Antigen-presenting Capacity Research Articles

Dendritic Cells: Cellular Mediators for Immunological Tolerance

In general, immunological tolerance is acquired upon treatment with non-specific immunosuppressive drugs. This indiscriminate immunosuppression of the patient often causes serious side-effects, such as opportunistic infectious diseases. Therefore, the need for antigen-specific modulation of pathogenic immune responses is of crucial importance in the treatment of inflammatory diseases. In this perspective, dendritic cells (DCs) can have an important immune-regulatory function, besides their notorious antigen-presenting capacity. DCs appear to be essential for both central and peripheral tolerance. In the thymus, DCs are involved in clonal deletion of autoreactive immature T cells by presenting self-antigens. Additionally, tolerance is achieved by their interactions with T cells in the periphery and subsequent induction of T cell anergy, T cell deletion, and induction of regulatory T cells (Treg). Various studies have described, modulation of DC characteristics with the purpose to induce antigen-specific tolerance in autoimmune diseases, graft-versus-host-disease (GVHD), and transplantations. Promising results in animal models have prompted researchers to initiate first-in-men clinical trials. The purpose of current review is to provide an overview of the role of DCs in the immunopathogenesis of autoimmunity, as well as recent concepts of dendritic cell-based therapeutic opportunities in autoimmune diseases.

Design of an Optimized Wilms’ Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo

Tumor antigen–encoding mRNA for dendritic cell (DC)-based vaccination has gained increasing popularity in recent years. Within this context, two main strategies have entered the clinical trial stage: the use of mRNA for ex vivo antigen loading of DCs and the direct application of mRNA as a source of antigen for DCs in vivo. DCs transfected with mRNA-encoding Wilms' tumor 1 (WT1) protein have shown promising clinical results. Using a stepwise approach, we re-engineered a WT1 cDNA-carrying transcription vector to improve the translational characteristics and immunogenicity of the transcribed mRNA. Different modifications were performed: (i) the WT1 sequence was flanked by the lysosomal targeting sequence of dendritic cell lysosomal-associated membrane protein to enhance cytoplasmic expression; (ii) the nuclear localization sequence (NLS) of WT1 was deleted to promote shuttling from the nucleus to the cytoplasm; (iii) the WT1 DNA sequence was optimized in silico to improve translational efficiency; and (iv) this WT1 sequence was cloned into an optimized RNA transcription vector. DCs electroporated with this optimized mRNA showed an improved ability to stimulate WT1-specific T-cell immunity. Furthermore, in a murine model, we were able to show the safety, immunogenicity, and therapeutic activity of this optimized mRNA. This work is relevant for the future development of improved mRNA-based vaccine strategies K.

The ratio between dendritic cells and T cells determines whether prostaglandin E2 has a stimulatory or inhibitory effect

Prostaglandin E2 has been shown to enhance the maturation, migration, and antigen-presenting capacity of DCs. It is therefore included in many maturation cocktails for the generation of monocyte-derived DCs. Paradoxically, PGE2 is also an important tumor-derived immunosuppressive factor and has inhibitory effects on DC differentiation and function. To further investigate these seemingly contradictory results we studied whether the DC:T cell ratio has an impact on the outcome of the interaction between PGE2-treated DCs and T cells. Surprisingly, at high DC:T cell ratios T cell proliferation was inhibited while at low ratios PGE2-treated DCs displayed enhanced T cell-stimulatory properties. The inhibitory function of PGE2-treated DCs depended primarily on the PGE2-induced induction of indoleamine 2,3-dioxygenase competence. In summary, we show that PGE2-treated DCs can have either an immunogenic or tolerogenic function depending on the DC:T cell ratio. This finding could explain the conflicting results regarding the influence of PGE2 on DC function.

The properties of human CD40-activated B cells as antigen-presenting cells are not affected by PGE2

Tumor vaccination represents a promising immuno-therapeutic strategy in cancer. However, the inherent ability of many tumors to evade immune responses by suppression of immune cell function represents a major barrier. ProstaglandinE2 (PGE2) has been shown to be a critical tumor-derived immunosuppressive factor. It affects a broad range of immune cells including T cells, macrophages and dendritic cells (DCs). CD40-activated Bcells are being studied as a potential alternative to DCs as antigen-presenting cells for immunotherapy. So far, it is not known whether PGE2 affects their antigen presenting capacity. We, therefore, investigated the influence of PGE2 on the phenotype, migratory potential and antigen-presenting function of CD40-activated human Bcells. Here, we demonstrate that the immunostimulatory properties of CD40-activated Bcells are not affected by PGE2. These results support the use of CD40-activated Bcells as cellular adjuvants, especially in settings where PGE2 is present in the tumor microenvironment.

