from a naïve patient were treated with exosomes from both established (U87) and primary (GBM2) glioma lines. Treatment with concentrated conditioned media from GBM cell culture, previously shown to induce robust PD-L1 expression in peripheral monocytes, served as an immunosuppressive positive control (Bloch et al., 2013; Parsa et al., 2014). Flow cytometry experiments were conducted in triplicate and measured in mean fluorescence intensities (MFI). PD-L1 surface expression was assessed by flow
Surface levels of HLA-DR on peripheral monocytes, typically increased in pro-inflammatory phenotypes and reduced in immunosuppressive phenotypes, was examined following 24 h of treatment with glioma-derived exosomes, and exhibited significantly lower levels of monocyte HLA-DR following treatment with GBM2-derived exosomes (MFI 451.8 ± 9.5, p = 0.009) or U87-derived exosomes (460.8 ± 7.1, p = 0.039), as compared to untreated controls (502.4 ± 9.2), reflecting a switch from inactivated or pro-inflammatory monocytic phenotypes to HLA-DRlow immunosuppressive phenotypes (Fig. 3B). These trends were not shared by monocytes treated with twice the concentration of glioma-derived exosomes (470.1 ± 8.1 from GBM2 exosomes, p = 0.186; and 462.3 ± 3.3 from U87 exosomes, p = 0.005). Condition media-treated controls displayed significantly lower HLA-DR expression than all other groups (262.1 ± 7.9, p b 0.001).
3.6. Glioma-derived exosomes failed to alter the activation of peripheral CD8+ T lymphocytes Activation of peripheral CD8 + cytotoxic T lymphocytes following 24 h of treatment with glioma-derived exosomes was assessed by flow cytometry for intracellularly stained interferon-γ (IFNγ). There were no significant differences in IFNγ levels following treatment of GBM patient peripheral T-cells with autologous GBM-derived exosomes, as compared to either untreated or NHA exosome-treated controls (Table 1, Fig. 4). T-cells from a naïve non-glioma patient provided an additional control, and also failed to alter IFNγ levels. There appeared to be limited activation of T-cells when treated with nonautologous exosomes (either derived from U251 or NHA), which likely represents T-cell recognition of foreign surface-bound antigens. IFNγ mRNA levels remained below threshold for detection by quantitative RT PCR, despite moderate 18S levels, for both untreated and gliomaderived exosome treated groups of naïve patient T-cells. Additionally, there were no significant differences in the proportions of CD8 + or CD4+ T-cells following treatment with glioma-derived exosomes.
Table 1 Cytotoxic T lymphocyte IFNγ levels following exosome treatment. Naïve
GBM1
GBM2
Treatment
MFI
SEM
p-value
MFI
SEM
p-value
MFI
SEM
p-value
Untreated (reference) Autologous GBM exosomes⁎ NHA exosomes U251 exosomes
7.1 7.8 8.1 8.7
0 0.3 0 0.6
1 0.62 0.17
7.8 8.1 10.4 11.9
0.4 0.4 0.2 0.7
1 0.87 0.02
8.2 7.9 8.3 8.6
0.3 0.1 0.5 0.6
1 1 1
MFI: mean fluorescence intensity, SEM: standard error of the mean. ⁎ Naïve patient peripheral T-cells were treated with GBM2-derived exosomes.
J.B. Iorgulescu et al. / Journal of Neuroimmunology 290 (2016) 103–108
107
Fig. 4. Cytotoxic T lymphocyte activation was not altered by glioma-derived exosomes. Treatment with glioma-derived exosomes demonstrated no change in IFNγ levels in CD8+ T-cells from (A) naïve non-glioma, (B) GBM1, and (C) GBM2 patients' PBLs, as compared to untreated and NHA-derived exosome-treated controls. * Naïve patient peripheral T-cells were treated with GBM2-derived exosomes.
