Exosome-loaded dendritic cells elicit tumor-specific CD8<Superscript>+</Superscript> cytotoxic T cells in patients with glioma

In this study, we demonstrate that tumor-derived exosome-loaded dendritic cells can elicit a specific CD8⁺ cytotoxic T-lymphocyte (CTL) response against autologous tumor cells in patients with malignant glioma. Exosomes were purified by ultrafiltration centrifugation and sucrose gradient ultracentrifugation. Exosomes had antigen-presenting molecules (MHC-I, HSP70), tumor antigen (MAGE-1) and adherent molecule (ICAM-1). After incubation with exosomes, the dendritic cells (DCs) could activate the T lymphocytes to become glioma-specialized CTL. The CTL had vigorous cytotoxicity to glioma cells as opposed to autologous lymphoblast cells. These data demonstrate that tumor exosome-loaded DC can be an effective tool in inducing glioma-specific CD8⁺ CTLs able to kill autologous glioma cells in vitro. In conclusion, exosomes are a natural and new source of tumor-rejection antigens, opening up new avenues for immunization against glioma.     

Induction of myeloid‐derived suppressor cells by tumor exosomes

Myeloid‐derived suppressor cells (MDSCs) promote tumor progression. The mechanisms of MDSC development during tumor growth remain unknown. Tumor exosomes (T‐exosomes) have been implicated to play a role in immune regulation, however the role of exosomes in the induction of MDSCs is unclear. Our previous work demonstrated that exosomes isolated from tumor cells are taken up by bone marrow myeloid cells. Here, we extend those findings showing that exosomes isolated from T‐exosomes switch the differentiation pathway of these myeloid cells to the MDSC pathway (CD11b⁺Gr‐1⁺). The resulting cells exhibit MDSC phenotypic and functional characteristics including promotion of tumor growth. Furthermore, we demonstrated that in vivo MDSC mediated promotion of tumor progression is dependent on T‐exosome prostaglandin E2 (PGE2) and TGF‐β molecules. T‐exosomes can induce the accumulation of MDSCs expressing Cox2, IL‐6, VEGF, and arginase‐1. Antibodies against exosomal PGE2 and TGF‐β block the activity of these exosomes on MDSC induction and therefore attenuate MDSC‐mediated tumor‐promoting ability. Exosomal PGE2 and TGF‐β are enriched in T‐exosomes when compared with exosomes isolated from the supernatants of cultured tumor cells (C‐exosomes). The tumor microenvironment has an effect on the potency of T‐exosome mediated induction of MDSCs by regulating the sorting and the amount of exosomal PGE2 and TGF‐β available. Together, these findings lend themselves to developing specific targetable therapeutic strategies to reduce or eliminate MDSC‐induced immunosuppression and hence enhance host antitumor immunotherapy efficacy. © 2008 Wiley‐Liss, Inc.     

CD4+ T cells support polyfunctionality of cytotoxic CD8+ T cells with memory potential in immunological control of tumor

Polyfunctionality/multifunctionality of effector T cells at the single cell level has been shown as an important parameter to predict the quality of T cell response and immunological control of infectious disease and malignancy. However, the fate of polyfunctional CD8⁺ CTLs and the factors that control the polyfunctionality of T cells remain largely unknown. Here we show that the acquisition of polyfunctionality on the initial stimulation is a sensitive immune correlate of CTL survival and memory formation. CD8⁺ T cells with high polyfunctionality, assessed with γ‐interferon and tumor necrosis factor‐α production and surface mobilization of the degranulation marker CD107a, showed enhanced Bcl‐2 expression, low apoptosis, and increased CD127^highKLRG1^low memory precursor phenotype. Consistent with these observations, CD8⁺ T cells were found to acquire high frequency of cells with polyfunctionality when stimulated in conditions known to enhance memory formation, such as the presence of CD4⁺ T cells, interleukin (IL)‐2, or IL‐21. Utilizing T‐cell receptor (TCR) transgenic mouse‐derived CD8⁺ T cells that express a TCR specific for a tumor‐derived neoantigen, we showed that polyfunctional tumor‐specific CTLs generated in the presence of CD4⁺ T cells showed long persistence in vivo and induced enhanced tumor regression when adoptively transferred into mice with progressing tumor. Acquisition of polyfunctionality thus impacts CTL survival and memory formation associated with immunological control of tumor.     

