Human dendritic cells activated via dectin-1 are efficient at priming Th17, cytotoxic CD8 T and B cell responses

Background

Dendritic cells capture antigens through PRRs and modulate adaptive immune responses. The type of adaptive immune T cell response generated is dependent upon the type of PRR activated by the microbes. Dectin-1 is a C-type lectin receptor present on dendritic cells.

Methodology/Principal Findings

Here we show that selective dectin-1 agonist Curdlan can activate human DCs and induce the secretion of large amounts of IL-23, IL-1β, IL-6 and low levels of IL-12p70 as determined by ELISA. The Curdlan-stimulated DCs are efficient at priming naïve CD4 cells to differentiate into Th17 and Th1 cells. Furthermore, these CD4 T cells induce differentiation of B cells to secrete IgG and IgA. In addition, Curdlan-stimulated DCs promote the expansion and differentiation of Granzyme and perforin expressing cytotoxic T lymphocyte that display high cytolytic activity against target tumor cells in vitro.

Conclusions/Significance

These data demonstrate that DCs stimulated through Dectin-1 can generate efficient Th, CTL and B cell responses and can therefore be used as effective mucosal and systemic adjuvants in humans.     

Tumor antigen-specific T helper cells in cancer immunity and immunotherapy

Historically, cancer-directed immune-based therapies have focused on eliciting a cytotoxic T cell (CTL) response, primarily due to the fact that CTL can directly kill tumors. In addition, many putative tumor antigens are intracellular proteins, and CTL respond to peptides presented in the context of MHC class I which are most often derived from intracellular proteins. Recently, increasing importance is being given to the stimulation of a CD4+ T helper cell (Th) response in cancer immunotherapy. Th cells are central to the development of an immune response by activating antigen-specific effector cells and recruiting cells of the innate immune system such as macrophages and mast cells. Two predominant Th cell subtypes exist, Th1 and Th2. Th1 cells, characterized by secretion of IFN- and TNF-, are primarily responsible for activating and regulating the development and persistence of CTL. In addition, Th1 cells activate antigen-presenting cells (APC) and induce limited production of the type of antibodies that can enhance the uptake of infected cells or tumor cells into APC. Th2 cells favor a predominantly humoral response. Particularly important during Th differentiation is the cytokine environment at the site of antigen deposition or in the local lymph node. Th1 commitment relies on the local production of IL-12, and Th2 development is promoted by IL-4 in the absence of IL-12. Specifically modulating the Th1 cell response against a tumor antigen may lead to effective immune-based therapies. Th1 cells are already widely implicated in the tissue-specific destruction that occurs during the pathogenesis of autoimmune diseases, such as diabetes mellitus and multiple sclerosis. Th1 cells directly kill tumor cells via release of cytokines that activate death receptors on the tumor cell surface. We now know that cross-priming of the tumor-specific response by potent APC is a major mechanism of the developing endogenous immune response; therefore, even intracellular proteins can be presented in the context of MHC class II. Indeed, recent studies demonstrate the importance of cross-priming in eliciting CTL. Many vaccine strategies aim to stimulate the Th response specific for a tumor antigen. Early clinical trials have shown that focus on the Th effector arm of the immune system can result in significant levels of both antigen-specific Th cells and CTL, the generation of long lasting immunity, and a Th1 phenotype resulting in the development of epitope spreading.     

Interplay between CD8α+ dendritic cells and monocytes in response to Listeria monocytogenes infection attenuates T cell responses

During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α(+) dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8(+) T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α(+) DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α(+) DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b(+) DCs primarily secrete low levels of TNFα while CD8α(+) DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α(+) DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming.     