4. Discussion GBM's proficiency at subverting local antitumoral immunity and globally suppressing immune effectors poses a key obstacle to the efficacy of immunotherapies (Crane et al., 2013; Han et al., 2012; Sayegh et al., 2014). We have previously demonstrated that dysregulation of PTEN and PI(3)K pathways in glioma leads to robust PD-L1 surface expression, which widely subdues the activation of tumor-infiltrating immune effectors upon binding of its cognate PD-1 receptor, as well as outcompetes T lymphocytes for the affinity of CD80 costimulatory signals (Han et al., 2009; Parsa et al., 2007; Waldron et al., 2010). We subsequently found that gliomas also secrete unidentified soluble factors which promote PD-L1 expression by monocytes circulating in the periphery (Bloch et al., 2013; Crane et al., 2012). With growing evidence that tumor-released exosomes contribute to immunosuppression in other tumor types and may play a role in glioma pathogenesis, we hypothesized that glioma-derived exosomes may comprise glioma's secreted soluble factors which promote immunosuppressive phenotypes (Valenti et al., 2006; Zhang and Grizzle, 2011). However, gliomaderived exosomes lacked the necessary antigen presentation machinery for priming immune responses and, other than promoting significant but limited immunosuppressive HLA-DRlow expression in peripheral monocytes, failed to directly induce phenotypic changes in peripheral blood immune effectors in vitro. It is likely that antigens carried by glioma-derived exosomes first require processing by antigen presenting cells (APCs), in order to appropriately prime an antigen-specific immune response, as observed in other tumor models (Wolfers et al., 2001; Yang et al., 2012). In fact, several in vitro experiments have harnessed APCs' capacity for processing antigens from glioma-derived and tumor-derived exosomes to successfully prime antigen-specific antitumoral immune responses (André et al., 2004; Bu et al., 2011; Mignot et al., 2006). Prophylactic vaccination of mouse glioma models with glioma-derived exosomes resulted in robust antitumoral immunity, complete rejection of subsequent glioma implantation, and increased overall survival (Graner et al., 2009). However, these successes were not mirrored by mice with pre-existing orthotopic gliomas, in which glioma-derived exosome-stimulated vaccines failed to induce tumor rejection or improve survival, partly as a consequence of the intact immunosuppression wrought by malignant gliomas. 5. Conclusions Our results suggest that malignant glioma-derived exosomes are restricted in their ability to directly prime peripheral immunosuppression. Although glioma-derived exosomes may play a limited direct role in immunosuppression, there is burgeoning promise in their diagnostic utility as a non-invasive means of typing gliomas by only using tumor exosomes from blood samples (Chen et al., 2010; Shao et al., 2012;
Skog et al., 2008). Likewise, as a circulating reservoir of tumor antigens, glioma-derived exosomes may help overcome a key hurdle in vaccine therapies for malignant glioma: the need for repeat tissue biopsies of recurrences in order to re-stimulate the vaccine against the newly selected-for glioma phenotypes (Bloch et al., 2014; Crane et al., 2013; Han et al., 2012; Sayegh et al., 2014). Analysis of circulating gliomaderived exosomes could obviate our reliance on delayed clinical or radiographic signs of recurrence, by permitting the real-time monitoring of glioma antigens. Exosomes are an exciting facet of cellular biology, one in which further inquiry is necessary for teasing apart what roles exosomes play in cancer pathophysiology