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The ubiquitin-proteasome system is elementary for cellular protein degradation and gained rising attention as a new target for cancer therapy due to promising clinical trials with bortezomib, the first-in class proteasome inhibitor meanwhile approved for multiple myeloma and mantle cell lymphoma. Both bortezomib and next-generation proteasome inhibitors mediate their effects by targeting the 20S core particle of the 26S proteasome. The novel small molecule inhibitor b-AP15 affects upstream elements of the ubiquitin-proteasome cascade by suppressing the deubiquitinase activity of both proteasomal regulatory 19S subunits and showed promising anticancer activity in preclinical models. Nonetheless, effects of inhibitors on the ubiquitin-proteasome system are not exclusively restricted to malignant cells: alteration of natural killer cell-mediated immune responses had already been described for drugs targeting either 19S or 20S proteasomal subunits. Moreover, it has been shown that bortezomib impairs dendritic cell (DC) phenotype and function at different levels. In the present study, we comparatively analyzed effects of bortezomib and b-AP15 on monocyte-derived DCs. In line with previous results, bortezomib exposure impaired maturation, antigen uptake, migration, cytokine secretion and immunostimulation, whereas treatment with b-AP15 had no compromising effects on these DC features. Our findings warrant the further investigation of b-AP15 as an alternative to clinically approved proteasome inhibitors in the therapy of malignancies, especially in the context of combinatorial treatment with DC-based immunotherapies.

In chronic myeloid leukemia (CML), the translocation t(9;22) results in the fusion protein BCR-ABL (breakpoint cluster region-abelson murine leukemia), a tyrosine kinase mediating oncogenic signaling which is successfully targeted by treatment with BCR-ABL inhibitors like imatinib. However, BCR-ABL inhibitors may also affect antitumor immunity. For instance, it was reported that imatinib impairs the function of dendritic cells (DCs) that play a central role in initiating and sustaining T cell responses. Meanwhile, second generation BCR-ABL inhibitors like nilotinib, which inhibits BCR-ABL with enhanced potency have become standard of treatment, at least in patients with BCR-ABL kinase domain mutations. In this study we analyzed the influence of therapeutic concentrations of nilotinib on human monocyte-derived DCs and compared its effects to imatinib. We found that both tyrosine kinase inhibitors (TKI) comparably and significantly impaired differentiation of monocytes to DCs as revealed by curtated downregulation of CD14 and reduced upregulation of CD1a and CD83. This was only partially restored after withdrawal of the TKI. Moreover, both TKI significantly reduced activation-induced IL-12p70 and C-C motif chemokine ligand (CCL) 3 secretion, while divergent TKI effects for CCL2 and CCL5 were observed. In contrast, only nilotinib significantly impaired the migratory capacity of DCs and their capacity to induce T-cell immune responses in MLRs. Our results indicate that imatinib and nilotinib may differ significantly with regard to their influence on antitumor immunity. Thus, for future combinatory approaches and particularly stop studies in CML treatment, choice of the most suitable BCR-ABL inhibitor requires careful consideration.

The first therapeutic proteasome inhibitor bortezomib has clinical efficacy in mantle cell lymphoma (MCL) which resulted in its incorporation in treatment algorithms for this disease. Impairment of proteasomal function by bortezomib is mediated via inhibition of the 20S core particle. However, proteasome function can also be modified by targeting upstream components of the ubiquitin–proteasome system. Recently, b-AP15 has been identified as a small molecule achieving proteasome inhibition by targeting the deubiquitinase (DUB) activity of the 19S regulatory subunit and was found to inhibit cancer cell growth in preclinical analyses. In the present study, both direct antitumor effects and the possibility to induce natural killer group 2 member D ligands (NKG2DL) to reinforce NK cell immunity with b-AP15 were investigated to provide a rational basis for clinical evaluation of this novel DUB inhibitor in MCL. Treatment with b-AP15 resulted in reduced viability as well as induction of apoptosis in a time- and dose-dependent manner, which could be attributed to caspase activation in MCL cells. In addition, treatment with b-AP15 differentially induced NKG2DL expression and subsequent NK cell lysis of MCL cells. These results indicate that the DUB inhibitor b-AP15 displays substantial antitumor activity in human MCL and suggest that b-AP15 might be a novel therapeutic option in the treatment of MCL that warrants clinical investigation.

The success of allogeneic stem cell transplantation depends upon the degree of human leukocyte antigen (HLA) compatibility between the donor and patient since T-cell responses to foreign HLA molecules can result in aggressive immune responses. The human HLA locus on chromosome 6 is highly polymorphic within the population. Due to the large number of allels, the exact determination of the individual HLA polymorphisms (HLA typing) is a technically challenging task requiring different molecular biological procedures.

Multiple approaches presently aim to combine targeted therapies using tyrosine kinase inhibitors with immunotherapy. Ex vivo-generated dendritic cells are frequently used in such strategies due to their unique ability to initiate primary T-cell immune responses. Besides governing tumor cell growth, many kinases targeted by tyrosine kinase inhibitors are involved in the development and function of dendritic cells and thus tyrosine kinase inhibitor therapy may cause immunoinhibitory side effects. We here report that exposure of developing human monocyte-derived dendritic cells to the BCR-ABL inhibitors imatinib, dasatinib, and nilotinib results in profound upregulation of the transmembrane glycoprotein osteoactivin that has recently been characterized as a negative regulator of T-cell activation. Thus, in line with osteoactivin upregulation, exposure to tyrosine kinase inhibitors resulted in significantly reduced stimulatory capacity of dendritic cells in mixed lymphocyte reactions that could be restored by the addition of blocking anti-osteoactivin antibody. Our data demonstrate that tyrosine kinase inhibitor-mediated inhibition of dendritic cell function is, at least in great part, mediated by upregulation of the immune inhibitory molecule osteoactivin.

Natural killer (NK) cells play an important role in the immunosurveillance of leukemia. Their reactivity is governed by a balance of activating and inhibitory receptors including various members of the tumor necrosis factor receptor (TNFR) family. Here we report that human NK cells acquire expression of the TNFR family member CD137 upon activation, and NK cells of acute myeloid leukemia (AML) patients display an activated phenotype with substantial CD137 expression. CD137 ligand (CD137L) was detectable on leukemic cells in 35% of 65 investigated AML patients, but not on healthy CD34+ cells, and expression was associated with monocytic differentiation. Bidirectional signaling following CD137-CD137L interaction induced the release of the immunomodulatory cytokines interleukin-10 and TNF by AML cells and directly diminished granule mobilization, cytotoxicity, and interferon-γ production of human NK cells, which was restored by blocking CD137. Cocultures of NK cells with CD137L transfectants confirmed that human CD137 inhibits NK-cell reactivity, while activating signals were transduced by its counterpart on NK cells in mice. Our data underline the necessity to study the function of seemingly analog immunoregulatory molecules in mice compared with men and demonstrate that CD137-CD137L interaction enables immune evasion of AML cells by impairing NK-cell tumor surveillance in humans.

In chronic myeloid leukemia (CML), BCR/ABL-mediated oncogenic signaling can be targeted with the BCR/ABL-inhibitors Imatinib, Nilotinib and Dasatinib. However, these agents may also affect anti-tumor immunity. Here, we analyzed the effects of the 3 BCR/ABL-inhibitors on natural killer (NK) cell reactivity. Exposure of CML cells (K562, Meg-01) to pharmacological concentrations of Imatinib, Nilotinib and Dasatinib diminished expression of ligands for the activating immunoreceptor NKG2D to a similar extent. This resulted in comparably reduced NK cell cytotoxicity and IFN-gamma production. When direct effects on NK cell responses to K562 and primary CML cells as well as activating cytokines were studied, Dasatinib was found to abrogate NK cytotoxicity and cytokine production. Nilotinib did not alter cytotoxicity but, at high levels, impaired NK cytokine production, while Imatinib had no direct influence on NK cell reactivity. Of note, Nilotinib, but not the other BCR/ABL-inhibitors increased cell death within the preferentially cytokine-secreting CD56(bright)CD16(-) NK cell subset, which may, at least in part, serve to explain the effect of Nilotinib on NK cytokine production. Analysis of NK cell signaling revealed that Dasatinib inhibited proximal signaling events leading to decreased phosphorylation of PI3K and ERK that are crucial for NK cell reactivity. Imatinib and Nilotinib, in contrast, showed no relevant effect on NK cell PI3K or ERK activity. In light of the potential role of NK cells in the immunesurveillance of residual leukemia and for future combinatory immunotherapeutic approaches, our data indicate that choice and dosing of the most suitable BCR/ABL-inhibitor for a given patient require careful consideration.