Tumor‐specific cytotoxic T cell generation and dendritic cell function are differentially regulated by interleukin 27 during development of anti‐tumor immunity

Interleukin (IL‐) 27 is a member of IL‐12 cytokine family with Th1‐promoting and anti‐inflammatory effects. IL‐27 has been shown to facilitate tumor‐specific cytotoxic T lymphocyte (CTL) induction against various tumors. However, IL‐27 suppresses cytokine production of lymphocytes and antigen‐presenting function of dendritic cells (DCs). To examine the in vivo role of IL‐27 in generation of anti‐tumor immunity, we examined IL‐27‐mediated antitumor‐effects using WSX‐1 (IL‐27 receptor α chain)‐deficient (WSX‐1^?/?) mice. In WSX‐1^?/? mice inoculated with B16 melanoma cells, tumor growth was higher than in wild‐type (WT) mice. Accordingly, tumor‐specific CTL generation was lower in WSX‐1^?/? mice than in WT mice. CTL induction in WSX‐1^?/? mice was not restored by transfer of WT DCs pulsed with TRP2 peptide, indicating that IL‐27 is directly required for generation of tumor‐specific CTLs. However, when transferred into tumor‐bearing mice, WSX‐1^?/? DCs pulsed with TRP2 peptide was more potent than WT DCs in tumor growth inhibition and generation of CTLs, indicating suppressive effects of IL‐27 on DC function. Finally, the combination of WT CD8⁺ T cells and KO DCs is more potent in generation of antigen‐specific CTLs than any other combinations. Expression of perforin gene and percentages of tumor‐specific CD8⁺ T cells were also the highest in the combination of WT CD8+ T cells and WSX‐1^?/? DCs. It was thus revealed that IL‐27 promotes CTL generation while suppressing DC function during generation of tumor immunity. The combination of WT T cells and IL‐27 signal‐defective DCs may have therapeutic potential against tumors. © 2008 Wiley‐Liss, Inc.     

Cancer‐associated fibroblast‐targeted strategy enhances antitumor immune responses in dendritic cell‐based vaccine

Given the close interaction between tumor cells and stromal cells in the tumor microenvironment (TME), TME‐targeted strategies would be promising for developing integrated cancer immunotherapy. Cancer‐associated fibroblasts (CAFs) are the dominant stromal component, playing critical roles in generation of the pro‐tumorigenic TME. We focused on the immunosuppressive trait of CAFs, and systematically explored the alteration of tumor‐associated immune responses by CAF‐targeted therapy. C57BL/6 mice s.c. bearing syngeneic E.G7 lymphoma, LLC1 Lewis lung cancer, or B16F1 melanoma were treated with an anti‐fibrotic agent, tranilast, to inhibit CAF function. The infiltration of immune suppressor cell types, including regulatory T cells and myeloid‐derived suppressor cells, in the TME was effectively decreased through reduction of stromal cell‐derived factor‐1, prostaglandin E₂, and transforming growth factor‐β. In tumor‐draining lymph nodes, these immune suppressor cell types were significantly decreased, leading to activation of tumor‐associated antigen‐specific CD8⁺ T cells. In addition, CAF‐targeted therapy synergistically enhanced multiple types of systemic antitumor immune responses such as the cytotoxic CD8⁺ T cell response, natural killer activity, and antitumor humoral immunity in combination with dendritic cell‐based vaccines; however, the suppressive effect on tumor growth was not observed in tumor‐bearing SCID mice. These data indicate that systemic antitumor immune responses by various immunologic cell types are required to bring out the efficacy of CAF‐targeted therapy, and these effects are enhanced when combined with effector‐stimulatory immunotherapy such as dendritic cell‐based vaccines. Our mouse model provides a novel rationale with TME‐targeted strategy for the development of cell‐based cancer immunotherapy.     