One-step spray-dried polyelectrolyte microparticles enhance the antigen cross-presentation capacity of porcine dendritic cells

Vaccination is regarded as the most efficient and cost-effective way to prevent infectious diseases. Vaccine design nowadays focuses on the implementation of safer recombinant subunit vaccines. However, these recombinant subunit antigens are often poor immunogens and several strategies are currently under investigation to enhance their immunogenicity. The encapsulation of antigens in biodegradable microparticulate delivery systems seems a promising strategy to boost their immunogenicity. Here, we evaluate the capacity of polyelectrolyte complex microparticles (PECMs), fabricated by single step spray-drying, to deliver antigens to porcine dendritic cells and how these particles affect the functional maturation of dendritic cells (DCs). As clinically relevant model antigen F4 fimbriae, a bacterial adhesin purified from a porcine-specific enterotoxigenic Escherichia coli strain was chosen. The resulting antigen-loaded PECMs are efficiently internalised by porcine monocyte-derived DCs. F4 fimbriae-loaded PECMs (F4-PECMs) enhanced CD40 and CD25 surface expression by DCs and this phenotypical maturation correlated with an increased secretion of IL-6 and IL-1β. More importantly, F4-PECMs enhance both the T cell stimulatory and antigen presentation capacity of DCs. Moreover, PECMs efficiently promoted the CD8+ T cell stimulatory capacity of dendritic cells, indicating an enhanced ability to cross-present the encapsulated antigens. These results could accelerate the development of veterinary and human subunit vaccines based on polyelectrolyte complex microparticles to induce protective immunity against a variety of extra- and intracellular pathogens.

Human plasmacytoid dendritic cells are equipped with antigen-presenting and tumoricidal capacities

AbstractHuman plasmacytoid dendritic cells (pDCs) represent a highly specialized naturally occurring dendritic-cell subset and are the main producers of type I interferons (IFNs) in response to viral infections. We show that human pDCs activated by the preventive vaccine FSME specifically up-regulate CD56 on their surface, a marker that was thought to be specific for NK cells and associated with cytolytic effector functions. We observed that FSME-activated pDCs specifically lysed NK target cells and expressed cytotoxic molecules, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and granzyme B. Elevated levels of these molecules coincided with the expression of CD56, indicative for skewing human pDCs toward an interferon-producing killer DC subset. Detailed phenotypical and functional analysis revealed that pDCs attained a mature phenotype, secreted proinflammatory cytokines, and had the capacity to present antigens and stimulate T cells. Here, we report on the generation of CD56+ human interferon producing killer pDCs with the capacity to present antigens. These findings aid in deciphering the role for pDCs in antitumor immunity and present a promising prospect of developing antitumor therapy using pDCs.

Analysis of dendritic cells in human lymphoid organs

Dendritic Cells (DCs) are important regulators of immune responses. In our previous studies we found differential antigen presentation capacities of murine DC subpopulations using an in vivo antigen targeting system [1]. In contrast to murine DCs, the functional role of human tissue DCs is largely unknown.

Antibodies attenuate the capacity of dendritic cells to stimulate HIV-specific cytotoxic T lymphocytes

Control of HIV is suggested to depend on potent effector functions of the virus-specific CD8(+) T-cell response. Antigen opsonization can modulate the capture of antigen, its presentation, and the priming of specific CD8(+) T-cell responses. We have previously shown that opsonization of retroviruses acts as an endogenous adjuvant for dendritic cell (DC)-mediated induction of specific cytotoxic T lymphocytes (CTLs). However, in some HIV-positive subjects, high levels of antibodies and low levels of complement fragments coat the HIV surface. Therefore we analyzed the effect of IgG opsonization on the antigen-presenting capacity of DCs by using CD8(+) T-cell proliferation assays after repeated prime boosting, by measuring the antiviral activity against HIV-infected autologous CD4(+) T cells, and by determining IFN-γ secretion from HIV-specific CTL clones. We find that DCs exposed to IgG-opsonized HIV significantly decreased the HIV-specific CD8(+) T-cell response compared with the earlier described efficient CD8(+) T-cell activation induced by DCs loaded with complement-opsonized HIV. DCs exposed to HIV bearing high surface IgG levels after incubation in plasma from HIV-infected subjects acted as weak stimulators for HIV-specific CTL clones. In contrast, HIV opsonized with plasma from patients exhibiting high complement and low IgG deposition on the viral surface favored significantly higher activation of HIV-specific CD8(+) T-cell clones. Our exvivo and invitro observations provide the first evidence that IgG opsonization of HIV is associated with a decreased CTL-stimulatory capacity of DCs.