Strategies for antigen choice and priming of dendritic cells influence the polarization and efficacy of antitumor T-cell responses in dendritic cell–based cancer vaccination

Dendritic cells (DCs) primed with tumor antigens (Ags) can stimulate tumor rejection. This study was aimed at evaluating the polarization of T-cell responses using various DC Ag-priming strategies for vaccination purposes. DCs cocultured with irradiated apoptotic tumor cells, DC-tumor fusions, and DCs pulsed with freeze-thaw tumor lysate Ags served as Ag-primed DCs, with EG7 tumor cells (class II negative) expressing OVA as the model Ag. DCs loaded with class I– and class II–restricted OVA synthetic peptides served as controls. Primed DCs were assessed by the in vitro activation of B3Z OVA-specific CD8 T cells and the proliferation of OVA-specific CD8 and CD4 T cells from OT-I and OT-II TCR transgenic mice, respectively. In vivo responses were measured by tumor regression following treatment with Ag-primed DCs and by CTL assays. Quantification of IL-2, IL-4, IL-5, IFN-, and TNF- by cytometric bead array (CBA) assay determined the polarization of TH1/TH2 responses, whereas H-2 K^b /SIINFEKL tetramers monitored the expansion of OVA-specific T cells. DC-EG7 hybrids stimulated both efficient class I and class II OVA responses, showing that DC-tumor hybrids are also capable of class II cross-presentation. The hybrids also induced the most potent CTLs, offered the highest protection against established EG7 tumors and also induced the highest stimulation of IFN- and TNF- production. DCs cocultured with irradiated EG7 were also effective at inducing OVA-specific responses, however with slightly reduced potency to those evoked by the hybrids. DCs loaded with lysates Ags were much less efficient at stimulating any of the OVA-specific T-cell responses, showed very little antitumor protection, and stimulated a weak TH1 response, overbalanced by an IL-5 TH2 response. The strategy of Ag-loading clearly influences the ability of DCs to polarize T cells for a TH1/TH2 response and thus determines the outcome of the elicited immune response, during various vaccination protocols.Abbreviations DC Dendritic cell - FSC Forward scatter - SSC Side scatter - TC Tumor cells This work was supported by Grant 9853 from the Leukaemia Research Fund, UK; a JRC studentship from GKT; and the Lewis Family Research Trust     

Specific microtubule-depolymerizing agents augment efficacy of dendritic cell-based cancer vaccines

Background

Damage-associated molecular patterns (DAMPs) are associated with immunogenic cell death and have the ability to enhance maturation and antigen presentation of dendritic cells (DCs). Specific microtubule-depolymerizing agents (MDAs) such as colchicine have been shown to confer anti-cancer activity and also trigger activation of DCs.

Methods

In this study, we evaluated the ability of three MDAs (colchicine and two 2-phenyl-4-quinolone analogues) to induce immunogenic cell death in test tumor cells, activate DCs, and augment T-cell proliferation activity. These MDAs were further evaluated for use as an adjuvant in a tumor cell lysate-pulsed DC vaccine.

Results

The three test phytochemicals considerably increased the expression of DAMPs including HSP70, HSP90 and HMGB1, but had no effect on expression of calreticulin (CRT). DC vaccines pulsed with MDA-treated tumor cell lysates had a significant effect on tumor growth, showed cytotoxic T-lymphocyte activity against tumors, and increased the survival rate of test mice. In vivo antibody depletion experiments suggested that CD8⁺ and NK cells, but not CD4⁺ cells, were the main effector cells responsible for the observed anti-tumor activity. In addition, culture of DCs with GM-CSF and IL-4 during the pulsing and stimulation period significantly increased the production of IL-12 and decreased production of IL-10. MDAs also induced phenotypic maturation of DCs and augmented CD4⁺ and CD8⁺ T-cell proliferation when co-cultured with DCs.

Conclusions

Specific MDAs including the clinical drug, colchicine, can induce immunogenic cell death in tumor cells, and DCs pulsed with MDA-treated tumor cell lysates (TCLs) can generate potent anti-tumor immunity in mice. This approach may warrant future clinical evaluation as a cancer vaccine.     