versus those instances where exosomes are merely physiologic byproducts of cellular garbage disposal systems. Acknowledgments This work was graciously supported by Doris Duke Clinical Research Fellowships (JBI and MS), the Clinical and Translational Science Institute at UCSF (JBI and MS), the National Research and Education Foundation (MEI), and the Michael J. Marchese Professor and Chair at Northwestern University (ATP). We thank O. Bloch for technical guidance and R. Kaur for technical assistance. We are incomparably grateful for Andew Parsa's mentorship, supervision, and stewardship of this study. References Albesiano, E., Han, J.E., Lim, M., 2010. Mechanisms of local immunoresistance in glioma. Neurosurg. Clin. N. Am. 21, 17–29. Al-Nedawi, K., Meehan, B., Micallef, J., Lhotak, V., May, L., Guha, A., Rak, J., 2008. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat. Cell Biol. 10, 619–624. André, F., Chaput, N., Schartz, N.E.C., Flament, C., Aubert, N., Bernard, J., Lemonnier, F., Raposo, G., Escudier, B., Hsu, D.-H., Tursz, T., Amigorena, S., Angevin, E., Zitvogel, L., 2004. Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells. J. Immunol. 1950 (172), 2126–2136. Bendz, H., Ruhland, S.C., Pandya, M.J., Hainzl, O., Riegelsberger, S., Braüchle, C., Mayer, M.P., Buchner, J., Issels, R.D., Noessner, E., 2007. Human heat shock protein 70 enhances tumor antigen presentation through complex formation and intracellular antigen delivery without innate immune signaling. J. Biol. Chem. 282, 31688–31702. Bloch, O., Crane, C.A., Kaur, R., Safaee, M., Rutkowski, M.J., Parsa, A.T., 2013. Gliomas promote immunosuppression through induction of B7-H1 expression in tumorassociated macrophages. Clin. Cancer Res. 19, 3165–3175. Bloch, O., Crane, C.A., Fuks, Y., Kaur, R., Aghi, M.K., Berger, M.S., Butowski, N.A., Chang, S.M., Clarke, J.L., McDermott, M.W., Prados, M.D., Sloan, A.E., Bruce, J.N., Parsa, A.T., 2014. Heat-shock protein peptide complex-96 vaccination for recurrent glioblastoma: a phase II, single-arm trial. Neuro-Oncology 16, 274–279. Bronisz, A., Wang, Y., Nowicki, M.O., Peruzzi, P., Ansari, K.I., Ogawa, D., Balaj, L., De Rienzo, G., Mineo, M., Nakano, I., Ostrowski, M.C., Hochberg, F., Weissleder, R., Lawler, S.E., Chiocca, E.A., Godlewski, J., 2014. Extracellular vesicles modulate the glioblastoma microenvironment via a tumor suppression signaling network directed by miR-1. Cancer Res. 74, 738–750. Bu, N., Wu, H., Sun, B., Zhang, G., Zhan, S., Zhang, R., Zhou, L., 2011. Exosome-loaded dendritic cells elicit tumor-specific CD8 + cytotoxic T cells in patients with glioma. J. Neuro-Oncol. 104, 659–667.
108
J.B. Iorgulescu et al. / Journal of Neuroimmunology 290 (2016) 103–108
Chalmin, F., Ladoire, S., Mignot, G., Vincent, J., Bruchard, M., Remy-Martin, J.-P., Boireau, W., Rouleau, A., Simon, B., Lanneau, D., De Thonel, A., Multhoff, G., Hamman, A., Martin, F., Chauffert, B., Solary, E., Zitvogel, L., Garrido, C., Ryffel, B., Borg, C., Apetoh, L., Rébé, C., Ghiringhelli, F., 2010. Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells. J. Clin. Invest. 120, 457–471. Chen, C., Skog, J., Hsu, C.-H., Lessard, R.T., Balaj, L., Wurdinger, T., Carter, B.S., Breakefield, X.O., Toner, M., Irimia, D., 2010. Microfluidic isolation and transcriptome analysis of serum microvesicles. Lab Chip 10, 505–511. Cho, J., Lee, Y.-S., Kim, S.-H., Ko, J.-K., Kim, C.