Dendritic cells (DCs) are key players in activation of the adaptive immune system by their ability of antigen presentation to and priming of T cells. An increasing body of evidence suggests that DCs may also play an important role in induction of tolerance, predominantly by induction of regulatory T cells (T(reg)). More recently, data have been published on how Toll-like receptor (TLR) ligands and cytokines affect DC differentiation, and how DC subsets might be involved in immunoregulation and tolerance rather than in T cell activation. The most important features of tolerance-inducing DCs appear to be their maturation state and their cytokine secretion pattern. The following types of tolerance-inducing DCs have been reported: immature DCs (DCs(im)) or DCs in the steady state (DCs(st)), DCs(IL-10), semi-mature DCs(TNF-alpha), semi-mature DCs(IL-6). With this review article we would like to discuss the aforementioned types of tolerogenic DCs with a focus on semi-mature DCs(IL-6) and discuss their potential role in maintenance of (hepatic or intestinal) immune homeostasis and inflammatory diseases such as inflammatory bowel disease.

Cancer immunotherapy aims at eliciting an immune response directed against tumor antigens to help fight off residual tumor cells and thereby improve survival and quality of life of cancer patients. Different immunotherapeutic approaches share the use of dendritic cells (DCs) to present tumor-associated antigens to T-lymphocytes. Ex vivo generated DCs can be loaded with antigens and re-infused to the patients, or they can be used for ex vivo expansion of antitumor lymphocytes. Alternatively, methods exist to target antigens to DCs in vivo without need for ex vivo cell manipulations. The clinical studies have shown that DC administration to patients is safe and induces antigen-specific immunity. However, it seldom elicits objective clinical responses in patients with advanced-stage malignancies. Novel insights into DC and lymphocyte regulation are expected to lead to more effective vaccines in the near future. Meanwhile, efforts are directed at identifying the most appropriate clinical targets for active specific immunotherapies. Data suggests that vaccinations may indeed be beneficial when given in the adjuvant setting rather than to treat metastatic cancers. These issues are discussed here together with an overview of the DC-based antitumor immunotherapy studies.

Advances in tumor immunology and Identification of tumor-associated antigens (TAAs) provide a basis for the development of novel immunotherapies to treat malignant diseases. In order to identify novel TAAs, we performed comparative microarray analysis of (heterogeneous) tissues and found regulator of G protein-signaling 1 (RGS1) extensively up-regulated in renal cell carcinoma (RCC) tissues. To examine the possible function of this molecule as a novel, broadly applicable TAA, synthetic full-length RGS1-mRNA was synthesized for the transfection of monocyte-derived dendritic cells (DCs). These modified antigen-presenting cells (APCs) were then used to induce RGS1-specific cytotoxic T cells (CTLs) in vitro. The CTLs generated from several healthy donors and a patient with chronic lymphocytic leukemia (CLL) elicited an antigen-specific and HLA-A2- and -A3-restricted cytolytic activity against tumor cells endogenously expressing the RGS1 protein including renal cell carcinomas (RCCs), melanoma, ovarian carcinoma and the primary autologous CLL-blasts. In conclusion, our study demonstrates that the in vitro induction of RGS1-specific CTLs by RNA-transfected DCs is feasible and highly effective. Since this molecule is (over-) expressed in a broad variety of malignancies it might represent an interesting novel TAA in the context of cancer vaccines designed to target RGS1 expressing tumor cells.

The delivery of protein fragments to major histocompatibility complex (MHC)-loading compartments of professional antigen-presenting cells is essential in the adaptive immune response against pathogens. Apart from the crucial role of the transporter associated with antigen processing (TAP) for peptide loading of MHC class I molecules in the endoplasmic reticulum, TAP-independent translocation pathways have been proposed but not identified so far. Based on its overlapping substrate specificity with TAP, we herein investigated the ABC transporter ABCB9, also named TAP-like (TAPL). Remarkably, TAPL expression is strongly induced during differentiation of monocytes to dendritic cells and to macrophages. TAPL does not, however, restore MHC class I surface expression in TAP-deficient cells, demonstrating that TAPL alone or in combination with single TAP subunits does not form a functional transport complex required for peptide loading of MHC I in the endoplasmic reticulum. In fact, by using quantitative immunofluorescence and subcellular fractionation, TAPL was detected in the lysosomal compartment co-localizing with the lysosome-associated membrane protein LAMP-2. By in vitro assays, we demonstrate a TAPL-specific translocation of peptides into isolated lysosomes, which strictly requires ATP hydrolysis. These results suggest a mechanism by which antigenic peptides have access to the lysosomal compartment in professional antigen-presenting cells.

Purpose: Histone deacetylases (HDAC) modulate gene transcription and chromatin assembly by modifying histones at the posttranscriptional level. HDAC inhibitors have promising antitumor activity and are presently explored in clinical studies. Cumulating evidence in animal models of immune disorders also suggests immunosuppressive properties for these small molecules, although the underlying mechanisms remain at present poorly understood. Here, we have evaluated the effects of two HDAC inhibitors currently in clinical use, sodium valproate and MS-275, on human monocyte-derived DCs.Experimental Design: DCs were generated from monocytes through incubation with granulocyte macrophage colony-stimulating factor and interleukin-4. DC maturation was induced by addition of polyinosinic-polycytidylic acid. DC phenotype, immunostimulatory capacity, cytokine secretion, and migratory capacity were determined by flow cytometry, mixed leukocyte reaction, ELISA, and Transwell migration assay, respectively. Nuclear translocation of RelB, IFN regulatory factor (IRF)-3, and IRF-8 were determined by immunoblotting.Results: HDAC inhibition skews DC differentiation by preventing the acquisition of the DC hallmark CD1a and by affecting the expression of costimulation and adhesion molecules. In addition, macrophage inflammatory protein-3β/chemokine, motif CC, ligand 19–induced migration, immunostimulatory capacity, and cytokine secretion by DCs are also profoundly impaired. The observed defects in DC function on exposure to HDAC inhibitors seem to reflect the obstruction of signaling through nuclear factor-κB, IRF-3, and IRF-8.Conclusions: HDAC inhibitors exhibit strong immunomodulatory properties in human DCs. Our results support the evaluation of HDAC inhibitors in inflammatory and autoimmune disorders.

Proteasome inhibitors are emerging as effective drugs for the treatment of multiple myeloma and possibly certain subtypes of non-Hodgkin's lymphoma. Bortezomib (Velcade) is the first proteasome inhibitor proven to be clinically useful and will soon be followed by a second generation of small molecule inhibitors with improved pharmacological properties. Although it is now understood that certain types of malignancies have an exquisite dependence on a functional proteasome for their survival, the underlying reason(s) remain unclear as of now. In this context, addiction to nuclear factor-kappaB (NF-kappaB)-induced survival signals, activation of the unfolded protein response as well as a reduced proteasomal activity in differentiated plasma cells have all been proposed to justify proteasome inhibitors' activity in susceptible tissues. In addition to their anticancer properties, bortezomib and related drugs modulate inflammatory and immune responses by affecting function and survival of immune cells such as lymphocytes and dendritic cells. The present review offers an overview of the biological effects that have been involved in proteasome inhibitors' antitumor activity and suggests prospective future applications for these drugs based on their recently characterized anti-inflammatory and immunomodulatory effects.