Combination of monocyte-derived dendritic cells and activated T cells which express CD40 ligand: a new approach to cancer immunotherapy

Interactions between dendritic cells (DCs) and activated T cells are critically important for the establishment of an effective immune response. To develop the basis for a new DC-based cancer vaccine, we investigated cell-to-cell interactions between human monocyte-derived DCs and autologous T cells that are activated to express the CD40 ligand (CD40L). Peripheral blood monocytes were cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) to induce differentiation of DCs. Activated T cells (ATs) consisted of autologous peripheral blood lymphocytes that had been activated with phytohemagglutinin (PHA) and then stimulated with calcium ionophore to up-regulate expression of CD40L. Coculture of these DCs and ATs induced significant production of interleukin 12 (IL-12) and also enhanced the production of interferon (IFN-). The production of IL-12 was blocked by an anti-CD40L antibody or by separation of the DC and AT fractions by a permeable membrane. Furthermore, coculture of DCs and ATs induced DCs to upregulate CD83 expression and stimulated migration of DCs toward the macrophage inflammatory protein 3- (MIP-3). ATs also migrated toward the MIP-3. These results suggest a combination of DCs and ATs as a potentially effective therapeutic strategy.     

Role of IL-13 in regulation of anti-tumor immunity and tumor growth

Major mediators of anti-tumor immunity are CD4⁺ T _h 1 cells and CD8⁺ cytotoxic T lymphocytes (CTLs). In tumor-bearing animals, the T _h 1- and CTL-mediated anti-tumor immunity is down-regulated in multiple ways. Better understanding of negative regulatory pathways of tumor immunity is crucial for the development of anti-tumor vaccines and immunotherapies. Since immune deviation toward T _h 2 suppresses T _h 1 development, it has been thought that induction affecting a T _h 2 immune response is one of the mechanisms that down-regulate effective tumor immune responses. Recent studies using T _h 2-deficient signal transducer and activator (Stat6) KO mice demonstrated that this hypothesis was the case. IL-13 is one of the T _h 2 cytokines that has very similar features to IL-4 through sharing some receptor components and Stat6 signal transduction. It has been thought that IL-13 is not as critical for immune deviation as IL-4 since it cannot directly act on T cells. However, recent studies of IL-13 reveal that this cytokine plays a critical role in many aspects of immune regulation. Studies from our lab and others indicate that IL-13 is central to a novel immunoregulatory pathway in which NKT cells suppress tumor immunosurveillance. Here we will describe biological properties and functions of IL-13, its role in the negative regulation of anti-tumor immunity, and effects of IL-13 on tumor cells themselves.This article forms part of the Symposium in Writing Inhibitors of immunosurveillance and anti-tumor immunity, published in Vol. 53.     

Complete tumour regressions induced by vaccination with IL-12 gene-transduced tumour cells in combination with IL-15 in a melanoma model in mice

In the present study, IL-12 gene-transduced B78-H1 melanoma cells (B78/IL-12) were used in combination with IL-15 to treat melanoma-bearing mice. Genetically modified B78/IL-12 cells, when injected subcutaneously, induced strong activation of antitumour mechanisms resulting in complete loss of tumourigenicity. In a therapeutic model, intratumoural injection of irradiated B78/IL-12 cells significantly delayed tumour growth and led to the regression of melanoma in one case. Similarly, consecutive daily injections of IL-15 markedly inhibited tumour progression with occasional curative effects. When used in combination, vaccination with B78/IL-12 cells and treatment with IL-15 caused eradication of established tumours in all treated mice. The combined treatment with B78/IL-12 cells and IL-15 activated not only a local response against tumour, but also induced systemic antitumour immunity that led to a delay or inhibition of tumour development at a distant site. In vitro studies demonstrated that when used together, B78/IL-12 cells and IL-15 induced a shift from a type Th2 to a type Th1 response. Activation of the antitumour immune response in double-treated mice resulted, in part, from stimulation of IFN- production and was accompanied by the development of cytotoxic effectors in the spleen. As shown in a macrophage tumouricidal assay, macrophages could also play a role in the antitumour effects. The results confirmed that vaccination with IL-12 gene-modified tumour cells is superior to the treatment with unmodified tumour cell vaccine and, additionally, showed that IL-15 is an excellent candidate for adjuvant therapy, inducing synergistically not only a delay of tumour growth but also its complete eradication.     

