Photochemically-Mediated Inflammation and Cross-Presentation of Mycobacterium bovis BCG Proteins Stimulates Strong CD4 and CD8 T-Cell Responses in Mice.

Conventional vaccines are very efficient in the prevention of bacterial infections caused by extracellular pathogens due to effective stimulation of pathogen-specific antibodies. In contrast, considering that intracellular surveillance by antibodies is not possible, they are typically less effective in preventing or treating infections caused by intracellular pathogens such as Mycobacterium tuberculosis. The objective of the current study was to use so-called photochemical internalization (PCI) to deliver a live bacterial vaccine to the cytosol of antigen-presenting cells (APCs) for the purpose of stimulating major histocompatibility complex (MHC) I-restricted CD8 T-cell responses. For this purpose, Mycobacterium bovis BCG (BCG) was combined with the photosensitiser tetraphenyl chlorine disulfonate (TPCS2a) and injected intradermally into mice. TPCS2a was then activated by illumination of the injection site with light of defined energy. Antigen-specific CD4 and CD8 T-cell responses were monitored in blood, spleen, and lymph nodes at different time points thereafter using flow cytometry, ELISA and ELISPOT. Finally, APCs were infected and PCI-treated in vitro for analysis of their activation of T cells in vitro or in vivo after autologous vaccination of mice. Combination of BCG with PCI induced stronger BCG-specific CD4 and CD8 T-cell responses than treatment with BCG only or with BCG and TPCS2a without light. The overall T-cell responses were multifunctional as characterized by the production of IFN-γ, TNF-α, IL-2 and IL-17. Importantly, PCI induced cross-presentation of BCG proteins for stimulation of antigen-specific CD8 T-cells that were particularly producing IFN-γ and TNF-α. PCI further facilitated antigen presentation by causing up-regulation of MHC and co-stimulatory proteins on the surface of APCs as well as their production of TNF-α and IL-1ß in vivo. Furthermore, PCI-based vaccination also caused local inflammation at the site of vaccination, showing strong infiltration of immune cells, which could contribute to the stimulation of antigen-specific immune responses. This study is the first to demonstrate that a live microbial vaccine can be combined with a photochemical compound and light for cross presentation of antigens to CD8 T cells. Moreover, the results revealed that PCI treatment strongly improved the immunogenicity of M. bovis BCG.

Photochemical internalization (PCI)-mediated activation of CD8 T cells involves antigen uptake and CCR7-mediated transport by migratory dendritic cells to draining lymph nodes.

Antigen cross-presentation to cytotoxic CD8+ T cells is crucial for the induction of anti-tumor and anti-viral immune responses. Recently, co-encapsulation of photosensitizers and antigens into microspheres and subsequent photochemical internalization (PCI) of antigens in antigen presenting cells has emerged as a promising new strategy for inducing antigen-specific CD8+ T cell responses in vitro and in vivo. However, the exact cellular mechanisms have hardly been investigated in vivo, i.e., which cell types take up antigen-loaded microspheres at the site of injection, or in which secondary lymphoid organ does T cell priming occur? We used spray-dried poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with ovalbumin and the photosensitizer tetraphenyl chlorine disulfonate (TPCS2a) to investigate these processes in vivo. Intravital microscopy and flow cytometric analysis of the murine ear skin revealed that dendritic cells (DCs) take up PLGA microspheres in peripheral tissues. Illumination then caused photoactivation of TPCS2a and induced local tissue inflammation that enhanced CCR7-dependent migration of microsphere-containing DCs to tissue-draining lymph nodes (LNs), i.e., the site of CD8+ T cell priming. The results contribute to a better understanding of the functional mechanism of PCI-mediated vaccination and highlight the importance of an active transport of vaccine microspheres by antigen presenting cells to draining LNs.

Combined Photosensitization and Vaccination Enable CD8 T-Cell Immunity and Tumor Suppression Independent of CD4 T-Cell Help.

