ALBERT

Description: 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.

Description: NK cell anti-tumor reactivity is governed by a balance of activating and inhibitory receptors including various TNF receptor (TNFR) family members. Here we report that human tumor cells release a soluble form of the TNF family member Glucocorticoid-Induced TNFR-Related Protein (GITR) ligand (sGITRL), which can be detected in cell culture supernatants. Tumor-derived sGITRL concentration-dependently reduced NK cell cytotoxicity and IFN-γ production, which could be overcome by neutralization of sGITRL using a GITR-Ig fusion protein. Although sGITRL did not induce apoptosis in NK cells, it diminished nuclear localized RelB, indicating that sGITRL negatively modulates NK cell NF-κB activity. Furthermore, we detected substantial levels of sGITRL in sera of patients with various malignancies, but not in healthy controls. Presence of sGITRL-containing patient serum in cocultures with tumor cells significantly reduced NK cell cytotoxicity and IFN-γ production, which could again be restored by neutralization of sGITRL. The strong correlation of tumor incidence and elevated sGITRL levels indicates that sGITRL is released from cancers in vivo, leading to impaired NK cell immunosurveillance of human tumors. Our data suggest that determination of sGITRL levels might be implemented as a tumor marker in patients, and GITRL neutralization may be used to improve immunotherapeutic strategies relying on NK cell reactivity.

Description: Human (h)Dectin-1 is a member of the C-type-lectin-like receptor family that was shown to be the major receptor for fungal β-glucans and to play an important role in cellular responses mediated by these carbohydrates. It is mainly expressed on human DCs and macrophages. In our study, we observed that activation of monocyte-derived dendritic cells (MDCs) with TLR3 ligand (poly I:C) but not with TLR ligand 7/8 (R848) resulted in down-regulation of hDectin-1 expression and this down-regulation correlated with a reduced uptake of apoptotic cells in phagocytosis assays. In order to analyze the possible cross-presentation of engulfed antigens we used CMV infected human fibroblasts (HFF). We found that hDectin-1 is involved in the uptake of CMV-infected HFF leading to cross-presentation of CMV-derived peptides on MHC class I molecules and activation of CMV pp65-specific CD8+ T-lymphocytes. To further delineate the pathway leading to presentation, we pretreated MDCs with compounds that inhibit processing of antigens at defined steps during presentation. Cytosolic protein degradation is performed by the proteasome, a large multicatalytic protease complex. Lactacystin specifically inhibits the 20S and 26S proteasome activity by targeting the catalytic subunit. In standard 51Cr-release assays, addition of lactacystin completely inhibited the presentation of pp65-derived peptides indicating the involvement of the proteasome in these process. The fungal product brefeldin A blocks the MHC class I processing pathway by specifically inhibiting the vesicular egress from the ER and the Golgi complex. In line with previous findings, incubation with brefeldin A almost completely abolished the lysis of MDCs incubated with CMV+ HFF. To further analyze whether the cross-presentation of CMV-derived peptides on HLA class I molecules was dependent on lysosomal proteases, MDCs that were co-incubated with HFF as above were treated with the lysosomotropic agent chloroquine that prevents acidification of the lysosomal compartment involved in the exogenous pathway of antigen presentation. The addition of chloroquine had no effect on the cross-presentation of CMV-derived epitopes on HLA class I-molecules. Summarized, the data reported here show that hDectin-1 functions not only as a pattern recognition receptor in innate immunity but is also involved in the clearing of apoptotic cells and cross-presentation of cellular antigens on MHC class I molecules to specific CTLs.

Description: 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.