Infiltration of tumor‐associated macrophages is involved in tumor programmed death‐ligand 1 expression in early lung adenocarcinoma

Programmed death‐ligand 1 (PD‐L1) is an immune modulator that promotes immunosuppression by binding to programmed death‐1 of T‐lymphocytes. Although tumor cell PD‐L1 expression has been shown to be associated with the clinical response to anti–PD‐L1 antibodies, its concise regulatory mechanisms remain elusive. In this study, we evaluated the associations of tumor PD‐L1 expression and immune cell infiltrating patterns in 146 cases of early lung adenocarcinoma (AC) to investigate the possible extrinsic regulation of tumor PD‐L1 by immune cells. Using immunohistochemistry, cell surface PD‐L1 expression in tumor cells was observed in 18.5% of stage 0‐IA lung AC patients. Tumor PD‐L1 positivity was significantly associated with stromal invasion, which was accompanied by increased tumor‐associated macrophages (TAM), CD8⁺ cytotoxic T cells and FoxP3⁺ regulatory T cells. Among these immune cells, TAM and CD8⁺ T cells significantly accumulated in PD‐L1‐positive carcinoma cell areas, which showed a tumor cell nest‐infiltrating pattern. Although CD8⁺ T cells are known to induce tumor PD‐L1 expression via interferon‐? production, the increased TAM within tumors were also associated with tumor cell PD‐L1 positivity, independently of CD8⁺ T cell infiltration. Our in vitro experiments revealed that PD‐L1 expression in lung cancer cell lines was significantly upregulated by co–culture with M2‐differentiated macrophages; expression of PD‐L1 was reduced to baseline levels following treatment with a transforming growth factor‐β inhibitor. These results demonstrated that tumor‐infiltrating TAM are extrinsic regulators of tumor PD‐L1 expression, indicating that combination therapy targeting both tumor PD‐L1 and stromal TAM might be a possible strategy for effective treatment of lung cancer.     

CD90+ stromal cells are the major source of IL‐6, which supports cancer stem‐like cells and inflammation in colorectal cancer

IL‐6 is a pleiotropic cytokine increased in CRC and known to directly promote tumor growth. Colonic myofibroblasts/fibroblasts (CMFs or stromal cells) are CD90⁺ innate immune cells representing up to 30% of normal colonic mucosal lamina propria cells. They are expanded in CRC tumor stroma, where they also known as a cancer associated fibroblasts (CAFs). Cells of mesenchymal origin, such as normal myofibroblasts/fibroblasts, are known to secrete IL‐6; however, their contribution to the increase in IL‐6 in CRC and to tumor‐promoting inflammation is not well defined. Using in situ, ex vivo and coculture analyses we have demonstrated that the number of IL‐6 producing CMFs is increased in CRC (C‐CMFs) and they represent the major source of IL‐6 in T2‐T3 CRC tumors. Activity/expression of stem cell markers‐aldehyde dehydrogenase and LGR5‐ was significantly up‐regulated in colon cancer cells (SW480, Caco‐2 or HT29) cultured in the presence of conditioned medium from tumor isolated C‐CMFs in an IL‐6 dependent manner. C‐CMF and its derived condition medium, but not normal CMF isolated from syngeneic normal colons, induced differentiation of tumor promoting inflammatory T helper 17 cells (Th17) cell responses in an IL‐6 dependent manner. Our study suggests that CD90⁺ fibroblasts/myofibroblasts may be the major source of IL‐6 in T2‐T3 CRC tumors, which supports the stemness of tumor cells and induces an immune adaptive inflammatory response (a.k.a. Th17) favoring tumor growth. Taken together our data supports the notion that IL‐6 producing CAFs (a.k.a. C‐CMFs) may provide a useful target for treating or preventing CRCs.     