In the present study, we analyzed the involvement of the BCR-ABL protein in the induction of antigen-specific CTL in order to develop an immunotherapeutic approach in patients with chronic myelogenous leukemia (CML). To accomplish this, we generated dendritic cells (DC) in vitro and electroporated them with various sources of RNA harboring the chimeric bcr-abl transcript. These genetically engineered DCs were used as antigen-presenting cells for the induction of CTLs. By applying this approach, we found that the CTLs induced by DCs transfected with RNA extracted from bcr-abl–positive K-562 cells or CML blasts lysed DCs transfected with the corresponding RNA, but failed to recognize epitopes derived from the chimeric BCR-ABL fusion protein in 51Cr-release assays. In contrast, they were able to lyse autologous DCs electroporated with RNA isolated from patients with acute myeloid leukemia, indicating that antigens shared among these malignant cells are involved and recognized by these CTLs. In patients with CML in complete cytogenetic remission during IFN-α treatment, we detected some reactivity of CD8+ T cells against BCR-ABL in IFN-γ ELISPOT assays, which was weaker as compared with proteinase 3 (PR3)- or prame-directed responses, suggesting that the BCR-ABL protein is less immunogenic as compared with other CML-derived antigens. (Cancer Res 2006; 66(11): 5892-900)

Transfection of dendritic cells (DCs) with RNA was shown to be effective in the generation of antigen-specific T cells, probably due to the induction of a polyclonal T cell response directed against multiple antigens. To verify this assumption we used DCs, generated from cytomegalovirus (CMV)-negative or -positive donors, that were electroporated with in vitro-transcribed RNA (in vitro transcript, IVT) coding for the CMV pp65 antigen. We found that transfection of DCs with pp65 IVT induces an expansion of polyclonal CD8(+) T lymphocytes that recognize peptide antigens presented on different HLA molecules. These T lymphocytes are able to lyse DCs pulsed with pp65-derived peptides or transfected with the cognate IVT. Furthermore, this approach allowed the identification of immunodominant epitopes presented upon IVT transfection. Interestingly, transfection of DCs with pp65 IVT resulted in the induction of CD4(+)-specific T cells. Cotransfection of DCs with IVTs coding for the CMV antigens pp65 and IE1 elicited polyclonal T lymphocytes specific for peptides derived from both antigens. More importantly, cytotoxic T cells could be generated in two of three CMV-negative donors. Finally, functional CMV-specific autologous cytotoxic T lymphocytes were successfully generated from immunosuppressed patients after allogeneic hematopoietic stem cell transplantation.

Recently, it was shown that dendritic cells (DCs) transfected with ribonucleic acid (RNA) coding for a tumour-associated antigen (TAA) or whole tumour RNA are able to induce potent antigen and tumour-specific T cell responses directed against multiple epitopes. The feasibility and efficacy of this approach was tested and analysed in different settings by administration of RNA-transfected DCs in patients with cancer or infectious diseases. In some cases, clinical responses were observed, but the overall effectiveness of this approach has to be demonstrated in further clinical trials.

Ribonucleic acid (RNA) transfection of dendritic cells (DCs) was shown to be highly efficient in eliciting CD8+ and CD4+ T-cell responses. We analyzed whether electroporation of DCs with RNA coding for a tumor-associated antigen (TAA) would elicit antigen-specific effector cytotoxic T lymphocyte (CTL) responses and whether these responses could be modulated by cotransfection with a second specific synthetic RNA. Therefore in vitro generated human monocyte-derived DCs were electroporated with in vitro transcribed RNA (in vitro transcript, IVT) encoding the TAA HER-2/neu. Additionally, these cells were cotransfected with IVT coding for human 4-1BBL. Transfection of DCs with 4-1BBL-IVT did not alter their typical phenotype. However, it increased the expression of the costimulatory molecules CD80 and CD40. Coadministration of HER-2/neu- and 4-1BBL-IVT resulted in an increased specific lysis of target cells by the in vitro induced CTL lines, indicating that 4-1BBL enhances their ability to elicit primary CTL responses. Interestingly, transfection of DCs with 4-1BBL-IVT did not augment their capacity to stimulate allogeneic lymphocyte responses. The here established approach of cotransfection of DCs with tumor-RNA and a second specific IVT could improve and optimize the in vitro manipulation of DCs for the induction of antigen-specific CTL responses.

Dendritic cells are the most powerful antigen-presenting cells playing a decisive role for the initiation and maintenance of primary immune responses. However, signaling pathways involved in the differentiation of these cells have not been fully determined. Imatinib is a novel tyrosine kinase inhibitor effective against Abl kinases, c-Kit, and platelet-derived growth factor receptor. Using this compound, we show that human monocyte-derived dendritic cells generated in the presence of therapeutic concentrations of imatinib show a reduced expression of CD1a, MHC class I and II, and costimulatory molecules as well as decreased secretion of chemokines and cytokines resulting in an impaired capacity of dendritic cells to elicit primary T-cell responses. Using Western blot analyses, we found that these effects are mediated by inhibition of phosphatidylinositol 3-kinase/Akt pathways and a pronounced down-regulation of nuclear localized protein levels of nuclear factor-κB family members. Importantly, using blocking antibodies and tyrosine kinase inhibitors, we show that the inhibitory effects of imatinib on dendritic cell differentiation are not mediated via platelet-derived growth factor receptor and c-Kit. Taken together, our study reveals that imatinib inhibits dendritic cell differentiation and function via Akt and nuclear factor-κB signal transduction. Importantly, we show that imatinib can inhibit the function of normal, nonmalignant cells that may result in immunosuppression of these patients.

Dendritic cells (DCs) are the most powerful antigen-presenting cells that induce and maintain primary immune responses in vitro and in vivo. The development of protocols for the ex vivo generation of DCs provided a rationale for designing and developing DC-based vaccination studies for the treatment of infectious and malignant diseases. Recently, it was shown that DCs transfected with ribonucleic acid (RNA) coding for a tumour-associated antigen or whole tumour RNA are able to induce potent antigen and tumour-specific T-cell responses directed against multiple epitopes. The first RNA-transfected-DC-based clinical studies have shown that this form of vaccination is feasible and safe. In some cases, clinical responses were observed, but the preliminary data require further extensive investigations that should address the technical and biological problems of manipulating human DCs, as well as the development of standardised protocols and definitions of clinical settings.

Recently, it was demonstrated that transfection of dendritic cells (DCs) with tumor-derived RNA can elicit effective T-cell responses. This technique does not require the definition of the tumor antigen or HLA haplotype of the patients. We applied this approach to induce HLA class I– and class II–restricted T-cell responses directed against malignant cells from patients with chronic lymphocytic leukemia (B-CLL). Here, we show that DCs generated from monocytes of patients with B-CLL induce leukemia-specific cytotoxic and proliferative T-cell responses on transfection with total RNA isolated from autologous leukemic B lymphocytes. Standard 51Cr-release assays showed specific major histocompatibility complex (MHC) class I–restricted cytotoxic activity against the autologous leukemic B cells and DCs transfected with CLL-RNA, whereas nonmalignant B cells were spared. The specificity of the cytotoxic T-lymphocyte (CTL) response was confirmed using cold target inhibition assays and by blocking HLA class I molecules. Furthermore, we established a protocol for the amplification of whole B-CLL mRNA. The use of DCs transfected with in vitro amplified B-CLL mRNA elicited specific T-cell responses similar to the results obtained with native mRNA. These data suggest that vaccinations using DCs transfected with RNA might be a potent new strategy in the treatment of CLL.