Mice lacking the IFN-γ receptor or fyn develop severe experimental autoimmune uveoretinitis characterized by different immune responses

Endogenous interferon (IFN)- negatively regulates experimental autoimmune uveoretinitis (EAU), a Th1-mediated disease. Although it is well known that IFN- exerts its effects by binding to the IFN- receptor (IFN-R), the role that IFN-R plays in the development of EAU has not been investigated. Fyn has been reported to inhibit Th2 differentiation. We aimed to investigate how endogenous IFN-R and fyn, which influence Th1/Th2 differentiation, participate in the development of EAU. Sex-matched 6- to 10-week-old C57BL/6 wild-type (WT), IFN-R knockout (GRKO) and fyn knockout (fyn KO) mice were compared. Mice were immunized subcutaneously with human interphotoreceptor retinoid-binding protein peptide 1–20 emulsified in Freunds complete adjuvant together with an intraperitoneal injection of Bordetella pertussis toxin. Three weeks later, mice were sacrificed, and their eyes and spleens were harvested for histopathologic analyses and examination of cellular immune responses, respectively. Cellular immune responses were evaluated by measuring the proliferative responses and cytokine production [interleukin (IL)-4, IL-5, IL-6, IL-13, IFN- and tumor necrosis factor (TNF)-] of splenocytes. The incidence of EAU was 40.0% in WT mice, 59.3% in GRKO mice and 78.6% in fyn KO mice. The average EAU score was 0.294 in WT mice, 0.917 in GRKO mice and 1.063 in fyn KO mice. Upon EAU induction, significant infiltration of eosinophils into the eyes was observed in GRKO and fyn KO mice compared to WT mice. Splenocytes from GRKO mice proliferated against the antigen and a mitogen more vigorously than those from WT and fyn KO mice. Stimulation of splenocytes with the antigen induced a higher production of IL-4, IL-6, IL-13 and IFN- in GRKO mice compared to WT and fyn KO mice. In contrast, IL-5 and TNF- were most abundantly produced by splenocytes from fyn KO mice compared to WT and GRKO mice. The incidence and mean severity of EAU were significantly higher in GRKO and fyn KO mice than in WT mice, suggesting that endogenous IFN-R and fyn negatively regulate the development of EAU. The different cytokine production patterns by the GRKO and fyn KO mice indicate that the negative regulatory mechanism mediated by IFN-R and fyn may differ.     

腺病毒携带IL-12增强抗原致敏树突细胞在肝癌基因治疗中的研究

目的:近年来通过应用白介素-12治疗肿瘤取得良好效果,因此对国内外应用腺病毒携带IL-12增强抗原致敏树突细胞在肝癌基因治疗中的研究进展进行总结,以探索更为可行治疗方法。方法:运用Pubmed、Elsevier Sciencedirect、CNKI及万方全文数据库检索系统,以腺病毒,IL-12,肝癌,树突细胞为关键词,检索2008-01至2012-11月发表的文献。纳入标准:1)IL-12的生物学特性,在抗肿瘤过程中的免疫作用,2)应用腺病毒携带IL-12对抗肝癌治疗研究,3)肿瘤抗原致敏树突细胞对肿瘤的影响。根据纳入标准分析文献26篇。结果:通过腺病毒携带IL-12可以增强肿瘤抗原致敏树突细胞的免疫应答。并通过诱导肿瘤细胞的凋亡,减少新生血管的生成而对肿瘤产生直接抑制,有效抑制肝癌的生长和转移。结论:本文通过对IL-12的生物学特征、抗肿瘤通路、作用机制及在腺病毒介导下肿瘤抗原致敏树突细胞研究进展的概述,为腺病毒携带IL-12作为肝癌的基因治疗进一步提供理论依据和探索,期待在将来应用IL-12为基础的基因治疗一定会为包括肝癌在内的肿瘤治疗提供新的途径。     