CD8+Foxp3+ tumor infiltrating lymphocytes accumulate in the context of an effective anti‐tumor response

The composition of tumor infiltrating lymphocytes (TIL) is heterogeneous. In addition, the ratio of various subpopulations in the tumor microenvironment is highly dependent on the nature of the host's immune response. Here, we characterize Foxp3‐expressing CD8⁺ T cells in the tumor that demonstrate effector function and accumulate in the context of an effective anti‐tumor response. CD8⁺Foxp3⁺ T cells are induced in TIL in regressing tumors of FVB/N mice treated with a GM‐CSF secreting HER‐2/neu targeted whole cell vaccine. Foxp3 expression in tumor antigen‐specific CD8 T cells is restricted to the tumor microenvironment and influenced by cues in the tumor. Interestingly, Foxp3⁺ and Foxp3^? CD8⁺ T cells have similar IFN‐γ production and antigen‐specific degranulation after stimulation with RNEU_420–429, the immunodominant HER‐2/neu (neu) epitope in this model. Adoptive transfer studies, using RNEU_(420–429)‐specific effector T cells into neu‐N mice (a model that results in immune tolerance to neu), confirm that CD8⁺Foxp3⁺ T cells are present in tumors only if there is an existing pool of tumor‐rejecting effector T cells. CD8⁺Foxp3⁺ TILs mark the presence of tumor‐rejecting antigen‐specific T cells and their accumulation serves as a marker for an effective T cell response.     

Exosomes from Murine-derived GL26 Cells Promote Glioblastoma Tumor Growth by Reducing Number and Function of CD8+T Cells

Aim: Brain tumors almost universally have fatal outcomes; new therapeutics are desperately needed andwill only come from improved understandins of glioma biology. Methods: Exosomes are endosomally derived30~100 nm membranous vesicles released from many cell types. Examples from GL26 cells were here purifiedusing density gradient ultracentrifugation and monitored for effects on GL26 tumor growth in C57BL/6j mice(H-2b). Lactate dehydrogenase release assays were used to detect the cytotoxic activity of CD8+T and NK cells.Percentages of immune cells producing intracellular cytokines were analyzed by FACS. Results: In this study,exosomes from murine-derived GL26 cells significantly promoted in vivo tumor growth in GL26-bearing B6mice. Then we further analyzed the effects of the GL26 cells-derived exosomes on immune cells including CD8+T,CD4+T and NK cells. Inhibition of CD8+T cell cytotoxic activity was demonstrated by CD8+T cell depletionassays in vivo and LDH release assays in vitro. The treatment of mice with exosomes also led to a reduction inthe percentages of CD8+T cells in splenocytes as determined by FACS analysis. Key features of CD8+T cellactivity were inhibited, including release of IFN-gamma and granzyme B. There were no effects of exosomeson CD4+T cells and NK cells. Conclusion: Based on our data, for the first time we demonstrated that exosomesfrom murine derived GL26 cells promote the tumor growth by inhibition of CD8+T cells in vivo and thus maybe a potential therapeutic target.     

The shared tumor associated antigen cyclin‐A2 is recognized by high‐avidity T‐cells

Cyclin‐A2, a key cell cycle regulator, has been shown to be overexpressed in various types of malignancies with little expression in normal tissue. Such tumor‐associated genes potentially are useful targets for cancer immunotherapy. However, high‐avidity cyclin‐specific T cells are considered to be thymically deleted. We identified at least one nonameric HLA‐A*0201 binding cyclin‐A2 epitope by a reverse immunology approach. Using a highly efficient T‐cell expansion system that is based on CD40‐activated B (CD40‐B) cells as sole antigen‐presenting cells we successfully generated cyclin‐A2 specific CTL from HLA‐A*0201⁺ donors. Interestingly, high‐avidity cyclin‐A2 specific CTL lines, which recognized peptide‐pulsed and antigen expressing target cells, were indeed generated by stimulation with CD40‐B cells when pulsed with low concentrations of peptide, whereas CD40‐B cells pulsed at saturating concentrations could only induce low‐avidity CTL, which recognized peptide‐pulsed target cells only. One high‐avidity CTL line was subcloned and CTL clones, whose peptide concentration required for half‐maximal lysis were less than 1 nM, could lyse cyclin‐A2 expressing tumor cells. Taken together, cyclin A2 is an attractive candidate for immune intervention in a significant number of cancer patients and high‐avidity T cells can be readily generated using CD40‐B cells as antigen‐presenting cells. © 2009 UICC     