Imatinib mesylate (STI571) is a competitive Bcr-Abl tyrosine kinase inhibitor and has yielded encouraging results in treatment of chronic myelogenous leukemia (CML) and gastrointestinal stroma tumors (GISTs). Apart from inhibition of the Abl protein tyrosine kinases, it also shows activity against platelet-derived growth factor receptor (PDGF-R), c-Kit, Abl-related gene (ARG), and their fusion proteins while sparing other kinases. In vitro studies have revealed that imatinib mesylate can inhibit growth of cell lines and primitive malignant progenitor cells in CML expressing Bcr-Abl. However, little is known about the effects of imatinib mesylate on nonmalignant hematopoietic cells. In the current study we demonstrate that in vitro exposure of mobilized human CD34+ progenitors to therapeutic concentrations of imatinib mesylate (1-5 μM) inhibits their differentiation into dendritic cells (DCs). DCs obtained after 10 to 16 days of culture in the presence of imatinib mesylate showed concentration-dependent reduced expression levels of CD1a and costimulatory molecules such as CD80 and CD40. Furthermore, exposure to imatinib mesylate inhibited the induction of primary cytotoxic T-lymphocyte (CTL) responses. The inhibitory effects of imatinib mesylate were accompanied by down-regulation of nuclear localized RelB protein. Our results demonstrate that imatinib mesylate can act on normal hematopoietic cells and inhibits the differentiation and function of DCs, which is in part mediated via the nuclear factor κB signal transduction pathway.

Survivin, a member of the inhibitor of apoptosis protein family, is expressed in almost all types of malignancies, making this protein a useful tool for the development of broadly applicable vaccination therapies. We used a recently identified HLA-A2 binding peptide and dendritic cells (DCs) from healthy donors to induce survivin-specific cytotoxic T lymphocytes (CTLs) in vitro. These T cells efficiently lysed target cells pulsed with the cognate peptide. Furthermore, survivin-specific CTLs recognized HLA-A2–matched tumor cell lines and primary malignant cells from patients with leukemia in an antigen-specific and HLA-restricted manner as demonstrated with the use of cold target inhibition assays and blocking antibodies. To validate the immunogenicity of survivin we performed the experiments in an autologous setting and used monocyte-derived DCs as targets. Interestingly, we found that DCs up-regulate survivin expression on stimulation with tumor necrosis factor α (TNF-α). However, these mature DCs were not recognized by survivin-specific CTLs, whereas they lysed autologous mature DCs pulsed with the antigenic peptide or transfected with whole tumor RNA purified from a survivin-expressing cell line. To further analyze the possible use of survivin-specific CTLs in cancer therapies, we induced survivin-specific CTLs using peripheral blood mononuclear cells (PBMNCs) and DCs from a patient with chronic lymphocytic leukemia (CLL). The in vitro–generated T cells efficiently recognized autologous malignant CLL cells, whereas they spared autologous-purified nonmalignant B cells or DCs. Our results demonstrate that survivin epitopes are presented on a broad variety of malignancies and can be applied in vaccination therapies.

Objective Dendritic cells (DC) are professional antigen-presenting cells playing a pivotal role in the induction of immunological responses. There is evidence that DC survival during ongoing immune responses is finite. However, little is known about the mechanisms regulating apoptosis in these cells. Here, we have investigated the effects of the anti-inflammatory cyclopentenone prostaglandins on human monocyte-derived DC. Materials and Methods Phenotype of DC was determined by flow cytometry and their allostimulatory potential in mixed leukocyte reaction. Induction of apoptosis in DC was monitored by staining with annexin-V-FITC and propidium iodide, propidium iodide staining of cell nuclei, and fluorimetric assay of caspase activity. Induction of maturation in DC was obtained by stimulation with TNF-α, LPS, IFN-γ, CD40-ligand, or different combinations of these stimuli. PPAR-γ expression in DC was determined by RT-PCR. Results Exposure of immature DC to cyclopentenone prostaglandins blunted their allostimulatory capacity and skewed their phenotype by downregulating CD1a and costimulatory molecules. These effects were due to activation of caspases and induction of apoptotic cell death in DC by cyclopentenone prostaglandins. Mature DC showed enhanced susceptibility to apoptosis via cyclopentenone prostaglandins as compared with immature DC. Although DC express PPAR-γ, the corresponding receptor for some of these metabolites, PPAR-γ activation by a synthetic high-affinity agonist failed to impair DC viability. Conclusions Cyclopentenone prostaglandins induce apoptosis of human DC by a PPAR-γ-independent mechanism. Since these compounds are released during an inflammatory event and show anti-inflammatory properties, they may contribute to the downregulation of DC function through apoptotic cell death.

Objectives Monoclonal antibody (mAb) 9C4 detects a surface antigen expressed on immature erythroid progenitor cells and epithelial tumor cell lines. The aim of this study was to identify the recognized surface antigen and to analyze a potential role of this molecule in early steps of erythropoiesis. Materials and Methods. A pituitary-derived retroviral cDNA library was used to generate viruses and infect NIH-3T3 fibroblasts. The transfected cells were stained with mAb 9C4; positive cells were sorted by FACS; and a clonal cell line binding mAb 9C4 was established. cDNA encoding the 9C4-binding protein was amplified by polymerase chain reaction and cloned. Reactivity of mAb 9C4 with human bone marrow (BM) cells was analyzed by flow cytometry. Results. Sequence analysis of the isolated cDNA uncovered a 100% identity with the epithelial cellular adhesion molecule (Ep-CAM). Two-color flow cytometric analysis revealed that almost 100% of Ep-CAM + BM cells coexpressed CD105, E-cadherin, and high levels of CD71. Fractions of Ep-CAM + BM cells also were CD34 + but lacked glycophorin A expression, suggesting that Ep-CAM + cells represent immature erythroid cells. Reverse transcriptase polymerase chain reaction analysis of BM mononuclear cells revealed that the 9C4 + erythroblast population but not the 9C4 − fraction expressed Ep-CAM mRNA. Peripheral blood CD34 + cells induced in vitro to differentiate into the erythroid lineage showed strong Ep-CAM expression on days 3 to 7 of culture. The addition of Ep-CAM–specific mAbs 9C4 or KS1/4 to the culture resulted in two- to three-fold up-regulation of Ep-CAM protein expression. Conclusion. mAb 9C4 recognizes Ep-CAM, a molecule expressed in the early steps of erythropoiesis.

High-Dose chemotherapy with peripheral blood progenitor cell transplantation (PBPCT) is a potentially curative treatment option for patients with both hematological malignancies and solid tumors, including breast cancer. However, based on a number of clinical studies, there is strong evidence that minimal residual disease (MRD) persists after high-dose chemotherapy in a number of patients, which eventually results in disease recurrence. Therefore, several approaches to the treatment of mrd are currently being evaluated, including treatment with dendritic cell (DC)-based cancer vaccines. DCs, which play a crucial role with regard to the initiation of T-lymphocyte responses, can be generated ex vivo either from CD34+ hematopoietic progenitor cells or from blood monocytes. They can be pulsed in vitro with tumor-derived peptides or proteins, and then used as a professional antigen-presenting cell (APC) vaccine for the induction of antigen-specific T-lymphocytes in vivo. This paper summarizes our preclinical studies on the induction of primary HER-2/neu specific cytotoxic T-lymphocyte (CTL) responses using peptide-pulsed DC. As HER-2/neu is overexpressed on 30-40% of breast and ovarian cancer cells, this novel vaccination approach might be particularly applicable to advanced breast or ovarian cancer patients after high-dose chemotherapy and autologous PBPCT.