Human Flt3L Generates Dendritic Cells from Canine Peripheral Blood Precursors: Implications for a Dog Glioma Clinical Trial

Background

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and carries a dismal prognosis. We have developed a conditional cytotoxic/immunotherapeutic approach using adenoviral vectors (Ads) encoding the immunostimulatory cytokine, human soluble fms-like tyrosine kinase 3 ligand (hsFlt3L) and the conditional cytotoxic molecule, i.e., Herpes Simplex Type 1- thymide kinase (TK). This therapy triggers an anti-tumor immune response that leads to tumor regression and anti-tumor immunological memory in intracranial rodent cancer models. We aim to test the efficacy of this immunotherapy in dogs bearing spontaneous GBM. In view of the controversy regarding the effect of human cytokines on dog immune cells, and considering that the efficacy of this treatment depends on hsFlt3L-stimulated dendritic cells (DCs), in the present work we tested the ability of Ad-encoded hsFlt3L to generate DCs from dog peripheral blood and compared its effects with canine IL-4 and GM-CSF.

Methodology/Principal Findings

Our results demonstrate that hsFlT3L expressed form an Ad vector, generated DCs from peripheral blood cultures with very similar morphological and phenotypic characteristics to canine IL-4 and GM-CSF-cultured DCs. These include phagocytic activity and expression of CD11c, MHCII, CD80 and CD14. Maturation of DCs cultured under both conditions resulted in increased secretion of IL-6, TNF-α and IFN-γ. Importantly, hsFlt3L-derived antigen presenting cells showed allostimulatory potential highlighting their ability to present antigen to T cells and elicit their proliferation.

Conclusions/Significance

These results demonstrate that hsFlt3L induces the proliferation of canine DCs and support its use in upcoming clinical trials for canine GBM. Our data further support the translation of hsFlt3L to be used for dendritic cells'' vaccination and gene therapeutic approaches from rodent models to canine patients and its future implementation in human clinical trials.     

Enhancement of anti-tumor CD8 immunity by IgG1-mediated targeting of Fc receptors

Dendritic cells (DCs) function as professional antigen presenting cells and are critical for linking innate immune responses to the induction of adaptive immunity. Many current cancer DC vaccine strategies rely on differentiating DCs, feeding them tumor antigens ex vivo, and infusing them into patients. Importantly, this strategy relies on prior knowledge of suitable “tumor-specific” antigens to prime an effective anti-tumor response. DCs express a variety of receptors specific for the Fc region of immunoglobulins, and antigen uptake via Fc receptors is highly efficient and facilitates antigen presentation to T cells. Therefore, we hypothesized that expression of the mouse IgG1 Fc region on the surface of tumors would enhance tumor cell uptake by DCs and other myeloid cells and promote the induction of anti-tumor T cell responses. To test this, we engineered a murine lymphoma cell line expressing surface IgG1 Fc and discovered that such tumor cells were taken up rapidly by DCs, leading to enhanced cross-presentation of tumor-derived antigen to CD8+ T cells. IgG1-Fc tumors failed to grow in vivo and prophylactic vaccination of mice with IgG1-Fc tumors resulted in rejection of unmanipulated tumor cells. Furthermore, IgG1-Fc tumor cells were able to slow the growth of an unmanipulated primary tumor when used as a therapeutic tumor vaccine. Our data demonstrate that engagement of Fc receptors by tumors expressing the Fc region of IgG1 is a viable strategy to induce efficient and protective anti-tumor CD8+ T cell responses without prior knowledge of tumor-specific antigens.     