Cytotoxic T lymphocytes (CTLs) are key players in fighting cancer, and their induction is a major focus in the design of therapeutic vaccines. Yet, therapeutic vaccine efficacy is limited, in part due to the suboptimal vaccine processing by antigen-presenting cells (APCs). Such processing typically takes place via the MHC class II pathway for CD4 T-cell activation and MHC class I pathway for activation of CD8 CTLs. We show that a combination of skin photochemical treatment and immunization, so-called photochemical internalization (PCI) facilitated CTL activation due to the photochemical adjuvant effect induced by photosensitizer, oxygen, and light. Mice were immunized intradermally with antigen and photosensitizer, followed by controlled light exposure. PCI-treated mice showed strong activation of CD8 T cells, with improved IFN-γ production and cytotoxicity, as compared to mice immunized without parallel PCI treatment. Surprisingly, the CD8 T-cell effector functions were not impaired in MHC class II- or CD4 T-cell-deficient mice. Moreover, PCI-based vaccination caused tumor regression independent of MHC class II or CD4 T cells presence in melanoma bearing mice. Together, the data demonstrate that PCI can act as a powerful adjuvant in cancer vaccines, even in hosts with impaired T-helper functions.

Photosensitizer and Light Pave the Way for Cytosolic Targeting and Generation of Cytosolic CD8 T Cells Using PLGA Vaccine Particles.

The generation of CTLs is crucial in the immunological fight against cancer and many infectious diseases. To achieve this, vaccine Ags need to be targeted to the cytosol of dendritic cells, which can activate CD8 T cells via MHC class I (MHCI). Therefore, such targeting has become one of the major objectives of vaccine research. In this study, we aimed to bypass the unwanted and default MHC class II Ag presentation and trigger MHCI presentation by using a photosensitizer that, upon light activation, would facilitate cytosolic targeting of codelivered Ag. Poly(lactide-co-glycolide) microparticles ∼1 µm size were loaded with OVA and the photosensitizer tetraphenyl chlorine disulphonate (TPCS2a) and administered intradermally in mice, which were illuminated 1 d later for activation of the photosensitizer. Immunization in the presence of TPCS2a significantly increased activation of CD8 T cells compared with immunization without TPCS2a and as measured by CD8 T cell proliferation, production of proinflammatory IFN-γ, TNF-α, and IL-2, and prevention of tumor growth. Cytotoxicity was demonstrated by granzyme B production in vitro and by in vivo killing of CFSE-labeled targets. CD4-dependent Ab responses were abrogated in mice immunized with TPCS2a-containing particles, suggesting that photosensitization facilitated a shift from default MHC class II toward MHCI Ag presentation. Hence, vaccine particles with Ag and photosensitizers proved an effective vehicle or adjuvant for stimulation of CTLs, and they may find potential application in therapeutic cancer vaccination and in prophylactic and therapeutic vaccination against intracellular infections.

Photosensitisation facilitates cross-priming of adjuvant-free protein vaccines and stimulation of tumour-suppressing CD8 T cells.

Cancer vaccines aim to induce CD8 T cells infiltrating the tumour. For protein-based vaccines, the main biological barrier to overcome is the default MHC class-II-pathway, with activation of CD4 T cells rather than CD8 T cells. The latter requires antigens to access the cytosol and MHC class I antigen presentation. We applied photosensitiser and light to trigger disruption of antigen-containing endosomes and thereby MHC class I cross-presentation of a model cancer vaccine. This "photochemical internalisation" resulted in activation, proliferation, and IFN-γ production of cytotoxic CD8 T cells, which suppressed tumour growth by infiltrating CD8 T cells and caspase-3-dependent apoptosis. The process was independent of MHC class II, MyD88, and TLR4 signalling, but dependent on trypsin- and caspase-like proteasome activity and partly also on chloroquine. This novel method of vaccination may find applications in cancer immunotherapy where the activation of CD8 T cells is important.

Intradermal photosensitisation facilitates stimulation of MHC class-I restricted CD8 T-cell responses of co-administered antigen.

The protection or treatment of several immunological disorders is dependent on the antigen-specific and cytotoxic CD8 T cells. However, vaccines aimed at stimulating CD8 T-cell responses are typically ineffective because vaccine antigens are primarily processed by the MHC class-II and not the MHC class-I pathway of antigen presentation: the latter requires cytosolic delivery of antigen. In order to facilitate targeting of antigen to cytosol, the antigen was combined with the photosensitiser TPCS2a (disulfonated tetraphenyl chlorin) and administered intradermally to mice. The photosensitiser was activated by illumination of the injection site. This photochemical internalization (PCI) strongly increased the stimulation of CD8 T-cell responses as measured by antigen-specific proliferation and secretion of pro-inflammatory cytokines. Fluorescence microscopy showed that delivery to cytosol was TPCS2a dependent and occurred by light-induced disruption of TPCS2a- and antigen-containing endosomes. PCI-based vaccination prevented growth of malignant B16 cells as compared with vaccination without PCI. In conclusion, PCI represents a potent tool for delivery of antigens to cytosol for stimulation of cytotoxic CD8 T-cell responses. This study demonstrated a first proof-of-principle for PCI-mediated immunisation with potential application in cancer immunotherapy.