-W., 2009. MHC independent anti-tumor immune responses induced by Hsp70-enriched exosomes generate tumor regression in murine models. Cancer Lett. 275, 256–265. Crane, C.A., Ahn, B.J., Han, S.J., Parsa, A.T., 2012. Soluble factors secreted by glioblastoma cell lines facilitate recruitment, survival, and expansion of regulatory T cells: implications for immunotherapy. Neuro-Oncology 14, 584–595. Crane, C.A., Han, S.J., Ahn, B., Oehlke, J., Kivett, V., Fedoroff, A., Butowski, N., Chang, S.M., Clarke, J., Berger, M.S., McDermott, M.W., Prados, M.D., Parsa, A.T., 2013. Individual patient-specific immunity against high-grade glioma after vaccination with autologous tumor derived peptides bound to the 96 KD chaperone protein. Clin. Cancer Res. 19, 205–214. Curran, M.A., Montalvo, W., Yagita, H., Allison, J.P., 2010. PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. Proc. Natl. Acad. Sci. U. S. A. 107, 4275–4280. Graner, M.W., Cumming, R.I., Bigner, D.D., 2007. The heat shock response and chaperones/ heat shock proteins in brain tumors: surface expression, release, and possible immune consequences. J. Neurosci. 27, 11214–11227. Graner, M.W., Alzate, O., Dechkovskaia, A.M., Keene, J.D., Sampson, J.H., Mitchell, D.A., Bigner, D.D., 2009. Proteomic and immunologic analyses of brain tumor exosomes. FASEB J. 23, 1541–1557. Han, S.J., Ahn, B.J., Waldron, J.S., Yang, I., Fang, S., Crane, C.A., Pieper, R.O., Parsa, A.T., 2009. Gamma interferon-mediated superinduction of B7-H1 in PTEN-deficient glioblastoma: a paradoxical mechanism of immune evasion. Neuroreport 20, 1597–1602. Han, S.J., Zygourakis, C., Lim, M., Parsa, A.T., 2012. Immunotherapy for glioma: promises and challenges. Neurosurg. Clin. N. Am. 23, 357–370. Henriksen, M., Johnsen, K.B., Olesen, P., Pilgaard, L., Duroux, M., 2014. MicroRNA expression signatures and their correlation with clinicopathological features in glioblastoma multiforme. Neruomol. Med. 16, 565–577. Kucharzewska, P., Christianson, H.C., Welch, J.E., Svensson, K.J., Fredlund, E., Ringnér, M., Mörgelin, M., Bourseau-Guilmain, E., Bengzon, J., Belting, M., 2013. Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development. Proc. Natl. Acad. Sci. U. S. A. 110, 7312–7317. Li, C.C.Y., Eaton, S.A., Young, P.E., Lee, M., Shuttleworth, R., Humphreys, D.T., Grau, G.E., Combes, V., Bebawy, M., Gong, J., Brammah, S., Buckland, M.E., Suter, C.M., 2013. Glioma microvesicles carry selectively packaged coding and non-coding RNAs which alter gene expression in recipient cells. RNA Biol. 10, 1333–1344. Manterola, L., Guruceaga, E., Gállego Pérez-Larraya, J., González-Huarriz, M., Jauregui, P., Tejada, S., Diez-Valle, R., Segura, V., Samprón, N., Barrena, C., Ruiz, I., Agirre, A., Ayuso, A., Rodríguez, J., González, A., Xipell, E., Matheu, A., López de Munain, A., Tuñón, T., Zazpe, I., García-Foncillas, J., Paris, S., Delattre, J.Y., Alonso, M.M., 2014. A small noncoding RNA signature found in exosomes of GBM patient serum as a diagnostic tool. Neuro-Oncology 16, 520–527. Mignot, G., Roux, S., Thery, C., Ségura, E., Zitvogel, L., 2006. Prospects for exosomes in immunotherapy of cancer. J. Cell. Mol. Med. 10, 376–388. Noerholm, M., Balaj, L., Limperg, T., Salehi, A., Zhu, L.D., Hochberg, F.H., Breakefield, X.O., Carter, B.S., Skog, J., 2012. RNA expression patterns in serum microvesicles from patients with glioblastoma multiforme and controls. BMC Cancer 12, 22. Ostrowski, M., Carmo, N.B., Krumeich, S., Fanget, I., Raposo, G., Savina, A., Moita, C.F., Schauer, K., Hume, A.N., Freitas, R.P., Goud, B., Benaroch, P., Hacohen, N., Fukuda, M., Desnos, C., Seabra, M.C., Darchen, F., Amigorena, S., Moita, L.F., Thery, C., 2010. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat. Cell Biol. 12, 19–30 (sup pp 1-13). Parsa, A.T., Waldron, J.S., Panner, A., Crane, C.A., Parney, I.F., Barry, J.J., Cachola, K.E., Murray, J.C., Tihan, T., Jensen, M.C., Mischel, P.S., Stokoe, D., Pieper, R.O., 2007.
Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in glioma. Nat. Med. 13, 84–88. Parsa, A.T., Bloch, O., Crane, C., Kaur, R., Safae, M., 2014. Gliomas promote induction of b7h1/pd-l1 expression on monocytes: clinical evidence of an immunosuppressive mechanism that can be targeted with antibody blockade. Neuro-Oncology 16 (Suppl. 3), iii41. Sayegh, E.T., Oh, T., Fakurnejad, S., Bloch, O., Parsa, A.T., 2014. Vaccine Therapies for Patients with Glioblastoma. J. Neurooncol. Shao, H., Chung, J., Balaj, L., Charest, A., Bigner, D.D., Carter, B.S., Hochberg, F.H., Breakefield, X.O., Weissleder, R., Lee, H., 2012. Protein typing of circulating microvesicles allows real-time monitoring of glioblastoma therapy. Nat. Med. 18, 1835–1840. Skog, J., Würdinger, T., van Rijn, S., Meijer, D.H., Gainche, L., Sena-Esteves, M., Curry, W.T., Carter, B.S., Krichevsky, A.M., Breakefield, X.O., 2008. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 10, 1470–1476. Stupp, R., Mason, W.P., van den Bent, M.J., Weller, M., Fisher, B., Taphoorn, M.J.B., Belanger, K., Brandes, A.A., Marosi, C., Bogdahn, U., Curschmann, J., Janzer, R.C., Ludwin, S.K., Gorlia, T., Allgeier, A., Lacombe, D., Cairncross, J.G., Eisenhauer, E., Mirimanoff, R.O., European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups, National Cancer Institute of Canada Clinical Trials Group, 2005. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N. Engl. J. Med. 352, 987–996. Svensson, K.J., Kucharzewska, P., Christianson, H.C., Sköld, S., Löfstedt, T., Johansson, M.C., Mörgelin, M., Bengzon, J., Ruf, W., Belting, M., 2011. Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells. Proc. Natl. Acad. Sci. U. S. A. 108, 13147–13152. Traugott, U., 1987. Multiple sclerosis: relevance of class I and class II MHC-expressing cells to lesion development. J. Neuroimmunol. 16, 283–302. Valenti, R., Huber, V., Filipazzi, P., Pilla, L., Sovena, G., Villa, A., Corbelli, A., Fais, S., Parmiani, G., Rivoltini, L., 2006. Human tumor-released microvesicles promote the differentiation of myeloid cells with transforming growth factor-beta-mediated suppressive activity on T lymphocytes. Cancer Res. 66, 9290–9298. Wachstein, J., Tischer, S., Figueiredo, C., Limbourg, A., Falk, C., Immenschuh, S., Blasczyk, R., Eiz-Vesper, B., 2012. HSP70 enhances immunosuppressive function of CD4(+)CD25(+)FoxP3(+) T regulatory cells and cytotoxicity in CD4(+)CD25(−) T cells. PLoS One 7, e51747. Waldron, J.S., Yang, I., Han, S., Tihan, T., Sughrue, M.E., Mills, S.A., Pieper, R.O., Parsa, A.T., 2010. Implications for immunotherapy of tumor-mediated T-cell apoptosis associated with loss of the tumor suppressor PTEN in glioblastoma. J. Clin. Neurosci. 17, 1543–1547. Waziri, A., 2010. Glioblastoma-derived mechanisms of systemic immunosuppression. Neurosurg. Clin. N. Am. 21, 31–42. Wolfers, J., Lozier, A., Raposo, G., Regnault, A., Théry, C., Masurier, C., Flament, C., Pouzieux, S., Faure, F., Tursz, T., Angevin, E., Amigorena, S., Zitvogel, L., 2001. Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat. Med. 7, 297–303. Yang, I., Fang, S., Parsa, A.T., 2010. Heat shock proteins in glioblastomas. Neurosurg. Clin. N. Am. 21, 111–123. Yang, C., Ruffner, M.A., Kim, S.-H., Robbins, P.D., 2012. Plasma-derived MHC class II+ exosomes from tumor-bearing mice suppress tumor antigen-specific immune responses. Eur. J. Immunol. 42, 1778–1784. Zhang, H.-G., Grizzle, W.E., 2011. Exosomes and cancer: a newly described pathway of immune suppression. Clin. Cancer Res. 17, 959–964. Zhang, H., Hu, H., Jiang, X., He, H., Cui, L., He, W., 2005. Membrane HSP70: the molecule triggering gammadelta T cells in the early stage of tumorigenesis. Immunol. Investig. 34, 453–468. Zou, J.P., Morford, L.A., Chougnet, C., Dix, A.R., Brooks, A.G., Torres, N., Shuman, J.D., Coligan, J.E., Brooks, W.H., Roszman, T.L., Shearer, G.M., 1999. Human gliomainduced immunosuppression involves soluble factor(s) that alters monocyte cytokine profile and surface markers. J. Immunol. 1950 (162), 4882–4892.