Description: Abstract 2169 Poster Board II-146 The tyrosine kinase inhibitors (TKIs) Imatinib mesylate (IM, Gleevec, Glivec) and nilotinib (Tasigna, AMN) are currently used in treatment of chronic myeloid leukaemia (CML). IM has been described to influence the function and differentiation of antigen presenting cells, to inhibit the effector function of T lymphocytes and to decrease the immunogenicity of CML cells by downregulation of tumor associated antigens. In the present study, we analyzed the effect of IM and AMN on proteasomal activity in IM-sensitive or IM/AMN- resistant CML cells as well as in patient samples using a biotinylated active site-directed probe, which, covalently binds and labels proteasomal subunits beta-1, beta-2 and beta-5 and their immunosubunit counterparts beta-1i, beta-2i and beta-5i, in an activity-dependent fashion. In addition, we analyzed the cleavage and processing of antigenic BCR-ABL derived peptides after degradation by the IM or AMN treated 20S immuno- and constitutive proteasome by massspectrometry. The analyzed epitopes KQSSKALQR and GFKQSSKAL are deduced from the fusion region of BCR-ABL and bind to HLA-A3/11 and HLA-B8, respectively. Both epitopes have been shown to be naturally presented by patient CML cells. So far, in vitro generation of these epitopes by proteasomal digestion experiments has been shown only for KQSSKLAQR. We found, that IM and AMN treatment in sensitive and resistant CML cells, as well as in primary patient samples and BCR-ABL-negative cells led to a concentration-dependent decrease of the MHC-class I expression in line with the decrease of proteasomal activity, indicating that the effects are BCR-ABL independent. This inhibitory effect was independent of the expression of proteasome subunits as analyzed by western blotting and it was not due to the induction of apoptosis. In vitro digestion experiments using purified proteasomes in the presence of AMN or IM showed little influence on the overall cleavage pattern observed. However, the generation of epitope-precursor peptides was significantly altered in the presence of AMN and IM for both peptides. Treatment of the immunoproteasome i20S with IM or AMN resulted in an almost complete reduction in the generation of the long precursor peptides for the HLA-A3/A11 and —B8 epitopes while the processing of the short peptide sequences significantly increased. In addition, we show for the first time that both epitopes KQSSKALQR and GFKQSSKAL can be generated as N-terminal elongated precursors in vitro by both constitutive and immuno-20S proteasomes. Interestingly, in all performed experiments AMN was more effective as compared to IM, while other TKIs such as Sunitinib, Sorafenib or the PI3K inhibitor LY294002 had no effect. Our results demonstrate that treatment with IM and AMN can affect the immunogenicity of malignant cells by affecting proteasomal degradation of cytosolic antigens, thereby modulating the repertoire of presented antigens. These strong effects of the TKIs IM and AMN on proteasomal activity might be a result of changes in the phosphorylation of proteasomal subunits akin to the recently showed endogenous phosphorylation sites of the mammalian 20S proteasome. Disclosures: No relevant conflicts of interest to declare.

Description: Dendritic cells play an inimitable role in the functioning immune system as they are the most potent antigen presenting cells being able to prime naive T-cells. Their characteristic properties that enable them to take up antigens and present them to leukocytes are due to an expression of specific genes and thus specific proteins that are unique to this subset of antigen-presenting cells. Using a substractive cDNA library based on suppression hybridization between DC cDNA and the reference monocyte cDNA, we identified in DC two differentially expressed genes p275 and p306. p275 codes for a membrane protein and represents a splice isoform of the transport protein NAT-1. The predicted structure of protein p306 is globular, suggesting that the protein is either intracellular or secreted. The expression of both genes was confirmed by RT-PCR using cDNA isolated from peripheral blood monocytes and DC, generated in vitro from monocytes or CD34+ progenitor cells. To further analyze the protein expression polyclonal antibodies were generated by immunization with synthetic peptides deduced from the identified sequences. Interestingly, inhibition of DC differentiation using IL-10 or STI571 (Imatinib) resulted in an impaired expression of both proteins. Utilizing specific primers for two recently described splice variants of p306 we identified a new splice form expressed in DC. While the gene of p306 contains eight exons, splice variant 1 consists of the exons 1,2,4,5,6, and 7 and splice variant 2 contains the exons 1,2,3,4,5,6, a shortened exon 7, and exon 8. The new identified splice form includes the exons 1–7. However, as the open reading frame starts in exon 4, the expressed protein is identical with the one corresponding to splice variant 1. Analyzing different DC populations in peripheral blood we show that p306 is expressed in plasmacytoid, but not myeloid DC. Interestingly, the activation of DC with Toll-like receptor ligands (TLRL) Pam3Cys (TLR2L), Poly I:C (TLR3L), LPS (TLR4L) and R848 (TLR7L) has no influence on the expression of p306. Although the functions of p275 and p306 in DC have yet to be determined, both genes play a role in DC differentiation and are found in different hematopoietic cell populations. Especially p306 might be an interesting marker of plasmacytoid DC as the predicted protein does not resemble any known protein structure.