Photochemical targeting of antigens to the cytosol for stimulation of MHC class-I-restricted T-cell responses.

Tumour chemotherapy with drugs is typically associated with severe systemic and local side effects for which reason immunotherapy represents a safer alternative. However, vaccination often fails to generate the required cytotoxic CD8 T-cell responses due to insufficient access of antigens to the cytosol and the MHC class I pathway of antigen presentation. One important issue of tumour research is therefore to develop strategies that allow cytosolic targeting or endosomal escape of tumour antigens. The objective of the current study was to test whether endocytosed antigen could be delivered to MHC class I by means of photochemical internalisation (PCI). Briefly, the antigen and the photosensitiser Amphinex were loaded in vitro onto bone-marrow-derived murine dendritic cells (DCs). After light activation, which is supposed to cause disruption of OVA- and Amphinex-containing endosomes, the DCs were cultured with OVA-specific CD8 T cells or used for immunisation of mice. PCI facilitated CD8 T-cell responses as measured by IFN-γ secretion in vitro and CD8 T-cell proliferation in vivo. In conclusion, the current proof-of-concept study is the first to describe PCI-mediated immunisation and the results revealed the feasibility of this novel technology in autologous vaccination for stimulation of CD8 T-cell responses.

The antihistamines clemastine and desloratadine inhibit STAT3 and c-Myc activities and induce apoptosis in cutaneous T-cell lymphoma cell lines.

Mycosis fungoides and its leukaemic counterpart Sézary syndrome are the most frequent cutaneous T-cell lymphomas (CTCL), and there is no cure for these diseases. We evaluated the effect of clinically approved antihistamines on the growth of CTCL cell lines. CTCL cell lines as well as blood lymphocytes from patients with Sézary syndrome were cultured with antihistamines, and the cell were analysed for proliferation, apoptosis and expression of programmed death molecules and transcription factors. The two antihistamines clemastine and desloratadine, currently used for symptom alleviation in allergy, induced potent reduction of the activities of the constitutively active transcription factors c-Myc, STAT3, STAT5a and STAT5b in mycosis fungoides and Sézary syndrome cell lines. This inhibition was followed by apoptosis and cell death, especially in the Sézary syndrome-derived cell line Hut78 that also showed increased expression of the programmed death-1 (PD-1) after clemastine treatment. In lymphocytes isolated from Sézary syndrome patients, the CD4-positive fraction underwent apoptosis after clemastine treatment, while CD4-negative lymphocytes were little affected. Because both c-Myc and STAT transcription factors are highly expressed in proliferating tumours, their inhibition by clemastine, desloratadine and other inhibitors could complement established chemotherapies not only for cutaneous T-cell lymphomas but perhaps also other cancers.

TLR4- and TRIF-dependent stimulation of B lymphocytes by peptide liposomes enables T cell-independent isotype switch in mice.

Immunoglobulin class switching from IgM to IgG in response to peptides is generally T cell-dependent and vaccination in T cell-deficient individuals is inefficient. We show that a vaccine consisting of a dense array of peptides on liposomes induced peptide-specific IgG responses totally independent of T-cell help. Independency was confirmed in mice lacking T cells and in mice deficient for MHC class II, CD40L, and CD28. The IgG titers were high, long-lived, and comparable with titers obtained in wild-type animals, and the antibody response was associated with germinal center formation, expression of activation-induced cytidine deaminase, and affinity maturation. The T cell-independent (TI) IgG response was strictly dependent on ligation of TLR4 receptors on B cells, and concomitant TLR4 and cognate B-cell receptor stimulation was required on a single-cell level. Surprisingly, the IgG class switch was mediated by TIR-domain-containing adapter inducing interferon-ß (TRIF), but not by MyD88. This study demonstrates that peptides can induce TI isotype switching when antigen and TLR ligand are assembled and appropriately presented directly to B lymphocytes. A TI vaccine could enable efficient prophylactic and therapeutic vaccination of patients with T-cell deficiencies and find application in diseases where induction of T-cell responses contraindicates vaccination, for example, in Alzheimer disease.