Programmed death ligand‐1 is associated with tumor infiltrating lymphocytes and poorer survival in urothelial cell carcinoma of the bladder

Drugs blocking programmed death ligand‐1 (PD‐L1) have shown unprecedented activity in metastatic and unresectable bladder cancer. The purpose of the present study was to investigate the expression, clinical significance and association of PD‐L1 with tumor‐infiltrating lymphocytes (TIL) in resectable urothelial cell carcinoma of the bladder (UCB). In this retrospective study, 248 UCB patients who received radical cystectomy or transurethral resection were examined. Immunohistochemistry was used to evaluate PD‐L1 expression and stromal CD8⁺ TIL, Th1 orientation T cell (T‐bet⁺) and PD‐1⁺ TIL densities within the intratumoral regions and associated stromal regions. Of the 248 specimens, 23% showed PD‐L1 expression in tumor cells and 55% in tumor‐infiltrating immune cells. CD8⁺ TIL, T‐bet⁺ TIL and PD‐1⁺ TIL were distributed throughout the tumor tissues and were more frequently distributed in stromal regions than in intratumoral regions. PD‐L1⁺ tumor cells and PD‐L1⁺ immune cells were positively associated with aggressive clinical features (all P < .05). Both PD‐L1⁺ tumor cells and PD‐L1⁺ immune cells were associated with poorer recurrence‐free and overall survival (all P < .05). Multivariate analysis showed that PD‐L1⁺ immune cells were an independent prognostic factor for overall (P = .001) and recurrence‐free survival (P = .024). Notably, high stromal CD8⁺ TIL and PD‐1⁺ TIL density were associated with poorer overall survival (P = .031 and P = .001, respectively). In the stroma, CD8⁺ TIL density has strong positive association with PD‐L1⁺ immune cells and PD‐1⁺ TIL density (all P < .0001). These results suggested that an exhausted immune state occurred in the tumor stroma in UCB. Further clinical development of immune‐checkpoint inhibitors may be effective for resectable patients with UCB.     

Exosomal microRNAs derived from colon cancer cells promote tumor progression by suppressing fibroblast TP53 expression

The tumor microenvironment offers favorable conditions for tumor progression, and activated fibroblasts, known as cancer‐associated fibroblasts, play a pivotal role. TP53‐deficient cancer cells are known to induce strong fibroblast activation. We aimed to elucidate the oncogenic role of exosomes derived from TP53‐deficient colon cancer cells in fibroblast proliferation and tumor growth. Cancer cell‐derived exosomes (CDEs) were isolated from the conditioned media of cancer cells using a sequential ultracentrifugation method. The effects of exosomes on tumor growth were evaluated using human cell lines (TP53‐WT colon cancer, HCT116; TP53‐mutant colon cancer, HT29; and fibroblasts, CCD‐18Co and WI‐38) and an immune‐deficient nude mouse xenograft model. HCT116 (HCT116^{sh p53}) cells deficient in TP53 accelerated cocultured fibroblast proliferation compared to TP53‐WT HCT116 (HCT116^{sh control}) cells in vitro. Exosomes from HCT116^{sh p53} cells suppressed TP53 expression of fibroblasts and promoted their proliferation. Xenografts of HCT116^{sh p53} cells grew significantly faster than those of HCT116^{sh control} cells in the presence of co‐injected fibroblasts, but this difference was diminished by CDE inhibition. Microarray analysis identified upregulation of several microRNAs (miR‐1249‐5p, miR‐6737‐5p, and miR‐6819‐5p) in TP53‐deficient CDEs, which were functionally proven to suppress TP53 expression in fibroblasts. Exosomes derived from TP53‐mutant HT29 cells also suppressed TP53 expression in fibroblasts and accelerated their growth. The proliferative effect of HT29 on cocultured fibroblasts was diminished by inhibition of these miRNAs in fibroblasts. Our results suggest that CDEs play a pivotal role in tumor progression by fibroblast modification. Cancer cell‐derived exosomes might, therefore, represent a novel therapeutic target in colon cancer.     