Angiogenic factors may play a role in the biology of neuroblastoma, a well vascularised tumour, which frequently spreads haematogenously. Therefore, we analysed expression of vascular endothelial growth factor (VEGF) in six human neuroblastoma cell lines and five primary neuroblastomas. High VEGF levels (1-3 ng/10(6) cells/day) were found in the supernatant of all cell lines examined (SK-N-LO, SK-N-SH, LS, SH-SY5Y, IMR-32, Kelly). VEGF peptide was also detected in tissue homogenates from four of five primary tumours. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that VEGF165 is the major isoform produced by neuroblastomas. In addition, all cell lines and primary tumours expressed the mitogenic VEGF receptor FLK-1, whilst the non-mitogenic receptor FLT-1 was less frequently positive, suggesting that the tyrosine kinase FLK-1 is involved in malignant transformation of neuroblastoma cells. However, neutralising antibodies to VEGF did not inhibit growth of neuroblastoma cell lines, which argues against a role of VEGF as an autocrine growth factor, at least for cell lines in vitro. We conclude that neuroblastoma cells produce VEGF, which may contribute to tumour vascularisation, growth and invasion.

Recently it has been shown that dendritic cells (DC) can develop from peripheral blood monocytes when grown in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). However, it is unclear whether DC can also develop from monocytes in absence of these cytokines. We therefore analyzed the effect of Flt-3 ligand (Flt3L) and of CD40 ligand on the development of human DC from blood monocytes in the absence of GM-CSF. Adherent peripheral blood mononuclear cells (PBMNC) were cultured in the presence of different cytokine combinations and analyzed for the expression of surface molecules and antigen presenting capacity. For functional analyses, cells were tested for their ability to stimulate allogeneic T lymphocytes in a mixed lymphocyte reaction (MLR), to present soluble antigens, and to induce primary HIV-peptide–specific cytotoxic T-cell (CTL) responses in vitro. Furthermore, expression of DC-CK1, a recently identified chemokine with specific expression in DC, and of IL-18 (IGIF), a growth and differentiation factor for Th 1 lymphocytes, was analyzed by reverse-transcription polymerase chain reaction (RT-PCR). In our study, Flt3L alone was not sufficient to generate DC and required addition of IL-4. DC generated with Flt3L and IL-4 underwent maturation after stimulation with tumor necrosis factor- (TNF-) or CD40L, characterized by CD83 expression, upregulation of MHC, adhesion, and costimulatory molecules as well as increased allogeneic proliferative response. In contrast, CD40 ligation alone promoted differentiation of adherent blood monocytes into functional DC in the absence of GM-CSF and IL-4. These cells displayed all phenotypic and functional characteristics of mature DC and were potent stimulatory cells in priming of major histocompatibility complex (MHC) class I–restricted CTL responses against an HIV-peptide, whereas their ability to present soluble protein antigens was reduced. Using a semiquantitative RT-PCR, DC-CK1 and IL-18 transcripts were detected in all generated DC populations, independent of growth factors used. Our findings provide further evidence for the importance of CD40-CD40L interaction for initiation and maintenance of T-cell responses and confirm the emerging concept that blood monocytes provide an additional source of DC depending on external stimuli.

Ligands of the prototypical activating NK receptor NKG2D render cancer cells susceptible to NK cell-mediated cytolysis if expressed at sufficiently high levels. However, malignant cells employ mechanisms to evade NKG2D-mediated immunosurveillance, such as NKG2D ligand (NKG2DL) shedding resulting in reduced surface expression levels. In addition, systemic downregulation of NKG2D on NK cells of cancer patients has been observed in many studies and was attributed to soluble NKG2DL (sNKG2DL), although there also are conflicting data. Likewise, relevant expression of NKG2DL in leukemia has been reported by some, but not all studies. Hence, we comprehensively studied expression, release, and function of the NKG2D ligands MHC class I chain-related molecules A and B and UL16-binding proteins 1–3 in 205 leukemia patients. Leukemia cells of most patients (75%) expressed at least one NKG2DL at the surface, and all investigated patient sera contained elevated sNKG2DL levels. Besides correlating NKG2DL levels with clinical data and outcome, we demonstrate that sNKG2DL in patient sera reduce NKG2D expression on NK cells, resulting in impaired antileukemia reactivity, which also critically depends on number and levels of surface-expressed NKG2DL. Together, we provide comprehensive data on the relevance of NKG2D/NKG2DL expression, release, and function for NK reactivity in leukemia, which exemplifies the mechanisms underlying NKG2D-mediated tumor immunosurveillance and escape.

Using dendritic cells (DCs) electroporated with whole RNA isolated from blasts of a patient with acute myeloid leukemia (AML), we were able to generate leukemia-specific cytotoxic T lymphocytes (CTLs) capable of recognizing the leucemic cells. To identify T-cell epitopes mediating lysis of malignant cells, peptides were eluted from the patient's blasts and analyzed by mass spectrometry (LC/MS)-based peptide sequencing. Using this approach, an HLA-A24-binding peptide derived from Bax inhibitor-1 (BI-1), a regulator of apoptosis pathways, was identified as an epitope recognized by the generated CTLs. To further characterize this novel antigenic peptide, CTLs were induced using DCs electroporated with RNA coding for BI-1 or pulsed with the cognate peptide. These CTLs generated from healthy donors in vitro efficiently lysed the patient's blasts as well as other HLA-matched leukemic cells. In conclusion, we identified a BI-1 peptide as a novel immunogenic tumor-associated antigen (TAA) in AML. In vitro induction of BI-1-specific CTLs by RNA transfection or pulsing of DCs with the synthetically generated peptide was a feasible and highly effective method to generate leukemia-specific CTLs. As BI-1 is (over-) expressed in a broad variety of malignancies, it may represent an interesting novel TAA in the context of cancer vaccines.

Members of the caudal (cdx) family of homeobox proteins are essential regulators of embryonic blood development in zebrafish. Previously, we reported that the murine homologues (Cdx1, Cdx2, and Cdx4) affect formation and differentiation of embryonic stem cell (ESC)–derived hematopoietic progenitor cells. Consistent with the notion that embryonic pathways can reactivate during adult oncogenesis, recent studies suggest involvement of CDX2 in human acute myeloid leukemia (AML). Here we study CDX2 in healthy and leukemic human lymphoid cells, and show that a majority of leukemic samples display various degrees of aberrant CDX2 expression. Analysis of a cohort of 37 childhood acute lymphoblastic leukemia (ALL) patients treated in our hospital reveals that high CDX2 expression levels at diagnosis correlate with persistence of minimal residual disease (MRD) during the course of treatment. Thus, CDX2 expression levels may serve as a marker for adverse prognosis in pediatric ALL.

To the Editor: Disparities in the polymorphic minor histocompatibility antigens (mHag) between donor and recipient are associated with the development of graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) reactions after allogeneic stem cell transplantation. The mHag HA-1 has a

Toll-like receptors (TLRs) act to sense the environment for microbial products and submit danger signals to antigen-presenting cells (APCs) resulting in activation of complex immune responses. In this study, we analyzed the function of human monocyte-derived APCs generated in vitro in the presence of interleukin (IL)-10 upon activation by TLR ligands. Exposure of these APCs to IL-10 resulted in a skewed phenotypic maturation in response to stimuli provided by the TLR ligands, a reduced cytokine production, such as IL-12, IL-6 or tumor necrosis factor-alpha, and impaired capacity to stimulate T-cell activation. Furthermore, CCR7 upregulation in APCs exposed to TLR stimulation as well as migration towards CCL19/MIP-3beta were strongly reduced. IL-10 was found to downregulate MyD88, IRAK1 (IL-1 receptor-associated kinase) and tumor necrosis factor receptor-associated factor 6, essential adaptor molecules for TLR signaling, and to decrease TLR-induced nuclear expression of the nuclear factor-kappaB transcription factors c-Rel and Rel-B as well as interferon regulatory factor (IRF)-3 and IRF-8. This was not due to the inhibition of the mitogen-activated protein kinase pathway, but was rather mediated by the blockage of the PI3K signaling cascade. Interestingly, the inhibition of proteins involved in TLR signaling, such as MyD88, IRAK1 and mammalian target of rapamycin, was due to a selective post-transcriptional regulation.

