Monocyte-derived dendritic cells display a highly activated phenotype and altered function in patients with familial Mediterranean fever.

Dendritic cells (DCs) are sentinels of the immune system that bridge innate and adaptive immunity. By capturing antigens in peripheral tissue, processing and presenting them with concurrent expression of co-stimulatory molecules and cytokine secretion they control and modulate immune reactions. Through pattern recognition receptors, DCs sense molecules that are associated with infection or tissue damage, frequently resulting in the formation of inflammasomes upon intracellular stimulation. The inherited autoinflammatory familial Mediterranean fever (FMF) is associated with deregulated activity of the pyrin inflammasome leading to acute inflammatory episodes. However, differentiation and function of DCs in this disease are as yet unclear. Therefore, we first determined DC subpopulation frequency in peripheral blood of a cohort of FMF patients. Joint evaluation without classification according to specific patient characteristics, such as mutational status, did not disclose significant differences compared to healthy controls. For the further examination of phenotype and function, we used immature and mature monocyte-derived DCs (imMo-DCs, mMo-DCs) that were generated in vitro from FMF patients. Immunophenotypical analysis of imMo-DCs revealed a significantly elevated expression of CD83, CD86 and human leukocyte antigen D-related (HLA-DR) as well as a significant down-regulation of CD206, CD209 and glycoprotein NMB (GPNMB) in our FMF patient group. Furthermore, FMF imMo-DCs presented a significantly higher capacity to migrate and to stimulate the proliferation of unmatched allogeneic T cells. Finally, the transition towards a more mature, and therefore activated, phenotype was additionally reinforced by the fact that peripheral blood DC populations in FMF patients exhibited significantly increased expression of the co-stimulatory molecule CD86.

Confirmation and next-generation sequencing of allele HLA-B*35:279 found in a family of a leukaemia patient with Western Asia origin.

The confirmation of novel allele HLA-B*35:279 in a family of a leukaemia patient with Western Asia origin is reported. Moreover, next-generation sequencing (NGS) resulted in whole-gene sequence data and revealed the inheritance of HLA-B*35:279 on the paternal haplotype.

Characterization of BAX inhibitor-1 as a novel leukemia-associated antigen.

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.

Post-transcriptional regulation of adapter molecules by IL-10 inhibits TLR-mediated activation of antigen-presenting cells.

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.

Zoledronic acid inhibits the function of Toll-like receptor 4 ligand activated monocyte-derived dendritic cells.

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.

Proteasome inhibitors: antitumor effects and beyond.

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.

Rituximab consolidation after high-dose chemotherapy and autologous blood stem cell transplantation in follicular and mantle cell lymphoma: a prospective, multicenter phase II study.

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 approximately 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 < 0.001). 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.

Delivery of tumor-derived RNA for the induction of cytotoxic T-lymphocytes.

Dendritic cells (DC) are professional antigen-presenting cells playing a central role in the induction of antigen-specific cytotoxic T-lymphocytes (CTL). We analyzed the efficiency of tumor RNA transfection into DC using different sources of RNA as well as delivery strategies including electroporation, lipofection and CD71-receptor-based delivery. To evaluate the sensitivity of these approaches, we utilized in vitro transcribed enhanced green fluorescence protein (EGFP)-RNA and whole tumor RNA from EGFP-transfected renal cell carcinoma cell line N43. We demonstrate that electroporation was the most effective way yielding about 30% EGFP positive cells while less than 1% of DC expressed EGFP using the transferrin receptor transfection system. Delivery of RNA with liposomes resulted in 17.5% of EGFP positive cells depending on the RNA amount. However, when these approaches were applied to transduce DC with RNA derived from the A498 cell line for T-cell priming, tumor-specific CTL could be induced using all delivery strategies suggesting that this technology has the potential to induce cytotoxic T-cell response even when low level of antigen is delivered. Furthermore, we demonstrate that amplification of whole tumor messenger RNA (mRNA) as well as the use of total instead of purified mRNA can be utilized for stimulating tumor-specific CTL responses.

The epithelial tumor antigen MUC1 is expressed in hematological malignancies and is recognized by MUC1-specific cytotoxic T-lymphocytes.