Silencing IDO in dendritic cells: A novel approach to enhance cancer immunotherapy in a murine breast cancer model

Cancer immunotherapeutic agents (vaccines) in the form of antigen‐loaded dendritic cells (DCs) reached an important milestone with the recent approval of Provenge, the first DC vaccine for treatment of prostate cancer. Although this heralds a new era of tumor immunotherapy, it also highlights the compelling need to optimize such DC‐based therapies as they are increasingly tested and used to treat human patients. In this study we sought to augment and enhance the antitumor activity of a DC‐based vaccine using siRNA to silence expression of immunosuppressive enzyme indoleamine 2,3‐dioxygenase (IDO) in DCs. We report here that DCs loaded with tumor antigens, but with siRNA‐silenced IDO expression, were introduced into 4T1 breast tumor‐bearing mice, the treatment: (i) lengthened the time required for tumor onset, (ii) decreased tumor size compared to tumors grown for equal lengths of time in mice treated with antigen‐loaded DCs without IDO silencing and (iii) reduced CD4⁺ and CD8⁺ T cell apoptosis. Furthermore, immunization with IDO‐silenced DCs enhanced tumor antigen‐specific T cell proliferation and CTL activity, and decreased numbers of CD4⁺CD25⁺Foxp3⁺ T_reg. This study provides evidence to support silencing of immunosuppressive genes (IDO) as an effective strategy to enhance the efficacy of DC‐based cancer immunotherapeutic.     

An ISCOM vaccine combined with a TLR9 agonist breaks immune evasion mediated by regulatory T cells in an orthotopic model of pancreatic carcinoma

Vaccines based on immune stimulatory complexes (ISCOM) induce T‐cell responses against tumor antigen (Ag). However, immune responses are impaired in pancreatic cancer patients. We investigated the efficacy of an ISCOM vaccine in a murine pancreatic carcinoma model. Panc02 cells expressing OVA as a model Ag were induced subcutaneously or orthotopically in the pancreas of C57BL/6 mice. Treatment consisted of an OVA containing ISCOM vaccine, either used alone or in combination with the TLR9 agonist CpG. The ISCOM vaccine effectively induced Ag‐specific CTL capable of killing tumor cells. However, in mice with established tumors CTL induction by the vaccine was inefficient and did not affect tumor growth. Lack of efficacy correlated with increased numbers of Treg. Depletion of Treg with anti‐CD25 mAb restored CTL induction and prolonged survival. Adding low‐dose CpG to the ISCOM vaccine reduced Treg numbers, enhanced CTL responses and induced regression of pancreatic tumors in a CD8⁺ T cell–dependent manner. Mice cured from the primary tumor mounted a memory T‐cell response against wild‐type Panc02 tumors, indicative of epitope spreading. Combining ISCOM vaccines with TLR agonists is a promising strategy for breaking tumor immune evasion and deserves further evaluation for the treatment of pancreatic carcinoma.     

Therapy‐induced antitumor vaccination in neuroblastomas by the combined targeting of IL‐2 and TNFα

L19‐IL2 and L19TNFα are fusion proteins composed of L19(scFv), specific for the angiogenesis‐associated ED‐B containing fibronectin isoform and IL‐2 or TNFα. Because of the tumor targeting properties of L19, IL‐2 and TNFα concentrate at therapeutic doses at the tumor vascular level. To evaluate the therapeutic effects of L19‐IL2 and L19mTNFα in neuroblastoma (NB)‐bearing mice, A/J mice bearing Neuro2A or NIE115 NB were systemically treated with L19‐IL2 and L19mTNFα, alone or in combination protocols. Seventy percent of Neuro2A‐ and 30% of NIE115‐bearing mice were cured by the combined treatment with L19‐IL2 and L19mTNFα, and further rejected a homologous tumor challenge, indicating specific antitumor immune memory. The immunological bases of tumor cure and rejection were studied. A highly efficient priming of CD4⁺ T helper cells and CD8⁺ CTL effectors was generated, paralleled by massive infiltration in the tumor tissue of CD4⁺ and CD8⁺ T cells at day 16 after tumor cell implantation, when, after therapy, tumor volume was drastically reduced and tumor necrosis reached about 80%. The curative treatment resulted in a long‐lasting antitumor immune memory, accompanied by a mixed Th1/Th2 type of response. Concluding, L19‐IL2 and L19mTNFα efficiently cooperate in determining a high percentage of NB cure that, in our experimental models, is strongly associated to the generation of adaptive immunity involving CD4⁺ and CD8⁺ T cells.     