Renal tubular PD-L1 (CD274) suppresses alloreactive human T-cell responses.

Renal proximal tubular epithelial cells, a target of infiltrating T cells during renal allograft rejection, may be protected from this injury by the cell surface protein CD274 (also termed PD-L1 for programmed death ligand 1). The co-inhibitory molecules PD-L1 (CD274) and PD-L2 (CD273) are ligands of PD-1 (programmed death 1; CD279). Here we determine the functional role of PD-1/PD-L pathways in human renal allograft rejection. Treatment of human primary tubular epithelial cells with interferon-beta and -gamma caused a dose-dependent and synergistic increase of PD-L1 and PD-L2 expression. Blockade of surface PD-L1, but not PD-L2, on interferon-treated tubular epithelial cells resulted in a significant increase in CD4+ T-cell proliferation and cytokine production by CD4+ and CD8+ T cells. The expression of PD-L1, PD-L2, and PD-1 mRNA and protein was upregulated in biopsies of patients with renal allograft rejection compared to the respective levels found in the pre-transplant biopsies. Induction of PD-L1 was significantly associated with acute vascular rejection. Our study suggests that the renal epithelial PD-1/PD-L1 pathway exerts an inhibitory effect of on alloreactive T-cell responses. The upregulation of PD-L1 on proximal tubular epithelial cells in patients with acute allograft rejection may reduce T-cell-mediated injury.

Limited costimulatory molecule expression on renal tubular epithelial cells impairs T cell activation.

BACKGROUND/AIMS: MHC molecules are upregulated on renal proximal tubular epithelial cells (TEC) under inflammatory conditions. This allows TEC to act as 'non-professional' antigen-presenting cells (APC). The aim of this study was to compare the costimulatory molecule expression pattern and the T cell activation capacity between renal TEC and professional APC, e.g. bone marrow-derived dendritic cells (BM-DC). METHODS: Flow cytometry analysis was used to study the costimulatory molecule surface expression on TEC or BM-DC. Ovalbumin-specific CD4 and CD8 T cell activation induced by TEC or BM-DC was compared, in terms of T cell proliferation, cytokine production and CTL activity. RESULTS: TEC did not constitutively express significant amounts of costimulatory molecules. Stimulation of TEC with IFN-beta or IFN-gamma, but not other tested cytokines, enhanced the expression of PD-L1, ICOS-L and CD40. Compared to BM-DC, TEC only induced suboptimal T cell activation. Blockade of PD-L1 on both APC strongly increased T cell activity. Furthermore, high PD-L1-expressing TEC were more resistant to the cytolysis by CTL. CONCLUSION: The low costimulatory molecule expression may explain the suboptimal T cell activation by TEC. The IFN-upregulated negative costimulatory molecule PD-L1 on TEC may play a protective role to limit tissue injury during renal parenchymal immune responses.

TGF-beta treatment modulates PD-L1 and CD40 expression in proximal renal tubular epithelial cells and enhances CD8 cytotoxic T-cell responses.

BACKGROUND/AIM: TGF-beta expression is increased in immune-mediated and fibrotic renal diseases and modulates the tubulointerstitial T-cell response. We examined whether TGF-beta changes the expression of PD-L1 and CD40 in the renal proximal tubular epithelial cell (TEC), and whether the activation of CD8(+) cytotoxic T cells (CTLs) is influenced by TGF-beta treatment of TECs. METHODS: Murine TECs were treated with TGF-beta or IFN-gamma. The expression of PD-L1 and CD40 was examined with RT-PCR and flow cytometry. To investigate if TGF-beta treatment influenced the antigen presentation capacity of TECs, OT-1 CTLs were co-incubated with TGF-beta-treated, OVA(257-264) peptide-pulsed congeneic TECs. The cytotoxicity of OT-1 CTLs was estimated by their capacity to produce IFN-gamma and their cytolytic activity. RESULTS: TGF-beta treatment lead to a transition in morphology of renal TECs and downregulated the basal and the IFN-gamma-stimulated PD-L1 expression in TECs. Interestingly, TGF-beta treatment of TECs increased the constitutive and IFN-gamma-induced CD40 expression. In contrast to IFN-gamma which reduced the CTL activity, TGF-beta treatment of TECs elevated the OVA-specific CTL response. CONCLUSION: Our data show that TGF-beta changed the cellular phenotype and the expression of PD-L1 and CD40 on TECs and enhanced the activity of OVA peptide-specific CD8(+) T cells. TGF-beta may thereby play an important role in the progression of renal tubulointerstitial damage in CD8(+) T-cell-mediated renal diseases.