Purpose: A prerequisite for the development of vaccination strategies is the identification and characterization of relevant tumor-associated antigen. Using microarray and reverse transcription-PCR analysis, we found matrix metalloproteinase (MMP)-7 to be extensively up-regulated in renal cell carcinomas and expressed in a broad variety of malignant cells. MMP-7 can promote cancer invasion and angiogenesis by proteolytic cleavage of extracellular matrix and basement membrane proteins, thus making it a promising target in the context of immunotherapies. Experimental Design: To analyze the possible use of MMP-7 as a tumor-associated antigen, specific CTLs were induced using monocyte-derived dendritic cells electroporated with MMP-7-mRNA. In addition, to better characterize the fine specificity of these CTLs, MMP-7 MHC class I ligands were isolated and characterized in renal cell carcinoma tissue, which overexpressed MMP-7, by mass spectrometry–based peptide sequencing. Using this approach, we identified a novel HLA-A3–binding antigenic MMP-7 peptide. CTLs generated from healthy donors by in vitro priming with dendritic cells, pulsed with the novel peptide, were used as effectors in 51Cr-release assays. Results: The induced CTLs elicited an antigen-specific and HLA-restricted cytolytic activity against tumor cells endogenously expressing the MMP-7 protein. Furthermore, we were able to induce MMP-7–specific CTLs using peripheral blood mononuclear cells from a patient with acute lymphoblastic leukemia capable of recognizing the autologous leukemic blasts while sparing nonmalignant cells. Conclusions: Our study describes the identification of a novel broadly expressed T-cell epitope derived from the MMP-7 protein that represents an interesting candidate to be applied in immunotherapies of human malignancies targeting both tumor cells and neovascularization.

Human Dectin-1 (hDectin-1) is a member of the C-type lectin–like receptor family that was shown to be the major receptor for fungal beta-glucans and to play an important role in the cellular responses mediated by these carbohydrates. In this study, we demonstrate that hDectin-1 is involved in the uptake and cross-presentation of cellular antigens. Furthermore, activation of monocyte-derived dendritic cells (MDCs) with toll-like receptor 3 (TLR3) ligand but not with TLR2 ligand or TLR7 ligand resulted in down-regulation of hDectin-1 expression and reduced phagocytosis of apoptotic tumor cells as well as presentation of pp65-derived T-cell epitopes upon engulfment of cytomegalovirus (CMV)–infected human foreskin fibroblasts.

Purpose: Identification of tumor-associated antigens and advances in tumor immunology resulted in the development of vaccination strategies to treat patients with malignant diseases. In a novel experimental approach that combined comparative mRNA expression analysis of defined cell types with the characterization of MHC ligands by mass spectrometry, we found that regulator of G protein signaling 5 (RGS5) is extensively up-regulated in a broad variety of malignant cells, and we identified two HLA-A2– and HLA-A3–binding peptides derived from the RGS5 protein. Interestingly, RGS5 was recently shown to be involved in tumor angiogenesis. Experimental Design: We used monocyte-derived dendritic cells pulsed with these novel antigenic peptides or transfected with RGS5-mRNA for the in vitro induction of CTLs, generated from healthy donors, to analyze the presentation of RGS5-deduced epitopes by malignant cells. Results: The generated CTL lines elicited an antigen-specific and HLA-restricted cytolytic activity against tumor cells endogenously expressing the RGS5 protein. Furthermore, we were able to induce RGS5-specific CTLs using peripheral blood mononuclear cells from a patient with acute myeloid leukemia capable of recognizing the autologous leukemic blasts while sparing nonmalignant cells. Conclusions: These results indicate that the RGS5 peptides represent interesting candidates for the development of cancer vaccines designed to target malignant cells and tumor vessels.

Zoledronic acid (ZA) is a nitrogen-containing bisphosphonate with antitumor activity used to treat patients with malignant diseases. ZA treatment induces, as a side effect, inflammatory responses, which are accompanied by expansion of gammadelta T cells. In our study, we analyzed the function and differentiation of monocyte-derived immature and lipopolysaccharide (LPS)-stimulated dendritic cells (moDCs) treated with different ZA concentrations, which are achieved in patients. We found that moDC activation with TLR4 ligand LPS is modulated by ZA. The expression of maturation markers was diminished with increasing ZA levels upon LPS activation. The migratory capacity, interleukin-12 secretion and generation of cytotoxic- T-cell responses were reduced at higher ZA levels. Increasing ZA concentrations downregulated nuclear factor-kappaB family members and interferon-regulatory factor (IRF)-3. Surprisingly, in immature moDCs, low ZA concentrations caused upregulation of RelB, c-Rel, IRF-3 and IRF-8. We conclude that ZA concentrations used to treat patients have inhibitory effects on DC activation. This might lead to immunosuppression or result in infectious complications.

Dendritic cells (DCs) have the unique ability to efficiently present T-cell epitopes from exogenous antigens on MHC class I molecules, a process called cross-presentation. In our study we demonstrate that stimulation of monocyte-derived DCs with Toll-like receptor (TLR) ligands differentially affects the uptake and cross-presentation of cellular antigens. Activation of DCs with TLR3 or TLR4 but not with TLR2 or TLR7/8 ligands inhibited phagocytosis of apoptotic tumor cells and resulted in a reduced cross-presentation of pp65-derived T-cell epitopes on MHC class I molecules upon engulfment of cytomegalovirus (CMV)–infected fibroblasts. These results have an important impact on the understanding of the interactions between the immune system and pathogens and the development of vaccination strategies to treat malignant diseases.

Dendritic cells (DCs) arise from hematopoietic stem cells and develop into a discrete cellular lineage distinct from other leucocytes. Mainly three phenotypically and functionally distinct DC subsets are described in the human peripheral blood (PB): plasmacytoid DCs (pDCs), which express the key marker CD303 (BDCA-2), and two myeloid DC subsets (CD1c(+) DC (mDC1) and CD141(+) DC (mDC2)), which express the key markers CD1c (BDCA-1) and CD141 (BDCA-3), respectively. In addition to these primary cell subsets, DCs can also be generated in vitro from either CD34(+) stem/progenitor cells in the presence of Flt3 (Fms-related tyrosine kinase 3) ligand or from CD14(+) monocytes (monocyte-derived DCs (mo-DCs)) in the presence of granulocyte-macrophage colony-stimulating factor+interleukin-4 (GM-CSF+IL-4). Here we compare the reactivity patterns of HLDA10 antibodies (monoclonal antibody (mAb)) with pDCs, CD1c(+) DCs and CD141(+) DCs, as well as with CD14(+)-derived mo-DCs cultured for 7 days in the presence of 100 ng/ml GM-CSF plus 20 ng/ml IL-4. A detailed profiling of these DC subsets based on immunophenotyping and multicolour flow cytometry analysis is presented. Using the panel of HLDA10 Workshop mAb, we could verify known targets selectively expressed on discrete DC subsets including CD370 as a selective marker for CD141(+) DCs and CD366 as a marker for both myeloid subsets. In addition, vimentin and other markers are heterogeneously expressed on all three subsets, suggesting the existence of so far not identified DC subsets.