Description: Imatinib mesylate (Gleevec®) is a specific tyrosine kinase inhibitor, which inhibits phosphorylation of downstream proteins involved in BCR-ABL signal transduction. In the treatment of chronic myelogenous leukemia (CML) it has become indispensable and shows few side effects. Recently, it was shown that patients treated with imatinib showed impaired CTL responses in comparison to patients treated with IFN-α, which might be due to a reduced immunogenicity of CML cells or result from an inhibitory effect of imatinib on the function of antigen presenting cells and T lymphocytes. In the present study, we show that imatinib treatment leads to a downregulation of immunogenic antigens on the CML cells, which in turn inhibits the development of CML-specific cytotoxic T lymphocytes (CTLs). To achieve this, we treated the CML cell line K562 and an imatinib-resistant K562 variant, K562R, with imatinib or DMSO, isolated the total RNA and used it to electroporate monocyte-derived dendritic cells (DCs). These cells were then used as antigen presenting cells (APCs) for the induction of polyclonal CTL responses. The cytolytic activity of the CTLs was assayed in standard 51Cr-release assays and their fine specificity in IFNγ-Elispot assays. CTLs generated using RNA from imatinib-treated K562 cells were completely incapable of specific killing and did not react in Elispot assays, whereas those CTLs induced using RNA from K562 cells subjected to DMSO treatment as well as RNA from imatinib-treated K562R cells showed specific cytolytic activity against targets electroporated with RNA from CML cells and were able to recognize several CML-associated antigens, like survivin, PRAME, WT-1 and PR3 in Elispot assays. To confirm that this effect is mediated by BCR-ABL inhibition, we used specific siRNA against the bcr-abl fusion site b3a2 to downregulate the protein expression and found essentially the same results. Even in K562R cells, that constitutively overexpress BCR-ABL, targeting the expression of the protein directly by specific siRNA leads to an impairment of CTL induction. In order to confirm and expand these studies, we additionally analyzed the expression of antigens connected to immune responses to CML in Western Blot and Real-time PCR experiments. We found, that imatinib-mediated inhibition of BCR-ABL in K562 cells leads to a decreased expression of tumor antigens and cellular proteins including survivin, adipophilin, hTERT, WT-1, Bcl-xL and Bcl-2 in correlation to the decreased development of specific CTLs. Matching the results of the 51Cr-release assays, these effects were not observed in K562R cells. In primary CML cells subjected to imatinib a downregulation of hTERT and survivin could be detected, which corresponded to a decreased lysis of DCs electroporated with RNA from these cells in standard 51Cr-release assays. Our results demonstrate, that BCR-ABL directly influences the expression of immunogenic tumor associated antigens by its uncontrolled tyrosine kinase activity and therefore substantially contributes to the immunogenicity of CML cells.