Human Cytomegalovirus Infection of M1 and M2 Macrophages Triggers Inflammation and Autologous T-Cell Proliferation

Macrophages (MΦ) are first targets during human cytomegalovirus (HCMV) infection and are thought to be crucial for viral persistence and dissemination. However, since MΦ are also a first line of defense and key modulators of the immune response, these cells are at the crossroad between protection and viral pathogenesis. To date, the MΦ-specific contribution to the immune response against HCMV is still poorly understood. In view of the opposite roles of M1 and M2 MΦ during initiation and resolution of the immune response, we characterized the effects of HCMV infection on classically activated M1 MΦ and alternatively activated M2 MΦ. Although HCMV susceptibility was higher in M2 MΦ, HCMV established a productive and persistent infection in both types of MΦ. Upon HCMV encounter, both types of MΦ acquired similar features of classical activation and secreted high levels of proinflammatory cytokines and chemokines. As a functional consequence, conditioned media obtained from HCMV-infected M1 and M2 MΦ potently activated freshly isolated monocytes. Finally, compared to HCMV-infected monocyte-derived dendritic cells, infected M1 and M2 MΦ were more efficient in stimulating proliferation of autologous T cells from HCMV-seropositive donors at early times (24 h) postinfection, while the MΦ immunostimulatory properties were reduced, but not abrogated, at later times (72 h postinfection). In summary, our findings indicate that MΦ preserve proper antigen presentation capacity upon HCMV infection while enhancing inflammation, thus suggesting that MΦ play a role in the maintenance of the large HCMV-specific T-cell repertoire in seropositive individuals.

Dendritic Cell-Based Immunotherapy in Mesothelioma

Mesothelioma is a rare thoracic malignancy with a dismal prognosis. Current treatment options are scarce and clinical outcomes are rather disappointing. Due to the immunogenic nature of mesothelioma, several studies have investigated immunotherapeutic strategies to improve the prognosis of patients with mesothelioma. In the last decade, progress in knowledge of the modulation of the immune system to attack the tumor has been remarkable, but the optimal strategy for immunotherapy has yet to be unraveled. Because of their potent antigen-presenting capacity, dendritic cells are acknowledged as a promising agent in immunotherapeutic approaches in a number of malignancies. This review gives an update and provides a future perspective in which immunotherapy may improve the outcome of mesothelioma therapy.

Enhanced protocol for CD14+ cell enrichment from equine peripheral blood via anti‐human CD14 mAb and automated magnetic activated cell sorting

CD14 positive (CD14+) cells are the precursor cells of monocyte-derived dendritic cells (DCs). In horses their potent antigen-presenting capacity and ability to induce an effective immune response classify these cells suitable for several therapeutic approaches such as for equine sarcoid. However, in horses, the generation efficiency of DCs from adherent peripheral blood mononuclear cells (PBMCs) is currently still poor. Establishment of a simple short protocol to enhance DC generation in horses by using a human CD14 monoclonal antibody (mAb) and an automated magnetic activated cell sorting (MACS) system. Peripheral blood mononuclear cells were isolated from fresh heparinised blood samples of 3 horses and primarily stained for flow cytometric analysis (FACS) with a mAb against human CD14 as well as a secondary phycoerythrin (PE) conjugated antibody to determine the initial percentage of CD14 cells in the sample. Peripheral blood mononuclear cells were used for automated MACS using the same primary and secondary antibodies and analysed by FACS. CD14+ selected cells were cultured for 4 days adding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to the culture media. Dendritic cell generation was assessed analysing cell morphology and surface marker expression (hCD83, hCD86, eqMHCII). Prior to selection, the mean percentage of CD14+ cells in the total cell population was 5.5%, further gaiting of this cell population resulted in 78.46% CD14+ monocytes. After our positive selection the mean percentage of CD14+ cells in the population was 98% without affecting viability. After culture, DC yield was 2-fold higher than in previous published outcomes. The additional CD14 cell separation step after PBMC isolation significantly amplified the number of CD14+ cells, increasing the number of generated DCs. The number of DCs available is critical for further use of these cells and the herein described protocol will therefore help to improved DC generation for therapeutic approaches in horses.