Evidence from the animal model suggests that proteasome inhibitors may have immunosuppressive properties; however, their effects on the human immune system remain poorly investigated. Here, we show that bortezomib, a proteasome inhibitor with anticancer activity, impairs several immune properties of human monocyte-derived dendritic cells (DCs). Namely, exposure of DCs to bortezomib reduces their phagocytic capacity, as shown by FITC-labeled dextran internalization and mannose-receptor CD206 down-regulation. DCs treated with bortezomib show skewed phenotypic maturation in response to stimuli of bacterial (lipopolysaccharide [LPS]) and endogenous sources (including TNF-α and CD40L), as well as reduced cytokine production and immunostimulatory capacity. LPS-induced CCL-2/MCP-1 and CCL5/RANTES secretions by DCs were prevented by DC treatment with bortezomib. Finally, CCR7 up-regulation in DCs exposed to LPS as well as migration toward CCL19/MIP-3β were strongly impaired. As a suitable mechanism for these effects, bortezomib was found to down-regulate MyD88, an essential adaptor for TLR signaling, and to relieve LPS-induced activation of NF-κB, IRF-3, and IRF-8 and of the MAP kinase pathway. In summary, inhibition of DC function may represent a novel mechanism by which proteasome inhibitors exert immunomodulatory effects. These compounds could prove useful for tuning TLR signaling and for the treatment of inflammatory and immune-mediated disorders.

Histone deacetylases (HDACs) modulate gene transcription and chromatin assembly by modifying histones at the post-transcriptional level. HDAC inhibitors have promising antitumor activity and are presently explored in clinical studies for malignancies of different histology. Preliminary evidence in animal models of immune disorders also suggests immunosuppressive properties for these small molecules, although the underlying mechanisms remain at present poorly understood. Here, we show that two HDAC inhibitors of clinical use, sodium valproate and MS-275, profoundly affect cytokine-induced differentiation of human monocytes to dendritic cells (DCs), and impair DC phenotypic maturation in response to the Toll-like receptor (TLR) ligand polyinosinic:polycytidylic acid (poly I:C) characterized by a reduced expression of HLA- and costimulatory molecules. HDAC inhibitors reduce cytokine secretion important for T-cell activation like IL-12 and immunostimulatory capacity in DCs and inhibit DC migratory capacity toward the chemoattractant CCL19/MIP-3β. The observed defects in DC function upon exposure to HDAC inhibitors were not due to an increased rate of apoptosis or production of IL-10. We finally investigated the effects of HDAC inhibition on intracellular signalling pathways that play a key role in controlling DC differentiation and function. HDAC inhibitors have been reported to cause a downregulation of the hematopoietic transcription factor PU.1, which contributes to the development of thymic and myeloid dendritic cells and of Langerhans cells in the mouse. We evaluated herein whether PU.1 expression was affected by VPA in monocyte-derived DCs, however, no significant reduction in protein levels could be detected. Therefore, our data rule out PU.1 shortening as a mechanism responsible for the functional defects observed in DCs that were exposed to HDAC inhibitors. Similarly, no significant effect of HDAC inhibitors was detected on Bcl-6, another known target of HDAC inhibitors that, besides its role in lymphomas, has also been linked to the regulation of cytokine and chemokine release in macrophages. In contrast, addition of VPA blocked RelB nuclear relocalization in response to poly I:C in a concentration-dependent manner. Interestingly, IRF-3 and IRF-8, which have been involved in DC differentiation, IL-12 production and DC migration, were also affected by HDAC inhibitors, since their nuclear levels were reduced by VPA in unstimulated as well as in poly I:C-activated DCs. On the other hand, IRAK-1 downregulation in response to poly I:C was not affected by VPA, suggesting that the effects of HDAC inhibition on RelB, IRF-3 and IRF-8 are probably mediated downstream of MyD88 and IRAK-1. In conclusion, HDAC inhibitors exhibit strong immunomodulatory properties on human DCs. Our results support the evaluation of HDAC inhibitors in inflammatory and autoimmune disorders.

Dendritic cells (DCs) are recognized as the most potent antigen-presenting cells of the immune system with the unique ability to initiate and maintain primary immune responses. In order to better characterize the functional and phenotypic features of DCs, a subtractive cDNA library to identify differentially expressed genes in monocyte-derived DCs (MDCs) was constructed. Using this approach, we found that the epithelial transcription factor ESE-3, which was previously shown to be exclusively expressed in cells of epithelial origin, is differentially expressed in MDCs. This was further confirmed by reverse transcriptase–polymerase chain reaction (RT-PCR) and Western blot analyses. The expression of ESE-3 is up-regulated upon maturation of MDCs and inhibited by treating the cells with IL-10 or IFN-γ. Knockdown experiments using siRNA suggest that ESE-3 plays an important role during MDC development. Our results might help to improve the phenotypic characterization of DCs and lead to a better understanding of the cellular mechanisms involved in antigen presentation and T-cell stimulation.

Dendritic cells (DCs) play an important role in initiating and maintaining primary immune responses. However, mechanisms involved in the resolution of these responses are elusive. We analyzed the effects of 15d-PGJ2 and the synthetic peroxisome proliferator-activated receptor (PPAR)-gamma ligand troglitazone (TGZ) on the immunogenicity of human monocyte-derived DCs upon stimulation with toll-like receptor (TLR) ligands. Activation of PPAR-gamma resulted in a reduced stimulation of DCs via the TLR ligands 2, 3, 4, and 7, characterized by down-regulation of costimulatory and adhesion molecules and reduced secretion of cytokines and chemokines involved in T-lymphocyte activation and recruitment. MCP-1 (monocyte chemotactic protein-1) production was increased due to PPAR-gamma activation. Furthermore, TGZ-treated DCs showed a significantly reduced capacity to stimulate T-cell proliferation, emphasizing the inhibitory effect of PPAR-gamma activation on TLR-induced DC maturation. Western blot analyses revealed that these inhibitory effects on TLR-induced DC activation were mediated via inhibition of the NF-kappaB and mitogen-activated protein (MAP) kinase pathways while not affecting the PI3 kinase/Akt signaling. Our data demonstrate that inhibition of the MAP kinase and NF-kappaB pathways is critically involved in the regulation of TLR and PPAR-gamma-mediated signaling in DCs.

RNA transfection of dendritic cells (DCs) was shown to be highly efficient in eliciting CD8+ and CD4+ T-cell responses. However, antigen presentation pathways involved in generation of human leukocyte antigen (HLA) class I and class II peptides have remained elusive. To analyze this we incubated mucin 1 (MUC1) RNA-transfected DCs with compounds known to inhibit HLA class I presentation and used these cells in chromium 51 (51Cr)–release assays. As effectors, we used cytotoxic T lymphocyte (CTL) lines specific for the MUC1 peptides M1.1 and M1.2. We observed that the presentation of HLA-A*02 epitopes is inhibited by brefeldin A and lactacystin. To determine the requirement of a functional transporter associated with antigen processing (TAP), we cotransfected DCs with MUC1 and infected cell peptide 47 (ICP47) RNA. ICP47 could only inhibit the presentation of the M1.1 but not the M1.2 peptide, indicating that this epitope derived from the signal sequence is presented independently of TAP. Cocultivation of MUC1 RNA-transfected DCs with MUC1-specific CD4+ T lymphocytes revealed that the presentation of HLA class II peptides is sensitive to proteasomal inhibitors and brefeldin A. Furthermore, the presentation pathway requires lysosomal and endosomal processing and is mediated by autophagy. Our results demonstrate that the efficient presentation of cytosolic proteins on major histocompatibility complex (MHC) class II combines the proteolytic and lysosomal pathways.

Dendritic cells (DC) are the most powerful antigen-presenting cells that induce and maintain primary immune responses in vitro and in vivo. The development of protocols for the ex vivo generation of DC provided a rationale to design and develop DC-based vaccination studies for the treatment of infectious and malignant diseases. The efficacy of antigen loading and delivery into DC is pivotal for the optimal induction of T-cell-mediated immune responses. Recently it was shown that DC transfected with RNA coding for a tumor-associated antigen (TAA) or whole-tumor RNA are able to induce potent antigen- and tumor-specific T-cell responses directed against multiple epitopes. The latter technique does not require the definition of the TAA or HLA haplotype of the patients and has the potential of broad clinical application. Such a polyvalent vaccine might be able to reduce the probability of clonal tumor escape and to elicit CTL responses directed against naturally processed and presented immuno-dominant tumor antigens. Additional targeting of HLA class II restricted epitopes may further amplify and prolong the induced T-cell responses.