Description: Abstract 1032 Myeloid derived suppressor cells (MDSC) play an important role in the regulation of immune responses by suppressing the function of antigen presenting cells and T cells. In humans several populations of MDSC with different phenotypes were characterized due to their distinct immunosuppressive function. However, the origin and development of these cells is not well understood. We observed that incubation of peripheral blood monocytes with IL-10 during their differentiation to dendritic cells (DC) in the presence of GM-CSF and IL-4 results in the generation of an APC population with dramatically reduced stimulatory capacity and a CD14+ and HLA-DR low phenotype (IL-10-APC) similar to the recently described human MDSC subpopulation. In coincubation experiments we found that the addition of these cells to immature or LPS activated DC generated from peripheral blood monocytes resulted in a cell number dependent inhibition of their stimulatory capacity in the mixed lymphocyte reaction assay. Furthermore, these IL-10-APCs reduced the expression of CD1a and costimulatory molecules on DC as well as their activation by LPS characterized by diminished expression of maturation markers including CD83, CD80, CD86 or CD40. IL-10-APC almost completely abolished the secretion of cytokines and chemokines by mature and immature DC involved in T cell stimulation and migration such as TNF-a, IL-6, MIP-1a or Rantes. These effects were not due to induction of IL-10 production and were not observed when purified CD14+ monocytes were used as a control in the experiments. In order to analyze the possible mechanisms and molecules involved in these inhibitory effects we found that IL-10-APC expressed higher levels of PD-1, GITR, GITRL and osteoactivin, an immunosuppressive molecule that was shown to inhibit the function of T cells. The effects mediated by these molecules were further confirmed by utilizing blocking antibodies. Interestingly, addition of IL-10-APC to mature or immature DC induced an increased expression of osteoactivin and its corresponding receptor syndecan 4 on DC thus demonstrating that osteoactivin mediates its effects by upregulating its own receptor. In the next set of experiments we isolated the CD14+ HLA-DR low cell population from buffy coats of healthy donors. We found, that these cells similar to the IL-10-APCs express high levels of osteoactivin and syndecan-4 that can be further upregulated by the addition of IL-10. Our results demonstrate that osteoactivin mediates its inhibitory effects by induction of its cognate receptor syndecan 4. In addition, cells with the phenotype and function of MDSC can differentiate from human peripheral blood monocytes in the presence of GM-CSF, IL-4 and IL-10. This immunosuppressive cell population can easily be generated in vitro and might be applied for the treatment of patients with GVHD or autoimmune disorders. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1645 Poster Board I-671 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 pathogens. Activation of Dectin-1 via extracts such as zymosan or more specifically with curdlan can lead to DC activation characterized by upregulation of surface molecules or secretion of cytokines. Futhermore, Dectin-1 is important for recruitment of leukocytes and production of inflammatory mediators at the site of infection.. Peroxisome proliferator-activated receptor (PPAR) and its ligands, cyclopentenone prostaglandins or thiazolidinediones, have been shown to have profound modulatory effects on B and T lymphocytes as well as DCs. Cyclopentenone prostaglandins are produced during the late phase of inflammation due to up-regulation of cyclooxygenase 2 (COX2), a key enzyme for the synthesis of cyclopentenone prostaglandins, that mediate their effects by activation of PPAR-gamma dependent and independent pathways. In our study we analyzed the effects of troglitazone (TGZ), a high affinity synthetic ligand of PPAR-gamma, on the Dectin-1 mediated activation of antigen presenting cells (APC). TGZ was added to the cell culture medium during the differention of monocyte derived DC. On day 5-6 of culture the cells were additionally treated with curdlan or zymosan and phenotypical and functional analyses were performed. Activation of PPAR-gamma resulted in a reduced stimulation of APC via Dectin-1 characterized by down-regulation of CD1a, CD83 and costimulatory molecules on the cell surface and reduced secretion of cytokines and chemokines involved in T-lymphocyte activation and recruitment including IL-1beta, TNF-a, IL-6, IL-12, MIP-1a and Rantes. Western blot analyses revealed that these inhibitory effects were mediated by the inhibition of the mitogen-activated protein (MAP) kinase pathways characterized by a reduced phosphorylation of ERK1/2 and p38. Furthermore, we found that Dectn-1 induced c-Jun phosphorylation was reduced by pretreatment with TGZ. In addition, Dectin-1 induced nuclear translocation of NF-kappaB family members RelB and cRel was dramatically reduced in APC incubated with TGZ. The observed inhibition of signaling pathways was not due to a reduced expression of Dectin-1, phosphorylation of Syk or increased secretion of IL-10 and TGF-beta. TGZ had no effect on the Dectin-1 induced phosphorylation and nuclear expression of NFAT. Our data demonstrate that PPAR-gamma ligands inhibit Dectin-1 mediated activation by interfering with the MAP kinase, c-Jun and NF-kappaB signaling pathways, thus confirming their important role as a negative feedback mechanisms in the regulation of potentially harmful inflammation signaling pathways. Disclosures No relevant conflicts of interest to declare.

Description: Dectin-1 is the major receptor for fungal β-glucans. The activation of Dectin-1 leads to the up-regulation of surface molecules on dendritic cells (DCs) and cytokine secretion. Furthermore, Dectin-1 is important for the recruitment of leukocytes and the production of inflammatory mediators. Peroxisome proliferator–activated receptor-γ (PPAR-γ) and its ligands, cyclopentenone prostaglandins or thiazolidinediones, have modulatory effects on B-cell, T-cell, and DC function. In the present study, we analyzed the effects of troglitazone (TGZ), a high-affinity synthetic PPAR-γ ligand, on the Dectin-1–mediated activation of monocyte-derived human DCs. Dectin-1–mediated activation of DCs was inhibited by TGZ, as shown by down-regulation of costimulatory molecules and reduced secretion of cytokines and chemokines involved in T-lymphocyte activation. Furthermore, TGZ inhibited the T-cell–stimulatory capacity of DCs. These effects were not due to a diminished expression of Dectin-1 or to a reduced phosphorylation of spleen tyrosine kinase; they were mediated by the inhibition of downstream signaling molecules such as mitogen-activated protein kinases and nuclear factor-κB. Furthermore, curdlan-mediated accumulation of caspase recruitment domain 9 (CARD9) in the cytosol was inhibited by TGZ. Our data demonstrate that the PPAR-γ ligand TGZ inhibits Dectin-1–mediated activation by interfering with CARD9, mitogen-activated protein kinase, and nuclear factor-κB signaling pathways. This confirms their important role as negative-feedback regulators of potentially harmful inflammatory responses.