Brain-resident microglia predominate over infiltrating myeloid cells in activation, phagocytosis and interaction with T-lymphocytes in the MPTP mouse model of Parkinson disease

Parkinson disease (PD) is characterized by dopaminergic neurodegeneration in the substantia nigra (SN). Recent evidence suggests that innate and adaptive immune responses can influence dopaminergic cell death in animal models of PD. However, the precise role of mononuclear phagocytes, key players in damaged tissue clearance and cross-talk with cells of adaptive immune system, remains open in PD. Mononuclear phagocytes in the brain occur as brain-resident microglia and as brain-infiltrating myeloid cells. To elucidate their differential contribution in the uptake of dopaminergic cell debris and antigen presentation capacity, we labeled nigral dopaminergic neurons retrogradely with inert rhodamine-conjugated latex retrobeads before inducing their degeneration by subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration. We used green fluorescent protein (GFP)-expressing bone marrow chimeric mice to differentiate brain-infiltrating from brain-resident myeloid cells. We found that half of both endogenous (GFP−) and exogenous (GFP+) microglia (Iba1+) in the SN incorporated the tracer from degenerating dopaminergic neurons 1d after MPTP intoxication. In absolute numbers, endogenous microglia were much more activated to macrophages compared to exogenous myeloid cells at 1d after MPTP. Mainly the endogenous, tracer-phagocytosing microglia expressed the major histocompatibility complex (MHC) class II molecule for antigen presentation. Additionally, T-lymphocytes (Iba1−/GFP+/CD3+), which infiltrate the MPTP-lesioned SN, were mainly in direct contact with MHCII+ endogenous microglia. Our data suggest that brain-resident microglia are predominantly implicated in the removal of dopaminergic cell debris and the cross-talk with infiltrating T-lymphocytes in the SN in the MPTP mouse model of PD.

Total glucosides of paeony attenuated functional maturation of dendritic cells via blocking TLR4/5 signaling in vivo

It is well known that dendritic cells (DCs) play a critical role in the initiation and development of an immune response. Inhibitory effect on DC maturation alters immune-mediated inflammatory reaction in vivo. Total glucosides of paeony (TGP) are active compounds extracted from the roots of Paeonia lactiflora and have been widely used to ameliorate inflammation in therapy for autoimmune diseases. However, whether TGP act on DC maturation remains unknown. In this study, we investigated the effect of TGP on DC maturation in ovalbumin (OVA) immunized mice. Ear inflammation was inhibited by TGP (150mgkg−1, i.p.×11days) obviously. The antigen presenting capacity of DC derived from TGP-treated mice was arrested. Meanwhile, OVA specific T cell proliferation was inhibited. In addition, we found that maturation of DCs was decreased by TGP treatment. Furthermore, OVA specific T cell proliferation was rescued by the adoptive transfer of mature DCs (mDCs) into TGP treated OVA-challenged mice. The research on the mechanism showed that TGP significantly inhibited activation of TLR4/5 singling. All these results demonstrated that TGP inhibited DC maturation and function by selectively blocking TLR4/5 activation in vivo, which in turn leads to reduce immune-mediated inflammation in vivo, adding a novel mechanism and therapeutic target of TGP for inflammatory and autoimmune disease treatment.