树突状细胞在抗感染免疫研究中的最新进展

树突状细胞(Dendritic cell,DC)是体内功能最强的抗原提呈细胞,也是介导机体固有免疫应答和适应性免疫应答的桥梁,其作用也越来越受到科研工作者的关注,而树突状细胞体外培养技术的发展成熟,为设计和发展DC依赖性疫苗提供了科学依据,也为感染性和肿瘤性疾病的预防和治疗展示了很好的应用前景。因此,对树突状细胞抗感染免疫方面研究的最新进展做一综述。     

Tumor mRNA-loaded dendritic cells elicit tumor-specific CD8(+) cytotoxic T cells in patients with malignant glioma

In this study, we demonstrate that tumor mRNA–loaded dendritic cells can elicit a specific CD8⁺ cytotoxic T-lymphocyte (CTL) response against autologous tumor cells in patients with malignant glioma. CTLs from three patients expressed strong cytolytic activity against autologous glioma cells, did not lyse autologous lymphoblasts or EBV-transformed cell lines, and were variably cytotoxic against the NK-sensitive cell line K-562. Also, DCs-pulsed normal brain mRNA failed to induce cytolytic activity against autologous glioma cells, suggesting the lack of autoimmune response. Two patients' CD8⁺ T cells expressed a modest cytotoxicity against autologous glioma cells. CD8⁺ T cells isolated during these ineffective primings secreted large amounts of IL-10 and smaller amounts of IFN- as detected by ELISA. Type 2 bias in the CD8⁺ T-cell response accounts for the lack of cytotoxic effector function from these patients. Cytotoxicity against autologous glioma cells could be significantly inhibited by anti-HLA class I antibody. These data demonstrate that tumor mRNA–loaded DC can be an effective tool in inducing glioma-specific CD8⁺ CTLs able to kill autologous glioma cells in vitro. However, high levels of tumor-specific tolerance in some patients may account for a significant barrier to therapeutic vaccination. These results may have important implications for the treatment of malignant glioma patients with immunotherapy. DCs transfected with total tumor RNA may represent a method for inducing immune responses against the entire repertoire of glioma antigens.     

Enhancement of anti-tumor CD8 immunity by IgG1-mediated targeting of Fc receptors

Dendritic cells (DCs) function as professional antigen presenting cells and are critical for linking innate immune responses to the induction of adaptive immunity. Many current cancer DC vaccine strategies rely on differentiating DCs, feeding them tumor antigens ex vivo, and infusing them into patients. Importantly, this strategy relies on prior knowledge of suitable “tumor-specific” antigens to prime an effective anti-tumor response. DCs express a variety of receptors specific for the Fc region of immunoglobulins, and antigen uptake via Fc receptors is highly efficient and facilitates antigen presentation to T cells. Therefore, we hypothesized that expression of the mouse IgG1 Fc region on the surface of tumors would enhance tumor cell uptake by DCs and other myeloid cells and promote the induction of anti-tumor T cell responses. To test this, we engineered a murine lymphoma cell line expressing surface IgG1 Fc and discovered that such tumor cells were taken up rapidly by DCs, leading to enhanced cross-presentation of tumor-derived antigen to CD8+ T cells. IgG1-Fc tumors failed to grow in vivo and prophylactic vaccination of mice with IgG1-Fc tumors resulted in rejection of unmanipulated tumor cells. Furthermore, IgG1-Fc tumor cells were able to slow the growth of an unmanipulated primary tumor when used as a therapeutic tumor vaccine. Our data demonstrate that engagement of Fc receptors by tumors expressing the Fc region of IgG1 is a viable strategy to induce efficient and protective anti-tumor CD8+ T cell responses without prior knowledge of tumor-specific antigens.     