The epithelial mucin MUC1 is overexpressed on the cell surface of many epithelial malignancies as well as on some B-cell lymphomas and multiple myelomas. Recently, we identified two HLA-A2-restricted T-cell epitopes derived from the MUC1 protein. To further extend the potential application of these peptides, we analyzed the expression of MUC1 on blast cells from patients with acute myelogenous leukemia (AML; n = 43) and several other hematological malignancies including acute lymphoblastic leukemia (n = 24), chronic lymphocytic leukemia (n = 36), hairy cell leukemia (n = 9), follicular lymphoma (n = 7), and multiple myeloma (n = 12). Using reverse transcription-PCR and MUC1-specific monoclonal antibodies, MUC1 expression was found in 67% of AML samples and 92% of myeloma samples. To analyze the presentation of MUC1 peptides by primary AML blasts, we induced MUC1-specific CTLs in vitro using peptide-pulsed dendritic cells from HLA-A2+ healthy donors as antigen-presenting cells. These CTLs efficiently lysed in an antigen-specific and HLA-A2-restricted manner not only target cells pulsed with the antigenic peptide but also tumor cell lines including multiple myeloma cells and primary AML blasts that constitutively expressed both MUC1 and HLA-A2. The specificity of the CTLs was confirmed in a cold target inhibition assay. Our data demonstrate that MUC1-derived peptides are tumor antigens in AML and several other hematological malignancies that could potentially be used for immunotherapeutic approaches.

Dendritic cell-based vaccines in patients with hematological malignancies.

The epithelial mucin MUC1 is overexpressed on many epithelial malignancies as well as on some B-cell lymphomas and multiple myelomas. In the present study, we described MUC1 expression also on primary AML blasts. To analyze the presentation of MUC1-derived HLA-A2 restricted peptides by primary AML blasts, we induced MUC1-specific cytotoxic T-lymphocytes (CTLs) in vitro using peptide pulsed dendritic cells from HLA-A2+ healthy donors as antigen-presenting cells. These CTLs efficiently lysed primary AML blasts that constitutively expressed both MUC1 and HLA-A2. The specificity of the CTLs was confirmed in a cold target inhibition assay. Our data demonstrate that MUC1-derived peptides are tumor antigens in AML which could potentially be used for immunotherapeutic approaches.

Ex vivo expansion of normal progenitor cells from acute myeloid leukemia cell-contaminated CD34+ peripheral blood progenitor cells after mafosfamide purging.

The rationale for purging of autologous acute myeloid leukemia (AML) grafts is to eradicate contaminating leukemic cells that might contribute to relapse. However, in vitro purging generally delays post-transplant hematopoietic recovery, thus increasing treatment-related complication rates. Theoretically, this prolonged aplasia might be shortened by the additional transplantation of ex vivo-generated progenitor cells. Therefore, we investigated whether nonleukemic progenitors could be expanded ex vivo from AML cell-contaminated CD34(+) peripheral blood progenitor cell (PBPC) preparations. Nonleukemic CD34(+)-selected PBPC and AML cells (Kasumi-1, KG-1, primary AML blasts) were cultured in cytokine-supplemented liquid culture for up to 19 days. Cells were used either unmanipulated or following in vitro purging with mafosfamide (30, 50, 75 microg/ml). Ex vivo-generated cells were assessed by flow cytometry, progenitor cell assays, and polymerase chain reaction. Without prior purging, ex vivo culture markedly amplified AML cells as well as nonleukemic CD34(+) PBPC (day 12: Kasumi-1, 18.5 +/- 0.6-fold; KG-1, 52.2 +/- 2.6-fold; CD34(+), 74.1 +/- 5.6-fold). Co-culture with leukemic cells did not affect CD34(+) cell growth and vice versa. Following in vitro purging, CD34(+) PBPC were expanded even at the highest mafosfamide dose (day 19: 25 +/- 15-fold), whereas leukemic cells were markedly depleted (approx. 1.5 log). Furthermore, normal colony-forming units (CFU) could be effectively recovered (day 19: 10 +/- 3.1% of prepurging input CFU), whereas CFU-L were depleted to undetectable levels in six of seven experiments. Finally, leukemic cells were undetectable following ex vivo co-culture of purged cells (CD34(+) PBPC plus 10% Kasumi-1 cells or primary blasts), but were clearly detectable without purging. Taken together, these data demonstrated that ex vivo expansion of normal progenitors from mafosfamide-purged AML cell-contaminated grafts might be feasible.

Quality assurance in RT-PCR-based BCR/ABL diagnostics--results of an interlaboratory test and a standardization approach.

Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.

Tumor necrosis factor alpha and CD40 ligand antagonize the inhibitory effects of interleukin 10 on T-cell stimulatory capacity of dendritic cells.