Agressions cérébrales, immunité et infections

Infections are a major cause of death and morbidity after acute injury of the central nervous system (CNS). Acute lesions of the CNS alter immune homeostasis contributing to the development of immunosuppression (IS), and making the bed of the infection. IS results in a decreased phagocytic functions of neutrophils and macrophages as well as monocyte deactivation (decreased capacity of antigen presentation to lymphocytes). The immune abnormalities occur very quickly and may last for weeks. The neurovegetative system is closely connected to the secondary lymphoid organs where cells of innate immunity receive information from injured organs inducing the long lasting adaptive immune response (immune synapse). The sympathetic system is critically involved in the IS through production of anti-inflammatory mediators like interleukin-10. This may prove important as all types of acute injury of the CNS can lead to direct damage to sympathetic centers. Specialized units of care for ischemic stroke, taking into account the risk of infection related to the IS, have improved the prognosis until 18th month after the initial damage of the SNC. It is now well recognized that the improved long-term prognosis is related with the secondary prevention of recurrent ischaemia as well as aggressive management of pulmonary infections. A better understanding of the pathophysiology of IS can be considered in the near future, opening the door to immunomodulatory therapeutic trials.

Abstract 1562: Efficient antigen presentation and induction of anti-tumor immunity by dendritic cells loaded with antigens by electroporation

Abstract For successful dendritic cell (DC) therapy of cancer, it is important to choose the optimal method to load DCs with antigens to induce higher anti-tumor immunity, particularly tumor-specific CTL responses. The co-incubation (Co) method is conventionally used to load DCs with tumor antigens in DC therapy. Although DCs could uptake tumor antigens by endocytosis in this method, they are thought to be processed in lysosome and presented mainly on MHC class II, but not efficiently on MHC class I. Electroporation (Ep) is one of the promising antigen-loading method because it is reported that DCs could uptake much larger amount of FITC-dextran by Ep in comparison with Co method. In this study, we performed side-by-side comparisons of these two antigen loading methods using murine bone marrow derived DCs. First, we compared antigen presentation using purified ovalbumin (OVA) and OVA-specific OT-I CD8 and OT-II CD4 T cells. To this end, DCs were loaded with OVA by either Co or Ep, and subsequently cultured with T cells. Two or three days later, T cell expansion and cytokine production were evaluated. As expected, OVA-Co-DCs induced moderate expansion and cytokine production of OT-II T cells, but had little effect on OT-I T cells. In contrast, OVA-Ep-DCs potently stimulated both OT-I and OT-II T cells to expand and produce cytokines, such as IL-2 and IFN-γ. Using monoclonal antibody specific for MHC class I/OVA peptide complex, we confirmed efficient presentation of the OVA peptide on MHC class I by OVA-Ep-DCs. Furthermore, when we used the lysate of EG7 tumor cell line (OVA gene-transfected) as antigen instead of purified OVA, Ep-DCs induced expansion of both OT-I and OT-II T cells efficiently compared with Co-DCs. In the case of Alexa488-OVA, unexpectedly, similar amounts of uptake were seen in Ep-DCs and Co-DCs by FCM analysis. By using confocal microscopy, however, there was a critical difference of intracellular localization of Alexa488-OVA. Although Alexa488-OVA was localized in intracellular vesicles of both DCs, the cytosolic localization was observed only in Ep-DCs, suggesting that antigen loaded by Ep entered cytosol of DCs and would be processed by proteasome and presented on MHC class I efficiently. Finally, to determine the ability of these DCs to induce anti-tumor immunity in vivo, mice were subcutaneously immunized twice at 7 days intervals with 3-5 x 105 cells of either OVA-Ep-DCs or OVA-Co-DCs and then challenged with 3 x 106 of EG7 cells at 7 days after the 2nd immunization. When OVA-Co-DCs were used for immunization, tumor formations were partially prevented. On the contrary, mice immunized with OVA-Ep-DCs were almost completely protected from tumor formations. These results demonstrate that DCs loaded with tumor antigens by Ep have greater antigen presentation capacity, especially through MHC class I pathway, and could induce stronger anti-tumor immunity in vivo than those loaded by Co. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1562. doi:1538-7445.AM2012-1562

Abstract 1599: Grape seed proanthocyanidins inhibit UV-induced tolerogenic T-cell response through DNA repair-dependent functional activation of Langerhans cells