肿瘤外泌体对CD8+T细胞功能的影响及机制研究进展

外泌体（exosomes）是介导细胞间通讯的细胞外囊泡。它携带来源细胞的多种生物活性分子,并可将其输送给受体细胞,进而影响细胞功能。肿瘤来源外泌体可通过多种机制介导肿瘤的免疫逃逸。本文就肿瘤外泌体对肿瘤杀伤主力军CD8+T细胞的调控作用进行总结,分析其相关作用机制,以期为肿瘤免疫治疗的研发提供新的思路。     

Role of exosomes in the immune microenvironment of ovarian cancer

Exosomes are excretory vesicles that can deliver a variety of bioactive cargo molecules to the extracellular environment. Accumulating evidence demonstrates exosome participation in intercellular communication, immune response, inflammatory response and they even play an essential role in affecting the tumor immune microenvironment. The role of exosomes in the immune microenvironment of ovarian cancer is mainly divided into suppression and stimulation. On one hand exosomes can stimulate the innate and adaptive immune systems by activating dendritic cells (DCs), natural killer cells and T cells, allowing these immune cells exert an antitumorigenic effect. On the other hand, ovarian cancer-derived exosomes initiate cross-talk with immunosuppressive effector cells, which subsequently cause immune evasion; one of the hallmarks of cancer. Exosomes induce the polarization of macrophages in M2 phenotype and induce apoptosis of lymphocytes and DCs. Exosomes further activate additional immunosuppressive effector cells (myeloid-derived suppressor cells and regulatory T cells) that induce fibroblasts to differentiate into cancer-associated fibroblasts. Exosomes also induce the tumorigenicity of mesenchymal stem cells to exert additional immune suppression. Furthermore, besides mediating the intercellular communication, exosomes carry microRNAs (miRNAs), proteins and lipids to the tumor microenvironment, which collectively promotes ovarian cancer cells to proliferate, invade and tumors to metastasize. Studying proteins, lipids and miRNAs carried by exosomes could potentially be used as an early diagnostic marker of ovarian cancer for designing treatment strategies.     

Binding of monosodium urate crystals with idiotype protein efficiently promote dendritic cells to induce cytotoxic T cells

Monosodium urate (MSU) crystals have been reported to evoke specific cell immunity and to work as an adjuvant in a mouse model. The crystals also have another unique characteristic to bind with positively charged proteins, which could help to deliver some antigens into human dendritic cells (DC). We focused on the application of MSU crystals as not only an adjuvant but also as a carrier of positively charged antigenic protein to induce human cytotoxic T cells (CTL) efficiently in vitro. We selected human leukocyte antigen (HLA)‐A2 expressing the multiple myeloma IM‐9 cell line and its product idiotype (Id) protein as one of the best pairs of target cells and positively charged tumor‐specific antigen, respectively. Following the sensitization of DC derived from HLA‐A2‐positive volunteers pulsed with tumor‐specific monoclonal immunoglobulin G‐Fab fragments (IM‐9 Fab) attached to MSU crystals, the DC‐stimulated CD8⁺ T cells killed significantly more target cells (40.1 ± 1.7%) than those stimulated by DC pulsed with MSU crystals alone (6.2 ± 8.6%, P < 0.01) or IM‐9 Fab alone (4.7 ± 8.1%, P < 0.01). These cytotoxic effects of the DC‐stimulated CD8⁺ cells were reduced when MSU crystals were precoated with fetal bovine serum. In addition, we confirmed that MSU crystals facilitated human DC to express the maturation marker, CD83 and deliver (Fab′)₂, attaching to the crystals by flow cytometer analysis. MSU crystals have distinct advantages of a protein carrier binding with positively charged proteins and delivering antigenic protein into DC, as well as an adjuvant promoting DC maturation and inducing CTL. (Cancer Sci 2008; 99: 2268–2273)