Interleukin (IL)-10 secretion by tumor cells was demonstrated to be one of the mechanisms by which tumor cells can escape immunological recognition and destruction. In dendritic cells (DCs), which are currently used for vaccination therapies for malignant diseases, IL-10 inhibits IL-12 production and induces a state of antigen-specific anergy in CD4- and CD8-positive T cells. We therefore analyzed the effects of different activation stimuli including lipopolysaccharide (LPS), tumor necrosis factor (TNF)-alpha, and CD40 ligation on IL-10 mediated inhibition of DC development and stimulatory capacity. In our study, the addition of IL-10 to the cultures containing granulocyte/macrophage-colony stimulating factor and IL-4 with or without LPS completely inhibited the generation of DCs from peripheral blood monocytes. These cells remained CD14 positive and expressed high levels of IL-10 receptor (IL-10R), suggesting that IL-10 mediates its effects by up-regulating the IL-10R. In contrast, the simultaneous incubation of monocytes with IL-10 and TNF-alpha or soluble CD40 ligand (sCD40L) resulted in the generation of CD83-positive DCs, induction of nuclear localized RelB, and inhibition of IL-10R up-regulation. DCs grown in the presence of IL-10 and TNF-alpha or sCD40L elicited efficient CTL responses against viral and tumor-associated peptide antigens, which, however, were reduced as compared with DC cultures generated without IL-10. IL-10 decreased the production of IL-6 and the expression of IL-12 in the presence of TNF-alpha or sCD40L, but it had no effect on IL-15, IL-18, and TNF-alpha secretion. Our results show that TNF-alpha or CD40 ligation can antagonize the IL-10-mediated inhibition on DC function, suggesting that depending on activation stimuli, the presence of IL-10 does not necessarily result in T-cell anergy.

Expression of vascular endothelial growth factor (VEGF) and its receptors in human neuroblastoma.

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.

Approaches to dendritic cell-based immunotherapy after peripheral blood stem cell transplantation.

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.

Expression of MUC-1 epitopes on normal bone marrow: implications for the detection of micrometastatic tumor cells.

PURPOSE: The expression of the carcinoma-associated mucin MUC-1 is thought to be restricted to epithelial cells and is used for micrometastatic tumor cell detection in patients with solid tumors, including those with breast cancer. Little is known, however, about the expression of MUC-1 epitopes in normal hematopoietic cells. MATERIALS AND METHODS: MUC-1 expression was analyzed by flow cytometry and immunocytology on bone marrow (BM) mononuclear cells and purified CD34+ cells from healthy volunteers, using different anti-MUC-1-specific monoclonal antibodies. In addition, Western blotting of MUC-1 proteins was performed. RESULTS: Surprisingly, 2% to 10% of normal human BM mononuclear cells expressed MUC-1, as defined by the anti-MUC-1 antibodies BM-2 (2E11), BM-7, 12H12, MAM-6, and HMFG-1. In contrast, two antibodies recognizing the BM-8 and the HMFG-2 epitopes of MUC-1 were not detected. MUC-1+ cells from normal BM consisted primarily of erythroblasts and normoblasts. In agreement with this, normal CD34+ cells cultured in vitro to differentiate into the erythroid lineage showed a strong MUC-1 expression on day 7 proerythroblasts. Western blotting of these cells confirmed that the reactive species is the known high molecular weight MUC-1 protein. CONCLUSION: Our data demonstrate that some MUC-1 epitopes are expressed on normal BM cells and particularly on cells of the erythroid lineage. Hence the application of anti-MUC-1 antibodies for disseminated tumor cell detection in BM or peripheral blood progenitor cells may provide false-positive results, and only carefully evaluated anti-MUC-1 antibodies (eg, HMFG-2) might be selected. Furthermore, MUC-1-targeted immunotherapy in cancer patients might be hampered by the suppression of erythropoiesis.

Immune regulatory loops determine productive interactions within human T lymphocyte-dendritic cell clusters.

Help for the induction of cytolytic T lymphocytes is mediated by dendritic cells (DC) that are conditioned by CD40 signaling. We identified tumor necrosis factor family member CD27L/CD70, which is expressed by cytolytic T lymphocytes on interaction with DC to control CD154 (CD40L) up-regulation on CD45RA+ helper T cells for subsequent DC stimulation. The results show that the initiation of a cytolytic immune response is determined by regulatory circuits, requiring simultaneous activation and differentiation of all cells involved in T lymphocyte-DC cluster formation.

Generation of functional human dendritic cells from adherent peripheral blood monocytes by CD40 ligation in the absence of granulocyte-macrophage colony-stimulating factor.

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.