Abstract It is well documented that ultraviolet (UV) radiation-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPD) diminishes the antigen presenting capacity of epidermal Langerhans cells (LCs) and this leads to the suppression of immune system. Previously, we have shown that dietary grape seed proanthocyanidins (GSPs) inhibit UVB-induced immunosuppression in mice via activation of CD8+ T cells. However, it is not understood whether (1) GSPs target LCs while exerting its immunomodulatory effects on T cells in response to the application of cutaneous contact sensitizer, 2,4-dinitrofluorobenzene (DNFB) onto the UV-exposed skin, and whether (2) the ability of GSPs to activate dendritic cells/LCs correlates with their DNA repair properties. To understand this mechanism, we first determined DNA repair ability of GSPs in UV-exposed dendritic cells for which CD11c+ cells were prepared from bone marrow of xeroderma pigmentosum complementation group A-knockout (XPA-KO) mice which are deficient in DNA repair, and their wild-type (WT) counterparts. CD11c+ cells were exposed to UVB radiation with or without pretreatment with GSPs. Treatment of GSPs resulted in significant reduction in the number of CPD-positive cells in CD11c+ cells from WT mice, but this DNA repair effect of GSPs was not seen in the CD11c+ cells from XPA-KO mice. We then analyzed effects of dietary GSPs (0.5%, w/w) on secretion of cytokines by CD11c+ cells that were isolated from lymph nodes and spleens of mice (WT and XPA-KO) that were exposed to UVB [150 mJ/cm2 (WT), 20 mJ/cm2 (XPA-KO)]. Our results showed that in comparison to CD11c+ cells isolated from WT control mice (GSP-untreated), CD11c+ cells isolated from mice that received GSPs in diet secreted a significantly higher level of Th1 cytokines (IL-12, IFNα), while the levels of Th2 cytokines (IL-4, IL-10) were significantly lower, but this effect of GSPs was not observed in CD11c+ cells isolated from XPA-KO mice. Finally, we used an adoptive transfer approach, wherein draining lymph nodes were harvested from mice that were exposed to UVB with and without GSPs supplementation in diet and sensitized by DNFB onto the UVB-exposed skin. CD11c+ cells were positively selected and transferred into naïve mice that were subsequently challenged by application of DNFB on the ear skin. Naïve recipients that received CD11c+ cells from WT, GSPs-treated, UVB-irradiated donors exhibited full contact hypersensitivity (CHS) response, whereas no significant CHS was observed in XPA-KO mice. These data suggest that dietary GSPs inhibit UVB-induced immunosuppression in mice by improving the ability of LCs to mount an immune response via activation of T cells in a manner which correlates with its capacity to enhance repair of UVB-induced DNA damage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1599. doi:1538-7445.AM2012-1599

An Obligatory Role for Lung Infiltrating B Cells in the Immunopathogenesis of Obliterative Airway Disease Induced by Antibodies to MHC Class I Molecules

Using a murine model, we demonstrated that endobronchial administration of antibodies (Abs) to major histocompatibility complex (MHC) class I results in cellular infiltration, epithelial metaplasia, fibrosis and obstruction of the small airways (obliterative airway disease [OAD]) mediated predominantly by Th17 responses to self-antigens. This resembles bronchiolitis obliterans syndrome developed following human lung transplantation. Since B cells play a crucial role in induction of autoimmune responses, we defined the role of B cells and its antigen presenting properties in induction of OAD in this study. Anti-MHC class I was administered endobronchially in B(-/-) and wild-type mice. In contrast to wild type, B(-/-) animals did not demonstrate cellular infiltration, epithelial metaplasia and obstruction of airways following anti-MHC. Frequency of K-α1 tubulin and CollagenV-specific IL-17 cells was significantly decreased in B(-/-) mice. As expected, Abs against self-antigens and germinal center formation were not developed in B(-/-) mice. Thus, we conclude that B cells and its antigen presenting capacity play an important role in induction of immune responses to self-antigens and immunopathogenesis of OAD following the administration of anti-MHC. Therefore, strategies to block B-cell and its antigen presenting functions should be considered for preventing the development of chronic rejection.