Dendritic cells (DC) are the most powerful antigen-presenting cells that induce and maintain primary immune responses in vitro and in vivo. The development of protocols for the ex vivo generation of DC provided a rationale to design and develop DC-based vaccination studies for the treatment of infectious and malignant diseases. The efficacy of antigen loading and delivery into DC is pivotal for the optimal induction of T-cell-mediated immune responses. Recently it was shown that DC transfected with RNA coding for a tumor-associated antigen (TAA) or whole-tumor RNA are able to induce potent antigen- and tumor-specific T-cell responses directed against multiple epitopes. The latter technique does not require the definition of the TAA or HLA haplotype of the patients and has the potential of broad clinical application. Such a polyvalent vaccine might be able to reduce the probability of clonal tumor escape and to elicit CTL responses directed against naturally processed and presented immuno-dominant tumor antigens. Additional targeting of HLA class II restricted epitopes may further amplify and prolong the induced T-cell responses.

Background Patients with follicular (FL) or mantle cell lymphoma (MCL) are incurable with conventional therapy. We investigated the safety and efficacy of rituximab consolidation after autologous stem cell transplantation (ASCT) in order to prevent relapse by clearance of minimal residual disease (MRD). Methods Rituximab was given ∼8 weeks after CD34+ cell enriched ASCT at 375 mg/m2, weekly for 4 weeks. Monitoring of MRD was performed by repetitive PCR analyses. Results Thirty-one patients were included; one died early after ASCT before rituximab administration. Thirty patients (20 FL, 10 MCL) were evaluable after rituximab consolidation, and 27 of these were assessable for MRD detection. Rituximab consolidation post-ASCT was safe, the most common toxicity being infection. At a median follow-up of 42 months (range 13–96) after ASCT, 25 patients were censored with an actuarial event-free survival (EFS) of 81% at 4 and 5 years. Four patients (two FL, two MCL) relapsed, and one additional MCL patient died unexpectedly in complete remission. PCR-negativity was observed in 22% of the patients before ASCT, 53% post-ASCT (P=0.0547), 72% after rituximab (P=0.0018) and 100% at 6 months post-transplant (P Conclusions One single course of rituximab consolidation given after ASCT is safe, may help to eliminate MRD and may translate into improved EFS in both FL and MCL patients.

Purpose: C-Met proto-oncogene is a receptor tyrosine kinase that mediates the oncogenic activities of the hepatocyte growth factor. Using a DNA chip analysis of tumor samples from patients with renal cell carcinoma and sequencing of peptides bound to the HLA-A*0201 molecules on tumor cells a peptide derived from the c-Met protein was identified recently. Experimental Design: We used this novel HLA-A*0201 peptide for the induction of specific CTLs to analyze the presentation of this epitope by malignant cells. Results: The induced CTL efficiently lysed target cells pulsed with the cognate peptide, as well as HLA-A*0201-matched tumor cell lines in an antigen-specific and HLA-restricted manner. Furthermore, the induced c-Met-specific CTLs recognized autologous dendritic cells (DCs) pulsed with the peptide or transfected with whole-tumor mRNA purified from c-Met-expressing cell lines. We next induced c-Met-specific CTLs using peripheral blood mononuclear cells and DC from an HLA-A*0201-positive patient with plasma cell leukemia to determine the recognition of primary autologous malignant cells. These CTLs lysed malignant plasma cells while sparing nonmalignant B- and T-lymphocytes, monocytes, and DCs. Conclusion: Our results demonstrate that c-Met oncogene is a novel tumor rejection antigen recognized by CTL and expressed on a broad variety of epithelial and hematopoietic malignant cells.

Identification of tumor-associated antigens and advances in tumor immunology resulted in the development of vaccination strategies to treat patients with malignant diseases. Using a novel approach that combines DNA chip analysis of tumor samples with isolation of peptides on the surface of tumor cells, a HLA-A*0201-binding peptide derived from the adipophilin protein was identified. Adipophilin is involved in lipid storage and was thought to be expressed only in adipocytes, but it can be found in other cell types such as macrophages or tumor cells. In the present study, we analyzed the possible use of this peptide as a T-cell epitope presented by malignant cells. To accomplish this, we induced CTL responses using this HLA-A*0201-binding peptide. The in vitro-induced CTLs efficiently lysed cells pulsed with the adipophilin peptide and HLA-matched tumor cell lines in an antigen-specific and HLA-restricted manner. Finally, the induced CTLs recognized autologous dendritic cells (DCs) pulsed with the antigenic peptide or transfected with tumor RNA purified from an adipophilin-expressing tumor cell line. To further analyze the possible use of this peptide in immunotherapies of human malignancies, we induced adipophilin-specific CTLs using peripheral blood mononuclear cells and DCs from HLA-A*0201-positive patients with chronic lymphatic leukemia and plasma cell leukemia. The in vitro-generated CTLs recognized autologous chronic lymphatic leukemia cells and malignant plasma cells, whereas they spared nonmalignant resting or activated B and T lymphocytes, monocytes, or DCs. Our results demonstrate that this peptide might represent an interesting candidate for the development of cancer vaccines designed to target adipophilin-derived epitopes in a wide range of malignancies.

Tumors often upregulate the expression of molecules that are normally suppressed or expressed at much lower levels in adult tissues, the so-named tumor-associated antigens (TAAs) [1]. Some ‘self-reactive’ T lymphocytes capable of T-cell receptor (TCR)-mediated recognition of these antigens and of mediating tumor rejection survive thymic selection and can be detected in peripheral blood and lymphoid tissues. Indeed, cases of spontaneous immune-mediated tumor regressions are reported, especially for melanoma, renal cell carcinoma and tumors presenting with neurological paraneoplastic syndromes, especially non-small-cell lung cancer [1–3]. However, in most cases, the immune system fails to recognize and destroy tumor cells, possibly due to the inefficiency of these as antigen-presenting cells (APCs), and to the lack of contact between tumor cells and the immune system [4, 5]. Now that knowledge of the immune system has improved, new perspectives for the development of active antitumor immunization strategies have been proposed. The main goal of these approaches is to expand in the patients CD8+ cytotoxic T lymphocytes (CTLs) capable of rejecting tumor cells via recognition of tumor-associated antigenic epitopes expressed by human leukocyte antigen (HLA) class I molecules on the cancer cells. However, optimal immunotherapeutic approaches should probably also prime CD4+ helper T cells, given the key role that these cells play in the control of immune responses [1]. The antigenic material used in anticancer vaccinations can be in the form of whole tumor material (either allogeneic or autologous) or of specific TAAs, which are delivered as DNA (naked or comprised in recombinant viruses), RNA, protein or HLA class I/II restricted peptide epitopes [1]. Many of these antigenic materials can be injected directly, often coupled to immunostimulatory cytokines or adjuvants, or used for ex vivo loading of APCs, usually dendritic cells (DCs) (Figure 1). These are specialized APCs with the capacity to initiate an immune response by presenting antigen-derived epitopes to T lymphocytes in a highly immunogenic fashion [6–9]. All of the antitumor vaccination approaches are believed to ultimately depend on the presentation of tumor-derived antigen(s) to T cells by DCs in the patient’s lymphoid tissues [10]. For the biology of DCs and the applications of ex vivo manipulated DCs to cancer immunotherapy we refer the reader to the review by Lesterhuis et al. [11] in this Supplement. Here, we mainly focus on the non-DC-based approaches that are currently evaluated for specific cancer immunotherapy. Autologous/allogeneic tumor cells as a tumor vaccine