Mitotic activation of Akt signalling pathway in Han:SPRD rats with polycystic kidney disease.

AIM: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by an imbalance between tubular epithelial cell proliferation and apoptosis. We have previously shown that the mammalian target of rapamycin (mTOR) signalling pathway is aberrantly activated in the cystic kidneys of Han:SPRD rats with ADPKD. Because the Akt kinase is an upstream regulator of mTOR, we hypothesized that the activity of Akt could be enhanced in the kidneys of Han:SPRD rats. METHODS: Reverse transcription-polymerase chain reaction, western blot, enzyme-linked immunosorbent assay and immunohistochemistry were used to analyse Akt expression in rat polycystic kidneys. RESULTS: Wild-type (+/+) and heterozygous (Cy/+) Han:SPRD rats showed constitutive expression of Akt-1, -2 and -3 mRNA by reverse transcription-polymerase chain reaction analysis with no significant difference between Cy/+ and +/+ kidneys. Western blotting and enzyme-linked immunosorbent assay showed a significant increase in phosphorylated Akt in Cy/+ compared with +/+ kidneys. The pattern of immunoreactivity for phosphorylated Akt in kidney sections was the same in +/+ and in Cy/+ rats, with very low levels in interphase cells, but extremely bright signals in mitotic cells, beginning with the onset of the prophase. The in vivo incorporation of bromo-deoxyuridine revealed approximately a ninefold higher rate of proliferation in Cy/+ cyst epithelia compared with normal tubule epithelia in +/+ rats, while the expression of the cell cycle marker Ki67 revealed approximately a sixfold higher rate of proliferation. In summary, enhanced phosphorylation of Akt can be demonstrated in Cy/+ kidneys which correlates with a markedly elevated proliferation rate of epithelial cells in cysts. Mitotic but not resting cells display strong phosphorylation of Akt. CONCLUSION: Because Akt is a proximal target of mTOR, its inhibition with specific antagonists could be useful to prevent or halt cystogenesis in ADPKD.

PD-L1 partially protects renal tubular epithelial cells from the attack of CD8+ cytotoxic T cells.

BACKGROUND: Activated infiltrating T cells play a crucial role in nephritic inflammation via the direct interaction with proximal tubular epithelial cells (TEC). Under inflammatory conditions, major histocompatibility complex class I and II molecules are upregulated on the surface of renal TEC, enabling them to function as "non-professional" antigen-presenting cells (APC) to activate T cells, and, in turn to be targeted by cytotoxic T lymphocytes (CTL) to cause tissue damage. It is known that co-stimulatory (e.g. B7/CD28) and co-inhibitory (e.g. PD-L1/PD-1) signals regulate and determine the magnitude of T cell responses. In this study, we examined the expression of co-stimulatory molecule PD-L1 by renal TEC and the functional role of renal PD-L1/PD-1 pathway in regulating CD8+ T cell responses induced by antigen-presenting renal TEC. METHODS: Renal TEC were treated with type I and type II interferons (IFN-alpha, IFN-beta or IFN-gamma). PD-L1 expression was then determined with flow cytometry and RT-PCR. To investigate the functional role of renal epithelial PD-L1 on CD8+ CTL responses, H-2K(b)-restricted, OVA(257-264) peptide-specific CD8+ T cells isolated from OT-1 T cell receptor transgenic mice were co-incubated with IFN-stimulated, OVA(257-264) peptide-pulsed congeneic TEC. The activation of OT-1 CD8+ CTL was estimated either by IFN-gamma production in the supernatants of co-cultures or by CTL activity. RESULTS: TECs do not constitutively express PD-L1 on their surface. However, a strong and dose-dependent upregulation of PD-L1 was observed on TEC after stimulation with IFN-beta or IFN-gamma, but not with IFN-alpha. OVA(257-264) peptide pulsed-TEC were able to activate OT-1 CD8+ T cells, indicated by the high amount of IFN-gamma production and cytolysis of TEC. Blockade of epithelial PD-L1 with specific mAb significantly increased OT-1 CD8+ T cell activity, indicating that the PD-L1 pathway has a negative effect on CD8+ T cell responses. Moreover, IFN- beta- or IFN-gamma-stimulated TEC with high surface PD-L1 expression were more resistant to the cytolysis by OT-1 CTL. CONCLUSION: Together our data reveal that the renal PD-L1/PD-1 pathway has a negative effect on CD8+ CTL activation. PD-L1 might, therefore, act as a protective molecule on TEC, downregulating the cytotoxic renal parenchymal immune response.