CD8+ T Cell Priming by Dendritic Cell Vaccines Requires Antigen Transfer to Endogenous Antigen Presenting Cells

Background

Immunotherapeutic strategies to stimulate anti-tumor immunity are promising approaches for cancer treatment. A major barrier to their success is the immunosuppressive microenvironment of tumors, which inhibits the functions of endogenous dendritic cells (DCs) that are necessary for the generation of anti-tumor CD8+ T cells. To overcome this problem, autologous DCs are generated ex vivo, loaded with tumor antigens, and activated in this non-suppressive environment before administration to patients. However, DC-based vaccines rarely induce tumor regression.

Methodology/Principal Findings

We examined the fate and function of these DCs following their injection using murine models, in order to better understand their interaction with the host immune system. Contrary to previous assumptions, we show that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells.

Conclusions/Significance

This reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches should focus on creating DC vaccines that are more effective at directly priming T cells, or abrogating the tumor induced suppression of endogenous DCs.     

Skewing effect of sulprostone on dendritic cell maturation compared with dinoprostone

Background

Dendritic cells (DCs) are the most efficient antigen-presenting cells and act at the center of the immune system owing to their ability to control both immune tolerance and immunity. In cancer immunotherapy, DCs play a key role in the regulation of the immune response against tumors and can be generated ex vivo with different cytokine cocktails. Methods. We evaluated the feasibility of dinoprostone (PGE₂) replacement with the molecular analog sulprostone, in our good manufacturing practice (GMP) protocol for the generation of DC-based cancer vaccine. We characterized the phenotype and the function of DCs matured in the presence of sulprostone as a potential substitute of dinoprostone in the pro-inflammatory maturation cocktail consisting of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and IL-6. Results. We found that sulprostone invariably reduces the recovery, but does not significantly modify the viability and the purity of DCs. The presence of sulprostone in the maturation cocktail increases the adhesion of single cells and of clusters of DCs to the flask, making them more similar to their immature counterpart in terms of adhesion and spreading proprieties. Moreover, we observed that sulprostone impairs the expression of co-stimulatory molecules and the spontaneous as well as the directed migration capacity of DCs.

Modification of natural killer activity of lymphocytes infiltrating human lung cancers

Summary The purpose of these studies was to compare local and systemic human lymphokine activated killer (LAK) and natural killer (NK) cytotoxic activity and to determine its modulation by biologic agents. Local immunity may be an important component in limiting local tumor growth. Therefore, as a model for studying immune function in the local compartment, we assessed NK activity of lymphocytes present at the site of human tumors and in peripheral blood (PBL). We extracted tumor infiltrating lymphocytes (TIL) and PBL from patients with pulmonary tumors and compared NK activity and response to the biological modifiers gamma interferon (IFN-), indomethacin (INDO), and interleukin 2 (IL-2). We also studied TIL and PBL LAK activity using the NK-resistant M14 target cells and determined the TIL response to IL-2, plus IFN-. Titration of K562 targets in a ⁵¹Cr release assay revealed that untreated TIL have low cytotoxicity (4.32%) compared to untreated PBL (34.3%, P=<0.001). This low level of TIL NK activity was not affected by IFN-, INDO, or IL-2 at 1 h. However, at 3 days of culture, IL-2 with or without exogenous IFN- significantly increased TIL NK ctotoxicity (20.5%, P=0.02 without IFN- and 32.52 lytic units (LU), P=<0.02 with IFN-). Untreated TIL and PBL both had low cytotoxicity against M14 targets (1.08 LU and 1.26 LU), respectively. After 3 days culture with IL-2 plus IFN-, both TIL and PBL LAK cytotoxicity were increased (14.34 LU and 40.63 LU). We conclude that local NK and LAK activity is intrinsically low. However, this activity can be modulated by biologic agents, thus giving hope for the development of local antitumor effectors capable of in vivo tumor control.