Activated Apoptotic Cells Induce Dendritic Cell Maturation via Engagement of Toll-like Receptor 4 (TLR4), Dendritic Cell-specific Intercellular Adhesion Molecule 3 (ICAM-3)-grabbing Nonintegrin (DC-SIGN), and β2 Integrins

Dendritic cells (DCs) are professional antigen-presenting cells playing a central role in connecting innate and adaptive immunity. Maturation signals are, however, required for DCs to undergo phenotypic and functional changes to acquire a fully competent antigen-presenting capacity. We previously reported that activated apoptotic peripheral lymphocytes (ActApo) provide activation/maturation signals to human monocyte-derived DCs. In this paper, we have characterized the signaling pathways and molecules involved in ActApo-mediated DC maturation. We found that both cellular and supernatant fractions from ActApo are required for DC maturation signaling. ActApoSup-induced CD80 and CD86 expression was significantly blocked in the presence of neutralizing antibodies against tumor necrosis factor-α (TNF-α). Cell-cell contact-dependent signaling involved β2 integrins, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), and TLR4 because ActApo-induced up-regulation of the maturation markers CD80 and CD86 was significantly inhibited in the presence of neutralizing antibodies against CD18, CD11a, CD11b, and DC-SIGN as well as TLR4. The role of TLR4 was further confirmed by silencing of TLR4 in DCs. In addition, the endogenous adjuvant effect exerted by activated apoptotic splenocytes (ActApoSp) was reduced after immunization with human serum albumin in TLR4(-/-) mice. We detected activation of multiple signaling pathways and transcription factors in DCs upon co-culture with ActApo, including p38, JNK, PI3K-Akt, Src family kinases, NFκB p65, and AP1 transcription factor family members c-Jun and c-Fos, demonstrating the complex interactions occurring between ActApo and DCs. These studies provide important mechanistic insight into the responses of DCs during encounter with cells undergoing immunogenic cell death.

Tumor-Associated Antigens for Specific Immunotherapy of Prostate Cancer

Prostate cancer (PCa) is the most common noncutaneous cancer diagnosis and the second leading cause of cancer-related deaths among men in the United States. Effective treatment modalities for advanced metastatic PCa are limited. Immunotherapeutic strategies based on T cells and antibodies represent interesting approaches to prevent progression from localized to advanced PCa and to improve survival outcomes for patients with advanced disease. CD8+ cytotoxic T lymphocytes (CTLs) efficiently recognize and destroy tumor cells. CD4+ T cells augment the antigen-presenting capacity of dendritic cells and promote the expansion of tumor-reactive CTLs. Antibodies mediate their antitumor effects via antibody-dependent cellular cytotoxicity, activation of the complement system, improving the uptake of coated tumor cells by phagocytes, and the functional interference of biological pathways essential for tumor growth. Consequently, several tumor-associated antigens (TAAs) have been identified that represent promising targets for T cell- or antibody-based immunotherapy. These TAAs comprise proteins preferentially expressed in normal and malignant prostate tissues and molecules which are not predominantly restricted to the prostate, but are overexpressed in various tumor entities including PCa. Clinical trials provide evidence that specific immunotherapeutic strategies using such TAAs represent safe and feasible concepts for the induction of immunological and clinical responses in PCa patients. However, further improvement of the current approaches is required which may be achieved by combining T cell- and/or antibody-based strategies with radio-, hormone-, chemo- or antiangiogenic therapy.

Diphenyl diselenide-modulation of macrophage activation: Down-regulation of classical and alternative activation markers

Diphenyl diselenide (PhSe)(2), a simple organoselenium compound, possesses interesting pharmacological properties that are under extensive research. As macrophages respond to microenvironmental stimuli and can display activities engaged in the initiation and the resolution of inflammation, in the present report we describe the ability of (PhSe)(2) to modulate the macrophage activation. Our data indicate that (PhSe)(2) could inhibit the NO production in a dose-dependent fashion in peritoneal macrophages activated by LPS or treated with vehicle alone. We could demonstrate that this effect correlated with a reduction in the expression of the inducible NO synthase in (PhSe)(2)-treated cells. Furthermore, (PhSe)(2) suppressed the production of reactive oxygen species, diminished the activity of the arginase enzyme, and the accumulation of nitrotyrosine modified proteins in LPS-stimulated macrophages. This compound also diminished the antigen presentation capacity of classically activated macrophages, as it reduced MHCII and CD86 expression. In addition, (PhSe)(2) modulated the alternative activation phenotype of macrophages. Dexamethasone-activated macrophages presented higher production of IL-10 and CD206, which were both down-regulated by the addition of (PhSe)(2). These results suggest that (PhSe)(2) possesses antioxidant and anti-inflammatory activities in classically-activated macrophages. We could demonstrate that (PhSe)(2) can be also utilized to modulate the alternative activation phenotype of macrophages.