Dendritic cell-based multi-epitope immunotherapy of hormone-refractory prostate carcinoma.

BACKGROUND: Dendritic cell (DC)-based immunotherapy is a promising approach to augment tumor antigen-specific T cell responses in cancer patients. However, tumor escape with down-regulation or complete loss of target antigens may limit the susceptibility of tumor cells to the immune attack. Concomitant generation of T cell responses against several immunodominant antigens may circumvent this potential drawback. In this trial, we determined the immunostimulatory capacity of autologous DC pulsed with multiple T cell epitopes derived from four different prostate-specific antigens in patients with advanced hormone-refractory prostate cancer. PATIENTS AND METHODS: Autologous DC of HLA-A*0201(+) patients with hormone-refractory prostate cancer were loaded with antigenic peptides derived from prostate stem cell antigen (PSCA(14-22)), prostatic acid phosphatase (PAP(299-307)), prostate-specific membrane antigen (PSMA(4-12)), and prostate-specific antigen (PSA(154-163)). DC were intradermally applied six times at biweekly intervals followed-in the case of an enhanced immune response-by monthly booster injections. Immune monitoring during the time of ongoing vaccinations (12-59 weeks) included ex vivo ELISPOT measurements, MHC tetramer analysis and in vitro cytotoxicity assays. RESULTS: Of the initial six patients, three qualified for long-term multi-epitope DC vaccination. This regime was tolerated well by all three patients. The vaccination elicited significant cytotoxic T cell responses against all prostate-specific antigens tested. In addition, memory T cell responses against the control peptides derived from influenza matrix protein and tetanus toxoid were efficiently boosted. Clinically, the long-term DC vaccination was associated with an increase in PSA doubling time. CONCLUSIONS: DC-based multi-epitope immunotherapy with repeated boosting in men with hormone-refractory prostate carcinoma is feasible and generates efficient cellular antitumor responses.

Encapsulation of proteins and peptides into biodegradable poly(D,L-lactide-co-glycolide) microspheres prolongs and enhances antigen presentation by human dendritic cells.

Dendritic cell (DC)-based immunotherapy has been hampered by the lack of suitable methods for antigen delivery. Here, we use biodegradable poly(D,L-lactide-co-glycolide) microspheres (PLGA-MS) as carriers of peptides and proteins for antigen delivery to human monocyte-derived DC (MoDC). Compared to soluble proteins, MHC classes I and II-restricted presentation of PLGA-MS-encapsulated proteins and peptides by MoDC was markedly prolonged and proteins were presented 50-fold more efficiently on class I molecules. The vaccination of mice with DC loaded with PLGA-MS-encapsulated proteins raised strong and persisting cytotoxic T cell responses. In conclusion, antigen delivery via PLGA-MS markedly enhanced the duration of antigen presentation by human MoDC and the potency of DC-based vaccination.

Dendritic cells generated from patients with androgen-independent prostate cancer are not impaired in migration and T-cell stimulation.

BACKGROUND: Dendritic cell (DC)-based vaccination has been investigated as immunotherapy for several types of cancer. A potential drawback to vaccination with autologous monocyte-derived DCs (MoDCs) could be that MoDCs from patients are functionally impaired. In case of androgen-independent prostate cancer (CaP), the cancer itself, diverse prior therapies, and the hormone manipulation may affect the immune system. METHODS: MoDCs from patients suffering from androgen-independent CaP were generated according to a clinically applicable protocol to evaluate the phenotype, maturation capacity, migration, and T-cell stimulation of these cells compared with those generated from tumor-free donors. RESULTS: MoDCs generated from CaP patients could be fully matured and efficiently migrated towards the chemokine CCL21. They had a strong potency to activate allogeneic CD4+ and CD8+ T-cells and to present antigens to specific CTL. CONCLUSIONS: Our data suggest that MoDCs from patients with androgen-independent CaP are functionally intact and hence qualify as cellular vaccines for immunotherapy of advanced stage CaP.

Delivery of tumor antigens to dendritic cells using biodegradable microspheres.

Poly(D,L-lactide-co-glycolide) (PLGA) polymers have been used for the production of biodegradable medical sutures and for controlled drug release for decades. Useful characteristics such as in vivo biodegradability, an adjustable release profile, and the very high encapsulation capacity have stimulated immunologists to explore PLGA microspheres (MS) as antigen delivery systems for vaccination for more than 15 yr. In previous studies aiming at the development of "single-dose" vaccines, direct immunization with PLGA MS containing various antigens induced strong and sustained immune responses. We have observed that human immature monocyte-derived dendritic cells (MoDC) prepared for clinical application are able to internalize high numbers of MS without negative effects on their pivotal properties. Furthermore, PLGA-MS-incorporated antigens are effectively processed for presentation on major histocompatibility complex (MHC) class I and MHC class II molecules by dendritic cells (DCs) in vitro and induced strong cytotoxic T-lymphocyte (CTL) responses in vivo. Taken together, PLGA MS is a promising delivery vehicle for the improvement of current DC-based tumor vaccine protocols.

PLGA microspheres for improved antigen delivery to dendritic cells as cellular vaccines.

Dendritic cells (DC) are currently employed as cellular vaccines in clinical trials of tumor immunotherapy. In most trials, peptide epitopes derived from tumor antigens are being exogenously loaded onto human DC for binding to MHC class I molecules. While this is a convenient method, it suffers from the drawback that the persistence of class I/peptide complexes on the cell surface is in the order of a few hours. This drawback limits the success of vaccination. We have investigated biodegradable poly(D,L-lactide-co-glycolide) microspheres (PLGA-MS) as delivery tools for antigen loading of human monocyte-derived DC (hMoDC). Immature hMoDC readily take up PLGA-MS and present epitopes from encapsulated proteins or peptides both on MHC class I and class II. Interestingly, antigen presentation by hMoDC was markedly prolonged when hMoDC were charged with PLGA-MS-encapsulated as opposed to soluble antigens. The properties of hMoDC with respect to migration, cytokine secretion, survival and allostimulation were not adversely affected by the uptake of PLGA-MS. In this article, we will review the properties of PLGA-MS as an adjuvant and summarize recent data on their potential for antigen delivery to dendritic cells.

Phenotype and functional analysis of human monocyte-derived dendritic cells loaded with biodegradable poly(lactide-co-glycolide) microspheres for immunotherapy.

Dendritic cells (DC) are increasingly explored as cellular vaccines for tumor immunotherapy. In most reported DC-based cancer vaccine trials, DC have been pulsed with soluble tumor antigen-derived peptide ligands of MHC molecules. Considering that the half-life of peptide/MHC complexes on the cell surface is relatively short and that soluble exogenous protein antigens cannot be efficiently processed via the MHC class I-processing pathway, the current vaccination procedure is not optimal for the induction of strong T cell responses aiming at tumor rejection. Recently, we have shown that antigen presentation can be prolonged when synthetic peptides were encapsulated in biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres (MS) for uptake by DC. In the present study, we investigated the phenotypic and functional consequences of MS uptake by human monocyte-derived dendritic cells (MoDC) in vitro. We found that immature MoDC that were prepared in serum free media suitable for clinical application were able to phagocytose high numbers of MS, while matured MoDC showed a reduced capacity for phagocytosis of MS. The ingestion of MS did not change the cell surface expression of CD80, CD83, CD86 and HLA-DR of immature and mature DC, suggesting that MS uptake did not induce DC maturation but that maturation by cytokines or LPS was unaltered in the presence of MS. Furthermore, MS-loaded mature MoDC expressed normal levels of the chemokine receptor CCR7 and migrated as efficiently towards CCL19 or CCL21 as unloaded MoDC. DC viability and the secretion of TNF-alpha and IL-12 was not significantly changed by MS loading. Taken together, our data indicate that PLGA-MS loading has no negative effects on the pivotal properties of MoDC in vitro. It should therefore be feasible to further develop this antigen loading strategy for clinical use in immunotherapy against viral infections and tumors.